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Sample records for high-throughput antimalarial drug

  1. Validating a Firefly Luciferase-Based High-Throughput Screening Assay for Antimalarial Drug Discovery

    PubMed Central

    Che, Pulin; Cui, Long; Kutsch, Olaf; Cui, Liwang

    2012-01-01

    Abstract The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the importance of developing novel antimalarial therapies. Using a previously generated stable transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a high-throughput screening assay in 384-well plates. Assay conditions, including the percentage of parasitemia and hematocrit, were optimized. Parameters of assay robustness, including Z′-value, coefficient variation (CV), and signal-to-background (S/B) ratio, were determined. The LOPAC1280 small-compound library was used to validate this assay. Our results demonstrated that this assay is robust and reliable, with an average Z′-value of >0.7 and CV of <10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay. It is evident that this assay is suitable for large-scale screening of chemical libraries for antimalarial drug discovery. PMID:22050430

  2. High-throughput matrix screening identifies synergistic and antagonistic antimalarial drug combinations

    PubMed Central

    Mott, Bryan T.; Eastman, Richard T.; Guha, Rajarshi; Sherlach, Katy S.; Siriwardana, Amila; Shinn, Paul; McKnight, Crystal; Michael, Sam; Lacerda-Queiroz, Norinne; Patel, Paresma R.; Khine, Pwint; Sun, Hongmao; Kasbekar, Monica; Aghdam, Nima; Fontaine, Shaun D.; Liu, Dongbo; Mierzwa, Tim; Mathews-Griner, Lesley A.; Ferrer, Marc; Renslo, Adam R.; Inglese, James; Yuan, Jing; Roepe, Paul D.; Su, Xin-zhuan; Thomas, Craig J.

    2015-01-01

    Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy. PMID:26403635

  3. High-throughput matrix screening identifies synergistic and antagonistic antimalarial drug combinations.

    PubMed

    Mott, Bryan T; Eastman, Richard T; Guha, Rajarshi; Sherlach, Katy S; Siriwardana, Amila; Shinn, Paul; McKnight, Crystal; Michael, Sam; Lacerda-Queiroz, Norinne; Patel, Paresma R; Khine, Pwint; Sun, Hongmao; Kasbekar, Monica; Aghdam, Nima; Fontaine, Shaun D; Liu, Dongbo; Mierzwa, Tim; Mathews-Griner, Lesley A; Ferrer, Marc; Renslo, Adam R; Inglese, James; Yuan, Jing; Roepe, Paul D; Su, Xin-Zhuan; Thomas, Craig J

    2015-01-01

    Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy. PMID:26403635

  4. Hematin Polymerization Assay as a High-Throughput Screen for Identification of New Antimalarial Pharmacophores

    PubMed Central

    Kurosawa, Yae; Dorn, Arnulf; Kitsuji-Shirane, Michiko; Shimada, Hisao; Satoh, Tomoko; Matile, Hugues; Hofheinz, Werner; Masciadri, Raffaello; Kansy, Manfred; Ridley, Robert G.

    2000-01-01

    Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of 14C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100,000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the “Catalyst” program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified “hit” compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program. PMID:10991837

  5. Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.

    PubMed

    Kurosawa, Y; Dorn, A; Kitsuji-Shirane, M; Shimada, H; Satoh, T; Matile, H; Hofheinz, W; Masciadri, R; Kansy, M; Ridley, R G

    2000-10-01

    Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program. PMID:10991837

  6. Automated High Throughput Drug Target Crystallography

    SciTech Connect

    Rupp, B

    2005-02-18

    The molecular structures of drug target proteins and receptors form the basis for 'rational' or structure guided drug design. The majority of target structures are experimentally determined by protein X-ray crystallography, which as evolved into a highly automated, high throughput drug discovery and screening tool. Process automation has accelerated tasks from parallel protein expression, fully automated crystallization, and rapid data collection to highly efficient structure determination methods. A thoroughly designed automation technology platform supported by a powerful informatics infrastructure forms the basis for optimal workflow implementation and the data mining and analysis tools to generate new leads from experimental protein drug target structures.

  7. Hemozoin and antimalarial drug discovery

    PubMed Central

    Fong, Kim Y; Wright, David W

    2014-01-01

    Recent initiatives to develop more effective and affordable drugs, controlling mosquitoes and development of a preventative vaccine have been launched with the goal of completely eradicating malaria. To this end, Novartis (Surrey, UK) and GlaxoSmithKline (Middlesex, UK) screened their chemical libraries of approximately two million small molecules for antimalarial properties, which resulted in a set of over 20,000 ‘highly druggable’ initial hits. Efforts in academia are centered on specific pathway targets. One such high-throughput screening effort has been focused on hemozoin formation, a unique heme detoxification pathway found in the malaria parasite. This review discusses the current approaches and limitations of high-throughput screening discovery of hemozoin inhibitors. In the future, new methods must be developed to validate the mechanism of action of these hit compounds within the parasite. PMID:23919553

  8. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

    PubMed Central

    Plouffe, David; Brinker, Achim; McNamara, Case; Henson, Kerstin; Kato, Nobutaka; Kuhen, Kelli; Nagle, Advait; Adrián, Francisco; Matzen, Jason T.; Anderson, Paul; Nam, Tae-gyu; Gray, Nathanael S.; Chatterjee, Arnab; Janes, Jeff; Yan, S. Frank; Trager, Richard; Caldwell, Jeremy S.; Schultz, Peter G.; Zhou, Yingyao; Winzeler, Elizabeth A.

    2008-01-01

    The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of ≈1.7 million compounds, we identified a diverse collection of ≈6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 μM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities. PMID:18579783

  9. Development and Optimization of a Novel 384-Well Anti-Malarial Imaging Assay Validated for High-Throughput Screening

    PubMed Central

    Duffy, Sandra; Avery, Vicky M.

    2012-01-01

    With the increasing occurrence of drug resistance in the malaria parasite, Plasmodium falciparum, there is a great need for new and novel anti-malarial drugs. We have developed a 384-well, high-throughput imaging assay for the detection of new anti-malarial compounds, which was initially validated by screening a marine natural product library, and subsequently used to screen more than 3 million data points from a variety of compound sources. Founded on another fluorescence-based P. falciparum growth inhibition assay, the DNA-intercalating dye 4′,6-diamidino-2-phenylindole, was used to monitor changes in parasite number. Fluorescent images were acquired on the PerkinElmer Opera High Throughput confocal imaging system and analyzed with a spot detection algorithm using the Acapella data processing software. Further optimization of this assay sought to increase throughput, assay stability, and compatibility with our high-throughput screening equipment platforms. The assay typically yielded Z'-factor values of 0.5–0.6, with signal-to-noise ratios of 12. PMID:22232455

  10. C. elegans in high-throughput drug discovery

    PubMed Central

    O’Reilly, Linda P.; Luke, Cliff J.; Perlmutter, David H.; Silverman, Gary A.; Pak, Stephen C.

    2014-01-01

    C. elegans has proven to be a useful model organism for investigating molecular and cellular aspects of numerous human diseases. More recently, investigators have explored the use of this organism as a tool for drug discovery. Although earlier drug screens were labor-intensive and low in throughput, recent advances in high-throughput liquid workflows, imaging platforms and data analysis software have made C. elegans a viable option for automated high-throughput drug screens. This review will outline the evolution of C. elegans-based drug screening, discuss the inherent challenges of using C. elegans, and highlight recent technological advances that have paved the way for future drug screens. PMID:24333896

  11. Identification of β-hematin inhibitors in a high-throughput screening effort reveals scaffolds with in vitro antimalarial activity

    PubMed Central

    Sandlin, Rebecca D.; Fong, Kim Y.; Wicht, Kathryn J.; Carrell, Holly M.; Egan, Timothy J.; Wright, David W.

    2014-01-01

    The emergence of drug resistant strains of Plasmodium spp. creates a critical need for the development of novel antimalarials. Formation of hemozoin, a crystalline heme detoxification process vital to parasite survival serves as an important drug target. The quinoline antimalarials including chloroquine and amodiaquine owe their antimalarial activity to inhibition of hemozoin formation. Though in vivo formation of hemozoin occurs within the presence of neutral lipids, the lipophilic detergent NP-40 was previously shown to serve as a surrogate in the β-hematin (synthetic hemozoin) formation process. Consequently, an NP-40 mediated β-hematin formation assay was developed for use in high-throughput screening. Here, the assay was utilized to screen 144,330 compounds for the identification of inhibitors of crystallization, resulting in 530 hits. To establish the effectiveness of these target-based β-hematin inhibitors against Plasmodiumfalciparum, each hit was further tested in cultures of parasitized red blood cells. This effort revealed that 171 of the β-hematin inhibitors are also active against the parasite. Dose–response data identified 73 of these β-hematin inhibitors have IC50 values ⩽5 μM, including 25 compounds with nanomolar activity against P. falciparum. A scaffold-based analysis of this data identified 14 primary scaffolds that represent 46% of the 530 total hits. Representative compounds from each of the classes were further assessed for hemozoin inhibitory activity in P. falciparum infected human erythrocytes. Each of the hit compounds tested were found to be positive inhibitors, while a negative control did not perturb this biological pathway in culture. PMID:25516843

  12. High throughput drug discovery with ESI-FTICR

    NASA Astrophysics Data System (ADS)

    Sannes-Lowery, Kristin A.; Cummins, Lendell L.; Chen, Shuo; Drader, Jared J.; Hofstadler, Steven A.

    2004-11-01

    Ribonucleic acids (RNA) are an attractive target for drug discovery since they play critical roles in cellular functions. Because small structured subdomains are known to mimic the behavior of the entire RNA, it is possible to design RNA drug targets that are amenable to interrogation by high performance mass spectrometry. We have developed a high throughput drug discovery platform that uses electrospray ionization Fourier transform ion cyclotron mass spectrometry to investigate ligand binding to structured RNA drug targets. This assay is called multitarget affinity/specificity screening (MASS). Using MASS, we show that it is possible to screen synthetic and natural product libraries in a high throughput and robust manner.

  13. Antimalarial drug resistance: An overview.

    PubMed

    Antony, Hiasindh Ashmi; Parija, Subhash Chandra

    2016-01-01

    Malaria is a major public health burden throughout the world. Resistance to the antimalarial drugs has increased the mortality and morbidity rate that is achieved so far through the malaria control program. Monitoring the drug resistance to the available antimalarial drugs helps to implement effective drug policy, through the in vivo efficacy studies, in vitro drug susceptibility tests and detection of molecular markers. It is important to understand the mechanism of the antimalarial drugs, as it is one of the key factors in the emergence and spread of drug resistance. This review summarizes the commonly used antimalarial drugs, their mechanism of action and the genetic markers validated so far for the detection of drug-resistant parasites. PMID:26998432

  14. Antimalarial drug resistance: An overview

    PubMed Central

    Antony, Hiasindh Ashmi; Parija, Subhash Chandra

    2016-01-01

    Malaria is a major public health burden throughout the world. Resistance to the antimalarial drugs has increased the mortality and morbidity rate that is achieved so far through the malaria control program. Monitoring the drug resistance to the available antimalarial drugs helps to implement effective drug policy, through the in vivo efficacy studies, in vitro drug susceptibility tests and detection of molecular markers. It is important to understand the mechanism of the antimalarial drugs, as it is one of the key factors in the emergence and spread of drug resistance. This review summarizes the commonly used antimalarial drugs, their mechanism of action and the genetic markers validated so far for the detection of drug-resistant parasites. PMID:26998432

  15. ANTIMALARIAL DRUG QUALITY IN AFRICA

    PubMed Central

    Amin, AA; Kokwaro, GO

    2009-01-01

    Background and objective There are several reports of sub-standard and counterfeit antimalarial drugs circulating in the markets of developing countries; we aimed to review the literature for the African continent. Methods A search was conducted in PubMED in English using the medical subject headings (MeSH) terms: “Antimalarials/analysis”[MeSH] OR “Antimalarials/standards”[MeSH] AND “Africa”[MeSH]” to include articles published up to and including 26/02/07. Data were augmented with reports on the quality of antimalarial drugs in Africa obtained from colleagues in the World Health Organization. We summarised the data under the following themes: content and dissolution; relative bioavalability of antimalarial products; antimalarial stability and shelf life; general tests on pharmaceutical dosage forms; and the presence of degradation or unidentifiable impurities in formulations. Results and discussion The search yielded 21 relevant peer-reviewed articles and three reports on the quality of antimalarial drugs in Africa. The literature was varied in the quality and breadth of data presented, with most bioavailability studies poorly designed and executed. The review highlights the common finding in drug quality studies that 1) most antimalarial products pass the basic tests for pharmaceutical dosage forms, such as the uniformity of weight for tablets 2) most antimalarial drugs pass the content test 3) in vitro product dissolution is the main problem area where most drugs fail to meet required pharmacopoeial specifications, especially with regard to sulfadoxine-pyrimethamine products. In addition, there are worryingly high quality failure rates for artemisinin monotherapies such as dihydroartemisin (DHA); for instance all five DHA sampled products in one study in Nairobi, Kenya, were reported to have failed the requisite tests. Conclusions There is an urgent need to strengthen pharmaceutical management systems such as post-marketing surveillance and the

  16. High-throughput analysis of behavior for drug discovery.

    PubMed

    Alexandrov, Vadim; Brunner, Dani; Hanania, Taleen; Leahy, Emer

    2015-03-01

    Drug testing with traditional behavioral assays constitutes a major bottleneck in the development of novel therapies. PsychoGenics developed three comprehensive high-throughput systems, SmartCube(®), NeuroCube(®) and PhenoCube(®) systems, to increase the efficiency of the drug screening and phenotyping in rodents. These three systems capture different domains of behavior, namely, cognitive, motor, circadian, social, anxiety-like, gait and others, using custom-built computer vision software and machine learning algorithms for analysis. This review exemplifies the use of the three systems and explains how they can advance drug screening with their applications to phenotyping of disease models, drug screening, selection of lead candidates, behavior-driven lead optimization, and drug repurposing. PMID:25592319

  17. [Resistance to the antimalarial drugs].

