Sample records for target molecules involved

  1. Cell signaling molecules as drug targets in lung cancer: an overview.

    PubMed

    Mukherjee, Tapan K; Paul, Karan; Mukhopadhyay, Srirupa

    2011-07-01

    Lung being one of the vital and essential organs in the body, lung cancer is a major cause of mortality in the modern human society. Lung cancer can be broadly subdivided into nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Although NSCLC is sometimes treated with surgery, the advanced and metastatic NSCLC and SCLC usually respond better to chemotherapy and radiation. The most important targets of these chemotherapeutic agents are various intracellular signaling molecules. The primary focus of this review article is to summarize the description of various cell signaling molecules involved in lung cancer development and their regulation by chemotherapeutic agents. Extensive research work in recent years has identified several cellular signaling molecules that may be intricately involved in the complexity of lung cancer. Some of these cell signaling molecules are epidermal growth factor receptors, vascular endothelial growth factor receptors, mammalian target of rapamycin, mitogen-activated protein kinase phosphatase-1, peroxisome proliferator-activated receptor-gamma, matrix metalloproteinases and receptor for advanced glycation end-products. The present review will strengthen our current knowledge regarding the efficacy of the above-mentioned cell signaling molecules as potential beneficial drug targets against lung cancer.

  2. Liver cell-targeted delivery of therapeutic molecules.

    PubMed

    Kang, Jeong-Hun; Toita, Riki; Murata, Masaharu

    2016-01-01

    The liver is the largest internal organ in mammals and is involved in metabolism, detoxification, synthesis of proteins and lipids, secretion of cytokines and growth factors and immune/inflammatory responses. Hepatitis, alcoholic or non-alcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. Safe and efficient delivery of therapeutic molecules (drugs, genes or proteins) into the liver is very important to increase the clinical efficacy of these molecules and to reduce their side effects in other organs. Several liver cell-targeted delivery systems have been developed and tested in vivo or ex vivo/in vitro. In this review, we discuss the literature concerning liver cell-targeted delivery systems, with a particular emphasis on the results of in vivo studies.

  3. Small molecules targeting viral RNA.

    PubMed

    Hermann, Thomas

    2016-11-01

    Highly conserved noncoding RNA (ncRNA) elements in viral genomes and transcripts offer new opportunities to expand the repertoire of drug targets for the development of antiinfective therapy. Ligands binding to ncRNA architectures are able to affect interactions, structural stability or conformational changes and thereby block processes essential for viral replication. Proof of concept for targeting functional RNA by small molecule inhibitors has been demonstrated for multiple viruses with RNA genomes. Strategies to identify antiviral compounds as inhibitors of ncRNA are increasingly emphasizing consideration of drug-like properties of candidate molecules emerging from screening and ligand design. Recent efforts of antiviral lead discovery for RNA targets have provided drug-like small molecules that inhibit viral replication and include inhibitors of human immunodeficiency virus (HIV), hepatitis C virus (HCV), severe respiratory syndrome coronavirus (SARS CoV), and influenza A virus. While target selectivity remains a challenge for the discovery of useful RNA-binding compounds, a better understanding is emerging of properties that define RNA targets amenable for inhibition by small molecule ligands. Insight from successful approaches of targeting viral ncRNA in HIV, HCV, SARS CoV, and influenza A will provide a basis for the future exploration of RNA targets for therapeutic intervention in other viral pathogens which create urgent, unmet medical needs. Viruses for which targeting ncRNA components in the genome or transcripts may be promising include insect-borne flaviviruses (Dengue, Zika, and West Nile) and filoviruses (Ebola and Marburg). WIREs RNA 2016, 7:726-743. doi: 10.1002/wrna.1373 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

  4. Single-Molecule View of Small RNA-Guided Target Search and Recognition.

    PubMed

    Globyte, Viktorija; Kim, Sung Hyun; Joo, Chirlmin

    2018-05-20

    Most everyday processes in life involve a necessity for an entity to locate its target. On a cellular level, many proteins have to find their target to perform their function. From gene-expression regulation to DNA repair to host defense, numerous nucleic acid-interacting proteins use distinct target search mechanisms. Several proteins achieve that with the help of short RNA strands known as guides. This review focuses on single-molecule advances studying the target search and recognition mechanism of Argonaute and CRISPR (clustered regularly interspaced short palindromic repeats) systems. We discuss different steps involved in search and recognition, from the initial complex prearrangement into the target-search competent state to the final proofreading steps. We focus on target search mechanisms that range from weak interactions, to one- and three-dimensional diffusion, to conformational proofreading. We compare the mechanisms of Argonaute and CRISPR with a well-studied target search system, RecA.

  5. Molecular targets for small-molecule modulators of circadian clocks

    PubMed Central

    He, Baokun; Chen, Zheng

    2016-01-01

    Background Circadian clocks are endogenous timing systems that regulate various aspects of mammalian metabolism, physiology and behavior. Traditional chronotherapy refers to the administration of drugs in a defined circadian time window to achieve optimal pharmacokinetic and therapeutic efficacies. In recent years, substantial efforts have been dedicated to developing novel small-molecule modulators of circadian clocks. Methods Here, we review the recent progress in the identification of molecular targets of small-molecule clock modulators and their efficacies in clock-related disorders. Specifically, we examine the clock components and regulatory factors as possible molecular targets of small molecules, and we review several key clock-related disorders as promising venues for testing the preventive/therapeutic efficacies of these small molecules. Finally, we also discuss circadian regulation of drug metabolism. Results Small molecules can modulate the period, phase and/or amplitude of the circadian cycle. Core clock proteins, nuclear hormone receptors, and clock-related kinases and other epigenetic regulators are promising molecular targets for small molecules. Through these targets small molecules exert protective effects against clock-related disorders including the metabolic syndrome, immune disorders, sleep disorders and cancer. Small molecules can also modulate circadian drug metabolism and response to existing therapeutics. Conclusion Small-molecule clock modulators target clock components or diverse cellular pathways that functionally impinge upon the clock. Target identification of new small-molecule modulators will deepen our understanding of key regulatory nodes in the circadian network. Studies of clock modulators will facilitate their therapeutic applications, alone or in combination, for clock-related diseases. PMID:26750111

  6. Contraceptive Vaccines Targeting Factors Involved in Establishment of Pregnancy

    PubMed Central

    Lemons, Angela R.; Naz, Rajesh K.

    2011-01-01

    Problem Current methods of contraception lack specificity and are accompanied with serious side effects. A more specific method of contraception is needed. Contraceptive vaccines can provide most, if not all, the desired characteristics of an ideal contraceptive. Approach This article reviews several factors involved in the establishment of pregnancy, focusing on those that are essential for successful implantation. Factors that are both essential and pregnancy-specific can provide potential targets for contraception. Conclusion Using database search, 76 factors (cytokines/chemokines/growth factors/others) were identified that are involved in various steps of the establishment of pregnancy. Among these factors, three, namely chorionic gonadotropin (CG), leukemia inhibitory factor (LIF), and preimplantation factor (PIF), are found to be unique and exciting molecules. Human CG is a well-known pregnancy-specific protein that has undergone phase I and phase II clinical trials, in women, as a contraceptive vaccine with encouraging results. LIF and PIF are pregnancy-specific and essential for successful implantation. These molecules are intriguing and may provide viable targets for immunocontraception. A multiepitope vaccine combining factors/antigens involved in various steps of the fertilization cascade and pregnancy establishment, may provide a highly immunogenic and efficacious modality for contraception in humans. PMID:21481058

  7. Human mesenchymal stem cells target adhesion molecules and receptors involved in T cell extravasation.

    PubMed

    Benvenuto, Federica; Voci, Adriana; Carminati, Enrico; Gualandi, Francesca; Mancardi, Gianluigi; Uccelli, Antonio; Vergani, Laura

    2015-12-10

    Systemic delivery of bone marrow-derived mesenchymal stem cells (MSC) seems to be of benefit in the treatment of multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) sustained by migration of T cells across the brain blood barrier (BBB) and subsequent induction of inflammatory lesions into CNS. MSC have been found to modulate several effector functions of T cells. In this study, we investigated the effects of MSC on adhesion molecules and receptors on T cell surface that sustain their transendothelial migration. We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate the expression both at the mRNA and at the plasma-membrane level of α4 integrin, β2 integrin, ICAM-1 and CXCR3. In parallel, we assessed if MSC are able to modulate expression of adhesion molecules on the endothelial cells that interact with T cells during their transendothelial migration. Our in vitro analyses revealed that MSC: (i) inhibit proliferation and activation of both peripheral blood mononuclear cells (PBMC) and CD3(+)-selected lymphocytes through the release of soluble factors; (ii) exert suppressive effects on those surface molecules highly expressed by activated lymphocytes and involved in transendothelial migration; (iii) inhibit CXCL10-driven chemotaxis of CD3(+) cells; (iv) down-regulated expression of adhesion molecules on endothelial cells. Taken together, these data demonstrate that the immunosuppressive effect of MSC does not exclusively depends on their anti-proliferative activity on T cells, but also on the impairment of leukocyte migratory potential through the inhibition of the adhesion molecules and receptors that are responsible for T cell trafficking across BBB. This could suggest a new mechanism through which MSC modulate T cell responses.

  8. Target molecules detection by waveguiding in a photonic silicon membrane

    DOEpatents

    Letant, Sonia E [Livermore, CA; Van Buuren, Anthony [Livermore, CA; Terminello, Louis [Danville, CA; Hart, Bradley R [Brentwood, CA

    2006-12-26

    Disclosed herein is a porous silicon filter capable of binding and detecting biological and chemical target molecules in liquid or gas samples. A photonic waveguiding silicon filter with chemical and/or biological anchors covalently attached to the pore walls bind target molecules. The system uses transmission curve engineering principles to allow measurements to be made in situ and in real time to detect the presence of various target molecules and calculate the concentration of bound target.

  9. Target molecules detection by waveguiding in a photonic silicon membrane

    DOEpatents

    Letant, Sonia; Van Buuren, Anthony; Terminello, Louis

    2004-08-31

    Disclosed herein is a photonic silicon filter capable of binding and detecting biological and chemical target molecules in liquid or gas samples. A photonic waveguiding silicon filter with chemical and/or biological anchors covalently attached to the pore walls selectively bind target molecules. The system uses transmission curve engineering principles to allow measurements to be made in situ and in real time to detect the presence of various target molecules and determine the concentration of bound target.

  10. Identification of Direct Protein Targets of Small Molecules

    PubMed Central

    2010-01-01

    Small-molecule target identification is a vital and daunting task for the chemical biology community as well as for researchers interested in applying the power of chemical genetics to impact biology and medicine. To overcome this “target ID” bottleneck, new technologies are being developed that analyze protein–drug interactions, such as drug affinity responsive target stability (DARTS), which aims to discover the direct binding targets (and off targets) of small molecules on a proteome scale without requiring chemical modification of the compound. Here, we review the DARTS method, discuss why it works, and provide new perspectives for future development in this area. PMID:21077692

  11. Nanostructure sensor of presence and concentration of a target molecule

    NASA Technical Reports Server (NTRS)

    Schipper, John F. (Inventor)

    2009-01-01

    Method and system (i) to determine when a selected target molecule is present or absent in a fluid, (2) to estimate concentration of the target molecule in the fluid and (3) estimate possible presence of a second (different) target molecule in the fluid, by analyzing differences in resonant frequencies of vibration of a thin beam suspended in the fluid, after the fluid has moved across the beam.

  12. Small molecules targeting heterotrimeric G proteins.

    PubMed

    Ayoub, Mohammed Akli

    2018-05-05

    G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors regulating many human and animal physiological functions. Their implication in human pathophysiology is obvious with almost 30-40% medical drugs commercialized today directly targeting GPCRs as molecular entities. However, upon ligand binding GPCRs signal inside the cell through many key signaling, adaptor and regulatory proteins, including various classes of heterotrimeric G proteins. Therefore, G proteins are considered interesting targets for the development of pharmacological tools that are able to modulate their interaction with the receptors, as well as their activation/deactivation processes. In this review, old attempts and recent advances in the development of small molecules that directly target G proteins will be described with an emphasis on their utilization as pharmacological tools to dissect the mechanisms of activation of GPCR-G protein complexes. These molecules constitute a further asset for research in the "hot" areas of GPCR biology, areas such as multiple G protein coupling/signaling, GPCR-G protein preassembly, and GPCR functional selectivity or bias. Moreover, this review gives a particular focus on studies in vitro and in vivo supporting the potential applications of such small molecules in various GPCR/G protein-related diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Targeting of adhesion molecules as a therapeutic strategy in multiple myeloma.

    PubMed

    Neri, Paola; Bahlis, Nizar J

    2012-09-01

    Multiple myeloma (MM) is a clonal disorder of plasma cells that remains, for the most part, incurable despite the advent of several novel therapeutic agents. Tumor cells in this disease are cradled within the bone marrow (BM) microenvironment by an array of adhesive interactions between the BM cellular residents, the surrounding extracellular matrix (ECM) components such as fibronectin (FN), laminin, vascular cell adhesion molecule-1 (VCAM-1), proteoglycans, collagens and hyaluronan, and a variety of adhesion molecules on the surface of MM cells including integrins, hyaluronan receptors (CD44 and RHAMM) and heparan sulfate proteoglycans. Several signaling responses are activated by these interactions, affecting the survival, proliferation and migration of MM cells. An important consequence of these direct adhesive interactions between the BM/ECM and MM cells is the development of drug resistance. This phenomenon is termed "cell adhesion-mediated drug resistance" (CAM-DR) and it is thought to be one of the major mechanisms by which MM cells escape the cytotoxic effects of therapeutic agents. This review will focus on the adhesion molecules involved in the cross-talk between MM cells and components of the BM microenvironment. The complex signaling networks downstream of these adhesive molecules mediated by direct ligand binding or inside-out soluble factors signaling will also be reviewed. Finally, novel therapeutic strategies targeting these molecules will be discussed. Identification of the mediators of MM-BM interaction is essential to understand MM biology and to elucidate novel therapeutic targets for this disease.

  14. Recent advances in developing small molecules targeting RNA.

    PubMed

    Guan, Lirui; Disney, Matthew D

    2012-01-20

    RNAs are underexploited targets for small molecule drugs or chemical probes of function. This may be due, in part, to a fundamental lack of understanding of the types of small molecules that bind RNA specifically and the types of RNA motifs that specifically bind small molecules. In this review, we describe recent advances in the development and design of small molecules that bind to RNA and modulate function that aim to fill this void.

  15. Development of a microcapillary column for detecting targeted messenger RNA molecules.

    PubMed

    Ohnishi, Michihiro

    2006-03-24

    A capillary column in a rapid-flow system has been developed for detecting targeted messenger RNA (mRNA) molecules. The column has a structure made of two beds-one bed of porous microbeads and one bed of microbeads with a polythymidine base sequence. The targeted eukaryotic mRNA molecules are detected by two-step hybridization (sandwich hybridization) composed of polyadenosine selection of mRNA molecules and formation of a probe-target (targeted mRNA) hybrid. The sandwich hybridization, which is accomplished within 1 h, was tested using synthetic polydeoxynucleotides. Ten picomoles of the targeted polydeoxynucleotide were detected.

  16. Chemical genetics-based development of small molecules targeting hepatitis C virus.

    PubMed

    Jin, Guanghai; Lee, Jisu; Lee, Kyeong

    2017-09-01

    Hepatitis C virus (HCV) infection is a major worldwide problem that has emerged as one of the most significant diseases affecting humans. There are currently no vaccines or efficient therapies without side effects, despite today's advanced medical technology. Currently, the common therapy for most patients (i.e. genotype 1) is combination of HCV-specific direct-acting antivirals (DAAs). Up to 2011, the standard of care (SOC) was a combination of peg-IFNα with ribavirin (RBV). After approval of NS3/4A protease inhibitor, SOC was peg-IFNα and RBV with either the first-generation DAAs boceprevir or telaprevir. In the past several years, various novel small molecules have been discovered and some of them (i.e., HCV polymerase, protease, helicase and entry inhibitors) have undergone clinical trials. Between 2013 and 2016, the second-generation DAA drugs simeprevir, asunaprevir, daclatasvir, dasabuvir, sofosbuvir, and elbasvir were approved, as well as the combinational drugs Harvoni ® , Zepatier ® , Technivie ® , and Epclusa ® . A number of reviews have been recently published describing the structure-activity relationship (SAR) in the development of HCV inhibitors and outlining current therapeutic approaches to hepatitis C infection. Target identification involves studying a drug's mechanism of action (MOA), and a variety of target identification methods have been developed in the past few years. Chemical biology has emerged as a powerful tool for studying biological processes using small molecules. The use of chemical genetic methods is a valuable strategy for studying the molecular mechanisms of the viral lifecycle and screening for anti-viral agents. Two general screening approaches have been employed: forward and reverse chemical genetics. This review reveals information on the small molecules in HCV drug discovery by using chemical genetics for targeting the HCV protein and describes successful examples of targets identified with these methods.

  17. Small mitochondria-targeting molecules as anti-cancer agents

    PubMed Central

    Wang, Feng; Ogasawara, Marcia A.; Huang, Peng

    2009-01-01

    Alterations in mitochondrial structure and functions have long been observed in cancer cells. Targeting mitochondria as a cancer therapeutic strategy has gained momentum in the recent years. The signaling pathways that govern mitochondrial function, apoptosis and molecules that affect mitochondrial integrity and cell viability have been important topics of the recent review in the literature. In this article, we first briefly summarize the rationale and biological basis for developing mitochondrial-targeted compounds as potential anticancer agents, and then provide key examples of small molecules that either directly impact mitochondria or functionally affect the metabolic alterations in cancer cells with mitochondrial dysfunction. The main focus is on the small molecular weight compounds with potential applications in cancer treatment. We also summarize information on the drug developmental stages of the key mitochondria-targeted compounds and their clinical trial status. The advantages and potential shortcomings of targeting the mitochondria for cancer treatment are also discussed. PMID:19995573

  18. Methods to enable the design of bioactive small molecules targeting RNA.

    PubMed

    Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

    2014-02-21

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome.

  19. Single-Molecule Analysis for RISC Assembly and Target Cleavage.

    PubMed

    Sasaki, Hiroshi M; Tadakuma, Hisashi; Tomari, Yukihide

    2018-01-01

    RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

  20. Urea transporter proteins as targets for small-molecule diuretics.

    PubMed

    Esteva-Font, Cristina; Anderson, Marc O; Verkman, Alan S

    2015-02-01

    Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

  1. Methods to enable the design of bioactive small molecules targeting RNA

    PubMed Central

    Disney, Matthew D.; Yildirim, Ilyas; Childs-Disney, Jessica L.

    2014-01-01

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including Structure-Activity Relationships Through Sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

  2. [Exploring New Drug Targets through the Identification of Target Molecules of Bioactive Natural Products].

    PubMed

    Arai, Masayoshi

    2016-01-01

    With the development of cell biology and microbiology, it has become easy to culture many types of animal cells and microbes, and they are frequently used for phenotypic screening to explore medicinal seeds. On the other hand, it is recognized that cells and pathogenic microbes present in pathologic sites and infected regions of the human body display unique properties different from those under general culture conditions. We isolated several bioactive compounds from marine medicinal resources using constructed bioassay-guided separation focusing on the unique changes in the characteristics of cells and pathogenic microbes (Mycobacterium spp.) in the human body under disease conditions. In addition, we also carried out identification studies of target molecules of the bioactive compounds by methods utilizing the gene expression profile, transformants of cells or microbes, synthetic probe molecules of the isolated compounds, etc., since bioactive compounds isolated from the phenotypic screening system often target new molecules. This review presents our phenotypic screening systems, isolation of bioactive compounds from marine medicinal resources, and target identification of bioactive compounds.

  3. Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

    PubMed

    Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

    2017-03-01

    Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by

  4. Market uptake of biologic and small-molecule--targeted oncology drugs in Europe.

    PubMed

    Obradovic, Marko; Mrhar, Ales; Kos, Mitja

    2009-12-01

    The aim of this study was to investigate the market uptake of biologic and small-molecule-targeted oncology drugs in Europe. Targeted oncology drugs that were used in one of the selected European countries before the end of 2007 were eligible for inclusion in the analysis. The following European countries were included: Austria, Croatia, France, Germany, Hungary, Italy, Slovenia, and the United Kingdom. Monetary market uptake of targeted oncology drugs was assessed by using sales data (in euros) obtained from 2 large data- bases for the period 1997-2007. Market uptake was assessed in terms of expenditures for specific drugs in euros per capita and in market shares. The monetary market uptake of targeted oncology drugs had an exponential growth from 1997 to 2007 in all comparison countries and reached 40% of the total oncology drug market in 2007. Although the various European countries allocate substantially different amounts of resources per capita for oncology drugs, the share of expenditures attributed to targeted oncology drugs did not differ substantially among the countries. Biologic molecules were used in clinical practice before the small-molecule-targeted oncology drugs. Targeted oncology drugs that were introduced first to clinical practice in most of the comparison countries (ie, rituximab, trastuzumab, imatinib mesylate) maintained the leading positions on the market throughout the period of the analysis. In 2007, approximately 25% of all expenditures for oncology drugs were attributed to biologic oncology drugs, and approximately 15% were spent on small-molecule-targeted oncology drugs. Expenditures on targeted oncology drugs have been increasing exponentially in Europe throughout the past decade and have reached a 40% share of the oncology drug market. As of 2007, the market share of biologic oncology drugs was higher than the market share of small-molecule-targeted oncology drugs. Copyright 2009 Excerpta Medica Inc. All rights reserved.

  5. Approaches to Validate and Manipulate RNA Targets with Small Molecules in Cells.

    PubMed

    Childs-Disney, Jessica L; Disney, Matthew D

    2016-01-01

    RNA has become an increasingly important target for therapeutic interventions and for chemical probes that dissect and manipulate its cellular function. Emerging targets include human RNAs that have been shown to directly cause cancer, metabolic disorders, and genetic disease. In this review, we describe various routes to obtain bioactive compounds that target RNA, with a particular emphasis on the development of small molecules. We use these cases to describe approaches that are being developed for target validation, which include target-directed cleavage, classic pull-down experiments, and covalent cross-linking. Thus, tools are available to design small molecules to target RNA and to identify the cellular RNAs that are their targets.

  6. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

    PubMed

    Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D

    2016-06-01

    Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example. Copyright © 2016. Published by Elsevier Ltd.

  7. Targeting RNA in mammalian systems with small molecules.

    PubMed

    Donlic, Anita; Hargrove, Amanda E

    2018-05-03

    The recognition of RNA functions beyond canonical protein synthesis has challenged the central dogma of molecular biology. Indeed, RNA is now known to directly regulate many important cellular processes, including transcription, splicing, translation, and epigenetic modifications. The misregulation of these processes in disease has led to an appreciation of RNA as a therapeutic target. This potential was first recognized in bacteria and viruses, but discoveries of new RNA classes following the sequencing of the human genome have invigorated exploration of its disease-related functions in mammals. As stable structure formation is evolving as a hallmark of mammalian RNAs, the prospect of utilizing small molecules to specifically probe the function of RNA structural domains and their interactions is gaining increased recognition. To date, researchers have discovered bioactive small molecules that modulate phenotypes by binding to expanded repeats, microRNAs, G-quadruplex structures, and RNA splice sites in neurological disorders, cancers, and other diseases. The lessons learned from achieving these successes both call for additional studies and encourage exploration of the plethora of mammalian RNAs whose precise mechanisms of action remain to be elucidated. Efforts toward understanding fundamental principles of small molecule-RNA recognition combined with advances in methodology development should pave the way toward targeting emerging RNA classes such as long noncoding RNAs. Together, these endeavors can unlock the full potential of small molecule-based probing of RNA-regulated processes and enable us to discover new biology and underexplored avenues for therapeutic intervention in human disease. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA in Disease and Development > RNA in Disease. © 2018 Wiley Periodicals, Inc.

  8. New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

    PubMed Central

    Maugg, Doris; Rothenaigner, Ina; Schorpp, Kenji; Potukuchi, Harish Kumar; Korsching, Eberhard; Baumhoer, Daniel; Hadian, Kamyar

    2015-01-01

    Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. PMID:26039064

  9. Rational Design of Small Molecules Targeting Oncogenic Noncoding RNAs from Sequence.

    PubMed

    Disney, Matthew D; Angelbello, Alicia J

    2016-12-20

    The discovery of RNA catalysis in the 1980s and the dissemination of the human genome sequence at the start of this century inspired investigations of the regulatory roles of noncoding RNAs in biology. In fact, the Encyclopedia of DNA Elements (ENCODE) project has shown that only 1-2% of the human genome encodes protein, yet 75% is transcribed into RNA. Functional studies both preceding and following the ENCODE project have shown that these noncoding RNAs have important roles in regulating gene expression, developmental timing, and other critical functions. RNA's diverse roles are often a consequence of the various folds that it adopts. The single-stranded nature of the biopolymer enables it to adopt intramolecular folds with noncanonical pairings to lower its free energy. These folds can be scaffolds to bind proteins or to form frameworks to interact with other RNAs. Not surprisingly, dysregulation of certain noncoding RNAs has been shown to be causative of disease. Given this as the background, it is easy to see why it would be useful to develop methods that target RNA and manipulate its biology in rational and predictable ways. The antisense approach has afforded strategies to target RNAs via Watson-Crick base pairing and has typically focused on targeting partially unstructured regions of RNA. Small molecule strategies to target RNA would be desirable not only because compounds could be lead optimized via medicinal chemistry but also because structured regions within an RNA of interest could be targeted to directly interfere with RNA folds that contribute to disease. Additionally, small molecules have historically been the most successful drug candidates. Until recently, the ability to design small molecules that target non-ribosomal RNAs has been elusive, creating the perception that they are "undruggable". In this Account, approaches to demystify targeting RNA with small molecules are described. Rather than bulk screening for compounds that bind to singular

  10. Molecules that target nucleophosmin for cancer treatment: an update

    PubMed Central

    Di Matteo, Adele; Franceschini, Mimma; Chiarella, Sara; Rocchio, Serena; Travaglini-Allocatelli, Carlo; Federici, Luca

    2016-01-01

    Nucleophosmin is a highly and ubiquitously expressed protein, mainly localized in nucleoli but able to shuttle between nucleus and cytoplasm. Nucleophosmin plays crucial roles in ribosome maturation and export, centrosome duplication, cell cycle progression, histone assembly and response to a variety of stress stimuli. Much interest in this protein has arisen in the past ten years, since the discovery of heterozygous mutations in the terminal exon of the NPM1 gene, which are the most frequent genetic alteration in acute myeloid leukemia. Nucleophosmin is also frequently overexpressed in solid tumours and, in many cases, its overexpression correlates with mitotic index and metastatization. Therefore it is considered as a promising target for the treatment of both haematologic and solid malignancies. NPM1 targeting molecules may suppress different functions of the protein, interfere with its subcellular localization, with its oligomerization properties or drive its degradation. In the recent years, several such molecules have been described and here we review what is currently known about them, their interaction with nucleophosmin and the mechanistic basis of their toxicity. Collectively, these molecules exemplify a number of different strategies that can be adopted to target nucleophosmin and we summarize them at the end of the review. PMID:27058426

  11. A small molecule nanodrug consisting of amphiphilic targeting ligand-chemotherapy drug conjugate for targeted cancer therapy.

    PubMed

    Mou, Quanbing; Ma, Yuan; Zhu, Xinyuan; Yan, Deyue

    2016-05-28

    Targeted drug delivery is a broadly applicable approach for cancer therapy. However, the nanocarrier-based targeted delivery system suffers from batch-to-batch variation, quality concerns and carrier-related toxicity issues. Thus, to develop a carrier-free targeted delivery system with nanoscale characteristics is very attractive. Here, a novel targeting small molecule nanodrug self-delivery system consisting of targeting ligand and chemotherapy drug was constructed, which combined the advantages of small molecules and nano-assemblies together and showed excellent targeting ability and long blood circulation time with well-defined structure, high drug loading ratio and on-demand drug release behavior. As a proof-of-concept, lactose (Lac) and doxorubicin (DOX) were chosen as the targeting ligand and chemotherapy drug, respectively. Lac and DOX were conjugated through a pH-responsive hydrazone group. For its intrinsic amphiphilic property, Lac-DOX conjugate could self-assemble into nanoparticles in water. Both in vitro and in vivo assays indicated that Lac-DOX nanoparticles exhibited enhanced anticancer activity and weak side effects. This novel active targeting nanodrug delivery system shows great potential in cancer therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules

    NASA Astrophysics Data System (ADS)

    Veetil, Aneesh T.; Chakraborty, Kasturi; Xiao, Kangni; Minter, Myles R.; Sisodia, Sangram S.; Krishnan, Yamuna

    2017-12-01

    Achieving triggered release of small molecules with spatial and temporal precision at designated cells within an organism remains a challenge. By combining a cell-targetable, icosahedral DNA-nanocapsule loaded with photoresponsive polymers, we show cytosolic delivery of small molecules with the spatial resolution of single endosomes in specific cells in Caenorhabditis elegans. Our technology can report on the extent of small molecules released after photoactivation as well as pinpoint the location at which uncaging of the molecules occurred. We apply this technology to release dehydroepiandrosterone (DHEA), a neurosteroid that promotes neurogenesis and neuron survival, and determined the timescale of neuronal activation by DHEA, using light-induced release of DHEA from targeted DNA nanocapsules. Importantly, sequestration inside the DNA capsule prevents photocaged DHEA from activating neurons prematurely. Our methodology can in principle be generalized to diverse neurostimulatory molecules.

  13. Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines.

    PubMed

    Zhang, Xinyuan; Zheng, Nan; Rosania, Gus R

    2008-09-01

    Cell-based molecular transport simulations are being developed to facilitate exploratory cheminformatic analysis of virtual libraries of small drug-like molecules. For this purpose, mathematical models of single cells are built from equations capturing the transport of small molecules across membranes. In turn, physicochemical properties of small molecules can be used as input to simulate intracellular drug distribution, through time. Here, with mathematical equations and biological parameters adjusted so as to mimic a leukocyte in the blood, simulations were performed to analyze steady state, relative accumulation of small molecules in lysosomes, mitochondria, and cytosol of this target cell, in the presence of a homogenous extracellular drug concentration. Similarly, with equations and parameters set to mimic an intestinal epithelial cell, simulations were also performed to analyze steady state, relative distribution and transcellular permeability in this non-target cell, in the presence of an apical-to-basolateral concentration gradient. With a test set of ninety-nine monobasic amines gathered from the scientific literature, simulation results helped analyze relationships between the chemical diversity of these molecules and their intracellular distributions.

  14. Target identification for small bioactive molecules: finding the needle in the haystack.

    PubMed

    Ziegler, Slava; Pries, Verena; Hedberg, Christian; Waldmann, Herbert

    2013-03-04

    Identification and confirmation of bioactive small-molecule targets is a crucial, often decisive step both in academic and pharmaceutical research. Through the development and availability of several new experimental techniques, target identification is, in principle, feasible, and the number of successful examples steadily grows. However, a generic methodology that can successfully be applied in the majority of the cases has not yet been established. Herein we summarize current methods for target identification of small molecules, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound. We describe the most frequently employed experimental approaches for target identification and provide several representative examples illustrating the state-of-the-art. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be most powerful and most frequently applied. To provide guidance for rapid entry into the field and based on our own experience we propose a typical workflow for target identification, which centers on the application of chemical proteomics as the key step to generate hypotheses for potential target proteins. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

    PubMed Central

    Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K.; Ekins, Sean

    2014-01-01

    We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules. PMID:25534741

  16. Design of a bioactive small molecule that targets r(AUUCU) repeats in spinocerebellar ataxia 10.

    PubMed

    Yang, Wang-Yong; Gao, Rui; Southern, Mark; Sarkar, Partha S; Disney, Matthew D

    2016-06-01

    RNA is an important target for chemical probes of function and lead therapeutics; however, it is difficult to target with small molecules. One approach to tackle this problem is to identify compounds that target RNA structures and utilize them to multivalently target RNA. Here we show that small molecules can be identified to selectively bind RNA base pairs by probing a library of RNA-focused small molecules. A small molecule that selectively binds AU base pairs informed design of a dimeric compound (2AU-2) that targets the pathogenic RNA, expanded r(AUUCU) repeats, that causes spinocerebellar ataxia type 10 (SCA10) in patient-derived cells. Indeed, 2AU-2 (50 nM) ameliorates various aspects of SCA10 pathology including improvement of mitochondrial dysfunction, reduced activation of caspase 3, and reduction of nuclear foci. These studies provide a first-in-class chemical probe to study SCA10 RNA toxicity and potentially define broadly applicable compounds targeting RNA AU base pairs in cells.

  17. TB Mobile: a mobile app for anti-tuberculosis molecules with known targets

    PubMed Central

    2013-01-01

    Background An increasing number of researchers are focused on strategies for developing inhibitors of Mycobacterium tuberculosis (Mtb) as tuberculosis (TB) drugs. Results In order to learn from prior work we have collated information on molecules screened versus Mtb and their targets which has been made available in the Collaborative Drug Discovery (CDD) database. This dataset contains published data on target, essentiality, links to PubMed, TBDB, TBCyc (which provides a pathway-based visualization of the entire cellular biochemical network) and human homolog information. The development of mobile cheminformatics apps could lower the barrier to drug discovery and promote collaboration. Therefore we have used this set of over 700 molecules screened versus Mtb and their targets to create a free mobile app (TB Mobile) that displays molecule structures and links to the bioinformatics data. By input of a molecular structures and performing a similarity search within the app we can infer potential targets or search by targets to retrieve compounds known to be active. Conclusions TB Mobile may assist researchers as part of their workflow in identifying potential targets for hits generated from phenotypic screening and in prioritizing them for further follow-up. The app is designed to lower the barriers to accessing this information, so that all researchers with an interest in combatting this deadly disease can use it freely to the benefit of their own efforts. PMID:23497706

  18. Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines

    PubMed Central

    Zhang, Xinyuan; Zheng, Nan

    2008-01-01

    Cell-based molecular transport simulations are being developed to facilitate exploratory cheminformatic analysis of virtual libraries of small drug-like molecules. For this purpose, mathematical models of single cells are built from equations capturing the transport of small molecules across membranes. In turn, physicochemical properties of small molecules can be used as input to simulate intracellular drug distribution, through time. Here, with mathematical equations and biological parameters adjusted so as to mimic a leukocyte in the blood, simulations were performed to analyze steady state, relative accumulation of small molecules in lysosomes, mitochondria, and cytosol of this target cell, in the presence of a homogenous extracellular drug concentration. Similarly, with equations and parameters set to mimic an intestinal epithelial cell, simulations were also performed to analyze steady state, relative distribution and transcellular permeability in this non-target cell, in the presence of an apical-to-basolateral concentration gradient. With a test set of ninety-nine monobasic amines gathered from the scientific literature, simulation results helped analyze relationships between the chemical diversity of these molecules and their intracellular distributions. Electronic supplementary material The online version of this article (doi:10.1007/s10822-008-9194-7) contains supplementary material, which is available to authorized users. PMID:18338229

  19. Anticancer molecules targeting fibroblast growth factor receptors.

    PubMed

    Liang, Guang; Liu, Zhiguo; Wu, Jianzhang; Cai, Yuepiao; Li, Xiaokun

    2012-10-01

    The fibroblast growth factor receptor (FGFR) family includes four highly conserved receptor tyrosine kinases: FGFR1-4. Upon ligand binding, FGFRs activate an array of downstream signaling pathways, such as the mitogen activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K)/Akt pathways. These FGFR cascades play crucial roles in tumor cell proliferation, angiogenesis, migration, and survival. The combination of knockdown studies and pharmaceutical inhibition in preclinical models demonstrates that FGFRs are attractive targets for therapeutic intervention in cancer. Multiple FGFR inhibitors with various structural skeletons have been designed, synthesized, and evaluated. Reviews on FGFRs have recently focused on FGFR signaling, pathophysiology, and functions in cancer or other diseases. In this article, we review recent advances in structure-activity relationships (SAR) of FGFR inhibitors, as well as the FGFR-targeting drug design strategies currently employed in targeting deregulated FGFRs by antibodies and small molecule inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. A structural biology perspective on bioactive small molecules and their plant targets.

    PubMed

    Kumari, Selva; van der Hoorn, Renier A L

    2011-10-01

    Structural biology efforts in recent years have generated numerous co-crystal structures of bioactive small molecules interacting with their plant targets. These studies include the targets of various phytohormones, pathogen-derived effectors, herbicides and other bioactive compounds. Here we discuss that this collection of structures contains excellent examples of nine collective observations: molecular glues, allostery, inhibitors, molecular mimicry, promiscuous binding sites, unexpected electron densities, natural selection at atomic resolution, and applications in structure-guided mutagenesis and small molecule design. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Importance of target-mediated drug disposition for small molecules.

    PubMed

    Smith, Dennis A; van Waterschoot, Robert A B; Parrott, Neil J; Olivares-Morales, Andrés; Lavé, Thierry; Rowland, Malcolm

    2018-06-18

    Target concentration is typically not considered in drug discovery. However, if targets are expressed at relatively high concentrations and compounds have high affinity, such that most of the drug is bound to its target, in vitro screens can give unreliable information on compound affinity. In vivo, a similar situation will generate pharmacokinetic (PK) profiles that deviate greatly from those normally expected, owing to target binding affecting drug distribution and clearance. Such target-mediated drug disposition (TMDD) effects on small molecules have received little attention and might only become apparent during clinical trials, with the potential for data misinterpretation. TMDD also confounds human microdosing approaches by providing therapeutically unrepresentative PK profiles. Being aware of these phenomena will improve the likelihood of successful drug discovery and development. Copyright © 2018. Published by Elsevier Ltd.

  2. Stress-induced molecules MICA as potential target for radioimmunotherapy of cancer

    NASA Astrophysics Data System (ADS)

    Abakushina, E. V.; Anokhin, Yu N.; Abakushin, D. N.; Kaprin, A. D.

    2017-01-01

    Improving the treatment of cancer, increasing their effectiveness and safety is the main objective in the medicine. Molecular nuclear medicine plays an important role in the therapy of cancer. Radioimmunotherapy (RIT) involves the use of antibodies conjugated with therapeutic radionuclides. More often for RIT use the radiolabeled monoclonal antibodies against tumor-associated antigens. Encouraging results have been achieved with this technology in the management of hematologic malignancies. On the contrary, solid tumors have been less responsive. Despite these encouraging results, new potential target for radioimmunodetection and RIT should be found. It was found to increase the level of tumor-associated molecules MICA in the serum of cancer patients. Use of anti-MICA monoclonal antibodies capable a specifically attach to cancer cell via NKG2D ligands and destroy it, is a very promising direction, both therapeutic and diagnostic standpoint.

  3. Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

    PubMed

    Disney, Matthew D; Winkelsas, Audrey M; Velagapudi, Sai Pradeep; Southern, Mark; Fallahi, Mohammad; Childs-Disney, Jessica L

    2016-06-17

    The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

  4. Single molecule targeted sequencing for cancer gene mutation detection.

    PubMed

    Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W; He, Jiankui

    2016-05-19

    With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.

  5. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid.

    PubMed

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php. © The Author(s) 2016. Published by Oxford University Press.

  6. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid

    PubMed Central

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php PMID:26896846

  7. Sequence-based design of bioactive small molecules that target precursor microRNAs.

    PubMed

    Velagapudi, Sai Pradeep; Gallo, Steven M; Disney, Matthew D

    2014-04-01

    Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.

  8. Sequence-based design of bioactive small molecules that target precursor microRNAs

    PubMed Central

    Velagapudi, Sai Pradeep; Gallo, Steven M.; Disney, Matthew D.

    2014-01-01

    Oligonucleotides are designed to target RNA using base pairing rules, however, they are hampered by poor cellular delivery and non-specific stimulation of the immune system. Small molecules are preferred as lead drugs or probes, but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA precursors and identified bioactive small molecules that inhibit biogenesis by binding to nuclease processing sites (41% hit rate). Amongst 29 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Importantly, microRNA profiling shows that 1 only significantly effects microRNA-96 biogenesis and is more selective than an oligonucleotide. PMID:24509821

  9. Targeting of the MYCN Protein with Small Molecule c-MYC Inhibitors

    PubMed Central

    Müller, Inga; Larsson, Karin; Frenzel, Anna; Oliynyk, Ganna; Zirath, Hanna; Prochownik, Edward V.; Westwood, Nicholas J.; Henriksson, Marie Arsenian

    2014-01-01

    Members of the MYC family are the most frequently deregulated oncogenes in human cancer and are often correlated with aggressive disease and/or poorly differentiated tumors. Since patients with MYCN-amplified neuroblastoma have a poor prognosis, targeting MYCN using small molecule inhibitors could represent a promising therapeutic approach. We have previously demonstrated that the small molecule 10058-F4, known to bind to the c-MYC bHLHZip dimerization domain and inhibiting the c-MYC/MAX interaction, also interferes with the MYCN/MAX dimerization in vitro and imparts anti-tumorigenic effects in neuroblastoma tumor models with MYCN overexpression. Our previous work also revealed that MYCN-inhibition leads to mitochondrial dysfunction resulting in accumulation of lipid droplets in neuroblastoma cells. To expand our understanding of how small molecules interfere with MYCN, we have now analyzed the direct binding of 10058-F4, as well as three of its analogs; #474, #764 and 10058-F4(7RH), one metabolite C-m/z 232, and a structurally unrelated c-MYC inhibitor 10074-G5, to the bHLHZip domain of MYCN. We also assessed their ability to induce apoptosis, neurite outgrowth and lipid accumulation in neuroblastoma cells. Interestingly, all c-MYC binding molecules tested also bind MYCN as assayed by surface plasmon resonance. Using a proximity ligation assay, we found reduced interaction between MYCN and MAX after treatment with all molecules except for the 10058-F4 metabolite C-m/z 232 and the non-binder 10058-F4(7RH). Importantly, 10074-G5 and 10058-F4 were the most efficient in inducing neuronal differentiation and lipid accumulation in MYCN-amplified neuroblastoma cells. Together our data demonstrate MYCN-binding properties for a selection of small molecules, and provide functional information that could be of importance for future development of targeted therapies against MYCN-amplified neuroblastoma. PMID:24859015

  10. Targeted Nanomaterials for Phototherapy

    PubMed Central

    Chitgupi, Upendra; Qin, Yiru; Lovell, Jonathan F.

    2017-01-01

    Phototherapies involve the irradiation of target tissues with light. To further enhance selectivity and potency, numerous molecularly targeted photosensitizers and photoactive nanoparticles have been developed. Active targeting typically involves harnessing the affinity between a ligand and a cell surface receptor for improved accumulation in the targeted tissue. Targeting ligands including peptides, proteins, aptamers and small molecules have been explored for phototherapy. In this review, recent examples of targeted nanomaterials used in phototherapy are summarized. PMID:29071178

  11. Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity.

    PubMed

    Ho, Patrick; Ede, Christopher; Chen, Yvonne Y

    2017-08-18

    Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen-responsive therapeutics that can complement surface-targeted therapies.

  12. Observation of anisotropic interactions between metastable atoms and target molecules by two-dimensional collisional ionization electron spectroscopy

    NASA Astrophysics Data System (ADS)

    Kishimoto, Naoki; Ohno, Koichi

    Excited metastable atoms colliding with target molecules can sensitively probe outer properties of molecules by chemi-ionization (Penning ionization) from molecular orbitals in the outer region, since metastable atoms cannot penetrate into the repulsive interaction wall around the molecules. By means of two-dimensional measurements using kinetic energy analysis of electrons combined with a velocity-resolved metastable beam, one can obtain information on the anisotropic interaction between the colliding particles without any control of orientation or alignment of target molecules. We have developed a classical trajectory method to calculate the collision energy dependence of partial ionization cross-sections (CEDPICS) on the anisotropic interaction potential energy surface, which has enabled us to study stereodynamics between metastable atoms and target molecules as well as the spatial distribution of molecular orbitals and electron ejection functions which have a relation with entrance and exit channels of the reaction. Based on the individual CEDPICS, the electronic structure of molecules can also be elucidated.

  13. Improved and targeted delivery of bioactive molecules to cells with magnetic layer-by-layer assembled microcapsules

    NASA Astrophysics Data System (ADS)

    Pavlov, Anton M.; Gabriel, Samantha A.; Sukhorukov, Gleb B.; Gould, David J.

    2015-05-01

    Despite our increasing knowledge of cell biology and the recognition of an increasing repertoire of druggable intracellular therapeutic targets, there remain a limited number of approaches to deliver bioactive molecules to cells and even fewer that enable targeted delivery. Layer-by-layer (LbL) microcapsules are assembled using alternate layers of oppositely charged molecules and are potential cell delivery vehicles for applications in nanomedicine. There are a wide variety of charged molecules that can be included in the microcapsule structure including metal nanoparticles that introduce physical attributes. Delivery of bioactive molecules to cells with LbL microcapsules has recently been demonstrated, so in this study we explore the delivery of bioactive molecules (luciferase enzyme and plasmid DNA) to cells using biodegradable microcapsules containing a layer of magnetite nanoparticles. Interestingly, significantly improved intracellular luciferase enzyme activity (25 fold) and increased transfection efficiency with plasmid DNA (3.4 fold) was observed with magnetic microcapsules. The use of a neodymium magnet enabled efficient targeting of magnetic microcapsules which further improved the delivery efficiency of the cargoes as a consequence of increased microcapsule concentration at the magnetic site. Microcapsules were well tolerated by cells in these experiments and only displayed signs of toxicity at a capsule : cell ratio of 100 : 1 and with extended exposure. These studies illustrate how multi-functionalization of LbL microcapsules can improve and target delivery of bioactive molecules to cells.

  14. Exploration of target molecules for molecular imaging of inflammatory bowel disease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Higashikawa, Kei; Akada, Naoki; Yagi, Katsuharu

    2011-07-08

    Highlights: {sup {yields}18}F-FDG PET could discriminate each inflamed area of IBD model mice clearly. {sup {yields}18}F-FDG PET could not discriminate the difference of pathogenic mechanism. {yields} Cytokines and cytokine receptors expression was different by pathogenic mechanism. {yields} Cytokines and cytokine receptors would be new target molecules for IBD imaging. -- Abstract: Molecular imaging technology is a powerful tool for the diagnosis of inflammatory bowel disease (IBD) and the efficacy evaluation of various drug therapies for it. However, it is difficult to elucidate directly the relationships between the responsible molecules and IBD using existing probes. Therefore, the development of an alternativemore » probe that is able to elucidate the pathogenic mechanism and provide information on the appropriate guidelines for treatment is earnestly awaited. In this study, we investigated pathognomonic molecules in the intestines of model mice. The accumulation of fluorine-18 fluorodeoxyglucose ({sup 18}F-FDG) in the inflamed area of the intestines of dextran sulfate sodium (DSS)- or indomethacin (IND)-induced IBD model mice was measured by positron emission tomography (PET) and autoradiography to confirm the inflamed area. The results suggested that the inflammation was selectively induced in the colons of mice by the administration of DSS, whereas it was induced mainly in the ilea and the proximal colons of mice by the administration of IND. To explore attractive target molecules for the molecular imaging of IBD, we evaluated the gene expression levels of cytokines and cytokine receptors in the inflamed area of the intestines of both model mice. We found that the expression levels of cytokines and cytokine receptors were significantly increased during the progression of IBD, whereas the expression levels were decreased as the mucosa began to heal. In particular, the expression levels of these molecules had already changed before the symptoms of IBD

  15. Differential targeting of Gbetagamma-subunit signaling with small molecules.

    PubMed

    Bonacci, Tabetha M; Mathews, Jennifer L; Yuan, Chujun; Lehmann, David M; Malik, Sundeep; Wu, Dianqing; Font, Jose L; Bidlack, Jean M; Smrcka, Alan V

    2006-04-21

    G protein betagamma subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gbetagamma subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gbetagamma subunit functions. Several compounds bound to Gbetagamma subunits with affinities from 0.1 to 60 muM and selectively modulated functional Gbetagamma-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

  16. The synthesis and biological evaluation of integrin receptor targeting molecules as potential radiopharmaceuticals

    NASA Astrophysics Data System (ADS)

    Pellegrini, Paul

    This thesis reports on the synthesis, characterisation and biological evaluation of a number of metal complexes designed to interact with the alphavbeta3 integrin receptor, an important biological target that is heavily involved in angiogenesis, and thus cancer related processes. Two approaches were used to synthesise the integrin-avid targets. The first was to attach a variety of bifunctional chelators (BFC's) for the incorporation of different metal centres to a known integrin antagonist, L-748,415, developed by Merck. The BFC's used were the hydrazinonicotinamide (HYNIC) and monoamine monoamide dithiol (MAMA) systems for coordination to Tc-99m and rhenium of which was used as a characterization surrogate for the unstable Tc core. The 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA) BFC was attached for the inclusion of copper and lutetium. This 'conjugate' approach was designed to yield information on how the BFC and the linker length would affect the affinity for the integrin receptor. The second approach was an 'integrated' method where the chelation moiety was integral to the biologically relevant part of the molecule, which in the case of the alphavbeta3 integrin receptor, is the arginine-glycine-aspartic acid (RGD) mimicking sequence. Two complexes were created with a modified MAMA derivative placed between a benzimidazole moiety (arginine mimick) and the aspartic acid mimicking terminal carboxylic acid to see how it would affect binding while keeping the molecular weight relatively low. The molecules were tested in vitro against purified human alphavbeta3 integrin receptor protein in a solid phase receptor binding assay to evaluate their inhibition constants against a molecule of known high affinity and selectivity in [I125]L-775,219, the I125 labelled alphavbeta3 integrin antagonist. The radiolabelled analogues were also tested in vivo against the A375 human melanoma cell line transplanted into balb/c nude mice as well as Fischer rats implanted

  17. Mechanisms of autophagy and relevant small-molecule compounds for targeted cancer therapy.

    PubMed

    Zhang, Jin; Wang, Guan; Zhou, Yuxin; Chen, Yi; Ouyang, Liang; Liu, Bo

    2018-05-01

    Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process for the clearance of damaged or superfluous proteins and organelles. Accumulating studies have recently revealed that autophagy is closely related to a variety of types of cancer; however, elucidation of its Janus role of either tumor-suppressive or tumor-promoting still remains to be discovered. In this review, we focus on summarizing the context-dependent role of autophagy and its complicated molecular mechanisms in different types of cancer. Moreover, we discuss a series of small-molecule compounds targeting autophagy-related proteins or the autophagic process for potential cancer therapy. Taken together, these findings would shed new light on exploiting the intricate mechanisms of autophagy and relevant small-molecule compounds as potential anti-cancer drugs to improve targeted cancer therapy.

  18. Highly sensitive detection of target molecules using a new fluorescence-based bead assay

    NASA Astrophysics Data System (ADS)

    Scheffler, Silvia; Strauß, Denis; Sauer, Markus

    2007-07-01

    Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

  19. Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics

    PubMed Central

    Tan, Dun-Xian; Manchester, Lucien C.; Qin, Lilan; Reiter, Russel J.

    2016-01-01

    Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT) is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s). Melatonin protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting the mitochondrial permeability transition pore (MPTP), and activating uncoupling proteins (UCPs). Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria. PMID:27999288

  20. Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer

    PubMed Central

    Kim, Ari; Yen, Paul; Mroczek, Marta; Nouri, Mannan; Lien, Scott; Axerio-Cilies, Peter; Dalal, Kush; Yau, Clement; Ghaidi, Fariba; Guo, Yubin; Yamazaki, Takeshi; Lawn, Sam; Gleave, Martin E.; Gregory-Evans, Cheryl Y.

    2017-01-01

    Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer. PMID:28465491

  1. A Fragment-Based Method of Creating Small-Molecule Libraries to Target the Aggregation of Intrinsically Disordered Proteins.

    PubMed

    Joshi, Priyanka; Chia, Sean; Habchi, Johnny; Knowles, Tuomas P J; Dobson, Christopher M; Vendruscolo, Michele

    2016-03-14

    The aggregation process of intrinsically disordered proteins (IDPs) has been associated with a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Currently, however, no drug in clinical use targets IDP aggregation. To facilitate drug discovery programs in this important and challenging area, we describe a fragment-based approach of generating small-molecule libraries that target specific IDPs. The method is based on the use of molecular fragments extracted from compounds reported in the literature to inhibit of the aggregation of IDPs. These fragments are used to screen existing large generic libraries of small molecules to form smaller libraries specific for given IDPs. We illustrate this approach by describing three distinct small-molecule libraries to target, Aβ, tau, and α-synuclein, which are three IDPs implicated in Alzheimer's and Parkinson's diseases. The strategy described here offers novel opportunities for the identification of effective molecular scaffolds for drug discovery for neurodegenerative disorders and to provide insights into the mechanism of small-molecule binding to IDPs.

  2. Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

    NASA Astrophysics Data System (ADS)

    Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

    2006-04-01

    G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

  3. Endothelial targeting of high-affinity multivalent polymer nanocarriers directed to intercellular adhesion molecule 1.

    PubMed

    Muro, Silvia; Dziubla, Thomas; Qiu, Weining; Leferovich, John; Cui, Xiumin; Berk, Erik; Muzykantov, Vladimir R

    2006-06-01

    Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (approximately 200 nm diameter spheres carrying approximately 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 approximately 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax approximately 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd approximately 80 pM versus approximately 8 nM) in cell culture and, probably because of this factor, higher value (185.3 +/- 24.2 versus 50.5 +/- 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 +/- 10.9 versus 2.1 +/- 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

  4. Using the QCM Biosensor-Based T7 Phage Display Combined with Bioinformatics Analysis for Target Identification of Bioactive Small Molecule.

    PubMed

    Takakusagi, Yoichi; Takakusagi, Kaori; Sugawara, Fumio; Sakaguchi, Kengo

    2018-01-01

    Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.

  5. Molecules and mechanisms involved in the generation and migration of cortical interneurons

    PubMed Central

    Hernández-Miranda, Luis R; Parnavelas, John G; Chiara, Francesca

    2010-01-01

    The GABA (γ-aminobutyric acid)-containing interneurons of the neocortex are largely derived from the ganglionic eminences in the subpallium. Numerous studies have previously defined the migratory paths travelled by these neurons from their origins to their destinations in the cortex. We review here results of studies that have identified many of the genes expressed in the subpallium that are involved in the specification of the subtypes of cortical interneurons, and the numerous transcription factors, motogenic factors and guidance molecules that are involved in their migration. PMID:20360946

  6. Soluble adhesion molecules in human cancers: sources and fates.

    PubMed

    van Kilsdonk, Jeroen W J; van Kempen, Léon C L T; van Muijen, Goos N P; Ruiter, Dirk J; Swart, Guido W M

    2010-06-01

    Adhesion molecules endow tumor cells with the necessary cell-cell contacts and cell-matrix interactions. As such, adhesion molecules are involved in cell signalling, proliferation and tumor growth. Rearrangements in the adhesion repertoire allow tumor cells to migrate, invade and form metastases. Besides these membrane-bound adhesion molecules several soluble adhesion molecules are detected in the supernatant of tumor cell lines and patient body fluids. Truncated soluble adhesion molecules can be generated by several conventional mechanisms, including alternative splicing of mRNA transcripts, chromosomal translocation, and extracellular proteolytic ectodomain shedding. Secretion of vesicles (ectosomes and exosomes) is an alternative mechanism mediating the release of full-length adhesion molecules. Soluble adhesion molecules function as modulators of cell adhesion, induce proteolytic activity and facilitate cell signalling. Additionally, adhesion molecules present on secreted vesicles might be involved in the vesicle-target cell interaction. Based on currently available data, released soluble adhesion molecules contribute to cancer progression and therefore should not be regarded as unrelated and non-functional side products of tumor progression. 2010 Elsevier GmbH. All rights reserved.

  7. INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1

    PubMed Central

    Ainsztein, Alexandra M.; Kandels-Lewis, Stefanie E.; Mackay, Alastair M.; Earnshaw, William C.

    1998-01-01

    The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1Hsα. Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle. PMID:9864353

  8. Target-molecule-triggered rupture of aptamer-encapsulated polyelectrolyte microcapsules.

    PubMed

    Zhang, Xueru; Chabot, Denise; Sultan, Yasir; Monreal, Carlos; DeRosa, Maria C

    2013-06-26

    Polyelectrolyte microcapsules have great potential for serving as carriers for the delivery of their contents when triggered by an external stimulus. Aptamers are synthetic ssDNA or RNA that can bind to specific targets with high affinity and selectivity. Aptamers may retain these superior molecular recognition properties after encapsulation within polymer microcapsules. In this work, stable polyelectrolyte microcapsules with encapsulated aptamers were obtained by the layer-by-layer (LbL) method. Polyelectrolyte films were deposited onto a CaCO3 template that had been predoped with polystyrene sulfonate (PSS) and aptamer sequences (SA) that have an affinity for the dye sulforhodamine B (SRB). The PSS and aptamers are thought to serve as an internal scaffold supporting the microcapsule walls. These microcapsules would present target-molecule-triggered rupture properties. Microcapsule collapse was triggered by the binding of SRB to the encapsulated aptamer. The specificity of microcapsule collapse was investigated using a similar dye, tetramethylrosamine (TMR), which does not have affinity for SA. A high concentration of TMR did not lead to the collapse of the microcapsules. The effect of target binding on the microcapsules was confirmed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). These microcapsules may have potential applications in targeted delivery systems for the controlled release of drugs, pesticides, or other payloads.

  9. Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation

    PubMed Central

    Bulatov, Emil; Ciulli, Alessio

    2015-01-01

    In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs. PMID:25886174

  10. Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

    PubMed

    Hengel, Sarah R; Spies, M Ashley; Spies, Maria

    2017-09-21

    To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Strategies that Target Leukocyte Traffic in IBD: Recent Developments

    PubMed Central

    Rivera-Nieves, Jesús

    2015-01-01

    Purpose of review We review the most recent developments regarding the targeting of molecules involved in the traffic of leukocytes for the treatment of IBD. Recent Findings We discuss the most important findings of one published phase II trial that targeted the β7 integrin (Etrolizumab), two phase II trials that targeted the α4β7 integrin ligand: Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1, PF-00547659), a phase II targeting the chemokine IP-10 (CXCL10) in Crohn’s and a phase II trial that targeted the sphingosine-1-phosphate receptor-1 (S1P1): ozanimod in patients with ulcerative colitis (UC). Summary Targeting molecules involved in leukocyte traffic has recently become an effective and safe strategy for the treatment of IBD. Novel approaches now not only target the integrins on the lymphocyte surface, but also its endothelial ligand: MAdCAM-1. As with vedolizumab, antibodies against MAdCAM-1 appear most effective in ulcerative colitis rather than in Crohn’s. Targeting chemokines or their receptors does not appear to have the same efficacy as those that target the most stable integrin:immunoglobulin superfamily interactions between the lymphocyte and endothelium. Preliminary results also suggest that the sphingosine-1-phosphate pathway might also be targeted therapeutically in IBD, no longer with parenterally administered antibodies but with orally administered small molecules. PMID:26398681

  12. Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA.

    PubMed

    Yang, Wang-Yong; Wilson, Henry D; Velagapudi, Sai Pradeep; Disney, Matthew D

    2015-04-29

    One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides.

  13. Plants and their active compounds: natural molecules to target angiogenesis.

    PubMed

    Lu, Kai; Bhat, Madhavi; Basu, Sujit

    2016-07-01

    Angiogenesis, or new blood vessel formation, is an important process in the pathogenesis of several diseases and thus has been targeted for the prevention and treatment for many disorders. However, the anti-angiogenic agents that are currently in use are mainly synthetic compounds and humanized monoclonal antibodies, which are either expensive or toxic, thereby limiting their use in many patients. Therefore, it is necessary to identify less toxic, inexpensive, novel and effective anti-angiogenic molecules. Several studies have indicated that natural plant products can meet these criteria. In this review, we discuss the anti-angiogenic properties of natural compounds isolated from plants and the molecular mechanisms by which these molecules act. Finally, we summarize the advantages of using plant products as anti-angiogenic agents. Compared with currently available anti-angiogenic drugs, plant products may not only have similar therapeutic potential but are also inexpensive, less toxic, and easy to administer. However, novel and effective strategies are necessary to improve their bioavailability for clinical use.