    PubMed

    Venanzi, E; López-Vélez, R

    2016-09-01

    Malaria is one of the most widespread infectious diseases around the world with 214 million cases and 438,000 deaths in 2015. In the early twentieth century it was described for the first time the resistance to quinine and, since then, drug resistance to antimalarial drugs has spread up to represent a global challenge in the fight and control of malaria. Understanding the mechanisms, geography and monitoring tools that we can act against resistance to antimalarial drugs is critical to prevent its expansion. PMID:27608319

  18. Microfluidic cell chips for high-throughput drug screening.

    PubMed

    Chi, Chun-Wei; Ahmed, Ah Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

    2016-05-01

    The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell-drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers. PMID:27071838

  19. Miniaturized Cultivation of Microbiota for Antimalarial Drug Discovery.

    PubMed

    Waterman, Carrie; Calcul, Laurent; Beau, Jeremy; Ma, Wai Sheung; Lebar, Matthew D; von Salm, Jacqueline L; Harter, Charles; Mutka, Tina; Morton, Lindsay C; Maignan, Patrick; Barisic, Betty; van Olphen, Alberto; Kyle, Dennis E; Vrijmoed, Lilian; Pang, Ka-Lai; Pearce, Cedric J; Baker, Bill J

    2016-01-01

    The ongoing search for effective antiplasmodial agents remains essential in the fight against malaria worldwide. Emerging parasitic drug resistance places an urgent need to explore chemotherapies with novel structures and mechanisms of action. Natural products have historically provided effective antimalarial drug scaffolds. In an effort to search nature's chemical potential for antiplasmodial agents, unconventionally sourced organisms coupled with innovative cultivation techniques were utilized. Approximately 60,000 niche microbes from various habitats (slow-growing terrestrial fungi, Antarctic microbes, and mangrove endophytes) were cultivated on a small-scale, extracted, and used in high-throughput screening to determine antimalarial activity. About 1% of crude extracts were considered active and 6% partially active (≥ 67% inhibition at 5 and 50 μg/mL, respectively). Active extracts (685) were cultivated on a large-scale, fractionated, and screened for both antimalarial activity and cytotoxicity. High interest fractions (397) with an IC50 < 1.11 μg/mL were identified and subjected to chromatographic separation for compound characterization and dereplication. Identifying active compounds with nanomolar antimalarial activity coupled with a selectivity index tenfold higher was accomplished with two of the 52 compounds isolated. This microscale, high-throughput screening project for antiplasmodial agents is discussed in the context of current natural product drug discovery efforts. PMID:25545963

  20. High Throughput Screening for Drugs that Modulate Intermediate Filament Proteins

    PubMed Central

    Sun, Jingyuan; Groppi, Vincent E.; Gui, Honglian; Chen, Lu; Xie, Qing; Liu, Li

    2016-01-01

    Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green-fluorescent-protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug ‘hits’ that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wildtype-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. ‘Hits’ of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant-IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients. PMID:26795471

  1. Silicon microphysiometer for high-throughput drug screening

    NASA Astrophysics Data System (ADS)

    Verhaegen, Katarina; Baert, Christiaan; Puers, Bob; Sansen, Willy; Simaels, Jeannine; Van Driessche, Veerle; Hermans, Lou; Mertens, Robert P.

    1999-06-01

    We report on a micromachined silicon chip that is capable of providing a high-throughput functional assay based on calorimetry. A prototype twin microcalorimeter based on the Seebeck effect has been fabricated by IC technology and micromachined postprocessing techniques. A biocompatible liquid rubber membrane supports two identical 0.5 X 2 cm2 measurement chambers, situated at the cold and hot junction of a 666-junction aluminum/p+-polysilicon thermopile. The chambers can house up to 106 eukaryotic cells cultured to confluence. The advantage of the device over microcalorimeters on the market, is the integration of the measurement channels on chip, rendering microvolume reaction vessels, ranging from 10 to 600 (mu) l, in the closest possible contact with the thermopile sensor (no springs are needed). Power and temperature sensitivity of the sensor are 23 V/W and 130 mV/K, respectively. The small thermal inertia of the microchannels results in the short response time of 70 s, when filled with 50 (mu) l of water. Biological experiments were done with cultured kidney cells of Xenopus laevis (A6). The thermal equilibration time of the device is 45 min. Stimulation of transport mechanisms by reducing bath osmolality by 50% increased metabolism by 20%. Our results show that it is feasible to apply this large-area, small- volume whole-cell biosensor for drug discovery, where the binding assays that are commonly used to provide high- throughput need to be complemented with a functional assay. Solutions are brought onto the sensor by a simple pipette, making the use of an industrial microtiterplate dispenser feasible on a nx96-array of the microcalorimeter biosensor. Such an array of biosensors has been designed based on a new set of requirements as set forth by people in the field as this project moved on. The results obtained from the prototype large-area sensor were used to obtain an accurate model of the calorimeter, checked for by the simulation software ANSYS. At

  2. Phenotypic Screens in Antimalarial Drug Discovery.

    PubMed

    Hovlid, Marisa L; Winzeler, Elizabeth A

    2016-09-01

    Phenotypic high-throughput screens are a valuable tool for identifying new chemical compounds with antimalarial activity. Traditionally, these screens have focused solely on the symptomatic asexual blood stage of the parasite life cycle; however, to discover new medicines for malaria treatment and prevention, robust screening technologies against other parasite life-cycle stages are required. This review highlights recent advances and progress toward phenotypic screening methodologies over the past several years, with a focus on exoerythrocytic stage screens. PMID:27247245

  3. Trioxaferroquines as new hybrid antimalarial drugs.

    PubMed

    Bellot, François; Coslédan, Frédéric; Vendier, Laure; Brocard, Jacques; Meunier, Bernard; Robert, Anne

    2010-05-27

    The synthesis, characterization, and antimalarial evaluation of a new series of potential antimalarial molecules, named trioxaferroquines, are reported. Trioxaferroquines are hybrid antimalarial drugs containing a 1,2,4-trioxane covalently linked to ferroquine (Fq), a synthetic ferrocenylquinoline derivative currently under clinical development. The aim was to combine, within a single structure, an iron(II) species, a 1,2,4-trioxane, as in artemisinin, and a substituted quinoline, as in chloroquine. PMID:20443628

  4. Emerging targets for antimalarial drugs.

    PubMed

    Padmanaban, Govinarajan; Rangarajan, Pundi N

    2001-08-01

    The absence of an effective vaccine against malaria and the ability of the parasite to develop resistance to known antimalarial drugs makes it mandatory to unravel newer drug targets with a view to developing newer pharmacophores. While conventional targets such as the purine, pyrimidine and folate pathways are still being investigated in the light of newer knowledge, a new opportunity has emerged from an understanding of certain unique features of the parasite biology. These include the food vacuole, haemoglobin catabolism, haeme biosynthesis, apicoplasts and their metabolism as well as macromolecular transactions, import of host proteins, parasite induced alterations in the red cell surface and transport phenomena. This review seeks to emphasise the new and emerging targets, while giving a brief account of the targets that have already been exploited. PMID:12540258

  5. A probabilistic approach to high throughput drug discovery.

    PubMed

    Labute, Paul; Nilar, Shahul; Williams, Christopher

    2002-03-01

    A methodology is presented in which high throughput screening experimental data are used to construct a probabilistic QSAR model which is subsequently used to select building blocks for a virtual combinatorial library. The methodology is based upon statistical probability estimation and not regression. The methodology is applied to the construction of two focused virtual combinatorial libraries: one for cyclic GMP phosphodiesterase type V inhibitors and one for acyl-CoA:cholesterol O-acyltransferase inhibitors. The results suggest that the methodology is capable of selecting combinatorial substituents that lead to active compounds starting with binary (pass/fail) activity measurements. PMID:11966422

  6. Searching for New Antimalarial Therapeutics amongst Known Drugs

    PubMed Central

    Weisman, Jennifer L.; Liou, Ally P.; Shelat, Anang A.; Cohen, Fred E.; Guy, R. Kiplin; DeRisi, Joseph L.

    2006-01-01

    The need to discover and develop new antimalarial therapeutics is severe. The annual mortality attributed to malaria, currently approximately 2.5 million, is increasing due primarily to widespread resistance to currently used drugs. One strategy to identify new treatment alternatives for malaria is to examine libraries of diverse compounds for the possible identification of novel scaffolds. Beginning with libraries of drug or drug-like compounds is an ideal starting point because, in the case of approved drugs, substantial pharmacokinetic and toxicologic data should be available for each compound series. We have employed a high throughput screen of the MicroSource Spectrum and Killer collections, a library of known drugs, bioactive compounds, and natural products. Our screening assay identifies compounds that inhibit growth of Plasmodium falciparum cultured in human erythrocytes. We have identified 36 novel inhibitors of P. falciparum, of which 19 are therapeutics, and five of these drugs exhibit effective 50% inhibitory concentrations within similar ranges to therapeutic serum concentrations for their currently indicated uses: propafenone, thioridazine, chlorprothixene, perhexiline and azlocillin. The findings we report here indicate that this is an effective strategy to identify novel scaffolds and therefore aid in antimalarial drug discovery efforts. PMID:16882315

  7. Searching for new antimalarial therapeutics amongst known drugs.

    PubMed

    Weisman, Jennifer L; Liou, Ally P; Shelat, Anang A; Cohen, Fred E; Guy, R Kiplin; DeRisi, Joseph L

    2006-06-01

    The need to discover and develop new antimalarial therapeutics is overwhelming. The annual mortality attributed to malaria, currently approximately 2.5 million, is increasing due primarily to widespread resistance to currently used drugs. One strategy to identify new treatment alternatives for malaria is to examine libraries of diverse compounds for the possible identification of novel scaffolds. Beginning with libraries of drug or drug-like compounds is an ideal starting point because, in the case of approved drugs, substantial pharmacokinetic and toxicologic data should be available for each compound series. We have employed a high-throughput screen of the MicroSource Spectrum and Killer Collections, a library of known drugs, bioactive compounds, and natural products. Our screening assay identifies compounds that inhibit growth of Plasmodium falciparum cultured in human erythrocytes. We have identified 36 novel inhibitors of P. falciparum, of which 19 are therapeutics, and five of these drugs exhibit effective 50% inhibitory concentrations within similar ranges to therapeutic serum concentrations for their recently indicated uses: propafenone, thioridazine, chlorprothixene, perhexiline, and azlocillin. The findings we report here indicate that this is an effective strategy to identify novel scaffolds and therefore aid in antimalarial drug discovery efforts. PMID:16882315

  8. A High-Throughput Screen for Antibiotic Drug Discovery

    PubMed Central

    Scanlon, Thomas C.; Dostal, Sarah M.; Griswold, Karl E.

    2014-01-01

    We describe an ultra-high-throughput screening platform enabling discovery and/or engineering of natural product antibiotics. The methodology involves creation of hydrogel-in-oil emulsions in which recombinant microorganisms are co-emulsified with bacterial pathogens; antibiotic activity is assayed by use of a fluorescent viability dye. We have successfully utilized both bulk emulsification and microfluidic technology for the generation of hydrogel microdroplets that are size-compatible with conventional flow cytometry. Hydrogel droplets are ~25 pL in volume, and can be synthesized and sorted at rates exceeding 3,000 drops/s. Using this technique, we have achieved screening throughputs exceeding 5 million clones/day. Proof-of-concept experiments demonstrate efficient selection of antibiotic-secreting yeast from a vast excess of negative controls. In addition, we have successfully used this technique to screen a metagenomic library for secreted antibiotics that kill the human pathogen Staphylococcus aureus. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules. PMID:23955804

  9. High-Throughput Screening of Ototoxic and Otoprotective Pharmacological Drugs

    ERIC Educational Resources Information Center

    Kalinec, Federico

    2005-01-01

    Drug ototoxicity research has relied traditionally on animal models for the discovery and development of therapeutic interventions. More than 50 years of research, however, has delivered few--if any--successful clinical strategies for preventing or ameliorating the ototoxic effects of common pharmacological drugs such as aminoglycoside…

  10. High-throughput imaging: Focusing in on drug discovery in 3D.

    PubMed

    Li, Linfeng; Zhou, Qiong; Voss, Ty C; Quick, Kevin L; LaBarbera, Daniel V

    2016-03-01

    3D organotypic culture models such as organoids and multicellular tumor spheroids (MCTS) are becoming more widely used for drug discovery and toxicology screening. As a result, 3D culture technologies adapted for high-throughput screening formats are prevalent. While a multitude of assays have been reported and validated for high-throughput imaging (HTI) and high-content screening (HCS) for novel drug discovery and toxicology, limited HTI/HCS with large compound libraries have been reported. Nonetheless, 3D HTI instrumentation technology is advancing and this technology is now on the verge of allowing for 3D HCS of thousands of samples. This review focuses on the state-of-the-art high-throughput imaging systems, including hardware and software, and recent literature examples of 3D organotypic culture models employing this technology for drug discovery and toxicology screening. PMID:26608110

  11. High throughput miniature drug-screening platform using bioprinting technology.

    PubMed

    Rodríguez-Dévora, Jorge I; Zhang, Bimeng; Reyna, Daniel; Shi, Zhi-dong; Xu, Tao

    2012-09-01

    In the pharmaceutical industry, new drugs are tested to find appropriate compounds for therapeutic purposes for contemporary diseases. Unfortunately, novel compounds emerge at expensive prices and current target evaluation processes have limited throughput, thus leading to an increase of cost and time for drug development. This work shows the development of the novel inkjet-based deposition method for assembling a miniature drug-screening platform, which can realistically and inexpensively evaluate biochemical reactions in a picoliter-scale volume at a high speed rate. As proof of concept, applying a modified Hewlett Packard model 5360 compact disc printer, green fluorescent protein expressing Escherichia coli cells along with alginate gel solution have been arrayed on a coverslip chip under a repeatable volume of 180% ± 26% picoliters per droplet; subsequently, different antibiotic droplets were patterned on the spots of cells to evaluate the inhibition of bacteria for antibiotic screening. The proposed platform was compared to the current screening process, validating its effectiveness. The viability and basic function of the printed cells were evaluated, resulting in cell viability above 98% and insignificant or no DNA damage to human kidney cells transfected. Based on the reduction of investment and compound volume used by this platform, this technique has the potential to improve the actual drug discovery process at its target evaluation stage. PMID:22728820

  12. The Tragedy Caused by Fake Antimalarial Drugs

    PubMed Central

    Ambroise-Thomas, Pierre

    2012-01-01

    Counterfeit antimalarials (mainly artemisinin derivatives) is a crucial health problem in developing countries, particularly in Africa. The illegal production, sale and distribution of fake drugs is a huge market evaluated to several billion of dollars and represents more than 50% of the pharmaceutical market in several African countries. Fake drugs have led to a very great number of deaths from untreated malaria or fatality provoked by toxic ingredients. These fake medicines increase the risk of artemisinin resistance developed by the use of sub therapeutic dosages of antimalarials. Tackling this criminal traffic is the objective of an international program created by WHO and involves the international police and custom organizations like INTERPOL. Several very important and encouraging results have been obtained, but the problem will be completely solved if genuine antimalarials, free-of-charge, are handed-over to populations in sub Sahara African countries. PMID:22708042

  13. High-throughput screening of drug-binding dynamics to HERG improves early drug safety assessment.

    PubMed

    Di Veroli, Giovanni Y; Davies, Mark R; Zhang, Henggui; Abi-Gerges, Najah; Boyett, Mark R

    2013-01-01

    The use of computational models to predict drug-induced changes in the action potential (AP) is a promising approach to reduce drug safety attrition but requires a better representation of more complex drug-target interactions to improve the quantitative prediction. The blockade of the human ether-a-go-go-related gene (HERG) channel is a major concern for QT prolongation and Torsade de Pointes risk. We aim to develop quantitative in-silico AP predictions based on a new electrophysiological protocol (suitable for high-throughput HERG screening) and mathematical modeling of ionic currents. Electrophysiological recordings using the IonWorks device were made from HERG channels stably expressed in Chinese hamster ovary cells. A new protocol that delineates inhibition over time was applied to assess dofetilide, cisapride, and almokalant effects. Dynamic effects displayed distinct profiles for these drugs compared with concentration-effects curves. Binding kinetics to specific states were identified using a new HERG Markov model. The model was then modified to represent the canine rapid delayed rectifier K(+) current at 37°C and carry out AP predictions. Predictions were compared with a simpler model based on conductance reduction and were found to be much closer to experimental data. Improved sensitivity to concentration and pacing frequency variables was obtained when including binding kinetics. Our new electrophysiological protocol is suitable for high-throughput screening and is able to distinguish drug-binding kinetics. The association of this protocol with our modeling approach indicates that quantitative predictions of AP modulation can be obtained, which is a significant improvement compared with traditional conductance reduction methods. PMID:23103500

  14. Pricing, distribution, and use of antimalarial drugs.

    PubMed

    Foster, S D

    1991-01-01

    Prices of new antimalarial drugs are targeted at the "travellers' market" in developed countries, which makes them unaffordable in malaria-endemic countries where the per capita annual drug expenditures are US$ 5 or less. Antimalarials are distributed through a variety of channels in both public and private sectors, the official malaria control programmes accounting for 25-30% of chloroquine distribution. The unofficial drug sellers in markets, streets, and village shops account for as much as half of antimalarials distributed in many developing countries. Use of antimalarials through the health services is often poor; drug shortages are common and overprescription and overuse of injections are significant problems. Anxiety over drug costs may prevent patients from getting the necessary treatment for malaria, especially because of the seasonal appearance of this disease when people's cash reserves are very low. The high costs may lead them to unofficial sources, which will sell a single tablet instead of a complete course of treatment, and subsequently to increased, often irrational demand for more drugs and more injections. Increasingly people are resorting to self-medication for malaria, which may cause delays in seeking proper treatment in cases of failure, especially in areas where chloroquine resistance has increased rapidly. Self-medication is now widespread, and measures to restrict the illicit sale of drugs have been unsuccessful. The "unofficial" channels thus represent an unacknowledged extension of the health services in many countries; suggestions are advanced to encourage better self-medication by increasing the knowledge base among the population at large (mothers, schoolchildren, market sellers, and shopkeepers), with an emphasis on correct dosing and on the importance of seeking further treatment without delay, if necessary. PMID:1893512

  15. Testing Tuberculosis Drug Efficacy in a Zebrafish High-Throughput Translational Medicine Screen

    PubMed Central

    Ordas, Anita; Raterink, Robert-Jan; Cunningham, Fraser; Jansen, Hans J.; Wiweger, Malgorzata I.; Jong-Raadsen, Susanne; Bos, Sabine; Bates, Robert H.; Barros, David; Meijer, Annemarie H.; Vreeken, Rob J.; Ballell-Pages, Lluís; Dirks, Ron P.