  14. Small Molecule Sequential Dual-Targeting Theragnostic Strategy (SMSDTTS): from Preclinical Experiments towards Possible Clinical Anticancer Applications

    PubMed Central

    Li, Junjie; Oyen, Raymond; Verbruggen, Alfons; Ni, Yicheng

    2013-01-01

    Hitting the evasive tumor cells proves challenging in targeted cancer therapies. A general and unconventional anticancer approach namely small molecule sequential dual-targeting theragnostic strategy (SMSDTTS) has recently been introduced with the aims to target and debulk the tumor mass, wipe out the residual tumor cells, and meanwhile enable cancer detectability. This dual targeting approach works in two steps for systemic delivery of two naturally derived drugs. First, an anti-tubulin vascular disrupting agent, e.g., combretastatin A4 phosphate (CA4P), is injected to selectively cut off tumor blood supply and to cause massive necrosis, which nevertheless always leaves peripheral tumor residues. Secondly, a necrosis-avid radiopharmaceutical, namely 131I-hypericin (131I-Hyp), is administered the next day, which accumulates in intratumoral necrosis and irradiates the residual cancer cells with beta particles. Theoretically, this complementary targeted approach may biologically and radioactively ablate solid tumors and reduce the risk of local recurrence, remote metastases, and thus cancer mortality. Meanwhile, the emitted gamma rays facilitate radio-scintigraphy to detect tumors and follow up the therapy, hence a simultaneous theragnostic approach. SMSDTTS has now shown promise from multicenter animal experiments and may demonstrate unique anticancer efficacy in upcoming preliminary clinical trials. In this short review article, information about the two involved agents, the rationale of SMSDTTS, its preclinical antitumor efficacy, multifocal targetability, simultaneous theragnostic property, and toxicities of the dose regimens are summarized. Meanwhile, possible drawbacks, practical challenges and future improvement with SMSDTTS are discussed, which hopefully may help to push forward this strategy from preclinical experiments towards possible clinical applications. PMID:23412554

  15. Small Molecule Sequential Dual-Targeting Theragnostic Strategy (SMSDTTS): from Preclinical Experiments towards Possible Clinical Anticancer Applications.

    PubMed

    Li, Junjie; Oyen, Raymond; Verbruggen, Alfons; Ni, Yicheng

    2013-01-01

    Hitting the evasive tumor cells proves challenging in targeted cancer therapies. A general and unconventional anticancer approach namely small molecule sequential dual-targeting theragnostic strategy (SMSDTTS) has recently been introduced with the aims to target and debulk the tumor mass, wipe out the residual tumor cells, and meanwhile enable cancer detectability. This dual targeting approach works in two steps for systemic delivery of two naturally derived drugs. First, an anti-tubulin vascular disrupting agent, e.g., combretastatin A4 phosphate (CA4P), is injected to selectively cut off tumor blood supply and to cause massive necrosis, which nevertheless always leaves peripheral tumor residues. Secondly, a necrosis-avid radiopharmaceutical, namely (131)I-hypericin ((131)I-Hyp), is administered the next day, which accumulates in intratumoral necrosis and irradiates the residual cancer cells with beta particles. Theoretically, this complementary targeted approach may biologically and radioactively ablate solid tumors and reduce the risk of local recurrence, remote metastases, and thus cancer mortality. Meanwhile, the emitted gamma rays facilitate radio-scintigraphy to detect tumors and follow up the therapy, hence a simultaneous theragnostic approach. SMSDTTS has now shown promise from multicenter animal experiments and may demonstrate unique anticancer efficacy in upcoming preliminary clinical trials. In this short review article, information about the two involved agents, the rationale of SMSDTTS, its preclinical antitumor efficacy, multifocal targetability, simultaneous theragnostic property, and toxicities of the dose regimens are summarized. Meanwhile, possible drawbacks, practical challenges and future improvement with SMSDTTS are discussed, which hopefully may help to push forward this strategy from preclinical experiments towards possible clinical applications.

  16. Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

    2014-02-01

    Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

  17. Alzheimer's Disease: A Systemic Review of Substantial Therapeutic Targets and the Leading Multi-functional Molecules.

    PubMed

    Umar, Tarana; Hoda, Nasimul

    2017-01-01

    Alzheimer's Disease (AD) is a fatal neurodegenerative disorder, having a complex aetiology with numerous possible drug targets. There are targets that have been known for years while more new targets and theories have also emerged. Beta amyloid and cholinesterases are the most significant biological targets for finding curative treatment of AD. The major class of drugs used for AD till now has been the Cholinesterase (ChE) inhibitors. Other prevailing models of molecular pathogenesis in AD include Neurofibrillary Tangles (NFTs) and amyloid deposition, tryptophan degradation pathway, kinase and phosphatase activity imbalance and neuroinflammation. The beta amyloid aggregation initiates flow of events resulting in neurotoxicity and finally clinical pathogenesis of AD. Furthermore, ApoE is another very significant entity involved in repairing and maintaining the neurons and has important role in neurodegeneration. Neuroinflammation being the primmest symptom for AD is essential to focus on. Multiple factors and complexity in interlinking disease progression pose huge challenge to find one complete curing drug. With so many promising molecules having multiform pharmacological profile from all over the world however facing failures in clinical trials indicates the need to consider all aspects of the old as well as new therapeutic targets of AD. Until the disease mechanism is better understood, it is likely that multiple targeting, symptomatic and diseasemodifying, is the way forward. Most recent approaches to find anti-Alzheimer's agents have focused on multi-target directed agents that include targeting all glorious targets hypothesized against AD. New identification of prototype candidates that could be starting point of a new way of thinking drug design has been done and many drug candidates are under preclinical evaluation. The main focus of this review is to discuss the recent understanding of key targets and the development of potential therapeutic agents for the

  18. Development of pharmacophore models for small molecules targeting RNA: Application to the RNA repeat expansion in myotonic dystrophy type 1.

    PubMed

    Angelbello, Alicia J; González, Àlex L; Rzuczek, Suzanne G; Disney, Matthew D

    2016-12-01

    RNA is an important drug target, but current approaches to identify bioactive small molecules have been engineered primarily for protein targets. Moreover, the identification of small molecules that bind a specific RNA target with sufficient potency remains a challenge. Computer-aided drug design (CADD) and, in particular, ligand-based drug design provide a myriad of tools to identify rapidly new chemical entities for modulating a target based on previous knowledge of active compounds without relying on a ligand complex. Herein we describe pharmacophore virtual screening based on previously reported active molecules that target the toxic RNA that causes myotonic dystrophy type 1 (DM1). DM1-associated defects are caused by sequestration of muscleblind-like 1 protein (MBNL1), an alternative splicing regulator, by expanded CUG repeats (r(CUG) exp ). Several small molecules have been found to disrupt the MBNL1-r(CUG) exp complex, ameliorating DM1 defects. Our pharmacophore model identified a number of potential lead compounds from which we selected 11 compounds to evaluate. Of the 11 compounds, several improved DM1 defects both in vitro and in cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Natural Products as Anticancerous Therapeutic Molecules with Special Reference to Enzymatic Targets Topoisomerase, COX, LOX and Aromatase.

    PubMed

    Singh, Swati; Awasthi, Manika; Pandey, Veda P; Dwivedi, Upendra N

    2018-01-01

    Cancer, characterized by uncontrolled growth and proliferation of cells, is affecting millions of people every year and estimated as the second leading cause of death. Its successful treatment yet remains a challenge due to the lack of selectivity, toxicity and the development of multidrug resistant cells to the currently available drugs. Plant derived natural products hold great promise for discovery and development of new pharmaceuticals against cancer as evident by the fact that out of 121 drugs prescribed for cancer treatment till date, 90 are derived from plant sources. Furthermore, the plant derived therapeutic molecules are also considered as safer substitutes to those of synthetic ones. In this review, the therapeutic potentials of plant derived natural products belonging to secondary metabolites, namely alkaloids, flavonoids and terpenoids as anticancer molecules, involving various strategies of treatment, have been discussed with special reference to topoisomerases (Topo), cyclooxygenases (COX), lipoxygenase (LOX) and aromatase as enzymatic targets for various types of cancers. Furthermore, in view of the recent advances made in the field of computer aided drug design, the present review also discusses the use of computational approaches such as ADMET, molecular docking, molecular dynamics simulation and QSAR to assess and predict the safety, efficacy, potency and identification of such potent anticancerous therapeutic molecules. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Targeting RSV with Vaccines and Small Molecule Drugs

    PubMed Central

    Costello, Heather M.; Ray, William C.; Chaiwatpongsakorn, Supranee; Peeples, Mark E.

    2012-01-01

    Respiratory syncytial virus (RSV) is the most significant cause of pediatric respiratory infections. Palivizumab (Synagis®), a humanized monoclonal antibody, has been used successfully for a number of years to prevent severe RSV disease in at-risk infants. However, despite intense efforts, there is no approved vaccine or small molecule drug for RSV. As an enveloped virus, RSV must fuse its envelope with the host cell membrane, which is accomplished through the actions of the fusion (F) glycoprotein, with attachment help from the G glycoprotein. Because of their integral role in initiation of infection and their accessibility outside the lipid bilayer, these proteins have been popular targets in the discovery and development of antiviral compounds and vaccines against RSV. This review examines advances in the development of antiviral compounds and vaccine candidates. PMID:22335496

  1. Targeting MDM2 by the small molecule RITA: towards the development of new multi-target drugs against cancer

    PubMed Central

    Espinoza-Fonseca, L Michel

    2005-01-01

    Background The use of low-molecular-weight, non-peptidic molecules that disrupt the interaction between the p53 tumor suppressor and its negative regulator MDM2 has provided a promising alternative for the treatment of different types of cancer. Among these compounds, RITA (reactivation of p53 and induction of tumor cell apoptosis) has been shown to be effective in the selective induction of apoptosis, and this effect is due to its binding to the p53 tumor suppressor. Since biological systems are highly dynamic and MDM2 may bind to different regions of p53, new alternatives should be explored. On this basis, the computational "blind docking" approach was employed in this study to see whether RITA would bind to MDM2. Results It was observed that RITA binds to the MDM2 p53 transactivation domain-binding cleft. Thus, RITA can be used as a lead compound for designing improved "multi-target" drugs. This novel strategy could provide enormous benefits to enable effective anti-cancer strategies. Conclusion This study has demonstrated that a single molecule can target at least two different proteins related to the same disease. PMID:16174299

  2. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets.

    PubMed

    Pinto-Fernandez, Adan; Kessler, Benedikt M

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  3. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets

    PubMed Central

    Pinto-Fernandez, Adan; Kessler, Benedikt M.

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors. PMID:27516771

  4. Interventions That Target Criminogenic Needs for Justice-Involved Persons With Serious Mental Illnesses: A Targeted Service Delivery Approach.

    PubMed

    Wilson, Amy Blank; Farkas, Kathleen; Bonfine, Natalie; Duda-Banwar, Janelle

    2018-05-01

    This research describes the development of a targeted service delivery approach that tailors the delivery of interventions that target criminogenic needs to the specific learning and treatment needs of justice-involved people with serious mental illnesses (SMIs). This targeted service delivery approach includes five service delivery strategies: repetition and summarizing, amplification, active coaching, low-demand practice, and maximizing participation. Examples of how to apply each strategy in session are provided, as well as recommendations on when to use each strategy during the delivery of interventions that target criminogenic needs. This targeted service delivery approach makes an important contribution to the development of interventions for justice-involved people with SMI by increasing the chances that people with SMI can participate fully in and benefit from these interventions that target criminogenic needs. These developments come at a critical time in the field as the next generation of services for justice-involved people with SMI are being developed.

  5. Small molecule therapeutics targeting F-box proteins in cancer.

    PubMed

    Liu, Yuan; Mallampalli, Rama K

    2016-02-01

    The ubiquitin proteasome system (UPS) plays vital roles in maintaining protein equilibrium mainly through proteolytic degradation of targeted substrates. The archetypical SCF ubiquitin E3 ligase complex contains a substrate recognition subunit F-box protein that recruits substrates to the catalytic ligase core for its polyubiquitylation and subsequent proteasomal degradation. Several well-characterized F-box proteins have been demonstrated that are tightly linked to neoplasia. There is mounting information characterizing F-box protein-substrate interactions with the rationale to develop unique therapeutics for cancer treatment. Here we review that how F-box proteins function in cancer and summarize potential small molecule inhibitors for cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Two-Way Gold Nanoparticle Label-Free Sensing of Specific Sequence and Small Molecule Targets Using Switchable Concatemers.

    PubMed

    Zhu, Longjiao; Shao, Xiangli; Luo, Yunbo; Huang, Kunlung; Xu, Wentao

    2017-05-19

    A two-way colorimetric biosensor based on unmodified gold nanoparticles (GNPs) and a switchable double-stranded DNA (dsDNA) concatemer have been demonstrated. Two hairpin probes (H1 and H2) were first designed that provided the fuels to assemble the dsDNA concatemers via hybridization chain reaction (HCR). A functional hairpin (FH) was rationally designed to recognize the target sequences. All the hairpins contained a single-stranded DNA (ssDNA) loop and sticky end to prevent GNPs from salt-induced aggregation. In the presence of target sequence, the capture probe blocked in the FH recognizes the target to form a duplex DNA, which causes the release of the initiator probe by FH conformational change. This process then starts the alternate-opening of H1 and H2 through HCR, and dsDNA concatemers grow from the target sequence. As a result, unmodified GNPs undergo salt-induced aggregation because the formed dsDNA concatemers are stiffer and provide less stabilization. A light purple-to-blue color variation was observed in the bulk solution, termed the light-off sensing way. Furthermore, H1 ingeniously inserted an aptamer sequence to generate dsDNA concatemers with multiple small molecule binding sites. In the presence of small molecule targets, concatemers can be disassembled into mixtures with ssDNA sticky ends. A blue-to-purple reverse color variation was observed due to the regeneration of the ssDNA, termed the light-on way. The two-way biosensor can detect both nucleic acids and small molecule targets with one sensing device. This switchable sensing element is label-free, enzyme-free, and sophisticated-instrumentation-free. The detection limits of both targets were below nanomolar.

  7. Eviction from the sanctuary: Development of targeted therapy against cell adhesion molecules in acute lymphoblastic leukemia.

    PubMed

    Barwe, Sonali P; Quagliano, Anthony; Gopalakrishnapillai, Anilkumar

    2017-04-01

    Acute lymphoblastic leukemia (ALL) is a malignant hematological disease afflicting hematopoiesis in the bone marrow. While 80%-90% of patients diagnosed with ALL will achieve complete remission at some point during treatment, ALL is associated with high relapse rate, with a 5-year overall survival rate of 68%. The initial remission failure and the high rate of relapse can be attributed to intrinsic chemoprotective mechanisms that allow persistence of ALL cells despite therapy. These mechanisms are mediated, at least in part, through the engagement of cell adhesion molecules (CAMs) within the bone marrow microenvironment. This review assembles CAMs implicated in protection of leukemic cells from chemotherapy. Such studies are limited in ALL. Therefore, CAMs that are associated with poor outcomes or are overexpressed in ALL and have been shown to be involved in chemoprotection in other hematological cancers are also included. It is likely that these molecules play parallel roles in ALL because the CAMs identified to be a factor in ALL chemoresistance also work similarly in other hematological malignancies. We review the signaling mechanisms activated by the engagement of CAMs that provide protection from chemotherapy. Development of targeted therapies against CAMs could improve outcome and raise the overall cure rate in ALL. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Strategies that target leukocyte traffic in inflammatory bowel diseases: recent developments.

    PubMed

    Rivera-Nieves, Jesús

    2015-11-01

    We review the most recent developments regarding the targeting of molecules involved in the traffic of leukocytes for the treatment of inflammatory bowel diseases (IBD). We discuss the most important findings of one published phase II trial that targeted the β7 integrin (etrolizumab), two phase II trials that targeted the α4β7 integrin ligand: mucosal addressin cell adhesion molecule 1 (MAdCAM-1, PF-00547659), a phase II trial targeting the chemokine IP-10 (CXCL10) in Crohn's, and a phase II trial that targeted the sphingosine-1-phosphate receptor-1: ozanimod in patients with ulcerative colitis. Targeting molecules involved in leukocyte traffic has recently become an effective and well tolerated strategy for the treatment of IBD. Novel approaches now not only target the integrins on the lymphocyte surface, but also its endothelial ligand: MAdCAM-1. As with vedolizumab, antibodies against MAdCAM-1 appear most effective in ulcerative colitis rather than in Crohn's. Targeting chemokines or their receptors does not appear to have the same efficacy as those that target the most stable integrin: immunoglobulin superfamily interactions between the lymphocyte and endothelium. Preliminary results also suggest that the sphingosine-1-phosphate pathway might also be targeted therapeutically in IBD, no longer with parenterally administered antibodies but with orally administered small molecules.

  9. Targeting Programmed Cell Death Using Small-Molecule Compounds to Improve Potential Cancer Therapy.

    PubMed

    Ke, Bowen; Tian, Mao; Li, Jingjing; Liu, Bo; He, Gu

    2016-11-01

    Evasion of cell death is one of the hallmarks of cancer cells, beginning with long-established apoptosis and extending to other new forms of cell death. An elaboration of cell death pathways thus will contribute to a better understanding of cancer pathogenesis and therapeutics. With the recent substantial biochemical and genetic explorations of cell death subroutines, their classification has switched from primarily morphological to more molecular definitions. According to their measurable biochemical features and intricate mechanisms, cell death subroutines can be divided into apoptosis, autophagic cell death, mitotic catastrophe, necroptosis, parthanatos, ferroptosis, pyroptosis, pyronecrosis, anoikis, cornification, entosis, and NETosis. Supportive evidence has gradually revealed the prime molecular mechanisms of each subroutine and thus providing series of possible targets in cancer therapy, while the intricate relationships between different cell death subroutines still remain to be clarified. Over the past decades, cancer drug discovery has significantly benefited from the use of small-molecule compounds to target classical modalities of cell death such as apoptosis, while newly identified cell death subroutines has also emerging their potential for cancer drug discovery in recent years. In this review, we comprehensively focus on summarizing 12 cell death subroutines and discussing their corresponding small-molecule compounds in potential cancer therapy. Together, these inspiring findings may provide more evidence to fill in the gaps between cell death subroutines and small-molecule compounds to better develop novel cancer therapeutic strategies. © 2016 Wiley Periodicals, Inc.

  10. Targeting neurotransmitter receptors with nanoparticles in vivo allows single-molecule tracking in acute brain slices

    NASA Astrophysics Data System (ADS)

    Varela, Juan A.; Dupuis, Julien P.; Etchepare, Laetitia; Espana, Agnès; Cognet, Laurent; Groc, Laurent

    2016-03-01

    Single-molecule imaging has changed the way we understand many biological mechanisms, particularly in neurobiology, by shedding light on intricate molecular events down to the nanoscale. However, current single-molecule studies in neuroscience have been limited to cultured neurons or organotypic slices, leaving as an open question the existence of fast receptor diffusion in intact brain tissue. Here, for the first time, we targeted dopamine receptors in vivo with functionalized quantum dots and were able to perform single-molecule tracking in acute rat brain slices. We propose a novel delocalized and non-inflammatory way of delivering nanoparticles (NPs) in vivo to the brain, which allowed us to label and track genetically engineered surface dopamine receptors in neocortical neurons, revealing inherent behaviour and receptor activity regulations. We thus propose a NP-based platform for single-molecule studies in the living brain, opening new avenues of research in physiological and pathological animal models.

  11. Osteoclast-targeting small molecules for the treatment of neoplastic bone metastases.

    PubMed

    Kawatani, Makoto; Osada, Hiroyuki

    2009-11-01

    Osteoclasts are highly specialized cells that resorb bone, and their abnormal activity is implicated in a variety of human bone diseases. In neoplastic bone metastasis, the bone destruction caused by osteoclasts is not only associated with the formation and progression of metastatic lesions, but also could contribute to frequent complications such as severe pain and pathological fractures, which greatly diminish the quality of life of patients. Bisphosphonates, potent antiresorptive drugs, have been shown to have efficacy for treating bone metastases in many types of cancer, and the development of various molecularly targeted agents is currently proceeding. Thus, inhibition of osteoclast function is now established as an important treatment strategy for bony metastases. This review focuses on promising small molecules that disrupt osteoclast function and introduces our chemical/biological approach for identifying osteoclast-targeting small molecular inhibitors.

  12. Signaling molecules involved in the transition of growth to development of Dictyostelium discoideum.

    PubMed

    Mir, Hina A; Rajawat, Jyotika; Pradhan, Shalmali; Begum, Rasheedunnisa

    2007-03-01

    The social amoeba Dictyostelium discoideum, a powerful paradigm provides clear insights into the regulation of growth and development. In addition to possessing complex individual cellular functions like a unicellular eukaryote, D. discoideum cells face the challenge of multicellular development. D. discoideum undergoes a relatively simple differentiation process mainly by cAMP mediated pathway. Despite this relative simplicity, the regulatory signaling pathways are as complex as those seen in metazoan development. However, the introduction of restriction-enzyme-mediated integration (REMI) technique to produce developmental gene knockouts has provided novel insights into the discovery of signaling molecules and their role in D. discoideum development. Cell cycle phase is an important aspect for differentiation of D. discoideum, as cells must reach a specific stage to enter into developmental phase and specific cell cycle regulators are involved in arresting growth phase genes and inducing the developmental genes. In this review, we present an overview of the signaling molecules involved in the regulation of growth to differentiation transition (GDT), molecular mechanism of early developmental events leading to generation of cAMP signal and components of cAMP relay system that operate in this paradigm.

  13. Acoustic input and efferent activity regulate the expression of molecules involved in cochlear micromechanics

    PubMed Central

    Lamas, Veronica; Arévalo, Juan C.; Juiz, José M.; Merchán, Miguel A.

    2015-01-01

    Electromotile activity in auditory outer hair cells (OHCs) is essential for sound amplification. It relies on the highly specialized membrane motor protein prestin, and its interactions with the cytoskeleton. It is believed that the expression of prestin and related molecules involved in OHC electromotility may be dynamically regulated by signals from the acoustic environment. However little is known about the nature of such signals and how they affect the expression of molecules involved in electromotility in OHCs. We show evidence that prestin oligomerization is regulated, both at short and relatively long term, by acoustic input and descending efferent activity originating in the cortex, likely acting in concert. Unilateral removal of the middle ear ossicular chain reduces levels of trimeric prestin, particularly in the cochlea from the side of the lesion, whereas monomeric and dimeric forms are maintained or even increased in particular in the contralateral side, as shown in Western blots. Unilateral removal of the auditory cortex (AC), which likely causes an imbalance in descending efferent activity on the cochlea, also reduces levels of trimeric and tetrameric forms of prestin in the side ipsilateral to the lesion, whereas in the contralateral side prestin remains unaffected, or even increased in the case of trimeric and tetrameric forms. As far as efferent inputs are concerned, unilateral ablation of the AC up-regulates the expression of α10 nicotinic Ach receptor (nAChR) transcripts in the cochlea, as shown by RT-Quantitative real-time PCR (qPCR). This suggests that homeostatic synaptic scaling mechanisms may be involved in dynamically regulating OHC electromotility by medial olivocochlear efferents. Limited, unbalanced efferent activity after unilateral AC removal, also affects prestin and β-actin mRNA levels. These findings support that the concerted action of acoustic and efferent inputs to the cochlea is needed to regulate the expression of major

  14. Cell adhesion molecules, the extracellular matrix and oral squamous carcinoma.

    PubMed

    Lyons, A J; Jones, J

    2007-08-01

    Carcinomas are characterized by invasion of malignant cells into the underlying connective tissue and migration of malignant cells to form metastases at distant sites. These processes require alterations in cell-cell and cell-extracellular matrix interactions. As cell adhesion molecules play a role in cell-cell and cell-extracellular matrix adhesion and interactions they are involved in the process of tumour invasion and metastases. In epithelial tissues, receptors of the integrin family mediate adhesion to the adjacent matrix whereas cadherins largely mediate intercellular adhesion. These and other cell adhesion molecules such as intercellular adhesion molecule-1, CD44, dystroglycans and selectins, are involved and undergo changes in carcinomas, which provide possible targets for anti-cancer drug treatments. In the extracellular matrix that is associated with tumours, laminin 5, oncofetal fibronectin and tenascin C appear. The degree of expression of some of these moieties indicates prognosis in oral cancer and offer targets for antibody-directed radiotherapy. Metalloproteases which degrade the extracellular matrix are increased in carcinomas, and their activity is necessary for tumour angiogenesis and consequent invasion and metastases. Metalloprotease inhibitors have begun to produce decreases in mortality in clinical trials. This report provides a brief overview of our current understanding of cell adhesion molecules, the extracellular matrix, tumour invasion and metastasis.

  15. Oriented and covalent immobilization of target molecules to solid supports: synthesis and application of a light-activatable and thiol-reactive cross-linking reagent.

    PubMed

    Collioud, A; Clémence, J F; Sänger, M; Sigrist, H

    1993-01-01

    Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.

  16. From Toxins Targeting Ligand Gated Ion Channels to Therapeutic Molecules

    PubMed Central

    Nasiripourdori, Adak; Taly, Valérie; Grutter, Thomas; Taly, Antoine

    2011-01-01

    Ligand-gated ion channels (LGIC) play a central role in inter-cellular communication. This key function has two consequences: (i) these receptor channels are major targets for drug discovery because of their potential involvement in numerous human brain diseases; (ii) they are often found to be the target of plant and animal toxins. Together this makes toxin/receptor interactions important to drug discovery projects. Therefore, toxins acting on LGIC are presented and their current/potential therapeutic uses highlighted. PMID:22069709

  17. Discovering Small Molecule Inhibitors Targeted to Ligand-Stimulated RAGE-DIAPH1 Signaling Transduction

    NASA Astrophysics Data System (ADS)

    Pan, Jinhong

    The receptor of advanced glycation end product (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules, which plays an important role in immune responses. Full-length RAGE includes three extracellular immunoglobulin domains, a transmembrane domain and an intracellular domain. It is a pattern recognition receptor that can bind diverse ligands. NMR spectroscopy and x-ray crystallization studies of the extracellular domains of RAGE indicate that RAGE ligands bind by distinct charge- and hydrophobicity-dependent mechanisms. It is found that calgranulin binding to the C1C2 domain or AGEs binding to the V domain activates extracellular signaling, which triggers interactions of the RAGE cytoplasmic tail (ctRAGE) with intracellular effector, such as diaphanous 1 (DIAPH1), to initiate signal transduction cascades. ctRAGE is essential for RAGE-ligand-mediated signal transduction and consequent modulation of gene expression and cellular properties. RAGE is over-expressed in diseased tissues of most RAGE-associated pathogenic conditions, such as complications of Alzheimer's diseases, diabetes, vascular diseases, inflammation, cancers and neurodegeneration. They are the major diseases affecting a large population worldwide. RAGE can function as a biomarker or drug target for these diseases. The cytoplasmic tail of RAGE can be used as a drug target to inhibit RAGE-induced intracellular signaling by small molecule inhibitors to treat RAGE-associated diseases. We developed a high throughput screening assay with which we probed a small molecule library of 58,000 compounds to find that 777 small molecules displayed 50% inhibition and 97 compounds demonstrated dose-dependent inhibition of the binding of ctRAGE-DIAPH1. Eventually, there were 13 compounds which displayed dose-dependent inhibition of ctRAGE binding to DIAPH1 and direct binding to ctRAGE analyzed by 15N HSQC-NMR and native tryptophan fluorescence titration experiments; thus, they were

  18. Anti-CD38 antibody therapy: windows of opportunity yielded by the functional characteristics of the target molecule.

    PubMed

    Chillemi, Antonella; Zaccarello, Gianluca; Quarona, Valeria; Ferracin, Manuela; Ghimenti, Chiara; Massaia, Massimo; Horenstein, Alberto L; Malavasi, Fabio

    2013-05-20

    In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule's regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

  19. A dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo.

    PubMed

    Mabry, Robert; Gilbertson, Debra G; Frank, Amanda; Vu, Tuyen; Ardourel, Dan; Ostrander, Craig; Stevens, Brenda; Julien, Susan; Franke, Secil; Meengs, Brent; Brody, Jennifer; Presnell, Scott; Hamacher, Nels B; Lantry, Megan; Wolf, Anitra; Bukowski, Tom; Rosler, Robert; Yen, Cindy; Anderson-Haley, Monica; Brasel, Kenneth; Pan, Qi; Franklin, Hank; Thompson, Penny; Dodds, Mike; Underwood, Sara; Peterson, Scott; Sivakumar, Pallavur V; Snavely, Mark

    2010-01-01

    Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRbeta and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.

  20. Characterizing protein domain associations by Small-molecule ligand binding

    PubMed Central

    Li, Qingliang; Cheng, Tiejun; Wang, Yanli; Bryant, Stephen H.

    2012-01-01

    Background Protein domains are evolutionarily conserved building blocks for protein structure and function, which are conventionally identified based on protein sequence or structure similarity. Small molecule binding domains are of great importance for the recognition of small molecules in biological systems and drug development. Many small molecules, including drugs, have been increasingly identified to bind to multiple targets, leading to promiscuous interactions with protein domains. Thus, a large scale characterization of the protein domains and their associations with respect to small-molecule binding is of particular interest to system biology research, drug target identification, as well as drug repurposing. Methods We compiled a collection of 13,822 physical interactions of small molecules and protein domains derived from the Protein Data Bank (PDB) structures. Based on the chemical similarity of these small molecules, we characterized pairwise associations of the protein domains and further investigated their global associations from a network point of view. Results We found that protein domains, despite lack of similarity in sequence and structure, were comprehensively associated through binding the same or similar small-molecule ligands. Moreover, we identified modules in the domain network that consisted of closely related protein domains by sharing similar biochemical mechanisms, being involved in relevant biological pathways, or being regulated by the same cognate cofactors. Conclusions A novel protein domain relationship was identified in the context of small-molecule binding, which is complementary to those identified by traditional sequence-based or structure-based approaches. The protein domain network constructed in the present study provides a novel perspective for chemogenomic study and network pharmacology, as well as target identification for drug repurposing. PMID:23745168

  1. Small Molecule Targeting of a MicroRNA Associated with Hepatocellular Carcinoma.

    PubMed

    Childs-Disney, Jessica L; Disney, Matthew D

    2016-02-19

    Development of precision therapeutics is of immense interest, particularly as applied to the treatment of cancer. By analyzing the preferred cellular RNA targets of small molecules, we discovered that 5"-azido neomycin B binds the Drosha processing site in the microRNA (miR)-525 precursor. MiR-525 confers invasive properties to hepatocellular carcinoma (HCC) cells. Although HCC is one of the most common cancers, treatment options are limited, making the disease often fatal. Herein, we find that addition of 5"-azido neomycin B and its FDA-approved precursor, neomycin B, to an HCC cell line selectively inhibits production of the mature miRNA, boosts a downstream protein, and inhibits invasion. Interestingly, neomycin B is a second-line agent for hepatic encephalopathy (HE) and bacterial infections due to cirrhosis. Our results provocatively suggest that neomycin B, or second-generation derivatives, may be dual functioning molecules to treat both HE and HCC. Collectively, these studies show that rational design approaches can be tailored to disease-associated RNAs to afford potential lead therapeutics.

  2. Homogeneous assay of target molecules based on chemiluminescence resonance energy transfer (CRET) using DNAzyme-linked aptamers.

    PubMed

    Mun, Hyoyoung; Jo, Eun-Jung; Li, Taihua; Joung, Hyou-Arm; Hong, Dong-Gu; Shim, Won-Bo; Jung, Cheulhee; Kim, Min-Gon

    2014-08-15

    We have designed a single-stranded DNAzyme-aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3' end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme-aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Identification of Host-Targeted Small Molecules That Restrict Intracellular Mycobacterium tuberculosis Growth

    PubMed Central

    Silvis, Melanie R.; Luo, Samantha S.; Sogi, Kimberly; Vokes, Martha; Bray, Mark-Anthony; Carpenter, Anne E.; Moore, Christopher B.; Siddiqi, Noman; Rubin, Eric J.; Hung, Deborah T.

    2014-01-01

    Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by

  4. Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease

    PubMed Central

    Yin, Xiangling; Manczak, Maria; Reddy, P. Hemachandra

    2016-01-01

    The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. PMID:26908605

  5. Chalcone-based small-molecule inhibitors attenuate malignant phenotype via targeting deubiquitinating enzymes

    PubMed Central

    Issaenko, Olga A.; Amerik, Alexander Yu

    2012-01-01

    The ubiquitin-proteasome system (UPS) is usurped by many if not all cancers to regulate their survival, proliferation, invasion, angiogenesis and metastasis. Bioflavonoids curcumin and chalcones exhibit anti-neoplastic selectivity through inhibition of the 26S proteasome-activity within the UPS. Here, we provide evidence for a novel mechanism of action of chalcone-based derivatives AM146, RA-9 and RA-14, which exert anticancer activity by targeting deubiquitinating enzymes (DUB) without affecting 20S proteasome catalytic-core activity. The presence of the α,β-unsaturated carbonyl group susceptible to nucleophilic attack from the sulfhydryl of cysteines in the active sites of DUB determines the capacity of novel small-molecules to act as cell-permeable, partly selective DUB inhibitors and induce rapid accumulation of polyubiquitinated proteins and deplete the pool of free ubiquitin. These chalcone-derivatives directly suppress activity of DUB UCH-L1, UCH-L3, USP2, USP5 and USP8, which are known to regulate the turnover and stability of key regulators of cell survival and proliferation. Inhibition of DUB-activity mediated by these compounds downregulates cell-cycle promoters, e.g., cyclin D1 and upregulates tumor suppressors p53, p27Kip1 and p16Ink4A. These changes are associated with arrest in S-G2/M, abrogated anchorage-dependent growth and onset of apoptosis in breast, ovarian and cervical cancer cells without noticeable alterations in primary human cells. Altogether, this work provides evidence of antitumor activity of novel chalcone-based derivatives mediated by their DUB-targeting capacity; supports the development of pharmaceuticals to directly target DUB as a most efficient strategy compared with proteasome inhibition and also provides a clear rationale for the clinical evaluation of these novel small-molecule DUB inhibitors. PMID:22510564

  6. Bispecific small molecule-antibody conjugate targeting prostate cancer.

    PubMed

    Kim, Chan Hyuk; Axup, Jun Y; Lawson, Brian R; Yun, Hwayoung; Tardif, Virginie; Choi, Sei Hyun; Zhou, Quan; Dubrovska, Anna; Biroc, Sandra L; Marsden, Robin; Pinstaff, Jason; Smider, Vaughn V; Schultz, Peter G

    2013-10-29

    Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.

  7. Mechanisms involved in the transport of mercuric ions in target tissues

    PubMed Central

    Bridges, Christy C.; Zalups, Rudolfs K.

    2016-01-01

    Mercury exists in the environment in various forms, all of which pose a risk to human health. Despite guidelines regulating the industrial release of mercury into the environment, humans continue to be exposed regularly to various forms of this metal via inhalation or ingestion. Following exposure, mercuric ions are taken up by and accumulate in numerous organs, including brain, intestine, kidney, liver, and placenta. In order to understand the toxicological effects of exposure to mercury, a thorough understanding of the mechanisms that facilitate entry of mercuric ions into target cells must first be obtained. A number of mechanisms for the transport of mercuric ions into target cells and organs have been proposed in recent years. However, the ability of these mechanisms to transport mercuric ions and the regulatory features of these carriers have not been characterized completely. The purpose of this review is to summarize the current findings related to the mechanisms that may be involved in the transport of inorganic and organic forms of mercury in target tissues and organs. This review will describe mechanisms known to be involved in the transport of mercury and will also propose additional mechanisms that may potentially be involved in the transport of mercuric ions into target cells. PMID:27422290

  8. Recent Advances in Aptamers Targeting Immune System.

    PubMed

    Hu, Piao-Ping

    2017-02-01

    The immune system plays important role in protecting the organism by recognizing non-self molecules from pathogen such as bacteria, parasitic worms, and viruses. When the balance of the host defense system is disturbed, immunodeficiency, autoimmunity, and inflammation occur. Nucleic acid aptamers are short single-stranded DNA (ssDNA) or RNA ligands that interact with complementary molecules with high specificity and affinity. Aptamers that target the molecules involved in immune system to modulate their function have great potential to be explored as new diagnostic and therapeutic agents for immune disorders. This review summarizes recent advances in the development of aptamers targeting immune system. The selection of aptamers with superior chemical and biological characteristics will facilitate their application in the diagnosis and treatment of immune disorders.

  9. Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease.

    PubMed

    Yin, Xiangling; Manczak, Maria; Reddy, P Hemachandra

    2016-05-01

    The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. K+ Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria.

    PubMed

    Groß, Christina J; Mishra, Ritu; Schneider, Katharina S; Médard, Guillaume; Wettmarshausen, Jennifer; Dittlein, Daniela C; Shi, Hexin; Gorka, Oliver; Koenig, Paul-Albert; Fromm, Stephan; Magnani, Giovanni; Ćiković, Tamara; Hartjes, Lara; Smollich, Joachim; Robertson, Avril A B; Cooper, Matthew A; Schmidt-Supprian, Marc; Schuster, Michael; Schroder, Kate; Broz, Petr; Traidl-Hoffmann, Claudia; Beutler, Bruce; Kuster, Bernhard; Ruland, Jürgen; Schneider, Sabine; Perocchi, Fabiana; Groß, Olaf

    2016-10-18

    Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K + efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K + efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila.

    PubMed

    Chang, Bin; Kura, Fumiaki; Amemura-Maekawa, Junko; Koizumi, Nobuo; Watanabe, Haruo

    2005-07-01

    Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.

  12. Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5*

    PubMed Central

    Liu, Liqiong; Richard, Jessica; Kim, Sunyoung; Wojcik, Edward J.

    2014-01-01

    Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable “next generation” target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are “druggable.” One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease. PMID:24737313

  13. Colorimetric Detection of Small Molecules in Complex Matrixes via Target-Mediated Growth of Aptamer-Functionalized Gold Nanoparticles.

    PubMed

    Soh, Jun Hui; Lin, Yiyang; Rana, Subinoy; Ying, Jackie Y; Stevens, Molly M

    2015-08-04

    A versatile and sensitive colorimetric assay that allows the rapid detection of small-molecule targets using the naked eye is demonstrated. The working principle of the assay integrates aptamer-target recognition and the aptamer-controlled growth of gold nanoparticles (Au NPs). Aptamer-target interactions modulate the amount of aptamer strands adsorbed on the surface of aptamer-functionalized Au NPs via desorption of the aptamer strands when target molecules bind with the aptamer. Depending on the resulting aptamer coverage, Au NPs grow into morphologically varied nanostructures, which give rise to different colored solutions. Au NPs with low aptamer coverage grow into spherical NPs, which produce red-colored solutions, whereas Au NPs with high aptamer coverage grow into branched NPs, which produce blue-colored solutions. We achieved visible colorimetric response and nanomolar detection limits for the detection of ochratoxin A (1 nM) in red wine samples, as well as cocaine (1 nM) and 17β-estradiol (0.2 nM) in spiked synthetic urine and saliva, respectively. The detection limits were well within clinically and physiologically relevant ranges, and below the maximum food safety limits. The assay is highly sensitive, specific, and able to detect an array of analytes rapidly without requiring sophisticated equipment, making it relevant for many applications, such as high-throughput drug and clinical screening, food sampling, and diagnostics. Furthermore, the assay is easily adapted as a chip-based platform for rapid and portable target detection.

  14. New small-molecule inhibitor class targeting human immunodeficiency virus type 1 virion maturation.

    PubMed

    Blair, Wade S; Cao, Joan; Fok-Seang, Juin; Griffin, Paul; Isaacson, Jason; Jackson, R Lynn; Murray, Edward; Patick, Amy K; Peng, Qinghai; Perros, Manos; Pickford, Chris; Wu, Hua; Butler, Scott L

    2009-12-01

    A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.

  15. Built-in adjuvanticity of genetically and protein-engineered chimeric molecules for targeting of influenza A peptide epitopes.

    PubMed

    Kerekov, Nikola S; Ivanova, Iva I; Mihaylova, Nikolina M; Nikolova, Maria; Prechl, Jozsef; Tchorbanov, Andrey I

    2014-10-01

    Highly purified, subunit, or synthetic viral antigens are known to be weakly immunogenic and potentate only the antibody, rather than cell-mediated immune responses. An alternative approach for inducing protective immunity with small viral peptides would be the direct targeting of viral epitopes to the immunocompetent cells by DNA vaccines encoding antibody fragments specific to activating cell surface co-receptor molecules. Here, we are exploring as a new genetic vaccine, a DNA chimeric molecule encoding a T and B cell epitope-containing influenza A virus hemagglutinin peptide joined to sequences encoding a single-chain variable fragment antibody fragment specific for the costimulatory B cell complement receptors 1 and 2. This recombinant DNA molecule was inserted into eukaryotic expression vector and used as a naked DNA vaccine in WT and CR1/2 KO mice. The intramuscular administration of the DNA construct resulted in the in vivo expression of an immunogenic chimeric protein, which cross-links cell surface receptors on influenza-specific B cells. The DNA vaccination was followed by prime-boosting with the protein-engineered replica of the DNA construct, thus delivering an activation intracellular signal. Immunization with an expression vector containing the described construct and boosting with the protein chimera induced a strong anti-influenza cytotoxic response, modulation of cytokine profile, and a weak antibody response in Balb/c mice. The same immunization scheme did not result in generation of influenza-specific response in mice lacking the target receptor, underlining the molecular adjuvant effect of receptor targeting.

  16. Proprotein convertase subtilisin/kexin type 9: a new target molecule for gene therapy.

    PubMed

    Banaszewska, Anna; Piechota, Michal; Plewa, Robert

    2012-06-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel target for controlling plasma levels of low-density lipoprotein cholesterol (LDL-C) and decreasing the risk of cardiovascular diseases. At present it is clear that the major classes of commonly prescribed lipid-lowering medications increase serum PCSK9 levels and fail to protect a significant percentage of patients from cardiovascular events. Therefore development of new LDL-C lowering medications that either do not increase circulating PCSK9 levels or work through inhibition of PCSK9 expression and protease activity is a highly desirable approach to overcome hypercholesterolemia. Since there are several agents which are being evaluated in human preclinical and clinical trials, this review summarizes current therapeutic strategies targeting PCSK9, including specific antibodies, antisense oligonucleotides, small interfering RNAs (siRNAs) and other small-molecule inhibitors.

  17. Pharmacophore based design of some multi-targeted compounds targeted against pathways of diabetic complications.

    PubMed

    Chadha, Navriti; Silakari, Om

    2017-09-01

    Diabetic complications is a complex metabolic disorder developed primarily due to prolonged hyperglycemia in the body. The complexity of the disease state as well as the unifying pathophysiology discussed in the literature reports exhibited that the use of multi-targeted agents with multiple complementary biological activities may offer promising therapy for the intervention of the disease over the single-target drugs. In the present study, novel thiazolidine-2,4-dione analogues were designed as multi-targeted agents implicated against the molecular pathways involved in diabetic complications using knowledge based as well as in-silico approaches such as pharmacophore mapping, molecular docking etc. The hit molecules were duly synthesized and biochemical estimation of these molecules against aldose reductase (ALR2), protein kinase Cβ (PKCβ) and poly (ADP-ribose) polymerase 1 (PARP-1) led to identification of compound 2 that showed good potency against PARP-1 and ALR2 enzymes. These positive results support the progress of a low cost multi-targeted agent with putative roles in diabetic complications. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Genetic polymorphisms of molecules involved in host immune response to dengue virus infection.

    PubMed

    Fang, Xin; Hu, Zhen; Shang, Weilong; Zhu, Junmin; Xu, Chuanshan; Rao, Xiancai

    2012-11-01

    The dengue virus (DENV) belongs to the flavivirus family. Each of the four distinct serotypes of this virus is capable of causing human disease, especially in tropical and subtropical areas. The majority of people infected with DENV manifest asymptomatic or dengue fever with flu-like self-limited symptoms. However, a small portion of patients emerge with severe manifestations referred to as dengue hemorrhagic fever, which has a high mortality rate if not treated promptly. The host immune system, which plays important roles throughout the whole process of DENV infection, has been confirmed to have double-edged effects on DENV infection. Recently, much attention has been paid to the genetic heterogeneity of molecules involved in the host immune response to DENV infection. This heterogeneity has been proved to be the determining factor for DENV disease orientation. The present review discusses the primary functions and single nucleotide polymorphisms of some critical molecules in the human DENV immunological defense, especially the polymorphism locus associated with the DENV pathogenesis and disease susceptibility. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  19. Screening of Small Molecule Interactor Library by Using In-Cell NMR Spectroscopy (SMILI-NMR)

    PubMed Central

    Xie, Jingjing; Thapa, Rajiv; Reverdatto, Sergey; Burz, David S.; Shekhtman, Alexander

    2011-01-01

    We developed an in-cell NMR assay for screening small molecule interactor libraries (SMILI-NMR) for compounds capable of disrupting or enhancing specific interactions between two or more components of a biomolecular complex. The method relies on the formation of a well-defined biocomplex and utilizes in-cell NMR spectroscopy to identify the molecular surfaces involved in the interaction at atomic scale resolution. Changes in the interaction surface caused by a small molecule interfering with complex formation are used as a read-out of the assay. The in-cell nature of the experimental protocol insures that the small molecule is capable of penetrating the cell membrane and specifically engaging the target molecule(s). Utility of the method was demonstrated by screening a small dipeptide library against the FKBP–FRB protein complex involved in cell cycle arrest. The dipeptide identified by SMILI-NMR showed biological activity in a functional assay in yeast. PMID:19422228

  20. (Pro)renin receptor: Involvement in diabetic retinopathy and development of molecular targeted therapy.

    PubMed

    Kanda, Atsuhiro; Ishida, Susumu

    2018-03-25

    The renin-angiotensin system (RAS), a crucial regulator of systemic blood pressure (circulatory RAS), plays distinct roles in pathological angiogenesis and inflammation in various organs (tissue RAS), such as diabetic microvascular complications. Using ocular clinical samples and animal disease models, we elucidated molecular mechanisms in which tissue RAS excites the expression of vascular endothelial growth factor (VEGF)-A responsible for retinal inflammation and angiogenesis, the two major pathological events in diabetic retinopathy (DR). Furthermore, we showed the involvement of (pro)renin receptor [(P)RR] in retinal RAS activation and its concurrent intracellular signal transduction (e.g., extracellular signal-regulated kinase); namely, the (P)RR-induced dual pathogenic bioactivity referred to as the receptor-associated prorenin system. Indeed, neovascular endothelial cells in the fibrovascular tissue collected from eyes with proliferative DR were immunoreactive for the receptor-associated prorenin system components including prorenin, (P)RR, phosphorylated extracellular signal-regulated kinase and VEGF-A. Protein levels of soluble (P)RR increased with its positive correlations with prorenin, renin enzymatic activity and VEGF in the vitreous of proliferative DR eyes, suggesting a close link between (P)RR and VEGF-A-driven angiogenic activity. Furthermore, we revealed an unsuspected, PAPS-independent role of (P)RR in glucose-induced oxidative stress. Recently, we developed an innovative single-strand ribonucleic acid interference molecule selectively targeting human and mouse (P)RR, and confirmed its efficacy in suppressing diabetes-induced retinal inflammation in mice. Our data using clinical samples and animal models suggested the significant implication of (P)RR in the pathogenesis of DR, and the potential usefulness of the ribonucleic acid interference molecule as a therapeutic agent to attenuate ocular inflammation and angiogenesis. © 2018 The Authors

  1. Small Molecule Chemical Probes of MicroRNA Function

    PubMed Central

    Velagapudi, Sai Pradeep; Vummidi, Balayeshwanth R.; Disney, Matthew D.

    2015-01-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as strides are made to understand small molecule recognition of RNA from a fundamental perspective. PMID:25500006

  2. Mitochondria-targeted molecules determine the redness of the zebra finch bill.

    PubMed

    Cantarero, Alejandro; Alonso-Alvarez, Carlos

    2017-10-01

    The evolution and production mechanisms of red carotenoid-based ornaments in animals are poorly understood. Recently, it has been suggested that enzymes transforming yellow carotenoids to red pigments (ketolases) in animal cells may be positioned in the inner mitochondrial membrane (IMM) intimately linked to the electron transport chain. These enzymes may mostly synthesize coenzyme Q 10 (coQ 10 ), a key redox-cycler antioxidant molecularly similar to yellow carotenoids. It has been hypothesized that this shared pathway favours the evolution of red traits as sexually selected individual quality indices by revealing a well-adjusted oxidative metabolism. We administered mitochondria-targeted molecules to male zebra finches ( Taeniopygia guttata ) measuring their bill redness, a trait produced by transforming yellow carotenoids. One molecule included coQ 10 (mitoquinone mesylate, MitoQ) and the other one (decyl-triphenylphosphonium; dTPP) has the same structure without the coQ 10 aromatic ring. At the highest dose, the bill colour of MitoQ and dTPP birds strongly differed: MitoQ birds' bills were redder and dTPP birds showed paler bills even compared to birds injected with saline only. These results suggest that ketolases are indeed placed at the IMM and that coQ 10 antioxidant properties may improve their efficiency. The implications for evolutionary theories of sexual signalling are discussed. © 2017 The Author(s).

  3. Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

    PubMed Central

    Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K.; Jiang, Shibo

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development. PMID:26324044

  4. Advances in nanotechnology-based carrier systems for targeted delivery of bioactive drug molecules with special emphasis on immunotherapy in drug resistant tuberculosis - a critical review.

    PubMed

    Singh, Jagdeep; Garg, Tarun; Rath, Goutam; Goyal, Amit K

    2016-06-01

    From the early sixteenth and seventeenth centuries to the present day of life, tuberculosis (TB) still is a global health threat with some new emergence of resistance. This type of emergence poses a vital challenge to control TB cases across the world. Mortality and morbidity rates are high due to this new face of TB. The newer nanotechnology-based drug-delivery approaches involving micro-metric and nano-metric carriers are much needed at this stage. These delivery systems would provide more advantages over conventional systems of treatment by producing enhanced therapeutic efficacy, uniform distribution of drug molecule to the target site, sustained and controlled release of drug molecules and lesser side effects. The main aim to develop these novel drug-delivery systems is to improve the patient compliance and reduce therapy time. This article reviews and elaborates the new concepts and drug-delivery approaches for the treatment of TB involving solid-lipid particulate drug-delivery systems (solid-lipid micro- and nanoparticles, nanostructured lipid carriers), vesicular drug-delivery systems (liposomes, niosomes and liposphere), emulsion-based drug-delivery systems (micro and nanoemulsion) and some other novel drug-delivery systems for the effective treatment of tuberculosis and role of immunomodulators as an adjuvant therapy for management of MDR-TB and XDR-TB.

  5. Advances in nanotechnology-based carrier systems for targeted delivery of bioactive drug molecules with special emphasis on immunotherapy in drug resistant tuberculosis - a critical review.

    PubMed

    Singh, Jagdeep; Garg, Tarun; Rath, Goutam; Goyal, Amit K

    2015-08-11

    From the early sixteenth and seventeenth centuries to the present day of life, tuberculosis (TB) still is a global health threat with some new emergence of resistance. This type of emergence poses a vital challenge to control TB cases across the world. Mortality and morbidity rates are high due to this new face of TB. The newer nanotechnology-based drug-delivery approaches involving micro-metric and nano-metric carriers are much needed at this stage. These delivery systems would provide more advantages over conventional systems of treatment by producing enhanced therapeutic efficacy, uniform distribution of drug molecule to the target site, sustained and controlled release of drug molecules and lesser side effects. The main aim to develop these novel drug-delivery systems is to improve the patient compliance and reduce therapy time. This article reviews and elaborates the new concepts and drug-delivery approaches for the treatment of TB involving solid-lipid particulate drug-delivery systems (solid-lipid micro- and nanoparticles, nanostructured lipid carriers), vesicular drug-delivery systems (liposomes, niosomes and liposphere), emulsion-based drug-delivery systems (micro and nanoemulsion) and some other novel drug-delivery systems for the effective treatment of tuberculosis and role of immunomodulators as an adjuvant therapy for management of MDR-TB and XDR-TB.

  6. Neural guidance molecules regulate vascular remodeling and vessel navigation.

    PubMed

    Eichmann, Anne; Makinen, Taija; Alitalo, Kari

    2005-05-01

    The development of the embryonic blood vascular and lymphatic systems requires the coordinated action of several transcription factors and growth factors that target endothelial and periendothelial cells. However, according to recent studies, the precise "wiring" of the vascular system does not occur without an ordered series of guidance decisions involving several molecules initially discovered for axons in the nervous system, including ephrins, netrins, slits, and semaphorins. Here, we summarize the new advances in our understanding of the roles of these axonal pathfinding molecules in vascular remodeling and vessel guidance, indicating that neuronal axons and vessel sprouts use common molecular mechanisms for navigation in the body.

  7. Targeting mitochondria with small molecules: the preparation of MitoB and MitoP as exomarkers of mitochondrial hydrogen peroxide.

    PubMed

    Cairns, Andrew G; McQuaker, Stephen J; Murphy, Michael P; Hartley, Richard C

    2015-01-01

    Small molecules can be physicochemically targeted to mitochondria using the lipophilic alkyltriphenylphosphonium (TPP) group. Once in the mitochondria the TPP-conjugate can detect or influence processes within the mitochondrial matrix directly. Alternatively, the conjugate can behave as a prodrug, which is activated by release from the TPP group either using an internal or external instruction. Small molecules can be designed that can be used in any cell line, tissue or whole organism, allow temporal control, and be applied in a reversible dose-dependent fashion. An example is the detection and quantification of hydrogen peroxide in mitochondria of whole living organisms by MitoB. Hydrogen peroxide produced within the mitochondrial matrix is involved in signalling and implicated in the oxidative damage associated with aging and a wide range of age-associated conditions including cardiovascular disease, neurodegeneration, and cancer. MitoB accumulates in mitochondria and is converted into the exomarker, MitoP, by hydrogen peroxide in the mitochondrial matrix. The hydrogen peroxide concentration is determined from the ratio of MitoP to MitoB after a period of incubation, and this ratio is determined by mass spectrometry using d15-MitoP and d15-MitoB as standard. Here we describe the synthesis of MitoB and MitoP and the deuterated standards necessary for this method of quantification.

  8. Small molecule chemical probes of microRNA function.

    PubMed

    Velagapudi, Sai Pradeep; Vummidi, Balayeshwanth R; Disney, Matthew D

    2015-02-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as progress is made in understanding small molecule recognition of RNA. Copyright © 2014. Published by Elsevier Ltd.

  9. A concise review on advances in development of small molecule anti-inflammatory therapeutics emphasising AMPK: An emerging target.

    PubMed

    Gejjalagere Honnappa, Chethan; Mazhuvancherry Kesavan, Unnikrishnan

    2016-12-01

    Inflammatory diseases are complex, multi-factorial outcomes of evolutionarily conserved tissue repair processes. For decades, non-steroidal anti-inflammatory drugs and cyclooxygenase inhibitors, the primary drugs of choice for the management of inflammatory diseases, addressed individual targets in the arachidonic acid pathway. Unsatisfactory safety and efficacy profiles of the above have necessitated the development of multi-target agents to treat complex inflammatory diseases. Current anti-inflammatory therapies still fall short of clinical needs and the clinical trial results of multi-target therapeutics are anticipated. Additionally, new drug targets are emerging with improved understanding of molecular mechanisms controlling the pathophysiology of inflammation. This review presents an outline of small molecules and drug targets in anti-inflammatory therapeutics with a summary of a newly identified target AMP-activated protein kinase, which constitutes a novel therapeutic pathway in inflammatory pathology. © The Author(s) 2016.

  10. GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice.

    PubMed

    Wang, Z; Gleichmann, H

    1998-01-01

    In mice, diabetes can be induced by multiple low doses of streptozotocin (MLD-STZ), i.e., 40 mg/kg body wt on each of 5 consecutive days. In this model, diabetes develops only when STZ induces both beta-cell toxicity and T-cell-dependent immune reactions. The target molecule(s) of MLD-STZ-induced beta-cell toxicity are not known, however. In this study, we report that GLUT2 is a target molecule for MLD-STZ toxicity. Ex vivo, a gradual decrement of both GLUT2 protein and mRNA expression was found in pancreatic islets isolated from MLD-STZ-treated C57BL/6 male mice, whereas mRNA expression of beta-actin, glucokinase, and proinsulin remained unaffected. Significant reduction of both GLUT2 protein and mRNA expression was first noted 1 day after the third STZ injection, clearly preceding the onset of hyperglycemia. The extent of reduction increased with the number of STZ injections administered and increased over time, after the last, i.e., fifth, STZ injection. The STZ-induced reduction of GLUT2 protein and mRNA was not due to an essential loss of beta-cells, because ex vivo, not only the total RNA yield and protein content in isolated islets, but also proinsulin mRNA expression, failed to differ significantly in the differently treated groups. Furthermore, islets isolated from MLD-STZ-treated donors responded to the nonglucose secretagogue arginine in a pattern similar to that of solvent-treated donors. Interestingly, the MLD-STZ-induced reduction of both GLUT2 protein and mRNA was prevented by preinjecting mice with 5-thio-D-glucose before each STZ injection. Apparently, GLUT2 is a crucial target molecule of MLD-STZ toxicity, and this toxicity seems to precede the immune reactions against beta-cells.

  11. The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer.

    PubMed

    Campodónico, Paola B; de Kier Joffé, Elisa D Bal; Urtreger, Alejandro J; Lauria, Lilia S; Lastiri, José M; Puricelli, Lydia I; Todaro, Laura B

    2010-04-01

    Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti-NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti-NCAM-treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM-140 knocked-down by siRNA in LP07 cells pretreated or not with anti-NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti-NCAM treatment could be mimicking homophilic trans-interactions and NCAM-140 knocked-down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression.

  12. Discovery of a biomarker and lead small molecules to target r(GGGGCC)-associated defects in c9FTD/ALS.

    PubMed

    Su, Zhaoming; Zhang, Yongjie; Gendron, Tania F; Bauer, Peter O; Chew, Jeannie; Yang, Wang-Yong; Fostvedt, Erik; Jansen-West, Karen; Belzil, Veronique V; Desaro, Pamela; Johnston, Amelia; Overstreet, Karen; Oh, Seok-Yoon; Todd, Peter K; Berry, James D; Cudkowicz, Merit E; Boeve, Bradley F; Dickson, Dennis; Floeter, Mary Kay; Traynor, Bryan J; Morelli, Claudia; Ratti, Antonia; Silani, Vincenzo; Rademakers, Rosa; Brown, Robert H; Rothstein, Jeffrey D; Boylan, Kevin B; Petrucelli, Leonard; Disney, Matthew D

    2014-09-03

    A repeat expansion in C9ORF72 causes frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). RNA of the expanded repeat (r(GGGGCC)exp) forms nuclear foci or undergoes repeat-associated non-ATG (RAN) translation, producing "c9RAN proteins." Since neutralizing r(GGGGCC)exp could inhibit these potentially toxic events, we sought to identify small-molecule binders of r(GGGGCC)exp. Chemical and enzymatic probing of r(GGGGCC)8 indicate that it adopts a hairpin structure in equilibrium with a quadruplex structure. Using this model, bioactive small molecules targeting r(GGGGCC)exp were designed and found to significantly inhibit RAN translation and foci formation in cultured cells expressing r(GGGGCC)66 and neurons transdifferentiated from fibroblasts of repeat expansion carriers. Finally, we show that poly(GP) c9RAN proteins are specifically detected in c9ALS patient cerebrospinal fluid. Our findings highlight r(GGGGCC)exp-binding small molecules as a possible c9FTD/ALS therapeutic and suggest that c9RAN proteins could potentially serve as a pharmacodynamic biomarker to assess efficacy of therapies that target r(GGGGCC)exp. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Discovery of a Biomarker and Lead Small Molecules to Target r(GGGGCC)-Associated Defects in c9FTD/ALS

    PubMed Central

    Su, Zhaoming; Zhang, Yongjie; Gendron, Tania F.; Bauer, Peter O.; Chew, Jeannie; Yang, Wang-Yong; Fostvedt, Erik; Jansen-West, Karen; Belzil, Veronique V.; Desaro, Pamela; Johnston, Amelia; Overstreet, Karen; Oh, Seok-Yoon; Todd, Peter K.; Berry, James D.; Cudkowicz, Merit E.; Boeve, Bradley F.; Dickson, Dennis; Floeter, Mary Kay; Traynor, Bryan J.; Morelli, Claudia; Ratti, Antonia; Silani, Vincenzo; Rademakers, Rosa; Brown, Robert H.; Rothstein, Jeffrey D.; Boylan, Kevin B.; Petrucelli, Leonard; Disney, Matthew D.