    2014-01-01

    The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans. PMID:25385118

  16. Evaluation of high-throughput assays for in vitro drug susceptibility testing of Tritrichomonas foetus trophozoites.

    PubMed

    Bader, Chris; Jesudoss Chelladurai, Jeba; Thompson, Kylie; Hall, Cindy; Carlson, Steve A; Brewer, Matthew T

    2016-06-15

    Tritrichomonas foetus is a sexually transmitted protozoan parasite that causes abortions in cattle and results in severe economic losses. In the United States, there are no safe and effective treatments for this parasite and infected animals are typically culled. In order to expedite drug discovery efforts, we investigated in vitro trophozoite killing assays amenable to high-throughput screening in 96 well plate formats. We evaluated the reduction of resorufin, incorporation of propidium iodide, and a luminescence-based ATP detection assay. Of these methods, reduction of resorufin was found to be the most reliable predictor of trophozoite concentrations. We further validated this method by conducting dose-response experiments suitable for calculation of EC50 values for two established compounds with known activity against trophozoites in vitro, namely, metronidazole and ronidazole. Our results demonstrate that the resorufin method is suitable for high-throughput screening and could be used to enhance efforts targeting new treatments for bovine trichomoniasis. PMID:27198774

  17. Engineering a Brain Cancer Chip for High-throughput Drug Screening

    PubMed Central

    Fan, Yantao; Nguyen, Duong Thanh; Akay, Yasemin; Xu, Feng; Akay, Metin

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common and malignant of all human primary brain cancers, in which drug treatment is still one of the most effective treatments. However, existing drug discovery and development methods rely on the use of conventional two-dimensional (2D) cell cultures, which have been proven to be poor representatives of native physiology. Here, we developed a novel three-dimensional (3D) brain cancer chip composed of photo-polymerizable poly(ethylene) glycol diacrylate (PEGDA) hydrogel for drug screening. This chip can be produced after a few seconds of photolithography and requires no silicon wafer, replica molding, and plasma bonding like microfluidic devices made of poly(dimethylsiloxane) (PDMS). We then cultured glioblastoma cells (U87), which formed 3D brain cancer tissues on the chip, and used the GBM chip to perform combinatorial treatment of Pitavastatin and Irinotecan. The results indicate that this chip is capable of high-throughput GBM cancer spheroids formation, multiple-simultaneous drug administration, and a massive parallel testing of drug response. Our approach is easily reproducible, and this chip has the potential to be a powerful platform in cases such as high-throughput drug screening and prolonged drug release. The chip is also commercially promising for other clinical applications, including 3D cell culture and micro-scale tissue engineering. PMID:27151082

  18. Engineering a Brain Cancer Chip for High-throughput Drug Screening.

    PubMed

    Fan, Yantao; Nguyen, Duong Thanh; Akay, Yasemin; Xu, Feng; Akay, Metin

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common and malignant of all human primary brain cancers, in which drug treatment is still one of the most effective treatments. However, existing drug discovery and development methods rely on the use of conventional two-dimensional (2D) cell cultures, which have been proven to be poor representatives of native physiology. Here, we developed a novel three-dimensional (3D) brain cancer chip composed of photo-polymerizable poly(ethylene) glycol diacrylate (PEGDA) hydrogel for drug screening. This chip can be produced after a few seconds of photolithography and requires no silicon wafer, replica molding, and plasma bonding like microfluidic devices made of poly(dimethylsiloxane) (PDMS). We then cultured glioblastoma cells (U87), which formed 3D brain cancer tissues on the chip, and used the GBM chip to perform combinatorial treatment of Pitavastatin and Irinotecan. The results indicate that this chip is capable of high-throughput GBM cancer spheroids formation, multiple-simultaneous drug administration, and a massive parallel testing of drug response. Our approach is easily reproducible, and this chip has the potential to be a powerful platform in cases such as high-throughput drug screening and prolonged drug release. The chip is also commercially promising for other clinical applications, including 3D cell culture and micro-scale tissue engineering. PMID:27151082

  19. Economic prospects for a new antimalarial drug.

    PubMed

    Foster, S

    1994-06-01

    The market for antimalarial drugs consists of the 2.8 billion (2.8 x 10(9) people living in malaria endemic areas and about 20-30 million people, mainly Europeans and North Americans, who travel or live in malarious areas, and the wealthy elite of malarious developing countries. Some of the largest markets include China, India, and Indonesia with a total of 1.9 billion people exposed; Latin America, with 119 million; and sub-Saharan Africa with 400 million. An estimated 200 million clinical cases occur each year, with around 2 million deaths annually, primarily in African children. Antimalarial drugs are distributed through several different networks and are purchased by governments and private individuals. At present the world market is dominated by chloroquine by virtue of its safety, wide availability and low price. Approximately 20% of the total production of chloroquine was distributed through national control programmes and 80% through other channels; chloroquine is probably the second or third most widely consumed drug in the world. The global market for antimalarial drugs is likely to be of the order of US$100-120 million, with chloroquine making up about US$64-80 m, sulfadoxine/pyrimethamine about US$20 m, and other drugs making up US$10-20 m. Chloroquine is rapidly losing its effectiveness against the malaria parasite, and a safe, effective, cheap replacement is urgently needed. Another product which was found to be safe and effective against malaria, with a price per treatment held at or near the present price of chloroquine, could quickly replace chloroquine as the first-line treatment against malaria.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8053030

  20. A multilayer microdevice for cell-based high-throughput drug screening

    NASA Astrophysics Data System (ADS)

    Liu, Chong; Wang, Lei; Xu, Zheng; Li, Jingmin; Ding, Xiping; Wang, Qi; Chunyu, Li

    2012-06-01

    A multilayer polydimethylsiloxane microdevice for cell-based high-throughput drug screening is described in this paper. This established microdevice was based on a modularization method and it integrated a drug/medium concentration gradient generator (CGG), pneumatic microvalves and a cell culture microchamber array. The CGG was able to generate five steps of linear concentrations with the same outlet flow rate. The medium/drug flowed through CGG and then into the pear-shaped cell culture microchambers vertically. This vertical perfusion mode was used to reduce the impact of the shear stress on the physiology of cells induced by the fluid flow in the microchambers. Pear-shaped microchambers with two arrays of miropillars at each outlet were adopted in this microdevice, which were beneficial to cell distribution. The chemotherapeutics Cisplatin (DDP)-induced Cisplatin-resistant cell line A549/DDP apoptotic experiments were performed well on this platform. The results showed that this novel microdevice could not only provide well-defined and stable conditions for cell culture, but was also useful for cell-based high-throughput drug screening with less reagents and time consumption.

  1. Quantitative high-throughput analysis of drugs in biological matrices by mass spectrometry.

    PubMed

    Hopfgartner, Gérard; Bourgogne, Emmanuel

    2003-01-01

    To support pharmacokinetic and drug metabolism studies, LC-MS/MS plays more and more an essential role for the quantitation of drugs and their metabolites in biological matrices. With the new challenges encountered in drug discovery and drug development, new strategies are put in place to achieve high-throughput analysis, using serial and parallel approaches. To speed-up method development and validation, generic approaches with the direct injection of biological fluids is highly desirable. Column-switching, using various packing materials for the extraction columns, is widely applied. Improvement of mass spectrometers performance, and in particular triple quadrupoles, also strongly influences sample preparation strategies, which remain a key element in the bioanalytical process. PMID:12838545

  2. Anticancer Properties of Distinct Antimalarial Drug Classes

    PubMed Central

    Hooft van Huijsduijnen, Rob; Guy, R. Kiplin; Chibale, Kelly; Haynes, Richard K.; Peitz, Ingmar; Kelter, Gerhard; Phillips, Margaret A.; Vennerstrom, Jonathan L.; Yuthavong, Yongyuth; Wells, Timothy N. C.

    2013-01-01

    We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC50s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC50s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings. PMID:24391728

  3. Protein Traffic Disorders: an Effective High-Throughput Fluorescence Microscopy Pipeline for Drug Discovery

    PubMed Central

    Botelho, Hugo M.; Uliyakina, Inna; Awatade, Nikhil T.; Proença, Maria C.; Tischer, Christian; Sirianant, Lalida; Kunzelmann, Karl; Pepperkok, Rainer; Amaral, Margarida D.

    2015-01-01

    Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking – Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts. PMID:25762484

  4. Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery.

    PubMed

    Botelho, Hugo M; Uliyakina, Inna; Awatade, Nikhil T; Proença, Maria C; Tischer, Christian; Sirianant, Lalida; Kunzelmann, Karl; Pepperkok, Rainer; Amaral, Margarida D

    2015-01-01

    Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking - Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts. PMID:25762484

  5. Moderate to high throughput in vitro binding kinetics for drug discovery.

    PubMed

    Zhang, Rumin; Barbieri, Christopher M; Garcia-Calvo, Margarita; Myers, Robert W; McLaren, David; Kavana, Michael

    2016-01-01

    This review provides a concise summary for state of the art, moderate to high throughput in vitro technologies being employed to study drug-target binding kinetics. These technologies cover a wide kinetic timescale spanning up to nine orders of magnitude from milliseconds to days. Automated stopped flow measures transient and (pre)steady state kinetics from milliseconds to seconds. For seconds to hours timescale kinetics we discuss surface plasmon resonance-based biosensor, global progress curve analysis for high throughput kinetic profiling of enzyme inhibitors and activators, and filtration plate-based radioligand or fluorescent binding assays for receptor binding kinetics. Jump dilution after pre-incubation is the preferred method for very slow kinetics lasting for days. The basic principles, best practices and simulated data for these technologies are described. Finally, the application of a universal label-free technology, liquid chromatography coupled tandem mass spectrometry (LC/MS/MS), is briefly reviewed. Select literature references are highlighted for in-depth understanding. A new reality is dawning wherein binding kinetics is an integral and routine part of mechanism of action elucidation and translational, quantitative pharmacology for drug discovery. PMID:27100706

  6. High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor

    PubMed Central

    Zhang, Le; Yang, Zhaoying; Granieri, Letizia; Pasculescu, Adrian; Datti, Alessandro; Asa, Sylvia L.; Xu, Zheli; Ezzat, Shereen

    2016-01-01

    We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer. PMID:26942566

  7. High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor.

    PubMed

    Zhang, Le; Yang, Zhaoying; Granieri, Letizia; Pasculescu, Adrian; Datti, Alessandro; Asa, Sylvia L; Xu, Zheli; Ezzat, Shereen

    2016-04-12

    We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer. PMID:26942566

  8. High-throughput flow cytometric screening of combinatorial chemistry bead libraries for proteomics and drug discovery

    NASA Astrophysics Data System (ADS)

    Leary, James F.; Reece, Lisa M.; Yang, Xian-Bin; Gorenstein, David

    2005-04-01

    For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

  9. High throughput physiological screening of iPSC-derived cardiomyocytes for drug development.

    PubMed

    Del Álamo, Juan C; Lemons, Derek; Serrano, Ricardo; Savchenko, Alex; Cerignoli, Fabio; Bodmer, Rolf; Mercola, Mark

    2016-07-01

    Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately identify new targets and test novel therapeutic strategies. Similarly, adverse drug effects on the heart are challenging to model, contributing to costly failure of drugs during development and even after market launch. Human induced pluripotent stem cell derived cardiac tissue represents a potentially powerful means to model aspects of heart physiology relevant to disease and adverse drug effects, providing both the human context and throughput needed to improve the efficiency of drug development. Here we review emerging technologies for high throughput measurements of cardiomyocyte physiology, and comment on the promises and challenges of using iPSC-derived cardiomyocytes to model disease and introduce the human context into early stages of drug discovery. This article is part of a Special Issue entitled: Cardiomyocyte biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. PMID:26952934

  10. Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics.

    PubMed

    Shembekar, Nachiket; Chaipan, Chawaree; Utharala, Ramesh; Merten, Christoph A

    2016-04-12

    Droplet-based microfluidics enables assays to be carried out at very high throughput (up to thousands of samples per second) and enables researchers to work with very limited material, such as primary cells, patient's biopsies or expensive reagents. An additional strength of the technology is the possibility to perform large-scale genotypic or phenotypic screens at the single-cell level. Here we critically review the latest developments in antibody screening, drug discovery and highly multiplexed genomic applications such as targeted genetic workflows, single-cell RNAseq and single-cell ChIPseq. Starting with a comprehensive introduction for non-experts, we pinpoint current limitations, analyze how they might be overcome and give an outlook on exciting future applications. PMID:27025767

  11. Novel Phenotypic Fluorescent Three-Dimensional Platforms for High-throughput Drug Screening and Personalized Chemotherapy.