    2014-01-01

    Summary A repeat expansion in C9ORF72 causes frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). RNA of the expanded repeat (r(GGGGCC)exp) forms nuclear foci or undergoes repeat-associated non-ATG (RAN) translation producing “c9RAN proteins”. Since neutralizing r(GGGGCC)exp could inhibit these potentially toxic events, we sought to identify small molecule binders of r(GGGGCC)exp. Chemical and enzymatic probing of r(GGGGCC)8 indicate it adopts a hairpin structure in equilibrium with a quadruplex structure. Using this model, bioactive small molecules targeting r(GGGGCC)exp were designed and found to significantly inhibit RAN translation and foci formation in cultured cells expressing r(GGGGCC)66 and neurons trans-differentiated from fibroblasts of repeat expansion carriers. Finally, we show that poly(GP) c9RAN proteins are specifically detected in c9ALS patient cerebrospinal fluid. Our findings highlight r(GGGGCC)exp-binding small molecules as a possible c9FTD/ALS therapeutic, and suggest c9RAN proteins could potentially serve as a pharmacodynamic biomarker to assess efficacy of therapies that target r(GGGGCC)exp. PMID:25132468

  14. Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

    PubMed

    Wu, Zining; Graybill, Todd L; Zeng, Xin; Platchek, Michael; Zhang, Jean; Bodmer, Vera Q; Wisnoski, David D; Deng, Jianghe; Coppo, Frank T; Yao, Gang; Tamburino, Alex; Scavello, Genaro; Franklin, G Joseph; Mataruse, Sibongile; Bedard, Katie L; Ding, Yun; Chai, Jing; Summerfield, Jennifer; Centrella, Paolo A; Messer, Jeffrey A; Pope, Andrew J; Israel, David I

    2015-12-14

    DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

  15. Alkali-ion microsolvation with benzene molecules.

    PubMed

    Marques, J M C; Llanio-Trujillo, J L; Albertí, M; Aguilar, A; Pirani, F

    2012-05-24

    The target of this investigation is to characterize by a recently developed methodology, the main features of the first solvation shells of alkaline ions in nonpolar environments due to aromatic rings, which is of crucial relevance to understand the selectivity of several biochemical phenomena. We employ an evolutionary algorithm to obtain putative global minima of clusters formed with alkali-ions (M(+)) solvated with n benzene (Bz) molecules, i.e., M(+)-(Bz)(n). The global intermolecular interaction has been decomposed in Bz-Bz and in M(+)-Bz contributions, using a potential model based on different decompositions of the molecular polarizability of benzene. Specifically, we have studied the microsolvation of Na(+), K(+), and Cs(+) with benzene molecules. Microsolvation clusters up to n = 21 benzene molecules are involved in this work and the achieved global minimum structures are reported and discussed in detail. We observe that the number of benzene molecules allocated in the first solvation shell increases with the size of the cation, showing three molecules for Na(+) and four for both K(+) and Cs(+). The structure of this solvation shell keeps approximately unchanged as more benzene molecules are added to the cluster, which is independent of the ion. Particularly stable structures, so-called "magic numbers", arise for various nuclearities of the three alkali-ions. Strong "magic numbers" appear at n = 2, 3, and 4 for Na(+), K(+), and Cs(+), respectively. In addition, another set of weaker "magic numbers" (three per alkali-ion) are reported for larger nuclearities.

  16. Small-molecule inhibitors of the receptor tyrosine kinases: promising tools for targeted cancer therapies.

    PubMed

    Hojjat-Farsangi, Mohammad

    2014-08-08

    Chemotherapeutic and cytotoxic drugs are widely used in the treatment of cancer. In spite of the improvements in the life quality of patients, their effectiveness is compromised by several disadvantages. This represents a demand for developing new effective strategies with focusing on tumor cells and minimum side effects. Targeted cancer therapies and personalized medicine have been defined as a new type of emerging treatments. Small molecule inhibitors (SMIs) are among the most effective drugs for targeted cancer therapy. The growing number of approved SMIs of receptor tyrosine kinases (RTKs) i.e., tyrosine kinase inhibitors (TKIs) in the clinical oncology imply the increasing attention and application of these therapeutic tools. Most of the current approved RTK-TKIs in preclinical and clinical settings are multi-targeted inhibitors with several side effects. Only a few specific/selective RTK-TKIs have been developed for the treatment of cancer patients. Specific/selective RTK-TKIs have shown less deleterious effects compared to multi-targeted inhibitors. This review intends to highlight the importance of specific/selective TKIs for future development with less side effects and more manageable agents. This article provides an overview of: (1) the characteristics and function of RTKs and TKIs; (2) the recent advances in the improvement of specific/selective RTK-TKIs in preclinical or clinical settings; and (3) emerging RTKs for targeted cancer therapies by TKIs.

  17. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-06-06

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  18. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-05-30

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  19. Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy

    PubMed Central

    Kuzuya, Akinori; Sakai, Yusuke; Yamazaki, Takahiro; Xu, Yan; Komiyama, Makoto

    2011-01-01

    DNA origami involves the folding of long single-stranded DNA into designed structures with the aid of short staple strands; such structures may enable the development of useful nanomechanical DNA devices. Here we develop versatile sensing systems for a variety of chemical and biological targets at molecular resolution. We have designed functional nanomechanical DNA origami devices that can be used as 'single-molecule beacons', and function as pinching devices. Using 'DNA origami pliers' and 'DNA origami forceps', which consist of two levers ~170 nm long connected at a fulcrum, various single-molecule inorganic and organic targets ranging from metal ions to proteins can be visually detected using atomic force microscopy by a shape transition of the origami devices. Any detection mechanism suitable for the target of interest, pinching, zipping or unzipping, can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform. PMID:21863016

  20. Targeting the Binding Interface on a Shared Receptor Subunit of a Cytokine Family Enables the Inhibition of Multiple Member Cytokines with Selectable Target Spectrum*

    PubMed Central

    Nata, Toshie; Basheer, Asjad; Cocchi, Fiorenza; van Besien, Richard; Massoud, Raya; Jacobson, Steven; Azimi, Nazli; Tagaya, Yutaka

    2015-01-01

    The common γ molecule (γc) is a shared signaling receptor subunit used by six γc-cytokines. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Moreover, recent studies suggest that multiple γc-cytokines are pathogenically involved in a single disease, thus making the shared γc-molecule a logical target for therapeutic intervention. However, the current therapeutic strategies seem to lack options to treat such cases, partly because of the lack of appropriate neutralizing antibodies recognizing the γc and, more importantly, because of the inherent and practical limitations in the use of monoclonal antibodies. By targeting the binding interface of the γc and cytokines, we successfully designed peptides that not only inhibit multiple γc-cytokines but with a selectable target spectrum. Notably, the lead peptide inhibited three γc-cytokines without affecting the other three or non-γc-cytokines. Biological and mutational analyses of our peptide provide new insights to our current understanding on the structural aspect of the binding of γc-cytokines the γc-molecule. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo, efficiently blocks the action of one of the target cytokines in animal models. Collectively, our technology can be expanded to target various combinations of γc-cytokines and thereby will provide a novel strategy to the current anti-cytokine therapies against immune, inflammatory, and malignant diseases. PMID:26183780

  1. Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive.

    PubMed

    Childs-Disney, Jessica L; Hoskins, Jason; Rzuczek, Suzanne G; Thornton, Charles A; Disney, Matthew D

    2012-05-18

    RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)(exp), is present in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)(exp) folds into a hairpin with regularly repeating 5'CUG/3'GUC motifs and sequesters muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1, including (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)(exp) were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5'CUG/3'GUC motif in r(CUG)(exp.) Therefore, we designed multivalent ligands to bind simultaneously multiple copies of this motif in r(CUG)(exp). Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence.

  2. Identification of alsterpaullone as a novel small molecule inhibitor to target group 3 medulloblastoma.

    PubMed

    Faria, Claudia C; Agnihotri, Sameer; Mack, Stephen C; Golbourn, Brian J; Diaz, Roberto J; Olsen, Samantha; Bryant, Melissa; Bebenek, Matthew; Wang, Xin; Bertrand, Kelsey C; Kushida, Michelle; Head, Renee; Clark, Ian; Dirks, Peter; Smith, Christian A; Taylor, Michael D; Rutka, James T

    2015-08-28

    Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma.

  3. Antibody-enabled small-molecule drug discovery.

    PubMed

    Lawson, Alastair D G

    2012-06-29

    Although antibody-based therapeutics have become firmly established as medicines for serious diseases, the value of antibodies as tools in the early stages of small-molecule drug discovery is only beginning to be realized. In particular, antibodies may provide information to reduce risk in small-molecule drug discovery by enabling the validation of targets and by providing insights into the design of small-molecule screening assays. Moreover, antibodies can act as guides in the quest for small molecules that have the ability to modulate protein-protein interactions, which have traditionally only been considered to be tractable targets for biological drugs. The development of small molecules that have similar therapeutic effects to current biologics has the potential to benefit a broader range of patients at earlier stages of disease.

  4. Targeted Phenotypic Screening in Plasmodium falciparum and Toxoplasma gondii Reveals Novel Modes of Action of Medicines for Malaria Venture Malaria Box Molecules

    PubMed Central

    2018-01-01

    ABSTRACT The Malaria Box collection includes 400 chemically diverse small molecules with documented potency against malaria parasite growth, but the underlying modes of action are largely unknown. Using complementary phenotypic screens against Plasmodium falciparum and Toxoplasma gondii, we report phenotype-specific hits based on inhibition of overall parasite growth, apicoplast segregation, and egress or host invasion, providing hitherto unavailable insights into the possible mechanisms affected. First, the Malaria Box library was screened against tachyzoite stage T. gondii and the half-maximal effective concentrations (EC50s) of molecules showing ≥80% growth inhibition at 10 µM were determined. Comparison of the EC50s for T. gondii and P. falciparum identified a subset of 24 molecules with nanomolar potency against both parasites. Thirty molecules that failed to induce acute growth inhibition in T. gondii tachyzoites in a 2-day assay caused delayed parasite death upon extended exposure, with at least three molecules interfering with apicoplast segregation during daughter cell formation. Using flow cytometry and microscopy-based examinations, we prioritized 26 molecules with the potential to inhibit host cell egress/invasion during asexual developmental stages of P. falciparum. None of the inhibitors affected digestive vacuole integrity, ruling out a mechanism mediated by broadly specific protease inhibitor activity. Interestingly, five of the plasmodial egress inhibitors inhibited ionophore-induced egress of T. gondii tachyzoites. These findings highlight the advantage of comparative and targeted phenotypic screens in related species as a means to identify lead molecules with a conserved mode of action. Further work on target identification and mechanism analysis will facilitate the development of antiparasitic compounds with cross-species efficacy. IMPORTANCE The phylum Apicomplexa includes many human and animal pathogens, such as Plasmodium falciparum

  5. Collaborative Enhancement of Endothelial Targeting of Nanocarriers by Modulating Platelet-Endothelial Cell Adhesion Molecule-1/CD31 Epitope Engagement.

    PubMed

    Chacko, Ann-Marie; Han, Jingyan; Greineder, Colin F; Zern, Blaine J; Mikitsh, John L; Nayak, Madhura; Menon, Divya; Johnston, Ian H; Poncz, Mortimer; Eckmann, David M; Davies, Peter F; Muzykantov, Vladimir R

    2015-07-28

    Nanocarriers (NCs) coated with antibodies (Abs) to extracellular epitopes of the transmembrane glycoprotein PECAM (platelet endothelial cell adhesion molecule-1/CD31) enable targeted drug delivery to vascular endothelial cells. Recent studies revealed that paired Abs directed to adjacent, yet distinct epitopes of PECAM stimulate each other's binding to endothelial cells in vitro and in vivo ("collaborative enhancement"). This phenomenon improves targeting of therapeutic fusion proteins, yet its potential role in targeting multivalent NCs has not been addressed. Herein, we studied the effects of Ab-mediated collaborative enhancement on multivalent NC spheres coated with PECAM Abs (Ab/NC, ∼180 nm diameter). We found that PECAM Abs do mutually enhance endothelial cell binding of Ab/NC coated by paired, but not "self" Ab. In vitro, collaborative enhancement of endothelial binding of Ab/NC by paired Abs is modulated by Ab/NC avidity, epitope selection, and flow. Cell fixation, but not blocking of endocytosis, obliterated collaborative enhancement of Ab/NC binding, indicating that the effect is mediated by molecular reorganization of PECAM molecules in the endothelial plasmalemma. The collaborative enhancement of Ab/NC binding was affirmed in vivo. Intravascular injection of paired Abs enhanced targeting of Ab/NC to pulmonary vasculature in mice by an order of magnitude. This stimulatory effect greatly exceeded enhancement of Ab targeting by paired Abs, indicating that '"collaborative enhancement"' effect is even more pronounced for relatively large multivalent carriers versus free Abs, likely due to more profound consequences of positive alteration of epitope accessibility. This phenomenon provides a potential paradigm for optimizing the endothelial-targeted nanocarrier delivery of therapeutic agents.

  6. Interaction of tumor and host cells with adhesion and extracellular matrix molecules in the development of multiple myeloma.

    PubMed

    Teoh, G; Anderson, K C

    1997-02-01

    Adhesion molecules play an important role in the growth regulation and migration of multiple myeloma (MM) cells. They mediate homing of MM cells to the bone marrow and MM cell to bone marrow stromal cell adhesion, with resultant interleukin-6 related autocrine and paracine growth and antiapoptotic affects. Their pattern of expression on tumor cells correlates with the development of plasma cell leukemia or extramedullary disease. Clinically, expression of adhesion molecules on tumor cells or in the serum has already shown prognostic utility. Finally, since adhesion molecules are involved at multiple steps in the pathogenesis of MM, therapeutic studies may target these molecules.

  7. Small Molecule Targeted Recruitment of a Nuclease to RNA.

    PubMed

    Costales, Matthew G; Matsumoto, Yasumasa; Velagapudi, Sai Pradeep; Disney, Matthew D

    2018-06-06

    The choreography between RNA synthesis and degradation is a key determinant in biology. Engineered systems such as CRISPR have been developed to rid a cell of RNAs. Here, we show that a small molecule can recruit a nuclease to a specific transcript, triggering its destruction. A small molecule that selectively binds the oncogenic microRNA(miR)-96 hairpin precursor was appended with a short 2'-5' poly(A) oligonucleotide. The conjugate locally activated endogenous, latent ribonuclease (RNase L), which selectively cleaved the miR-96 precursor in cancer cells in a catalytic and sub-stoichiometric fashion. Silencing miR-96 derepressed pro-apoptotic FOXO1 transcription factor, triggering apoptosis in breast cancer, but not healthy breast, cells. These results demonstrate that small molecules can be programmed to selectively cleave RNA via nuclease recruitment and has broad implications.

  8. MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development.

    PubMed

    Lu, Cecilia S; Zhai, Bo; Mauss, Alex; Landgraf, Matthias; Gygi, Stephen; Van Vactor, David

    2014-09-26

    Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.

  9. MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

    PubMed Central

    Lu, Cecilia S.; Zhai, Bo; Mauss, Alex; Landgraf, Matthias; Gygi, Stephen; Van Vactor, David

    2014-01-01

    Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins. PMID:25135978

  10. A Small Molecule that Targets r(CGG)exp and Improves Defects in Fragile X-Associated Tremor Ataxia Syndrome

    PubMed Central

    Disney, Matthew D.; Liu, Biao; Yang, Wang-Yong; Sellier, Chantal; Tran, Tuan; Charlet-Berguerand, Nicolas; Childs-Disney, Jessica L.

    2012-01-01

    The development of small molecule chemical probes or therapeutics that target RNA remains a significant challenge despite the great interest in such compounds. The most significant barrier to compound development is a lack of knowledge of the chemical and RNA motif spaces that interact specifically. Herein, we describe a bioactive small molecule probe that targets expanded r(CGG) repeats, or r(CGG)exp , that causes Fragile X-associated Tremor Ataxia Syndrome (FXTAS). The compound was identified by using information on the chemotypes and RNA motifs that interact. Specifically, 9-hydroxy-5,11-dimethyl-2-(2-(piperidin-1-yl)ethyl)-6H-pyrido[4,3-b]carbazol-2-ium, binds the 5’CGG/3’GGC motifs in r(CGG)exp and disrupts a toxic r(CGG)exp -protein complex in vitro. Structure-activity relationships (SAR) studies determined that the alkylated pyridyl and phenolic side chains are important chemotypes that drive molecular recognition to r(CGG)exp . Importantly, the compound is efficacious in FXTAS model cellular systems as evidenced by its ability to improve FXTAS-associated pre-mRNA splicing defects and to reduce the size and number of r(CGG)exp -protein aggregates. This approach may establish a general strategy to identify lead ligands that target RNA while also providing a chemical probe to dissect the varied mechanisms by which r(CGG)exp promotes toxicity. PMID:22948243

  11. A small molecule that targets r(CGG)(exp) and improves defects in fragile X-associated tremor ataxia syndrome.

    PubMed

    Disney, Matthew D; Liu, Biao; Yang, Wang-Yong; Sellier, Chantal; Tran, Tuan; Charlet-Berguerand, Nicolas; Childs-Disney, Jessica L

    2012-10-19

    The development of small molecule chemical probes or therapeutics that target RNA remains a significant challenge despite the great interest in such compounds. The most significant barrier to compound development is defining which chemical and RNA motif spaces interact specifically. Herein, we describe a bioactive small molecule probe that targets expanded r(CGG) repeats, or r(CGG)(exp), that causes Fragile X-associated Tremor Ataxia Syndrome (FXTAS). The compound was identified by using information on the chemotypes and RNA motifs that interact. Specifically, 9-hydroxy-5,11-dimethyl-2-(2-(piperidin-1-yl)ethyl)-6H-pyrido[4,3-b]carbazol-2-ium binds the 5'CGG/3'GGC motifs in r(CGG)(exp) and disrupts a toxic r(CGG)(exp)-protein complex in vitro. Structure-activity relationship studies determined that the alkylated pyridyl and phenolic side chains are important chemotypes that drive molecular recognition of r(CGG)(exp). Importantly, the compound is efficacious in FXTAS model cellular systems as evidenced by its ability to improve FXTAS-associated pre-mRNA splicing defects and to reduce the size and number of r(CGG)(exp)-containing nuclear foci. This approach may establish a general strategy to identify lead ligands that target RNA while also providing a chemical probe to dissect the varied mechanisms by which r(CGG)(exp) promotes toxicity.

  12. Small-molecule intramimics of formin autoinhibition: a new strategy to target the cytoskeletal remodeling machinery in cancer cells.

    PubMed

    Lash, L Leanne; Wallar, Bradley J; Turner, Julie D; Vroegop, Steven M; Kilkuskie, Robert E; Kitchen-Goosen, Susan M; Xu, H Eric; Alberts, Arthur S

    2013-11-15

    Although the cancer cell cytoskeleton is a clinically validated target, few new strategies have emerged for selectively targeting cell division by modulating the cytoskeletal structure, particularly ways that could avoid the cardiotoxic and neurotoxic effects of current agents such as taxanes. We address this gap by describing a novel class of small-molecule agonists of the mammalian Diaphanous (mDia)-related formins, which act downstream of Rho GTPases to assemble actin filaments, and their organization with microfilaments to establish and maintain cell polarity during migration and asymmetric division. GTP-bound Rho activates mDia family members by disrupting the interaction between the DID and DAD autoregulatory domains, which releases the FH2 domain to modulate actin and microtubule dynamics. In screening for DID-DAD disruptors that activate mDia, we identified two molecules called intramimics (IMM-01 and -02) that were sufficient to trigger actin assembly and microtubule stabilization, serum response factor-mediated gene expression, cell-cycle arrest, and apoptosis. In vivo analysis of IMM-01 and -02 established their ability to slow tumor growth in a mouse xenograft model of colon cancer. Taken together, our work establishes the use of intramimics and mDia-related formins as a new general strategy for therapeutic targeting of the cytoskeletal remodeling machinery of cancer cells. ©2013 AACR

  13. Sample Analysis at Mars (SAM) and Mars Organic Molecule Analyzer (MOMA) as Critical In Situ Investigation for Targeting Mars Returned Samples

    NASA Astrophysics Data System (ADS)

    Freissinet, C.; Glavin, D. P.; Mahaffy, P. R.; Szopa, C.; Buch, A.; Goesmann, F.; Goetz, W.; Raulin, F.; SAM Science Team; MOMA Science Team

    2018-04-01

    SAM (Curiosity) and MOMA (ExoMars) Mars instruments, seeking for organics and biosignatures, are essential to establish taphonomic windows of preservation of molecules, in order to target the most interesting samples to return from Mars.

  14. A Forward Genetic Screen for Molecules Involved in Pheromone-Induced Dauer Formation in Caenorhabditis elegans.

    PubMed

    Neal, Scott J; Park, JiSoo; DiTirro, Danielle; Yoon, Jason; Shibuya, Mayumi; Choi, Woochan; Schroeder, Frank C; Butcher, Rebecca A; Kim, Kyuhyung; Sengupta, Piali

    2016-05-03

    Animals must constantly assess their surroundings and integrate sensory cues to make appropriate behavioral and developmental decisions. Pheromones produced by conspecific individuals provide critical information regarding environmental conditions. Ascaroside pheromone concentration and composition are instructive in the decision of Caenorhabditis elegans to either develop into a reproductive adult or enter into the stress-resistant alternate dauer developmental stage. Pheromones are sensed by a small set of sensory neurons, and integrated with additional environmental cues, to regulate neuroendocrine signaling and dauer formation. To identify molecules required for pheromone-induced dauer formation, we performed an unbiased forward genetic screen and identified phd (pheromone response-defective dauer) mutants. Here, we describe new roles in dauer formation for previously identified neuronal molecules such as the WD40 domain protein QUI-1 and MACO-1 Macoilin, report new roles for nociceptive neurons in modulating pheromone-induced dauer formation, and identify tau tubulin kinases as new genes involved in dauer formation. Thus, phd mutants define loci required for the detection, transmission, or integration of pheromone signals in the regulation of dauer formation. Copyright © 2016 Neal et al.

  15. High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

    PubMed

    Stockwell, B R; Haggarty, S J; Schreiber, S L

    1999-02-01

    Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

  16. Involvement of sulfates from cruzipain, a major antigen of Trypanosoma cruzi, in the interaction with immunomodulatory molecule Siglec-E.

    PubMed

    Ferrero, Maximiliano R; Heins, Anja M; Soprano, Luciana L; Acosta, Diana M; Esteva, Mónica I; Jacobs, Thomas; Duschak, Vilma G

    2016-02-01

    In order to investigate the involvement of sulfated groups in the Trypanosoma cruzi host-parasite relationship, we studied the interaction between the major cysteine proteinase of T. cruzi, cruzipain (Cz), a sulfate-containing sialylated molecule and the sialic acid-binding immunoglobulin like lectin-E (Siglec-E). To this aim, ELISA, indirect immunofluorescence assays and flow cytometry, using mouse Siglec-E-Fc fusion molecules and glycoproteins of parasites, were performed. Competition assays verified that the lectins, Maackia amurensis II (Mal II) and Siglec-E-Fc, compete for the same binding sites. Taking into account that Mal II binding remains unaltered by sulfation, we established this lectin as sialylation degree control. Proteins of an enriched microsomal fraction showed the highest binding to Siglec-E as compared with those from the other parasite subcellular fractions. ELISA assays and the affinity purification of Cz by a Siglec-E column confirmed the interaction between both molecules. The significant decrease in binding of Siglec-E-Fc to Cz and to its C-terminal domain (C-T) after desulfation of these molecules suggests that sulfates contribute to the interaction between Siglec-E-Fc and these glycoproteins. Competitive ELISA assays confirmed the involvement of sulfated epitopes in the affinity between Siglec-E and Cz, probably modified by natural protein environment. Interestingly, data from flow cytometry of untreated and chlorate-treated parasites suggested that sulfates are not primary receptors, but enhance the binding of Siglec-E to trypomastigotic forms. Altogether, our findings support the notion that sulfate-containing sialylated glycoproteins interact with Siglec-E, an ortholog protein of human Siglec-9, and might modulate the immune response of the host, favoring parasitemia and persistence of the parasite.

  17. PAT: predictor for structured units and its application for the optimization of target molecules for the generation of synthetic antibodies.

    PubMed

    Jeon, Jouhyun; Arnold, Roland; Singh, Fateh; Teyra, Joan; Braun, Tatjana; Kim, Philip M

    2016-04-01

    The identification of structured units in a protein sequence is an important first step for most biochemical studies. Importantly for this study, the identification of stable structured region is a crucial first step to generate novel synthetic antibodies. While many approaches to find domains or predict structured regions exist, important limitations remain, such as the optimization of domain boundaries and the lack of identification of non-domain structured units. Moreover, no integrated tool exists to find and optimize structural domains within protein sequences. Here, we describe a new tool, PAT ( http://www.kimlab.org/software/pat ) that can efficiently identify both domains (with optimized boundaries) and non-domain putative structured units. PAT automatically analyzes various structural properties, evaluates the folding stability, and reports possible structural domains in a given protein sequence. For reliability evaluation of PAT, we applied PAT to identify antibody target molecules based on the notion that soluble and well-defined protein secondary and tertiary structures are appropriate target molecules for synthetic antibodies. PAT is an efficient and sensitive tool to identify structured units. A performance analysis shows that PAT can characterize structurally well-defined regions in a given sequence and outperforms other efforts to define reliable boundaries of domains. Specially, PAT successfully identifies experimentally confirmed target molecules for antibody generation. PAT also offers the pre-calculated results of 20,210 human proteins to accelerate common queries. PAT can therefore help to investigate large-scale structured domains and improve the success rate for synthetic antibody generation.

  18. Neural cell adhesion molecule-deficient beta-cell tumorigenesis results in diminished extracellular matrix molecule expression and tumour cell-matrix adhesion.

    PubMed

    Håkansson, Joakim; Xian, Xiaojie; He, Liqun; Ståhlberg, Anders; Nelander, Sven; Samuelsson, Tore; Kubista, Mikael; Semb, Henrik

    2005-01-01

    To understand by which mechanism neural cell adhesion molecule (N-CAM) limits beta tumour cell disaggregation and dissemination, we searched for potential downstream genes of N-CAM during beta tumour cell progression by gene expression profiling. Here, we show that N-CAM-deficient beta-cell tumorigenesis is associated with changes in the expression of genes involved in cell-matrix adhesion and cytoskeletal dynamics, biological processes known to affect the invasive and metastatic behaviour of tumour cells. The extracellular matrix (ECM) molecules emerged as the primary target, i.e. N-CAM deficiency resulted in down-regulated mRNA expression of a broad range of ECM molecules. Consistent with this result, deficient deposition of major ECM stromal components, such as fibronectin, laminin 1 and collagen IV, was observed. Moreover, N-CAM-deficient tumour cells displayed defective matrix adhesion. These results offer a potential mechanism for tumour cell disaggregation during N-CAM-deficient beta tumour cell progression. Prospective consequences of these findings for the role of N-CAM in beta tumour cell dissemination are discussed.

  19. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    PubMed

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  20. Identification of small molecule compounds targeting the interaction of HIV-1 Vif and human APOBEC3G by virtual screening and biological evaluation.

    PubMed

    Ma, Ling; Zhang, Zhixin; Liu, Zhenlong; Pan, Qinghua; Wang, Jing; Li, Xiaoyu; Guo, Fei; Liang, Chen; Hu, Laixing; Zhou, Jinming; Cen, Shan

    2018-05-23

    Human APOBEC3G (hA3G) is a restriction factor that inhibits human immunodeficiency 1 virus (HIV-1) replication. The virally encoded protein Vif binds to hA3G and induces its degradation, thereby counteracting the antiviral activity of hA3G. Vif-mediated hA3G degradation clearly represents a potential target for anti-HIV drug development. Herein, we have performed virtual screening to discover small molecule inhibitors that target the binding interface of the Vif/hA3G complex. Subsequent biochemical studies have led to the identification of a small molecule inhibitor, IMB-301 that binds to hA3G, interrupts the hA3G-Vif interaction and inhibits Vif-mediated degradation of hA3G. As a result, IMB-301 strongly inhibits HIV-1 replication in a hA3G-dependent manner. Our study further demonstrates the feasibility of inhibiting HIV replication by abrogating the Vif-hA3G interaction with small molecules.

  1. The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules

    PubMed Central

    Antoniou, Antony N.; Ford, Stuart; Alphey, Magnus; Osborne, Andrew; Elliott, Tim; Powis, Simon J.

    2002-01-01

    The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone-assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide-loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N-terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation. PMID:12032078

  2. Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein-Protein Interaction.

    PubMed

    Sail, Vibhavari; Rizzo, Alessandro A; Chatterjee, Nimrat; Dash, Radha C; Ozen, Zuleyha; Walker, Graham C; Korzhnev, Dmitry M; Hadden, M Kyle

    2017-07-21

    Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

  3. Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

    PubMed

    Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

    2013-06-14

    Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

  4. Costimulatory molecule-targeted immunotherapy of cutaneous graft-versus-host disease.

    PubMed

    Kim, Juyang; Kim, Hye J; Park, Keunhee; Kim, Jiyoung; Choi, Hye-Jeong; Yagita, Hideo; Nam, Seok H; Cho, Hong R; Kwon, Byungsuk

    2007-07-15

    Chronic graft-versus-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Current therapies for cGVHD reduce symptoms but are not cures. The B10.D2-->Balb/c (H-2(d)) minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD, was used in this study. We demonstrated that a single injection of an agonistic monoclonal antibody (mAb) against CD137, a member of the tumor necrosis factor receptor superfamily, reverses skin fibrosis, ulceration, and alopecia, a dominant feature of cGVHD (cutaneous GVHD), ultimately improving general health conditions. The reversal is associated with markedly reduced CD4(+) T-cell cytokines and increased apoptosis of donor CD4(+) T cells. The Fas pathway is required for ameliorating cutaneous GVHD by anti-CD137 mAb. Taken together, these data indicate that the anti-CD137 mAb has a therapeutic effect on cutaneous GVHD by removing donor CD4(+) T cells that cause cutaneous GVHD. Thus, our study demonstrates an agonistic mAb, specific for a costimulatory molecule, as a possible target for therapeutic intervention in cutaneous GVHD.

  5. Targeting cell division: Small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality

    PubMed Central

    Margalit, Danielle N.; Romberg, Laura; Mets, Rebecca B.; Hebert, Alan M.; Mitchison, Timothy J.; Kirschner, Marc W.; RayChaudhuri, Debabrata

    2004-01-01

    FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target. PMID:15289600

  6. Targeting the r(CGG) repeats that cause FXTAS with modularly assembled small molecules and oligonucleotides.

    PubMed

    Tran, Tuan; Childs-Disney, Jessica L; Liu, Biao; Guan, Lirui; Rzuczek, Suzanne; Disney, Matthew D

    2014-04-18

    We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)(exp)) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)(exp) toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)(exp) in vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)(exp)'s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2'-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.

  7. Targeting the r(CGG) Repeats That Cause FXTAS with Modularly Assembled Small Molecules and Oligonucleotides

    PubMed Central

    2015-01-01

    We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)exp) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)exp toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)expin vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)exp’s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2′-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide. PMID:24506227

  8. Drugs and Targets in Fibrosis

    PubMed Central

    Li, Xiaoyi; Zhu, Lixin; Wang, Beibei; Yuan, Meifei; Zhu, Ruixin

    2017-01-01

    Fibrosis contributes to the development of many diseases and many target molecules are involved in fibrosis. Currently, the majority of fibrosis treatment strategies are limited to specific diseases or organs. However, accumulating evidence demonstrates great similarities among fibroproliferative diseases, and more and more drugs are proved to be effective anti-fibrotic therapies across different diseases and organs. Here we comprehensively review the current knowledge on the pathological mechanisms of fibrosis, and divide factors mediating fibrosis progression into extracellular and intracellular groups. Furthermore, we systematically summarize both single and multiple component drugs that target fibrosis. Future directions of fibrosis drug discovery are also proposed. PMID:29218009

  9. Targeting of Streptococcus mutans Biofilms by a Novel Small Molecule Prevents Dental Caries and Preserves the Oral Microbiome.

    PubMed

    Garcia, S S; Blackledge, M S; Michalek, S; Su, L; Ptacek, T; Eipers, P; Morrow, C; Lefkowitz, E J; Melander, C; Wu, H

    2017-07-01

    Dental caries is a costly and prevalent disease characterized by the demineralization of the tooth's enamel. Disease outcome is influenced by host factors, dietary intake, cariogenic bacteria, and other microbes. The cariogenic bacterial species Streptococcus mutans metabolizes sucrose to initiate biofilm formation on the tooth surface and consequently produces lactic acid to degrade the tooth's enamel. Persistence of S. mutans biofilms in the oral cavity can lead to tooth decay. To date, no anticaries therapies that specifically target S. mutans biofilms but do not disturb the overall oral microbiome are available. We screened a library of 2-aminoimidazole antibiofilm compounds with a biofilm dispersion assay and identified a small molecule that specifically targets S. mutans biofilms. At 5 µM, the small molecule annotated 3F1 dispersed 50% of the established S. mutans biofilm but did not disperse biofilms formed by the commensal species Streptococcus sanguinis or Streptococcus gordonii. 3F1 dispersed S. mutans biofilms independently of biofilm-related factors such as antigen I/II and glucosyltransferases. 3F1 treatment effectively prevented dental caries by controlling S. mutans in a rat caries model without perturbing the oral microbiota. Our study demonstrates that selective targeting of S. mutans biofilms by 3F1 was able to effectively reduce dental caries in vivo without affecting the overall oral microbiota shaped by the intake of dietary sugars, suggesting that the pathogenic biofilm-specific treatment is a viable strategy for disease prevention.

  10. A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis.

    PubMed

    Kerr, Georgina; Sheldon, Helen; Chaikuad, Apirat; Alfano, Ivan; von Delft, Frank; Bullock, Alex N; Harris, Adrian L

    2015-04-01

    Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-β receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology.

  11. Defining RNA-Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA.

    PubMed

    Velagapudi, Sai Pradeep; Luo, Yiling; Tran, Tuan; Haniff, Hafeez S; Nakai, Yoshio; Fallahi, Mohammad; Martinez, Gustavo J; Childs-Disney, Jessica L; Disney, Matthew D

    2017-03-22

    RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif-small molecule interactions identified via selection. Named High Throughput Structure-Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif-small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule-RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.

  12. Anti-Inflammatory Targets for the Treatment of Reperfusion Injury in Stroke

    PubMed Central

    Mizuma, Atsushi; Yenari, Midori A.

    2017-01-01

    While the mainstay of acute stroke treatment includes revascularization via recombinant tissue plasminogen activator or mechanical thrombectomy, only a minority of stroke patients are eligible for treatment, as delayed treatment can lead to worsened outcome. This worsened outcome at the experimental level has been attributed to an entity known as reperfusion injury (R/I). R/I is occurred when revascularization is delayed after critical brain and vascular injury has occurred, so that when oxygenated blood is restored, ischemic damage is increased, rather than decreased. R/I can increase lesion size and also worsen blood barrier breakdown and lead to brain edema and hemorrhage. A major mechanism underlying R/I is that of poststroke inflammation. The poststroke immune response consists of the aberrant activation of glial cell, infiltration of peripheral leukocytes, and the release of damage-associated molecular pattern (DAMP) molecules elaborated by ischemic cells of the brain. Inflammatory mediators involved in this response include cytokines, chemokines, adhesion molecules, and several immune molecule effectors such as matrix metalloproteinases-9, inducible nitric oxide synthase, nitric oxide, and reactive oxygen species. Several experimental studies over the years have characterized these molecules and have shown that their inhibition improves neurological outcome. Yet, numerous clinical studies failed to demonstrate any positive outcomes in stroke patients. However, many of these clinical trials were carried out before the routine use of revascularization therapies. In this review, we cover mechanisms of inflammation involved in R/I, therapeutic targets, and relevant experimental and clinical studies, which might stimulate renewed interest in designing clinical trials to specifically target R/I. We propose that by targeting anti-inflammatory targets in R/I as a combined therapy, it may be possible to further improve outcomes from pharmacological thrombolysis or

  13. Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation

    PubMed Central

    Soto, Lilian; Ferrier, Ashley; Aravena, Octavio; Fonseca, Elianet; Berendsen, Jorge; Biere, Andrea; Bueno, Daniel; Ramos, Verónica; Aguillón, Juan Carlos; Catalán, Diego

    2015-01-01

    The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis. PMID:26483788

  14. Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery.

    PubMed

    Zech, Stephan G; Kohlmann, Anna; Zhou, Tianjun; Li, Feng; Squillace, Rachel M; Parillon, Lois E; Greenfield, Matthew T; Miller, David P; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Xu, Yongjin; Miret, Juan J; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

    2016-01-28

    Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

  15. Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation.

    PubMed

    Hiatt, Joseph B; Pritchard, Colin C; Salipante, Stephen J; O'Roak, Brian J; Shendure, Jay

    2013-05-01

    The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10(-6) in cell lines and 2.6 × 10(-5) in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%-4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%-1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings.

  16. Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

    PubMed

    Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

    2014-01-01

    Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

  17. Covalent small-molecule-RNA complex formation enables cellular profiling of small-molecule-RNA interactions.

    PubMed

    Guan, Lirui; Disney, Matthew D

    2013-09-16

    Won't let you go! A strategy is described to design small molecules that react with their cellular RNA targets. This approach not only improves the activity of compounds targeting RNA in cell culture by a factor of about 2500 but also enables cell-wide profiling of its RNA targets. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Therapeutic targeting and rapid mobilization of endosteal HSC using a small molecule integrin antagonist

    PubMed Central

    Cao, Benjamin; Zhang, Zhen; Grassinger, Jochen; Williams, Brenda; Heazlewood, Chad K.; Churches, Quentin I.; James, Simon A.; Li, Songhui; Papayannopoulou, Thalia; Nilsson, Susan K.

    2016-01-01

    The inherent disadvantages of using granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell (HSC) mobilization have driven efforts to identify alternate strategies based on single doses of small molecules. Here, we show targeting α9β1/α4β1 integrins with a single dose of a small molecule antagonist (BOP (N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine)) rapidly mobilizes long-term multi-lineage reconstituting HSC. Synergistic engraftment augmentation is observed when BOP is co-administered with AMD3100. Impressively, HSC in equal volumes of peripheral blood (PB) mobilized with this combination effectively out-competes PB mobilized with G-CSF. The enhanced mobilization observed using BOP and AMD3100 is recapitulated in a humanized NODSCIDIL2Rγ−/− model, demonstrated by a significant increase in PB CD34+ cells. Using a related fluorescent analogue of BOP (R-BC154), we show that this class of antagonists preferentially bind human and mouse HSC and progenitors via endogenously primed/activated α9β1/α4β1 within the endosteal niche. These results support using dual α9β1/α4β1 inhibitors as effective, rapid and transient mobilization agents with promising clinical applications. PMID:26975966

  19. A novel oncogenic mechanism in Ewing sarcoma involving IGF pathway targeting by EWS/Fli1-regulated microRNAs

    PubMed Central

    McKinsey, EL; Parrish, JK; Irwin, AE; Niemeyer, BF; Kern, HB; Birks, DK; Jedlicka, P

    2015-01-01

    MicroRNAs (miRs) are a novel class of cellular bioactive molecules with critical functions in the regulation of gene expression in normal biology and disease. MiRs are frequently misexpressed in cancer, with potent biological consequences. However, relatively little is known about miRs in pediatric cancers, including sarcomas. Moreover, the mechanisms behind aberrant miR expression in cancer are poorly understood. Ewing sarcoma is an aggressive pediatric malignancy driven by EWS/Ets fusion oncoproteins, which are gain-of-function transcriptional regulators. We employed stable silencing of EWS/Fli1, the most common of the oncogenic fusions, and global miR profiling to identify EWS/Fli1-regulated miRs with oncogenesis-modifying roles in Ewing sarcoma. In this report, we characterize a group of miRs (100, 125b, 22, 221/222, 27a and 29a) strongly repressed by EWS/Fli1. Strikingly, all of these miRs have predicted targets in the insulin-like growth factor (IGF) signaling pathway, a pivotal driver of Ewing sarcoma oncogenesis. We demonstrate that miRs in this group negatively regulate the expression of multiple pro-oncogenic components of the IGF pathway, namely IGF-1, IGF-1 receptor, mammalian/mechanistic target of rapamycin and ribosomal protein S6 kinase A1. Consistent with tumor-suppressive functions, these miRs manifest growth inhibitory properties in Ewing sarcoma cells. Our studies thus uncover a novel oncogenic mechanism in Ewing sarcoma, involving post-transcriptional derepression of IGF signaling by the EWS/Fli1 fusion oncoprotein via miRs. This novel pathway may be amenable to innovative therapeutic targeting in Ewing sarcoma and other malignancies with activated IGF signaling. PMID:21643012

  20. A novel oncogenic mechanism in Ewing sarcoma involving IGF pathway targeting by EWS/Fli1-regulated microRNAs.

    PubMed

    McKinsey, E L; Parrish, J K; Irwin, A E; Niemeyer, B F; Kern, H B; Birks, D K; Jedlicka, P

    2011-12-08

    MicroRNAs (miRs) are a novel class of cellular bioactive molecules with critical functions in the regulation of gene expression in normal biology and disease. MiRs are frequently misexpressed in cancer, with potent biological consequences. However, relatively little is known about miRs in pediatric cancers, including sarcomas. Moreover, the mechanisms behind aberrant miR expression in cancer are poorly understood. Ewing sarcoma is an aggressive pediatric malignancy driven by EWS/Ets fusion oncoproteins, which are gain-of-function transcriptional regulators. We employed stable silencing of EWS/Fli1, the most common of the oncogenic fusions, and global miR profiling to identify EWS/Fli1-regulated miRs with oncogenesis-modifying roles in Ewing sarcoma. In this report, we characterize a group of miRs (100, 125b, 22, 221/222, 27a and 29a) strongly repressed by EWS/Fli1. Strikingly, all of these miRs have predicted targets in the insulin-like growth factor (IGF) signaling pathway, a pivotal driver of Ewing sarcoma oncogenesis. We demonstrate that miRs in this group negatively regulate the expression of multiple pro-oncogenic components of the IGF pathway, namely IGF-1, IGF-1 receptor, mammalian/mechanistic target of rapamycin and ribosomal protein S6 kinase A1. Consistent with tumor-suppressive functions, these miRs manifest growth inhibitory properties in Ewing sarcoma cells. Our studies thus uncover a novel oncogenic mechanism in Ewing sarcoma, involving post-transcriptional derepression of IGF signaling by the EWS/Fli1 fusion oncoprotein via miRs. This novel pathway may be amenable to innovative therapeutic targeting in Ewing sarcoma and other malignancies with activated IGF signaling.

  1. CD163-Macrophages Are Involved in Rhabdomyolysis-Induced Kidney Injury and May Be Detected by MRI with Targeted Gold-Coated Iron Oxide Nanoparticles.

    PubMed

    Rubio-Navarro, Alfonso; Carril, Mónica; Padro, Daniel; Guerrero-Hue, Melanie; Tarín, Carlos; Samaniego, Rafael; Cannata, Pablo; Cano, Ainhoa; Villalobos, Juan Manuel Amaro; Sevillano, Ángel Manuel; Yuste, Claudia; Gutiérrez, Eduardo; Praga, Manuel; Egido, Jesús; Moreno, Juan Antonio

    2016-01-01

    Macrophages play an important role in rhabdomyolysis-acute kidney injury (AKI), although the molecular mechanisms involved in macrophage differentiation are poorly understood. We analyzed the expression and regulation of CD163, a membrane receptor mainly expressed by anti-inflammatory M2 macrophages, in rhabdomyolysis-AKI and developed targeted probes for its specific detection in vivo by MRI. Intramuscular injection of glycerol in mice promoted an early inflammatory response, with elevated proportion of M1 macrophages, and partial differentiation towards a M2 phenotype in later stages, where increased CD163 expression was observed. Immunohistological studies confirmed the presence of CD163-macrophages in human rhabdomyolysis-AKI. In cultured macrophages, myoglobin upregulated CD163 expression via HO-1/IL-10 axis. Moreover, we developed gold-coated iron oxide nanoparticles vectorized with an anti-CD163 antibody that specifically targeted CD163 in kidneys from glycerol-injected mice, as determined by MRI studies, and confirmed by electron microscopy and immunological analysis. Our findings are the first to demonstrate that CD163 is present in both human and experimental rhabdomyolysis-induced AKI, suggesting an important role of this molecule in this pathological condition. Therefore, the use of probes targeting CD163-macrophages by MRI may provide important information about the cellular composition of renal lesion in rhabdomyolysis.

  2. PPAR Ligands Function as Suppressors That Target Biological Actions of HMGB1

    PubMed Central

    Chen, Tianhui

    2016-01-01

    High mobility group box 1 (HMGB1), which has become one of the most intriguing molecules in inflammatory disorders and cancers and with which ligand-activated peroxisome proliferator-activated receptors (PPARs) are highly associated, is considered as a therapeutic target. Of particular interest is the fact that certain PPAR ligands have demonstrated their potent anti-inflammatory activities and potential anticancer effects. In this review article we summarize recent experimental evidence that PPAR ligands function as suppressors that target biological actions of HMGB1, including intracellular expression, receptor signaling cascades, and extracellular secretion of HMGB1 in cell lines and/or animal models. We also propose the possible mechanisms underlying PPAR involvement in inflammatory disorders and discuss the future therapeutic value of PPAR ligands targeting HMGB1 molecule for cancer prevention and treatment. PMID:27563308

  3. Prediction of novel target genes and pathways involved in bevacizumab-resistant colorectal cancer

    PubMed Central

    Makondi, Precious Takondwa; Lee, Chia-Hwa; Huang, Chien-Yu; Chu, Chi-Ming; Chang, Yu-Jia

    2018-01-01

    Bevacizumab combined with cytotoxic chemotherapy is the backbone of metastatic colorectal cancer (mCRC) therapy; however, its treatment efficacy is hampered by therapeutic resistance. Therefore, understanding the mechanisms underlying bevacizumab resistance is crucial to increasing the therapeutic efficacy of bevacizumab. The Gene Expression Omnibus (GEO) database (dataset, GSE86525) was used to identify the key genes and pathways involved in bevacizumab-resistant mCRC. The GEO2R web tool was used to identify differentially expressed genes (DEGs). Functional and pathway enrichment analyses of the DEGs were performed using the Database for Annotation, Visualization, and Integrated Discovery(DAVID). Protein–protein interaction (PPI) networks were established using the Search Tool for the Retrieval of Interacting Genes/Proteins database(STRING) and visualized using Cytoscape software. A total of 124 DEGs were obtained, 57 of which upregulated and 67 were downregulated. PPI network analysis showed that seven upregulated genes and nine downregulated genes exhibited high PPI degrees. In the functional enrichment, the DEGs were mainly enriched in negative regulation of phosphate metabolic process and positive regulation of cell cycle process gene ontologies (GOs); the enriched pathways were the phosphoinositide 3-kinase-serine/threonine kinase signaling pathway, bladder cancer, and microRNAs in cancer. Cyclin-dependent kinase inhibitor 1A(CDKN1A), toll-like receptor 4 (TLR4), CD19 molecule (CD19), breast cancer 1, early onset (BRCA1), platelet-derived growth factor subunit A (PDGFA), and matrix metallopeptidase 1 (MMP1) were the DEGs involved in the pathways and the PPIs. The clinical validation of the DEGs in mCRC (TNM clinical stages 3 and 4) revealed that high PDGFA expression levels were associated with poor overall survival, whereas high BRCA1 and MMP1 expression levels were associated with favorable progress free survival(PFS). The identified genes and pathways

  4. High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

    PubMed

    Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

    2018-05-01

    K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Generating Focused Molecule Libraries for Drug Discovery with Recurrent Neural Networks

    PubMed Central

    2017-01-01

    In de novo drug design, computational strategies are used to generate novel molecules with good affinity to the desired biological target. In this work, we show that recurrent neural networks can be trained as generative models for molecular structures, similar to statistical language models in natural language processing. We demonstrate that the properties of the generated molecules correlate very well with the properties of the molecules used to train the model. In order to enrich libraries with molecules active toward a given biological target, we propose to fine-tune the model with small sets of molecules, which are known to be active against that target. Against Staphylococcus aureus, the model reproduced 14% of 6051 hold-out test molecules that medicinal chemists designed, whereas against Plasmodium falciparum (Malaria), it reproduced 28% of 1240 test molecules. When coupled with a scoring function, our model can perform the complete de novo drug design cycle to generate large sets of novel molecules for drug discovery. PMID:29392184

  6. Biosensor-based small molecule fragment screening with biolayer interferometry

    NASA Astrophysics Data System (ADS)

    Wartchow, Charles A.; Podlaski, Frank; Li, Shirley; Rowan, Karen; Zhang, Xiaolei; Mark, David; Huang, Kuo-Sen

    2011-07-01

    Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions. The BLI format is based on a disposable sensor that is immersed in 96-well or 384-well plates. BLI has been validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with surface plasmon resonsance (SPR). Screens with challenging targets involved in protein-protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.

  7. Signaling lymphocytic activation molecules Slam and cancers: friends or foes?

    PubMed

    Fouquet, Gregory; Marcq, Ingrid; Debuysscher, Véronique; Bayry, Jagadeesh; Rabbind Singh, Amrathlal; Bengrine, Abderrahmane; Nguyen-Khac, Eric; Naassila, Mickael; Bouhlal, Hicham

    2018-03-23

    Signaling Lymphocytic Activation Molecules (SLAM) family receptors are initially described in immune cells. These receptors recruit both activating and inhibitory SH2 domain containing proteins through their Immunoreceptor Tyrosine based Switch Motifs (ITSMs). Accumulating evidence suggest that the members of this family are intimately involved in different physiological and pathophysiological events such as regulation of immune responses and entry pathways of certain viruses. Recently, other functions of SLAM, principally in the pathophysiology of neoplastic transformations have also been deciphered. These new findings may prompt SLAM to be considered as new tumor markers, diagnostic tools or potential therapeutic targets for controlling the tumor progression. In this review, we summarize the major observations describing the implications and features of SLAM in oncology and discuss the therapeutic potential attributed to these molecules.

  8. Visualization and Analysis of MiRNA-Targets Interactions Networks.

    PubMed

    León, Luis E; Calligaris, Sebastián D

    2017-01-01

    MicroRNAs are a class of small, noncoding RNA molecules of 21-25 nucleotides in length that regulate the gene expression by base-pairing with the target mRNAs, mainly leading to down-regulation or repression of the target genes. MicroRNAs are involved in diverse regulatory pathways in normal and pathological conditions. In this context, it is highly important to identify the targets of specific microRNA in order to understand the mechanism of its regulation and consequently its involvement in disease. However, the microRNA target identification is experimentally laborious and time-consuming. The in silico prediction of microRNA targets is an extremely useful approach because you can identify potential mRNA targets, reduce the number of possibilities and then, validate a few microRNA-mRNA interactions in an in vitro experimental model. In this chapter, we describe, in a simple way, bioinformatics guidelines to use miRWalk database and Cytoscape software for analyzing microRNA-mRNA interactions through their visualization as a network.

  9. Defining RNA–Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA

    PubMed Central

    2017-01-01

    RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif–small molecule interactions identified via selection. Named High Throughput Structure–Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif–small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule–RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs. PMID:28386598

  10. Small-molecule inhibitors directly target CARD9 and mimic its protective variant in inflammatory bowel disease.

    PubMed

    Leshchiner, Elizaveta S; Rush, Jason S; Durney, Michael A; Cao, Zhifang; Dančík, Vlado; Chittick, Benjamin; Wu, Huixian; Petrone, Adam; Bittker, Joshua A; Phillips, Andrew; Perez, Jose R; Shamji, Alykhan F; Kaushik, Virendar K; Daly, Mark J; Graham, Daniel B; Schreiber, Stuart L; Xavier, Ramnik J

    2017-10-24

    Advances in human genetics have dramatically expanded our understanding of complex heritable diseases. Genome-wide association studies have identified an allelic series of CARD9 variants associated with increased risk of or protection from inflammatory bowel disease (IBD). The predisposing variant of CARD9 is associated with increased NF-κB-mediated cytokine production. Conversely, the protective variant lacks a functional C-terminal domain and is unable to recruit the E3 ubiquitin ligase TRIM62. Here, we used biochemical insights into CARD9 variant proteins to create a blueprint for IBD therapeutics and recapitulated the mechanism of the CARD9 protective variant using small molecules. We developed a multiplexed bead-based technology to screen compounds for disruption of the CARD9-TRIM62 interaction. We identified compounds that directly and selectively bind CARD9, disrupt TRIM62 recruitment, inhibit TRIM62-mediated ubiquitinylation of CARD9, and demonstrate cellular activity and selectivity in CARD9-dependent pathways. Taken together, small molecules targeting CARD9 illustrate a path toward improved IBD therapeutics. Published under the PNAS license.

  11. A novel small-molecule compound targeting CD147 inhibits the motility and invasion of hepatocellular carcinoma cells

    PubMed Central

    Peng, Jian-long; Wang, Shi-jie; Geng, Jie-jie; Liu, Ji-de; Feng, Fei; Song, Fei; Li, Ling; Zhu, Ping; Jiang, Jian-li; Chen, Zhi-nan

    2016-01-01

    CD147, a type I transmembrane glycoprotein, is highly expressed in various cancer types and plays important roles in tumor progression, especially by promoting the motility and invasion of hepatocellular carcinoma (HCC) cells. These crucial roles make CD147 an attractive target for therapeutic intervention in HCC, but no small-molecule inhibitors of CD147 have been developed to date. To identify a candidate inhibitor, we used a pharmacophore model derived from the structure of CD147 to virtually screen over 300,000 compounds. The 100 highest-ranked compounds were subjected to biological assays, and the most potent one, dubbed AC-73 (ID number: AN-465/42834501), was studied further. We confirmed that AC-73 targeted CD147 and further demonstrated it can specifically disrupt CD147 dimerization. Moreover, molecular docking and mutagenesis experiments showed that the possible binding sites of AC-73 on CD147 included Glu64 and Glu73 in the N-terminal IgC2 domain, which two residues are located in the dimer interface of CD147. Functional assays revealed that AC-73 inhibited the motility and invasion of typical HCC cells, but not HCC cells that lacked the CD147 gene, demonstrating on-target action. Further, AC-73 reduced HCC metastasis by suppressing matrix metalloproteinase (MMP)-2 via down-regulation of the CD147/ERK1/2/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Finally, AC-73 attenuated progression in an orthotopic nude mouse model of liver metastasis, suggesting that AC-73 or its derivatives have potential for use in HCC intervention. We conclude that the novel small-molecule inhibitor AC-73 inhibits HCC mobility and invasion, probably by disrupting CD147 dimerization and thereby mainly suppressing the CD147/ERK1/2/STAT3/MMP-2 pathways, which are crucial for cancer progression. PMID:26882566

  12. A novel small-molecule compound targeting CD147 inhibits the motility and invasion of hepatocellular carcinoma cells.

    PubMed

    Fu, Zhi-guang; Wang, Li; Cui, Hong-yong; Peng, Jian-long; Wang, Shi-jie; Geng, Jie-jie; Liu, Ji-de; Feng, Fei; Song, Fei; Li, Ling; Zhu, Ping; Jiang, Jian-li; Chen, Zhi-nan

    2016-02-23

    CD147, a type I transmembrane glycoprotein, is highly expressed in various cancer types and plays important roles in tumor progression, especially by promoting the motility and invasion of hepatocellular carcinoma (HCC) cells. These crucial roles make CD147 an attractive target for therapeutic intervention in HCC, but no small-molecule inhibitors of CD147 have been developed to date. To identify a candidate inhibitor, we used a pharmacophore model derived from the structure of CD147 to virtually screen over 300,000 compounds. The 100 highest-ranked compounds were subjected to biological assays, and the most potent one, dubbed AC-73 (ID number: AN-465/42834501), was studied further. We confirmed that AC-73 targeted CD147 and further demonstrated it can specifically disrupt CD147 dimerization. Moreover, molecular docking and mutagenesis experiments showed that the possible binding sites of AC-73 on CD147 included Glu64 and Glu73 in the N-terminal IgC2 domain, which two residues are located in the dimer interface of CD147. Functional assays revealed that AC-73 inhibited the motility and invasion of typical HCC cells, but not HCC cells that lacked the CD147 gene, demonstrating on-target action. Further, AC-73 reduced HCC metastasis by suppressing matrix metalloproteinase (MMP)-2 via down-regulation of the CD147/ERK1/2/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Finally, AC-73 attenuated progression in an orthotopic nude mouse model of liver metastasis, suggesting that AC-73 or its derivatives have potential for use in HCC intervention. We conclude that the novel small-molecule inhibitor AC-73 inhibits HCC mobility and invasion, probably by disrupting CD147 dimerization and thereby mainly suppressing the CD147/ERK1/2/STAT3/MMP-2 pathways, which are crucial for cancer progression.

  13. Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation

    PubMed Central

    Hiatt, Joseph B.; Pritchard, Colin C.; Salipante, Stephen J.; O'Roak, Brian J.; Shendure, Jay

    2013-01-01

    The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10−6 in cell lines and 2.6 × 10−5 in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%–4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%–1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings. PMID:23382536

  14. Microtubule-Targeting Therapy for Prostate Cancer

    DTIC Science & Technology

    2007-02-01

    that were done to achieve the above specific goals. 1. Biological effects of ribozyme -carrying adenoviruses that target stathmin mRNA in human...prostate cancer cells: A ribozyme is a small RNA molecule that acts stoichiometrically to cleave multiple target RNA molecules [1]. This unique ability...of a ribozyme to degrade multiple target RNA molecules is a more efficient approach for down regulating genes that are expressed at very high levels

  15. Small-Molecule Hormones: Molecular Mechanisms of Action

    PubMed Central

    Budzińska, Monika

    2013-01-01

    Small-molecule hormones play crucial roles in the development and in the maintenance of an adult mammalian organism. On the molecular level, they regulate a plethora of biological pathways. Part of their actions depends on their transcription-regulating properties, exerted by highly specific nuclear receptors which are hormone-dependent transcription factors. Nuclear hormone receptors interact with coactivators, corepressors, basal transcription factors, and other transcription factors in order to modulate the activity of target genes in a manner that is dependent on tissue, age and developmental and pathophysiological states. The biological effect of this mechanism becomes apparent not earlier than 30–60 minutes after hormonal stimulus. In addition, small-molecule hormones modify the function of the cell by a number of nongenomic mechanisms, involving interaction with proteins localized in the plasma membrane, in the cytoplasm, as well as with proteins localized in other cellular membranes and in nonnuclear cellular compartments. The identity of such proteins is still under investigation; however, it seems that extranuclear fractions of nuclear hormone receptors commonly serve this function. A direct interaction of small-molecule hormones with membrane phospholipids and with mRNA is also postulated. In these mechanisms, the reaction to hormonal stimulus appears within seconds or minutes. PMID:23533406

  16. Epidermal growth factor receptor and K-Ras in non-small cell lung cancer-molecular pathways involved and targeted therapies

    PubMed Central

    de Mello, Ramon Andrade; Marques, Dânia Sofia; Medeiros, Rui; Araújo, António MF

    2011-01-01

    Lung cancer is currently the leading cause of cancer death in Western nations. Non-small cell lung cancer (NSCLC) represents 80% of all lung cancers, and adenocarcinoma is the predominant histological type. Despite the intensive research carried out on this field and therapeutic advances, the overall prognosis of these patients remains unsatisfactory, with a 5-year overall survival rate of less than 15%. Nowadays, pharmacogenetics and pharmacogenomics represent the key to successful treatment. Recent studies suggest the existence of two distinct molecular pathways in the carcinogenesis of lung adenocarcinoma: one associated with smoking and activation of the K-Ras oncogene and the other not associated with smoking and activation of the epidermal growth factor receptor (EGFR). The K-ras mutation is mainly responsible for primary resistance to new molecules which inhibit tyrosine kinase EGFR (erlotinib and gefitinib) and most of the EGFR mutations are responsible for increased tumor sensitivity to these drugs. This article aims to conduct a systematic review of the literature regarding the molecular pathways involving the EGFR, K-Ras and EGFR targeted therapies in NSCLC tumor behavior. PMID:22087435

  17. Visualization of nanoconstructions with DNA-Aptamers for targeted molecules binding on the surface of screen-printed electrodes

    NASA Astrophysics Data System (ADS)

    Lapin, Ivan N.; Shabalina, Anastasiia V.; Svetlichyi, Valery A.; Kolovskaya, Olga S.

    2018-04-01

    Nanoconstructions of gold nanoparticles (NPs) obtained via pulsed laser ablation in liquid with DNA-aptamer specific to protein tumor marker were visualized on the surface of screen-printed electrode using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). AuNPs/aptamer nanoconstuctions distribution on the solid surface was studied. More uniform coverage of the carbon electrode surface with the nanoconstuctions was showed in comparison with DNA-aptamer alone on the golden electrode surface. Targeted binding of the tumor marker molecules with the AuNPs/DNA-aptamer nanoconstuctions was approved.

  18. Dietary curcumin supplementation counteracts reduction in levels of molecules involved in energy homeostasis after brain trauma.

    PubMed

    Sharma, S; Zhuang, Y; Ying, Z; Wu, A; Gomez-Pinilla, F

    2009-07-21

    Traumatic brain injury (TBI) is followed by an energy crisis that compromises the capacity of the brain to cope with challenges, and often reduces cognitive ability. New research indicates that events that regulate energy homeostasis crucially impact synaptic function and this can compromise the capacity of the brain to respond to challenges during the acute and chronic phases of TBI. The goal of the present study is to determine the influence of the phenolic yellow curry pigment curcumin on molecular systems involved with the monitoring, balance, and transduction of cellular energy, in the hippocampus of animals exposed to mild fluid percussion injury (FPI). Young adult rats were exposed to a regular diet (RD) without or with 500 ppm curcumin (Cur) for four weeks, before an FPI was performed. The rats were assigned to four groups: RD/Sham, Cur/Sham, RD/FPI, and Cur/FPI. We found that FPI decreased the levels of AMP-activated protein kinase (AMPK), ubiquitous mitochondrial creatine kinase (uMtCK) and cytochrome c oxidase II (COX-II) in RD/FPI rats as compared to the RD/sham rats. The curcumin diet counteracted the effects of FPI and elevated the levels of AMPK, uMtCK, COX-II in Cur/FPI rats as compared to RD/sham rats. In addition, in the Cur/sham rats, AMPK and uMtCK increased compared to the RD/sham. Results show the potential of curcumin to regulate molecules involved in energy homeostasis following TBI. These studies may foster a new line of therapeutic treatments for TBI patients by endogenous upregulation of molecules important for functional recovery.

  19. Targeting of small molecule anticancer drugs to the tumour and its vasculature using cationic liposomes: lessons from gene therapy

    PubMed Central

    Dass, Crispin R; Choong, Peter FM

    2006-01-01

    Cationic (positively charged) liposomes have been tested in various gene therapy clinical trials for neoplastic and other diseases. They have demonstrated selectivity for tumour vascular endothelial cells raising hopes for both antiangiogenic and antivascular therapies. They are also capable of being selectively delivered to the lungs and liver when administered intravenously. These vesicles are being targeted to the tumour in various parts of the body by using advanced liposomal systems such as ligand-receptor and antibody-antigen combinations. At present, the transferrin receptor is commonly used for cancer-targeted drug delivery systems including cationic liposomes. This review looks at the growing utility of these vesicles for delivery of small molecule anticancer drugs. PMID:16792817

  20. Involvement of HLA class I molecules in the immune escape of urologic tumors.

    PubMed

    Carretero, R; Gil-Julio, H; Vázquez-Alonso, F; Garrido, F; Castiñeiras, J; Cózar, J M

    2014-04-01

    To analyze the influence of different alterations in human leukocyte antigen class I molecules (HLA I) in renal cell carcinoma, as well as in bladder and prostate cancer. We also study the correlation between HLA I expression and the progression of the disease and the response after immunotherapy protocols. It has been shown, experimentally, that the immune system can recognize and kill neoplastic cells. By analyzing the expression of HLA I molecules on the surface of cancer cells, we were able to study the tumor escape mechanisms against the immune system. Alteration or irreversible damage in HLA I molecules is used by the neoplastic cells to escape the immune system. The function of these molecules is to recognize endogenous peptides and present them to T cells of the immune system. There is a clear relationship between HLA I reversible alterations and success of therapy. Irreversible lesions also imply a lack of response to treatment. The immune system activation can reverse HLA I molecules expression in tumors with reversible lesions, whereas tumors with irreversible ones do not respond to such activation. Determine the type of altered HLA I molecules in tumors is of paramount importance when choosing the type of treatment to keep looking for therapeutic success. Those tumors with reversible lesions can be treated with traditional immunotherapy; however, tumour with irreversible alterations should follow alternative protocols, such as the use of viral vectors carrying the HLA genes to achieve damaged re-expression of the protein. From studies in urologic tumors, we can conclude that the HLA I molecules play a key role in these tumors escape to the immune system. Copyright © 2013 AEU. Published by Elsevier Espana. All rights reserved.

  1. Small-molecule inhibitors of DNA damage-repair pathways: an approach to overcome tumor resistance to alkylating anticancer drugs

    PubMed Central

    Srinivasan, Ajay; Gold, Barry

    2013-01-01

    A major challenge in the future development of cancer therapeutics is the identification of biological targets and pathways, and the subsequent design of molecules to combat the drug-resistant cells hiding in virtually all cancers. This therapeutic approach is justified based upon the limited advances in cancer cures over the past 30 years, despite the development of many novel chemotherapies and earlier detection, which often fail due to drug resistance. Among the various targets to overcome tumor resistance are the DNA repair systems that can reverse the cytotoxicity of many clinically used DNA-damaging agents. Some progress has already been made but much remains to be done. We explore some components of the DNA-repair process, which are involved in repair of alkylation damage of DNA, as targets for the development of novel and effective molecules designed to improve the efficacy of existing anticancer drugs. PMID:22709253

  2. A small molecule inhibitor of Rheb selectively targets mTORC1 signaling.

    PubMed

    Mahoney, Sarah J; Narayan, Sridhar; Molz, Lisa; Berstler, Lauren A; Kang, Seong A; Vlasuk, George P; Saiah, Eddine

    2018-02-07

    The small G-protein Rheb activates the mechanistic target of rapamycin complex 1 (mTORC1) in response to growth factor signals. mTORC1 is a master regulator of cellular growth and metabolism; aberrant mTORC1 signaling is associated with fibrotic, metabolic, and neurodegenerative diseases, cancers, and rare disorders. Point mutations in the Rheb switch II domain impair its ability to activate mTORC1. Here, we report the discovery of a small molecule (NR1) that binds Rheb in the switch II domain and selectively blocks mTORC1 signaling. NR1 potently inhibits mTORC1 driven phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) but does not inhibit phosphorylation of AKT or ERK. In contrast to rapamycin, NR1 does not cause inhibition of mTORC2 upon prolonged treatment. Furthermore, NR1 potently and selectively inhibits mTORC1 in mouse kidney and muscle in vivo. The data presented herein suggest that pharmacological inhibition of Rheb is an effective approach for selective inhibition of mTORC1 with therapeutic potential.

  3. Targeting DDX3 with a small molecule inhibitor for lung cancer therapy

    PubMed Central

    Bol, Guus M; Vesuna, Farhad; Xie, Min; Zeng, Jing; Aziz, Khaled; Gandhi, Nishant; Levine, Anne; Irving, Ashley; Korz, Dorian; Tantravedi, Saritha; Heerma van Voss, Marise R; Gabrielson, Kathleen; Bordt, Evan A; Polster, Brian M; Cope, Leslie; van der Groep, Petra; Kondaskar, Atul; Rudek, Michelle A; Hosmane, Ramachandra S; van der Wall, Elsken; van Diest, Paul J; Tran, Phuoc T; Raman, Venu

    2015-01-01

    Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3–β-catenin axis and inhibited non-homologous end joining—the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy. PMID:25820276

  4. Small Molecules from Nature Targeting G-Protein Coupled Cannabinoid Receptors: Potential Leads for Drug Discovery and Development

    PubMed Central

    Sharma, Charu; Sadek, Bassem; Goyal, Sameer N.; Sinha, Satyesh; Ojha, Shreesh

    2015-01-01

    The cannabinoid molecules are derived from Cannabis sativa plant which acts on the cannabinoid receptors types 1 and 2 (CB1 and CB2) which have been explored as potential therapeutic targets for drug discovery and development. Currently, there are numerous cannabinoid based synthetic drugs used in clinical practice like the popular ones such as nabilone, dronabinol, and Δ9-tetrahydrocannabinol mediates its action through CB1/CB2 receptors. However, these synthetic based Cannabis derived compounds are known to exert adverse psychiatric effect and have also been exploited for drug abuse. This encourages us to find out an alternative and safe drug with the least psychiatric adverse effects. In recent years, many phytocannabinoids have been isolated from plants other than Cannabis. Several studies have shown that these phytocannabinoids show affinity, potency, selectivity, and efficacy towards cannabinoid receptors and inhibit endocannabinoid metabolizing enzymes, thus reducing hyperactivity of endocannabinoid systems. Also, these naturally derived molecules possess the least adverse effects opposed to the synthetically derived cannabinoids. Therefore, the plant based cannabinoid molecules proved to be promising and emerging therapeutic alternative. The present review provides an overview of therapeutic potential of ligands and plants modulating cannabinoid receptors that may be of interest to pharmaceutical industry in search of new and safer drug discovery and development for future therapeutics. PMID:26664449

  5. Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

    PubMed

    Wang, Weifeng; Zhou, Jing; Halambage, Upul D; Jurado, Kellie A; Jamin, Augusta V; Wang, Yujie; Engelman, Alan N; Aiken, Christopher

    2017-05-01

    The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle. IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein

  6. Concepts of Protein Sorting or Targeting Signals and Membrane Topology in Undergraduate Teaching

    ERIC Educational Resources Information Center

    Tang, Bor Luen; Teng, Felicia Yu Hsuan

    2005-01-01

    The process of protein biogenesis culminates in its correct targeting to specific subcellular locations where it serves a function. Contemporary molecular and cell biology investigations often involve the exogenous expression of epitope- or fluorescent protein-tagged recombinant molecules as well as subsequent analysis of protein-protein…

  7. Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications

    PubMed Central

    Ruscito, Annamaria; DeRosa, Maria C.

    2016-01-01

    Aptamers are single-stranded, synthetic oligonucleotides that fold into 3-dimensional shapes capable of binding non-covalently with high affinity and specificity to a target molecule. They are generated via an in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment, from which candidates are screened and characterized, and then used in various applications. These applications range from therapeutic uses to biosensors for target detection. Aptamers for small molecule targets such as toxins, antibiotics, molecular markers, drugs, and heavy metals will be the focus of this review. Their accurate detection is needed for the protection and wellbeing of humans and animals. However, the small molecular weights of these targets, including the drastic size difference between the target and the oligonucleotides, make it challenging to select, characterize, and apply aptamers for their detection. Thus, recent (since 2012) notable advances in small molecule aptamers, which have overcome some of these challenges, are presented here, while defining challenges that still exist are discussed. PMID:27242994

  8. A comparative modeling and molecular docking study on Mycobacterium tuberculosis targets involved in peptidoglycan biosynthesis.

    PubMed

    Fakhar, Zeynab; Naiker, Suhashni; Alves, Claudio N; Govender, Thavendran; Maguire, Glenn E M; Lameira, Jeronimo; Lamichhane, Gyanu; Kruger, Hendrik G; Honarparvar, Bahareh

    2016-11-01

    An alarming rise of multidrug-resistant Mycobacterium tuberculosis strains and the continuous high global morbidity of tuberculosis have reinvigorated the need to identify novel targets to combat the disease. The enzymes that catalyze the biosynthesis of peptidoglycan in M. tuberculosis are essential and noteworthy therapeutic targets. In this study, the biochemical function and homology modeling of MurI, MurG, MraY, DapE, DapA, Alr, and Ddl enzymes of the CDC1551 M. tuberculosis strain involved in the biosynthesis of peptidoglycan cell wall are reported. Generation of the 3D structures was achieved with Modeller 9.13. To assess the structural quality of the obtained homology modeled targets, the models were validated using PROCHECK, PDBsum, QMEAN, and ERRAT scores. Molecular dynamics simulations were performed to calculate root mean square deviation (RMSD) and radius of gyration (Rg) of MurI and MurG target proteins and their corresponding templates. For further model validation, RMSD and Rg for selected targets/templates were investigated to compare the close proximity of their dynamic behavior in terms of protein stability and average distances. To identify the potential binding mode required for molecular docking, binding site information of all modeled targets was obtained using two prediction algorithms. A docking study was performed for MurI to determine the potential mode of interaction between the inhibitor and the active site residues. This study presents the first accounts of the 3D structural information for the selected M. tuberculosis targets involved in peptidoglycan biosynthesis.

  9. CD163-Macrophages Are Involved in Rhabdomyolysis-Induced Kidney Injury and May Be Detected by MRI with Targeted Gold-Coated Iron Oxide Nanoparticles

    PubMed Central

    Rubio-Navarro, Alfonso; Carril, Mónica; Padro, Daniel; Guerrero-Hue, Melanie; Tarín, Carlos; Samaniego, Rafael; Cannata, Pablo; Cano, Ainhoa; Villalobos, Juan Manuel Amaro; Sevillano, Ángel Manuel; Yuste, Claudia; Gutiérrez, Eduardo; Praga, Manuel; Egido, Jesús; Moreno, Juan Antonio

    2016-01-01

    Macrophages play an important role in rhabdomyolysis-acute kidney injury (AKI), although the molecular mechanisms involved in macrophage differentiation are poorly understood. We analyzed the expression and regulation of CD163, a membrane receptor mainly expressed by anti-inflammatory M2 macrophages, in rhabdomyolysis-AKI and developed targeted probes for its specific detection in vivo by MRI. Intramuscular injection of glycerol in mice promoted an early inflammatory response, with elevated proportion of M1 macrophages, and partial differentiation towards a M2 phenotype in later stages, where increased CD163 expression was observed. Immunohistological studies confirmed the presence of CD163-macrophages in human rhabdomyolysis-AKI. In cultured macrophages, myoglobin upregulated CD163 expression via HO-1/IL-10 axis. Moreover, we developed gold-coated iron oxide nanoparticles vectorized with an anti-CD163 antibody that specifically targeted CD163 in kidneys from glycerol-injected mice, as determined by MRI studies, and confirmed by electron microscopy and immunological analysis. Our findings are the first to demonstrate that CD163 is present in both human and experimental rhabdomyolysis-induced AKI, suggesting an important role of this molecule in this pathological condition. Therefore, the use of probes targeting CD163-macrophages by MRI may provide important information about the cellular composition of renal lesion in rhabdomyolysis. PMID:27162559

  10. Antibody-Mediated Small Molecule Detection Using Programmable DNA-Switches.

    PubMed

    Rossetti, Marianna; Ippodrino, Rudy; Marini, Bruna; Palleschi, Giuseppe; Porchetta, Alessandro

    2018-06-13

    The development of rapid, cost-effective, and single-step methods for the detection of small molecules is crucial for improving the quality and efficiency of many applications ranging from life science to environmental analysis. Unfortunately, current methodologies still require multiple complex, time-consuming washing and incubation steps, which limit their applicability. In this work we present a competitive DNA-based platform that makes use of both programmable DNA-switches and antibodies to detect small target molecules. The strategy exploits both the advantages of proximity-based methods and structure-switching DNA-probes. The platform is modular and versatile and it can potentially be applied for the detection of any small target molecule that can be conjugated to a nucleic acid sequence. Here the rational design of programmable DNA-switches is discussed, and the sensitive, rapid, and single-step detection of different environmentally relevant small target molecules is demonstrated.

  11. Molecular Strategies for Targeting Antioxidants to Mitochondria: Therapeutic Implications

    PubMed Central

    2015-01-01

    Abstract Mitochondrial function and specifically its implication in cellular redox/oxidative balance is fundamental in controlling the life and death of cells, and has been implicated in a wide range of human pathologies. In this context, mitochondrial therapeutics, particularly those involving mitochondria-targeted antioxidants, have attracted increasing interest as potentially effective therapies for several human diseases. For the past 10 years, great progress has been made in the development and functional testing of molecules that specifically target mitochondria, and there has been special focus on compounds with antioxidant properties. In this review, we will discuss several such strategies, including molecules conjugated with lipophilic cations (e.g., triphenylphosphonium) or rhodamine, conjugates of plant alkaloids, amino-acid- and peptide-based compounds, and liposomes. This area has several major challenges that need to be confronted. Apart from antioxidants and other redox active molecules, current research aims at developing compounds that are capable of modulating other mitochondria-controlled processes, such as apoptosis and autophagy. Multiple chemically different molecular strategies have been developed as delivery tools that offer broad opportunities for mitochondrial manipulation. Additional studies, and particularly in vivo approaches under physiologically relevant conditions, are necessary to confirm the clinical usefulness of these molecules. Antioxid. Redox Signal. 22, 686–729. PMID:25546574

  12. Breast cancer stem cells, EMT and therapeutic targets

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kotiyal, Srishti; Bhattacharya, Susinjan, E-mail: s.bhattacharya@jiit.ac.in

    Highlights: • Therapeutic targeting or inhibition of the key molecules of signaling pathways can control growth of breast cancer stem cells (BCSCs). • Development of BCSCs also involves miRNA interactions. • Therapeutic achievement can be done by targeting identified targets in the BCSC pathways. - Abstract: A small heterogeneous population of breast cancer cells acts as seeds to induce new tumor growth. These seeds or breast cancer stem cells (BCSCs) exhibit great phenotypical plasticity which allows them to undergo “epithelial to mesenchymal transition” (EMT) at the site of primary tumor and a future reverse transition. Apart from metastasis they aremore » also responsible for maintaining the tumor and conferring it with drug and radiation resistance and a tendency for post-treatment relapse. Many of the signaling pathways involved in induction of EMT are involved in CSC generation and regulation. Here we are briefly reviewing the mechanism of TGF-β, Wnt, Notch, TNF-α, NF-κB, RTK signalling pathways which are involved in EMT as well as BCSCs maintenance. Therapeutic targeting or inhibition of the key/accessory players of these pathways could control growth of BCSCs and hence malignant cancer. Additionally several miRNAs are dysregulated in cancer stem cells indicating their roles as oncogenes or tumor suppressors. This review also lists the miRNA interactions identified in BCSCs and discusses on some newly identified targets in the BCSC regulatory pathways like SHIP2, nicastrin, Pin 1, IGF-1R, pro-inflammatory cytokines and syndecan which can be targeted for therapeutic achievements.« less

  13. Identification of Cell Surface Molecules Involved in Dystroglycan-Independent Lassa Virus Cell Entry

    PubMed Central

    Ströher, Ute; Ebihara, Hideki; Feldmann, Heinz

    2012-01-01

    Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry. PMID:22156524

  14. The role of water molecules in computational drug design.

    PubMed

    de Beer, Stephanie B A; Vermeulen, Nico P E; Oostenbrink, Chris

    2010-01-01

    Although water molecules are small and only consist of two different atom types, they play various roles in cellular systems. This review discusses their influence on the binding process between biomacromolecular targets and small molecule ligands and how this influence can be modeled in computational drug design approaches. Both the structure and the thermodynamics of active site waters will be discussed as these influence the binding process significantly. Structurally conserved waters cannot always be determined experimentally and if observed, it is not clear if they will be replaced upon ligand binding, even if sufficient space is available. Methods to predict the presence of water in protein-ligand complexes will be reviewed. Subsequently, we will discuss methods to include water in computational drug research. Either as an additional factor in automated docking experiments, or explicitly in detailed molecular dynamics simulations, the effect of water on the quality of the simulations is significant, but not easily predicted. The most detailed calculations involve estimates of the free energy contribution of water molecules to protein-ligand complexes. These calculations are computationally demanding, but give insight in the versatility and importance of water in ligand binding.

  15. A Small-molecule Inhibitor, 5′-O-Tritylthymidine, targets FAK and Mdm-2 Interaction, and Blocks Breast and Colon Tumorigenesis in vivo

    PubMed Central

    Golubovskaya, Vita; Palma, Nadia L.; Zheng, Min; Ho, Baotran; Magis, Andrew; Ostrov, David; Cance, William G.

    2013-01-01

    Focal Adhesion Kinase (FAK) is overexpressed in many types of tumors and plays an important role in survival. We developed a novel approach, targeting FAK-protein interactions by computer modeling and screening of NCI small molecule drug database. In this report we targeted FAK and Mdm-2 protein interaction to decrease tumor growth. By macromolecular modeling we found a model of FAK and Mdm-2 interaction and performed screening of >200,000 small molecule compounds from NCI database with drug-like characteristics, targeting the FAK-Mdm-2 interaction. We identified 5′-O-Tritylthymidine, called M13 compound that significantly decreased viability in different cancer cells. M13 was docked into the pocket of FAK and Mdm-2 interaction and was directly bound to the FAK-N terminal domain by ForteBio Octet assay. In addition, M13 compound affected FAK and Mdm-2 levels and decreased complex of FAK and Mdm-2 proteins in breast and colon cancer cells. M13 re-activated p53 activity inhibited by FAK with Mdm-2 promoter. M13 decreased viability, clonogenicity, increased detachment and apoptosis in a dose-dependent manner in BT474 breast and in HCT116 colon cancer cells in vitro. M13 decreased FAK, activated p53 and caspase-8 in both cell lines. In addition, M13 decreased breast and colon tumor growth in vivo. M13 activated p53 and decreased FAK in tumor samples consistent with decreased tumor growth. The data demonstrate a novel approach for targeting FAK and Mdm-2 protein interaction, provide a model of FAK and Mdm-2 interaction, identify M13 compound targeting this interaction and decreasing tumor growth that is critical for future targeted therapeutics. PMID:22292771

  16. Targeted studies on the interaction of nicotine and morin molecules with amyloid β-protein.

    PubMed

    Boopathi, Subramaniam; Kolandaivel, Ponmalai

    2014-03-01

    Alzheimer's disease (AD) is a neurodegenerative disorder that occurs due to progressive deposition of amyloid β-protein (Aβ) in the brain. Stable conformations of solvated Aβ₁₋₄₂ protein were predicted by molecular dynamics (MD) simulation using the OPLSAA force field. The seven residue peptide (Lys-Leu-Val-Phe-Phe-Ala-Glu) Aβ₁₆₋₂₂ associated with AD was studied and reported in this paper. Since effective therapeutic agents have not yet been studied in detail, attention has focused on the use of natural products as effective anti-aggregation compounds, targeting the Aβ₁₋₄₂ protein directly. Experimental and theoretical investigation suggests that some compounds extracted from natural products might be useful, but detailed insights into the mechanism by which they might act remains elusive. The molecules nicotine and morin are found in cigarettes and beverages. Here, we report the results of interaction studies of these compounds at each hydrophobic residue of Aβ₁₆₋₂₂ peptide using the hybrid ONIOM (B3LYP/6-31G**:UFF) method. It was found that interaction with nicotine produced higher deformation in the Aβ₁₆₋₂₂ peptide than interaction with morin. MD simulation studies revealed that interaction of the nicotine molecule with the β-sheet of Aβ₁₆₋₂₂ peptide transforms the β-sheet to an α-helical structure, which helps prohibit the aggregation of Aβ-protein.

  17. The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis*

    PubMed Central

    Cwiklinski, Krystyna; de la Torre-Escudero, Eduardo; Trelis, Maria; Bernal, Dolores; Dufresne, Philippe J.; Brennan, Gerard P.; O'Neill, Sandra; Tort, Jose; Paterson, Steve; Marcilla, Antonio; Dalton, John P.; Robinson, Mark W.

    2015-01-01

    Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack. PMID:26486420

  18. Small Molecule Inhibitors Target the Tissue Transglutaminase and Fibronectin Interaction

    PubMed Central

    Yakubov, Bakhtiyor; Chen, Lan; Belkin, Alexey M.; Zhang, Sheng; Chelladurai, Bhadrani; Zhang, Zhong-Yin; Matei, Daniela

    2014-01-01

    Tissue transglutaminase (TG2) mediates protein crosslinking through generation of ε−(γ-glutamyl) lysine isopeptide bonds and promotes cell adhesion through interaction with fibronectin (FN) and integrins. Cell adhesion to the peritoneal matrix regulated by TG2 facilitates ovarian cancer dissemination. Therefore, disruption of the TG2-FN complex by small molecules may inhibit cell adhesion and metastasis. A novel high throughput screening (HTS) assay based on AlphaLISA™ technology was developed to measure the formation of a complex between His-TG2 and the biotinylated FN fragment that binds TG2 and to discover small molecules that inhibit this protein-protein interaction. Several hits were identified from 10,000 compounds screened. The top candidates selected based on >70% inhibition of the TG2/FN complex formation were confirmed by using ELISA and bioassays measuring cell adhesion, migration, invasion, and proliferation. In conclusion, the AlphaLISA bead format assay measuring the TG2-FN interaction is robust and suitable for HTS of small molecules. One compound identified from the screen (TG53) potently inhibited ovarian cancer cell adhesion to FN, cell migration, and invasion and could be further developed as a potential inhibitor for ovarian cancer dissemination. PMID:24586660

  19. Pyrrole-Based Macrocyclic Small-Molecule Inhibitors That Target Oocyte Maturation.

    PubMed

    Gunasekaran, Pethaiah; Lee, So-Rim; Jeong, Seung-Min; Kwon, Jeong-Woo; Takei, Toshiki; Asahina, Yuya; Bang, Geul; Kim, Seongnyeon; Ahn, Mija; Ryu, Eun Kyung; Kim, Hak Nam; Nam, Ki-Yub; Shin, Song Yub; Hojo, Hironobu; Namgoong, Suk; Kim, Nam-Hyung; Bang, Jeong Kyu

    2017-04-20

    Polo-like kinase 1 (PLK1) plays crucial roles in various stages of oocyte maturation. Recently, we reported that the peptidomimetic compound AB103-8, which targets the polo box domain (PBD) of PLK1, affects oocyte meiotic maturation and the resumption of meiosis. However, to overcome the drawbacks of peptidic compounds, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and tested them for their effects on the rates of porcine oocyte maturation. Among them, the macrocyclic compound (E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4) showed the highest inhibitory activity with enhanced inhibition against embryonic blastocyst formation. Furthermore, the addition of this compound to culture media efficiently blocked the maturation of porcine and mouse oocytes, indicating its ability to penetrate the zona pellucida and cell membrane. We investigated mouse oocytes treated with compound 4, and the resulting impairment of spindle formation confirmed PLK1 inhibition. Finally, molecular modeling studies with PLK1 PBD also confirmed the presence of significant interactions between compound 4 and PLK1 PBD binding pocket residues, including those in the phosphate, tyrosine-rich, and pyrrolidine binding pockets. Collectively, these results suggest that the macrocyclic compound 4 may serve as a promising template for the development of novel contraceptive agents. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. A semantic web ontology for small molecules and their biological targets.

    PubMed

    Choi, Jooyoung; Davis, Melissa J; Newman, Andrew F; Ragan, Mark A

    2010-05-24

    A wide range of data on sequences, structures, pathways, and networks of genes and gene products is available for hypothesis testing and discovery in biological and biomedical research. However, data describing the physical, chemical, and biological properties of small molecules have not been well-integrated with these resources. Semantically rich representations of chemical data, combined with Semantic Web technologies, have the potential to enable the integration of small molecule and biomolecular data resources, expanding the scope and power of biomedical and pharmacological research. We employed the Semantic Web technologies Resource Description Framework (RDF) and Web Ontology Language (OWL) to generate a Small Molecule Ontology (SMO) that represents concepts and provides unique identifiers for biologically relevant properties of small molecules and their interactions with biomolecules, such as proteins. We instanced SMO using data from three public data sources, i.e., DrugBank, PubChem and UniProt, and converted to RDF triples. Evaluation of SMO by use of predetermined competency questions implemented as SPARQL queries demonstrated that data from chemical and biomolecular data sources were effectively represented and that useful knowledge can be extracted. These results illustrate the potential of Semantic Web technologies in chemical, biological, and pharmacological research and in drug discovery.

  1. The Molecule Pages database

    PubMed Central

    Saunders, Brian; Lyon, Stephen; Day, Matthew; Riley, Brenda; Chenette, Emily; Subramaniam, Shankar

    2008-01-01

    The UCSD-Nature Signaling Gateway Molecule Pages (http://www.signaling-gateway.org/molecule) provides essential information on more than 3800 mammalian proteins involved in cellular signaling. The Molecule Pages contain expert-authored and peer-reviewed information based on the published literature, complemented by regularly updated information derived from public data source references and sequence analysis. The expert-authored data includes both a full-text review about the molecule, with citations, and highly structured data for bioinformatics interrogation, including information on protein interactions and states, transitions between states and protein function. The expert-authored pages are anonymously peer reviewed by the Nature Publishing Group. The Molecule Pages data is present in an object-relational database format and is freely accessible to the authors, the reviewers and the public from a web browser that serves as a presentation layer. The Molecule Pages are supported by several applications that along with the database and the interfaces form a multi-tier architecture. The Molecule Pages and the Signaling Gateway are routinely accessed by a very large research community. PMID:17965093

  2. The Molecule Pages database.

    PubMed

    Saunders, Brian; Lyon, Stephen; Day, Matthew; Riley, Brenda; Chenette, Emily; Subramaniam, Shankar; Vadivelu, Ilango

    2008-01-01

    The UCSD-Nature Signaling Gateway Molecule Pages (http://www.signaling-gateway.org/molecule) provides essential information on more than 3800 mammalian proteins involved in cellular signaling. The Molecule Pages contain expert-authored and peer-reviewed information based on the published literature, complemented by regularly updated information derived from public data source references and sequence analysis. The expert-authored data includes both a full-text review about the molecule, with citations, and highly structured data for bioinformatics interrogation, including information on protein interactions and states, transitions between states and protein function. The expert-authored pages are anonymously peer reviewed by the Nature Publishing Group. The Molecule Pages data is present in an object-relational database format and is freely accessible to the authors, the reviewers and the public from a web browser that serves as a presentation layer. The Molecule Pages are supported by several applications that along with the database and the interfaces form a multi-tier architecture. The Molecule Pages and the Signaling Gateway are routinely accessed by a very large research community.

  3. Skeletal muscle plasticity induced by seasonal acclimatization in carp involves differential expression of rRNA and molecules that epigenetically regulate its synthesis.

    PubMed

    Fuentes, Eduardo N; Zuloaga, Rodrigo; Nardocci, Gino; Fernandez de la Reguera, Catalina; Simonet, Nicolas; Fumeron, Robinson; Valdes, Juan Antonio; Molina, Alfredo; Alvarez, Marco

    2014-01-01

    Ribosomal biogenesis controls cellular growth in living organisms, with the rate-limiting step of this process being the transcription of ribosomal DNA (rDNA). Considering that epigenetic mechanisms allow an organism to respond to environmental changes, the expression in muscle of several molecules that regulate epigenetic rRNA synthesis, as well as rDNA transcription, were evaluated during the seasonal acclimatization of the carp. First, the nucleotide sequences encoding the components forming the NoRC (ttf-I, tip5) and eNoSC (sirt1, nml, suv39h1), two chromatin remodeling complexes that silence rRNA synthesis, as well as the sequence of ubf1, a key regulator of rDNA transcription, were obtained. Subsequently the transcriptional regulation of the aforementioned molecules, and other key molecules involved in rRNA synthesis (mh2a1, mh2a2, h2a.z, h2a.z.7, nuc, p80), was assessed. The carp sequences for TTF-I, TIP5, SIRT1, NML, SUV39H1, and UBF1 showed a high conservation of domains and key amino acids in comparison with other fish and higher vertebrates. The mRNA contents in muscle for ttf-I, tip5, sirt1, nml, suv39h1, mh2a1, mh2a.z, and nuc were up-regulated during winter in comparison with summer, whereas the mRNA levels of mh2a2, ubf1, and p80 were down-regulated. Also, the contents of molecules involved in processing the rRNA (snoRNAs) and pRNA, a stabilizer of NoRC complex, were analyzed, finding that these non-coding RNAs were not affected by seasonal acclimatization. These results suggest that variations in the expression of rRNA and the molecules that epigenetically regulate its synthesis are contributing to the muscle plasticity induced by seasonal acclimatization in carp. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Targeting polyamine metabolism for cancer therapy and prevention

    PubMed Central

    Murray-Stewart, Tracy R.; Woster, Patrick M.; Casero, Robert A.

    2017-01-01

    The chemically simple, biologically complex eukaryotic polyamines, spermidine and spermine, are positively charged alkylamines involved in many crucial cellular processes. Along with their diamine precursor putrescine, their normally high intracellular concentrations require fine attenuation by multiple regulatory mechanisms to keep these essential molecules within strict physiologic ranges. Since the metabolism of and requirement for polyamines are frequently dysregulated in neoplastic disease, the metabolic pathway and functions of polyamines provide rational drug targets; however, these targets have been difficult to exploit for chemotherapy. It is the goal of this article to review the latest findings in the field that demonstrate the potential utility of targeting the metabolism and function of polyamines as strategies for both chemotherapy and, possibly more importantly, chemoprevention. PMID:27679855

  5. Identifying relationships between unrelated pharmaceutical target proteins on the basis of shared active compounds.

    PubMed

    Miljković, Filip; Kunimoto, Ryo; Bajorath, Jürgen

    2017-08-01

    Computational exploration of small-molecule-based relationships between target proteins from different families. Target annotations of drugs and other bioactive compounds were systematically analyzed on the basis of high-confidence activity data. A total of 286 novel chemical links were established between distantly related or unrelated target proteins. These relationships involved a total of 1859 bioactive compounds including 147 drugs and 141 targets. Computational analysis of large amounts of compounds and activity data has revealed unexpected relationships between diverse target proteins on the basis of compounds they share. These relationships are relevant for drug discovery efforts. Target pairs that we have identified and associated compound information are made freely available.

  6. Influence of DOTA chelator position on biodistribution and targeting properties of (111)In-labeled synthetic anti-HER2 affibody molecules.

    PubMed

    Perols, Anna; Honarvar, Hadis; Strand, Joanna; Selvaraju, Ramkumar; Orlova, Anna; Karlström, Amelie Eriksson; Tolmachev, Vladimir

    2012-08-15

    Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with (99m)Tc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K(D) values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1)-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z(HER2:S1) (64 °C) and DOTA-K58-Z(HER2:S1) (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with (111)In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1)and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is

  7. Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Noninternalizing Receptors.

    PubMed

    Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J

    2017-03-28

    Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ∼3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or noninternalizing receptor (CD45). When lymphocytes were preloaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell antitumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors.

  8. The immunological synapse as a pharmacological target.

    PubMed

    Francesca, Finetti; Baldari, Cosima T

    2018-06-10

    The development of T cell mediated immunity relies on the assembly of a highly specialized interface between T cell and antigen presenting cell (APC), known as the immunological synapse (IS). IS assembly is triggered when the T cell receptor (TCR) binds to specific peptide antigen presented in association to the major histocompatibility complex (MHC) by the APC, and is followed by the spatiotemporal dynamic redistribution of TCR, integrins, co-stimulatory receptors and signaling molecules, allowing for the fine-tuning and integration of the signals that lead to T cell activation. The knowledge acquired to date about the mechanisms of IS assembly underscores this structure as a robust pharmacological target. The activity of molecules involved in IS assembly and function can be targeted by specific compounds to modulate the immune response in a number of disorders, including cancers and autoimmune diseases, or in transplanted patients. Here, we will review the state-of-the art of the current therapies which exploit the IS to modulate the immune response. Copyright © 2018. Published by Elsevier Ltd.

  9. Bilirubin Nanoparticle-Assisted Delivery of a Small Molecule-Drug Conjugate for Targeted Cancer Therapy.

    PubMed

    Lee, Soyoung; Lee, Yonghyun; Kim, Hyungjun; Lee, Dong Yun; Jon, Sangyong

    2018-06-11

    Despite growing interest in targeted cancer therapy with small molecule drug conjugates (SMDCs), the short half-life of these conjugates in blood associated with their small size has limited their efficacy in cancer therapy. In this report, we propose a new approach for improving the antitumor efficacy of SMDCs based on nanoparticle-assisted delivery. Ideally, a nanoparticle-based delivery vehicle would prolong the half-life of an SMDC in blood and then release it in response to stimuli in the tumor microenvironment (TME). In this study, PEGylated bilirubin-based nanoparticles (BRNPs) were chosen as an appropriate delivery carrier because of their ability to release drugs in response to TME-associated reactive oxygen species (ROS) through rapid particle disruption. As a model SMDC, ACUPA-SN38 was synthesized by linking the prostate-specific membrane antigen (PSMA)-targeting ligand, ACUPA, to the chemotherapeutic agent, SN38. ACUPA-SN38 was loaded into BRNPs using a film-formation and rehydration method. The resulting ACUPA-SN38@BRNPs exhibited ROS-mediated particle disruption and rapid release of the SMDC, resulting in greater cytotoxicity toward PSMA-overexpressing prostate cancer cells (LNCaP) than toward ROS-unresponsive ACUPA-SN38@Liposomes. In a pharmacokinetic study, the circulation time of ACUPA-SN38@BRNPs in blood was prolonged by approximately 2-fold compared with that of the SMDC-based micellar nanoparticles. Finally, ACUPA-SN38@BRNPs showed greater antitumor efficacy in a PSMA-overexpressing human prostate xenograft tumor model than SN38@BRNPs or the SMDC alone. Collectively, these findings suggest that BRNPs are a viable delivery carrier option for various cancer-targeting SMDCs that suffer from short circulation half-life and limited therapeutic efficacy.

  10. Avidin-conjugated calcium phosphate nanoparticles as a modular targeting system for the attachment of biotinylated molecules in vitro and in vivo.

    PubMed

    van der Meer, Selina Beatrice; Knuschke, Torben; Frede, Annika; Schulze, Nina; Westendorf, Astrid M; Epple, Matthias

    2017-07-15

    Avidin was covalently conjugated to the surface of calcium phosphate nanoparticles, coated with a thin silica shell and terminated by sulfhydryl groups (diameter of the solid core about 50nm), with a bifunctional crosslinker connecting the amino groups of avidin to the sulfhydryl group on the nanoparticle surface. This led to a versatile nanoparticle system where all kinds of biotinylated (bio-)molecules can be easily attached to the surface by the non-covalent avidin-biotin-complex formation. It also permits the attachment of different biomolecules on the same nanoparticle (heteroavidity), creating a modular system for specific applications in medicine and biology. The variability of the binding to the nanoparticle surface of the was demonstrated with various biotinylated molecules, i.e. fluorescent dyes and antibodies. The accessibility of the conjugated avidin was demonstrated by a fluorescence-quenching assay. About 2.6 binding sites for biotin were accessible on each avidin tetramer. Together with a number of about 240 avidin tetramer units per nanoparticle, this offers about 600 binding sites for biotin on each nanoparticle. The uptake of fluorescently labelled avidin-conjugated calcium phosphate nanoparticles by HeLa cells showed the co-localization of fluorescent avidin and fluorescent biotin, indicating the stability of the complex under cell culture conditions. CD11c-antibody functionalized nanoparticles specifically targeted antigen-presenting immune cells (dendritic cells; DCs) in vitro and in vivo (mice) with high efficiency. Calcium phosphate nanoparticles have turned out to be very useful transporters for biomolecules into cells, both in vitro and in vivo. However, their covalent surface functionalization with antibodies, fluorescent dyes, or proteins requires a separate chemical synthesis for each kind of surface molecule. We have therefore developed avidin-terminated calcium phosphate nanoparticles to which all kinds of biotinylated molecules can

  11. Globular domain of adiponectin: promising target molecule for detection of atherosclerotic lesions

    PubMed Central

    Almer, Gunter; Saba-Lepek, Matthias; Haj-Yahya, Samih; Rohde, Eva; Strunk, Dirk; Fröhlich, Eleonore; Prassl, Ruth; Mangge, Harald

    2011-01-01

    Background: Adiponectin, an adipocyte-specific plasma protein, has been shown to accumulate in injured endothelial cells during development of atherosclerotic lesions. In this study, we investigated the potential of different adiponectin subfractions with special emphasis on globular adiponectin (gAd) to recognize and visualize atherosclerotic lesions. Methods: Recombinant mouse gAd and subfractions of full-length adiponectin (ie, trimeric, hexameric, and oligomeric forms) were fluorescence-labeled. Aortas of wild-type and apoprotein E-deficient mice fed a high cholesterol diet were dissected and incubated with the labeled biomarkers. Imaging was performed using confocal laser scanning microscopy. Results: Confocal laser scanning microscopic images showed that gAd binds more strongly to atherosclerotic plaques than full-length adiponectin subfractions. Further, we showed that gAd accumulates preferentially in endothelial cells and the fibrous cap area of plaques. Here we demonstrate for the first time that gAd recognizes atherosclerotic plaques on aortic sections of apoprotein E-deficient mice. Conclusion: These results suggest that gAd, in addition to its physiological properties, is also suitable as a target molecule for prospective diagnostic strategies in imaging atherosclerotic lesions. PMID:22022204

  12. Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

    PubMed

    Bol, Guus M; Vesuna, Farhad; Xie, Min; Zeng, Jing; Aziz, Khaled; Gandhi, Nishant; Levine, Anne; Irving, Ashley; Korz, Dorian; Tantravedi, Saritha; Heerma van Voss, Marise R; Gabrielson, Kathleen; Bordt, Evan A; Polster, Brian M; Cope, Leslie; van der Groep, Petra; Kondaskar, Atul; Rudek, Michelle A; Hosmane, Ramachandra S; van der Wall, Elsken; van Diest, Paul J; Tran, Phuoc T; Raman, Venu

    2015-05-01

    Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  13. Measurement of Ligand–Target Residence Times by 1H Relaxation Dispersion NMR Spectroscopy

    PubMed Central

    2016-01-01

    A ligand-observed 1H NMR relaxation experiment is introduced for measuring the binding kinetics of low-molecular-weight compounds to their biomolecular targets. We show that this approach, which does not require any isotope labeling, is applicable to ligand–target systems involving proteins and nucleic acids of variable molecular size. The experiment is particularly useful for the systematic investigation of low affinity molecules with residence times in the micro- to millisecond time regime. PMID:27933946

  14. MicroRNAs – Important Molecules in Lung Cancer Research

    PubMed Central

    Leidinger, Petra; Keller, Andreas; Meese, Eckart

    2011-01-01

    MicroRNAs (miRNA) are important regulators of gene expression. They are involved in many physiological processes ensuring the cellular homeostasis of human cells. Alterations of the miRNA expression have increasingly been associated with pathophysiologic changes of cancer cells making miRNAs currently to one of the most analyzed molecules in cancer research. Here, we provide an overview of miRNAs in lung cancer. Specifically, we address biological functions of miRNAs in lung cancer cells, miRNA signatures generated from tumor tissue and from patients’ body fluids, the potential of miRNAs as diagnostic and prognostic biomarker for lung cancer, and its role as therapeutic target. PMID:22303398

  15. Single Molecule Sensing by Nanopores and Nanopore Devices

    PubMed Central

    Gu, Li-Qun; Shim, Ji Wook

    2010-01-01

    Molecular-scale pore structures, called nanopores, can be assembled by protein ion channels through genetic engineering or be artificially fabricated on solid substrates using fashion nanotechnology. When target molecules interact with the functionalized lumen of a nanopore, they characteristically block the ion pathway. The resulting conductance changes allow for identification of single molecules and quantification of target species in the mixture. In this review, we first overview nanopore-based sensory techniques that have been created for the detection of myriad biomedical targets, from metal ions, drug compounds, and cellular second messengers to proteins and DNA. Then we introduce our recent discoveries in nanopore single molecule detection: (1) using the protein nanopore to study folding/unfolding of the G-quadruplex aptamer; (2) creating a portable and durable biochip that is integrated with a single-protein pore sensor (this chip is compared with recently developed protein pore sensors based on stabilized bilayers on glass nanopore membranes and droplet interface bilayer); and (3) creating a glass nanopore-terminated probe for single-molecule DNA detection, chiral enantiomer discrimination, and identification of the bioterrorist agent ricin with an aptamer-encoded nanopore. PMID:20174694

  16. Nitrogen modulation of legume root architecture signaling pathways involves phytohormones and small regulatory molecules.

    PubMed

    Mohd-Radzman, Nadiatul A; Djordjevic, Michael A; Imin, Nijat

    2013-10-01

    Nitrogen, particularly nitrate is an important yield determinant for crops. However, current agricultural practice with excessive fertilizer usage has detrimental effects on the environment. Therefore, legumes have been suggested as a sustainable alternative for replenishing soil nitrogen. Legumes can uniquely form nitrogen-fixing nodules through symbiotic interaction with specialized soil bacteria. Legumes possess a highly plastic root system which modulates its architecture according to the nitrogen availability in the soil. Understanding how legumes regulate root development in response to nitrogen availability is an important step to improving root architecture. The nitrogen-mediated root development pathway starts with sensing soil nitrogen level followed by subsequent signal transduction pathways involving phytohormones, microRNAs and regulatory peptides that collectively modulate the growth and shape of the root system. This review focuses on the current understanding of nitrogen-mediated legume root architecture including local and systemic regulations by different N-sources and the modulations by phytohormones and small regulatory molecules.

  17. Progress in Computational Electron-Molecule Collisions

    NASA Astrophysics Data System (ADS)

    Rescigno, Tn

    1997-10-01

    The past few years have witnessed tremendous progress in the development of sophisticated ab initio methods for treating collisions of slow electrons with isolated small molecules. Researchers in this area have benefited greatly from advances in computer technology; indeed, the advent of parallel computers has made it possible to carry out calculations at a level of sophistication inconceivable a decade ago. But bigger and faster computers are only part of the picture. Even with today's computers, the practical need to study electron collisions with the kinds of complex molecules and fragments encountered in real-world plasma processing environments is taxing present methods beyond their current capabilities. Since extrapolation of existing methods to handle increasingly larger targets will ultimately fail as it would require computational resources beyond any imagined, continued progress must also be linked to new theoretical developments. Some of the techniques recently introduced to address these problems will be discussed and illustrated with examples of electron-molecule collision calculations we have carried out on some fairly complex target gases encountered in processing plasmas. Electron-molecule scattering continues to pose many formidable theoretical and computational challenges. I will touch on some of the outstanding open questions.

  18. One Novel Multiple-Target Plasmid Reference Molecule Targeting Eight Genetically Modified Canola Events for Genetically Modified Canola Detection.

    PubMed

    Li, Zhuqing; Li, Xiang; Wang, Canhua; Song, Guiwen; Pi, Liqun; Zheng, Lan; Zhang, Dabing; Yang, Litao

    2017-09-27

    Multiple-target plasmid DNA reference materials have been generated and utilized as good substitutes of matrix-based reference materials in the analysis of genetically modified organisms (GMOs). Herein, we report the construction of one multiple-target plasmid reference molecule, pCAN, which harbors eight GM canola event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) and a partial sequence of the canola endogenous reference gene PEP. The applicability of this plasmid reference material in qualitative and quantitative PCR assays of the eight GM canola events was evaluated, including the analysis of specificity, limit of detection (LOD), limit of quantification (LOQ), and performance of pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2, MS1, and RT73 assays using pCAN as the calibrator and 10 genome copies for the other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and PEP assays are between 91% and 97%, and the squared regression coefficients (R 2 ) are all higher than 0.99. The quantification bias values varied from 0.47% to 20.68% with relative standard deviation (RSD) from 1.06% to 24.61% in the quantification of simulated samples. Furthermore, 10 practical canola samples sampled from imported shipments in the port of Shanghai, China, were analyzed employing pCAN as the calibrator, and the results were comparable with those assays using commercial certified materials as the calibrator. Concluding from these results, we believe that this newly developed pCAN plasmid is one good candidate for being a plasmid DNA reference material in the detection and quantification of the eight GM canola events in routine analysis.

  19. Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

    PubMed Central

    David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick

    2018-01-01

    Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998

  20. Computer-assisted identification of novel small molecule inhibitors targeting GLUT1

    NASA Astrophysics Data System (ADS)

    Wan, Zhining; Li, Xin; Sun, Rong; Li, Yuanyuan; Wang, Xiaoyun; Li, Xinru; Rong, Li; Shi, Zheng; Bao, Jinku

    2015-12-01

    Glucose transporters (GLUTs) are the main carriers of glucose that facilitate the diffusion of glucose in mammalian cells, especially GLUT1. Notably, GLUT1 is a rate-limiting transporter for glucose uptake, and its overexpression is a common characteristic in most cancers. Thus, the inhibition of GLUT1 by novel small compounds to lower glucose levels for cancer cells has become an emerging strategy. Herein, we employed high-throughput screening approaches to identify potential inhibitors against the sugar-binding site of GLUT1. Firstly, molecular docking screening was launched against the specs products, and three molecules (ZINC19909927, ZINC19908826, and ZINC19815451) were selected as candidate GLUT1 inhibitors for further analysis. Then, taking the initial ligand β-NG as a reference, molecular dynamic (MD) simulations and molecular mechanics/generalized born surface area (MM/GBSA) method were applied to evaluate the binding stability and affinity of the three candidates towards GLUT1. Finally, we found that ZINC19909927 might have the highest affinity to occupy the binding site of GLUT1. Meanwhile, energy decomposition analysis identified several residues located in substrate-binding site that might provide clues for future inhibitor discovery towards GLUT1. Taken together, these results in our study may provide valuable information for identifying new inhibitors targeting GLUT1-mediated glucose transport and metabolism for cancer therapeutics.

  1. Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

    2014-03-01

    Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

  2. Targeted therapies in gastric cancer and future perspectives.

    PubMed

    Yazici, Ozan; Sendur, M Ali Nahit; Ozdemir, Nuriye; Aksoy, Sercan

    2016-01-14

    Advanced gastric cancer (AGC) is associated with a high mortality rate and, despite multiple new chemotherapy options, the survival rates of patients with AGC remains poor. After the discovery of targeted therapies, research has focused on the new treatment options for AGC. In the last two decades, many targeted molecules were developed against AGC. Currently, two targeted therapy molecules have been approved for patients with AGC. In 2010, trastuzumab was the first molecule shown to improve survival in patients with HER2-positive AGC as part of a first-line combination regimen. In 2014, ramucirumab was the second targeted molecule to improve survival rates and was suggested as treatment for patients with AGC who had progressed after first-line platinum plus fluoropyrimidine with or without anthracycline chemotherapy. Ramucirumab was the first targeted therapy acting as a single agent in patients with advanced gastroesophageal cancers. Although these two molecules were introduced into clinical use, many other promising molecules have been tested in phase I-II trials. It is obvious that in the near future many different targeted therapies will be in use for treatment of AGC. In this review, the current status of targeted therapies in the treatment of AGC and gastroesophageal junction tumors, including HER (2-3) inhibitors, epidermal growth factor receptor inhibitors, tyrosine kinase inhibitors, antiangiogenic agents, c-MET inhibitors, mammalian target of rapamycin inhibitors, agents against other molecular pathways fibroblast growth factor, Claudins, insulin-like growth factor, heat shock proteins, and immunotherapy, will be discussed.

  3. A systematic approach to prioritize drug targets using machine learning, a molecular descriptor-based classification model, and high-throughput screening of plant derived molecules: a case study in oral cancer.

    PubMed

    Randhawa, Vinay; Kumar Singh, Anil; Acharya, Vishal

    2015-12-01

    Systems-biology inspired identification of drug targets and machine learning-based screening of small molecules which modulate their activity have the potential to revolutionize modern drug discovery by complementing conventional methods. To utilize the effectiveness of such pipelines, we first analyzed the dysregulated gene pairs between control and tumor samples and then implemented an ensemble-based feature selection approach to prioritize targets in oral squamous cell carcinoma (OSCC) for therapeutic exploration. Based on the structural information of known inhibitors of CXCR4-one of the best targets identified in this study-a feature selection was implemented for the identification of optimal structural features (molecular descriptor) based on which a classification model was generated. Furthermore, the CXCR4-centered descriptor-based classification model was finally utilized to screen a repository of plant derived small-molecules to obtain potential inhibitors. The application of our methodology may assist effective selection of the best targets which may have previously been overlooked, that in turn will lead to the development of new oral cancer medications. The small molecules identified in this study can be ideal candidates for trials as potential novel anti-oral cancer agents. Importantly, distinct steps of this whole study may provide reference for the analysis of other complex human diseases.

  4. Small molecule membrane transporters in the mammalian podocyte: a pathogenic and therapeutic target.

    PubMed

    Zennaro, Cristina; Artero, Mary; Di Maso, Vittorio; Carraro, Michele

    2014-11-18

    The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development.

  5. Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

    PubMed

    Nakatsuka, Nako; Cao, Huan H; Deshayes, Stephanie; Melkonian, Arin Lucy; Kasko, Andrea M; Weiss, Paul S; Andrews, Anne M

    2018-05-31

    Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or L-tryptophan were selectively recognized by previously identified dopamine or L-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though slightly greater than the previously determined solution dissociation constant. Using pre-functionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with L-DOPA, L-DOPS, and L-5-HTP enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons, and future identification and characterization of novel aptamers targeting neurotransmitters or other important small-molecules.

  6. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

    NASA Astrophysics Data System (ADS)

    Neira, José L.; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L.; Abián, Olga

    2017-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

  7. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

    PubMed Central

    Neira, José L.; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L.; Abián, Olga

    2017-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs. PMID:28054562

  8. Optoporation of impermeable molecules and genes for visualization and activation of cells

    NASA Astrophysics Data System (ADS)

    Dhakal, Kamal; Batbyal, Subrata; Kim, Young-Tae; Mohanty, Samarendra

    2015-03-01

    Visualization, activation, and detection of the cell(s) and their electrical activity require delivery of exogenous impermeable molecules and targeted expression of genes encoding labeling proteins, ion-channels and voltage indicators. While genes can be delivered by viral vector to cells, delivery of other impermeable molecules into the cytoplasm of targeted cells requires microinjection by mechanical needle or microelectrodes, which pose significant challenge to the viability of the cells. Further, it will be useful to localize the expression of the targeted molecules not only in specific cell types, but to specific cells in restricted spatial regions. Here, we report use of focused near-infrared (NIR) femtosecond laser beam to transiently perforate targeted cell membrane to insert genes encoding blue light activatable channelrhodopsin-2 (ChR2) and red-shifted opsin (ReachR). Optoporation of nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons) is also achieved allowing imaging of dynamics and intact morphology of cellular structures without requiring fixation.

  9. Investigating Small-Molecule Ligand Binding to G Protein-Coupled Receptors with Biased or Unbiased Molecular Dynamics Simulations.

    PubMed

    Marino, Kristen A; Filizola, Marta

    2018-01-01

    An increasing number of G protein-coupled receptor (GPCR) crystal structures provide important-albeit static-pictures of how small molecules or peptides interact with their receptors. These high-resolution structures represent a tremendous opportunity to apply molecular dynamics (MD) simulations to capture atomic-level dynamical information that is not easy to obtain experimentally. Understanding ligand binding and unbinding processes, as well as the related responses of the receptor, is crucial to the design of better drugs targeting GPCRs. Here, we discuss possible ways to study the dynamics involved in the binding of small molecules to GPCRs, using long timescale MD simulations or metadynamics-based approaches.

  10. Investigating Small-Molecule Ligand Binding to G Protein-Coupled Receptors with Biased or Unbiased Molecular Dynamics Simulations

    PubMed Central

    Marino, Kristen A.; Filizola, Marta

    2017-01-01

    An increasing number of G protein-coupled receptor (GPCR) crystal structures provide important—albeit static—pictures of how small molecules or peptides interact with their receptors. These high-resolution structures represent a tremendous opportunity to apply molecular dynamics (MD) simulations to capture atomic-level dynamical information that is not easy to obtain experimentally. Understanding ligand binding and unbinding processes, as well as the related responses of the receptor, is crucial to the design of better drugs targeting GPCRs. Here, we discuss possible ways to study the dynamics involved in the binding of small molecules to GPCRs, using long timescale MD simulations or metadynamics-based approaches. PMID:29188572

  11. Toward Small-Molecule Inhibition of Protein-Protein Interactions: General Aspects and Recent Progress in Targeting Costimulatory and Coinhibitory (Immune Checkpoint) Interactions.

    PubMed

    Bojadzic, Damir; Buchwald, Peter

    2018-05-30

    Protein-protein interactions (PPIs) that are part of the costimulatory and coinhibitory (immune checkpoint) signaling are critical for adequate T cell response and are important therapeutic targets for immunomodulation. Biologics targeting them have already achieved considerable clinical success in the treatment of autoimmune diseases or transplant recipients (e.g., abatacept, belatacept, and belimumab) as well as cancer (e.g., ipilimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab). In view of such progress, there have been only relatively limited efforts toward developing small-molecule PPI inhibitors (SMPPIIs) targeting these cosignaling interactions, possibly because they, as all other PPIs, are difficult to target by small molecules and were not considered druggable. Nevertheless, substantial progress has been achieved during the last decade. SMPPIIs proving the feasibility of such approaches have been identified through various strategies for a number of cosignaling interactions including CD40-CD40L, OX40-OX40L, BAFFR-BAFF, CD80-CD28, and PD-1-PD-L1s. Here, after an overview of the general aspects and challenges of SMPPII-focused drug discovery, we review them briefly together with relevant structural, immune-signaling, physicochemical, and medicinal chemistry aspects. While so far only a few of these SMPPIIs have shown activity in animal models (DRI-C21045 for CD40-D40L, KR33426 for BAFFR-BAFF) or reached clinical development (RhuDex for CD80-CD28, CA-170 for PD-1-PD-L1), there is proof-of-principle evidence for the feasibility of such approaches in immunomodulation. They can result in products that are easier to develop/manufacture and are less likely to be immunogenic or encounter postmarket safety events than corresponding biologics, and, contrary to them, can even become orally bioavailable. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Designing small molecules to target cryptic pockets yields both positive and negative allosteric modulators

    PubMed Central

    Moeder, Katelyn E.; Ho, Chris M. W.; Zimmerman, Maxwell I.; Frederick, Thomas E.; Bowman, Gregory R.

    2017-01-01

    Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered “undruggable” and to develop new therapies that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such “cryptic pockets,” and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM β-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators. PMID:28570708

  13. Turmeric (Curcuma longa): miRNAs and their regulating targets are involved in development and secondary metabolite pathways.

    PubMed

    Singh, Noopur; Sharma, Ashok

    Turmeric has been used as a therapeutic herb over centuries in traditional medicinal systems due to the presence of several secondary metabolite compounds. microRNAs are known to regulate gene expression at the post-transcriptional level by transcriptional cleavage or translation repression. miRNAs have been demonstrated to play an active role in secondary metabolism regulation. The present work was focused on the identification of the miRNAs involved in the regulation of secondary metabolite and development process of turmeric. Eighteen miRNA families were identified for turmeric. Sixteen miRNA families were observed to regulate 238 target transcripts. LncRNAs targets of the putative miRNA candidates were also predicted. Our results indicated their role in binding, reproduction, stress, and other developmental processes. Gene annotation and pathway analysis illustrated the biological function of the targets regulated by the putative miRNAs. The miRNA-mediated gene regulatory network also revealed co-regulated targets that were regulated by two or more miRNA families. miR156 and miR5015 were observed to be involved in rhizome development. miR5021 showed regulation for terpenoid backbone biosynthesis and isoquinoline alkaloid biosynthesis pathways. The flavonoid biosynthesis pathway was observed to be regulated by miR2919. The analysis revealed the probable involvement of three miRNAs (miR1168.2, miR156b and miR1858) in curcumin biosynthesis. Other miRNAs were found to be involved in the growth and developmental process of turmeric. Phylogenetic analysis of selective miRNAs was also performed. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

  14. Activating transcription factor 3 is a target molecule linking hepatic steatosis to impaired glucose homeostasis.

    PubMed

    Kim, Ji Yeon; Park, Keon Jae; Hwang, Joo-Yeon; Kim, Gyu Hee; Lee, DaeYeon; Lee, Yoo Jeong; Song, Eun Hyun; Yoo, Min-Gyu; Kim, Bong-Jo; Suh, Young Ho; Roh, Gu Seob; Gao, Bin; Kim, Won; Kim, Won-Ho

    2017-08-01

    Non-alcoholic fatty liver disease (NAFLD) contributes to impaired glucose tolerance, leading to type 2 diabetes (T2D); however, the precise mechanisms and target molecules that are involved remain unclear. Activating transcription factor 3 (ATF3) is associated with β-cell dysfunction that is induced by severe stress signals in T2D. We aimed to explore the exact functional role of ATF3 as a mechanistic link between hepatic steatosis and T2D development. Zucker diabetic fatty (ZDF) rats were utilized for animal experiments. An in vivo-jetPEI siRNA delivery system against ATF3 was used for loss-of-function experiments. We analyzed the baseline cross-sectional data derived from the biopsy-proven NAFLD registry (n=322). Human sera and liver tissues were obtained from 43 patients with biopsy-proven NAFLD and from seven healthy participants. ATF3 was highly expressed in the livers of ZDF rats and in human participants with NAFLD and/or T2D. Insulin resistance and hepatic steatosis were associated with increased ATF3 expression and decreased fatty acid oxidation via mitochondrial dysfunction and were attenuated by in vivo ATF3 silencing. Knockdown of ATF3 also ameliorated glucose intolerance, impaired insulin action, and inflammatory responses in ZDF rats. In patients with NAFLD and/or T2D, a significant positive correlation was observed between hepatic ATF3 expression and surrogate markers of T2D, mitochondrial dysfunction, and macrophage infiltration. Increased hepatic ATF3 expression is closely associated with hepatic steatosis and incident T2D; therefore, ATF3 may serve as a potential therapeutic target for NAFLD and hepatic steatosis-induced T2D. Hepatic activating transcription factor 3 (ATF3) may play an important role in oxidative stress-mediated hepatic steatosis and the development of type 2 diabetes (T2D) in a Zucker diabetic fatty (ZDF) rat model and in human patients with non-alcoholic fatty liver disease (NAFLD). Therefore, ATF3 may be a useful biomarker for

  15. Target mimics: an embedded layer of microRNA-involved gene regulatory networks in plants.

    PubMed

    Meng, Yijun; Shao, Chaogang; Wang, Huizhong; Jin, Yongfeng

    2012-05-21

    MicroRNAs (miRNAs) play an essential role in gene regulation in plants. At the same time, the expression of miRNA genes is also tightly controlled. Recently, a novel mechanism called "target mimicry" was discovered, providing another layer for modulating miRNA activities. However, except for the artificial target mimics manipulated for functional studies on certain miRNA genes, only one example, IPS1 (Induced by Phosphate Starvation 1)-miR399 was experimentally confirmed in planta. To date, few analyses for comprehensive identification of natural target mimics have been performed in plants. Thus, limited evidences are available to provide detailed information for interrogating the questionable issue whether target mimicry was widespread in planta, and implicated in certain biological processes. In this study, genome-wide computational prediction of endogenous miRNA mimics was performed in Arabidopsis and rice, and dozens of target mimics were identified. In contrast to a recent report, the densities of target mimic sites were found to be much higher within the untranslated regions (UTRs) when compared to those within the coding sequences (CDSs) in both plants. Some novel sequence characteristics were observed for the miRNAs that were potentially regulated by the target mimics. GO (Gene Ontology) term enrichment analysis revealed some functional insights into the predicted mimics. After degradome sequencing data-based identification of miRNA targets, the regulatory networks constituted by target mimics, miRNAs and their downstream targets were constructed, and some intriguing subnetworks were further exploited. These results together suggest that target mimicry may be widely implicated in regulating miRNA activities in planta, and we hope this study could expand the current understanding of miRNA-involved regulatory networks.