    PubMed

    Fang, Changge; Avis, Ingalill; Salomon, David; Cuttitta, Frank

    2013-01-01

    We have developed novel phenotypic fluorescent three-dimensional co-culture platforms that efficiently and economically screen anti-angiogenic/anti-metastatic drugs on a high-throughput scale. Individual cell populations can be identified and isolated for protein/gene expression profiling studies and cellular movement/interactions can be tracked by time-lapse cinematography. More importantly, these platforms closely parallel the in vivo angiogenic and metastatic outcomes of a given tumor xenograft in the nude mouse model but, unlike in vivo models, our co-culture platforms produce comparable results in five to nine days. Potentially, by incorporating cancer patient biopsies, the co-culture platforms should greatly improve the effectiveness and efficiency of personalized chemotherapy. PMID:23833685

  12. Unlocking the Potential of High-Throughput Drug Combination Assays Using Acoustic Dispensing.

    PubMed

    Chan, Grace Ka Yan; Wilson, Stacy; Schmidt, Stephen; Moffat, John G

    2016-02-01

    Assessment of synergistic effects of drug combinations in vitro is a critical part of anticancer drug research. However, the complexities of dosing and analyzing two drugs over the appropriate range of doses have generally led to compromises in experimental design that restrict the quality and robustness of the data. In particular, the use of a single dose response of combined drugs, rather than a full two-way matrix of varying doses, has predominated in higher-throughput studies. Acoustic dispensing unlocks the potential of high-throughput dose matrix analysis. We have developed acoustic dispensing protocols that enable compound synergy assays in a 384-well format. This experimental design is considerably more efficient and flexible with respect to time, reagent usage, and labware than is achievable using traditional serial-dilution approaches. Data analysis tools integrated in Genedata Screener were used to efficiently deconvolute the combination compound mapping scheme and calculate compound potency and synergy metrics. We have applied this workflow to evaluate interactions among drugs targeting different nodes of the mitogen-activated protein kinase pathway in a panel of cancer cell lines. PMID:26160862

  13. Open Access High Throughput Drug Discovery in the Public Domain: A Mount Everest in the Making

    PubMed Central

    Roy, Anuradha; McDonald, Peter R.; Sittampalam, Sitta; Chaguturu, Rathnam

    2013-01-01

    High throughput screening (HTS) facilitates screening large numbers of compounds against a biochemical target of interest using validated biological or biophysical assays. In recent years, a significant number of drugs in clinical trails originated from HTS campaigns, validating HTS as a bona fide mechanism for hit finding. In the current drug discovery landscape, the pharmaceutical industry is embracing open innovation strategies with academia to maximize their research capabilities and to feed their drug discovery pipeline. The goals of academic research have therefore expanded from target identification and validation to probe discovery, chemical genomics, and compound library screening. This trend is reflected in the emergence of HTS centers in the public domain over the past decade, ranging in size from modestly equipped academic screening centers to well endowed Molecular Libraries Probe Centers Network (MLPCN) centers funded by the NIH Roadmap initiative. These centers facilitate a comprehensive approach to probe discovery in academia and utilize both classical and cutting-edge assay technologies for executing primary and secondary screening campaigns. The various facets of academic HTS centers as well as their implications on technology transfer and drug discovery are discussed, and a roadmap for successful drug discovery in the public domain is presented. New lead discovery against therapeutic targets, especially those involving the rare and neglected diseases, is indeed a Mount Everestonian size task, and requires diligent implementation of pharmaceutical industry’s best practices for a successful outcome. PMID:20809896

  14. Synergistic Drug Combinations for Tuberculosis Therapy Identified by a Novel High-Throughput Screen▿†

    PubMed Central

    Ramón-García, Santiago; Ng, Carol; Anderson, Hilary; Chao, Joseph D.; Zheng, Xingji; Pfeifer, Tom; Av-Gay, Yossef; Roberge, Michel; Thompson, Charles J.

    2011-01-01

    Therapeutic options for tuberculosis (TB) are limited and notoriously ineffective despite the wide variety of potent antibiotics available for treating other bacterial infections. We investigated an approach that enables an expansion of TB therapeutic strategies by using synergistic combinations of drugs. To achieve this, we devised a high-throughput synergy screen (HTSS) of chemical libraries having known pharmaceutical properties, including thousands that are clinically approved. Spectinomycin was used to test the concept that clinically available antibiotics with limited efficacy against Mycobacterium tuberculosis might be used for TB treatment when coadministered with a synergistic partner compound used as a sensitizer. Screens using Mycobacterium smegmatis revealed many compounds in our libraries that acted synergistically with spectinomycin. Among them, several families of antimicrobial compounds, including macrolides and azoles, were also synergistic against M. tuberculosis in vitro and in a macrophage model of M. tuberculosis infection. Strikingly, each sensitizer identified for synergy with spectinomycin uniquely enhanced the activities of other clinically used antibiotics, revealing a remarkable number of unexplored synergistic drug combinations. HTSS also revealed a novel activity for bromperidol, a butyrophenone used as an antipsychotic drug, which was discovered to be bactericidal and greatly enhanced the activities of several antibiotics and drug combinations against M. tuberculosis. Our results suggest that many compounds in the currently available pharmacopoeia could be readily mobilized for TB treatment, including disease caused by multi- and extensively drug-resistant strains for which there are no effective therapies. PMID:21576426

  15. Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen.

    PubMed

    Debnath, Anjan; Tunac, Josefino B; Galindo-Gómez, Silvia; Silva-Olivares, Angélica; Shibayama, Mineko; McKerrow, James H

    2012-11-01

    Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM. PMID:22869574

  16. Quantitative High-Throughput Identification of Drugs as Modulators of Human Constitutive Androstane Receptor

    PubMed Central

    Lynch, Caitlin; Zhao, Jinghua; Huang, Ruili; Xiao, Jingwei; Li, Linhao; Heyward, Scott; Xia, Menghang; Wang, Hongbing

    2015-01-01

    The constitutive androstane receptor (CAR, NR1I3) plays a key role in governing the transcription of numerous hepatic genes that involve xenobiotic metabolism/clearance, energy homeostasis, and cell proliferation. Thus, identification of novel human CAR (hCAR) modulators may not only enhance early prediction of drug-drug interactions but also offer potentially novel therapeutics for diseases such as metabolic disorders and cancer. In this study, we have generated a double stable cell line expressing both hCAR and a CYP2B6-driven luciferase reporter for quantitative high-throughput screening (qHTS) of hCAR modulators. Approximately 2800 compounds from the NIH Chemical Genomics Center Pharmaceutical Collection were screened employing both the activation and deactivation modes of the qHTS. Activators (115) and deactivators (152) of hCAR were identified from the primary qHTS, among which 10 agonists and 10 antagonists were further validated in the physiologically relevant human primary hepatocytes for compound-mediated hCAR nuclear translocation and target gene expression. Collectively, our results reveal that hCAR modulators can be efficiently identified through this newly established qHTS assay. Profiling drug collections for hCAR activity would facilitate the prediction of metabolism-based drug-drug interactions, and may lead to the identification of potential novel therapeutics. PMID:25993555

  17. Open access high throughput drug discovery in the public domain: a Mount Everest in the making.

    PubMed

    Roy, Anuradha; McDonald, Peter R; Sittampalam, Sitta; Chaguturu, Rathnam

    2010-11-01

    High throughput screening (HTS) facilitates screening large numbers of compounds against a biochemical target of interest using validated biological or biophysical assays. In recent years, a significant number of drugs in clinical trails originated from HTS campaigns, validating HTS as a bona fide mechanism for hit finding. In the current drug discovery landscape, the pharmaceutical industry is embracing open innovation strategies with academia to maximize their research capabilities and to feed their drug discovery pipeline. The goals of academic research have therefore expanded from target identification and validation to probe discovery, chemical genomics, and compound library screening. This trend is reflected in the emergence of HTS centers in the public domain over the past decade, ranging in size from modestly equipped academic screening centers to well endowed Molecular Libraries Probe Centers Network (MLPCN) centers funded by the NIH Roadmap initiative. These centers facilitate a comprehensive approach to probe discovery in academia and utilize both classical and cutting-edge assay technologies for executing primary and secondary screening campaigns. The various facets of academic HTS centers as well as their implications on technology transfer and drug discovery are discussed, and a roadmap for successful drug discovery in the public domain is presented. New lead discovery against therapeutic targets, especially those involving the rare and neglected diseases, is indeed a Mount Everestonian size task, and requires diligent implementation of pharmaceutical industry's best practices for a successful outcome. PMID:20809896

  18. Quantitative high-throughput identification of drugs as modulators of human constitutive androstane receptor.

    PubMed

    Lynch, Caitlin; Zhao, Jinghua; Huang, Ruili; Xiao, Jingwei; Li, Linhao; Heyward, Scott; Xia, Menghang; Wang, Hongbing

    2015-01-01

    The constitutive androstane receptor (CAR, NR1I3) plays a key role in governing the transcription of numerous hepatic genes that involve xenobiotic metabolism/clearance, energy homeostasis, and cell proliferation. Thus, identification of novel human CAR (hCAR) modulators may not only enhance early prediction of drug-drug interactions but also offer potentially novel therapeutics for diseases such as metabolic disorders and cancer. In this study, we have generated a double stable cell line expressing both hCAR and a CYP2B6-driven luciferase reporter for quantitative high-throughput screening (qHTS) of hCAR modulators. Approximately 2800 compounds from the NIH Chemical Genomics Center Pharmaceutical Collection were screened employing both the activation and deactivation modes of the qHTS. Activators (115) and deactivators (152) of hCAR were identified from the primary qHTS, among which 10 agonists and 10 antagonists were further validated in the physiologically relevant human primary hepatocytes for compound-mediated hCAR nuclear translocation and target gene expression. Collectively, our results reveal that hCAR modulators can be efficiently identified through this newly established qHTS assay. Profiling drug collections for hCAR activity would facilitate the prediction of metabolism-based drug-drug interactions, and may lead to the identification of potential novel therapeutics. PMID:25993555

  19. High-throughput microplate assay for the determination of drug partition coefficients.

    PubMed

    Magalhães, Luís M; Nunes, Cláudia; Lúcio, Marlene; Segundo, Marcela A; Reis, Salette; Lima, José L F C

    2010-11-01

    Partition coefficients (K(p)) of drugs between the phospholipid bilayer and the aqueous phase provide useful information in quantitative structure-activity relationship studies. Hexadecylphosphocholine (HePC) micelles, composed of a zwitterionic hydrophilic surface and a hydrophobic core, mimic the biomembranes and have several advantages over other lipid structures to assess K(p) values. Their preparation is easy, fast and avoids the use of toxic organic solvents, and the output has fewer spectroscopic interferences. Here, we describe a high-throughput microplate protocol for assessing the K(p) of drugs using HePC micelles as membrane models and derivative spectrophotometry as the detection technique. Moreover, the time-consuming data treatment to assess K(p) values is easily performed by a dedicated Excel routine developed here and described in detail. The K(p) values of nonsteroidal anti-inflammatory drugs (acemetacin, clonixin, diclofenac and indomethacin) were determined to show the simplicity of the method and to validate this protocol, which provides K(p) values (n = 3) of two drugs in ∼ 2 h. PMID:21030957

  20. Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

    PubMed Central

    Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

    2011-01-01

    Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

  1. CRIMALDDI: a prioritized research agenda to expedite the discovery of new anti-malarial drugs

    PubMed Central

    2013-01-01

    The CRIMALDDI Consortium has been a three-year project funded by the EU Framework Seven Programme. It aimed to develop a prioritized set of recommendations to speed up anti-malarial drug discovery research and contribute to the setting of the global research agenda. It has attempted to align thinking on the high priority issues and then to develop action plans and strategies to address these issues. Through a series of facilitated and interactive workshops, it has concluded that these priorities can be grouped under five key themes: attacking artemisinin resistance; creating and sharing community resources; delivering enabling technologies; exploiting high throughput screening hits quickly; and, identifying novel targets. Recommendations have been prioritized into one of four levels: quick wins; removing key roadblocks to future progress; speeding-up drug discovery; and, nice to have (but not essential). Use of this prioritization allows efforts and resources to be focused on the lines of work that will contribute most to expediting anti-malarial drug discovery. Estimates of the time and finances required to implement the recommendations have also been made, along with indications of when recommendations within each theme will make an impact. All of this has been collected into an indicative roadmap that, it is hoped, will guide decisions about the direction and focus of European anti-malarial drug discovery research and contribute to the setting of the global research agenda. PMID:24191947

  2. World Antimalarial Resistance Network (WARN) II: in vitro antimalarial drug susceptibility.

    PubMed

    Bacon, David J; Jambou, Ronan; Fandeur, Thierry; Le Bras, Jacques; Wongsrichanalai, Chansuda; Fukuda, Mark M; Ringwald, Pascal; Sibley, Carol Hopkins; Kyle, Dennis E

    2007-01-01

    Intrinsic resistance of Plasmodium falciparum is clearly a major determinant of the clinical failure of antimalarial drugs. However, complex interactions between the host, the parasite and the drug obscure the ability to define parasite drug resistance in vivo. The in vitro antimalarial drug susceptibility assay determines ex-vivo growth of parasite in the presence of serial drug concentrations and, thus, eliminates host effects, such as drug metabolism and immunity. Although the sensitivity of the parasite to various antimalarials provided by such a test provides an important indicator of intrinsic parasite susceptibility, there are fundamental methodological issues that undermine comparison of in vitro susceptibility both between laboratories and within a single laboratory over time. A network of laboratories is proposed that will agree on the basic parameters of the in vitro test and associated measures of quality control. The aim of the network would be to establish baseline values of sensitivity to commonly used antimalarial agents from key regions of the world, and create a global database, linked to clinical, molecular and pharmacology databases, to support active surveillance to monitor temporal trends in parasite susceptibility. Such a network would facilitate the rapid detection of strains with novel antimalarial resistance profiles and investigate suitable alternative treatments with retained efficacy. PMID:17822533

  3. Thermo-responsive polymer aided spheroid culture in cryogel based platform for high throughput drug screening.

    PubMed

    Sarkar, J; Kumar, A

    2016-04-21

    In high throughput cell culture, the paradigm is now shifting from 2D to 3D systems. However, in 3D cell culture systems, it is important that the cells form spheroids with robust cell-cell interactions. We fabricated poly(N-isopropylacrylamide-co-gelatin) cryogel scaffolds for cell culture and inserted them into open-ended 96-well plates that formed a drainage and leakage protected, easy to handle high throughput platform. This platform was used to screen for the optimal concentration of poly(N-isopropylacrylamide) (pNIPAAm) as an external aid to the formation of cellular spheroids. It was found that hepatic cells (Hep G2) seeded in the presence of 0.03% pNIPAAm formed better hepatic spheroids in terms of morphology (as assessed by microscopic analysis and formation of bile canaliculi-like structures) and functionality by day 5 of culture. An increase of 44.22%, 15.75%, 36.44%, 32.05% and 27.02% was observed in glucose consumption (1.925 mM per day per 10(4) cells), albumin synthesis (164.18 ng per day per 10(4) cells), CYP1A1 (304.92 pg per min per 10(4) cells), CYP2A6 (441.23 nM per min per 10(4) cells) and phase II metabolic activity (386.18 nM per min(-1) per 10(4) cells), respectively, upon using 0.03% pNIPAAm, as compared to the 3D control. The platform was tested with other cells such as breast and lung cancer cells and found to be compatible. The cell spheroids were subjected to drug toxicity screening in cryogel based open-ended platforms. It was observed that the spheroids were more resistant to anticancer drugs, as compared to 2D and 3D controls, with approximately 11%-67% increase in the IC50 values of tamoxifen and paclitaxel. The platform also showed dose dependent and reproducible responses to drugs. PMID:27027476

  4. High-Throughput Nano-Biofilm Microarray for Antifungal Drug Discovery

    PubMed Central

    Srinivasan, Anand; Leung, Kai P.; Lopez-Ribot, Jose L.; Ramasubramanian, Anand K.