  16. Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces

    PubMed Central

    Briesewitz, Roger; Ray, Gregory T.; Wandless, Thomas J.; Crabtree, Gerald R.

    1999-01-01

    A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein–protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds. PMID:10051576

  17. Multimodal effects of small molecule ROCK and LIMK inhibitors on mitosis, and their implication as anti-leukemia agents.

    PubMed

    Oku, Yusuke; Tareyanagi, Chiaki; Takaya, Shinichi; Osaka, Sayaka; Ujiie, Haruki; Yoshida, Kentaro; Nishiya, Naoyuki; Uehara, Yoshimasa

    2014-01-01

    Accurate chromosome segregation is vital for cell viability. Many cancer cells show chromosome instability (CIN) due to aberrant expression of the genes involved in chromosome segregation. The induction of massive chromosome segregation errors in such cancer cells by small molecule inhibitors is an emerging strategy to kill these cells selectively. Here we screened and characterized small molecule inhibitors which cause mitotic chromosome segregation errors to target cancer cell growth. We screened about 300 chemicals with known targets, and found that Rho-associated coiled-coil kinase (ROCK) inhibitors bypassed the spindle assembly checkpoint (SAC), which delays anaphase onset until proper kinetochore-microtubule interactions are established. We investigated how ROCK inhibitors affect chromosome segregation, and found that they induced microtubule-dependent centrosome fragmentation. Knockdown of ROCK1 and ROCK2 revealed their additive roles in centrosome integrity. Pharmacological inhibition of LIMK also induced centrosome fragmentation similar to that by ROCK inhibitors. Inhibition of ROCK or LIMK hyper-stabilized mitotic spindles and impaired Aurora-A activation. These results suggested that ROCK and LIMK are directly or indirectly involved in microtubule dynamics and activation of Aurora-A. Furthermore, inhibition of ROCK or LIMK suppressed T cell leukemia growth in vitro, but not peripheral blood mononuclear cells. They induced centrosome fragmentation and apoptosis in T cell leukemia cells. These results suggested that ROCK and LIMK can be a potential target for anti-cancer drugs.

  18. Ejection-ionization of molecules from free standing graphene

    NASA Astrophysics Data System (ADS)

    Verkhoturov, Stanislav V.; Czerwinski, Bartlomiej; Verkhoturov, Dmitriy S.; Geng, Sheng; Delcorte, Arnaud; Schweikert, Emile A.

    2017-02-01

    We present the first data on emission of -C60 stimulated by single impacts of 50 keV C60+2 on the self-assembled molecular layer of C60 deposited on free standing 2 layer graphene. The yield, Y, of -C60 emitted in the transmission direction is 1.7%. To characterize the ejection and ionization of molecules, we have measured the emission of -C60 from the surface of bulk C60 (Y = 3.7%) and from a single layer of C60 deposited on bulk pyrolytic graphite (Y = 3.3%). To gain insight into the mechanism(s) of ejection, molecular dynamic simulations were performed. The scenario of the energy deposition and ejection of molecules is different for the case of graphene due to the confined volume of projectile-analyte interaction. In the case of 50 keV C60+2 impacts on graphene plus C60, the C atoms of the projectile collide with those of the target. The knocked-on atoms take on a part of the kinetic energy of the projectile atoms. Another part of the kinetic energy is deposited into the rim around the impact site. The ejection of molecules from the rim is a result of collective movement of the molecules and graphene membrane, where the membrane movement provides the impulse for ejection. The efficient emission of the intact molecular ions implies an effective ionization probability of intact C60. The proposed mechanism of ionization involves the tunneling of electrons from the vibrationally exited area around the hole to the ejecta.

  19. Biological Nanopores: Confined Spaces for Electrochemical Single-Molecule Analysis.

    PubMed

    Cao, Chan; Long, Yi-Tao

    2018-02-20

    Nanopore sensing is developing into a powerful single-molecule approach to investigate the features of biomolecules that are not accessible by studying ensemble systems. When a target molecule is transported through a nanopore, the ions occupying the pore are excluded, resulting in an electrical signal from the intermittent ionic blockade event. By statistical analysis of the amplitudes, duration, frequencies, and shapes of the blockade events, many properties of the target molecule can be obtained in real time at the single-molecule level, including its size, conformation, structure, charge, geometry, and interactions with other molecules. With the development of the use of α-hemolysin to characterize individual polynucleotides, nanopore technology has attracted a wide range of research interest in the fields of biology, physics, chemistry, and nanoscience. As a powerful single-molecule analytical method, nanopore technology has been applied for the detection of various biomolecules, including oligonucleotides, peptides, oligosaccharides, organic molecules, and disease-related proteins. In this Account, we highlight recent developments of biological nanopores in DNA-based sensing and in studying the conformational structures of DNA and RNA. Furthermore, we introduce the application of biological nanopores to investigate the conformations of peptides affected by charge, length, and dipole moment and to study disease-related proteins' structures and aggregation transitions influenced by an inhibitor, a promoter, or an applied voltage. To improve the sensing ability of biological nanopores and further extend their application to a wider range of molecular sensing, we focus on exploring novel biological nanopores, such as aerolysin and Stable Protein 1. Aerolysin exhibits an especially high sensitivity for the detection of single oligonucleotides both in current separation and duration. Finally, to facilitate the use of nanopore measurements and statistical analysis

  20. Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Non-Internalizing Receptors

    PubMed Central

    Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J.

    2017-01-01

    Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ~3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or non-internalizing receptor (CD45). When lymphocytes were pre-loaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell anti-tumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors. PMID:28231431

  1. Small Molecule Docking from Theoretical Structural Models

    NASA Astrophysics Data System (ADS)

    Novoa, Eva Maria; de Pouplana, Lluis Ribas; Orozco, Modesto

    Structural approaches to rational drug design rely on the basic assumption that pharmacological activity requires, as necessary but not sufficient condition, the binding of a drug to one or several cellular targets, proteins in most cases. The traditional paradigm assumes that drugs that interact only with a single cellular target are specific and accordingly have little secondary effects, while promiscuous molecules are more likely to generate undesirable side effects. However, current examples indicate that often efficient drugs are able to interact with several biological targets [1] and in fact some dirty drugs, such as chlorpromazine, dextromethorphan, and ibogaine exhibit desired pharmacological properties [2]. These considerations highlight the tremendous difficulty of designing small molecules that both have satisfactory ADME properties and the ability of interacting with a limited set of target proteins with a high affinity, avoiding at the same time undesirable interactions with other proteins. In this complex and challenging scenario, computer simulations emerge as the basic tool to guide medicinal chemists during the drug discovery process.

  2. Mapping small molecule binding data to structural domains

    PubMed Central

    2012-01-01

    Background Large-scale bioactivity/SAR Open Data has recently become available, and this has allowed new analyses and approaches to be developed to help address the productivity and translational gaps of current drug discovery. One of the current limitations of these data is the relative sparsity of reported interactions per protein target, and complexities in establishing clear relationships between bioactivity and targets using bioinformatics tools. We detail in this paper the indexing of targets by the structural domains that bind (or are likely to bind) the ligand within a full-length protein. Specifically, we present a simple heuristic to map small molecule binding to Pfam domains. This profiling can be applied to all proteins within a genome to give some indications of the potential pharmacological modulation and regulation of all proteins. Results In this implementation of our heuristic, ligand binding to protein targets from the ChEMBL database was mapped to structural domains as defined by profiles contained within the Pfam-A database. Our mapping suggests that the majority of assay targets within the current version of the ChEMBL database bind ligands through a small number of highly prevalent domains, and conversely the majority of Pfam domains sampled by our data play no currently established role in ligand binding. Validation studies, carried out firstly against Uniprot entries with expert binding-site annotation and secondly against entries in the wwPDB repository of crystallographic protein structures, demonstrate that our simple heuristic maps ligand binding to the correct domain in about 90 percent of all assessed cases. Using the mappings obtained with our heuristic, we have assembled ligand sets associated with each Pfam domain. Conclusions Small molecule binding has been mapped to Pfam-A domains of protein targets in the ChEMBL bioactivity database. The result of this mapping is an enriched annotation of small molecule bioactivity data and a

  3. Current trends in small molecule discovery targeting key cellular signaling events towards the combined management of diabetes and obesity

    PubMed Central

    Sangeetha, Kadapakkam Nandabalan; Sujatha, Sundaresan; Muthusamy, Velusamy Shanmuganathan; Anand, Singaravel; Shilpa, Kusampudi; kumari, Posa Jyothi; Sarathkumar, Baskaran; Thiyagarajan, Gopal; Lakshmi, Baddireddi Subhadra

    2017-01-01

    transport in L6 myotubes through the involvement of GLUT4 via the PI3K-independent pathway. However, the activation of glucose storage was effective in the presence of 3β-taraxerol and aloe emodin though inhibition of GSK3β activity. Therefore, the mechanism of improvement of glucose and lipid metabolism exhibited by the small molecules isolated from our lab is discussed. However, Obesity is a major risk factor for type-2 diabetes leading to destruction of insulin receptors causing insulin resistance. Identification of compounds with dual activity (anti-diabetic and antiadipogenic activity) is of current interest. The protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of the insulin and leptin-signaling pathway is of significance in target definition and discovery. PMID:29379255

  4. Current trends in small molecule discovery targeting key cellular signaling events towards the combined management of diabetes and obesity.

    PubMed

    Sangeetha, Kadapakkam Nandabalan; Sujatha, Sundaresan; Muthusamy, Velusamy Shanmuganathan; Anand, Singaravel; Shilpa, Kusampudi; Kumari, Posa Jyothi; Sarathkumar, Baskaran; Thiyagarajan, Gopal; Lakshmi, Baddireddi Subhadra

    2017-01-01

    transport in L6 myotubes through the involvement of GLUT4 via the PI3K-independent pathway. However, the activation of glucose storage was effective in the presence of 3β-taraxerol and aloe emodin though inhibition of GSK3β activity. Therefore, the mechanism of improvement of glucose and lipid metabolism exhibited by the small molecules isolated from our lab is discussed. However, Obesity is a major risk factor for type-2 diabetes leading to destruction of insulin receptors causing insulin resistance. Identification of compounds with dual activity (anti-diabetic and antiadipogenic activity) is of current interest. The protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of the insulin and leptin-signaling pathway is of significance in target definition and discovery.

  5. Cytokine-targeting biologics for allergic diseases.

    PubMed

    Lawrence, Monica G; Steinke, John W; Borish, Larry

    2018-04-01

    Asthma and allergic diseases continue to increase in prevalence, creating a financial burden on the health care system and affecting the quality of life for those who have these diseases. Many intrinsic and extrinsic factors are involved in the initiation and maintenance of the allergic response. Cytokines are proteins with growth, differentiation, and activation functions that regulate and direct the nature of immune responses. clinicaltrials.gov and PubMed. Relevant clinical trials and recent basic science studies were chosen for discussion. Many cytokines have been implicated in the development and perpetuation of the allergic response. Biologics have been and are continuing to be developed that target these molecules for use in patients with asthma and atopic dermatitis where standard treatment options fail. The current state of cytokine-targeting therapies is discussed. This review focused on cytokines involved in the allergic response with an emphasis on those for which therapies are being or have been developed. Copyright © 2018 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Multiplex detection of quality indicator molecule targets in urine using programmable hairpin probes based on a simple double-T type microchip electrophoresis platform and isothermal polymerase-catalyzed target recycling.

    PubMed

    Zhou, Lingying; Gan, Ning; Wu, Yongxiang; Hu, Futao; Lin, Jianyuan; Cao, Yuting; Wu, Dazhen

    2018-05-29

    Recently, it has been crucial to be able to detect and quantify small molecular targets simultaneously in biological samples. Herein, a simple and conventional double-T type microchip electrophoresis (MCE) based platform for the multiplex detection of quality indicator molecule targets in urine, using ampicillin (AMPI), adenosine triphosphate (ATP) and estradiol (E2) as models, was developed. Several programmable hairpin probes (PHPs) were designed for detecting different targets and triggering isothermal polymerase-catalyzed target recycling (IPCTR) for signal amplification. Based on the target-responsive aptamer structure of PHP (Domain I), target recognition can induce PHP conformational transition and produce extension duplex DNA (dsDNA), assisted by primers & Bst polymerase. Afterwards, the target can be displaced to react with another PHP and initiate the next cycle. After several rounds of reaction, the dsDNA can be produced in large amounts by IPCTR. Three targets can be simultaneously converted to dsDNA fragments with different lengths, which can be separated and detected using MCE. Thus, a simple double-T type MCE based platform was successfully built for the homogeneous detection of multiplex targets in one channel. Under optimal conditions, the assay exhibited high throughput (48 samples per hour at most, not including reaction time) and sensitivity to three targets in urine with a detection limit of 1 nM (ATP), 0.05 nM (AMPI) and 0.1 nM (E2) respectively. The multiplex assay was successfully employed for the above three targets in several urine samples and combined the advantages of the high specificity of programmable hairpin probes, the excellent signal amplification of IPCTR, and the high through-put of MCE which can be employed for screening in biochemical analysis.

  7. A 20-Amino Acid Module of Protein Kinase Cϵ Involved in Translocation and Selective Targeting at Cell-Cell Contacts*

    PubMed Central

    Diouf, Barthélémy; Collazos, Alejandra; Labesse, Gilles; Macari, Françoise; Choquet, Armelle; Clair, Philippe; Gauthier-Rouvière, Cécile; Guérineau, Nathalie C.; Jay, Philippe; Hollande, Frédéric; Joubert, Dominique

    2009-01-01

    In the pituitary gland, activated protein kinase C (PKC) isoforms accumulate either selectively at the cell-cell contact (α and ϵ) or at the entire plasma membrane (β1 and δ). The molecular mechanisms underlying these various subcellular locations are not known. Here, we demonstrate the existence within PKCϵ of a cell-cell contact targeting sequence (3CTS) that, upon stimulation, is capable of targeting PKCδ, chimerin-α1, and the PKCϵ C1 domain to the cell-cell contact. We show that this selective targeting of PKCϵ is lost upon overexpression of 3CTS fused to a (R-Ahx-R)4 (where Ahx is 6-aminohexanoic acid) vectorization peptide, reflecting a dominant-negative effect of the overexpressed 3CTS on targeting selectivity. 3CTS contains a putative amphipathic α-helix, a 14-3-3-binding site, and the Glu-374 amino acid, involved in targeting selectivity. We show that the integrity of the α-helix is important for translocation but that 14-3-3 is not involved in targeting selectivity. However, PKCϵ translocation is increased when PKCϵ/14-3-3 interaction is abolished, suggesting that phorbol 12-myristate 13-acetate activation may initiate two sets of PKCϵ functions, those depending on 14-3-3 and those depending on translocation to cell-cell contacts. Thus, 3CTS is involved in the modulation of translocation via its 14-3-3-binding site, in cytoplasmic desequestration via the α-helix, and in selective PKCϵ targeting at the cell-cell contact via Glu-374. PMID:19429675

  8. Use of mRNA expression signatures to discover small molecule inhibitors of skeletal muscle atrophy

    PubMed Central

    Adams, Christopher M.; Ebert, Scott M.; Dyle, Michael C.

    2017-01-01

    Purpose of review Here, we discuss a recently developed experimental strategy for discovering small molecules with potential to prevent and treat skeletal muscle atrophy. Recent findings Muscle atrophy involves and requires widespread changes in skeletal muscle gene expression, which generate complex but measurable patterns of positive and negative changes in skeletal muscle mRNA levels (a.k.a. mRNA expression signatures of muscle atrophy). Many bioactive small molecules generate their own characteristic mRNA expression signatures, and by identifying small molecules whose signatures approximate mirror images of muscle atrophy signatures, one may identify small molecules with potential to prevent and/or reverse muscle atrophy. Unlike a conventional drug discovery approach, this strategy does not rely on a predefined molecular target but rather exploits the complexity of muscle atrophy to identify small molecules that counter the entire spectrum of pathological changes in atrophic muscle. We discuss how this strategy has been used to identify two natural compounds, ursolic acid and tomatidine, that reduce muscle atrophy and improve skeletal muscle function. Summary Discovery strategies based on mRNA expression signatures can elucidate new approaches for preserving and restoring muscle mass and function. PMID:25807353

  9. Use of mRNA expression signatures to discover small molecule inhibitors of skeletal muscle atrophy.

    PubMed

    Adams, Christopher M; Ebert, Scott M; Dyle, Michael C

    2015-05-01

    Here, we discuss a recently developed experimental strategy for discovering small molecules with potential to prevent and treat skeletal muscle atrophy. Muscle atrophy involves and requires widespread changes in skeletal muscle gene expression, which generate complex but measurable patterns of positive and negative changes in skeletal muscle mRNA levels (a.k.a. mRNA expression signatures of muscle atrophy). Many bioactive small molecules generate their own characteristic mRNA expression signatures, and by identifying small molecules whose signatures approximate mirror images of muscle atrophy signatures, one may identify small molecules with potential to prevent and/or reverse muscle atrophy. Unlike a conventional drug discovery approach, this strategy does not rely on a predefined molecular target but rather exploits the complexity of muscle atrophy to identify small molecules that counter the entire spectrum of pathological changes in atrophic muscle. We discuss how this strategy has been used to identify two natural compounds, ursolic acid and tomatidine, that reduce muscle atrophy and improve skeletal muscle function. Discovery strategies based on mRNA expression signatures can elucidate new approaches for preserving and restoring muscle mass and function.

  10. Three H₂O₂ molecules are involved in the "Fenton-like" reaction between Co(H₂O)₆²⁺ and H₂O₂.

    PubMed

    Burg, Ariela; Shusterman, Inna; Kornweitz, Haya; Meyerstein, Dan

    2014-06-28

    Co(II) complexes and Co(H2O)6(2+) are used as catalysts in advanced oxidation processes. Therefore it was decided to study the kinetics of reaction of Co(H2O)6(2+) with H2O2. Surprisingly, the kinetic results point out that the process involves three consecutive reactions, each of them requiring an H2O2 molecule, i.e. three H2O2 molecules ligate to the central cobalt cation prior to the formation of radicals. DFT analysis suggests that the transient (H2O)3Co(II)(OOH)2(H2O2) decomposes via: (H2O)3Co(II)(OOH)2(H2O2) → (H2O)3Co(II)(OOH)(˙OOH)(OH) + ˙OH ΔG(0) = -5.975 kcal mol(-1), with no evidence for the formation of a Co(III) transient. It is proposed that analogous mechanisms are involved whenever the redox potential of the central cation is too high to enable the reaction: M(H2O)6(n+) + H2O2 → M((n+1)+)aq + ˙OH + OH(-).

  11. Delivery of acid sphingomyelinase in normal and niemann-pick disease mice using intercellular adhesion molecule-1-targeted polymer nanocarriers.

    PubMed

    Garnacho, Carmen; Dhami, Rajwinder; Simone, Eric; Dziubla, Thomas; Leferovich, John; Schuchman, Edward H; Muzykantov, Vladimir; Muro, Silvia

    2008-05-01

    Type B Niemann-Pick disease (NPD) is a multiorgan system disorder caused by a genetic deficiency of acid sphingomyelinase (ASM), for which lung is an important and challenging therapeutic target. In this study, we designed and evaluated new delivery vehicles for enzyme replacement therapy of type B NPD, consisting of polystyrene and poly(lactic-coglycolic) acid polymer nanocarriers targeted to intercellular adhesion molecule (ICAM)-1, an endothelial surface protein up-regulated in many pathologies, including type B NPD. Real-time vascular imaging using intravital microscopy and postmortem imaging of mouse organs showed rapid, uniform, and efficient binding of fluorescently labeled ICAM-1-targeted ASM nanocarriers (anti-ICAM/ASM nanocarriers) to endothelium after i.v. injection in mice. Fluorescence microscopy of lung alveoli actin, tissue histology, and 125I-albumin blood-to-lung transport showed that anti-ICAM nanocarriers cause neither detectable lung injury, nor abnormal vascular permeability in animals. Radioisotope tracing showed rapid disappearance from the circulation and enhanced accumulation of anti-ICAM/125I-ASM nanocarriers over the nontargeted naked enzyme in kidney, heart, liver, spleen, and primarily lung, both in wild-type and ASM knockout mice. These data demonstrate that ICAM-1-targeted nanocarriers may enhance enzyme replacement therapy for type B NPD and perhaps other lysosomal storage disorders.

  12. Targeted protein degradation by PROTACs.

    PubMed

    Neklesa, Taavi K; Winkler, James D; Crews, Craig M

    2017-06-01

    Targeted protein degradation using the PROTAC technology is emerging as a novel therapeutic method to address diseases driven by the aberrant expression of a disease-causing protein. PROTAC molecules are bifunctional small molecules that simultaneously bind a target protein and an E3-ubiquitin ligase, thus causing ubiquitination and degradation of the target protein by the proteasome. Like small molecules, PROTAC molecules possess good tissue distribution and the ability to target intracellular proteins. Herein, we highlight the advantages of protein degradation using PROTACs, and provide specific examples where degradation offers therapeutic benefit over classical enzyme inhibition. Foremost, PROTACs can degrade proteins regardless of their function. This includes the currently "undruggable" proteome, which comprises approximately 85% of all human proteins. Other beneficial aspects of protein degradation include the ability to target overexpressed and mutated proteins, as well as the potential to demonstrate prolonged pharmacodynamics effect beyond drug exposure. Lastly, due to their catalytic nature and the pre-requisite ubiquitination step, an exquisitely potent molecules with a high degree of degradation selectivity can be designed. Impressive preclinical in vitro and in vivo PROTAC data have been published, and these data have propelled the development of clinically viable PROTACs. With the molecular weight falling in the 700-1000Da range, the delivery and bioavailability of PROTACs remain the largest hurdles on the way to the clinic. Solving these issues and demonstrating proof of concept clinical data will be the focus of many labs over the next few years. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Advancing Biological Understanding and Therapeutics Discovery with Small Molecule Probes

    PubMed Central

    Schreiber, Stuart L.; Kotz, Joanne D.; Li, Min; Aubé, Jeffrey; Austin, Christopher P.; Reed, John C.; Rosen, Hugh; White, E. Lucile; Sklar, Larry A.; Lindsley, Craig W.; Alexander, Benjamin R.; Bittker, Joshua A.; Clemons, Paul A.; de Souza, Andrea; Foley, Michael A.; Palmer, Michelle; Shamji, Alykhan F.; Wawer, Mathias J.; McManus, Owen; Wu, Meng; Zou, Beiyan; Yu, Haibo; Golden, Jennifer E.; Schoenen, Frank J.; Simeonov, Anton; Jadhav, Ajit; Jackson, Michael R.; Pinkerton, Anthony B.; Chung, Thomas D.Y.; Griffin, Patrick R.; Cravatt, Benjamin F.; Hodder, Peter S.; Roush, William R.; Roberts, Edward; Chung, Dong-Hoon; Jonsson, Colleen B.; Noah, James W.; Severson, William E.; Ananthan, Subramaniam; Edwards, Bruce; Oprea, Tudor I.; Conn, P. Jeffrey; Hopkins, Corey R.; Wood, Michael R.; Stauffer, Shaun R.; Emmitte, Kyle A.

    2015-01-01

    Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the U.S. National Institutes of Health launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines, but also highlight the need to innovate the science of therapeutic discovery. PMID:26046436

  14. Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples.

    PubMed

    Cui, Liang; Zou, Yuan; Lin, Ninghang; Zhu, Zhi; Jenkins, Gareth; Yang, Chaoyong James

    2012-07-03

    Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective

  15. Surface enhanced single-molecule magnetism involving 4f spin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Yachao, E-mail: yachao.zhang@pku.edu.cn

    2016-03-28

    We study the magnetic anisotropy energy (MAE) of the isolated and deposited Eu(C{sub 8}H{sub 8}){sub 2} by first-principles calculations considering the van der Waals correction and the strong correlation effects. We find that both the molecular spin moment and the easy-axis magnetic anisotropy are enhanced upon deposition on Cu(111). We propose a mechanism in terms of the weakened spin polarization of the π-2p orbitals and the induced anisotropic occupations of the 4f orbitals. Our findings pave the way for raising the MAE of 4f-element single-molecule magnets by tailoring the molecule–surface contacts.

  16. Exploring Polypharmacology Using a ROCS-Based Target Fishing Approach

    DTIC Science & Technology

    2012-01-01

    target representatives. Target profiles were then generated for a given query molecule by computing maximal shape/ chemistry overlap between the query...molecule and the drug sets assigned to each protein target. The overlap was computed using the program ROCS (Rapid Overlay of Chemical Structures ). We...approaches in off-target prediction has been reviewed.9,10 Many structure -based target fishing (SBTF) approaches, such as INVDOCK11 and Target Fishing Dock

  17. Analyzing Single-Molecule Protein Transportation Experiments via Hierarchical Hidden Markov Models

    PubMed Central

    Chen, Yang; Shen, Kuang

    2017-01-01

    To maintain proper cellular functions, over 50% of proteins encoded in the genome need to be transported to cellular membranes. The molecular mechanism behind such a process, often referred to as protein targeting, is not well understood. Single-molecule experiments are designed to unveil the detailed mechanisms and reveal the functions of different molecular machineries involved in the process. The experimental data consist of hundreds of stochastic time traces from the fluorescence recordings of the experimental system. We introduce a Bayesian hierarchical model on top of hidden Markov models (HMMs) to analyze these data and use the statistical results to answer the biological questions. In addition to resolving the biological puzzles and delineating the regulating roles of different molecular complexes, our statistical results enable us to propose a more detailed mechanism for the late stages of the protein targeting process. PMID:28943680

  18. Single-Molecule Interfacial Electron Transfer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lu, H. Peter

    This project is focused on the use of single-molecule high spatial and temporal resolved techniques to study molecular dynamics in condensed phase and at interfaces, especially, the complex reaction dynamics associated with electron and energy transfer rate processes. The complexity and inhomogeneity of the interfacial ET dynamics often present a major challenge for a molecular level comprehension of the intrinsically complex systems, which calls for both higher spatial and temporal resolutions at ultimate single-molecule and single-particle sensitivities. Combined single-molecule spectroscopy and electrochemical atomic force microscopy approaches are unique for heterogeneous and complex interfacial electron transfer systems because the static andmore » dynamic inhomogeneities can be identified and characterized by studying one molecule at a specific nanoscale surface site at a time. The goal of our project is to integrate and apply these spectroscopic imaging and topographic scanning techniques to measure the energy flow and electron flow between molecules and substrate surfaces as a function of surface site geometry and molecular structure. We have been primarily focusing on studying interfacial electron transfer under ambient condition and electrolyte solution involving both single crystal and colloidal TiO 2 and related substrates. The resulting molecular level understanding of the fundamental interfacial electron transfer processes will be important for developing efficient light harvesting systems and broadly applicable to problems in fundamental chemistry and physics. We have made significant advancement on deciphering the underlying mechanism of the complex and inhomogeneous interfacial electron transfer dynamics in dyesensitized TiO 2 nanoparticle systems that strongly involves with and regulated by molecule-surface interactions. We have studied interfacial electron transfer on TiO 2 nanoparticle surfaces by using ultrafast single-molecule spectroscopy

  19. Biased and unbiased strategies to identify biologically active small molecules.

    PubMed

    Abet, Valentina; Mariani, Angelica; Truscott, Fiona R; Britton, Sébastien; Rodriguez, Raphaël

    2014-08-15

    Small molecules are central players in chemical biology studies. They promote the perturbation of cellular processes underlying diseases and enable the identification of biological targets that can be validated for therapeutic intervention. Small molecules have been shown to accurately tune a single function of pluripotent proteins in a reversible manner with exceptional temporal resolution. The identification of molecular probes and drugs remains a worthy challenge that can be addressed by the use of biased and unbiased strategies. Hypothesis-driven methodologies employs a known biological target to synthesize complementary hits while discovery-driven strategies offer the additional means of identifying previously unanticipated biological targets. This review article provides a general overview of recent synthetic frameworks that gave rise to an impressive arsenal of biologically active small molecules with unprecedented cellular mechanisms. Copyright © 2014. Published by Elsevier Ltd.

  20. Chemical Structural Novelty: On-Targets and Off-Targets

    PubMed Central

    Yera, Emmanuel R.; Cleves, Ann. E.; Jain, Ajay N.

    2011-01-01

    Drug structures may be quantitatively compared based on 2D topological structural considerations and based on 3D characteristics directly related to binding. A framework for combining multiple similarity computations is presented along with its systematic application to 358 drugs with overlapping pharmacology. Given a new molecule along with a set of molecules sharing some biological effect, a single score based on comparison to the known set is produced, reflecting either 2D similarity, 3D similarity, or their combination. For prediction of primary targets, the benefit of 3D over 2D was relatively small, but for prediction of off-targets, the added benefit was large. In addition to assessing prediction, the relationship between chemical similarity and pharmacological novelty was studied. Drug pairs that shared high 3D similarity but low 2D similarity (i.e. a novel scaffold) were shown to be much more likely to exhibit pharmacologically relevant differences in terms of specific protein target modulation. PMID:21916467

  1. Docking into Mycobacterium tuberculosis Thioredoxin Reductase Protein Yields Pyrazolone Lead Molecules for Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Sweeney, Noreena L.; Lipker, Lauren; Hanson, Alicia M.; Bohl, Chris J.; Engel, Katie E.; Kalous, Kelsey S.; Stemper, Mary E.; Sem, Daniel S.; Schwan, William R.

    2017-01-01

    The thioredoxin/thioredoxin reductase system (Trx/TrxR) is an attractive drug target because of its involvement in a number of important physiological processes, from DNA synthesis to regulating signal transduction. This study describes the finding of pyrazolone compounds that are active against Staphylococcus aureus. Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the Mycobacterium tuberculosis thioredoxin reductase. This led to the discovery of a pyrazolone scaffold-containing compound series that showed bactericidal capability against S. aureus strains, including drug-resistant clinical isolates. The findings support continued development of the pyrazolone compounds as potential anti-S. aureus antibiotics. PMID:28134858

  2. miR-4725-3p targeting Stim1 signaling is involved in xanthohumol inhibition of glioma cell invasion.

    PubMed

    Ho, Kuo-Hao; Chang, Cheng-Kuei; Chen, Peng-Hsu; Wang, Yu-Jia; Chang, Wei-Chiao; Chen, Ku-Chung

    2018-05-10

    Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Due to its highly invasive nature, it is not easy to treat, resulting in high mortality rates. Stromal interacting molecule 1 (Stim1) plays important roles in regulating store-operated Ca 2+ entry (SOCE), and controls invasion by cancer cells. However, the mechanisms and functions of Stim1 in glioma progression are still unclear. In this study, we investigated the effects of targeting Stim1 expression on glioma cell invasion. By analyzing profiles of GBM patients from RNA-sequencing data in The Cancer Genome Atlas (TCGA), higher expression levels of STIM1 were correlated with the poor survival. Furthermore, signaling pathways associated with tumor malignancy, including the epithelial-to-mesenchymal transition (EMT), were activated in patients with high STIM1 expression according to gene set enrichment analyses. Higher Stim1 levels were found in glioma cells compared to human astrocytes, and these higher levels enhanced glioma cell invasion. Xanthohumol (XN), a prenylated flavonoid extracted from the hop plant Humulus lupulus L. (Cannabaceae), significantly reduced cell invasion through inhibiting Stim1 expression. From an micro(mi)RNA array analysis, miR-4725-3p was upregulated by XN treatment. Overexpression of miR-4725-3p inhibited glioma cell invasion via directly targeting the 3'-untranslated region of STIM1. The extracellular signal-regulated kinase/c-Fos pathway was also validated to participate in XN-upregulated miR-4725-3p expression according to promoter and chromatin immunoprecipitation assays. These results emphasize that miR-4725-3p-inhibited STIM1 signaling is involved in XN-attenuated glioma cell invasion. These findings may provide insights into novel therapeutic strategies for future glioblastoma therapy and drug development. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. Functionalization of Probe Tips and Supports for Single-Molecule Recognition Force Microscopy

    NASA Astrophysics Data System (ADS)

    Ebner, Andreas; Wildling, Linda; Zhu, Rong; Rankl, Christian; Haselgrübler, Thomas; Hinterdorfer, Peter; Gruber, Hermann J.

    The measuring tip of a force microscope can be converted into a monomolecular sensor if one or few "ligand" molecules are attached to the apex of the tip while maintaining ligand function. Functionalized tips are used to study fine details of receptor-ligand interaction by force spectroscopy or to map cognate "receptor" molecules on the sample surface. The receptor (or target) molecules can be present on the surface of a biological specimen; alternatively, soluble target molecules must be immobilized on ultraflat supports. This review describes the methods of tip functionalization, as well as target molecule immobilization. Silicon nitride tips, silicon chips, and mica have usually been functionalized in three steps: (1) aminofunctionalization, (2) crosslinker attachment, and (3) ligand/receptor coupling, whereby numerous crosslinkers are available to couple widely different ligand molecules. Gold-covered tips and/or supports have usually been coated with a self-assembled monolayer, on top of which the ligand/receptor molecule has been coupled either directly or via a crosslinker molecule. Apart from these general strategies, many simplified methods have been used for tip and/or support functionalization, even single-step methods such as adsorption or chemisorption being very efficient under suitable circumstances. All methods are described with the same explicitness and critical parameters are discussed. In conclusion, this review should help to find suitable methods for specific problems of tip and support functionalization.

  4. Small Molecule Membrane Transporters in the Mammalian Podocyte: A Pathogenic and Therapeutic Target

    PubMed Central

    Zennaro, Cristina; Artero, Mary; Di Maso, Vittorio; Carraro, Michele

    2014-01-01

    The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development. PMID:25411800

  5. Small molecules targeting LapB protein prevent Listeria attachment to catfish muscle

    PubMed Central

    Das, Bhaskar; Lawrence, Mark

    2017-01-01

    Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listeriosis. L. monocytogenes lapB gene encodes a cell wall surface anchor protein, and mutation of this gene causes Listeria attenuation in mice. In this work, the potential role of Listeria LapB protein in catfish fillet attachment was investigated. To achieve this, boron-based small molecules designed to interfere with the active site of the L. monocytogenes LapB protein were developed, and their ability to prevent L. monocytogenes attachment to fish fillet was tested. Results indicated that seven out of nine different small molecules were effective in reducing the Listeria attachment to catfish fillets. Of these, three small molecules (SM3, SM5, and SM7) were highly effective in blocking Listeria attachment to catfish fillets. This study suggests an alternative strategy for reduction of L. monocytogenes contamination in fresh and frozen fish products. PMID:29253892

  6. Single-molecule comparison of DNA Pol I activity with native and analog nucleotides

    NASA Astrophysics Data System (ADS)

    Gul, Osman; Olsen, Tivoli; Choi, Yongki; Corso, Brad; Weiss, Gregory; Collins, Philip

    2014-03-01

    DNA polymerases are critical enzymes for DNA replication, and because of their complex catalytic cycle they are excellent targets for investigation by single-molecule experimental techniques. Recently, we studied the Klenow fragment (KF) of DNA polymerase I using a label-free, electronic technique involving single KF molecules attached to carbon nanotube transistors. The electronic technique allowed long-duration monitoring of a single KF molecule while processing thousands of template strands. Processivity of up to 42 nucleotide bases was directly observed, and statistical analysis of the recordings determined key kinetic parameters for the enzyme's open and closed conformations. Subsequently, we have used the same technique to compare the incorporation of canonical nucleotides like dATP to analogs like 1-thio-2'-dATP. The analog had almost no affect on duration of the closed conformation, during which the nucleotide is incorporated. On the other hand, the analog increased the rate-limiting duration of the open conformation by almost 40%. We propose that the thiolated analog interferes with KF's recognition and binding, two key steps that determine its ensemble turnover rate.

  7. Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules

    DOE PAGES

    Lee, Somin Eunice; Chen, Qian; Bhat, Ramray; ...

    2015-06-03

    Plasmon rulers, consisting of pairs of gold nanoparticles, allow single-molecule analysis without photobleaching or blinking; however, current plasmon rulers are irreversible, restricting detection to only single events. Here, we present a reversible plasmon ruler, comprised of coupled gold nanoparticles linked by a single aptamer, capable of binding individual secreted molecules with high specificity. We show that the binding of target secreted molecules to the reversible plasmon ruler is characterized by single-molecule sensitivity, high specificity, and reversibility. Lastly, such reversible plasmon rulers should enable dynamic and adaptive live-cell measurement of secreted single molecules in their local microenvironment.

  8. Chloride channels as drug targets

    PubMed Central

    Verkman, Alan S.; Galietta, Luis J. V.

    2013-01-01

    Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABAA (γ-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery. PMID:19153558

  9. Nano-fabrication of molecular electronic junctions by targeted modification of metal-molecule bonds

    NASA Astrophysics Data System (ADS)

    Jafri, S. Hassan M.; Löfås, Henrik; Blom, Tobias; Wallner, Andreas; Grigoriev, Anton; Ahuja, Rajeev; Ottosson, Henrik; Leifer, Klaus

    2015-09-01

    Reproducibility, stability and the coupling between electrical and molecular properties are central challenges in the field of molecular electronics. The field not only needs devices that fulfill these criteria but they also need to be up-scalable to application size. In this work, few-molecule based electronics devices with reproducible electrical characteristics are demonstrated. Our previously reported 5 nm gold nanoparticles (AuNP) coated with ω-triphenylmethyl (trityl) protected 1,8-octanedithiol molecules are trapped in between sub-20 nm gap spacing gold nanoelectrodes forming AuNP-molecule network. When the trityl groups are removed, reproducible devices and stable Au-thiol junctions are established on both ends of the alkane segment. The resistance of more than 50 devices is reduced by orders of magnitude as well as a reduction of the spread in the resistance histogram is observed. By density functional theory calculations the orders of magnitude decrease in resistance can be explained and supported by TEM observations thus indicating that the resistance changes and strongly improved resistance spread are related to the establishment of reproducible and stable metal-molecule bonds. The same experimental sequence is carried out using 1,6-hexanedithiol functionalized AuNPs. The average resistances as a function of molecular length, demonstrated herein, are comparable to the one found in single molecule devices.

  10. Nano-fabrication of molecular electronic junctions by targeted modification of metal-molecule bonds

    PubMed Central

    Jafri, S. Hassan M.; Löfås, Henrik; Blom, Tobias; Wallner, Andreas; Grigoriev, Anton; Ahuja, Rajeev; Ottosson, Henrik; Leifer, Klaus

    2015-01-01

    Reproducibility, stability and the coupling between electrical and molecular properties are central challenges in the field of molecular electronics. The field not only needs devices that fulfill these criteria but they also need to be up-scalable to application size. In this work, few-molecule based electronics devices with reproducible electrical characteristics are demonstrated. Our previously reported 5 nm gold nanoparticles (AuNP) coated with ω-triphenylmethyl (trityl) protected 1,8-octanedithiol molecules are trapped in between sub-20 nm gap spacing gold nanoelectrodes forming AuNP-molecule network. When the trityl groups are removed, reproducible devices and stable Au-thiol junctions are established on both ends of the alkane segment. The resistance of more than 50 devices is reduced by orders of magnitude as well as a reduction of the spread in the resistance histogram is observed. By density functional theory calculations the orders of magnitude decrease in resistance can be explained and supported by TEM observations thus indicating that the resistance changes and strongly improved resistance spread are related to the establishment of reproducible and stable metal-molecule bonds. The same experimental sequence is carried out using 1,6-hexanedithiol functionalized AuNPs. The average resistances as a function of molecular length, demonstrated herein, are comparable to the one found in single molecule devices. PMID:26395225

  11. Proteoform-specific protein binding of small molecules in complex matrices

    USDA-ARS?s Scientific Manuscript database

    Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original ...

  12. Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

    PubMed Central

    Sloan, Derek D.; Lam, Chia-Ying Kao; Irrinki, Alivelu; Liu, Liqin; Tsai, Angela; Pace, Craig S.; Kaur, Jasmine; Murry, Jeffrey P.; Balakrishnan, Mini; Moore, Paul A.; Johnson, Syd; Nordstrom, Jeffrey L.; Cihlar, Tomas; Koenig, Scott

    2015-01-01

    HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. PMID:26539983

  13. Challenges for single molecule electronic devices with nanographene and organic molecules. Do single molecules offer potential as elements of electronic devices in the next generation?

    NASA Astrophysics Data System (ADS)

    Enoki, Toshiaki; Kiguchi, Manabu

    2018-03-01

    Interest in utilizing organic molecules to fabricate electronic materials has existed ever since organic (molecular) semiconductors were first discovered in the 1950s. Since then, scientists have devoted serious effort to the creation of various molecule-based electronic systems, such as molecular metals and molecular superconductors. Single-molecule electronics and the associated basic science have emerged over the past two decades and provided hope for the development of highly integrated molecule-based electronic devices in the future (after the Si-based technology era has ended). Here, nanographenes (nano-sized graphene) with atomically precise structures are among the most promising molecules that can be utilized for electronic/spintronic devices. To manipulate single small molecules for an electronic device, a single molecular junction has been developed. It is a powerful tool that allows even small molecules to be utilized. External electric, magnetic, chemical, and mechanical perturbations can change the physical and chemical properties of molecules in a way that is different from bulk materials. Therefore, the various functionalities of molecules, along with changes induced by external perturbations, allows us to create electronic devices that we cannot create using current top-down Si-based technology. Future challenges that involve the incorporation of condensed matter physics, quantum chemistry calculations, organic synthetic chemistry, and electronic device engineering are expected to open a new era in single-molecule device electronic technology.

  14. Quenching methods for background reduction in luminescence-based probe-target binding assays

    DOEpatents

    Cai, Hong [Los Alamos, NM; Goodwin, Peter M [Los Alamos, NM; Keller, Richard A [Los Alamos, NM; Nolan, Rhiannon L [Santa Fe, NM

    2007-04-10

    Background luminescence is reduced from a solution containing unbound luminescent probes, each having a first molecule that attaches to a target molecule and having an attached luminescent moiety, and luminescent probe/target adducts. Quenching capture reagent molecules are formed that are capable of forming an adduct with the unbound luminescent probes and having an attached quencher material effective to quench luminescence of the luminescent moiety. The quencher material of the capture reagent molecules is added to a solution of the luminescent probe/target adducts and binds in a proximity to the luminescent moiety of the unbound luminescent probes to quench luminescence from the luminescent moiety when the luminescent moiety is exposed to exciting illumination. The quencher capture reagent does not bind to probe molecules that are bound to target molecules and the probe/target adduct emission is not quenched.

  15. Electron Impact Excitation-Ionization of Molecules

    NASA Astrophysics Data System (ADS)

    Ali, Esam Abobakr A.

    In the last few decades, the study of atomic collisions by electron-impact has made significant advances. The most difficult case to study is electron impact ionization of molecules for which many approximations have to be made and the validity of these approximations can only be checked by comparing with experiment. In this thesis, I have examined the Molecular three-body distorted wave (M3DW) or Molecular four-body distorted wave (M4DW) approximations for electron-impact ionization. These models use a fully quantum mechanical approach where all particles are treated quantum mechanically and the post collision interaction (PCI) is treated to all orders of perturbation. These electron impact ionization collisions play central roles in the physics and chemistry of upper atmosphere, biofuel, the operation of discharges and lasers, radiation induced damage in biological material like damage to DNA by secondary electrons, and plasma etching processes. For the M3DW model, I will present results for electron impact single ionization of small molecules such as Water, Ethane, and Carbon Dioxide and the much larger molecules Tetrahydrofuran, phenol, furfural, 1-4 Benzoquinone. I will also present results for the four-body problem in which there are two target electrons involved in the collision. M4DW results will be presented for dissociative excitation-ionization of orientated D2. I will show that M4DW calculations using a variational wave function for the ground state that included s- and p- orbital states give better agreement to the experimental measurements than a ground state approximated as a product of two 1s-type Dyson orbitals.

  16. Hierarchical virtual screening approaches in small molecule drug discovery.

    PubMed

    Kumar, Ashutosh; Zhang, Kam Y J

    2015-01-01

    Virtual screening has played a significant role in the discovery of small molecule inhibitors of therapeutic targets in last two decades. Various ligand and structure-based virtual screening approaches are employed to identify small molecule ligands for proteins of interest. These approaches are often combined in either hierarchical or parallel manner to take advantage of the strength and avoid the limitations associated with individual methods. Hierarchical combination of ligand and structure-based virtual screening approaches has received noteworthy success in numerous drug discovery campaigns. In hierarchical virtual screening, several filters using ligand and structure-based approaches are sequentially applied to reduce a large screening library to a number small enough for experimental testing. In this review, we focus on different hierarchical virtual screening strategies and their application in the discovery of small molecule modulators of important drug targets. Several virtual screening studies are discussed to demonstrate the successful application of hierarchical virtual screening in small molecule drug discovery. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide

    PubMed Central

    Morizono, Kouki; Xie, Yiming; Helguera, Gustavo; Daniels, Tracy R.; Lane, Timothy F.; Penichet, Manuel L.; Chen, Irvin S. Y.

    2010-01-01

    Background Targeted gene transduction in vivo is the ultimate preferred method for gene delivery. We previously developed targeting lentiviral vectors that specifically recognize cell surface molecules with conjugated antibodies and mediate targeted gene transduction both in vitro and in vivo. Although effective in some experimental settings, the conjugation of virus with antibodies is mediated by the interaction between protein A and the Fc region of antibodies, which is not as stable as covalent conjugation. We have now developed a more stable conjugation strategy utilizing the interaction between avidin and biotin. Methods We inserted the biotin-adaptor-peptide, which was biotinylated by secretory biotin ligase at specific sites, into our targeting envelope proteins, enabling conjugation of the pseudotyped virus with avidin, streptavidin or neutravidin. Results When conjugated with avidin-antibody fusion proteins or the complex of avidin and biotinylated targeting molecules, the vectors could mediate specific transduction to targeted cells recognized by the targeting molecules. When conjugated with streptavidin-coated magnetic beads, transduction by the vectors was targeted to the locations of magnets. Conclusions This targeting vector system can be used for broad applications of targeted gene transduction using biotinylated targeting molecules or targeting molecules fused with avidin. PMID:19455593

  18. Precise small molecule recognition of a toxic CUG RNA repeat expansion

    PubMed Central

    Rzuczek, Suzanne G; Colgan, Lesley A; Nakai, Yoshio; Cameron, Michael D; Furling, Denis; Yasuda, Ryohei; Disney, Matthew D

    2017-01-01

    Excluding the ribosome and riboswitches, developing small molecules that selectively target RNA is a longstanding problem in chemical biology. A typical cellular RNA is difficult to target because it has little tertiary, but abundant secondary structure. We designed allele-selective compounds that target such an RNA, the toxic noncoding repeat expansion (r(CUG)exp) that causes myotonic dystrophy type 1 (DM1). We developed several strategies to generate allele-selective small molecules, including non-covalent binding, covalent binding, cleavage and on-site probe synthesis. Covalent binding and cleavage enabled target profiling in cells derived from individuals with DM1, showing precise recognition of r(CUG)exp. In the on-site probe synthesis approach, small molecules bound adjacent sites in r(CUG)exp and reacted to afford picomolar inhibitors via a proximity-based click reaction only in DM1-affected cells. We expanded this approach to image r(CUG)exp in its natural context. PMID:27941760

  19. Precise small-molecule recognition of a toxic CUG RNA repeat expansion.

    PubMed

    Rzuczek, Suzanne G; Colgan, Lesley A; Nakai, Yoshio; Cameron, Michael D; Furling, Denis; Yasuda, Ryohei; Disney, Matthew D

    2017-02-01

    Excluding the ribosome and riboswitches, developing small molecules that selectively target RNA is a longstanding problem in chemical biology. A typical cellular RNA is difficult to target because it has little tertiary, but abundant secondary structure. We designed allele-selective compounds that target such an RNA, the toxic noncoding repeat expansion (r(CUG) exp ) that causes myotonic dystrophy type 1 (DM1). We developed several strategies to generate allele-selective small molecules, including non-covalent binding, covalent binding, cleavage and on-site probe synthesis. Covalent binding and cleavage enabled target profiling in cells derived from individuals with DM1, showing precise recognition of r(CUG) exp . In the on-site probe synthesis approach, small molecules bound adjacent sites in r(CUG) exp and reacted to afford picomolar inhibitors via a proximity-based click reaction only in DM1-affected cells. We expanded this approach to image r(CUG) exp in its natural context.

  20. Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer

    PubMed Central

    O'Grady, Michael; Raha, Debasish; Hanson, Bonnie J; Bunting, Michaeline; Hanson, George T

    2005-01-01

    Background The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. Methods AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. Results Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a β-lactamase reporter gene, a 10–12 fold shift or 2.5–3 fold shift toward greater potency in the IC50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. Conclusion EGFR

  1. Immunotherapy Targets in Pediatric Cancer

    PubMed Central

    Orentas, Rimas J.; Lee, Daniel W.; Mackall, Crystal

    2011-01-01

    Immunotherapy for cancer has shown increasing success and there is ample evidence to expect that progress gleaned in immune targeting of adult cancers can be translated to pediatric oncology. This manuscript reviews principles that guide selection of targets for immunotherapy of cancer, emphasizing the similarities and distinctions between oncogene-inhibition targets and immune targets. It follows with a detailed review of molecules expressed by pediatric tumors that are already under study as immune targets or are good candidates for future studies of immune targeting. Distinctions are made between cell surface antigens that can be targeted in an MHC independent manner using antibodies, antibody derivatives, or chimeric antigen receptors versus intracellular antigens which must be targeted with MHC restricted T cell therapies. Among the most advanced immune targets for childhood cancer are CD19 and CD22 on hematologic malignancies, GD2 on solid tumors, and NY-ESO-1 expressed by a majority of synovial sarcomas, but several other molecules reviewed here also have properties which suggest that they too could serve as effective targets for immunotherapy of childhood cancer. PMID:22645714

  2. On the Several Molecules and Nanostructures of Water

    PubMed Central

    Whitney, Cynthia Kolb

    2012-01-01

    This paper investigates the water molecule from a variety of viewpoints. Water can involve different isotopes of Hydrogen and Oxygen, it can form differently shaped isomer molecules, and, when frozen, it occupies space differently than most other substances do. The tool for conducting the investigation of all this is called ‘Algebraic Chemistry’. This tool is a quantitative model for predicting the energy budget for all sorts of changes between different ionization states of atoms that are involved in chemical reactions and in changes of physical state. The model is based on consistent patterns seen in empirical data about ionization potentials, together with rational scaling laws that can interpolate and extrapolate for situations where no data are available. The results of the investigation of the water molecule include comments, both positive and negative, about technologies involving heavy water, poly water, Brown’s gas, and cold fusion. PMID:22312305

  3. On the several molecules and nanostructures of water.

    PubMed

    Whitney, Cynthia Kolb

    2012-01-01

    This paper investigates the water molecule from a variety of viewpoints. Water can involve different isotopes of Hydrogen and Oxygen, it can form differently shaped isomer molecules, and, when frozen, it occupies space differently than most other substances do. The tool for conducting the investigation of all this is called 'Algebraic Chemistry'. This tool is a quantitative model for predicting the energy budget for all sorts of changes between different ionization states of atoms that are involved in chemical reactions and in changes of physical state. The model is based on consistent patterns seen in empirical data about ionization potentials, together with rational scaling laws that can interpolate and extrapolate for situations where no data are available. The results of the investigation of the water molecule include comments, both positive and negative, about technologies involving heavy water, poly water, Brown's gas, and cold fusion.

  4. Threading the Needle: Small-Molecule Targeting of a Xenobiotic Receptor to Ablate Escherichia coli Polysaccharide Capsule Expression Without Altering Antibiotic Resistance.

    PubMed

    Arshad, Mehreen; Goller, Carlos C; Pilla, Danielle; Schoenen, Frank J; Seed, Patrick C

    2016-04-15

    Uropathogenic Escherichia coli (UPEC), a leading cause of urinary tract and invasive infections worldwide, is rapidly acquiring multidrug resistance, hastening the need for selective new anti-infective agents. Here we demonstrate the molecular target of DU011, our previously discovered potent, nontoxic, small-molecule inhibitor of UPEC polysaccharide capsule biogenesis and virulence. Real-time polymerase chain reaction analysis and a target-overexpression drug-suppressor screen were used to localize the putative inhibitor target. A thermal shift assay quantified interactions between the target protein and the inhibitor, and a novel DNase protection assay measured chemical inhibition of protein-DNA interactions. Virulence of a regulatory target mutant was assessed in a murine sepsis model. MprA, a MarR family transcriptional repressor, was identified as the putative target of the DU011 inhibitor. Thermal shift measurements indicated the formation of a stable DU011-MprA complex, and DU011 abrogated MprA binding to its DNA promoter site. Knockout of mprA had effects similar to that of DU011 treatment of wild-type bacteria: a loss of encapsulation and complete attenuation in a murine sepsis model, without any negative change in antibiotic resistance. MprA regulates UPEC polysaccharide encapsulation, is essential for UPEC virulence, and can be targeted without inducing antibiotic resistance. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  5. [The cancer paradigm in pulmonary arterial hypertension: towards anti-remodeling therapies targeting metabolic dysfunction?

    PubMed

    Dumas, Sébastien J; Humbert, Marc; Cohen-Kaminsky, Sylvia

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a rare, complex and multifactorial disease in which pulmonary vascular remodeling plays a major role ending in right heart failure and death. Current specific therapies of PAH that mainly target the vasoconstriction/vasodilatation imbalance are not curative. Bi-pulmonary transplantation remains the only option in patients resistant to current therapies. It is thus crucial to identify novel vascular anti-remodeling therapeutic targets. This remodeling displays several properties of cancer cells, especially overproliferation and apoptosis resistance of pulmonary vascular cells, hallmarks of cancer related to the metabolic shift known as the "Warburg effect". The latter is characterized by a shift of ATP production, from oxidative phosphorylation to low rate aerobic glycolysis. In compensation, the cancer cells exhibit exacerbated glutaminolysis thus resulting in glutamine addiction, necessary to their overproliferation. Glutamine intake results in glutamate production, a molecule at the crossroads of energy metabolism and cancer cell communication, thus contributing to cell proliferation. Accordingly, therapeutic strategies targeting glutamate production, its release into the extracellular space and its membrane receptors have been suggested to treat different types of cancers, not only in the central nervous system but also in the periphery. We propose that similar strategies targeting glutamatergic signaling may be considered in PAH, especially as they could affect not only the vascular remodeling but also the right heart hypertrophy known to involve the glutaminolysis pathway. Ongoing studies aim to characterize the involvement of the glutamate pathway and its receptors in vascular remodeling, and the therapeutic potential of specific molecules targeting this pathway. © Société de Biologie, 2017.

  6. miR-133 involves in lung adenocarcinoma cell metastasis by targeting FLOT2.

    PubMed

    Wei, Guangxia; Xu, Yahuan; Peng, Tao; Yan, Jie

    2018-03-01

    Dysregulated microRNAs (miRNAs) reported to involve into the oncogenesis and progression in various human cancers. However, the roles and mechanism of miR-133 in lung adenocarcinoma remain largely unclear. In this study, qPCR assay and western blot were used to detect the expression levels of miR-133, Akt and FLOT2. Luciferase reporter assay was used to identify the target role of miR-133 on FLOT2. The cell invasion and the migration capability were performed using the transwell invasion assay and wound healing assay. We found that miR-133 expression levels were downregulated in human lung adenocarcinoma specimens and cell lines compared with the adjacent normal tissues and normal human bronchial epithelial cell. miR-133 significantly suppressed metastasis of lung adenocarcinoma cells in vitro. Furthermore, FLOT2 (flotillin-2) identified as a direct target of miR-133, and FLOT2 expression levels were inversely correlated with miR-133 expression levels in human lung adenocarcinoma specimens. And the restoration studies suggested FGF2 as a downstream effector of miR-133 which acted through Akt signalling pathway. Our study revealed the mechanism that miR-133 suppresses lung adenocarcinoma metastasis by targeting FLOT2 via Akt signalling pathway, implicating a potential prognostic biomarker and therapeutic target for lung adenocarcinoma treatment.

  7. Reviewing Hit Discovery Literature for Difficult Targets: Glutathione Transferase Omega-1 as an Example.

    PubMed

    Xie, Yiyue; Dahlin, Jayme L; Oakley, Aaron J; Casarotto, Marco G; Board, Philip G; Baell, Jonathan B

    2018-05-10

    Early stage drug discovery reporting on relatively new or difficult targets is often associated with insufficient hit triage. Literature reviews of such targets seldom delve into the detail required to critically analyze the associated screening hits reported. Here we take the enzyme glutathione transferase omega-1 (GSTO1-1) as an example of a relatively difficult target and review the associated literature involving small-molecule inhibitors. As part of this process we deliberately pay closer-than-usual attention to assay interference and hit quality aspects. We believe this Perspective will be a useful guide for future development of GSTO1-1 inhibitors, as well serving as a template for future review formats of new or difficult targets.

  8. Method of detecting luminescent target ions with modified magnetic microspheres

    DOEpatents

    Shkrob, Ilya A; Kaminski, Michael D

    2014-05-13

    This invention provides methods of using modified magnetic microspheres to extract target ions from a sample in order to detect their presence in a microfluidic environment. In one or more embodiments, the microspheres are modified with molecules on the surface that allow the target ions in the sample to form complexes with specific ligand molecules on the microsphere surface. In one or more embodiments, the microspheres are modified with molecules that sequester the target ions from the sample, but specific ligand molecules in solution subsequently re-extract the target ions from the microspheres into the solution, where the complexes form independent of the microsphere surface. Once the complexes form, they are exposed to an excitation wavelength light source suitable for exciting the target ion to emit a luminescent signal pattern. Detection of the luminescent signal pattern allows for determination of the presence of the target ions in the sample.

  9. [Search for biofunctional constituents from medicinal foods-elucidation of constituents with anti-proliferation effects and the target molecule from Citrullus colocynthis].

    PubMed

    Nakamura, Seikou

    2012-01-01

    Many foods are known to have not only nutritive and taste values but also medicinal effects. In Egypt, many medicinal foods have been used for the prevention and treatment of various diseases since ancient. However, in most cases, their effective constituents as well as the mechanism of action remained uncharacterized. In the course of our characterization studies on Egyptian medicinal foods and plants, cucurbitane-type triterpene and related compounds such as cucurbitacin E from the fruit of Citrullus colocynthis and the roots of Bryonia cretica were found to show anti-proliferation effects. We therefore synthesized a biotin-linked cucurbitacin E to isolate target proteins based on affinity for the molecule. As a result, cofilin, which regulates the depolymerization of actin, was isolated and suggested to be a target.

  10. Quantitative self-assembly prediction yields targeted nanomedicines

    NASA Astrophysics Data System (ADS)

    Shamay, Yosi; Shah, Janki; Işık, Mehtap; Mizrachi, Aviram; Leibold, Josef; Tschaharganeh, Darjus F.; Roxbury, Daniel; Budhathoki-Uprety, Januka; Nawaly, Karla; Sugarman, James L.; Baut, Emily; Neiman, Michelle R.; Dacek, Megan; Ganesh, Kripa S.; Johnson, Darren C.; Sridharan, Ramya; Chu, Karen L.; Rajasekhar, Vinagolu K.; Lowe, Scott W.; Chodera, John D.; Heller, Daniel A.

    2018-02-01

    Development of targeted nanoparticle drug carriers often requires complex synthetic schemes involving both supramolecular self-assembly and chemical modification. These processes are generally difficult to predict, execute, and control. We describe herein a targeted drug delivery system that is accurately and quantitatively predicted to self-assemble into nanoparticles based on the molecular structures of precursor molecules, which are the drugs themselves. The drugs assemble with the aid of sulfated indocyanines into particles with ultrahigh drug loadings of up to 90%. We devised quantitative structure-nanoparticle assembly prediction (QSNAP) models to identify and validate electrotopological molecular descriptors as highly predictive indicators of nano-assembly and nanoparticle size. The resulting nanoparticles selectively targeted kinase inhibitors to caveolin-1-expressing human colon cancer and autochthonous liver cancer models to yield striking therapeutic effects while avoiding pERK inhibition in healthy skin. This finding enables the computational design of nanomedicines based on quantitative models for drug payload selection.

  11. Role of epithelial-mesenchymal transition involved molecules in the progression of cutaneous melanoma.

    PubMed

    Murtas, Daniela; Maxia, Cristina; Diana, Andrea; Pilloni, Luca; Corda, Claudia; Minerba, Luigi; Tomei, Sara; Piras, Franca; Ferreli, Caterina; Perra, Maria Teresa

    2017-12-01

    Epithelial-mesenchymal transition (EMT) has been suggested to have a driving role in the acquisition of a metastatic potential by melanoma cells. Important hallmarks of EMT include both E-cadherin downregulation and increased expression of N-cadherin. This switch in distinct classes of adhesion molecules leads melanoma cells to lose contact with adjacent keratinocytes and interact instead with stromal fibroblasts and endothelial cells, thus promoting dermal and vascular melanoma invasion. Consequently, tumor cells migrate to distant host tissues and establish metastases. A key regulator in the induction of EMT in melanoma is the Notch1 signaling pathway that, when activated, is prompt to upregulate N-cadherin expression. By means of this strategy, melanoma cells gain enhanced survival, proliferation and invasion properties, driving the tumor toward a more aggressive phenotype. On the basis of these statements, the present study aimed to investigate the possible association between N-cadherin and Notch1 presence in primary cutaneous melanomas and lymph node metastases. Our results from immunohistochemical analysis confirmed a positive correlation between N-cadherin and Notch1 presence in the same tumor samples. Moreover, this study highlighted that a concomitant high expression of N-cadherin and Notch1, both in primary lesions and in lymph node metastases, predicts an adverse clinical outcome in melanoma patients. Therefore, N-cadherin and Notch1 co-presence can be monitored as a predictive factor in early- and advanced-stage melanomas and open additional therapeutic targets for the restraint of melanoma metastasis.

  12. Structures, Bonding, and Energetics of Potential Triatomic Circumstellar Molecules Containing Group 15 and 16 Elements.

    PubMed

    Turner, Walter E; Agarwal, Jay; Schaefer, Henry F

    2015-12-03

    The recent discovery of PN in the oxygen-rich shell of the supergiant star VY Canis Majoris points to the formation of several triatomic molecules involving oxygen, nitrogen, and phosphorus; these are also intriguing targets for main-group synthetic inorganic chemistry. In this research, high-level ab initio electronic structure computations were conducted on the potential circumstellar molecule OPN and several of its heavier group 15 and 16 congeners (SPN, SePN, TePN, OPP, OPAs, and OPSb). For each congener, four isomers were examined. Optimized geometries were obtained with coupled cluster theory [CCSD(T)] using large Dunning basis sets [aug-cc-pVQZ, aug-cc-pV(Q+d)Z, and aug-cc-pVQZ-PP], and relative energies were determined at the complete basis set limit of CCSDT(Q) from focal point analyses. The linear phosphorus-centered molecules were consistently the lowest in energy of the group 15 congeners by at least 6 kcal mol(-1), resulting from double-triple and single-double bond resonances within the molecule. The linear nitrogen-centered molecules were consistently the lowest in energy of the group 16 congeners by at least 5 kcal mol(-1), due to the electronegative central nitrogen atom encouraging electron delocalization throughout the molecule. For OPN, OPP, and SPN, anharmonic vibrational frequencies and vibrationally corrected rotational constants are predicted; good agreement with available experimental data is observed.

  13. Involvement of MicroRNA in T-Cell Differentiation and Malignancy

    PubMed Central

    Saki, Najmaldin; Soleimani, Masoud; Hajizamani, Saeideh; Shahjahani, Mohammad; Kast, Richard E.; Mortazavi, Yousef

    2015-01-01

    ABSTRACT MicroRNAs are 19–22 nucleotide RNAs involved in such important processes as development, proliferation, differentiation and apoptosis. Different miRNAs are uniquely expressed in lymphoid T cells, and play a role indevelopment and differentiation of various subtypes by targeting their target genes. Recent studies have shown that aberrant miRNA expression may be involved in T cell leukemogenesis and lymphogenesis, and may function as tumor suppressor (such as miR-451, miR-31, miR-150, and miR-29a) or oncogene (e.g. miR-222, miR-223, miR-17-92, miR-155). MiRNAs can be used as new biomarkers for prognosis and diagnosis or as an index of disease severity in T-cell leukemia and lymphoma. This article presents a review of studies in recent years on the role of miRNAs in T-cell development and their aberrant expression in pathogenesis of T-cell leukemia and lymphoma. Characterizing miRNAs can help recognize their role as new important molecules with prognostic and therapeutic applications. PMID:25802699

  14. Smart multifunctional nanoagents for in situ monitoring of small molecules with a switchable affinity towards biomedical targets

    NASA Astrophysics Data System (ADS)

    Shevchenko, Konstantin G.; Cherkasov, Vladimir R.; Nikitina, Irina L.; Babenyshev, Andrey V.; Nikitin, Maxim P.

    2018-02-01

    The great diversity of nanomaterials provides ample opportunities for constructing effective agents for biomedical applications ranging from biosensing to drug delivery. Multifunctional nanoagents that combine several features in a single particle are of special interest due to capabilities that substantially exceed those of molecular drugs. An ideal theranostic agent should simultaneously be an advanced biosensor to identify a disease and report the diagnosis and a biomedical actuator to treat the disease. While many approaches were developed to load a nanoparticle with various drugs for actuation of the diseased cells (e.g., to kill them), the nanoparticle-based approaches for the localized biosensing with real-time reporting of the marker concentration severely lag behind. Here, we show a smart in situ nanoparticle-based biosensor/actuator system that dynamically and reversibly changes its structural and optical properties in response to a small molecule marker to allow real-time monitoring of the marker concentration and adjustment of the system ability to bind its biomedical target. Using the synergistic combination of signal readout based on the localized surface plasmon resonance and an original method of fabrication of smart ON/OFF-switchable nanoagents, we demonstrate reversible responsiveness of the system to a model small molecule marker (antibiotic chloramphenicol) in a wide concentration range. The proposed approach can be used for the development of advanced multifunctional nanoagents for theranostic applications.

  15. The future of small molecule inhibitors in lymphoma.

    PubMed

    Gerecitano, John

    2009-09-01

    For the many patients with lymphoma that has relapsed after and/or has become refractory to existing treatments, the development of novel therapeutics is imperative. Investigation into intracellular processes that are dysregulated during lymphomagenesis has uncovered several new potential targets for anticancer agents. Although monoclonal antibodies and other immunotherapeutics have led to dramatic advances in the treatment of patients with lymphoma, the parallel development of small molecule inhibitors has been equally exciting. These agents, whose small size allows direct entry into tumor cells, can target distinct proteins or complexes, thereby disrupting molecular processes on which neoplastic cells depend for survival and growth. This review surveys the published literature on many of these new targeted molecules, focusing on some of the most promising agents for which phase 2 data currently exist. It also explores the potential for incorporating these agents into broader multidrug regimens.

  16. Tools for in silico target fishing.

    PubMed

    Cereto-Massagué, Adrià; Ojeda, María José; Valls, Cristina; Mulero, Miquel; Pujadas, Gerard; Garcia-Vallve, Santiago

    2015-01-01

    Computational target fishing methods are designed to identify the most probable target of a query molecule. This process may allow the prediction of the bioactivity of a compound, the identification of the mode of action of known drugs, the detection of drug polypharmacology, drug repositioning or the prediction of the adverse effects of a compound. The large amount of information regarding the bioactivity of thousands of small molecules now allows the development of these types of methods. In recent years, we have witnessed the emergence of many methods for in silico target fishing. Most of these methods are based on the similarity principle, i.e., that similar molecules might bind to the same targets and have similar bioactivities. However, the difficult validation of target fishing methods hinders comparisons of the performance of each method. In this review, we describe the different methods developed for target prediction, the bioactivity databases most frequently used by these methods, and the publicly available programs and servers that enable non-specialist users to obtain these types of predictions. It is expected that target prediction will have a large impact on drug development and on the functional food industry. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

    PubMed

    Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre; Bouvier, Michel; Benovic, Jeffrey L; Shukla, Arun K

    2018-05-04

    G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Exploring biology with small organic molecules

    PubMed Central

    Stockwell, Brent R.

    2011-01-01

    Small organic molecules have proven to be invaluable tools for investigating biological systems, but there is still much to learn from their use. To discover and to use more effectively new chemical tools to understand biology, strategies are needed that allow us to systematically explore ‘biological-activity space’. Such strategies involve analysing both protein binding of, and phenotypic responses to, small organic molecules. The mapping of biological-activity space using small molecules is akin to mapping the stars — uncharted territory is explored using a system of coordinates that describes where each new feature lies. PMID:15602550

  19. Novel Small Molecule Therapeutics for Sickle Cell Disease: Nitric Oxide, Carbon Monoxide, Nitrite, and Apolipoprotein A-I

    PubMed Central

    Kato, Gregory J.

    2009-01-01

    A hemolysis-linked subphenotype of sickle cell disease (SCD), characterized by pulmonary hypertension, stroke, priapism and leg ulcers, is associated with decreased nitric oxide bioavailability and vasculopathy. Vasculopathy appears to have a multifactorial etiology, including mechanisms primarily that involve deficient nitric oxide (NO) signaling, but also involving altered function of NO synthase related to substrate availability and cooperating factors such as apolipoproteins. Improved understanding of the vascular pathophysiology of SCD has led to new vascular targets for translational research in SCD. This growing vascular therapeutics field in SCD is complementary to the ongoing efforts to reduce the morbidity of vaso-occlusive pain crisis. This presentation will review the current biology and translational clinical development of novel small molecules targeting sickle cell vasculopathy. Strategies targeting the heme-oxygenase-carbon monoxide pathway, the arginine-NO synthase-cGMP-phosphodiesterase 5 pathway, the nitrate-nitrite-NO pathway, and the apolipoprotein A-I pathways will be reviewed. In this context, current clinical trials of inhaled NO, CO, nitrite, sildenafil and apoA-I mimetics will be discussed. PMID:19074079

  20. Diversity in Biological Molecules

    ERIC Educational Resources Information Center

    Newbury, H. John

    2010-01-01

    One of the striking characteristics of fundamental biological processes, such as genetic inheritance, development and primary metabolism, is the limited amount of variation in the molecules involved. Natural selective pressures act strongly on these core processes and individuals carrying mutations and producing slightly sub-optimal versions of…

  1. Non-coding RNAs in cardiac fibrosis: emerging biomarkers and therapeutic targets.

    PubMed

    Chen, Zhongxiu; Li, Chen; Lin, Ke; Cai, Huawei; Ruan, Weiqiang; Han, Junyang; Rao, Li

    2017-12-14

    Non-coding RNAs (ncRNAs) are a class of RNA molecules that do not encode proteins. ncRNAs are involved in cell proliferation, apoptosis, differentiation, metabolism, and other physiological processes as well as the pathogenesis of diseases. Cardiac fibrosis is increasingly recognized as a common final pathway in advanced heart diseases. Many studies have shown that the occurrence and development of cardiac fibrosis is closely related to the regulation of ncRNAs. This review will highlight recent updates regarding the involvement of ncRNAs in cardiac fibrosis, and their potential as emerging biomarkers and therapeutic targets.

  2. Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33

    PubMed Central

    Azoitei, Ninel; Hoffmann, Christopher M.; Ellegast, Jana M.; Ball, Claudia R.; Obermayer, Kerstin; Gößele, Ulrike; Koch, Britta; Faber, Katrin; Genze, Felicitas; Schrader, Mark; Kestler, Hans A.; Döhner, Hartmut; Chiosis, Gabriela; Glimm, Hanno

    2012-01-01

    Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential. PMID:22451720

  3. Structural basis of AMPK regulation by small molecule activators

    NASA Astrophysics Data System (ADS)

    Xiao, Bing; Sanders, Matthew J.; Carmena, David; Bright, Nicola J.; Haire, Lesley F.; Underwood, Elizabeth; Patel, Bhakti R.; Heath, Richard B.; Walker, Philip A.; Hallen, Stefan; Giordanetto, Fabrizio; Martin, Stephen R.; Carling, David; Gamblin, Steven J.

    2013-12-01

    AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.

  4. Involvement of Receptor-like Protein Tyrosine Phosphatase ζ/RPTPβ and Its Ligand Pleiotrophin/Heparin-binding Growth-associated Molecule (HB-GAM) in Neuronal Migration

    PubMed Central

    Maeda, Nobuaki; Noda, Masaharu

    1998-01-01

    Pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) is a specific ligand of protein tyrosine phosphatase ζ (PTPζ)/receptor-like protein tyrosine phosphatase β (RPTPβ) expressed in the brain as a chondroitin sulfate proteoglycan. Pleiotrophin and PTPζ isoforms are localized along the radial glial fibers, a scaffold for neuronal migration, suggesting that these molecules are involved in migratory processes of neurons during brain development. In this study, we examined the roles of pleiotrophin-PTPζ interaction in the neuronal migration using cell migration assay systems with glass fibers and Boyden chambers. Pleiotrophin and poly-l-lysine coated on the substratums stimulated cell migration of cortical neurons, while laminin, fibronectin, and tenascin exerted almost no effect. Pleiotrophin-induced and poly-l-lysine–induced neuronal migrations showed significant differences in sensitivity to various molecules and reagents. Polyclonal antibodies against the extracellular domain of PTPζ, PTPζ-S, an extracellular secreted form of PTPζ, and sodium vanadate, a protein tyrosine phosphatase inhibitor, added into the culture medium strongly suppressed specifically the pleiotrophin-induced neuronal migration. Furthermore, chondroitin sulfate C but not chondroitin sulfate A inhibited pleiotrophin-induced neuronal migration, in good accordance with our previous findings that chondroitin sulfate constitutes a part of the pleiotrophin-binding site of PTPζ, and PTPζ-pleiotrophin binding is inhibited by chondroitin sulfate C but not by chondroitin sulfate A. Immunocytochemical analysis indicated that the transmembrane forms of PTPζ are expressed on the migrating neurons especially at the lamellipodia along the leading processes. These results suggest that PTPζ is involved in the neuronal migration as a neuronal receptor of pleiotrophin distributed along radial glial fibers. PMID:9660874

  5. P2X Receptors as Drug Targets

    PubMed Central

    Jarvis, Michael F.

    2013-01-01

    The study of P2X receptors has long been handicapped by a poverty of small-molecule tools that serve as selective agonists and antagonists. There has been progress, particularly in the past 10 years, as cell-based high-throughput screening methods were applied, together with large chemical libraries. This has delivered some drug-like molecules in several chemical classes that selectively target P2X1, P2X3, or P2X7 receptors. Some of these are, or have been, in clinical trials for rheumatoid arthritis, pain, and cough. Current preclinical research programs are studying P2X receptor involvement in pain, inflammation, osteoporosis, multiple sclerosis, spinal cord injury, and bladder dysfunction. The determination of the atomic structure of P2X receptors in closed and open (ATP-bound) states by X-ray crystallography is now allowing new approaches by molecular modeling. This is supported by a large body of previous work using mutagenesis and functional expression, and is now being supplemented by molecular dynamic simulations and in silico ligand docking. These approaches should lead to P2X receptors soon taking their place alongside other ion channel proteins as therapeutically important drug targets. PMID:23253448

  6. Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

    PubMed

    Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

    2014-03-04

    Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

  7. Exchange and correlation in positronium-molecule scattering

    NASA Astrophysics Data System (ADS)

    Fabrikant, I. I.; Wilde, R. S.

    2018-05-01

    Exchange and correlations play a particularly important role in positronium (Ps) collisions with atoms and molecules, since the static potential for Ps interaction with a neutral system is zero. Theoretical description of both effects is a very challenging task. In the present work we use the free-electron-gas model to describe exchange and correlations in Ps collisions with molecules similar to the approach widely used in the theory of electron-molecule collisions. The results for exchange and correlation energies are presented as functions of the Fermi momentum of the electron gas and the Ps incident energy. Using the Thomas-Fermi model, these functions can be converted into exchange and correlation potentials for Ps interaction with molecules as functions of the distance between the projectile and the target.

  8. Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

    PubMed

    Kong, Muwen; Beckwitt, Emily C; Springall, Luke; Kad, Neil M; Van Houten, Bennett

    2017-01-01

    Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair. © 2017 Elsevier Inc. All rights reserved.

  9. Planning paths to multiple targets: memory involvement and planning heuristics in spatial problem solving.

    PubMed

    Wiener, J M; Ehbauer, N N; Mallot, H A

    2009-09-01

    For large numbers of targets, path planning is a complex and computationally expensive task. Humans, however, usually solve such tasks quickly and efficiently. We present experiments studying human path planning performance and the cognitive processes and heuristics involved. Twenty-five places were arranged on a regular grid in a large room. Participants were repeatedly asked to solve traveling salesman problems (TSP), i.e., to find the shortest closed loop connecting a start location with multiple target locations. In Experiment 1, we tested whether humans employed the nearest neighbor (NN) strategy when solving the TSP. Results showed that subjects outperform the NN-strategy, suggesting that it is not sufficient to explain human route planning behavior. As a second possible strategy we tested a hierarchical planning heuristic in Experiment 2, demonstrating that participants first plan a coarse route on the region level that is refined during navigation. To test for the relevance of spatial working memory (SWM) and spatial long-term memory (LTM) for planning performance and the planning heuristics applied, we varied the memory demands between conditions in Experiment 2. In one condition the target locations were directly marked, such that no memory was required; a second condition required participants to memorize the target locations during path planning (SWM); in a third condition, additionally, the locations of targets had to retrieved from LTM (SWM and LTM). Results showed that navigation performance decreased with increasing memory demands while the dependence on the hierarchical planning heuristic increased.

  10. Synthesis and characterization of novel 1,2-oxazine-based small molecules that targets acetylcholinesterase.

    PubMed

    Sukhorukov, Alexey Yu; Nirvanappa, Anilkumar C; Swamy, Jagadish; Ioffe, Sema L; Nanjunda Swamy, Shivananju; Basappa; Rangappa, Kanchugarakoppal S

    2014-08-01

    Thirteen 2-oxazine-based small molecules were synthesized targeting 5-lipoxygenase (LOX), and acetylcholinesterase (AChE). The test revealed that the newly synthesized compounds had potent inhibition towards both 5-LOX and AChE in lower micro molar concentration. Among the tested compounds, the most active compound, 2-[(2-acetyl-6,6-dimethyl-4-phenyl-5,6-dihydro-2H-1,2-oxazin-3-yl)methyl]-1H-isoindole-1,3(2H)-dione (2a) showed inhibitory activity towards 5-LOX and AChE with an IC50 values of 1.88, and 2.5 μM, respectively. Further, the in silico molecular docking studies revealed that the compound 2a bound to the catalytic domain of AChE strongly with a highest CDOCKER score of -1.18 kcal/mol when compared to other compounds of the same series. Additionally, 2a showed a good lipophilicity (logP=2.66), suggesting a potential ability to penetrate the blood-brain-barrier. These initial pharmacological data revealed that the compound 2a could serve as a drug-seed in developing anti-Alzheimer's agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Epithelial cell adhesion molecule aptamer functionalized PLGA-lecithin-curcumin-PEG nanoparticles for targeted drug delivery to human colorectal adenocarcinoma cells.

    PubMed

    Li, Lei; Xiang, Dongxi; Shigdar, Sarah; Yang, Wenrong; Li, Qiong; Lin, Jia; Liu, Kexin; Duan, Wei

    2014-01-01

    To improve the efficacy of drug delivery, active targeted nanotechnology-based drug delivery systems are gaining considerable attention as they have the potential to reduce side effects, minimize toxicity, and improve efficacy of anticancer treatment. In this work CUR-NPs (curcumin-loaded lipid-polymer-lecithin hybrid nanoparticles) were synthesized and functionalized with ribonucleic acid (RNA) Aptamers (Apts) against epithelial cell adhesion molecule (EpCAM) for targeted delivery to colorectal adenocarcinoma cells. These CUR-encapsulated bioconjugates (Apt-CUR-NPs) were characterized for particle size, zeta potential, drug encapsulation, stability, and release. The in vitro specific cell binding, cellular uptake, and cytotoxicity of Apt-CUR-NPs were also studied. The Apt-CUR-NP bioconjugates exhibited increased binding to HT29 colon cancer cells and enhancement in cellular uptake when compared to CUR-NPs functionalized with a control Apt (P<0.01). Furthermore, a substantial improvement in cytotoxicity was achieved toward HT29 cells with Apt-CUR-NP bioconjugates. The encapsulation of CUR in Apt-CUR-NPs resulted in the increased bioavailability of delivered CUR over a period of 24 hours compared to that of free CUR in vivo. These results show that the EpCAM Apt-functionalized CUR-NPs enhance the targeting and drug delivery of CUR to colorectal cancer cells. Further development of CUR-encapsulated, nanosized carriers will lead to improved targeted delivery of novel chemotherapeutic agents to colorectal cancer cells.

  12. Fragment-Based Phenotypic Lead Discovery: Cell-Based Assay to Target Leishmaniasis.

    PubMed

    Ayotte, Yann; Bilodeau, François; Descoteaux, Albert; LaPlante, Steven R

    2018-05-02

    A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment-based phenotypic lead discovery (FPLD) combines aspects of traditional fragment-based lead discovery (FBLD), which involves the screening of small-molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug-like compounds in cell-based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low-molecular-weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone-marrow-derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

    PubMed

    Landry, James P; Fei, Yiyan; Zhu, X D

    2011-12-01

    Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

  14. "Trampoline" ejection of organic molecules from graphene and graphite via keV cluster ions impacts.

    PubMed

    Verkhoturov, Stanislav V; Gołuński, Mikołaj; Verkhoturov, Dmitriy S; Geng, Sheng; Postawa, Zbigniew; Schweikert, Emile A

    2018-04-14

    We present the data on ejection of molecules and emission of molecular ions caused by single impacts of 50 keV C 60 2+ on a molecular layer of deuterated phenylalanine (D8Phe) deposited on free standing, 2-layer graphene. The projectile impacts on the graphene side stimulate the abundant ejection of intact molecules and the emission of molecular ions in the transmission direction. To gain insight into the mechanism of ejection, Molecular Dynamic simulations were performed. It was found that the projectile penetrates the thin layer of graphene, partially depositing the projectile's kinetic energy, and molecules are ejected from the hot area around the hole that is made by the projectile. The yield, Y, of negative ions of deprotonated phenylalanine, (D8Phe-H) - , emitted in the transmission direction is 0.1 ions per projectile impact. To characterize the ejection and ionization of molecules, we have performed the experiments on emission of (D8Phe-H) - from the surface of bulk D8Phe (Y = 0.13) and from the single molecular layer of D8Phe deposited on bulk pyrolytic graphite (Y = 0.15). We show that, despite the similar yields of molecular ions, the scenario of the energy deposition and ejection of molecules is different for the case of graphene due to the confined volume of projectile-analyte interaction. The projectile impact on the graphene-D8Phe sample stimulates the collective radial movement of analyte atoms, which compresses the D8Phe layer radially from the hole. At the same time, this compression bends and stretches the graphene membrane around the hole thus accumulating potential energy. The accumulated potential energy is transformed into the kinetic energy of correlated movement upward for membrane atoms, thus the membrane acts as a trampoline for the molecules. The ejected molecules are effectively ionized; the ionization probability is ∼30× higher compared to that obtained for the bulk D8Phe target. The proposed mechanism of ionization involves

  15. "Trampoline" ejection of organic molecules from graphene and graphite via keV cluster ions impacts

    NASA Astrophysics Data System (ADS)

    Verkhoturov, Stanislav V.; Gołuński, Mikołaj; Verkhoturov, Dmitriy S.; Geng, Sheng; Postawa, Zbigniew; Schweikert, Emile A.

    2018-04-01

    We present the data on ejection of molecules and emission of molecular ions caused by single impacts of 50 keV C602+ on a molecular layer of deuterated phenylalanine (D8Phe) deposited on free standing, 2-layer graphene. The projectile impacts on the graphene side stimulate the abundant ejection of intact molecules and the emission of molecular ions in the transmission direction. To gain insight into the mechanism of ejection, Molecular Dynamic simulations were performed. It was found that the projectile penetrates the thin layer of graphene, partially depositing the projectile's kinetic energy, and molecules are ejected from the hot area around the hole that is made by the projectile. The yield, Y, of negative ions of deprotonated phenylalanine, (D8Phe-H)-, emitted in the transmission direction is 0.1 ions per projectile impact. To characterize the ejection and ionization of molecules, we have performed the experiments on emission of (D8Phe-H)- from the surface of bulk D8Phe (Y = 0.13) and from the single molecular layer of D8Phe deposited on bulk pyrolytic graphite (Y = 0.15). We show that, despite the similar yields of molecular ions, the scenario of the energy deposition and ejection of molecules is different for the case of graphene due to the confined volume of projectile-analyte interaction. The projectile impact on the graphene-D8Phe sample stimulates the collective radial movement of analyte atoms, which compresses the D8Phe layer radially from the hole. At the same time, this compression bends and stretches the graphene membrane around the hole thus accumulating potential energy. The accumulated potential energy is transformed into the kinetic energy of correlated movement upward for membrane atoms, thus the membrane acts as a trampoline for the molecules. The ejected molecules are effectively ionized; the ionization probability is ˜30× higher compared to that obtained for the bulk D8Phe target. The proposed mechanism of ionization involves tunneling of

  16. Rational design of chemical genetic probes of RNA function and lead therapeutics targeting repeating transcripts.

    PubMed

    Disney, Matthew D

    2013-12-01

    RNA is an important yet vastly underexploited target for small molecule chemical probes or lead therapeutics. Small molecules have been used successfully to modulate the function of the bacterial ribosome, viral RNAs and riboswitches. These RNAs are either highly expressed or can be targeted using substrate mimicry, a mainstay in the design of enzyme inhibitors. However, most cellular RNAs are neither highly expressed nor have a lead small molecule inhibitor, a significant challenge for drug discovery efforts. Herein, I describe the design of small molecules targeting expanded repeating transcripts that cause myotonic muscular dystrophy (DM). These test cases illustrate the challenges of designing small molecules that target RNA and the advantages of targeting repeating transcripts. Lastly, I discuss how small molecules might be more advantageous than oligonucleotides for targeting RNA. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. SM-TF: A structural database of small molecule-transcription factor complexes.

    PubMed

    Xu, Xianjin; Ma, Zhiwei; Sun, Hongmin; Zou, Xiaoqin

    2016-06-30

    Transcription factors (TFs) are the proteins involved in the transcription process, ensuring the correct expression of specific genes. Numerous diseases arise from the dysfunction of specific TFs. In fact, over 30 TFs have been identified as therapeutic targets of about 9% of the approved drugs. In this study, we created a structural database of small molecule-transcription factor (SM-TF) complexes, available online at http://zoulab.dalton.missouri.edu/SM-TF. The 3D structures of the co-bound small molecule and the corresponding binding sites on TFs are provided in the database, serving as a valuable resource to assist structure-based drug design related to TFs. Currently, the SM-TF database contains 934 entries covering 176 TFs from a variety of species. The database is further classified into several subsets by species and organisms. The entries in the SM-TF database are linked to the UniProt database and other sequence-based TF databases. Furthermore, the druggable TFs from human and the corresponding approved drugs are linked to the DrugBank. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. The Molecular Industrial Revolution: Automated Synthesis of Small Molecules

    PubMed Central

    Trobe, Melanie; Burke, Martin D.

    2018-01-01

    The eighteenth and nineteenth centuries marked a sweeping transition from manual to automated manufacturing on the macroscopic scale. This enabled an unmatched period of human innovation that helped drive the Industrial Revolution. The impact on society was transformative, ultimately yielding substantial improvements in living conditions and lifespan in many parts of the world. During the same time period, the first manual syntheses of organic molecules was achieved. Now, two centuries later, we are poised for an analogous transition from highly customized crafting of specific molecular targets by hand to the increasingly general and automated assembly of many different types of molecules with the push of a button. Automation of customized small molecule synthesis pathways is already enabling safer, more reproducible, and readily scalable production of specific targets, and general machines now exist for the synthesis of a wide range of different peptides, oligonucleotides, and oligosaccharides. Creating general machines that are similarly capable of making many different types of small molecules on-demand, akin to that which has been achieved on the macroscopic scale with 3D printers, has proven to be substantially more challenging. Yet important progress is being made toward this potentially transformative objective with two complementary approaches: (1) automation of customized synthesis routes to different targets via machines that enable use of many different reactions and starting materials, and (2) automation of generalized platforms that make many different targets using common coupling chemistry and building blocks. Continued progress in these exciting directions has the potential to shift the bottleneck in molecular innovation from synthesis to imagination, and thereby help drive a new industrial revolution on the molecular scale. PMID:29513400

  19. Prospecting for Novel Plant-Derived Molecules of Rauvolfia serpentina as Inhibitors of Aldose Reductase, a Potent Drug Target for Diabetes and Its Complications

    PubMed Central

    Pathania, Shivalika; Randhawa, Vinay; Bagler, Ganesh

    2013-01-01

    Aldose Reductase (AR) is implicated in the development of secondary complications of diabetes, providing an interesting target for therapeutic intervention. Extracts of Rauvolfia serpentina, a medicinal plant endemic to the Himalayan mountain range, have been known to be effective in alleviating diabetes and its complications. In this study, we aim to prospect for novel plant-derived inhibitors from R. serpentina and to understand structural basis of their interactions. An extensive library of R. serpentina molecules was compiled and computationally screened for inhibitory action against AR. The stability of complexes, with docked leads, was verified using molecular dynamics simulations. Two structurally distinct plant-derived leads were identified as inhibitors: indobine and indobinine. Further, using these two leads as templates, 16 more leads were identified through ligand-based screening of their structural analogs, from a small molecules database. Thus, we obtained plant-derived indole alkaloids, and their structural analogs, as potential AR inhibitors from a manually curated dataset of R. serpentina molecules. Indole alkaloids reported herein, as a novel structural class unreported hitherto, may provide better insights for designing potential AR inhibitors with improved efficacy and fewer side effects. PMID:23613832

  20. Prospecting for novel plant-derived molecules of Rauvolfia serpentina as inhibitors of Aldose Reductase, a potent drug target for diabetes and its complications.

    PubMed

    Pathania, Shivalika; Randhawa, Vinay; Bagler, Ganesh

    2013-01-01

    Aldose Reductase (AR) is implicated in the development of secondary complications of diabetes, providing an interesting target for therapeutic intervention. Extracts of Rauvolfia serpentina, a medicinal plant endemic to the Himalayan mountain range, have been known to be effective in alleviating diabetes and its complications. In this study, we aim to prospect for novel plant-derived inhibitors from R. serpentina and to understand structural basis of their interactions. An extensive library of R. serpentina molecules was compiled and computationally screened for inhibitory action against AR. The stability of complexes, with docked leads, was verified using molecular dynamics simulations. Two structurally distinct plant-derived leads were identified as inhibitors: indobine and indobinine. Further, using these two leads as templates, 16 more leads were identified through ligand-based screening of their structural analogs, from a small molecules database. Thus, we obtained plant-derived indole alkaloids, and their structural analogs, as potential AR inhibitors from a manually curated dataset of R. serpentina molecules. Indole alkaloids reported herein, as a novel structural class unreported hitherto, may provide better insights for designing potential AR inhibitors with improved efficacy and fewer side effects.

  1. Recent Progress and Advances in HGF/MET-Targeted Therapeutic Agents for Cancer Treatment

    PubMed Central

    Zhang, Yilong; Jain, Rajul K.; Zhu, Min

    2015-01-01

    The hepatocyte growth factor (HGF): MET axis is a ligand-mediated receptor tyrosine kinase pathway that is involved in multiple cellular functions, including proliferation, survival, motility, and morphogenesis. Aberrancy in the HGF/MET pathway has been reported in multiple tumor types and is associated with tumor stage and prognosis. Thus, targeting the HGF/MET pathway has become a potential therapeutic strategy in oncology development in the last two decades. A number of novel therapeutic agents—either as therapeutic proteins or small molecules that target the HGF/MET pathway—have been tested in patients with different tumor types in clinical studies. In this review, recent progress in HGF/MET pathway-targeted therapy for cancer treatment, the therapeutic potential of HGF/MET-targeted agents, and challenges in the development of such agents will be discussed. PMID:28536405

  2. A quinolinol-based small molecule with anti-MRSA activity that targets bacterial membrane and promotes fermentative metabolism.

    PubMed

    Nair, Dhanalakshmi R; Chen, Ji; Monteiro, João M; Josten, Michaele; Pinho, Mariana G; Sahl, Hans-Georg; Wu, Jimmy; Cheung, Ambrose

    2017-10-01

    In a loss-of-viability screen of small molecules against methicillin-resistant Staphylococcus aureus (MRSA) USA300, we found a small molecule, designated DNAC-2, which has an MIC of 8 μg ml -1 . DNAC-2 is a quinolinol derivative that is bactericidal at 2X MIC. Macromolecular synthesis assays at 2 × MIC of DNAC-2 revealed inhibition of DNA, cell wall, RNA and protein synthesis within fifteen to thirty minutes of treatment when compared to the untreated control. Transmission electron microscopy of DNAC-2-treated cells revealed a significantly thicker cell wall and impaired daughter cell separation. Exposure of USA300 cells to 1 × MIC of DNAC-2 resulted in mislocalization of PBP2 away from the septum in an FtsZ-independent manner. In addition, membrane localization with FM4-64, as well as depolarization study with DiOC 2 and lipophilic cation TPP+ displayed membrane irregularities and rapid membrane depolarization, respectively, in DNAC-2-treated cells vs -untreated control. However, DNAC-2 exhibited almost no toxicity toward eukaryotic membranes. Notably, DNAC-2 drives energy generation toward substrate level phosphorylation and the bacteria become more sensitive to DNAC-2 under anaerobic conditions. We propose that DNAC-2 affects USA300 by targeting the membrane, leading to partial membrane depolarization and subsequently affecting aerobic respiration and energy-dependent functional organization of macromolecular biosynthetic pathways. The multiple effects may have the desirable consequence of limiting the emergence of resistance to DNAC-2.

  3. Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter

    2012-04-01

    Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

  4. Aptamer-Encoded Nanopore for Ultrasensitive Detection of Bioterrorist Agent Ricin at Single-Molecule Resolution

    PubMed Central

    Gu, Li-Qun; Ding, Shu; Gao, Changlu

    2011-01-01

    The molecular-scale pore structure, called nanopore, can be formed from protein ion channels by genetic engineering or fabricated on solid substrates using fashion nanotechnology. Target molecules in interaction with the functionalized lumen of nanopore, can produce characteristic changes in the pore conductance, which act as fingerprints, allowing us to identify single molecules and simultaneously quantify each target species in the mixture. Nanopore sensors have been created for tremendous biomedical detections, with targets ranging from metal ions, drug compounds and cellular second messengers, to proteins and DNAs. Here we will review our recent discoveries with a lab-in-hand glass nanopore: single-molecule discrimination of chiral enantiomers with a trapped cyclodextrin, and sensing of bioterrorist agent ricin. PMID:19964179

  5. Target identification of small molecules based on chemical biology approaches.

    PubMed

    Futamura, Yushi; Muroi, Makoto; Osada, Hiroyuki

    2013-05-01

    Recently, a phenotypic approach-screens that assess the effects of compounds on cells, tissues, or whole organisms-has been reconsidered and reintroduced as a complementary strategy of a target-based approach for drug discovery. Although the finding of novel bioactive compounds from large chemical libraries has become routine, the identification of their molecular targets is still a time-consuming and difficult process, making this step rate-limiting in drug development. In the last decade, we and other researchers have amassed a large amount of phenotypic data through progress in omics research and advances in instrumentation. Accordingly, the profiling methodologies using these datasets expertly have emerged to identify and validate specific molecular targets of drug candidates, attaining some progress in current drug discovery (e.g., eribulin). In the case of a compound that shows an unprecedented phenotype likely by inhibiting a first-in-class target, however, such phenotypic profiling is invalid. Under the circumstances, a photo-crosslinking affinity approach should be beneficial. In this review, we describe and summarize recent progress in both affinity-based (direct) and phenotypic profiling (indirect) approaches for chemical biology target identification.

  6. Non-Invasive Monitoring of CNS MHC-I Molecules in Ischemic Stroke Mice.

    PubMed

    Xia, Jing; Zhang, Ying; Zhao, Huanhuan; Wang, Jie; Gao, Xueren; Chen, Jinpeng; Fu, Bo; Shen, Yuqing; Miao, Fengqin; Zhang, Jianqiong; Teng, Gaojun

    2017-01-01

    Ischemic stroke is one of the leading causes of morbidity and mortality worldwide. The expression of major histocompatibility complex class I (MHC-I) molecules in the central nervous system, which are silenced under normal physiological conditions, have been reported to be induced by injury stimulation. The purpose of this study was to determine whether MHC-I molecules could serve as molecular targets for the acute phase of ischemic stroke and to assess whether a high-affinity peptide specific for MHC-I molecules could be applied in the near-infrared imaging of cerebral ischemic mice. Quantitative real-time PCR and Western blotting were used to detect the expression of MHC-I molecules in two mouse models of cerebral ischemic stroke and an in vitro model of ischemia. The NetMHC 4.0 server was used to screen a high-affinity peptide specific for mouse MHC-I molecules. The Rosetta program was used to identify the specificity and affinity of the screened peptide (histocompatibility-2 binding peptide, H2BP). The results demonstrated that MHC-I molecules could serve as molecular targets for the acute phase of ischemic stroke. Cy5.5-H2BP molecular probes could be applied in the near-infrared imaging of cerebral ischemic mice. Research on the expression of MHC-I molecules in the acute phase after ischemia and MHC-I-targeted imaging may not only be helpful for understanding the mechanism of ischemic and hypoxic brain injury and repair but also has potential application value in the imaging of ischemic stroke.

  7. Targeting Innate Immunity for Antiviral Therapy through Small Molecule Agonists of the RLR Pathway

    PubMed Central

    Pattabhi, Sowmya; Wilkins, Courtney R.; Dong, Ran; Knoll, Megan L.; Posakony, Jeffrey; Kaiser, Shari; Mire, Chad E.; Wang, Myra L.; Ireton, Renee C.; Geisbert, Thomas W.; Bedard, Kristin M.; Iadonato, Shawn P.

    2015-01-01

    ABSTRACT The cellular response to virus infection is initiated when pathogen recognition receptors (PRR) engage viral pathogen-associated molecular patterns (PAMPs). This process results in induction of downstream signaling pathways that activate the transcription factor interferon regulatory factor 3 (IRF3). IRF3 plays a critical role in antiviral immunity to drive the expression of innate immune response genes, including those encoding antiviral factors, type 1 interferon, and immune modulatory cytokines, that act in concert to restrict virus replication. Thus, small molecule agonists that can promote IRF3 activation and induce innate immune gene expression could serve as antivirals to induce tissue-wide innate immunity for effective control of virus infection. We identified small molecule compounds that activate IRF3 to differentially induce discrete subsets of antiviral genes. We tested a lead compound and derivatives for the ability to suppress infections caused by a broad range of RNA viruses. Compound administration significantly decreased the viral RNA load in cultured cells that were infected with viruses of the family Flaviviridae, including West Nile virus, dengue virus, and hepatitis C virus, as well as viruses of the families Filoviridae (Ebola virus), Orthomyxoviridae (influenza A virus), Arenaviridae (Lassa virus), and Paramyxoviridae (respiratory syncytial virus, Nipah virus) to suppress infectious virus production. Knockdown studies mapped this response to the RIG-I-like receptor pathway. This work identifies a novel class of host-directed immune modulatory molecules that activate IRF3 to promote host antiviral responses to broadly suppress infections caused by RNA viruses of distinct genera. IMPORTANCE Incidences of emerging and reemerging RNA viruses highlight a desperate need for broad-spectrum antiviral agents that can effectively control infections caused by viruses of distinct genera. We identified small molecule compounds that can

  8. Target-protecting dumbbell molecular probe against exonucleases digestion for sensitive detection of ATP and streptavidin.

    PubMed

    Chen, Jinyang; Liu, Yucheng; Ji, Xinghu; He, Zhike

    2016-09-15

    In this work, a versatile dumbbell molecular (DM) probe was designed and employed in the sensitively homogeneous bioassay. In the presence of target molecule, the DM probe was protected from the digestion of exonucleases. Subsequently, the protected DM probe specifically bound to the intercalation dye and resulted in obvious fluorescence signal which was used to determine the target molecule in return. This design allows specific and versatile detection of diverse targets with easy operation and no sophisticated fluorescence labeling. Integrating the idea of target-protecting DM probe with adenosine triphosphate (ATP) involved ligation reaction, the DM probe with 5'-end phosphorylation was successfully constructed for ATP detection, and the limitation of detection was found to be 4.8 pM. Thanks to its excellent selectivity and sensitivity, this sensing strategy was used to detect ATP spiked in human serum as well as cellular ATP. Moreover, the proposed strategy was also applied in the visual detection of ATP in droplet-based microfluidic platform with satisfactory results. Similarly, combining the principle of target-protecting DM probe with streptavidin (SA)-biotin interaction, the DM probe with 3'-end biotinylation was developed for selective and sensitive SA determination, which demonstrated the robustness and versatility of this design. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Exporters for Production of Amino Acids and Other Small Molecules.

    PubMed

    Eggeling, Lothar

    Microbes are talented catalysts to synthesize valuable small molecules in their cytosol. However, to make full use of their skills - and that of metabolic engineers - the export of intracellularly synthesized molecules to the culture medium has to be considered. This step is as essential as is each step for the synthesis of the favorite molecule of the metabolic engineer, but is frequently not taken into account. To export small molecules via the microbial cell envelope, a range of different types of carrier proteins is recognized to be involved, which are primary active carriers, secondary active carriers, or proteins increasing diffusion. Relevant export may require just one carrier as is the case with L-lysine export by Corynebacterium glutamicum or involve up to four carriers as known for L-cysteine excretion by Escherichia coli. Meanwhile carriers for a number of small molecules of biotechnological interest are recognized, like for production of peptides, nucleosides, diamines, organic acids, or biofuels. In addition to carriers involved in amino acid excretion, such carriers and their impact on product formation are described, as well as the relatedness of export carriers which may serve as a hint to identify further carriers required to improve product formation by engineering export.

  10. Water-assisted dehalogenation of thionyl chloride in the presence of water molecules.

    PubMed

    Yeung, Chi Shun; Ng, Ping Leung; Guan, Xiangguo; Phillips, David Lee

    2010-04-01

    A second-order Møller-Plesset perturbation theory (MP2) and density functional theory (DFT) investigation of the dehalogenation reactions of thionyl chloride is reported, in which water molecules (up to seven) were explicitly involved in the reaction complex. The dehalogenation processes of thionyl chloride were found to be dramatically catalyzed by water molecules. The reaction rate became significantly faster as more water molecules became involved in the reaction complex. The dehalogenation processes can be reasonably simulated by the gas-phase water cluster models, which reveals that water molecules can help to solvate the thionyl chloride molecules and activate the release of the Cl(-) leaving group. The computed activation energies were used to compare the calculations to available experimental data.

  11. Differential Expression of Osteo-Modulatory Molecules in Periodontal Ligament Stem Cells in Response to Modified Titanium Surfaces

    PubMed Central

    Kim, So Yeon; Yoo, Ji-Yeon; Ohe, Joo-Young; Lee, Jung-Woo; Moon, Ji-Hoi; Kwon, Yong-Dae; Heo, Jung Sun

    2014-01-01

    This study assessed differential gene expression of signaling molecules involved in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) subjected to different titanium (Ti) surface types. PDLSCs were cultured on tissue culture polystyrene (TCPS), and four types of Ti discs (PT, SLA, hydrophilic PT (pmodPT), and hydrophilic SLA (modSLA)) with no osteoinductive factor and then osteogenic activity, including alkaline phosphatase (ALP) activity, mRNA expression of runt-related gene 2, osterix, FOSB, FRA1, and protein levels of osteopontin and collagen type IA, were examined. The highest osteogenic activity appeared in PDLSCs cultured on SLA, compared with the TCPS and other Ti surfaces. The role of surface properties in affecting signaling molecules to modulate PDLSC behavior was determined by examining the regulation of Wnt pathways. mRNA expression of the canonical Wnt signaling molecules, Wnt3a and β-catenin, was higher on SLA and modSLA than on smooth surfaces, but gene expression of the calcium-dependent Wnt signaling molecules Wnt5a, calmodulin, and NFATc1 was increased significantly on PT and pmodPT. Moreover, integrin α2/β1, sonic hedgehog, and Notch signaling molecules were affected differently by each surface modification. In conclusion, surface roughness and hydrophilicity can affect differential Wnt pathways and signaling molecules, targeting the osteogenic differentiation of PDLSCs. PMID:25057487

  12. Differential expression of osteo-modulatory molecules in periodontal ligament stem cells in response to modified titanium surfaces.

    PubMed

    Kim, So Yeon; Yoo, Ji-Yeon; Ohe, Joo-Young; Lee, Jung-Woo; Moon, Ji-Hoi; Kwon, Yong-Dae; Heo, Jung Sun

    2014-01-01

    This study assessed differential gene expression of signaling molecules involved in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) subjected to different titanium (Ti) surface types. PDLSCs were cultured on tissue culture polystyrene (TCPS), and four types of Ti discs (PT, SLA, hydrophilic PT (pmodPT), and hydrophilic SLA (modSLA)) with no osteoinductive factor and then osteogenic activity, including alkaline phosphatase (ALP) activity, mRNA expression of runt-related gene 2, osterix, FOSB, FRA1, and protein levels of osteopontin and collagen type IA, were examined. The highest osteogenic activity appeared in PDLSCs cultured on SLA, compared with the TCPS and other Ti surfaces. The role of surface properties in affecting signaling molecules to modulate PDLSC behavior was determined by examining the regulation of Wnt pathways. mRNA expression of the canonical Wnt signaling molecules, Wnt3a and β-catenin, was higher on SLA and modSLA than on smooth surfaces, but gene expression of the calcium-dependent Wnt signaling molecules Wnt5a, calmodulin, and NFATc1 was increased significantly on PT and pmodPT. Moreover, integrin α2/β1, sonic hedgehog, and Notch signaling molecules were affected differently by each surface modification. In conclusion, surface roughness and hydrophilicity can affect differential Wnt pathways and signaling molecules, targeting the osteogenic differentiation of PDLSCs.

  13. SMMRNA: a database of small molecule modulators of RNA

    PubMed Central

    Mehta, Ankita; Sonam, Surabhi; Gouri, Isha; Loharch, Saurabh; Sharma, Deepak K.; Parkesh, Raman

    2014-01-01

    We have developed SMMRNA, an interactive database, available at http://www.smmrna.org, with special focus on small molecule ligands targeting RNA. Currently, SMMRNA consists of ∼770 unique ligands along with structural images of RNA molecules. Each ligand in the SMMRNA contains information such as Kd, Ki, IC50, ΔTm, molecular weight (MW), hydrogen donor and acceptor count, XlogP, number of rotatable bonds, number of aromatic rings and 2D and 3D structures. These parameters can be explored using text search, advanced search, substructure and similarity-based analysis tools that are embedded in SMMRNA. A structure editor is provided for 3D visualization of ligands. Advance analysis can be performed using substructure and OpenBabel-based chemical similarity fingerprints. Upload facility for both RNA and ligands is also provided. The physicochemical properties of the ligands were further examined using OpenBabel descriptors, hierarchical clustering, binning partition and multidimensional scaling. We have also generated a 3D conformation database of ligands to support the structure and ligand-based screening. SMMRNA provides comprehensive resource for further design, development and refinement of small molecule modulators for selective targeting of RNA molecules. PMID:24163098

  14. Human natural killer cell receptors for HLA-class I molecules. Evidence that the Kp43 (CD94) molecule functions as receptor for HLA-B alleles

    PubMed Central

    1994-01-01

    GL183 or EB6 (p58) molecules have been shown to function as receptors for different HLA-C alleles and to deliver an inhibitory signal to natural killer (NK) cells, thus preventing lysis of target cells. In this study, we analyzed a subset of NK cells characterized by a p58- negative surface phenotype. We show that p58-negative clones, although specific for class I molecules do not recognize HLA-C alleles. In addition, by the use of appropriate target cells transfected with different HLA-class I alleles we identified HLA-B7 as the protective element recognized by a fraction of p58-negative clones. In an attempt to identify the receptor molecules expressed by HLA-B7-specific clones, monoclonal antibodies (mAbs) were selected after mice immunization with such clones. Two of these mAbs, termed XA-88 and XA-185, and their F(ab')2 fragments, were found to reconstitute lysis of B7+ target cells by B7-specific NK clones. Both mAbs were shown to be directed against the recently clustered Kp43 molecule (CD94). Thus, mAb-mediated masking of Kp43 molecules interferes with recognition of HLA-B7 and results in target cell lysis. Moreover, in a redirected killing assay, the cross- linking of Kp43 molecules mediated by the XA185 mAb strongly inhibited the cytolytic activity of HLA-B7-specific NK clones, thus mimicking the functional effect of B7 molecules. Taken together, these data strongly suggest that Kp43 molecules function as receptors for HLA-B7 and that this receptor/ligand interaction results in inhibition of the NK- mediated cytolytic activity. Indirect immunofluorescence and FACS analysis of a large number of random NK clones showed that Kp43 molecules (a) were brightly expressed on a subset of p58-negative clones, corresponding to those specific for HLA-B7; (b) displayed a medium/low fluorescence in the p58-negative clones that are not B7- specific as well as in most p58+ NK clones; and (c) were brightly expressed as in the p58+ clone ET34 (GL183-/EB6+, Cw4-specific

  15. A common minimal motif for the ligands of HLA-B*27 class I molecules.

    PubMed

    Barriga, Alejandro; Lorente, Elena; Johnstone, Carolina; Mir, Carmen; del Val, Margarita; López, Daniel

    2014-01-01

    CD8(+) T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development.

  16. Epithelial cell adhesion molecule aptamer functionalized PLGA-lecithin-curcumin-PEG nanoparticles for targeted drug delivery to human colorectal adenocarcinoma cells

    PubMed Central

    Li, Lei; Xiang, Dongxi; Shigdar, Sarah; Yang, Wenrong; Li, Qiong; Lin, Jia; Liu, Kexin; Duan, Wei

    2014-01-01

    To improve the efficacy of drug delivery, active targeted nanotechnology-based drug delivery systems are gaining considerable attention as they have the potential to reduce side effects, minimize toxicity, and improve efficacy of anticancer treatment. In this work CUR-NPs (curcumin-loaded lipid-polymer-lecithin hybrid nanoparticles) were synthesized and functionalized with ribonucleic acid (RNA) Aptamers (Apts) against epithelial cell adhesion molecule (EpCAM) for targeted delivery to colorectal adenocarcinoma cells. These CUR-encapsulated bioconjugates (Apt-CUR-NPs) were characterized for particle size, zeta potential, drug encapsulation, stability, and release. The in vitro specific cell binding, cellular uptake, and cytotoxicity of Apt-CUR-NPs were also studied. The Apt-CUR-NP bioconjugates exhibited increased binding to HT29 colon cancer cells and enhancement in cellular uptake when compared to CUR-NPs functionalized with a control Apt (P<0.01). Furthermore, a substantial improvement in cytotoxicity was achieved toward HT29 cells with Apt-CUR-NP bioconjugates. The encapsulation of CUR in Apt-CUR-NPs resulted in the increased bioavailability of delivered CUR over a period of 24 hours compared to that of free CUR in vivo. These results show that the EpCAM Apt-functionalized CUR-NPs enhance the targeting and drug delivery of CUR to colorectal cancer cells. Further development of CUR-encapsulated, nanosized carriers will lead to improved targeted delivery of novel chemotherapeutic agents to colorectal cancer cells. PMID:24591829

  17. Dynamics of molecules in extreme rotational states

    PubMed Central

    Yuan, Liwei; Teitelbaum, Samuel W.; Robinson, Allison; Mullin, Amy S.

    2011-01-01

    We have constructed an optical centrifuge with a pulse energy that is more than 2 orders of magnitude larger than previously reported instruments. This high pulse energy enables us to create large enough number densities of molecules in extreme rotational states to perform high-resolution state-resolved transient IR absorption measurements. Here we report the first studies of energy transfer dynamics involving molecules in extreme rotational states. In these studies, the optical centrifuge drives CO2 molecules into states with J ∼ 220 and we use transient IR probing to monitor the subsequent rotational, translational, and vibrational energy flow dynamics. The results reported here provide the first molecular insights into the relaxation of molecules with rotational energy that is comparable to that of a chemical bond.

  18. Bitter and sweet tasting molecules: It's complicated.

    PubMed

    Di Pizio, Antonella; Ben Shoshan-Galeczki, Yaron; Hayes, John E; Niv, Masha Y

    2018-04-19

    "Bitter" and "sweet" are frequently framed in opposition, both functionally and metaphorically, in regard to affective responses, emotion, and nutrition. This oppositional relationship is complicated by the fact that some molecules are simultaneously bitter and sweet. In some cases, a small chemical modification, or a chirality switch, flips the taste from sweet to bitter. Molecules humans describe as bitter are recognized by a 25-member subfamily of class A G-protein coupled receptors (GPCRs) known as TAS2Rs. Molecules humans describe as sweet are recognized by a TAS1R2/TAS1R3 heterodimer of class C GPCRs. Here we characterize the chemical space of bitter and sweet molecules: the majority of bitter compounds show higher hydrophobicity compared to sweet compounds, while sweet molecules have a wider range of sizes. Importantly, recent evidence indicates that TAS1Rs and TAS2Rs are not limited to the oral cavity; moreover, some bitterants are pharmacologically promiscuous, with the hERG potassium channel, cytochrome P450 enzymes, and carbonic anhydrases as common off-targets. Further focus on polypharmacology may unravel new physiological roles for tastant molecules. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Reactive oxygen species, essential molecules, during plant-pathogen interactions.

    PubMed

    Camejo, Daymi; Guzmán-Cedeño, Ángel; Moreno, Alexander

    2016-06-01

    Reactive oxygen species (ROS) are continually generated as a consequence of the normal metabolism in aerobic organisms. Accumulation and release of ROS into cell take place in response to a wide variety of adverse environmental conditions including salt, temperature, cold stresses and pathogen attack, among others. In plants, peroxidases class III, NADPH oxidase (NOX) locates in cell wall and plasma membrane, respectively, may be mainly enzymatic systems involving ROS generation. It is well documented that ROS play a dual role into cells, acting as important signal transduction molecules and as toxic molecules with strong oxidant power, however some aspects related to its function during plant-pathogen interactions remain unclear. This review focuses on the principal enzymatic systems involving ROS generation addressing the role of ROS as signal molecules during plant-pathogen interactions. We described how the chloroplasts, mitochondria and peroxisomes perceive the external stimuli as pathogen invasion, and trigger resistance response using ROS as signal molecule. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. NBBA, a synthetic small molecule, inhibits TNF-{alpha}-induced angiogenesis by suppressing the NF-{kappa}B signaling pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Nam Hee; Jung, Hye Jin; Shibasaki, Futoshi

    2010-01-15

    Nuclear factor-{kappa}B (NF-{kappa}B) is a crucial transcription factor that contributes to cancer development by regulating a number of genes involved in angiogenesis and tumorigenesis. Here, we describe (Z)-N-(3-(7-nitro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)benzylidene) propan-2-amine oxide (NBBA) as a new anti-angiogenic small molecule that targets NF-{kappa}B activity. NBBA showed stronger growth inhibition on human umbilical vein endothelial cells (HUVECs) than on the cancer cell lines we tested. Moreover, NBBA inhibited tumor necrosis factor-alpha (TNF-{alpha})-induced tube formation and invasion of HUVECs. In addition, NBBA suppressed the neovascularization of chorioallantonic membrane from growing chick embryos in vivo. To address the mode of action of the compound, the effectmore » of NBBA on TNF-{alpha}-induced NF-{kappa}B transcription activity was investigated. NBBA suppressed TNF-{alpha}-induced c-Jun N-terminal kinase phosphorylation, which resulted in suppression of transcription of NF-{kappa}B and its target genes, including interleukin-8, interleukin-1{alpha}, and epidermal growth factor. Collectively, these results demonstrated that NBBA is a new anti-angiogenic small molecule that targets the NF-{kappa}B signaling pathway.« less

  1. Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit.

    PubMed

    Costales, Matthew G; Haga, Christopher L; Velagapudi, Sai Pradeep; Childs-Disney, Jessica L; Phinney, Donald G; Disney, Matthew D

    2017-03-08

    A hypoxic state is critical to the metastatic and invasive characteristics of cancer. Numerous pathways play critical roles in cancer maintenance, many of which include noncoding RNAs such as microRNA (miR)-210 that regulates hypoxia inducible factors (HIFs). Herein, we describe the identification of a small molecule named Targapremir-210 that binds to the Dicer site of the miR-210 hairpin precursor. This interaction inhibits production of the mature miRNA, derepresses glycerol-3-phosphate dehydrogenase 1-like enzyme (GPD1L), a hypoxia-associated protein negatively regulated by miR-210, decreases HIF-1α, and triggers apoptosis of triple negative breast cancer cells only under hypoxic conditions. Further, Targapremir-210 inhibits tumorigenesis in a mouse xenograft model of hypoxic triple negative breast cancer. Many factors govern molecular recognition of biological targets by small molecules. For protein, chemoproteomics and activity-based protein profiling are invaluable tools to study small molecule target engagement and selectivity in cells. Such approaches are lacking for RNA, leaving a void in the understanding of its druggability. We applied Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP) to study the cellular selectivity and the on- and off-targets of Targapremir-210. Targapremir-210 selectively recognizes the miR-210 precursor and can differentially recognize RNAs in cells that have the same target motif but have different expression levels, revealing this important feature for selectively drugging RNAs for the first time. These studies show that small molecules can be rapidly designed to selectively target RNAs and affect cellular responses to environmental conditions, resulting in favorable benefits against cancer. Further, they help define rules for identifying druggable targets in the transcriptome.

  2. Combining functional genomics and chemical biology to identify targets of bioactive compounds.

    PubMed

    Ho, Cheuk Hei; Piotrowski, Jeff; Dixon, Scott J; Baryshnikova, Anastasia; Costanzo, Michael; Boone, Charles

    2011-02-01

    Genome sequencing projects have revealed thousands of suspected genes, challenging researchers to develop efficient large-scale functional analysis methodologies. Determining the function of a gene product generally requires a means to alter its function. Genetically tractable model organisms have been widely exploited for the isolation and characterization of activating and inactivating mutations in genes encoding proteins of interest. Chemical genetics represents a complementary approach involving the use of small molecules capable of either inactivating or activating their targets. Saccharomyces cerevisiae has been an important test bed for the development and application of chemical genomic assays aimed at identifying targets and modes of action of known and uncharacterized compounds. Here we review yeast chemical genomic assays strategies for drug target identification. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Stabilizing photoassociated Cs2 molecules by optimal control

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Xie, Ting; Huang, Yin; Wang, Gao-Ren; Cong, Shu-Lin

    2013-01-01

    We demonstrate theoretically that photoassociated molecules can be stabilized to deeply bound states. This process is achieved by transferring the population from the outer well to the inner well using the optimal control theory, the Cs2 molecule is taken as an example. Numerical calculations show that weakly bound molecules formed in the outer well by a pump pulse can be compressed to the inner well via a vibrational level of the ground electronic state as an intermediary by an additionally optimized laser pulse. The positively chirped pulse can enhance the population of the target state. With a transform-limited dump pulse, nearly all the photoassociated molecules in the inner well of the excited electronic state can be transferred to the deeply vibrational level of the ground electronic state.

  4. MicroRNA-320 involves in the cardioprotective effect of insulin against myocardial ischemia by targeting survivin.

    PubMed

    Yang, Ni; Wu, Liuzhong; Zhao, Ying; Zou, Ning; Liu, Chunfeng

    2018-04-01

    It is generally accepted that insulin exerts an antiapoptotic effect against ischemia/reperfusion through the activation of PI3K/Akt/mTOR pathway. MicroRNAs involve in multiple cardiac pathophysiological processes, including ischemia/reperfusion-induced cardiac injury. However, the regulation of microRNAs in the cardioprotective effect of insulin is rarely discussed. In this study, using a cell model of ischemia through culturing H9C2 cardiac myocytes in serum-free medium with hypoxia, we demonstrated that pretreatment with insulin significantly inhibited cell apoptosis and downregulated microRNA-320 (miR-320) expression. Interestingly, miR-320 mimic impaired the cardioprotective effect of insulin against myocardial ischemia injury by targeting survivin, which is a member of the family of inhibitor of apoptosis proteins. Suppression miR-320 expression by miR-320 inhibitor in H9C2 cells with myocardial ischemia mimics the cardioprotective effect of insulin by maintaining survivin expression. Taken together, miR-320-mediated survivin expression involves in cardioprotective effect of insulin against myocardial ischemia injury. Myocardial ischemia/reperfusion (I/R) injury remains an important clinical problem with extremely deficient clinical therapies. Insulin exerts an antiapoptotic effect against I/R through the activation of PI3K/Akt/mTOR pathway. Here, we provided evidences to show that microRNA-320 involves in the cardioprotective effect of insulin by targeting survivin, which is an inhibitor of apoptosis protein and functions as a key regulator in cell apoptosis and involves in the tumour genesis and progression. Our findings may provide a new potential therapeutic strategy for I/R injury and ischemic heart disease. Copyright © 2018 John Wiley & Sons, Ltd.

  5. Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules.

    PubMed

    Chapman, Daniel C; Stocki, Pawel; Williams, David B

    2015-01-01

    Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α1-antitrypsin.

  6. Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules

    PubMed Central

    Chapman, Daniel C.; Stocki, Pawel; Williams, David B.