    2013-01-01

    ABSTRACT Micro- and nanoscale technologies have radically transformed biological research from genomics to tissue engineering, with the relative exception of microbial cell culture, which is still largely performed in microtiter plates and petri dishes. Here, we present nanoscale culture of the opportunistic fungal pathogen Candida albicans on a microarray platform. The microarray consists of 1,200 individual cultures of 30 nl of C. albicans biofilms (“nano-biofilms”) encapsulated in an inert alginate matrix. We demonstrate that these nano-biofilms are similar to conventional macroscopic biofilms in their morphological, architectural, growth, and phenotypic characteristics. We also demonstrate that the nano-biofilm microarray is a robust and efficient tool for accelerating the drug discovery process: (i) combinatorial screening against a collection of 28 antifungal compounds in the presence of immunosuppressant FK506 (tacrolimus) identified six drugs that showed synergistic antifungal activity, and (ii) screening against the NCI challenge set small-molecule library identified three heretofore-unknown hits. This cell-based microarray platform allows for miniaturization of microbial cell culture and is fully compatible with other high-throughput screening technologies. PMID:23800397

  5. Adaptation of High-Throughput Screening in Drug Discovery—Toxicological Screening Tests

    PubMed Central

    Szymański, Paweł; Markowicz, Magdalena; Mikiciuk-Olasik, Elżbieta

    2012-01-01

    High-throughput screening (HTS) is one of the newest techniques used in drug design and may be applied in biological and chemical sciences. This method, due to utilization of robots, detectors and software that regulate the whole process, enables a series of analyses of chemical compounds to be conducted in a short time and the affinity of biological structures which is often related to toxicity to be defined. Since 2008 we have implemented the automation of this technique and as a consequence, the possibility to examine 100,000 compounds per day. The HTS method is more frequently utilized in conjunction with analytical techniques such as NMR or coupled methods e.g., LC-MS/MS. Series of studies enable the establishment of the rate of affinity for targets or the level of toxicity. Moreover, researches are conducted concerning conjugation of nanoparticles with drugs and the determination of the toxicity of such structures. For these purposes there are frequently used cell lines. Due to the miniaturization of all systems, it is possible to examine the compound’s toxicity having only 1–3 mg of this compound. Determination of cytotoxicity in this way leads to a significant decrease in the expenditure and to a reduction in the length of the study. PMID:22312262

  6. Ophthalmologic safety profile of antimalarial drugs.

    PubMed

    Rynes, R I; Bernstein, H N

    1993-02-01

    The ophthalmologic safety of antimalarial drugs is well established, but absolute safety cannot be assured. Three types of side effects may develop. Corneal deposits and neuromuscular-associated blurred vision are always reversible and therefore benign. Visual loss has occurred in patients with retinopathy. Retinopathy may be divided into true retinopathy and premaculopathy. It is true retinopathy that may be associated with visual loss, while premaculopathy consists of subtle visual field and funduscopic abnormalities. These premaculopathic changes are generally completely reversible with drug discontinuation and have not been shown to progress. Hydroxychloroquine appears safer than chloroquine when currently accepted equivalent doses are used. Fewer than 20 patients with true retinopathy caused by hydroxychloroquine have been reported; more patients have developed true retinopathy when taking chloroquine. The safety profile is most dependent on low daily dose and regular ophthalmologic monitoring. The optimal strategy of ophthalmologic testing has not yet been determined, but visual acuity, funduscopic examination and visual field examination should be monitored. Self-administered ophthalmologic testing with Amsler grids may contribute additional safety but is not a replacement for physician testing. PMID:8485566

  7. Anti-malarial Drug Design by Targeting Apicoplasts: New Perspectives

    PubMed Central

    Mukherjee, Avinaba; Sadhukhan, Gobinda Chandra

    2016-01-01

    Objectives: Malaria has been a major global health problem in recent times with increasing mortality. Current treatment methods include parasiticidal drugs and vaccinations. However, resistance among malarial parasites to the existing drugs has emerged as a significant area of concern in anti-malarial drug design. Researchers are now desperately looking for new targets to develop anti-malarials drug which is more target specific. Malarial parasites harbor a plastid-like organelle known as the ‘apicoplast’, which is thought to provide an exciting new outlook for the development of drugs to be used against the parasite. This review elaborates on the current state of development of novel compounds targeted againstemerging malaria parasites. Methods: The apicoplast, originates by an endosymbiotic process, contains a range of metabolic pathways and housekeeping processes that differ from the host body and thereby presents ideal strategies for anti-malarial drug therapy. Drugs are designed by targeting the unique mechanism of the apicoplasts genetic machinery. Several anabolic and catabolic processes, like fatty acid, isopenetyl diphosphate and heme synthess in this organelle, have also been targeted by drugs. Results: Apicoplasts offer exciting opportunities for the development of malarial treatment specific drugs have been found to act by disrupting this organelle’s function, which wouldimpede the survival of the parasite. Conclusion: Recent advanced drugs, their modes of action, and their advantages in the treatment of malaria by using apicoplasts as a target are discussed in this review which thought to be very useful in desigining anti-malarial drugs. Targetting the genetic machinery of apicoplast shows a great advantange regarding anti-malarial drug design. Critical knowledge of these new drugs would give a healthier understanding for deciphering the mechanism of action of anti-malarial drugs when targeting apicoplasts to overcome drug resistance. PMID

  8. Assay development and high throughput antiviral drug screening against Bluetongue virus

    PubMed Central

    Li, Qianjun; Maddox, Clinton; Rasmussen, Lynn; Hobrath, Judith V.; White, Lucile E.

    2009-01-01

    Bluetongue virus (BTV) infection is one of the most important diseases of domestic livestock. There are no antivirals available against BTV disease. In this paper, we present the development, optimization and validation of an in vitro cell-based high-throughput screening (HTS) assay using the luminescent-based CellTiter-Glo reagent to identify novel antivirals against BTV. Conditions of the cytopathic effect (CPE)-based assay were optimized at cell density of 5 000 cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z'-values ≥ 0.70, Coefficient of Variations ≥ 5.68 and signal-to-background ratio ≥ 7.10. This assay was further validated using a 9 532 compound library. The fully validated assay was then used to screen the 194 950 compound collection, which identified 693 compounds with > 30% CPE inhibition. The ten-concentration dose response assay identified 185 structures with IC50 ≤ 100 μM, out of which 42 compounds were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. The CPE-based assay development demonstrated its robustness and reliability, and its application in the HTS campaign will make significant contribution to the antiviral drug discovery against BTV disease. PMID:19559054

  9. Fluorescence polarization assays in high-throughput screening and drug discovery: a review

    NASA Astrophysics Data System (ADS)

    Hall, Matthew D.; Yasgar, Adam; Peryea, Tyler; Braisted, John C.; Jadhav, Ajit; Simeonov, Anton; Coussens, Nathan P.

    2016-06-01

    The sensitivity of fluorescence polarization (FP) and fluorescence anisotropy (FA) to molecular weight changes has enabled the interrogation of diverse biological mechanisms, ranging from molecular interactions to enzymatic activity. Assays based on FP/FA technology have been widely utilized in high-throughput screening (HTS) and drug discovery due to the homogenous format, robust performance and relative insensitivity to some types of interferences, such as inner filter effects. Advancements in assay design, fluorescent probes, and technology have enabled the application of FP assays to increasingly complex biological processes. Herein we discuss different types of FP/FA assays developed for HTS, with examples to emphasize the diversity of applicable targets. Furthermore, trends in target and fluorophore selection, as well as assay type and format, are examined using annotated HTS assays within the PubChem database. Finally, practical considerations for the successful development and implementation of FP/FA assays for HTS are provided based on experience at our center and examples from the literature, including strategies for flagging interference compounds among a list of hits.

  10. Ecotoxicological evaluation of the antimalarial drug chloroquine.

    PubMed

    Zurita, Jorge L; Jos, Angeles; del Peso, Ana; Salguero, Manuel; López-Artíguez, Miguel; Repetto, Guillermo

    2005-10-15

    There is limited information available about the potential environmental effects of chloroquine (CQ), a widely used antimalarial agent and a promising inexpensive drug in the management of HIV disease. The acute effects of CQ were studied using four ecotoxicological model systems. The most sensitive bioindicator was the immobilization of the cladoceran Daphnia magna, with an EC50 of 12 microM CQ at 72 h and a non-observed adverse effect level of 2.5 microM CQ, followed very closely by the decrease of the uptake of neutral red and the reduction of the lysosomal function in the fish cell line PLHC-1 derived from the top minnow Poeciliopsis lucida, probably due to the selective accumulation of the drug into the lysosomes. There was significant cellular stress as indicated by the increases on metallothionein and glucose-6P dehydrogenase levels after 24 h of exposure and succinate dehydrogenase activity mainly after 48 h. No changes were observed for ethoxyresorufin-O-deethylase (EROD) activity. The least sensitive model was the inhibition of bioluminescence in the bacterium Vibrio fischeri. An increase of more than five-fold in the toxicity from 24 to 72 h of exposure was observed for the inhibition of the growth in the alga Chlorella vulgaris and the content of total protein and MTS tetrazolium salt metabolization in PLHC-1 cells. At the morphological level, the most evident alterations in PLHC-1 cultures were hydropic degeneration from 25 microM CQ after 24h of exposure and the presence of many cells with pyknotic nuclei, condensed cytoplasm and apoptosis with concentrations higher than 50 microM CQ after 48 h of exposure. In conclusion, CQ should be classified as harmful to aquatic organisms. PMID:16153718

  11. Antimalarial drug policy in India: Past, present & future

    PubMed Central

    Anvikar, Anupkumar R.; Arora, Usha; Sonal, G.S.; Mishra, Neelima; Shahi, Bharatendu; Savargaonkar, Deepali; Kumar, Navin; Shah, Naman K.; Valecha, Neena

    2014-01-01

    The use of antimalarial drugs in India has evolved since the introduction of quinine in the 17th century. Since the formal establishment of a malaria control programme in 1953, shortly after independence, treatments provided by the public sector ranged from chloroquine, the mainstay drug for many decades, to the newer, recently introduced artemisinin based combination therapy. The complexity of considerations in antimalarial treatment led to the formulation of a National Antimalarial Drug Policy to guide procurement as well as communicate best practices to both public and private healthcare providers. Challenges addressed in the policy include the use of presumptive treatment, the introduction of alternate treatments for drug-resistant malaria, the duration of primaquine therapy to prevent relapses of vivax malaria, the treatment of malaria in pregnancy, and the choice of drugs for chemoprophylaxis. While data on antimalarial drug resistance and both public and private sector treatment practices have been recently reviewed, the policy process of setting national standards has not. In this perspective on antimalarial drug policy, this review highlights its relevant history, analyzes the current policy, and examines future directions. PMID:24718394

  12. High Throughput Screening in Duchenne Muscular Dystrophy: From Drug Discovery to Functional Genomics

    PubMed Central

    Gintjee, Thomas J.J.; Magh, Alvin S.H.; Bertoni, Carmen

    2014-01-01

    Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD), the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS) technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD. PMID:25405319

  13. Selected Approaches for Rational Drug Design and High Throughput Screening to Identify Anti-Cancer Molecules

    PubMed Central

    Hedvat, Michael; Emdad, Luni; Das, Swadesh K.; Kim, Keetae; Dasgupta, Santanu; Thomas, Shibu; Hu, Bin; Zhu, Shan; Dash, Rupesh; Quinn, Bridget A.; Oyesanya, Regina A.; Kegelman, Timothy P.; Sokhi, Upneet K.; Sarkar, Siddik; Erdogan, Eda; Menezes, Mitchell E.; Bhoopathi, Praveen; Wang, Xiang-Yang; Pomper, Martin G.; Wei, Jun; Wu, Bainan; Stebbins, John L.; Diaz, Paul W.; Reed, John C.; Pellecchia, Maurizio; Sarkar, Devanand; Fisher, Paul B.

    2013-01-01

    Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specific-promoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy. PMID:22931411

  14. Novel drug candidates for blast phase chronic myeloid leukemia from high-throughput drug sensitivity and resistance testing

    PubMed Central

    Pietarinen, P O; Pemovska, T; Kontro, M; Yadav, B; Mpindi, J P; Andersson, E I; Majumder, M M; Kuusanmäki, H; Koskenvesa, P; Kallioniemi, O; Wennerberg, K; Heckman, C A; Mustjoki, S; Porkka, K

    2015-01-01

    Chronic myeloid leukemia in blast crisis (CML BC) remains a challenging disease to treat despite the introduction and advances in tyrosine kinase inhibitor (TKI) therapy. In this study we set out to identify novel candidate drugs for CML BC by using an unbiased high-throughput drug testing platform. We used three CML cell lines representing different types of CML blast phases (K562, EM-2 and MOLM-1) and primary leukemic cells from three CML BC patients. Profiling of drug responses was performed with a drug sensitivity and resistance testing platform comprising 295 anticancer agents. Overall, drug sensitivity scores and the drug response profiles of cell line and primary cell samples correlated well and were distinct from other types of leukemia samples. The cell lines were highly sensitive to TKIs and the clinically TKI-resistant patient samples were also resistant ex vivo. Comparison of cell line and patient sample data identified new candidate drugs for CML BC, such as vascular endothelial growth factor receptor and nicotinamide phosphoribosyltransferase inhibitors. Our results indicate that these drugs in particular warrant further evaluation by analyzing a larger set of primary patient samples. The results also pave way for designing rational combination therapies. PMID:25933373

  15. Candida albicans biofilm chip (CaBChip) for high-throughput antifungal drug screening.

    PubMed

    Srinivasan, Anand; Lopez-Ribot, Jose L; Ramasubramanian, Anand K

    2012-01-01

    Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 °C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance). Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation

  16. Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening.

    PubMed

    Pothineni, Venkata Raveendra; Wagh, Dhananjay; Babar, Mustafeez Mujtaba; Inayathullah, Mohammed; Solow-Cordero, David; Kim, Kwang-Min; Samineni, Aneesh V; Parekh, Mansi B; Tayebi, Lobat; Rajadas, Jayakumar

    2016-01-01

    Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%-20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved) that were screened are Library of Pharmacologically Active Compounds (LOPAC1280), the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150 unique compounds, which inhibited >90% of B. burgdorferi growth at a concentration of <25 µM. These 150 unique compounds comprise many safe antibiotics, chemical compounds, and also small molecules from plant sources. Of the 150 unique compounds, 101 compounds are FDA approved. We selected the top 20 FDA-approved molecules based on safety and potency and studied their minimum inhibitory concentration and minimum bactericidal concentration. The promising safe FDA-approved candidates that show low minimum inhibitory concentration and minimum bactericidal concentration values can be chosen as lead

  17. Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening

    PubMed Central

    Pothineni, Venkata Raveendra; Wagh, Dhananjay; Babar, Mustafeez Mujtaba; Inayathullah, Mohammed; Solow-Cordero, David; Kim, Kwang-Min; Samineni, Aneesh V; Parekh, Mansi B; Tayebi, Lobat; Rajadas, Jayakumar

    2016-01-01

    Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%–20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved) that were screened are Library of Pharmacologically Active Compounds (LOPAC1280), the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150 unique compounds, which inhibited >90% of B. burgdorferi growth at a concentration of <25 µM. These 150 unique compounds comprise many safe antibiotics, chemical compounds, and also small molecules from plant sources. Of the 150 unique compounds, 101 compounds are FDA approved. We selected the top 20 FDA-approved molecules based on safety and potency and studied their minimum inhibitory concentration and minimum bactericidal concentration. The promising safe FDA-approved candidates that show low minimum inhibitory concentration and minimum bactericidal concentration values can be chosen as

  18. Antimalarial Drug Discovery: From Quinine to the Dream of Eradication

    PubMed Central

    2013-01-01

    The search for antimalarial remedies predates modern medicine and the concept of small molecule chemotherapy, yet has played a central role in the development of both. This history is reviewed in the context of the current renaissance in antimalarial drug discovery, which is seeing modern drug discovery approaches applied to the problem for the first time. Great strides have been made in the past decade, but further innovations from the drug discovery community will be required if the ultimate dream of eradication is to be achieved. PMID:24790706