    2015-01-01

    Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α1-antitrypsin. PMID:26691022

  7. Small Molecules Targeting the miRNA-Binding Domain of Argonaute 2: From Computer-Aided Molecular Design to RNA Immunoprecipitation.

    PubMed

    Bellissimo, Teresa; Masciarelli, Silvia; Poser, Elena; Genovese, Ilaria; Del Rio, Alberto; Colotti, Gianni; Fazi, Francesco

    2017-01-01

    The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein. Here, we describe these two methodologies that we recently used to select a specific compound able to interfere with the AGO2 functional activity and able to improve the retinoic acid-dependent myeloid differentiation of leukemic cells.

  8. Single molecule techniques in DNA repair: A primer

    PubMed Central

    Hughes, Craig D.; Simons, Michelle; Mackenzie, Cassidy E.; Van Houten, Bennett; Kad, Neil M.

    2016-01-01

    A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair. PMID:24819596

  9. HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi

    PubMed Central

    Palmeira, Vanila F.; Alviano, Daniela S.; Braga-Silva, Lys A.; Goulart, Fátima R. V.; Granato, Marcela Q.; Rozental, Sonia; Alviano, Celuta S.; Santos, André L. S.; Kneipp, Lucimar F.

    2017-01-01

    Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics. PMID:28579986

  10. HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi.

    PubMed

    Palmeira, Vanila F; Alviano, Daniela S; Braga-Silva, Lys A; Goulart, Fátima R V; Granato, Marcela Q; Rozental, Sonia; Alviano, Celuta S; Santos, André L S; Kneipp, Lucimar F

    2017-01-01

    Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti- F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi . In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics.

  11. Comprehensive peptidomimetic libraries targeting protein-protein interactions.

    PubMed

    Whitby, Landon R; Boger, Dale L

    2012-10-16

    Transient protein-protein interactions (PPIs) are essential components in cellular signaling pathways as well as in important processes such as viral infection, replication, and immune suppression. The unknown or uncharacterized PPIs involved in such interaction networks often represent compelling therapeutic targets for drug discovery. To date, however, the main strategies for discovery of small molecule modulators of PPIs are typically limited to structurally characterized targets. Recent developments in molecular scaffolds that mimic the side chain display of peptide secondary structures have yielded effective designs, but few screening libraries of such mimetics are available to interrogate PPI targets. We initiated a program to prepare a comprehensive small molecule library designed to mimic the three major recognition motifs that mediate PPIs (α-helix, β-turn, and β-strand). Three libraries would be built around templates designed to mimic each such secondary structure and substituted with all triplet combinations of groups representing the 20 natural amino acid side chains. When combined, the three libraries would contain a member capable of mimicking the key interaction and recognition residues of most targetable PPIs. In this Account, we summarize the results of the design, synthesis, and validation of an 8000 member α-helix mimetic library and a 4200 member β-turn mimetic library. We expect that the screening of these libraries will not only provide lead structures against α-helix- or β-turn-mediated protein-protein or peptide-receptor interactions, even if the nature of the interaction is unknown, but also yield key insights into the recognition motif (α-helix or β-turn) and identify the key residues mediating the interaction. Consistent with this expectation, the screening of the libraries against p53/MDM2 and HIV-1 gp41 (α-helix mimetic library) or the opioid receptors (β-turn mimetic library) led to the discovery of library members expected

  12. A general strategy to construct small molecule biosensors in eukaryotes.

    PubMed

    Feng, Justin; Jester, Benjamin W; Tinberg, Christine E; Mandell, Daniel J; Antunes, Mauricio S; Chari, Raj; Morey, Kevin J; Rios, Xavier; Medford, June I; Church, George M; Fields, Stanley; Baker, David

    2015-12-29

    Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

  13. Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters

    PubMed Central

    Doliwa, Christelle; Escotte-Binet, Sandie; Aubert, Dominique; Sauvage, Virginie; Velard, Frédéric; Schmid, Aline; Villena, Isabelle

    2013-01-01

    Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-RSDZ and ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied. PMID:23707894

  14. Design of small-molecule epigenetic modulators

    PubMed Central

    Pachaiyappan, Boobalan

    2013-01-01

    The field of epigenetics has expanded rapidly to reveal multiple new targets for drug discovery. The functional elements of the epigenomic machinery can be catagorized as writers, erasers and readers, and together these elements control cellular gene expression and homeostasis. It is increasingly clear that aberrations in the epigenome can underly a variety of diseases, and thus discovery of small molecules that modulate the epigenome in a specific manner is a viable approach to the discovery of new therapeutic agents. In this Digest, the components of epigenetic control of gene expression will be briefly summarized, and efforts to identify small molecules that modulate epigenetic processes will be described. PMID:24300735

  15. Utilizing random Forest QSAR models with optimized parameters for target identification and its application to target-fishing server.

    PubMed

    Lee, Kyoungyeul; Lee, Minho; Kim, Dongsup

    2017-12-28

    The identification of target molecules is important for understanding the mechanism of "target deconvolution" in phenotypic screening and "polypharmacology" of drugs. Because conventional methods of identifying targets require time and cost, in-silico target identification has been considered an alternative solution. One of the well-known in-silico methods of identifying targets involves structure activity relationships (SARs). SARs have advantages such as low computational cost and high feasibility; however, the data dependency in the SAR approach causes imbalance of active data and ambiguity of inactive data throughout targets. We developed a ligand-based virtual screening model comprising 1121 target SAR models built using a random forest algorithm. The performance of each target model was tested by employing the ROC curve and the mean score using an internal five-fold cross validation. Moreover, recall rates for top-k targets were calculated to assess the performance of target ranking. A benchmark model using an optimized sampling method and parameters was examined via external validation set. The result shows recall rates of 67.6% and 73.9% for top-11 (1% of the total targets) and top-33, respectively. We provide a website for users to search the top-k targets for query ligands available publicly at http://rfqsar.kaist.ac.kr . The target models that we built can be used for both predicting the activity of ligands toward each target and ranking candidate targets for a query ligand using a unified scoring scheme. The scores are additionally fitted to the probability so that users can estimate how likely a ligand-target interaction is active. The user interface of our web site is user friendly and intuitive, offering useful information and cross references.

  16. Optimization of anti-cancer drugs and a targeting molecule on multifunctional gold nanoparticles.

    PubMed

    Rizk, Nahla; Christoforou, Nicolas; Lee, Sungmun

    2016-05-06

    Breast cancer is the most common and deadly cancer among women worldwide. Currently, nanotechnology-based drug delivery systems are useful for cancer treatment; however, strategic planning is critical in order to enhance the anti-cancer properties and reduce the side effects of cancer therapy. Here, we designed multifunctional gold nanoparticles (AuNPs) conjugated with two anti-cancer drugs, TGF-β1 antibody and methotrexate, and a cancer-targeting molecule, folic acid. First, optimum size and shape of AuNPs was selected by the highest uptake of AuNPs by MDA-MB-231, a metastatic human breast cancer cell line. It was 100 nm spherical AuNPs (S-AuNPs) that were used for further studies. A fixed amount (900 μl) of S-AuNP (3.8 × 10(8) particles/ml) was conjugated with folic acid-BSA or methotrexate-BSA. Methotrexate on S-AuNP induced cellular toxicity and the optimum amount of methotrexate-BSA (2.83 mM) was 500 μl. Uptake of S-AuNPs was enhanced by folate conjugation that binds to folate receptors overexpressed by MDA-MB-231 and the optimum uptake was at 500 μl of folic acid-BSA (2.83 mM). TGF-β1 antibody on S-AuNP reduced extracellular TGF-β1 of cancer cells by 30%. Due to their efficacy and tunable properties, we anticipate numerous clinical applications of multifunctional gold nanospheres in treating breast cancer.

  17. Optimization of anti-cancer drugs and a targeting molecule on multifunctional gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Rizk, Nahla; Christoforou, Nicolas; Lee, Sungmun

    2016-05-01

    Breast cancer is the most common and deadly cancer among women worldwide. Currently, nanotechnology-based drug delivery systems are useful for cancer treatment; however, strategic planning is critical in order to enhance the anti-cancer properties and reduce the side effects of cancer therapy. Here, we designed multifunctional gold nanoparticles (AuNPs) conjugated with two anti-cancer drugs, TGF-β1 antibody and methotrexate, and a cancer-targeting molecule, folic acid. First, optimum size and shape of AuNPs was selected by the highest uptake of AuNPs by MDA-MB-231, a metastatic human breast cancer cell line. It was 100 nm spherical AuNPs (S-AuNPs) that were used for further studies. A fixed amount (900 μl) of S-AuNP (3.8 × 108 particles/ml) was conjugated with folic acid-BSA or methotrexate-BSA. Methotrexate on S-AuNP induced cellular toxicity and the optimum amount of methotrexate-BSA (2.83 mM) was 500 μl. Uptake of S-AuNPs was enhanced by folate conjugation that binds to folate receptors overexpressed by MDA-MB-231 and the optimum uptake was at 500 μl of folic acid-BSA (2.83 mM). TGF-β1 antibody on S-AuNP reduced extracellular TGF-β1 of cancer cells by 30%. Due to their efficacy and tunable properties, we anticipate numerous clinical applications of multifunctional gold nanospheres in treating breast cancer.

  18. Spectroscopic Characterization of Key Aromatic Molecules: A Route toward The Origin of Life.

    PubMed

    Puzzarini, Cristina; Baiardi, Alberto; Bloino, Julien; Barone, Vincenzo; Murphy, Thomas E; Drew, Dennis; Ali, Ashraf

    2017-08-04

    To gain information on the abiotic synthesis of the building blocks of life from simple molecules, and their subsequent chemical evolution to biological systems, the starting point is the identification of target species in Titan-like planets, i.e., planets that resemble the primitive Earth, as well as in Earth-like planets in the habitable zone of their star, namely planets where life can be already originated. In this scenario, molecular spectroscopy plays a crucial role because spectroscopic signatures are at the basis of an unequivocal proof for the presence of these target molecules. Thanks to the advances in many different techniques and to the NASA successful Kepler exoplanet transit mission, thousands of diverse planets outside of our solar system have been discovered. The James Webb Space Telescope (JWST), scheduled to be launched in 2018, will be very helpful in the identification of biosignature gases in Earth-like planets' atmospheres and of prebiotic molecule signatures in Titan-like atmospheres by observing their absorption during transits. While the search for key-target molecules in exoplanet atmospheres can be carried out by the JWST Transit Spectroscopy in the infrared (IR) region (0.6 - 29 µm wavelength range), opportunities for their detection in protostellar cores, protoplanetary disks and on Titan are also offered by the interferometric high spectral and spatial resolution observations using the Atacama Large Millimeter/submillimeter Array (ALMA). In the present work, target molecules have been selected and their spectroscopic characterization presented in view of supporting their infrared and complementary millimeter/submillimeter-wave spectral observations. In detail, the selected target molecules include: (1) the three-membered oxygen-containing heterocycles: oxirane and protonated oxirane, (2) the cyclopropenyl cation and its methyl derivative, (3) two examples of ortho- and peri-fused tri-cyclic aromatic rings, i.e., the phenalenyl

  19. Inhibiting NF-κB Activation by Small Molecules As a Therapeutic Strategy

    PubMed Central

    Gupta, Subash C; Sundaram, Chitra; Reuter, Simone; Aggarwal, Bharat B

    2010-01-01

    Because nuclear factor-κB (NF-κB) is a ubiquitously expressed proinflammatory transcription factor that regulates the expression of over 500 genes involved in cellular transformation, survival, proliferation, invasion, angiogenesis, metastasis, and inflammation, the NF-κB signaling pathway has become a potential target for pharmacological intervention. A wide variety of agents can activate NF-κB through canonical and noncanonical pathways. Canonical pathway involves various steps including the phosphorylation, ubiquitnation, and degradation of the inhibitor of NF-κB (IκBα), which leads to the nuclear translocation of the p50- p65 subunits of NF-κB followed by p65 phosphorylation, acetylation and methylation, DNA binding, and gene transcription. Thus, agents that can inhibit protein kinases, protein phosphatases, proteasomes, ubiquitnation, acetylation, methylation, and DNA binding steps have been identified as NF-κB inhibitors. Here, we review the small molecules that suppress NF-κB activation and thus may have therapeutic potential. PMID:20493977

  20. Multivalent small molecule pan-RAS inhibitors

    PubMed Central

    Welsch, Matthew E.; Kaplan, Anna; Chambers, Jennifer M.; Stokes, Michael E.; Bos, Pieter H.; Zask, Arie; Zhang, Yan; Sanchez-Martin, Marta; Badgley, Michael A.; Huang, Christine S.; Tran, Timothy H.; Akkiraju, Hemanth; Brown, Lewis M.; Nandakumar, Renu; Cremers, Serge; Yang, Wan S.; Tong, Liang; Olive, Kenneth P.; Ferrando, Adolfo; Stockwell, Brent R.

    2017-01-01

    SUMMARY Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, have potential use as chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers, and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins. PMID:28235199

  1. MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors

    PubMed Central

    Birsoy, Kivanc; Wang, Tim; Possemato, Richard; Yilmaz, Omer H.; Koch, Catherine E.; Chen, Walter W.; Hutchins, Amanda W.; Gultekin, Yetis; Peterson, Tim R.; Carette, Jan E.; Brummelkamp, Thijn R.; Clish, Clary B.; Sabatini, David M.

    2012-01-01

    SUMMARY There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anti-cancer therapy are underway. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, MCT1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Lastly, forced MCT1 expression in 3-BrPA resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors. PMID:23202129

  2. Design of a small molecule against an oncogenic noncoding RNA.

    PubMed

    Velagapudi, Sai Pradeep; Cameron, Michael D; Haga, Christopher L; Rosenberg, Laura H; Lafitte, Marie; Duckett, Derek R; Phinney, Donald G; Disney, Matthew D

    2016-05-24

    The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.

  3. Design of a small molecule against an oncogenic noncoding RNA

    PubMed Central

    Velagapudi, Sai Pradeep; Cameron, Michael D.; Haga, Christopher L.; Rosenberg, Laura H.; Lafitte, Marie; Duckett, Derek R.; Phinney, Donald G.; Disney, Matthew D.

    2016-01-01

    The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif–small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm. PMID:27170187

  4. Development of a reference material of a single DNA molecule for the quality control of PCR testing.

    PubMed

    Mano, Junichi; Hatano, Shuko; Futo, Satoshi; Yoshii, Junji; Nakae, Hiroki; Naito, Shigehiro; Takabatake, Reona; Kitta, Kazumi

    2014-09-02

    We developed a reference material of a single DNA molecule with a specific nucleotide sequence. The double-strand linear DNA which has PCR target sequences at the both ends was prepared as a reference DNA molecule, and we named the PCR targets on each side as confirmation sequence and standard sequence. The highly diluted solution of the reference molecule was dispensed into 96 wells of a plastic PCR plate to make the average number of molecules in a well below one. Subsequently, the presence or absence of the reference molecule in each well was checked by real-time PCR targeting for the confirmation sequence. After an enzymatic treatment of the reaction mixture in the positive wells for the digestion of PCR products, the resultant solution was used as the reference material of a single DNA molecule with the standard sequence. PCR analyses revealed that the prepared samples included only one reference molecule with high probability. The single-molecule reference material developed in this study will be useful for the absolute evaluation of a detection limit of PCR-based testing methods, the quality control of PCR analyses, performance evaluations of PCR reagents and instruments, and the preparation of an accurate calibration curve for real-time PCR quantitation.

  5. The Molecular Industrial Revolution: Automated Synthesis of Small Molecules.

    PubMed

    Trobe, Melanie; Burke, Martin D

    2018-04-09

    Today we are poised for a transition from the highly customized crafting of specific molecular targets by hand to the increasingly general and automated assembly of different types of molecules with the push of a button. Creating machines that are capable of making many different types of small molecules on demand, akin to that which has been achieved on the macroscale with 3D printers, is challenging. Yet important progress is being made toward this objective with two complementary approaches: 1) Automation of customized synthesis routes to different targets by machines that enable the use of many reactions and starting materials, and 2) automation of generalized platforms that make many different targets using common coupling chemistry and building blocks. Continued progress in these directions has the potential to shift the bottleneck in molecular innovation from synthesis to imagination, and thereby help drive a new industrial revolution on the molecular scale. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Catalytic in vivo protein knockdown by small-molecule PROTACs

    PubMed Central

    Bondeson, Daniel P; Mares, Alina; Smith, Ian E D; Ko, Eunhwa; Campos, Sebastien; Miah, Afjal H; Mulholland, Katie E; Routly, Natasha; Buckley, Dennis L; Gustafson, Jeffrey L; Zinn, Nico; Grandi, Paola; Shimamura, Satoko; Bergamini, Giovanna; Faelth-Savitski, Maria; Bantscheff, Marcus; Cox, Carly; Gordon, Deborah A; Willard, Ryan R; Flanagan, John J; Casillas, Linda N; Votta, Bartholomew J; den Besten, Willem; Famm, Kristoffer; Kruidenier, Laurens; Carter, Paul S; Harling, John D; Churcher, Ian; Crews, Craig M

    2015-01-01

    The current predominant theapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target’s ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR. PMID:26075522

  7. TargetNet: a web service for predicting potential drug-target interaction profiling via multi-target SAR models.

    PubMed

    Yao, Zhi-Jiang; Dong, Jie; Che, Yu-Jing; Zhu, Min-Feng; Wen, Ming; Wang, Ning-Ning; Wang, Shan; Lu, Ai-Ping; Cao, Dong-Sheng

    2016-05-01

    Drug-target interactions (DTIs) are central to current drug discovery processes and public health fields. Analyzing the DTI profiling of the drugs helps to infer drug indications, adverse drug reactions, drug-drug interactions, and drug mode of actions. Therefore, it is of high importance to reliably and fast predict DTI profiling of the drugs on a genome-scale level. Here, we develop the TargetNet server, which can make real-time DTI predictions based only on molecular structures, following the spirit of multi-target SAR methodology. Naïve Bayes models together with various molecular fingerprints were employed to construct prediction models. Ensemble learning from these fingerprints was also provided to improve the prediction ability. When the user submits a molecule, the server will predict the activity of the user's molecule across 623 human proteins by the established high quality SAR model, thus generating a DTI profiling that can be used as a feature vector of chemicals for wide applications. The 623 SAR models related to 623 human proteins were strictly evaluated and validated by several model validation strategies, resulting in the AUC scores of 75-100 %. We applied the generated DTI profiling to successfully predict potential targets, toxicity classification, drug-drug interactions, and drug mode of action, which sufficiently demonstrated the wide application value of the potential DTI profiling. The TargetNet webserver is designed based on the Django framework in Python, and is freely accessible at http://targetnet.scbdd.com .

  8. TargetNet: a web service for predicting potential drug-target interaction profiling via multi-target SAR models

    NASA Astrophysics Data System (ADS)

    Yao, Zhi-Jiang; Dong, Jie; Che, Yu-Jing; Zhu, Min-Feng; Wen, Ming; Wang, Ning-Ning; Wang, Shan; Lu, Ai-Ping; Cao, Dong-Sheng

    2016-05-01

    Drug-target interactions (DTIs) are central to current drug discovery processes and public health fields. Analyzing the DTI profiling of the drugs helps to infer drug indications, adverse drug reactions, drug-drug interactions, and drug mode of actions. Therefore, it is of high importance to reliably and fast predict DTI profiling of the drugs on a genome-scale level. Here, we develop the TargetNet server, which can make real-time DTI predictions based only on molecular structures, following the spirit of multi-target SAR methodology. Naïve Bayes models together with various molecular fingerprints were employed to construct prediction models. Ensemble learning from these fingerprints was also provided to improve the prediction ability. When the user submits a molecule, the server will predict the activity of the user's molecule across 623 human proteins by the established high quality SAR model, thus generating a DTI profiling that can be used as a feature vector of chemicals for wide applications. The 623 SAR models related to 623 human proteins were strictly evaluated and validated by several model validation strategies, resulting in the AUC scores of 75-100 %. We applied the generated DTI profiling to successfully predict potential targets, toxicity classification, drug-drug interactions, and drug mode of action, which sufficiently demonstrated the wide application value of the potential DTI profiling. The TargetNet webserver is designed based on the Django framework in Python, and is freely accessible at http://targetnet.scbdd.com.

  9. Probing Electronic and Thermoelectric Properties of Single Molecule Junctions

    NASA Astrophysics Data System (ADS)

    Widawsky, Jonathan R.

    In an effort to further understand electronic and thermoelectric phenomenon at the nanometer scale, we have studied the transport properties of single molecule junctions. To carry out these transport measurements, we use the scanning tunneling microscope-break junction (STM-BJ) technique, which involves the repeated formation and breakage of a metal point contact in an environment of the target molecule. Using this technique, we are able to create gaps that can trap the molecules, allowing us to sequentially and reproducibly create a large number of junctions. By applying a small bias across the junction, we can measure its conductance and learn about the transport mechanisms at the nanoscale. The experimental work presented here directly probes the transmission properties of single molecules through the systematic measurement of junction conductance (at low and high bias) and thermopower. We present measurements on a variety of molecular families and study how conductance depends on the character of the linkage (metal-molecule bond) and the nature of the molecular backbone. We start by describing a novel way to construct single molecule junctions by covalently connecting the molecular backbone to the electrodes. This eliminates the use of linking substituents, and as a result, the junction conductance increases substantially. Then, we compare transport across silicon chains (silanes) and saturated carbon chains (alkanes) while keeping the linkers the same and find a stark difference in their electronic transport properties. We extend our studies of molecular junctions by looking at two additional aspects of quantum transport -- molecular thermopower and molecular current-voltage characteristics. Each of these additional parameters gives us further insight into transport properties at the nanoscale. Evaluating the junction thermopower allows us to determine the nature of charge carriers in the system and we demonstrate this by contrasting the measurement of amine

  10. Transferrin receptors and the targeted delivery of therapeutic agents against cancer

    PubMed Central

    Daniels, Tracy R.; Bernabeu, Ezequiel; Rodríguez, José A.; Patel, Shabnum; Kozman, Maggie; Chiappetta, Diego A.; Holler, Eggehard; Ljubimova, Julia Y.; Helguera, Gustavo; Penichet, Manuel L.

    2012-01-01

    Background Traditional cancer therapy can be successful in destroying tumors, but can also cause dangerous side effects. Therefore, many targeted therapies are in development. The transferrin receptor (TfR) functions in cellular iron uptake through its interaction with transferrin. This receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and is efficiently internalized. This receptor can be targeted in two ways: 1) for the delivery of therapeutic molecules into malignant cells or 2) to block the natural function of the receptor leading directly to cancer cell death. Scope of review In the present article we discuss the strategies used to target the TfR for the delivery of therapeutic agents into cancer cells. We provide a summary of the vast types of anti-cancer drugs that have been delivered into cancer cells employing a variety of receptor binding molecules including Tf, anti-TfR antibodies, or TfR-binding peptides alone or in combination with carrier molecules including nanoparticles and viruses. Major conclusions Targeting the TfR has been shown to be effective in delivering many different therapeutic agents and causing cytotoxic effects in cancer cells in vitro and in vivo. General significance The extensive use of TfR for targeted therapy attests to the versatility of targeting this receptor for therapeutic purposes against malignant cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR for cancer therapy leading to an increase in the number of clinical trials of molecules targeting this receptor. PMID:21851850

  11. Electron transmission through a class of anthracene aldehyde molecules

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Petreska, Irina, E-mail: irina.petreska@pmf.ukim.mk; Ohanesjan, Vladimir, E-mail: ohanesjan.vladimir@gmail.com; Pejov, Ljupco, E-mail: ljupcop@pmf.ukim.mk

    2016-03-25

    Transmission of electrons via metal-molecule-metal junctions, involving rotor-stator anthracene aldehyde molecules is investigated. Two model barriers having input parameters evaluated from accurate ab initio calculations are proposed and the transmission coefficients are obtained by using the quasiclassical approximation. Transmission coefficients further enter in the integral for the net current, utilizing Simmons’ method. Conformational dependence of the tunneling processes is evident and the presence of the side groups enhances the functionality of the future single-molecule based electronic devices.

  12. Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

    PubMed Central

    Zhen, Chao Yu; Tatavosian, Roubina; Huynh, Thao Ngoc; Duc, Huy Nguyen; Das, Raibatak; Kokotovic, Marko; Grimm, Jonathan B; Lavis, Luke D; Lee, Jun; Mejia, Frances J; Li, Yang; Yao, Tingting; Ren, Xiaojun

    2016-01-01

    The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes. DOI: http://dx.doi.org/10.7554/eLife.17667.001 PMID:27723458

  13. Therapeutic antibodies: market considerations, disease targets and bioprocessing.

    PubMed

    Elvin, John G; Couston, Ruairidh G; van der Walle, Christopher F

    2013-01-02

    Antibodies are well established in mainstream clinical practice and present an exciting area for collaborative research and development in industry and academia alike. In this review, we will provide an overview of the current market and an outlook to 2015, focussing on whole antibody molecules while acknowledging the next generation scaffolds containing variable fragments. The market will be discussed in the context of disease targets, particularly in the areas of oncology and immune disorders which generate the greatest revenue by a wide margin. Emerging targets include central nervous system disorders which will also stimulate new delivery strategies. It is becoming increasingly apparent that a better understanding of bioprocessing is required in order to optimize the steps involved in the preparation of a protein prior to formulation. The latter is outside the scope of this review and nor is it our intention to discuss protein delivery and pharmacokinetics. The challenges that lie ahead include the discovery of new disease targets and the development of robust bioprocessing operations. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  14. Ligand cluster-based protein network and ePlatton, a multi-target ligand finder.

    PubMed

    Du, Yu; Shi, Tieliu

    2016-01-01

    Small molecules are information carriers that make cells aware of external changes and couple internal metabolic and signalling pathway systems with each other. In some specific physiological status, natural or artificial molecules are used to interact with selective biological targets to activate or inhibit their functions to achieve expected biological and physiological output. Millions of years of evolution have optimized biological processes and pathways and now the endocrine and immune system cannot work properly without some key small molecules. In the past thousands of years, the human race has managed to find many medicines against diseases by trail-and-error experience. In the recent decades, with the deepening understanding of life and the progress of molecular biology, researchers spare no effort to design molecules targeting one or two key enzymes and receptors related to corresponding diseases. But recent studies in pharmacogenomics have shown that polypharmacology may be necessary for the effects of drugs, which challenge the paradigm, 'one drug, one target, one disease'. Nowadays, cheminformatics and structural biology can help us reasonably take advantage of the polypharmacology to design next-generation promiscuous drugs and drug combination therapies. 234,591 protein-ligand interactions were extracted from ChEMBL. By the 2D structure similarity, 13,769 ligand emerged from 156,151 distinct ligands which were recognized by 1477 proteins. Ligand cluster- and sequence-based protein networks (LCBN, SBN) were constructed, compared and analysed. For assisting compound designing, exploring polypharmacology and finding possible drug combination, we integrated the pathway, disease, drug adverse reaction and the relationship of targets and ligand clusters into the web platform, ePlatton, which is available at http://www.megabionet.org/eplatton. Although there were some disagreements between the LCBN and SBN, communities in both networks were largely the same

  15. In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

    PubMed

    Martini, Laura; Meyer, Adam J; Ellefson, Jared W; Milligan, John N; Forlin, Michele; Ellington, Andrew D; Mansy, Sheref S

    2015-10-16

    An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

  16. A general strategy to construct small molecule biosensors in eukaryotes

    DOE PAGES

    Feng, Justin; Jester, Benjamin W.; Tinberg, Christine E.; ...

    2015-12-29

    Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activatesmore » transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. As a result, this work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.« less

  17. A general strategy to construct small molecule biosensors in eukaryotes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Feng, Justin; Jester, Benjamin W.; Tinberg, Christine E.

    Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activatesmore » transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. As a result, this work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.« less

  18. A general strategy to construct small molecule biosensors in eukaryotes

    PubMed Central

    Feng, Justin; Jester, Benjamin W; Tinberg, Christine E; Mandell, Daniel J; Antunes, Mauricio S; Chari, Raj; Morey, Kevin J; Rios, Xavier; Medford, June I; Church, George M; Fields, Stanley; Baker, David

    2015-01-01

    Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.10606.001 PMID:26714111

  19. How reliable are ligand-centric methods for Target Fishing?

    NASA Astrophysics Data System (ADS)

    Peon, Antonio; Dang, Cuong; Ballester, Pedro

    2016-04-01

    Computational methods for Target Fishing (TF), also known as Target Prediction or Polypharmacology Prediction, can be used to discover new targets for small-molecule drugs. This may result in repositioning the drug in a new indication or improving our current understanding of its efficacy and side effects. While there is a substantial body of research on TF methods, there is still a need to improve their validation, which is often limited to a small part of the available targets and not easily interpretable by the user. Here we discuss how target-centric TF methods are inherently limited by the number of targets that can possibly predict (this number is by construction much larger in ligand-centric techniques). We also propose a new benchmark to validate TF methods, which is particularly suited to analyse how predictive performance varies with the query molecule. On average over approved drugs, we estimate that only five predicted targets will have to be tested to find two true targets with submicromolar potency (a strong variability in performance is however observed). In addition, we find that an approved drug has currently an average of eight known targets, which reinforces the notion that polypharmacology is a common and strong event. Furthermore, with the assistance of a control group of randomly-selected molecules, we show that the targets of approved drugs are generally harder to predict.

  20. Imbalanced immune responses involving inflammatory molecules and immune-related pathways in the lung of acute and subchronic arsenic-exposed mice.

    PubMed

    Li, Jinlong; Zhao, Lu; Zhang, Yang; Li, Wei; Duan, Xiaoxu; Chen, Jinli; Guo, Yuanyuan; Yang, Shan; Sun, Guifan; Li, Bing

    2017-11-01

    treatment. Conclusively, our present study demonstrated both acute and subchronic oral administration of arsenic triggers multiple pulmonary immune responses involving inflammatory molecules and T-cell differentiation, which might be closely associated with the imbalanced redox status and activation of immune-related MAPKs, NF-κB and anti-inflammatory NRF2 pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Targeted endothelial nanomedicine for common acute pathological conditions

    PubMed Central

    Shuvaev, Vladimir V.; Brenner, Jacob S.; Muzykantov, Vladimir R.

    2017-01-01

    Endothelium, a thin monolayer of specialized cells lining the lumen of blood vessels is the key regulatory interface between blood and tissues. Endothelial abnormalities are implicated in many diseases, including common acute conditions with high morbidity and mortality lacking therapy, in part because drugs and drug carriers have no natural endothelial affinity. Precise endothelial drug delivery may improve management of these conditions. Using ligands of molecules exposed to the bloodstream on the endothelial surface enables design of diverse targeted endothelial nanomedicine agents. Target molecules and binding epitopes must be accessible to drug carriers, carriers must be free of harmful effects, and targeting should provide desirable sub-cellular addressing of the drug cargo. The roster of current candidate target molecules for endothelial nanomedicine includes peptidases and other enzymes, cell adhesion molecules and integrins, localized in different domains of the endothelial plasmalemma and differentially distributed throughout the vasculature. Endowing carriers with an affinity to specific endothelial epitopes enables an unprecedented level of precision of control of drug delivery: binding to selected endothelial cell phenotypes, cellular addressing and duration of therapeutic effects. Features of nanocarrier design such as choice of epitope and ligand control delivery and effect of targeted endothelial nanomedicine agents. Pathological factors modulate endothelial targeting and uptake of nanocarriers. Selection of optimal binding sites and design features of nanocarriers are key controllable factors that can be iteratively engineered based on their performance from in vitro to pre-clinical in vivo experimental models. Targeted endothelial nanomedicine agents provide antioxidant, anti-inflammatory and other therapeutic effects unattainable by non-targeted counterparts in animal models of common acute severe human disease conditions. The results of animal

  2. Advances in aptamer screening and small molecule aptasensors.

    PubMed

    Kim, Yeon Seok; Gu, Man Bock

    2014-01-01

    It has been 20 years since aptamer and SELEX (systematic evolution of ligands by exponential enrichment) were described independently by Andrew Ellington and Larry Gold. Based on the great advantages of aptamers, there have been numerous isolated aptamers for various targets that have actively been applied as therapeutic and analytical tools. Over 2,000 papers related to aptamers or SELEX have been published, attesting to their wide usefulness and the applicability of aptamers. SELEX methods have been modified or re-created over the years to enable aptamer isolation with higher affinity and selectivity in more labor- and time-efficient manners, including automation. Initially, most of the studies about aptamers have focused on the protein targets, which have physiological functions in the body, and their applications as therapeutic agents or receptors for diagnostics. However, aptamers for small molecules such as organic or inorganic compounds, drugs, antibiotics, or metabolites have not been studied sufficiently, despite the ever-increasing need for rapid and simple analytical methods for various chemical targets in the fields of medical diagnostics, environmental monitoring, food safety, and national defense against targets including chemical warfare. This review focuses on not only recent advances in aptamer screening methods but also its analytical application for small molecules.

  3. Small Molecule Inhibitors in Acute Myeloid Leukemia: From the Bench to the Clinic

    PubMed Central

    Al-Hussaini, Muneera; DiPersio, John F.

    2014-01-01

    Many patients with acute myeloid leukemia (AML) will eventually develop refractory or relapsed disease. In the absence of standard therapy for this population, there is currently an urgent unmet need for novel therapeutic agents. Targeted therapy with small molecule inhibitors (SMIs) represents a new therapeutic intervention that has been successful for the treatment of multiple tumors (e.g., gastrointestinal stromal tumors, chronic myelogenous leukemia). Hence, there has been great interest in generating selective small molecule inhibitors targeting critical pathways of proliferation and survival in AML. This review highlights a selective group of intriguing therapeutic agents and their presumed targets in both preclinical models and in early human clinical trials. PMID:25025370

  4. Spectroscopic Characterization of Key Aromatic and Heterocyclic Molecules: A Route toward the Origin of Life

    NASA Astrophysics Data System (ADS)

    Puzzarini, Cristina; Baiardi, Alberto; Bloino, Julien; Barone, Vincenzo; Murphy, Thomas E.; Drew, H. Dennis; Ali, Ashraf

    2017-09-01

    To gain information on the abiotic synthesis of the building blocks of life from simple molecules, and their subsequent chemical evolution to biological systems, the starting point is the identification of target species in Titan-like planets; I.e., planets that resemble the primitive Earth, as well as in Earth-like planets in the habitable zone of their star, namely planets where life can be already originated. In this scenario, molecular spectroscopy plays a crucial role because spectroscopic signatures are at the basis of an unequivocal proof for the presence of these target molecules. Thanks to advances in many different techniques and NASA’s successful Kepler exoplanet transit mission, thousands of diverse planets outside of our solar system have been discovered. The James Webb Space Telescope (JWST), scheduled to be launched in 2018, will be very helpful in the identification of biosignature gases in Earth-like planets’ atmospheres and prebiotic molecule signatures in Titan-like atmospheres, by observing their absorption during transits. Although the search for key-target molecules in exoplanet atmospheres can be carried out by the JWST Transit Spectroscopy in the infrared (IR) region (0.6-29 μm wavelength range), opportunities for their detection in protostellar cores, protoplanetary disks, and on Titan are also offered by interferometric high spectral and spatial resolution observations using the Atacama Large Millimeter/submillimeter Array. In the present work, target molecules have been selected, and their spectroscopic characterization presented in view of supporting their infrared and complementary millimeter/submillimeter-wave spectral observations. In detail, the selected target molecules include: (1) the three-membered oxygen-containing heterocycles, oxirane and protonated oxirane; (2) the cyclopropenyl cation and its methyl derivative; (3) two examples of ortho- and peri-fused tri-cyclic aromatic rings, I.e., the phenalenyl cation ({{{C}}}13

  5. The neural cell adhesion molecule promotes FGFR-dependent phosphorylation and membrane targeting of the exocyst complex to induce exocytosis in growth cones.

    PubMed

    Chernyshova, Yana; Leshchyns'ka, Iryna; Hsu, Shu-Chan; Schachner, Melitta; Sytnyk, Vladimir

    2011-03-09

    The exocyst complex is an essential regulator of polarized exocytosis involved in morphogenesis of neurons. We show that this complex binds to the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes FGF receptor-mediated phosphorylation of two tyrosine residues in the sec8 subunit of the exocyst complex and is required for efficient recruitment of the exocyst complex to growth cones. NCAM at the surface of growth cones induces Ca(2+)-dependent vesicle exocytosis, which is blocked by an inhibitor of L-type voltage-dependent Ca(2+) channels and tetanus toxin. Preferential exocytosis in growth cones underlying neurite outgrowth is inhibited in NCAM-deficient neurons as well as in neurons transfected with phosphorylation-deficient sec8 and dominant-negative peptides derived from the intracellular domain of NCAM. Thus, we reveal a novel role for a cell adhesion molecule in that it regulates addition of the new membrane to the cell surface of growth cones in developing neurons.

  6. Trends in GPCR drug discovery: new agents, targets and indications.

    PubMed

    Hauser, Alexander S; Attwood, Misty M; Rask-Andersen, Mathias; Schiöth, Helgi B; Gloriam, David E

    2017-12-01

    G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, mostly due to their substantial involvement in human pathophysiology and their pharmacological tractability. Here, we report an up-to-date analysis of all GPCR drugs and agents in clinical trials, which reveals current trends across molecule types, drug targets and therapeutic indications, including showing that 475 drugs (~34% of all drugs approved by the US Food and Drug Administration (FDA)) act at 108 unique GPCRs. Approximately 321 agents are currently in clinical trials, of which ~20% target 66 potentially novel GPCR targets without an approved drug, and the number of biological drugs, allosteric modulators and biased agonists has increased. The major disease indications for GPCR modulators show a shift towards diabetes, obesity and Alzheimer disease, although several central nervous system disorders are also highly represented. The 224 (56%) non-olfactory GPCRs that have not yet been explored in clinical trials have broad untapped therapeutic potential, particularly in genetic and immune system disorders. Finally, we provide an interactive online resource to analyse and infer trends in GPCR drug discovery.

  7. Targeting Micrornas With Small Molecules: A Novel Approach to Treating Breast Cancer

    DTIC Science & Technology

    2010-10-01

    ribozymes and the DNAzymes, small interfering RNAs and short hairpin RNAs, and anti-miRNA agents such as antisense oligo- nucleotides, locked nucleic...of the antagomir Preclinical studies Ribozymes or DNAzymes A ribozyme , or RNA enzyme, is an RNA molecule that can catalyze a chemical reaction. A

  8. CRISPR Approaches to Small Molecule Target Identification. | Office of Cancer Genomics

    Cancer.gov

    A long-standing challenge in drug development is the identification of the mechanisms of action of small molecules with therapeutic potential. A number of methods have been developed to address this challenge, each with inherent strengths and limitations. We here provide a brief review of these methods with a focus on chemical-genetic methods that are based on systematically profiling the effects of genetic perturbations on drug sensitivity.

  9. Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

    PubMed Central

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; Ramanathan, Arvind; Zhang, Weixing; Shelat, Anang A.; Zuo, Jian; Kriwacki, Richard W.

    2015-01-01

    Disordered proteins are highly prevalent in biological systems, they control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule:disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of-principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A). PMID:26507530

  10. Ccdc3: A New P63 Target Involved in Regulation Of Liver Lipid Metabolism.

    PubMed

    Liao, Wenjuan; Liu, Hongbing; Zhang, Yiwei; Jung, Ji Hoon; Chen, Jiaxiang; Su, Xiaohua; Kim, Yeong C; Flores, Elsa R; Wang, San Ming; Czarny-Ratajczak, Malwina; Li, Wen; Zeng, Shelya X; Lu, Hua

    2017-08-21

    TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, upregulates CCDC3 expression by directly binding to its enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity, but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but conversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance and steatosis formation in transgenic CCDC3 mice on high-fat diet (HFD) by reducing the expression of hepatic PPARγ and its target gene CIDEA as well as other genes involved in de novo lipogenesis. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on HFD. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.

  11. Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis.

    PubMed

    Chen, Jia; He, Qing-Yu; Yuen, Anthony Po-Wing; Chiu, Jeng-Fu

    2004-08-01

    Squamous cell carcinoma (SCC) of the buccal mucosa is an aggressive oral cancer. It mainly occurs in Central and Southeast Asia, and is closely related to the practice of tobacco smoking and betel squid chewing. The high recurrence and low survival rates of buccal SCC require our continued efforts to understand the pathogenesis of the disease for designing better therapeutic strategies. We used proteomic technology to analyze buccal SCC tissues aiming at identifying tumor-associated proteins for the utilization as biomarkers or molecular targets. With the exception of alpha B-crystallin being substantially reduced, a number of proteins were found to be significantly over-expressed in cancer tissues. These increased proteins included glycolytic enzymes, heat-shock proteins, tumor antigens, cytoskeleton proteins, enzymes involved in detoxification and anti-oxidation systems, and proteins involved in mitochondrial and intracellular signaling pathways. These extensive protein variations indicate that multiple cellular pathways were involved in the process of tumorigenesis, and suggest that multiple protein molecules should be simultaneously targeted as an effective strategy to counter the disease. At least, SCC antigen, G protein, glutathione S-transferase, manganese superoxide dismutase, annexins, voltage-dependent anion channel, cyclophilin A, stratifin and galectin 7 are candidates for targeted proteins. The present findings also demonstrated that rich protein information can be produced by means of proteomic analysis for a better understanding of the oncogenesis and pathogenesis in a global way, which in turn is a basis for the rational designs of diagnostic and therapeutic methods.

  12. Therapeutic Targeting of Eosinophil Adhesion and Accumulation in Allergic Conjunctivitis

    PubMed Central

    Baiula, Monica; Bedini, Andrea; Carbonari, Gioia; Dattoli, Samantha Deianira; Spampinato, Santi

    2012-01-01

    Considerable evidence indicates that eosinophils are important effectors of ocular allergy. Increased worldwide prevalence of allergic eye pathologies has stimulated the identification of novel drug targets, including eosinophils and adhesion molecules. Accumulation of eosinophils in the eye is a key event in the onset and maintenance of allergic inflammation and is mediated by different adhesion molecules. Antihistamines with multiple mechanisms of action can be effective during the early and late phases of allergic conjunctivitis by blocking the interaction between β1 integrins and vascular cell adhesion molecule (VCAM)-1. Small molecule antagonists that target key elements in the process of eosinophil recruitment have been identified and reinforce the validity of α4β1 integrin as a therapeutic target. Glucocorticoids are among the most effective drugs for ocular allergy, but their use is limited by adverse effects. Novel dissociated glucocorticoids can prevent eosinophil accumulation and induce apoptosis of eosinophils, making them promising candidates for ophthalmic drugs. This article reviews recent understanding of the role of adhesion molecules in eosinophil recruitment in the inflamed conjunctiva along with effective treatments for allergic conjunctivitis. PMID:23271999

  13. Hydrogen transfer reactions of interstellar Complex Organic Molecules

    NASA Astrophysics Data System (ADS)

    Álvarez-Barcia, S.; Russ, P.; Kästner, J.; Lamberts, T.

    2018-06-01

    Radical recombination has been proposed to lead to the formation of complex organic molecules (COMs) in CO-rich ices in the early stages of star formation. These COMs can then undergo hydrogen addition and abstraction reactions leading to a higher or lower degree of saturation. Here, we have studied 14 hydrogen transfer reactions for the molecules glyoxal, glycoaldehyde, ethylene glycol, and methylformate and an additional three reactions where CHnO fragments are involved. Over-the-barrier reactions are possible only if tunneling is invoked in the description at low temperature. Therefore the rate constants for the studied reactions are calculated using instanton theory that takes quantum effects into account inherently. The reactions were characterized in the gas phase, but this is expected to yield meaningful results for CO-rich ices due to the minimal alteration of reaction landscapes by the CO molecules. We found that rate constants should not be extrapolated based on the height of the barrier alone, since the shape of the barrier plays an increasingly larger role at decreasing temperature. It is neither possible to predict rate constants based only on considering the type of reaction, the specific reactants and functional groups play a crucial role. Within a single molecule, though, hydrogen abstraction from an aldehyde group seems to be always faster than hydrogen addition to the same carbon atom. Reactions that involve heavy-atom tunneling, e.g., breaking or forming a C-C or C-O bond, have rate constants that are much lower than those where H transfer is involved.

  14. The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

    PubMed

    Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

    2013-04-25

    The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

  15. Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

    PubMed Central

    Panjarian, Shoghag; Iacob, Roxana E.; Chen, Shugui; Wales, Thomas E.; Engen, John R.; Smithgall, Thomas E.

    2013-01-01

    Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control. PMID:23303187

  16. Cell-adhesion molecules in memory formation.

    PubMed

    Schmidt, R

    1995-01-23

    After learning events the CNS of higher organisms selects, which acquired informations are permanently stored as a memory trace. This period of memory consolidation is susceptible to interference by biochemical inhibitors of transcription and translation. Ependymin is a specific CNS glycoprotein functionally involved in memory consolidation in goldfish: after active shock-avoidance conditioning ependymin mRNA is rapidly induced in meningeal fibroblasts followed by enhanced synthesis and secretion of several closely related forms of the protein. Intracranial injections of anti-ependymin antisera or antisense oligodeoxynucleotides interfere specifically with memory consolidation, indicating that only de novo synthesized ependymin molecules are involved. Ependymin is capable of directing the growth of central axons in vitro and participates in neuronal regeneration in situ, presumably by its HNK-1 cell-adhesion epitope. Experiments reviewed in this article suggest a model that involves two regulation mechanisms for the function of ependymin in behavioural plasticity: while hormones appear to determine, how much of this cell adhesion molecule is synthesized after learning, local changes of metal cation concentrations in the micro-environment of activated neurons may polymerize ependymin at those synapses, that have to be consolidated to improve their efficacy for future use.

  17. Coherent electron emission beyond Young-type interference from diatomic molecules

    NASA Astrophysics Data System (ADS)

    Agueny, H.; Makhoute, A.; Dubois, A.; Hansen, J. P.

    2016-01-01

    It has been known for more than 15 years that the differential cross section of electrons emitted from diatomic molecules during interaction with energetic charged particles oscillates as a function of electron momentum. The origin of the phenomenon is two-center interference, which naturally relates it back to the Young double-slit experiment. In addition to a characteristic frequency which can be described by lowest-order perturbation theories, the observation and origin of higher-order harmonics of the basic oscillation frequency has been much discussed. Here, we show that high harmonics of the fundamental Young-type oscillation frequency observed in electron spectra in fast ion-molecule collisions can be clearly exposed in numerical solutions of the time-dependent Schrödinger equation within a one-dimensional model. Momentum distribution of the ejected electron is analyzed and shows that the phenomenon emerges when the charged particle beam collides with diatomic molecules with substantial large internuclear distance. Frequency spectra from nonperturbative calculations for electron emission from Rb2+ and Cs2+ exhibit a pronounced high-order oscillation in contrast to similar close-coupling calculations performed on H2 targets. The electron emission from these heavy molecules contains second- and third-order harmonics which are fully reproduced in an analytic model based on the Born series. Extending to triatomic molecular targets displays an increased range of harmonics. This suggests that electron emission spectra from new experiments on heavy diatomic and linear polyatomic molecular targets may provide a unique insight into competing coherent emission mechanisms and their relative strength.

  18. Photoelectron spectroscopy of heavy atoms and molecules

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    White, M.G.

    1979-07-01

    The importance of relativistic interactions in the photoionization of heavy atoms and molecules has been investigated by the technique of photoelectron spectroscopy. In particular, experiments are reported which illustrate the effects of the spin-orbit interaction in the neutral ground state, final ionic states and continuum states of the photoionization target.

  19. Molecular basis of Williams-Beuren syndrome: TFII-I regulated targets involved in craniofacial development.

    PubMed

    Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

    2011-01-01

    The aim of this study is to identify gene targets of TFII-I transcription factors involved in craniofacial development. Recent findings in individuals with Williams-Beuren syndrome who show facial dysmorphism and cognitive defects have pointed to TFII-I genes (GTF2I and GTF2IRD1) as the prime candidates responsible for these clinical features. However, TFII-I proteins are multifunctional transcriptional factors regulating a number of genes during development, and how their haploinsufficiency leads to the Williams-Beuren syndrome phenotype is currently unknown. Here we report the identification of three genes with a well-established relevance to craniofacial development as direct TFII-I targets. These genes, craniofacial development protein 1 (Cfdp1), Sec23 homolog A (Sec23a), and nuclear receptor binding SET domain protein 1 (Nsd1), contain consensus TFII-I binding sites in their proximal promoters; the chromatin immunoprecipitation analysis showed that TFII-I transcription factors are recruited to these sites in vivo. The results suggest that transcriptional regulation of these genes by TFII-I proteins could provide a possible genotype-phenotype link in Williams-Beuren syndrome.

  20. Emodin targets mitochondrial cyclophilin D to induce apoptosis in HepG2 cells.

    PubMed

    Zhang, Ling; He, Dian; Li, Kun; Liu, Hongli; Wang, Baitao; Zheng, Lifang; Li, Jiazhong

    2017-06-01

    Emodin has demonstrated potent anticancer activity in human hepatocarcinoma cells and animal models, however, the cellular targets of emodin have not been fully defined. Here we report that emodin induces the dysfunction of mitochondria and the apoptosis in HepG2 cells through an enrichment in mitochondria. Specifically, A mitochondrial matrix protein (cyclophilin D, CyPD) is involved in emodin-induced apoptosis, and the inhibitor of CyPD (cyclosporin A) could almost completely suppressing the apoptosis; Moreover, as the expression of CyPD could be effectively inhibited by antioxidant N-acetyl-l-cysteine and epidermal growth factor (the activator of ERK), reactive oxygen species and ERK might be involved in the relevant role of CyPD. A further molecule-docking discloses the existence of three hydrogen-bonds in CyPD-emodin complex. Thus, target localization and CyPD in mitochondria provides an insight into the action of emodin in the treatment of liver cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. Hitting the sweet spot-glycans as targets of fungal defense effector proteins.

    PubMed

    Künzler, Markus

    2015-05-06

    Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparably low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous) fungi against microbial competitors and animal predators.

  2. The prelude on novel receptor and ligand targets involved in the treatment of diabetes mellitus.

    PubMed

    Jonnalagadda, Venu Gopal; Ram Raju, Allam Venkata Sita; Pittala, Srinivas; Shaik, Afsar; Selkar, Nilakash Annaji

    2014-01-01

    Metabolic disorders are a group of disorders, due to the disruption of the normal metabolic process at a cellular level. Diabetes Mellitus and Tyrosinaemia are the majorly reported metabolic disorders. Among them, Diabetes Mellitus is a one of the leading metabolic syndrome, affecting 5 to 7 % of the population worldwide and mainly characterised by elevated levels of glucose and is associated with two types of physiological event disturbances such as impaired insulin secretion and insulin resistance. Up to now, various treatment strategies are like insulin, alphaglucosidase inhibitors, biguanides, incretins were being followed. Concurrently, various novel therapeutic strategies are required to advance the therapy of Diabetes mellitus. For the last few decades, there has been an extensive research in understanding the metabolic pathways involved in Diabetes Mellitus at the cellular level and having the profound knowledge on cell-growth, cell-cycle, and apoptosis at a molecular level provides new targets for the treatment of Diabetes Mellitus. Receptor signalling has been involved in these mechanisms, to translate the information coming from outside. To understand the various receptors involved in these pathways, we must have a sound knowledge on receptors and ligands involved in it. This review mainly summarises the receptors and ligands which are involved the Diabetes Mellitus. Finally, researchers have to develop the alternative chemical moieties that retain their affinity to receptors and efficacy. Diabetes Mellitus being a metabolic disorder due to the glucose surfeit, demands the need for regular exercise along with dietary changes.

  3. A method to obtain static potential for electron-molecule scattering

    NASA Astrophysics Data System (ADS)

    Srivastava, Rajesh; Das, Tapasi; Stauffer, Allan

    2014-05-01

    Electron scattering from molecules is complicated by the fact that molecules are a multi-centered target with the nuclei of the constituent atoms being a center of charge. One of the most important parts of a scattering calculation is to obtain the static potential which represents the interaction of the incident electron with the unperturbed charge distribution of the molecule. A common way to represent the charge distribution of molecules is with Gaussian orbitals centered on the various nuclei. We have derived a way to calculate spherically-averaged molecular static potentials using this form of molecular wave function which is mostly analytic. This method has been applied to elastic electron scattering from water molecules and we obtained differential cross sections which are compared with previous experimental and theoretical results. The method can be extended to more complex molecules. One of us (RS) is thankful to IAEA, Vienna, Austria and DAE-BRNS, Mumbai, India for financial support.

  4. Effects of 5-h multimodal stress on the molecules and pathways involved in dendritic morphology and cognitive function.

    PubMed

    Xu, Yiran; Cheng, Xiaorui; Cui, Xiuliang; Wang, Tongxing; Liu, Gang; Yang, Ruishang; Wang, Jianhui; Bo, Xiaochen; Wang, Shengqi; Zhou, Wenxia; Zhang, Yongxiang

    2015-09-01

    Stress induces cognitive impairments, which are likely related to the damaged dendritic morphology in the brain. Treatments for stress-induced impairments remain limited because the molecules and pathways underlying these impairments are unknown. Therefore, the aim of this study was to find the potential molecules and pathways related to damage of the dendritic morphology induced by stress. To do this, we detected gene expression, constructed a protein-protein interaction (PPI) network, and analyzed the molecular pathways in the brains of mice exposed to 5-h multimodal stress. The results showed that stress increased plasma corticosterone concentration, decreased cognitive function, damaged dendritic morphologies, and altered APBB1, CLSTN1, KCNA4, NOTCH3, PLAU, RPS6KA1, SYP, TGFB1, KCNA1, NTRK3, and SNCA expression in the brains of mice. Further analyses found that the abnormal expressions of CLSTN1, PLAU, NOTCH3, and TGFB1 induced by stress were related to alterations in the dendritic morphology. These four genes demonstrated interactions with 55 other genes, and configured a closed PPI network. Molecular pathway analysis use the Database for Annotation, Visualization, and Integrated Discovery (DAVID), specifically the gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG), each identified three pathways that were significantly enriched in the gene list of the PPI network, with genes belonging to the Notch and transforming growth factor-beta (TGF-B) signaling pathways being the most enriched. Our results suggest that TGFB1, PLAU, NOTCH3, and CLSTN1 may be related to the alterations in dendritic morphology induced by stress, and imply that the Notch and TGF-B signaling pathways may be involved. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Identification of chemogenomic features from drug–target interaction networks using interpretable classifiers

    PubMed Central

    Tabei, Yasuo; Pauwels, Edouard; Stoven, Véronique; Takemoto, Kazuhiro; Yamanishi, Yoshihiro

    2012-01-01

    Motivation: Drug effects are mainly caused by the interactions between drug molecules and their target proteins including primary targets and off-targets. Identification of the molecular mechanisms behind overall drug–target interactions is crucial in the drug design process. Results: We develop a classifier-based approach to identify chemogenomic features (the underlying associations between drug chemical substructures and protein domains) that are involved in drug–target interaction networks. We propose a novel algorithm for extracting informative chemogenomic features by using L1 regularized classifiers over the tensor product space of possible drug–target pairs. It is shown that the proposed method can extract a very limited number of chemogenomic features without loosing the performance of predicting drug–target interactions and the extracted features are biologically meaningful. The extracted substructure–domain association network enables us to suggest ligand chemical fragments specific for each protein domain and ligand core substructures important for a wide range of protein families. Availability: Softwares are available at the supplemental website. Contact: yamanishi@bioreg.kyushu-u.ac.jp Supplementary Information: Datasets and all results are available at http://cbio.ensmp.fr/~yyamanishi/l1binary/ . PMID:22962471

  6. Nanocarrier-mediated drugs targeting cancer stem cells: an emerging delivery approach.

    PubMed

    Malhi, Sarandeep; Gu, Xiaochen

    2015-07-01

    Cancer stem cells (CSCs) play an important role in the development of drug resistance, metastasis and recurrence. Current conventional therapies do not commonly target CSCs. Nanocarrier-based delivery systems targeting cancer cells have entered a new era of treatment, where specific targeting to CSCs may offer superior outcomes to efficient cancer therapies. This review discusses the involvement of CSCs in tumor progression and relevant mechanisms associated with CSCs resistance to conventional chemo- and radio-therapies. It highlights CSCs-targeted strategies that are either under evaluation or could be explored in the near future, with a focus on various nanocarrier-based delivery systems of drugs and nucleic acids to CSCs. Novel nanocarriers targeting CSCs are presented in a cancer-specific way to provide a current perspective on anti-CSCs therapeutics. The field of CSCs-targeted therapeutics is still emerging with a few small molecules and macromolecules currently proving efficacy in clinical trials. However considering the complexities of CSCs and existing delivery difficulties in conventional anticancer therapies, CSC-specific delivery systems would face tremendous technical and clinical challenges. Nanocarrier-based approaches have demonstrated significant potential in specific drug delivery and targeting; their success in CSCs-targeted drug delivery would not only significantly enhance anticancer treatment but also address current difficulties associated with cancer resistance, metastasis and recurrence.

  7. Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining.

    PubMed

    Tang, Xiaoyu; Li, Jie; Millán-Aguiñaga, Natalie; Zhang, Jia Jia; O'Neill, Ellis C; Ugalde, Juan A; Jensen, Paul R; Mantovani, Simone M; Moore, Bradley S

    2015-12-18

    Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or "orphaned" secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs and (ii) how to rapidly connect genes to biosynthesized small molecules. Here, we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes colocalized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related ttm BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and autoresistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized but also paves the way for the investigation of novel enzymology involved in thiotetronic acid natural product biosynthesis.

  8. Femtogram-scale photothermal spectroscopy of explosive molecules on nanostrings.

    PubMed

    Biswas, T S; Miriyala, N; Doolin, C; Liu, X; Thundat, T; Davis, J P

    2014-11-18

    We demonstrate detection of femtogram-scale quantities of the explosive molecule 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) via combined nanomechanical photothermal spectroscopy and mass desorption. Photothermal spectroscopy provides a spectroscopic fingerprint of the molecule, which is unavailable using mass adsorption/desorption alone. Our measurement, based on thermomechanical measurement of silicon nitride nanostrings, represents the highest mass resolution ever demonstrated via nanomechanical photothermal spectroscopy. This detection scheme is quick, label-free, and is compatible with parallelized molecular analysis of multicomponent targets.

  9. Mapping the Small Molecule Interactome by Mass Spectrometry.

    PubMed

    Flaxman, Hope A; Woo, Christina M

    2018-01-16

    Mapping small molecule interactions throughout the proteome provides the critical structural basis for functional analysis of their impact on biochemistry. However, translation of mass spectrometry-based proteomics methods to directly profile the interaction between a small molecule and the whole proteome is challenging because of the substoichiometric nature of many interactions, the diversity of covalent and noncovalent interactions involved, and the subsequent computational complexity associated with their spectral assignment. Recent advances in chemical proteomics have begun fill this gap to provide a structural basis for the breadth of small molecule-protein interactions in the whole proteome. Innovations enabling direct characterization of the small molecule interactome include faster, more sensitive instrumentation coupled to chemical conjugation, enrichment, and labeling methods that facilitate detection and assignment. These methods have started to measure molecular interaction hotspots due to inherent differences in local amino acid reactivity and binding affinity throughout the proteome. Measurement of the small molecule interactome is producing structural insights and methods for probing and engineering protein biochemistry. Direct structural characterization of the small molecule interactome is a rapidly emerging area pushing new frontiers in biochemistry at the interface of small molecules and the proteome.

  10. Epigenetic drugs that do not target enzyme activity.

    PubMed

    Owen, Dafydd R; Trzupek, John D

    2014-06-01

    While the installation and removal of epigenetic post-translational modifications or ‘marks’ on both DNA and histone proteins are the tangible outcome of enzymatically catalyzed processes, the role of the epigenetic reader proteins looks, at first, less obvious. As they do not catalyze a chemical transformation or process as such, their role is not enzymatic. However, this does not preclude them from being potential targets for drug discovery as their function is clearly correlated to transcriptional activity and as a class of proteins, they appear to have binding sites of sufficient definition and size to be inhibited by small molecules. This suggests that this third class of epigenetic proteins that are involved in the interpretation of post-translational marks (as opposed to the creation or deletion of marks) may represent attractive targets for drug discovery efforts. This review mainly summarizes selected publications, patent literature and company disclosures on these non-enzymatic epigenetic reader proteins from 2009 to the present. © 2014 Elsevier Ltd . All rights reserved.

  11. Raman spectroelectrochemistry of molecules within individual electromagnetic hot spots.

    PubMed

    Shegai, Timur; Vaskevich, Alexander; Rubinstein, Israel; Haran, Gilad

    2009-10-14

    The role of chemical enhancement in surface-enhanced Raman scattering (SERS) remains a contested subject. We study SERS spectra of 4-mercaptopyridine molecules excited far from the molecular resonance, which are collected from individual electromagnetic hot spots at concentrations close to the single-molecule limit. The hot spots are created by depositing Tollen's silver island films on a transparent electrode incorporated within an electrochemical cell. Analysis of the intensity of the spectra relative to those obtained from individual rhodamine 6G molecules on the same surface provides a lower limit of approximately 3 orders of magnitude for the chemical enhancement. This large enhancement is likely to be due to a charge transfer resonance involving the transfer of an electron from the metal to an adsorbed molecule. Excitation at three different wavelengths, as well as variation of electrode potential from 0 to -1.2 V, lead to significant changes in the relative intensities of bands in the spectrum. It is suggested that while the bulk of the enhancement is due to an Albrecht A-term resonance Raman effect (involving the charge transfer transition), vibronic coupling provides additional enhancement which is sensitive to electrode potential. The measurement of potential-dependent SERS spectra from individual hot spots opens the way to a thorough characterization of chemical enhancement, as well to studies of redox phenomena at the single-molecule level.