  19. Treatment of Murine Cerebral Malaria by Artemisone in Combination with Conventional Antimalarial Drugs: Antiplasmodial Effects and Immune Responses

    PubMed Central

    Guiguemde, W. Armand; Hunt, Nicholas H.; Guo, Jintao; Marciano, Annael; Haynes, Richard K.; Clark, Julie; Guy, R. Kiplin

    2014-01-01

    The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response. PMID:24913162

  20. Drug resistance genomics of the antimalarial drug artemisinin.

    PubMed

    Winzeler, Elizabeth A; Manary, Micah J

    2014-01-01

    Across the globe, over 200 million annual malaria infections result in up to 660,000 deaths, 77% of which occur in children under the age of five years. Although prevention is important, malaria deaths are typically prevented by using antimalarial drugs that eliminate symptoms and clear parasites from the blood. Artemisinins are one of the few remaining compound classes that can be used to cure multidrug-resistant Plasmodium falciparum infections. Unfortunately, clinical trials from Southeast Asia are showing that artemisinin-based treatments are beginning to lose their effectiveness, adding renewed urgency to the search for the genetic determinants of parasite resistance to this important drug class. We review the genetic and genomic approaches that have led to an improved understanding of artemisinin resistance, including the identification of resistance-conferring mutations in the P. falciparum kelch13 gene. PMID:25470531

  1. From hybrid compounds to targeted drug delivery in antimalarial therapy.

    PubMed

    Oliveira, Rudi; Miranda, Daniela; Magalhães, Joana; Capela, Rita; Perry, Maria J; O'Neill, Paul M; Moreira, Rui; Lopes, Francisca

    2015-08-15

    The discovery of new drugs to treat malaria is a continuous effort for medicinal chemists due to the emergence and spread of resistant strains of Plasmodium falciparum to nearly all used antimalarials. The rapid adaptation of the malaria parasite remains a major limitation to disease control. Development of hybrid antimalarial agents has been actively pursued as a promising strategy to overcome the emergence of resistant parasite strains. This review presents the journey that started with simple combinations of two active moieties into one chemical entity and progressed into a delivery/targeted system based on major antimalarial classes of drugs. The rationale for providing different mechanisms of action against a single or additional targets involved in the multiple stages of the parasite's life-cycle is highlighted. Finally, a perspective for this polypharmacologic approach is presented. PMID:25913864

  2. High-throughput microsomal stability assay for screening new chemical entities in drug discovery.

    PubMed

    Fonsi, Massimiliano; Orsale, Maria V; Monteagudo, Edith

    2008-10-01

    In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities. PMID:18812573

  3. Artemisinin anti-malarial drugs in China.

    PubMed

    Guo, Zongru

    2016-03-01

    Discovered by Youyou Tu, one of the 2015 Nobel Prize winners in Physiology or Medicine, together with many other Chinese scientists, artemisinin, artemether and artesunate, as well as other artemisinins, have brought the global anti-malarial treatment to a new era, saving millions of lives all around the world for the past 40 years. The discoveries of artemisinins were carried out beginning from the 1970s, a special period in China, by hundreds of scientists all together under the "whole nation" system. This article focusing on medicinal chemistry research, briefly introduced the discovery and invention course of the scientists according to the published papers, and highlighted their academic contribution and achievements. PMID:27006895

  4. Artemisinin anti-malarial drugs in China

    PubMed Central

    Guo, Zongru

    2016-01-01

    Discovered by Youyou Tu, one of the 2015 Nobel Prize winners in Physiology or Medicine, together with many other Chinese scientists, artemisinin, artemether and artesunate, as well as other artemisinins, have brought the global anti-malarial treatment to a new era, saving millions of lives all around the world for the past 40 years. The discoveries of artemisinins were carried out beginning from the 1970s, a special period in China, by hundreds of scientists all together under the “whole nation” system. This article focusing on medicinal chemistry research, briefly introduced the discovery and invention course of the scientists according to the published papers, and highlighted their academic contribution and achievements. PMID:27006895

  5. Facile and rapid generation of 3D chemical gradients within hydrogels for high-throughput drug screening applications.

    PubMed

    Ahadian, Samad; Ramón-Azcón, Javier; Estili, Mehdi; Obregón, Raquel; Shiku, Hitoshi; Matsue, Tomokazu

    2014-09-15

    We propose a novel application of dielectrophoresis (DEP) to make three-dimensional (3D) methacrylated gelatin (GelMA) hydrogels with gradients of micro- and nanoparticles. DEP forces were able to manipulate micro- and nanoparticles of different sizes and materials (i.e., C2C12 myoblasts, polystyrene beads, gold microparticles, and carbon nanotubes) within GelMA hydrogels in a rapid and facile way and create 3D gradients of these particles in a microchamber. Immobilization of drugs, such as fluorescein isothiocyanate-dextran (FITC-dextran) and 6-hydroxydopamine (6-OHDA), on gold microparticles allowed us to investigate the high-throughput release of these drugs from GelMA-gold microparticle gradient systems. The latter gradient constructs were incubated with C2C12 myoblasts for 24h to examine the cell viability through the release of 6-OHDA. The drug was released from the microparticles in a gradient manner, inducing a cell viability gradient. This novel approach to create 3D chemical gradients within hydrogels is scalable to any arbitrary length scale. It is useful for making anisotropic biomimetic materials and high-throughput platforms to investigate cell-microenvironment interactions in a rapid, simple, cost-effective, and reproducible manner. PMID:24727602

  6. Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1) for antimalarial drug development

    PubMed Central

    Deniskin, Roman; Frame, I.J.; Sosa, Yvett; Akabas, Myles H.

    2015-01-01

    Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([3H]adenosine) and pyrimidines ([3H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 μM), compared to guanosine (14.9 μM) and adenosine (142 μM). For pyrimidines, thymidine had an IC50 of 183 μM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 μM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial drugs

  7. Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1) for antimalarial drug development.

    PubMed

    Deniskin, Roman; Frame, I J; Sosa, Yvett; Akabas, Myles H

    2016-04-01

    Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([(3)H]adenosine) and pyrimidines ([(3)H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 μM), compared to guanosine (14.9 μM) and adenosine (142 μM). For pyrimidines, thymidine had an IC50 of 183 μM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 μM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial

  8. High-throughput identification of off-targets for the mechanistic study of severe adverse drug reactions induced by analgesics

    SciTech Connect

    Pan, Jian-Bo; Ji, Nan; Pan, Wen; Hong, Ru; Wang, Hao; Ji, Zhi-Liang

    2014-01-01

    Drugs may induce adverse drug reactions (ADRs) when they unexpectedly bind to proteins other than their therapeutic targets. Identification of these undesired protein binding partners, called off-targets, can facilitate toxicity assessment in the early stages of drug development. In this study, a computational framework was introduced for the exploration of idiosyncratic mechanisms underlying analgesic-induced severe adverse drug reactions (SADRs). The putative analgesic-target interactions were predicted by performing reverse docking of analgesics or their active metabolites against human/mammal protein structures in a high-throughput manner. Subsequently, bioinformatics analyses were undertaken to identify ADR-associated proteins (ADRAPs) and pathways. Using the pathways and ADRAPs that this analysis identified, the mechanisms of SADRs such as cardiac disorders were explored. For instance, 53 putative ADRAPs and 24 pathways were linked with cardiac disorders, of which 10 ADRAPs were confirmed by previous experiments. Moreover, it was inferred that pathways such as base excision repair, glycolysis/glyconeogenesis, ErbB signaling, calcium signaling, and phosphatidyl inositol signaling likely play pivotal roles in drug-induced cardiac disorders. In conclusion, our framework offers an opportunity to globally understand SADRs at the molecular level, which has been difficult to realize through experiments. It also provides some valuable clues for drug repurposing. - Highlights: • A novel computational framework was developed for mechanistic study of SADRs. • Off-targets of drugs were identified in large scale and in a high-throughput manner. • SADRs like cardiac disorders were systematically explored in molecular networks. • A number of ADR-associated proteins were identified.

  9. Evolution from double to triple-antimalarial drug combinations.

    PubMed

    Shanks, G Dennis; Edstein, Michael D; Jacobus, David

    2015-03-01

    Drug combinations are used to treat multiple-drug resistant malaria parasites and to attempt to further delay the evolution of drug resistance. Most current antimalarial combinations are binary but it is likely that new triple drug combinations will be required in the future. A review of previous triple combinations of antimalarial drugs was done to focus attention on past problems and possible future combinations. The advantages of such triple drug combinations include greater efficacy against multiple-drug resistant strains, synergistic action between the different medications and simplification of the regimen so that it could be administered under direct observation and possibly as single-dose therapy. The disadvantages of poly-pharmacy include increased cost of medication, difficulty preparing robust regulatory packages and problems constructing combined formulations due to drug-drug interactions. Given the arrival of artemisinin tolerance/resistance in Southeast Asia, it is likely that new drugs introduced for malaria treatment will be in triple drug combinations. PMID:25549631

  10. A high throughput solubility assay for drug discovery using microscale shake-flask and rapid UHPLC-UV-CLND quantification.

    PubMed

    Lin, Baiwei; Pease, Joseph H

    2016-04-15

    The rapid determination of key physical properties of lead compounds is essential to the drug discovery process. Solubility is one of the most important properties since good solubility is needed not only for obtaining reliable in vitro and in vivo assay results in early discovery but also to ensure sufficient concentration of the drug being in circulation to get the desired therapeutic exposure at the target of interest. In order for medicinal chemists to tune solubility of lead compounds, a rapid assay is needed to provide solubility data that is accurate and predictive so that it can be reliably used for designing the next generation of compounds with improved properties. To ensure speed and data quality, we developed a high throughput solubility assay that utilizes a single calibration UHPLC-UV-CLND method and a 24h shake-flask format for rapid quantification. A set of 46 model compounds was used to demonstrate that the method is accurate, reproducible and predictive. Here we present development of the assay, including evaluation of quantification method, filtration membranes, equilibrium times, DMSO concentrations, and buffer conditions. A comparison of thermodynamic solubility results to our high throughput 24h shake-flask solubility assay results is also discussed. PMID:26855285

  11. Antimicrobial peptides: a new class of antimalarial drugs?

    PubMed Central

    Vale, Nuno; Aguiar, Luísa; Gomes, Paula

    2014-01-01

    A range of antimicrobial peptides (AMP) exhibit activity on malaria parasites, Plasmodium spp., in their blood or mosquito stages, or both. These peptides include a diverse array of both natural and synthetic molecules varying greatly in size, charge, hydrophobicity, and secondary structure features. Along with an overview of relevant literature reports regarding AMP that display antiplasmodial activity, this review makes a few considerations about those molecules as a potential new class of antimalarial drugs. PMID:25566072

  12. An implantable microdevice to perform high-throughput in vivo drug sensitivity testing in tumors

    PubMed Central

    Jonas, Oliver; Landry, Heather M.; Fuller, Jason E.; Santini, John T.; Baselga, Jose; Tepper, Robert I.; Cima, Michael J.; Langer, Robert

    2016-01-01

    Current anticancer chemotherapy relies on a limited set of in vitro or indirect prognostic markers of tumor response to available drugs. A more accurate analysis of drug sensitivity would involve studying tumor response in vivo. To this end, we have developed an implantable device that can perform drug sensitivity testing of several anticancer agents simultaneously inside the living tumor. The device contained reservoirs that released microdoses of single agents or drug combinations into spatially distinct regions of the tumor. The local drug concentrations were chosen to be representative of concentrations achieved during systemic treatment. Local efficacy and drug concentration profiles were evaluated for each drug or drug combination on the device, and the local efficacy was confirmed to be a predictor of systemic efficacy in vivo for multiple drugs and tumor models. Currently, up to 16 individual drugs or combinations can be assessed independently, without systemic drug exposure, through minimally invasive biopsy of a small region of a single tumor. This assay takes into consideration physiologic effects that contribute to drug response by allowing drugs to interact with the living tumor in its native microenvironment. Because these effects are crucial to predicting drug response, we envision that these devices will help identify optimal drug therapy before systemic treatment is initiated and could improve drug response prediction beyond the biomarkers and in vitro and ex vivo studies used today. These devices may also be used in clinical drug development to safely gather efficacy data on new compounds before pharmacological optimization. PMID:25904741

  13. An implantable microdevice to perform high-throughput in vivo drug sensitivity testing in tumors.

    PubMed

    Jonas, Oliver; Landry, Heather M; Fuller, Jason E; Santini, John T; Baselga, Jose; Tepper, Robert I; Cima, Michael J; Langer, Robert

    2015-04-22

    Current anticancer chemotherapy relies on a limited set of in vitro or indirect prognostic markers of tumor response to available drugs. A more accurate analysis of drug sensitivity would involve studying tumor response in vivo. To this end, we have developed an implantable device that can perform drug sensitivity testing of several anticancer agents simultaneously inside the living tumor. The device contained reservoirs that released microdoses of single agents or drug combinations into spatially distinct regions of the tumor. The local drug concentrations were chosen to be representative of concentrations achieved during systemic treatment. Local efficacy and drug concentration profiles were evaluated for each drug or drug combination on the device, and the local efficacy was confirmed to be a predictor of systemic efficacy in vivo for multiple drugs and tumor models. Currently, up to 16 individual drugs or combinations can be assessed independently, without systemic drug exposure, through minimally invasive biopsy of a small region of a single tumor. This assay takes into consideration physiologic effects that contribute to drug response by allowing drugs to interact with the living tumor in its native microenvironment. Because these effects are crucial to predicting drug response, we envision that these devices will help identify optimal drug therapy before systemic treatment is initiated and could improve drug response prediction beyond the biomarkers and in vitro and ex vivo studies used today. These devices may also be used in clinical drug development to safely gather efficacy data on new compounds before pharmacological optimization. PMID:25904741

  14. Resistance to antimalarial drugs: molecular, pharmacological and clinical considerations

    PubMed Central

    Travassos, Mark A.; Laufer, Miriam K.

    2009-01-01

    One of the greatest obstacles to the control of malaria has been the spread of resistance to drugs used on a large scale. This review provides an update of the current understanding of the molecular basis for antimalarial drug resistance. Parasite intrinsic resistance is just one component that determines the in vivo efficacy of a drug. Human immune responses and pharmacological properties play important roles in determining the clinical outcome of treatment. The emergence and spread of resistance also results from an interplay of these factors. Current efforts to characterize and deter resistance to new combination therapy are also discussed. PMID:19918214

  15. Fighting fire with fire: mass antimalarial drug administrations in an era of antimalarial resistance.

    PubMed

    von Seidlein, Lorenz; Dondorp, Arjen

    2015-06-01

    The emergence and spread of antimalarial resistance has been a major liability for malaria control. The spread of chloroquine-resistant Plasmodium falciparum strains had catastrophic consequences for people in malaria-endemic regions, particularly in sub-Saharan Africa. The recent emergence of artemisinin-resistant P. falciparum strains is of highest concern. Current efforts to contain artemisinin resistance have yet to show success. In the absence of more promising plans, it has been suggested to eliminate falciparum malaria from foci of artemisinin resistance using a multipronged approach, including mass drug administrations. The use of mass drug administrations is controversial as it increases drug pressure. Based on current knowledge it is difficult to conceptualize how targeted malaria elimination could contribute to artemisinin resistance, provided a full treatment course is ensured. PMID:25831482

  16. In Vitro High-Throughput RNAi Screening to Accelerate the Process of Target Identification and Drug Development.

    PubMed

    Yin, Hongwei; Kassner, Michelle

    2016-01-01

    High-throughput RNA interference (HT-RNAi) is a powerful tool that can be used to knock down gene expression in order to identify novel genes and pathways involved in many cellular processes. It is a systematic, yet unbiased, approach to identify essential or synthetic lethal genes that promote cell survival in diseased cells as well as genes that confer resistance or sensitivity to drug treatment. This information serves as a foundation for enhancing current treatments for cancer and other diseases by identifying new drug targets, uncovering potential combination therapies, and helping clinicians match patients with the most effective treatment based on genetic information. Here, we describe the method of performing an in vitro HT-RNAi screen using chemically synthesized siRNA. PMID:27581290

  17. Peptide deformylase: a new target in antibacterial, antimalarial and anticancer drug discovery.

    PubMed

    Sangshetti, Jaiprakash N; Khan, Firoz A Kalam; Shinde, Devanand B

    2015-01-01

    Peptide deformylase (PDF) is a class of metalloenzyme responsible for catalyzing the removal of the N-formyl group from N-terminal methionine following translation. PDF inhibitors are moving into new phase of drug development. Initially, PDF was considered as an important target in antibacterial drug discovery; however genome database searches have revealed PDF-like sequences in parasites (P. falciparum) and human, widening the utility of this target in antimalarial and anticancer drug discovery along with antibacterial. Using structural and mechanistic information together with high throughput screening, several types of chemical classes of PDF inhibitors with improved efficacy and specificity have been identified. Various drugs like, GSK-1322322 (Phase II), BB-83698 (Phase I), and LBM-415 (Phase I) have entered into clinical developments. Developments in the field have prompted us to review the current aspects of PDFs, especially their structures, different classes of PDF inhibitors, and molecular modeling studies. In nut shell, this review enlightens PDF as a versatile target along with its inhibitors and future perspectives of different PDF inhibitors. PMID:25174923

  18. Quantifying the pharmacology of antimalarial drug combination therapy.

    PubMed

    Hastings, Ian M; Hodel, Eva Maria; Kay, Katherine

    2016-01-01

    Most current antimalarial drugs are combinations of an artemisinin plus a 'partner' drug from another class, and are known as artemisinin-based combination therapies (ACTs). They are the frontline drugs in treating human malaria infections. They also have a public-health role as an essential component of recent, comprehensive scale-ups of malaria interventions and containment efforts conceived as part of longer term malaria elimination efforts. Recent reports that resistance has arisen to artemisinins has caused considerable concern. We investigate the likely impact of artemisinin resistance by quantifying the contribution artemisinins make to the overall therapeutic capacity of ACTs. We achieve this using a simple, easily understood, algebraic approach and by more sophisticated pharmacokinetic/pharmacodynamic analyses of drug action; the two approaches gave consistent results. Surprisingly, the artemisinin component typically makes a negligible contribution (≪0.0001%) to the therapeutic capacity of the most widely used ACTs and only starts to make a significant contribution to therapeutic outcome once resistance has started to evolve to the partner drugs. The main threat to antimalarial drug effectiveness and control comes from resistance evolving to the partner drugs. We therefore argue that public health policies be re-focussed to maximise the likely long-term effectiveness of the partner drugs. PMID:27604175

  19. Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

    PubMed Central

    Li, Gang; Diogo, Dorothée; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J.; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L.; Siminovitch, Katherine A.; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K.; Worthington, Jane; Gupta, Namrata; Clemons, Paul A.; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M.

    2013-01-01

    Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10−9). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10−9), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in

  20. Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

    PubMed

    Li, Gang; Diogo, Dorothée; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L; Siminovitch, Katherine A; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K; Worthington, Jane; Gupta, Namrata; Clemons, Paul A; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M

    2013-05-01

    Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in

  1. The Development of Protein Chips for High Throughput Screening (HTS) of Chemically Labeling Small Molecular Drugs.

    PubMed

    Feng, Yingzhu; Wang, Bochu; Chu, Xinxin; Wang, Yazhou; Zhu, Liancai

    2016-01-01

    How to construct protein chips and chemically labeling drug molecules without disrupting structures for HTS is still a challenging area. There are two main obstacles, one is that human multitrans membrane receptors, which are major drug targets, exhibit distinct motifs, and fold structures, and they will collapse unfold without membrane support in vitro; another one is that there still lack effective chemical labeling method for small drugs for detection. Therefore, how to acquire high detecting sensitivity for small molecules and to immobilize membrane protein receptors in native conformation with uniform direction on the chip, need to be solved for drug HTS. This paper reviews drug HTS trends in recent years, proposed a new virion-chip model and a feasible C-H activation method for CY-5 labeling drugs. It is expected to provide a good platform for future drug HTS. PMID:25963567

  2. High-Throughput 3D Screening Reveals Differences in Drug Sensitivities between Culture Models of JIMT1 Breast Cancer Cells

    PubMed Central

    Fey, Vidal; Mpindi, John-Patrick; Kleivi Sahlberg, Kristine; Kallioniemi, Olli; Perälä, Merja

    2013-01-01

    The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional monolayers on plastic. However, many cellular features are impaired in these artificial conditions, and large changes in gene expression compared to tumors have been reported. Three-dimensional cell culture models have become increasingly popular and are suggested to be better models than two-dimensional monolayers due to improved cell-to-cell contact and structures that resemble in vivo architecture. The aim of this study was to develop a simple high-throughput three-dimensional drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in two dimensions, in poly(2-hydroxyethyl methacrylate) induced anchorage-independent three-dimensional models, and in Matrigel three-dimensional cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating, or they were allowed to grow in three-dimensional cultures for 4 days before the drug treatment. Large variations in drug responses were observed between the models indicating that comparisons of culture model-influenced drug sensitivities cannot be made based on the effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in two-dimensional cultures, while the responses of cells grown in poly(2-hydroxyethyl methacrylate) resembled those of the two-dimensional cultures. Furthermore, comparing the gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study, we also suggest that three-dimensional cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode

  3. Quality of antimalarials at the epicenter of antimalarial drug resistance: results from an overt and mystery client survey in Cambodia.

    PubMed

    Yeung, Shunmay; Lawford, Harriet L S; Tabernero, Patricia; Nguon, Chea; van Wyk, Albert; Malik, Naiela; DeSousa, Mikhael; Rada, Ouk; Boravann, Mam; Dwivedi, Prabha; Hostetler, Dana M; Swamidoss, Isabel; Green, Michael D; Fernandez, Facundo M; Kaur, Harparkash

    2015-06-01

    Widespread availability of monotherapies and falsified antimalarials is thought to have contributed to the historical development of multidrug-resistant malaria in Cambodia. This study aimed to document the quality of artemisinin-containing antimalarials (ACAs) and to compare two methods of collecting antimalarials from drug outlets: through open surveyors and mystery clients (MCs). Few oral artemisinin-based monotherapies and no suspected falsified medicines were found. All 291 samples contained the stated active pharmaceutical ingredient (API) of which 69% were considered good quality by chemical analysis. Overall, medicine quality did not differ by collection method, although open surveyors were less likely to obtain oral artemisinin-based monotherapies than MCs. The results are an encouraging indication of the positive impact of the country's efforts to tackle falsified antimalarials and artemisinin-based monotherapies. However, poor-quality medicines remain an ongoing challenge that demands sustained political will and investment of human and financial resources. PMID:25897063

  4. Quality of Antimalarials at the Epicenter of Antimalarial Drug Resistance: Results from an Overt and Mystery Client Survey in Cambodia

    PubMed Central

    Yeung, Shunmay; Lawford, Harriet L. S.; Tabernero, Patricia; Nguon, Chea; van Wyk, Albert; Malik, Naiela; DeSousa, Mikhael; Rada, Ouk; Boravann, Mam; Dwivedi, Prabha; Hostetler, Dana M.; Swamidoss, Isabel; Green, Michael D.; Fernandez, Facundo M.; Kaur, Harparkash

    2015-01-01

    Widespread availability of monotherapies and falsified antimalarials is thought to have contributed to the historical development of multidrug-resistant malaria in Cambodia. This study aimed to document the quality of artemisinin-containing antimalarials (ACAs) and to compare two methods of collecting antimalarials from drug outlets: through open surveyors and mystery clients (MCs). Few oral artemisinin-based monotherapies and no suspected falsified medicines were found. All 291 samples contained the stated active pharmaceutical ingredient (API) of which 69% were considered good quality by chemical analysis. Overall, medicine quality did not differ by collection method, although open surveyors were less likely to obtain oral artemisinin-based monotherapies than MCs. The results are an encouraging indication of the positive impact of the country's efforts to tackle falsified antimalarials and artemisinin-based monotherapies. However, poor-quality medicines remain an ongoing challenge that demands sustained political will and investment of human and financial resources. PMID:25897063

  5. Investigation of the incidence of "undesirable" molecular moieties for high-throughput screening compound libraries in marketed drug compounds.

    PubMed

    Axerio-Cilies, Peter; Castañeda, Ivan P; Mirza, Amin; Reynisson, Jóhannes

    2009-03-01

    A database of 1070 marketed drug compounds was compiled and analyzed in order to assess the occurrence of moieties described in the literature as "undesirable" for high-throughput screening compound libraries due to their ability to perturb assay formats. The study revealed a total of 277 compounds, 26% of the database, contained at least one of the moieties. As some of the drug compounds contained more than one "undesirable" moiety, the total number was 352. Electrophilic reactive groups, particularly aliphatic esters, were the most abundant type with 55% of the total. Half of the drug compounds incorporating the "undesirable" moieties were synthetic organic molecules. These findings suggest that "undesirable" moieties do not pose a major hindrance during clinical trials, the most expensive phase of drug development. In addition, their early elimination in the preclinical stage excludes large regions of known drug space due to the reliance on biochemical and cell-based assays. In general, it can be concluded that compounds with "undesirable" moieties should not simply be eliminated from compound screening libraries but rather be flagged as potentially problematic. A possible solution is to segregate the compounds containing suspect moieties and screen them when deemed appropriate. PMID:18692938

  6. Biomimetic three-dimensional tissue models for advanced high-throughput drug screening

    PubMed Central

    Nam, Ki-Hwan; Smith, Alec S.T.; Lone, Saifullah; Kwon, Sunghoon; Kim, Deok-Ho

    2015-01-01

    Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately recreate the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when utilizing such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models which accurately mimic the physiological properties of native tissue samples, and highlight the advantages of using such 3D micro-tissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based-on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models. PMID:25385716

  7. A New Set of Chemical Starting Points with Plasmodium falciparum Transmission-Blocking Potential for Antimalarial Drug Discovery

    PubMed Central

    Almela, Maria Jesus; Lozano, Sonia; Lelièvre, Joël; Colmenarejo, Gonzalo; Coterón, José Miguel; Rodrigues, Janneth; Gonzalez, Carolina; Herreros, Esperanza

    2015-01-01

    The discovery of new antimalarials with transmission blocking activity remains a key issue in efforts to control malaria and eventually eradicate the disease. Recently, high-throughput screening (HTS) assays have been successfully applied to Plasmodium falciparum asexual stages to screen millions of compounds, with the identification of thousands of new active molecules, some of which are already in clinical phases. The same approach has now been applied to identify compounds that are active against P. falciparum gametocytes, the parasite stage responsible for transmission. This study reports screening results for the Tres Cantos Antimalarial Set (TCAMS), of approximately 13,533 molecules, against P. falciparum stage V gametocytes. Secondary confirmation and cytotoxicity assays led to the identification of 98 selective molecules with dual activity against gametocytes and asexual stages. Hit compounds were chemically clustered and analyzed for appropriate physicochemical properties. The TCAMS chemical space around the prioritized hits was also studied. A selection of hit compounds was assessed ex vivo in the standard membrane feeding assay and demonstrated complete block in transmission. As a result of this effort, new chemical structures not connected to previously described antimalarials have been identified. This new set of compounds may serve as starting points for future drug discovery programs as well as tool compounds for identifying new modes of action involved in malaria transmission. PMID:26317851

  8. Precision multidimensional assay for high-throughput microRNA drug discovery

    PubMed Central

    Haefliger, Benjamin; Prochazka, Laura; Angelici, Bartolomeo; Benenson, Yaakov

    2016-01-01

    Development of drug discovery assays that combine high content with throughput is challenging. Information-processing gene networks can address this challenge by integrating multiple potential targets of drug candidates' activities into a small number of informative readouts, reporting simultaneously on specific and non-specific effects. Here we show a family of networks implementing this concept in a cell-based drug discovery assay for miRNA drug targets. The networks comprise multiple modules reporting on specific effects towards an intended miRNA target, together with non-specific effects on gene expression, off-target miRNAs and RNA interference pathway. We validate the assays using known perturbations of on- and off-target miRNAs, and evaluate an ∼700 compound library in an automated screen with a follow-up on specific and non-specific hits. We further customize and validate assays for additional drug targets and non-specific inputs. Our study offers a novel framework for precision drug discovery assays applicable to diverse target families. PMID:26880188

  9. Precision multidimensional assay for high-throughput microRNA drug discovery.

    PubMed

    Haefliger, Benjamin; Prochazka, Laura; Angelici, Bartolomeo; Benenson, Yaakov

    2016-01-01

    Development of drug discovery assays that combine high content with throughput is challenging. Information-processing gene networks can address this challenge by integrating multiple potential targets of drug candidates' activities into a small number of informative readouts, reporting simultaneously on specific and non-specific effects. Here we show a family of networks implementing this concept in a cell-based drug discovery assay for miRNA drug targets. The networks comprise multiple modules reporting on specific effects towards an intended miRNA target, together with non-specific effects on gene expression, off-target miRNAs and RNA interference pathway. We validate the assays using known perturbations of on- and off-target miRNAs, and evaluate an ∼700 compound library in an automated screen with a follow-up on specific and non-specific hits. We further customize and validate assays for additional drug targets and non-specific inputs. Our study offers a novel framework for precision drug discovery assays applicable to diverse target families. PMID:26880188

  10. Genotype-Selective Combination Therapies for Melanoma Identified by High Throughput Drug Screening

    PubMed Central

    Held, Matthew A.; Langdon, Casey G.; Platt, James T.; Graham-Steed, Tisheeka; Liu, Zongzhi; Chakraborty, Ashok; Bacchiocchi, Antonella; Koo, Andrew; Bosenberg, Marcus W.; Stern, David F.

    2012-01-01

    Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small molecule inhibitors on early passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGFR and AKT sensitized treatment-resistant BRAF-mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results demonstrate the utility of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF and RAS driven melanomas. PMID:23239741

  11. Antimalarial mass drug administration: ethical considerations.

    PubMed

    Cheah, Phaik Yeong; White, Nicholas J

    2016-07-01

    Falciparum malaria is a major cause of death and illness in tropical countries, particularly in childhood. In endemic countries, a significant proportion of the community is infected with malaria asymptomatically. One promising way to eliminate malaria is to give the entire population malaria treatment. This is called mass drug administration (MDA) and it raises a number of ethical issues, as possible long-term benefits are uncertain. The effectiveness of MDA is critically dependent on level of participation, so the promised benefits to the community can be annulled by non-participation of a small number of individuals. These potential benefits range a wide spectrum, from the permanent elimination of malaria (success) to a transient reduction in the prevalence of infection and the incidence of illness (failure). The drawbacks of MDA are: inconvenience, potential toxicity, loss of confidence in the elimination campaign, possible drug resistance (though highly unlikely), and the potential for a rebound of malaria illness (if immunity is lost and malaria is reintroduced later). Other ethical issues are related to balancing individual and public health interests, and potentially limiting individual autonomy by making MDA compulsory. PMID:27481834

  12. An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells.

    PubMed

    Silva, Ariosto; Jacobson, Timothy; Meads, Mark; Distler, Allison; Shain, Kenneth

    2015-01-01

    In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response. PMID:26274375

  13. The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei

    PubMed Central

    Carvalho, Luis; Martínez-García, Marta; Pérez-Victoria, Ignacio; Manzano, José Ignacio; Yardley, Vanessa

    2015-01-01

    The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca2+, and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei. PMID:26195527

  14. The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei.

    PubMed

    Carvalho, Luis; Martínez-García, Marta; Pérez-Victoria, Ignacio; Manzano, José Ignacio; Yardley, Vanessa; Gamarro, Francisco; Pérez-Victoria, José M

    2015-10-01

    The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca(2+), and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei. PMID:26195527

  15. Large-scale drug screening against Babesia divergens parasite using a fluorescence-based high-throughput screening assay.