  12. A single-molecule view of gene regulation in cancer

    NASA Astrophysics Data System (ADS)

    Larson, Daniel

    2013-03-01

    Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. Steroid receptors coordinate a diverse range of responses in higher eukaryotes and are involved in a wide range of human diseases, including cancer. Steroid receptor response elements are present throughout the human genome and modulate chromatin remodeling and transcription in both a local and long-range fashion. As such, steroid receptor-mediated transcription is a paradigm of genetic control in the metazoan nucleus. Moreover, the ligand-dependent nature of these transcription factors makes them appealing targets for therapeutic intervention, necessitating a quantitative understanding of how receptors control output from target genes. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single gene and follow dynamic synthesis of RNA from the activated locus. The response delay is a measure of time required for chromatin remodeling at a single gene.

  13. Use of biotin targeted methotrexate–human serum albumin conjugated nanoparticles to enhance methotrexate antitumor efficacy

    PubMed Central

    Taheri, Azade; Dinarvand, Rassoul; Nouri, Faranak Salman; Khorramizadeh, Mohammad Reza; Borougeni, Atefeh Taheri; Mansoori, Pooria; Atyabi, Fatemeh

    2011-01-01

    Biotin molecules could be used as suitable targeting moieties in targeted drug delivery systems against tumors. To develop a biotin targeted drug delivery system, we employed human serum albumin (HSA) as a carrier. Methotrexate (MTX) molecules were conjugated to HSA. MTX-HSA nanoparticles (MTX-HSA NPs) were prepared from these conjugates by cross-linking the HSA molecules. Biotin molecules were then conjugated on the surface of MTX-HSA NPs. The anticancer efficacy of biotin targeted MTX-HSA NPs was evaluated in mice bearing 4T1 breast carcinoma. A single dose of biotin targeted MTX-HSA NPs showed stronger in vivo antitumor activity than non-targeted MTX-HSA NPs and free MTX. By 7 days after treatment, average tumor volume in the biotin targeted MTX-HSA NPs-treated group decreased to 17.6% of the initial tumor volume when the number of attached biotin molecules on MTX-HSA-NPs was the highest. Average tumor volume in non-targeted MTX-HSA NPs-treated mice grew rapidly and reached 250.7% of the initial tumor volume. Biotin targeted MTX-HSA NPs increased the survival of tumor-bearing mice to 47.5 ± 0.71 days and increased their life span up to 216.7%. Mice treated with biotin targeted MTX-HSA NPs showed slight body weight loss (8%) 21 days after treatment, whereas non-targeted MTX-HSA NPs treatment at the same dose caused a body weight loss of 27.05% ± 3.1%. PMID:21931482

  14. Targeted Radiotherapy of Estrogen Receptor Positive Tumors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Raghavan Rajagopalan

    The overall objectives of the proposal were to develop estrogen receptor (ER) binding small molecule radiopharmaceuticals for targeted radiotherapy of ER positive (ER+) tumors. In particular, this proposal focused on embedding a {sup 186,188}Re or a {sup 32}P radionuclide into an estrogen steroidal framework by isosteric substitution such that the resulting structure is topologically similar to the estrogen (estrogen mimic). The estrogen mimic molecules expected to bind to the ER and exhibit biodistribution akin to that of native estrogen due to structural mimicry. It is anticipated that the {sup 186,188}Re- or a {sup 32}P-containing estrogen mimics will be useful formore » targeted molecular radiotherapy of ER+ tumors. It is well established that the in vivo target tissue uptake of estrogen like steroidal molecules is related to the binding of the steroids to sex hormone binding globulin (SHBG). SHBG is important in the uptake of estrogens and testosterone in target tissues by SHBG receptors on the cell surface. However, hitherto the design of estrogen like small molecule radiopharmaceuticals was focused on optimizing ER binding characteristics without emphasis on SHBG binding properties. Consequently, even the molecules with good ER affinity in vitro, performed poorly in biodistribution studies. Based on molecular modeling studies the proposal focused on developing estrogen mimics 1-3 which were topologically similar to native estrogens, and form hydrogen bonds in ER and SHBG in the same manner as those of native estrogens. To this end the technical objectives of the proposal focused on synthesizing the rhenium-estrone and estradiol mimics 1 and 2 respectively, and phosphorous estradiol mimic 3 and to assess their stability and in vitro binding characteristics to ER and SHBG.« less

  15. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    PubMed

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  16. Ultrasensitive detection and characterization of molecules with infrared plasmonic metamaterials

    PubMed Central

    Cheng, Fei; Yang, Xiaodong; Gao, Jie

    2015-01-01

    Infrared vibrational spectroscopy is an effective technique which enables the direct probe of molecular fingerprints, and such detection can be further enhanced by the emerging engineered plasmonic metamaterials. Here we experimentally demonstrate ultrasensitive detection and characterization of polymer molecules based on an asymmetric infrared plasmonic metamaterial, and quantitatively analyze the molecule detection sensitivity and molecule-structure interactions. A sharp, non-radiative Fano resonance supported by the plasmonic metamaterial exhibits strongly enhanced near-field, and the resonance frequency is tailored to match the vibrational fingerprint of the target molecule. By utilizing the near-field nature of the plasmonic excitation, significantly enhanced absorption signal of molecules in the infrared spectroscopy are obtained, enabling ultrasensitive detection of only minute quantities of organic molecules. The enhancement of molecular absorption up to 105 fold is obtained, and sensitive detection of molecules at zeptomole levels (corresponding to a few tens of molecules within a unit cell) is achieved with high signal-to-noise ratio in our experiment. The demonstrated infrared plasmonic metamaterial sensing platform offers great potential for improving the specificity and sensitivity of label-free, biochemical detection. PMID:26388404

  17. Design of small molecule epigenetic modulators.

    PubMed

    Pachaiyappan, Boobalan; Woster, Patrick M

    2014-01-01

    The field of epigenetics has expanded rapidly to reveal multiple new targets for drug discovery. The functional elements of the epigenomic machinery can be categorized as writers, erasers and readers, and together these elements control cellular gene expression and homeostasis. It is increasingly clear that aberrations in the epigenome can underly a variety of diseases, and thus discovery of small molecules that modulate the epigenome in a specific manner is a viable approach to the discovery of new therapeutic agents. In this Digest, the components of epigenetic control of gene expression will be briefly summarized, and efforts to identify small molecules that modulate epigenetic processes will be described. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. [Molecules involved in sperm-zona pellucida interaction in mammals. Role in human fertility].

    PubMed

    Serres, Catherine; Auer, Jana; Petit, François; Patrat, Catherine; Jouannet, Pierre

    2008-01-01

    Fertilization in mammals requires an initial interaction of sperm with the oocyte envelope, the zona pellucida (ZP), before it reaches the oocyte. ZP is a highly glycosylated structure, composed of three (mouse) or four (rabbit, boar, bovine, humans...) glycoproteins. The presence of ZP around the oocyte does not allow heterospecific fertilization. This barrier is principally due to the presence of species-specific glycosylations on ZP proteins. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Upon initial attachment, spermatozoa bind ZP3/ZP4 which induces the sperm acrosome exocytosis followed by a secondary binding of acrosome reacted spermatozoa to ZP2 and by ZP penetration. The sperm receptors are adhesive proteins or integral plasma membrane proteins linked to intraspermatic signalling pathways activating the acrosome reaction. Over the last twenty years, numerous studies have been carried out to identify sperm receptors to ZP in several species, but the data in humans are still incomplete. Work initiated in our research group has identified several proteins interacting with recombinant human ZP2, ZP3 and ZP4, among which are glycolytic enzymes. These enzymes are involved in the gamete interaction by means of their affinity to sugars and not by their catalytic properties. From a clinical point of view, an observed lack or weak expression of some sperm receptors to ZP3 in cases of idiopathic infertility associated with in vitro fertilization failure suggests that knowing the molecular mechanism driving the gamete recognition can be important at the diagnostic level. Furthermore, it has been shown that proteins that mediate gamete recognition diverge rapidly, as a result of positive darwinian selection. A sexual conflict can drive co-evolution of reproductive molecules in both sexes resulting in reproductive isolation and species emergence.

  19. Examining small molecule: HIV RNA interactions using arrayed imaging reflectometry

    NASA Astrophysics Data System (ADS)

    Chaimayo, Wanaruk; Miller, Benjamin L.

    2014-03-01

    Human Immunodeficiency Virus (HIV) has been the subject of intense research for more than three decades as it causes an uncurable disease: Acquired Immunodeficiency Syndrome, AIDS. In the pursuit of a medical treatment, RNAtargeted small molecules are emerging as promising targets. In order to understand the binding kinetics of small molecules and HIV RNA, association (ka) and dissociation (kd) kinetic constants must be obtained, ideally for a large number of sequences to assess selectivity. We have developed Aqueous Array Imaged Reflectometry (Aq-AIR) to address this challenge. Using a simple light interference phenomenon, Aq-AIR provides real-time high-throughput multiplex capabilities to detect binding of targets to surface-immobilized probes in a label-free microarray format. The second generation of Aq-AIR consisting of high-sensitivity CCD camera and 12-μL flow cell was fabricated. The system performance was assessed by real-time detection of MBNL1-(CUG)10 and neomycin B - HIV RNA bindings. The results establish this second-generation Aq-AIR to be able to examine small molecules binding to RNA sequences specific to HIV.

  20. Protein kinases: mechanisms and downstream targets in inflammation-mediated obesity and insulin resistance.

    PubMed

    Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

    2017-02-01

    Obesity-induced low-grade inflammation (metaflammation) impairs insulin receptor signaling. This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, inhibitor of NF-kB kinase complex β (IKKβ), AMP-activated protein kinase, protein kinase C, Rho-associated coiled-coil containing protein kinase, and RNA-activated protein kinase. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in type 2 diabetes mellitus (T2-DM). Identifying the specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity-induced T2-DM.

  1. Single Molecule Nano-Metronome

    PubMed Central

    Buranachai, Chittanon; McKinney, Sean A.; Ha, Taekjip

    2008-01-01

    We constructed a DNA-based nano-mechanical device called the nano-metronome. Our device is made by introducing complementary single stranded overhangs at the two arms of the DNA four-way junction. The ticking rates of this stochastic metronome depend on ion concentrations and can be changed by a set of DNA-based switches to deactivate/reactivate the sticky end. Since the device displays clearly distinguishable responses even with a single basepair difference, it may lead to a single molecule sensor of minute sequence differences of a target DNA. PMID:16522050

  2. Features of Modularly Assembled Compounds That Impart Bioactivity Against an RNA Target

    PubMed Central

    Rzuczek, Suzanne G.; Gao, Yu; Tang, Zhen-Zhi; Thornton, Charles A.; Kodadek, Thomas; Disney, Matthew D.

    2013-01-01

    Transcriptomes provide a myriad of potential RNAs that could be the targets of therapeutics or chemical genetic probes of function. Cell permeable small molecules, however, generally do not exploit these targets, owing to the difficulty in the design of high affinity, specific small molecules targeting RNA. As part of a general program to study RNA function using small molecules, we designed bioactive, modularly assembled small molecules that target the non-coding expanded RNA repeat that causes myotonic dystrophy type 1 (DM1), r(CUG)exp. Herein, we present a rigorous study to elucidate features in modularly assembled compounds that afford bioactivity. Different modular assembly scaffolds were investigated including polyamines, α-peptides, β-peptides, and peptide tertiary amides (PTAs). Based on activity as assessed by improvement of DM1-associated defects, stability against proteases, cellular permeability, and toxicity, we discovered that constrained backbones, namely PTAs, are optimal. Notably, we determined that r(CUG)exp is the target of the optimal PTA in cellular models and that the optimal PTA improves DM1-associated defects in a mouse model. Biophysical analyses were employed to investigate potential sources of bioactivity. These investigations show that modularly assembled compounds have increased residence times on their targets and faster on rates than the RNA-binding modules from which they were derived and faster on rates than the protein that binds r(CUG)exp, the inactivation of which gives rise to DM1-associated defects. These studies provide information about features of small molecules that are programmable for targeting RNA, allowing for the facile optimization of therapeutics or chemical probes against other cellular RNA targets. PMID:24032410

  3. Features of modularly assembled compounds that impart bioactivity against an RNA target.

    PubMed

    Rzuczek, Suzanne G; Gao, Yu; Tang, Zhen-Zhi; Thornton, Charles A; Kodadek, Thomas; Disney, Matthew D

    2013-10-18

    Transcriptomes provide a myriad of potential RNAs that could be the targets of therapeutics or chemical genetic probes of function. Cell-permeable small molecules, however, generally do not exploit these targets, owing to the difficulty in the design of high affinity, specific small molecules targeting RNA. As part of a general program to study RNA function using small molecules, we designed bioactive, modularly assembled small molecules that target the noncoding expanded RNA repeat that causes myotonic dystrophy type 1 (DM1), r(CUG)(exp). Herein, we present a rigorous study to elucidate features in modularly assembled compounds that afford bioactivity. Different modular assembly scaffolds were investigated, including polyamines, α-peptides, β-peptides, and peptide tertiary amides (PTAs). On the basis of activity as assessed by improvement of DM1-associated defects, stability against proteases, cellular permeability, and toxicity, we discovered that constrained backbones, namely, PTAs, are optimal. Notably, we determined that r(CUG)(exp) is the target of the optimal PTA in cellular models and that the optimal PTA improves DM1-associated defects in a mouse model. Biophysical analyses were employed to investigate potential sources of bioactivity. These investigations show that modularly assembled compounds have increased residence times on their targets and faster on rates than the RNA-binding modules from which they were derived. Moreover, they have faster on rates than the protein that binds r(CUG)(exp), the inactivation of which gives rise to DM1-associated defects. These studies provide information about features of small molecules that are programmable for targeting RNA, allowing for the facile optimization of therapeutics or chemical probes against other cellular RNA targets.

  4. Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting.

    PubMed

    Gerlai, R; Williams, S P; Cairns, B; Van Bruggen, N; Moran, P; Shih, A; Caras, I; Sauer, H; Phillips, H S; Winslow, J W

    1998-11-01

    Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location

  5. Free fatty acid receptors: emerging targets for treatment of diabetes and its complications

    PubMed Central

    Vangaveti, Venkat; Shashidhar, Venkatesh; Jarrod, Ghassan; Baune, Bernhard T.; Kennedy, R. Lee

    2010-01-01

    Fatty acids (FAs) are important as metabolic substrates and as structural components of biological membranes. However, they also function as signalling molecules. Recently, a series of G protein-coupled receptors (GPRs) for FAs has been described and characterized. These receptors have differing specificities for FAs of differing chain length and degree of saturation, for FA derivatives such as oleoylethanolamide, and for oxidized FAs. They are a critical component of the body's nutrient sensing apparatus, and small molecule agonists and antagonists of these receptors show considerable promise in the management of diabetes and its complications. Agonists of the long-chain free fatty acid receptors FFAR1 and GPR119 act as insulin secretagogues, both directly and by increasing incretins. Although, drugs acting at short-chain FFA receptors (FFAR2 and FFAR3) have not yet been developed, they are attractive targets as they regulate nutrient balance through effects in the intestine and adipose tissue. These include regulation of the secretion of cholecystokinin, peptide YY and leptin. Finally, GPR132 is a receptor for oxidized FAs, which may be a sensor of lipid overload and oxidative stress, and which is involved in atherosclerosis. Regulation of its signalling pathways with drugs may decrease the macrovascular risk experienced by diabetic patients. In summary, FA receptors are emerging drug targets that are involved in the regulation of nutrient status and carbohydrate tolerance, and modulators of these receptors may well figure prominently in the next generation of antidiabetic drugs. PMID:23148161

  6. Implementation of quantum logic gates using polar molecules in pendular states.

    PubMed

    Zhu, Jing; Kais, Sabre; Wei, Qi; Herschbach, Dudley; Friedrich, Bretislav

    2013-01-14

    We present a systematic approach to implementation of basic quantum logic gates operating on polar molecules in pendular states as qubits for a quantum computer. A static electric field prevents quenching of the dipole moments by rotation, thereby creating the pendular states; also, the field gradient enables distinguishing among qubit sites. Multi-target optimal control theory is used as a means of optimizing the initial-to-target transition probability via a laser field. We give detailed calculations for the SrO molecule, a favorite candidate for proposed quantum computers. Our simulation results indicate that NOT, Hadamard and CNOT gates can be realized with high fidelity, as high as 0.985, for such pendular qubit states.

  7. Discovery of Small Molecules that Inhibit the Disordered Protein, p27 Kip1

    DOE PAGES

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; ...

    2015-10-28

    In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27 Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groupsmore » of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).« less

  8. Hindsight regulates photoreceptor axon targeting through transcriptional control of jitterbug/Filamin and multiple genes involved in axon guidance in Drosophila.

    PubMed

    Oliva, Carlos; Molina-Fernandez, Claudia; Maureira, Miguel; Candia, Noemi; López, Estefanía; Hassan, Bassem; Aerts, Stein; Cánovas, José; Olguín, Patricio; Sierralta, Jimena

    2015-09-01

    During axon targeting, a stereotyped pattern of connectivity is achieved by the integration of intrinsic genetic programs and the response to extrinsic long and short-range directional cues. How this coordination occurs is the subject of intense study. Transcription factors play a central role due to their ability to regulate the expression of multiple genes required to sense and respond to these cues during development. Here we show that the transcription factor HNT regulates layer-specific photoreceptor axon targeting in Drosophila through transcriptional control of jbug/Filamin and multiple genes involved in axon guidance and cytoskeleton organization.Using a microarray analysis we identified 235 genes whose expression levels were changed by HNT overexpression in the eye primordia. We analyzed nine candidate genes involved in cytoskeleton regulation and axon guidance, six of which displayed significantly altered gene expression levels in hnt mutant retinas. Functional analysis confirmed the role of OTK/PTK7 in photoreceptor axon targeting and uncovered Tiggrin, an integrin ligand, and Jbug/Filamin, a conserved actin- binding protein, as new factors that participate of photoreceptor axon targeting. Moreover, we provided in silico and molecular evidence that supports jbug/Filamin as a direct transcriptional target of HNT and that HNT acts partially through Jbug/Filamin in vivo to regulate axon guidance. Our work broadens the understanding of how HNT regulates the coordinated expression of a group of genes to achieve the correct connectivity pattern in the Drosophila visual system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1018-1032, 2015. © 2015 Wiley Periodicals, Inc.

  9. Dust-forming molecules in VY Canis Majoris (and Betelgeuse)

    NASA Astrophysics Data System (ADS)

    Kamiński, T.; Gottlieb, C. A.; Schmidt, M. R.; Patel, N. A.; Young, K. H.; Menten, K. M.; Brünken, S.; Müller, H. S. P.; Winters, J. M.; McCarthy, M. C.

    2013-05-01

    The formation of inorganic dust in circumstellar environments of evolved stars is poorly understood. Spectra of molecules thought to be most important for the nucleation, i.e. AlO, TiO, and TiO2, have been recently detected in the red supergiant VY CMa. These molecules are effectively formed in VY CMa and the observations suggest that non-equilibrium chemistry must be involved in their formation and nucleation into dust. In addition to exploring the recent observations of VY CMa, we briefly discuss the possibility of detecting these molecules in the "dust-poor" circumstellar environment of Betelgeuse.

  10. Application of the R-matrix method to photoionization of molecules.

    PubMed

    Tashiro, Motomichi

    2010-04-07

    The R-matrix method has been used for theoretical calculation of electron collision with atoms and molecules for long years. The method was also formulated to treat photoionization process, however, its application has been mostly limited to photoionization of atoms. In this work, we implement the R-matrix method to treat molecular photoionization problem based on the UK R-matrix codes. This method can be used for diatomic as well as polyatomic molecules, with multiconfigurational description for electronic states of both target neutral molecule and product molecular ion. Test calculations were performed for valence electron photoionization of nitrogen (N(2)) as well as nitric oxide (NO) molecules. Calculated photoionization cross sections and asymmetry parameters agree reasonably well with the available experimental results, suggesting usefulness of the method for molecular photoionization.

  11. Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rudenko, Gabby

    Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, formingtrans-complexes spanning the synaptic cleft orcis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affectmore » their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics.« less

  12. Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

    PubMed Central

    2017-01-01

    Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, forming trans-complexes spanning the synaptic cleft or cis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affect their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics. PMID:28255461

  13. Development of small molecules targeting the pseudokinase Her3.

    PubMed

    Lim, Sang Min; Xie, Ting; Westover, Kenneth D; Ficarro, Scott B; Tae, Hyun Seop; Gurbani, Deepak; Sim, Taebo; Marto, Jarrod A; Jänne, Pasi A; Crews, Craig M; Gray, Nathanael S

    2015-08-15

    Her3 is a member of the human epidermal growth factor receptor (EGFR) tyrosine kinase family, and it is often either overexpressed or deregulated in many types of human cancer. Her3 has not been the subject of small-molecule inhibitor development because it is a pseudokinase and does not possess appreciable kinase activity. We recently reported on the development of the first selective irreversible Her3 ligand (TX1-85-1) that forms a covalent bond with cysteine 721 which is unique to Her3 among all kinases. We also developed a bi-functional compound (TX2-121-1) containing a hydrophobic adamantane moiety and the same warhead of TX1-85-1 that is capable of inhibiting Her3-dependent signaling and growth. Here we report on the structure-based medicinal chemistry effort that resulted in the discovery of these two compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Application of chemical biology in target identification and drug discovery.

    PubMed

    Zhu, Yue; Xiao, Ting; Lei, Saifei; Zhou, Fulai; Wang, Ming-Wei

    2015-09-01

    Drug discovery and development is vital to the well-being of mankind and sustainability of the pharmaceutical industry. Using chemical biology approaches to discover drug leads has become a widely accepted path partially because of the completion of the Human Genome Project. Chemical biology mainly solves biological problems through searching previously unknown targets for pharmacologically active small molecules or finding ligands for well-defined drug targets. It is a powerful tool to study how these small molecules interact with their respective targets, as well as their roles in signal transduction, molecular recognition and cell functions. There have been an increasing number of new therapeutic targets being identified and subsequently validated as a result of advances in functional genomics, which in turn led to the discovery of numerous active small molecules via a variety of high-throughput screening initiatives. In this review, we highlight some applications of chemical biology in the context of drug discovery.

  15. Application of Chemical Genomics to Plant-Bacteria Communication: A High-Throughput System to Identify Novel Molecules Modulating the Induction of Bacterial Virulence Genes by Plant Signals.

    PubMed

    Vandelle, Elodie; Puttilli, Maria Rita; Chini, Andrea; Devescovi, Giulia; Venturi, Vittorio; Polverari, Annalisa

    2017-01-01

    The life cycle of bacterial phytopathogens consists of a benign epiphytic phase, during which the bacteria grow in the soil or on the plant surface, and a virulent endophytic phase involving the penetration of host defenses and the colonization of plant tissues. Innovative strategies are urgently required to integrate copper treatments that control the epiphytic phase with complementary tools that control the virulent endophytic phase, thus reducing the quantity of chemicals applied to economically and ecologically acceptable levels. Such strategies include targeted treatments that weaken bacterial pathogens, particularly those inhibiting early infection steps rather than tackling established infections. This chapter describes a reporter gene-based chemical genomic high-throughput screen for the induction of bacterial virulence by plant molecules. Specifically, we describe a chemical genomic screening method to identify agonist and antagonist molecules for the induction of targeted bacterial virulence genes by plant extracts, focusing on the experimental controls required to avoid false positives and thus ensuring the results are reliable and reproducible.

  16. Small-molecule targeting of signal transducer and activator of transcription (STAT) 3 to treat non-small cell lung cancer.

    PubMed

    Lewis, Katherine M; Bharadwaj, Uddalak; Eckols, T Kris; Kolosov, Mikhail; Kasembeli, Moses M; Fridley, Colleen; Siller, Ricardo; Tweardy, David J

    2015-11-01

    Lung cancer is the leading cause of cancer death in both men and women. Non-small cell lung cancer (NSCLC) has an overall 5-year survival rate of 15%. While aberrant STAT3 activation has previously been observed in NSCLC, the scope of its contribution is uncertain and agents that target STAT3 for treatment are not available clinically. We determined levels of activated STAT3 (STAT3 phosphorylated on Y705, pSTAT3) and the two major isoforms of STAT3 (α and β) in protein extracts of 8 NSCLC cell lines, as well as the effects of targeting STAT3 in vitro and in vivo in NSCLC cells using short hairpin (sh) RNA and two novel small-molecule STAT3 inhibitors, C188-9 and piperlongumine (PL). Levels of pSTAT3, STAT3α, and STATβ were increased in 7 of 8 NSCLC cell lines. Of note, levels of pSTAT3 were tightly correlated with levels of STAT3β, but not STAT3α. Targeting of STAT3 in A549 cells using shRNA decreased tSTAT3 by 75%; this was accompanied by a 47-78% reduction in anchorage-dependent and anchorage-independent growth and a 28-45% reduction in mRNA levels for anti-apoptotic STAT3 gene targets. C188-9 and PL (@30 μM) each reduced pSTAT3 levels in all NSCLC cell lines tested by ≥50%, reduced anti-apoptotic protein mRNA levels by 25-60%, and reduced both anchorage-dependent and anchorage-independent growth of NSCLC cell lines with IC50 values ranging from 3.06 to 52.44 μM and 0.86 to 11.66 μM, respectively. Treatment of nude mice bearing A549 tumor xenografts with C188-9 or PL blocked tumor growth and reduced levels of pSTAT3 and mRNA encoding anti-apoptotic proteins. STAT3 is essential for growth of NSCLC cell lines and tumors and its targeting using C188-9 or PL may be a useful strategy for treatment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Temporal identity in axonal target layer recognition.

    PubMed

    Petrovic, Milan; Hummel, Thomas

    2008-12-11

    The segregation of axon and dendrite projections into distinct synaptic layers is a fundamental principle of nervous system organization and the structural basis for information processing in the brain. Layer-specific recognition molecules that allow projecting neurons to stabilize transient contacts and initiate synaptogenesis have been identified. However, most of the neuronal cell-surface molecules critical for layer organization are expressed broadly in the developing nervous system, raising the question of how these so-called permissive adhesion molecules support synaptic specificity. Here we show that the temporal expression dynamics of the zinc-finger protein sequoia is the major determinant of Drosophila photoreceptor connectivity into distinct synaptic layers. Neighbouring R8 and R7 photoreceptors show consecutive peaks of elevated sequoia expression, which correspond to their sequential target-layer innervation. Loss of sequoia in R7 leads to a projection switch into the R8 recipient layer, whereas a prolonged expression in R8 induces a redirection of their axons into the R7 layer. The sequoia-induced axon targeting is mediated through the ubiquitously expressed Cadherin-N cell adhesion molecule. Our data support a model in which recognition specificity during synaptic layer formation is generated through a temporally restricted axonal competence to respond to broadly expressed adhesion molecules. Because developing neurons innervating the same target area often project in a distinct, birth-order-dependent sequence, temporal identity seems to contain crucial information in generating not only cell type diversity during neuronal division but also connection diversity of projecting neurons.

  18. Laser-triggered release of encapsulated molecules from polylactic-co-glycolic acid microcapsules

    NASA Astrophysics Data System (ADS)

    Ariyasu, Kazumasa; Ishii, Atsuhiro; Umemoto, Taiga; Terakawa, Mitsuhiro

    2016-08-01

    The controlled release of encapsulated molecules from a microcapsule is a promising method of targeted drug delivery. Laser-triggered methods for the release of encapsulated molecules have the advantage of spatial and temporal controllability. In this study, we demonstrated the release of encapsulated molecules from biodegradable polymer-based microcapsules using near-infrared femtosecond laser pulses. The polylactic-co-glycolic acid microcapsules encapsulating fluorescein isothiocyanate-dextran molecules were fabricated using a dual-coaxial nozzle system. Irradiation of femtosecond laser pulses enhanced the release of the molecules from the microcapsules, which was accompanied by a decrease in the residual ratio of the microcapsules. The laser-induced modification of the surface of the shell of the microcapsules indicated the potential for sustained release as well as burst release.

  19. Theoretical Investigation of Single-Molecule Sensing Using Nanotube-Enhanced Circular Dichroism.

    PubMed

    Silva, Jaime; Milne, Bruce F; Nogueira, Fernando

    2018-06-19

    First-principles calculations have been used to investigate the potential use of circular dichroism (CD) spectroscopy in single-molecule sensing. Using a real-space implementation of time-dependent density functional theory (TDDFT), several systems involving single-walled carbon nanotubes (SWCNT) and small molecules have been studied to evaluate their CD response. Large induced CD (ICD) effects, differing for each test molecule, were observed in all SWCNT-molecule complexes. As the SWCNT used in this study shows no intrinsic CD response, the ICD spectra are the result of interaction with the small molecules. This finding is general and independent of the (a)chiral nature of the adsorbed molecule. Our results indicate that it is possible to design a system that uses SWCNT for detection of molecules using the change in CD spectrum of the system induced by adsorption of the molecule onto the SWCNT surface.

  20. Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Weighill, Deborah; Jones, Piet; Shah, Manesh

    Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant's sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes usemore » of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. Lastly, the resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for

  1. Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

    DOE PAGES

    Weighill, Deborah; Jones, Piet; Shah, Manesh; ...

    2018-05-11

    Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant's sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes usemore » of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. Lastly, the resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for

  2. RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

    PubMed

    Takahashi, Hirokazu; Ohkawachi, Masahiko; Horio, Kyohei; Kobori, Toshiro; Aki, Tsunehiro; Matsumura, Yukihiko; Nakashimada, Yutaka; Okamura, Yoshiko

    2018-05-17

    RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

  3. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

    PubMed

    Xu, Hong-Tao; Colby-Germinario, Susan P; Hassounah, Said; Quashie, Peter K; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R; Wainberg, Mark A

    2016-01-01

    The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Structure–function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matho, Michael H.; Schlossman, Andrew; Gilchuk, Iuliia M.

    Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here in this paper, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, andmore » VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138– and VACV-304–binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.« less

  5. Structure–function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8

    DOE PAGES

    Matho, Michael H.; Schlossman, Andrew; Gilchuk, Iuliia M.; ...

    2017-11-09

    Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here in this paper, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, andmore » VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138– and VACV-304–binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.« less

  6. High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

    PubMed Central

    Gerli, R; Muscat, C; Bistoni, O; Falini, B; Tomassini, C; Agea, E; Tognellini, R; Biagini, P; Bertotto, A

    1995-01-01

    The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA. PMID:8536371

  7. HIV and Drug Resistance: Hitting a Moving Target | Center for Cancer Research

    Cancer.gov

    Prior research revealed how HIV-1 makes its destructive entry into the target cell by fusing together the cholesterol-rich lipid bilayer of the viral envelope—made with key glycoproteins gp120 and gp41—and the host cell’s plasma membrane. Cell-viral interactions begin with the binding of gp120 to the CD4 receptor molecule on the target cell, followed by gp120 binding to coreceptors. These coreceptors likely reside in structures called lipid rafts—areas in the cell plasma membrane that are rich in cholesterol, saturated fatty acids, and certain proteins that facilitate the entry of viruses into host cells. Finally, sequences in gp41 trigger the fusion of the viral and cellular lipid bilayers. The lipid rafts are then involved in the production of new viral particles.

  8. Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules.

    PubMed

    Childs-Disney, Jessica L; Stepniak-Konieczna, Ewa; Tran, Tuan; Yildirim, Ilyas; Park, HaJeung; Chen, Catherine Z; Hoskins, Jason; Southall, Noel; Marugan, Juan J; Patnaik, Samarjit; Zheng, Wei; Austin, Chris P; Schatz, George C; Sobczak, Krzysztof; Thornton, Charles A; Disney, Matthew D

    2013-01-01

    The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)(exp)). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. A thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)(exp) and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)(exp). This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)(exp) and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of myotonic dystrophy type 1.

  9. Induction and Reversal of Myotonic Dystrophy Type 1 Pre-mRNA Splicing Defects by Small Molecules

    PubMed Central

    Childs-Disney, Jessica L.; Stepniak-Konieczna, Ewa; Tran, Tuan; Yildirim, Ilyas; Park, HaJeung; Chen, Catherine Z.; Hoskins, Jason; Southall, Noel; Marugan, Juan J.; Patnaik, Samarjit; Zheng, Wei; Austin, Chris P.; Schatz, George C.; Sobczak, Krzysztof; Thornton, Charles A.; Disney, Matthew D.

    2013-01-01

    The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 (DM1) is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)exp). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. The thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)exp and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)exp. This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)exp and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of DM1. PMID:23806903

  10. Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

    PubMed Central

    Kim, Dongwook; Yan, Yitang; Valencia, C. Alexander; Liu, Rihe

    2012-01-01

    Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2, were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric ZEGFR and ZHER2 ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents.. PMID:22912791

  11. The future impacts of non-targeted effects.

    PubMed

    Bright, Scott; Kadhim, Munira

    2018-04-11

    Ionizing radiation was traditionally thought to exert its detrimental effects through interaction with sensitive cellular targets, nuclear DNA being of most importance. This theory has since merged with a more recently described radiation response called non-targeted effects (NTE). This review will briefly look at the various types of NTE and the potential implications they may have for radiobiology research and its applications. The most well-known NTE are genomic instability (GI) and bystander effects (BE). Other NTE include abscopal effects, which are similar to bystander effects but are generally based in a clinical environment with immune involvement as the defining feature. Currently, our understanding of NTE is limited to certain signaling pathways/molecules, and as yet there is no theory that describes or can accurately predict the occurrence or outcome of these NTE. There are numerous groups investigating these processes in vitro and in vivo, and thus steady progress is being made. Developing a deeper understanding of NTE has potential impacts for therapy and diagnosis, safer occupational exposures, space flight and our general understanding of radiation biology.

  12. Computational modeling of chemical reactions and interstitial growth and remodeling involving charged solutes and solid-bound molecules

    PubMed Central

    Nims, Robert J.; Maas, Steve; Weiss, Jeffrey A.

    2014-01-01

    Mechanobiological processes are rooted in mechanics and chemistry, and such processes may be modeled in a framework that couples their governing equations starting from fundamental principles. In many biological applications, the reactants and products of chemical reactions may be electrically charged, and these charge effects may produce driving forces and constraints that significantly influence outcomes. In this study, a novel formulation and computational implementation are presented for modeling chemical reactions in biological tissues that involve charged solutes and solid-bound molecules within a deformable porous hydrated solid matrix, coupling mechanics with chemistry while accounting for electric charges. The deposition or removal of solid-bound molecules contributes to the growth and remodeling of the solid matrix; in particular, volumetric growth may be driven by Donnan osmotic swelling, resulting from charged molecular species fixed to the solid matrix. This formulation incorporates the state of strain as a state variable in the production rate of chemical reactions, explicitly tying chemistry with mechanics for the purpose of modeling mechanobiology. To achieve these objectives, this treatment identifies the specific theoretical and computational challenges faced in modeling complex systems of interacting neutral and charged constituents while accommodating any number of simultaneous reactions where reactants and products may be modeled explicitly or implicitly. Several finite element verification problems are shown to agree with closed-form analytical solutions. An illustrative tissue engineering analysis demonstrates tissue growth and swelling resulting from the deposition of chondroitin sulfate, a charged solid-bound molecular species. This implementation is released in the open-source program FEBio (www.febio.org). The availability of this framework may be particularly beneficial to optimizing tissue engineering culture systems by examining the

  13. Computational modeling of chemical reactions and interstitial growth and remodeling involving charged solutes and solid-bound molecules.

    PubMed

    Ateshian, Gerard A; Nims, Robert J; Maas, Steve; Weiss, Jeffrey A

    2014-10-01

    Mechanobiological processes are rooted in mechanics and chemistry, and such processes may be modeled in a framework that couples their governing equations starting from fundamental principles. In many biological applications, the reactants and products of chemical reactions may be electrically charged, and these charge effects may produce driving forces and constraints that significantly influence outcomes. In this study, a novel formulation and computational implementation are presented for modeling chemical reactions in biological tissues that involve charged solutes and solid-bound molecules within a deformable porous hydrated solid matrix, coupling mechanics with chemistry while accounting for electric charges. The deposition or removal of solid-bound molecules contributes to the growth and remodeling of the solid matrix; in particular, volumetric growth may be driven by Donnan osmotic swelling, resulting from charged molecular species fixed to the solid matrix. This formulation incorporates the state of strain as a state variable in the production rate of chemical reactions, explicitly tying chemistry with mechanics for the purpose of modeling mechanobiology. To achieve these objectives, this treatment identifies the specific theoretical and computational challenges faced in modeling complex systems of interacting neutral and charged constituents while accommodating any number of simultaneous reactions where reactants and products may be modeled explicitly or implicitly. Several finite element verification problems are shown to agree with closed-form analytical solutions. An illustrative tissue engineering analysis demonstrates tissue growth and swelling resulting from the deposition of chondroitin sulfate, a charged solid-bound molecular species. This implementation is released in the open-source program FEBio ( www.febio.org ). The availability of this framework may be particularly beneficial to optimizing tissue engineering culture systems by examining the

  14. Targeting MicroRNAs with Small Molecules a Novel Approach to Treating Breast Cancer

    DTIC Science & Technology

    2011-10-01

    pathogenesis of a disease. To date, the main RNA inhibition agents used in pre- clinical and clinical studies include antisense oligonucleotides, ribozymes ...antagomir Preclinical studies Ribozymes or DNAzymes A ribozyme , or RNA enzyme, is an RNA molecule that can catalyze a chemical reaction. A DNAzyme

  15. Neuron membrane trafficking and protein kinases involved in autism and ADHD.

    PubMed

    Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

    2015-01-30

    A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  16. Multifunctional magnetic nanoparticles for targeted imaging and therapy

    PubMed Central

    McCarthy, Jason R.; Weissleder, Ralph

    2008-01-01

    Magnetic nanoparticles have become important tools for the imaging of prevalent diseases, such as cancer, atherosclerosis, diabetes, and others. While first generation nanoparticles were fairly nonspecific, newer generations have been targeted to specific cell types and molecular targets via affinity ligands. Commonly, these ligands emerge from phage or small molecule screens, or are based on antibodies or aptamers. Secondary reporters and combined therapeutic molecules have further opened potential clinical applications of these materials. This review summarizes some of the recent biomedical applications of these newer magnetic nanomaterials. PMID:18508157

  17. Targeting of drugs and nanoparticles to tumors

    PubMed Central

    Bhatia, Sangeeta N.; Sailor, Michael J.

    2010-01-01

    The various types of cells that comprise the tumor mass all carry molecular markers that are not expressed or are expressed at much lower levels in normal cells. These differentially expressed molecules can be used as docking sites to concentrate drug conjugates and nanoparticles at tumors. Specific markers in tumor vessels are particularly well suited for targeting because molecules at the surface of blood vessels are readily accessible to circulating compounds. The increased concentration of a drug in the site of disease made possible by targeted delivery can be used to increase efficacy, reduce side effects, or achieve some of both. We review the recent advances in this delivery approach with a focus on the use of molecular markers of tumor vasculature as the primary target and nanoparticles as the delivery vehicle. PMID:20231381

  18. Oligodendroglial p130Cas Is a Target of Fyn Kinase Involved in Process Formation, Cell Migration and Survival

    PubMed Central

    Gonsior, Constantin; Binamé, Fabien; Frühbeis, Carsten; Bauer, Nina M.; Hoch-Kraft, Peter; Luhmann, Heiko J.; Trotter, Jacqueline; White, Robin

    2014-01-01

    Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation. PMID:24586768

  19. Future therapeutic targets for the treatment and prevention of cholesterol gallstones.

    PubMed

    Castro-Torres, Ibrahim Guillermo; de Jesús Cárdenas-Vázquez, René; Velázquez-González, Claudia; Ventura-Martínez, Rosa; De la O-Arciniega, Minarda; Naranjo-Rodríguez, Elia Brosla; Martínez-Vázquez, Mariano

    2015-10-15

    The formation of cholesterol gallstones involves very complex imbalances, such as alterations in the secretion of biliary lipids (which involves the ABCG5, ABCG8, ABCB4 and ABCB11 transporters), biochemical and immunological reactions in the gallbladder that produce biliary sludge (mucins), physicochemical changes in the structure of cholesterol (crystallization), alterations in gallbladder motility, changes in the intestinal absorption of cholesterol (ABCG5/8 transporters and Niemann-Pick C1L1 protein) and alterations in small intestine motility. Some of these proteins have been studied at the clinical and experimental levels, but more research is required. In this review, we discuss the results of studies on some molecules involved in the pathophysiology of gallstones that may be future therapeutic targets to prevent the development of this disease, and possible sites for treatment based mainly on the absorption of intestinal cholesterol (Niemann-Pick C1L1 and ABCG5/8 proteins). Copyright © 2015. Published by Elsevier B.V.

  20. Targeted pancreatic cancer therapy with the small molecule drug conjugate SW IV-134.

    PubMed

    Hashim, Yassar M; Spitzer, Dirk; Vangveravong, Suwanna; Hornick, Mary C; Garg, Gunjal; Hornick, John R; Goedegebuure, Peter; Mach, Robert H; Hawkins, William G

    2014-07-01

    Pancreatic adenocarcinoma is highly resistant to conventional therapeutics and has been shown to evade apoptosis by deregulation of the X-linked and cellular inhibitors of apoptosis proteins (XIAP and cIAP). Second mitochondria-derived activator of caspases (Smac) induces and amplifies cell death by reversing the anti-apoptotic activity of IAPs. Thus, Smac-derived peptide analogues (peptidomimetics) have been developed and shown to represent promising cancer therapeutics. Sigma-2 receptors are overexpressed in many proliferating tumor cells including pancreatic cancer. Selected ligands to this receptor are rapidly internalized by cancer cells. These characteristics have made the sigma-2 receptor an attractive target for drug delivery because selective delivery to cancer cells has the potential to increase therapeutic efficacy while minimizing toxicity to normal tissues. Here, we describe the initial characterization of SW IV-134, a chemically linked drug conjugate between the sigma-2 ligand SW43 and the Smac mimetic SW IV-52 as a novel treatment option for pancreatic adenocarcinoma. The tumor killing characteristics of our dual-domain therapeutic SW IV-134 was far greater than either component in isolation or in an equimolar mix and suggests enhanced cellular delivery when chemically linked to the sigma-2 ligand. One of the key findings was that SW IV-134 retained target selectivity of the Smac cargo with the involvement of the NF-κB/TNFα signaling pathway. Importantly, SW IV-134 slowed tumor growth and improved survival in murine models of pancreatic cancer. Our data support further study of this novel therapeutic and this drug delivery strategy because it may eventually benefit patients with pancreatic cancer. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. Screening for small molecule inhibitors of Toxoplasma gondii.

    PubMed

    Kortagere, Sandhya

    2012-12-01

    Toxoplasma gondii, the agent that causes toxoplasmosis, is an opportunistic parasite that infects many mammalian species. It is an obligate intracellular parasite that causes severe congenital neurological and ocular disease mostly in immunocompromised humans. The current regimen of therapy includes only a few medications that often lead to hypersensitivity and toxicity. In addition, there are no vaccines available to prevent the transmission of this agent. Therefore, safer and more effective medicines to treat toxoplasmosis are urgently needed. The author presents in silico and in vitro strategies that are currently used to screen for novel targets and unique chemotypes against T. gondii. Furthermore, this review highlights the screening technologies and characterization of some novel targets and new chemical entities that could be developed into highly efficacious treatments for toxoplasmosis. A number of diverse methods are being used to design inhibitors against T. gondii. These include ligand-based methods, in which drugs that have been shown to be efficacious against other Apicomplexa parasites can be repurposed to identify lead molecules against T. gondii. In addition, structure-based methods use currently available repertoire of structural information in various databases to rationally design small-molecule inhibitors of T. gondii. Whereas the screening methods have their advantages and limitations, a combination of methods is ideally suited to design small-molecule inhibitors of complex parasites such as T. gondii.

  2. Targeted drug delivery to the brain using magnetic nanoparticles.

    PubMed

    Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Moos, Torben

    2015-01-01

    Brain capillary endothelial cells denote the blood-brain barrier (BBB), and conjugation of nanoparticles with antibodies that target molecules expressed by these endothelial cells may facilitate their uptake and transport into the brain. Magnetic nanoparticles can be encapsulated in liposomes and carry large molecules with therapeutic potential, for example, siRNA, cDNA and polypeptides. An additional approach to enhance the transport of magnetic nanoparticles across the BBB is the application of extracranially applied magnetic force. Stepwise targeting of magnetic nanoparticles to brain capillary endothelial cells followed by transport through the BBB using magnetic force may prove a novel mechanism for targeted therapy of macromolecules to the brain.

  3. The immune synapse clears and excludes molecules above a size threshold

    PubMed Central

    Cartwright, Adam N. R.; Griggs, Jeremy; Davis, Daniel M.

    2014-01-01

    Natural killer cells assess target cell health via interactions at the immune synapse (IS) that facilitates signal integration and directed secretion. Here we test whether the IS also functions as a gasket. Quantitative fluorescence microscopy of nanometer-scale dextrans within synapses formed by various effector-target cell conjugates reveal that molecules are excluded in a size-dependent manner at activating synapses. Dextran sized ≤4 nm move in and out of the IS, but access is significantly reduced (by >50%) for dextran sized 10–13 nm, and dextran ≥32 nm is almost entirely excluded. Depolymerization of F-actin abrogated exclusion. Unexpectedly, larger-sized dextrans are cleared as the IS assembles in a zipper-like manner. Monoclonal antibodies are also excluded from the IS but smaller single-domain antibodies are able to penetrate. Therefore, the IS can clear and exclude molecules above a size threshold, and drugs designed to target synaptic cytokines or cytotoxic proteins must fit these dimensions. PMID:25407222

  4. Improved Dye Stability in Single-Molecule Fluorescence Experiments

    NASA Astrophysics Data System (ADS)

    EcheverrÍa Aitken, Colin; Marshall, R. Andrew; Pugi, Joseph D.

    Complex biological systems challenge existing single-molecule methods. In particular, dye stability limits observation time in singlemolecule fluorescence applications. Current approaches to improving dye performance involve the addition of enzymatic oxygen scavenging systems and small molecule additives. We present an enzymatic oxygen scavenging system that improves dye stability in single-molecule experiments. Compared to the currently-employed glucose-oxidase/catalase system, the protocatechuate-3,4-dioxygenase system achieves lower dissolved oxygen concentration and stabilizes single Cy3, Cy5, and Alexa488 fluorophores. Moreover, this system possesses none of the limitations associated with the glucose oxidase/catalase system. We also tested the effects of small molecule additives in this system. Biological reducing agents significantly destabilize the Cy5 fluorophore as a function of reducing potential. In contrast, anti-oxidants stabilize the Cy3 and Alexa488 fluorophores. We recommend use of the protocatechuate-3,4,-dioxygenase system with antioxidant additives, and in the absence of biological reducing agents. This system should have wide application to single-molecule fluorescence experiments.

  5. Rational Combinations of Targeted Agents in AML

    PubMed Central

    Bose, Prithviraj; Grant, Steven

    2015-01-01

    Despite modest improvements in survival over the last several decades, the treatment of AML continues to present a formidable challenge. Most patients are elderly, and these individuals, as well as those with secondary, therapy-related, or relapsed/refractory AML, are particularly difficult to treat, owing to both aggressive disease biology and the high toxicity of current chemotherapeutic regimens. It has become increasingly apparent in recent years that coordinated interruption of cooperative survival signaling pathways in malignant cells is necessary for optimal therapeutic results. The modest efficacy of monotherapy with both cytotoxic and targeted agents in AML testifies to this. As the complex biology of AML continues to be elucidated, many “synthetic lethal” strategies involving rational combinations of targeted agents have been developed. Unfortunately, relatively few of these have been tested clinically, although there is growing interest in this area. In this article, the preclinical and, where available, clinical data on some of the most promising rational combinations of targeted agents in AML are summarized. While new molecules should continue to be combined with conventional genotoxic drugs of proven efficacy, there is perhaps a need to rethink traditional philosophies of clinical trial development and regulatory approval with a focus on mechanism-based, synergistic strategies. PMID:26113989

  6. Unraveling secrets of telomeres: one molecule at a time

    PubMed Central

    Lin, Jiangguo; Kaur, Parminder; Countryman, Preston; Opresko, Patricia L.; Wang, Hong

    2016-01-01

    Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins. PMID:24569170

  7. Tomatidine Is a Lead Antibiotic Molecule That Targets Staphylococcus aureus ATP Synthase Subunit C.

    PubMed

    Lamontagne Boulet, Maxime; Isabelle, Charles; Guay, Isabelle; Brouillette, Eric; Langlois, Jean-Philippe; Jacques, Pierre-Étienne; Rodrigue, Sébastien; Brzezinski, Ryszard; Beauregard, Pascale B; Bouarab, Kamal; Boyapelly, Kumaraswamy; Boudreault, Pierre-Luc; Marsault, Éric; Malouin, François

    2018-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti- Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro -generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutants into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >10 5 -fold for FC04-100. Copyright © 2018 American Society for Microbiology.

  8. Lysine Acetylation in Sexual Stage Malaria Parasites Is a Target for Antimalarial Small Molecules

    PubMed Central

    Trenholme, Katharine; Marek, Linda; Duffy, Sandra; Pradel, Gabriele; Fisher, Gillian; Hansen, Finn K.; Skinner-Adams, Tina S.; Butterworth, Alice; Ngwa, Che Julius; Moecking, Jonas; Goodman, Christopher D.; McFadden, Geoffrey I.; Sumanadasa, Subathdrage D. M.; Fairlie, David P.; Avery, Vicky M.

    2014-01-01

    Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in Plasmodium falciparum gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in P. falciparum gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology. PMID:24733477

  9. Chemical wiring and soldering toward all-molecule electronic circuitry.

    PubMed

    Okawa, Yuji; Mandal, Swapan K; Hu, Chunping; Tateyama, Yoshitaka; Goedecker, Stefan; Tsukamoto, Shigeru; Hasegawa, Tsuyoshi; Gimzewski, James K; Aono, Masakazu

    2011-06-01

    Key to single-molecule electronics is connecting functional molecules to each other using conductive nanowires. This involves two issues: how to create conductive nanowires at designated positions, and how to ensure chemical bonding between the nanowires and functional molecules. Here, we present a novel method that solves both issues. Relevant functional molecules are placed on a self-assembled monolayer of diacetylene compound. A probe tip of a scanning tunneling microscope is then positioned on the molecular row of the diacetylene compound to which the functional molecule is adsorbed, and a conductive polydiacetylene nanowire is fabricated by initiating chain polymerization by stimulation with the tip. Since the front edge of chain polymerization necessarily has a reactive chemical species, the created polymer nanowire forms chemical bonding with an encountered molecular element. We name this spontaneous reaction "chemical soldering". First-principles theoretical calculations are used to investigate the structures and electronic properties of the connection. We demonstrate that two conductive polymer nanowires are connected to a single phthalocyanine molecule. A resonant tunneling diode formed by this method is discussed. © 2011 American Chemical Society

  10. Emerging targets in lipid-based therapy☆

    PubMed Central

    Tucker, Stephanie C.; Honn, Kenneth V.

    2013-01-01

    The use of prostaglandins and NSAIDS in the clinic has proven that lipid mediators and their associated pathways make attractive therapeutic targets. When contemplating therapies involving lipid pathways, several basic agents come to mind. There are the enzymes and accessory proteins that lead to the metabolism of lipid substrates, provided through diet or through actions of lipases, the subsequent lipid products, and finally the lipid sensors or receptors. There is abundant evidence that molecules along this lipid continuum can serve as prognostic and diagnostic indicators and are in fact viable therapeutic targets. Furthermore, lipids themselves can be used as therapeutics. Despite this, the vernacular dialog pertaining to “biomarkers” does not routinely include mention of lipids, though this is rapidly changing. Collectively these agents are becoming more appreciated for their respective roles in diverse disease processes from cancer to preterm labor and are receiving their due appreciation after decades of ground work in the lipid field. By relating examples of disease processes that result from dysfunction along the lipid continuum, as well as examples of lipid therapies and emerging technologies, this review is meant to inspire further reading and discovery. PMID:23261527

  11. Identifying Mechanisms of Interfacial Dynamics Using Single-Molecule Tracking

    PubMed Central

    Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2012-01-01

    The “soft” (i.e. non-covalent) interactions between molecules and surfaces are complex and highly-varied (e.g. hydrophobic, hydrogen bonding, ionic) often leading to heterogeneous interfacial behavior. Heterogeneity can arise either from spatial variation of the surface/interface itself or from molecular configurations (i.e. conformation, orientation, aggregation state, etc.). By observing adsorption, diffusion, and desorption of individual fluorescent molecules, single-molecule tracking can characterize these types of heterogeneous interfacial behavior in ways that are inaccessible to traditional ensemble-averaged methods. Moreover, the fluorescence intensity or emission wavelength (in resonance energy transfer experiments) can be used to simultaneously track molecular configuration and directly relate this to the resulting interfacial mobility or affinity. In this feature article, we review recent advances involving the use of single-molecule tracking to characterize heterogeneous molecule-surface interactions including: multiple modes of diffusion and desorption associated with both internal and external molecular configuration, Arrhenius activated interfacial transport, spatially dependent interactions, and many more. PMID:22716995

  12. Low energy electron-molecule scattering using the R-matrix method

    NASA Astrophysics Data System (ADS)

    Gorfinkiel, Jimena

    2014-10-01

    The study of electron-molecule collisions continues to attract significant interest stimulated, in no small part, by the need for collisional data to model a number of physical environments and applied processes (e.g. the modelling of focused electron beam induced deposition and the description of the interaction of radiation with biological matter). This need for electron scattering data (cross sections but also information on the temporary negative ions, TNI, that can be formed) has motivated the renewed development of theoretical methodology and their computational implementation. I will present the latest developments in the study of low energy electron scattering from molecules and molecular clusters using the R-matrix method. Recent calculations on electron collisions with biologically relevant molecules have shed light on the formation of core-excited TNI these larger targets. The picture that emerges is much more complex than previously thought. I will discuss some examples as well as current and future developments of the methodology and software in order to provide more accurate collisional data (in particular cross sections) for bigger targets. In collaboration with Zdenek Masin, The Open University. This work was partially supported by EPSRC.

  13. Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation

    PubMed Central

    Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y.; Tontonoz, Peter; Park, Kye Won

    2016-01-01

    Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

  14. One-by-one single-molecule detection of mutated nucleobases by monitoring tunneling current using a DNA tip.

    PubMed

    Bui, Phuc Tan; Nishino, Tomoaki; Shiigi, Hiroshi; Nagaoka, Tsutomu

    2015-01-31

    A DNA molecule was utilized as a probe tip to achieve single-molecule genetic diagnoses. Hybridization of the probe and target DNAs resulted in electron tunneling along the emergent double-stranded DNA. Simple stationary monitoring of the tunneling current leads to single-molecule DNA detection and discovery of base mismatches and methylation.

  15. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  16. A novel approach to the simultaneous extraction and non-targeted analysis of the small molecules metabolome and lipidome using 96-well solid phase extraction plates with column-switching technology.

    PubMed

    Li, Yubo; Zhang, Zhenzhu; Liu, Xinyu; Li, Aizhu; Hou, Zhiguo; Wang, Yuming; Zhang, Yanjun

    2015-08-28

    This study combines solid phase extraction (SPE) using 96-well plates with column-switching technology to construct a rapid and high-throughput method for the simultaneous extraction and non-targeted analysis of small molecules metabolome and lipidome based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry. This study first investigated the columns and analytical conditions for small molecules metabolome and lipidome, separated by an HSS T3 and BEH C18 columns, respectively. Next, the loading capacity and actuation duration of SPE were further optimized. Subsequently, SPE and column switching were used together to rapidly and comprehensively analyze the biological samples. The experimental results showed that the new analytical procedure had good precision and maintained sample stability (RSD<15%). The method was then satisfactorily applied to more widely analyze the small molecules metabolome and lipidome to test the throughput. The resulting method represents a new analytical approach for biological samples, and a highly useful tool for researches in metabolomics and lipidomics. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. 3D flexible alignment using 2D maximum common substructure: dependence of prediction accuracy on target-reference chemical similarity.

    PubMed

    Kawabata, Takeshi; Nakamura, Haruki

    2014-07-28

    A protein-bound conformation of a target molecule can be predicted by aligning the target molecule on the reference molecule obtained from the 3D structure of the compound-protein complex. This strategy is called "similarity-based docking". For this purpose, we develop the flexible alignment program fkcombu, which aligns the target molecule based on atomic correspondences with the reference molecule. The correspondences are obtained by the maximum common substructure (MCS) of 2D chemical structures, using our program kcombu. The prediction performance was evaluated using many target-reference pairs of superimposed ligand 3D structures on the same protein in the PDB, with different ranges of chemical similarity. The details of atomic correspondence largely affected the prediction success. We found that topologically constrained disconnected MCS (TD-MCS) with the simple element-based atomic classification provides the best prediction. The crashing potential energy with the receptor protein improved the performance. We also found that the RMSD between the predicted and correct target conformations significantly correlates with the chemical similarities between target-reference molecules. Generally speaking, if the reference and target compounds have more than 70% chemical similarity, then the average RMSD of 3D conformations is <2.0 Å. We compared the performance with a rigid-body molecular alignment program based on volume-overlap scores (ShaEP). Our MCS-based flexible alignment program performed better than the rigid-body alignment program, especially when the target and reference molecules were sufficiently similar.

  18. Sulforaphane suppresses vascular adhesion molecule-1 expression in TNF-α-stimulated mouse vascular smooth muscle cells: involvement of the MAPK, NF-κB and AP-1 signaling pathways.

    PubMed

    Kim, Ji-Yun; Park, Hye-Jin; Um, Sung Hee; Sohn, Eun-Hwa; Kim, Byung-Oh; Moon, Eun-Yi; Rhee, Dong-Kwon; Pyo, Suhkneung

    2012-01-01

    Atherosclerosis is a long-term inflammatory disease of the arterial wall. Increased expression of the cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) is associated with increased proliferation of vascular smooth muscle cells (VSMCs), leading to increased neointima or atherosclerotic lesion formation. Therefore, the functional inhibition of adhesion molecules could be a critical therapeutic target of inflammatory disease. In the present study, we investigate the effect of sulforaphane on the expression of VCAM-1 induced by TNF-α in cultured mouse vascular smooth muscle cell lines. Pretreatment of VSMCs for 2h with sulforaphane (1-5μg/ml) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and protein expression of VCAM-1. Sulforaphane also suppressed TNF-α-induced production of intracellular reactive oxygen species (ROS) and activation of p38, ERK and JNK. Furthermore, sulforaphane inhibited NK-κB and AP-1 activation induced by TNF-α. Sulforaphane inhibited TNF-α-induced ΙκΒ kinase activation, subsequent degradation of ΙκΒα and nuclear translocation of p65 NF-κB and decreased c-Jun and c-Fos protein level. This study suggests that sulforaphane inhibits the adhesive capacity of VSMC and downregulates the TNF-α-mediated induction of VCAM-1 in VSMC by inhibiting the MAPK, NF-κB and AP-1 signaling pathways and intracellular ROS production. Thus, sulforaphane may have beneficial effects to suppress inflammation within the atherosclerotic lesion. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Recent development of small molecule glutaminase inhibitors.

    PubMed

    Song, Minsoo; Kim, Soong-Hyun; Im, Chun Young; Hwang, Hee-Jong

    2018-05-24

    Glutaminase (GLS) which is responsible for the conversion of glutamine to glutamate plays vital role in up-regulating cell metabolism for tumor cell growth, and is considered as a valuable therapeutic target for cancer treatment. Based on this important function of glutaminase in cancer, several GLS inhibitors have been developed from both academia and industries. Most importantly, Calithera Biosciences Inc. is actively developing glutaminase inhibitor CB-839 for the treatment of various cancers in phase 1 and 2 clinical trials at present. In this review, it is discussed about recent efforts to develop small molecule glutaminase inhibitors targeting glutamine metabolism both in the preclinical and clinical studies. In particular, more emphasis is placed on CB-839 since it is the only small molecule GLS inhibitor being studied in clinical setting. Inhibition mechanism is discussed based on x-ray structure study of thiadiazole derivatives as well. Finally, recent medicinal chemistry efforts to develop a new class of GLS inhibitors are given herein in the hope of providing useful information for GLS inhibitors of the next generation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.

    PubMed

    Lin, Ran; Zhang, Pengcheng; Cheetham, Andrew G; Walston, Jeremy; Abadir, Peter; Cui, Honggang

    2015-01-21

    Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.