    PubMed

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; AbouLaila, Mahmoud; Tuvshintulga, Bumduuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-08-30

    The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the efficacies of large chemical libraries against Babesia divergens (bovine strain) in in vitro cultures was evaluated in this study. Hematocrits (HCTs) of 2.5%, 5%, and 10% were used for the in vitro culture at 1% parasitemia without daily replacement of the medium. Linearity and HTS assay results revealed that the best HCTs were 5% and 10%. The obtained IC50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence- and microscopy-based methods, did not differ significantly at 5% HCT. Actinonin and chloroquine diphosphate were the most effective drugs against the in vitro growth of B. divergens, followed by pyronaridine tetraphosphate- and luteolin-treated cultures. On contrary, tetracycline hydrochloride and (-)-epigallocatechin-3-gallate from green tea exhibited poor activity as compared with diminazene aceturate (positive control drug). The data indicated that 5% HCT without daily replacement of the culture medium mixed with bovine serum in vitro using a fluorescence-based HTS assay creates the best conditions for large-scale drug screening against B. divergens that infect cattle. PMID:27523944

  16. Discovering novel neuroactive drugs through high-throughput behavior-based chemical screening in the zebrafish

    PubMed Central

    Bruni, Giancarlo; Lakhani, Parth; Kokel, David

    2014-01-01

    Most neuroactive drugs were discovered through unexpected behavioral observations. Systematic behavioral screening is inefficient in most model organisms. But, automated technologies are enabling a new phase of discovery-based research in central nervous system (CNS) pharmacology. Researchers are using large-scale behavior-based chemical screens in zebrafish to discover compounds with new structures, targets, and functions. These compounds are powerful tools for understanding CNS signaling pathways. Substantial differences between human and zebrafish biology will make it difficult to translate these discoveries to clinical medicine. However, given the molecular genetic similarities between humans and zebrafish, it is likely that some of these compounds will have translational utility. We predict that the greatest new successes in CNS drug discovery will leverage many model systems, including in vitro assays, cells, rodents, and zebrafish. PMID:25104936

  17. Elucidating Hyperconjugation from Electronegativity to Predict Drug Conformational Energy in a High Throughput Manner.

    PubMed

    Liu, Zhaomin; Pottel, Joshua; Shahamat, Moeed; Tomberg, Anna; Labute, Paul; Moitessier, Nicolas

    2016-04-25

    Computational chemists use structure-based drug design and molecular dynamics of drug/protein complexes which require an accurate description of the conformational space of drugs. Organic chemists use qualitative chemical principles such as the effect of electronegativity on hyperconjugation, the impact of steric clashes on stereochemical outcome of reactions, and the consequence of resonance on the shape of molecules to rationalize experimental observations. While computational chemists speak about electron densities and molecular orbitals, organic chemists speak about partial charges and localized molecular orbitals. Attempts to reconcile these two parallel approaches such as programs for natural bond orbitals and intrinsic atomic orbitals computing Lewis structures-like orbitals and reaction mechanism have appeared. In the past, we have shown that encoding and quantifying chemistry knowledge and qualitative principles can lead to predictive methods. In the same vein, we thought to understand the conformational behaviors of molecules and to encode this knowledge back into a molecular mechanics tool computing conformational potential energy and to develop an alternative to atom types and training of force fields on large sets of molecules. Herein, we describe a conceptually new approach to model torsion energies based on fundamental chemistry principles. To demonstrate our approach, torsional energy parameters were derived on-the-fly from atomic properties. When the torsional energy terms implemented in GAFF, Parm@Frosst, and MMFF94 were substituted by our method, the accuracy of these force fields to reproduce MP2-derived torsional energy profiles and their transferability to a variety of functional groups and drug fragments were overall improved. In addition, our method did not rely on atom types and consequently did not suffer from poor automated atom type assignments. PMID:27028941

  18. Poor quality drugs: grand challenges in high throughput detection, countrywide sampling, and forensics in developing countries†

    PubMed Central

    Fernandez, Facundo M.; Hostetler, Dana; Powell, Kristen; Kaur, Harparkash; Green, Michael D.; Mildenhall, Dallas C.; Newton, Paul N.

    2012-01-01

    Throughout history, poor quality medicines have been a persistent problem, with periodical crises in the supply of antimicrobials, such as fake cinchona bark in the 1600s and fake quinine in the 1800s. Regrettably, this problem seems to have grown in the last decade, especially afflicting unsuspecting patients and those seeking medicines via on-line pharmacies. Here we discuss some of the challenges related to the fight against poor quality drugs, and counterfeits in particular, with an emphasis on the analytical tools available, their relative performance, and the necessary workflows needed for distinguishing between genuine, substandard, degraded and counterfeit medicines. PMID:21107455

  19. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity.

    PubMed

    Astashkina, Anna; Mann, Brenda; Grainger, David W

    2012-04-01

    Drug candidate and toxicity screening processes currently rely on results from early-stage in vitro cell-based assays expected to faithfully represent essential aspects of in vivo pharmacology and toxicology. Several in vitro designs are optimized for high throughput to benefit screening efficiencies, allowing the entire libraries of potential pharmacologically relevant or possible toxin molecules to be screened for different types of cell signals relevant to tissue damage or to therapeutic goals. Creative approaches to multiplexed cell-based assay designs that select specific cell types, signaling pathways and reporters are routine. However, substantial percentages of new chemical and biological entities (NCEs/NBEs) that fail late-stage human drug testing, or receive regulatory "black box" warnings, or that are removed from the market for safety reasons after regulatory approvals all provide strong evidence that in vitro cell-based assays and subsequent preclinical in vivo studies do not yet provide sufficient pharmacological and toxicity data or reliable predictive capacity for understanding drug candidate performance in vivo. Without a reliable translational assay tool kit for pharmacology and toxicology, the drug development process is costly and inefficient in taking initial in vitro cell-based screens to in vivo testing and subsequent clinical approvals. Commonly employed methods of in vitro testing, including dissociated, organotypic, organ/explant, and 3-D cultures, are reviewed here with specific focus on retaining cell and molecular interactions and physiological parameters that determine cell phenotypes and their corresponding responses to bioactive agents. Distinct advantages and performance challenges for these models pertinent to cell-based assay and their predictive capabilities required for accurate correlations to in vivo mechanisms of drug toxicity are compared. PMID:22252140

  20. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.

    PubMed

    Gao, Hui; Korn, Joshua M; Ferretti, Stéphane; Monahan, John E; Wang, Youzhen; Singh, Mallika; Zhang, Chao; Schnell, Christian; Yang, Guizhi; Zhang, Yun; Balbin, O Alejandro; Barbe, Stéphanie; Cai, Hongbo; Casey, Fergal; Chatterjee, Susmita; Chiang, Derek Y; Chuai, Shannon; Cogan, Shawn M; Collins, Scott D; Dammassa, Ernesta; Ebel, Nicolas; Embry, Millicent; Green, John; Kauffmann, Audrey; Kowal, Colleen; Leary, Rebecca J; Lehar, Joseph; Liang, Ying; Loo, Alice; Lorenzana, Edward; Robert McDonald, E; McLaughlin, Margaret E; Merkin, Jason; Meyer, Ronald; Naylor, Tara L; Patawaran, Montesa; Reddy, Anupama; Röelli, Claudia; Ruddy, David A; Salangsang, Fernando; Santacroce, Francesca; Singh, Angad P; Tang, Yan; Tinetto, Walter; Tobler, Sonja; Velazquez, Roberto; Venkatesan, Kavitha; Von Arx, Fabian; Wang, Hui Qin; Wang, Zongyao; Wiesmann, Marion; Wyss, Daniel; Xu, Fiona; Bitter, Hans; Atadja, Peter; Lees, Emma; Hofmann, Francesco; Li, En; Keen, Nicholas; Cozens, Robert; Jensen, Michael Rugaard; Pryer, Nancy K; Williams, Juliet A; Sellers, William R

    2015-11-01

    Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses. PMID:26479923

  1. A Novel Reporter System for Molecular Imaging and High Throughput Screening of Anticancer Drugs

    PubMed Central

    Wang, Chunxia; Virostko, John; Manning, H. Charles; Pham, Wellington; Bauer, Joshua

    2013-01-01

    Apoptosis is irreversible programmed cell death characterized by a cellular cascade activation of caspase-3, which subsequently degrades proteins and other components of cells with a motif sequence. Here we report a novel reporter system to detect apoptosis, growth arrest and cell death based on controlled and self-amplified protein degradation. The key element of the reporter system is an apoptotic sensor chimerical protein which consists of three components, the procaspase 3, ubiquitin (Ub), and a strong consensus sequence of N-degron. Between each of these units, there is a DEVD (Asp-Glu-Val-Asp) sequence, which acts as the cleavage target of caspase-3. This non-conventional signal loss approach is much more sensitive than other native methods based on signal gain. The superior sensitivity is demonstrated by its effective application in 386-well HTS with lower drug concentrations and short incubation time. The HTS selection process using this reporter system is very simple and economic. The simplicity eliminates potential errors introduced by multiple steps; there is no need for any substrate. Furthermore the cells in the assay need not be disrupted and the morphology of the cells can provide additional information on mechanisms; the intact cells after HTS can also be used for other analytic analysis. This system thus has a potentially important role in the discovery and development of new anticancer drugs, it also appears to be very versatile, can be used both in vitro and in vivo, with different linked reporter genes, it can be used for a variety of imaging applications. PMID:23881799

  2. High-throughput screening of drug-lipid membrane interactions via counter-propagating second harmonic generation imaging.

    PubMed

    Nguyen, Trang T; Conboy, John C

    2011-08-01

    Here we report the use of counter-propagating second harmonic generation (SHG) to image the interactions between the local anesthetic tetracaine and a multicomponent planar supported lipid bilayer array in a label-free manner. The lipid bilayer arrays, prepared using a 3D continuous flow microspotter, allow the effects of lipid phase and cholesterol content on tetracaine binding to be examined simultaneously. SHG images show that tetracaine has a higher binding affinity to liquid-crystalline phase lipids than to solid-gel phase lipids. The presence of 28 mol % cholesterol decreased the binding affinity of tetracaine to bilayers composed of the mixed chain lipid, 1-steroyl-2-oleoyl-sn-glycero-3-phophocholine (SOPC), and the saturated lipids 1,2-dimyristoyl-sn-glycero-3-phophocholine (DMPC) and 1,2-dipamitoyl-sn-glycero-3-phophocholine (DPPC) while having no effect on diunsaturated 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC). The maximum surface excess of tetracaine increases with the degree of unsaturation of the phospholipids and decreases with cholesterol in the lipid bilayers. The paper demonstrates that SHG imaging is a sensitive technique that can directly image and quantitatively measure the association of a drug to a multicomponent lipid bilayer array, providing a high-throughput means to assess drug-membrane interactions. PMID:21696170

  3. High-Throughput Screening of Drug-Lipid Membrane Interactions via Counter-Propagating Second Harmonic Generation Imaging

    PubMed Central

    Nguyen, Trang T.; Conboy, John C.

    2011-01-01

    Here we report the use of counter-propagating second harmonic generation (SHG) to image the interactions between the local anesthetic tetracaine and a multi-component planar supported lipid bilayer array in a label-free manner. The lipid bilayer arrays, prepared using a 3D continuous flow microspotter, allow the effects of lipid phase and cholesterol content on tetracaine binding to be examined simultaneously. SHG images show that tetracaine has a higher binding affinity to liquid-crystalline phase lipids than to solid-gel phase lipids. The presence of 28 mol % cholesterol decreased the binding affinity of tetracaine to bilayers composed of the mixed chain lipid, 1-steroyl-2-oleoyl-sn-glycero-3-phophocholine (SOPC) and the saturated lipids 1,2-dimyristoyl-sn-glycero-3-phophocholine (DMPC) and 1,2-dipamitoyl-sn-glycero-3-phophocholine (DPPC) while having no effect on di-unsaturated 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC). The maximum surface excess of tetracaine increases with the degree of unsaturation of the phospholipids and decreases with cholesterol in the lipid bilayers. The paper demonstrates that SHG imaging is a sensitive technique that can directly image and quantitatively measure the association of a drug to a multi-component lipid bilayer array, providing a high-throughput means to assess drug-membrane interactions. PMID:21696170

  4. Quantitative High-Throughput Profiling of Environmental Chemicals and Drugs that Modulate Farnesoid X Receptor

    PubMed Central

    Hsu, Chia-Wen; Zhao, Jinghua; Huang, Ruili; Hsieh, Jui-Hua; Hamm, Jon; Chang, Xiaoqing; Houck, Keith; Xia, Menghang

    2014-01-01

    The farnesoid X receptor (FXR) regulates the homeostasis of bile acids, lipids, and glucose. Because endogenous chemicals bind and activate FXR, it is important to examine which xenobiotic compounds would disrupt normal receptor function. We used a cell-based human FXR β-lactamase (Bla) reporter gene assay to profile the Tox21 10K compound collection of environmental chemicals and drugs. Structure-activity relationships of FXR-active compounds revealed by this screening were then compared against the androgen receptor, estrogen receptor α, peroxisome proliferator-activated receptors δ and γ, and the vitamin D receptor. We identified several FXR-active structural classes including anthracyclines, benzimidazoles, dihydropyridines, pyrethroids, retinoic acids, and vinca alkaloids. Microtubule inhibitors potently decreased FXR reporter gene activity. Pyrethroids specifically antagonized FXR transactivation. Anthracyclines affected reporter activity in all tested assays, suggesting non-specific activity. These results provide important information to prioritize chemicals for further investigation, and suggest possible modes of action of compounds in FXR signaling. PMID:25257666

  5. Agarose microwell based neuronal micro-circuit arrays on microelectrode arrays for high throughput drug testing.

    PubMed

    Kang, Gyumin; Lee, Ji-Hye; Lee, Chang-Soo; Nam, Yoonkey

    2009-11-21

    For cell-based biosensor applications, dissociated neurons have been cultured on planar microelectrode arrays (MEAs) to measure the network activity with substrate-embedded microelectrodes. There has been a need for a multi-well type platform to reduce the data collection time and increase the statistical power for data analysis. This study presents a novel method to convert a conventional MEA into a multi-well MEA with an array of micrometre-sized neuronal culture ('neuronal micro-circuit array'). An MEA was coated first with cell-adhesive layer (poly-D-lysine) which was subsequently patterned with a cell-repulsive layer (agarose hydrogel) to both pattern the cell adhesive region and isolate neuronal micro-circuits from each other. For a few weeks, primary hippocampal neurons were cultured on the agarose microwell MEA and the development of spontaneous electrical activities were characterized with extracellular action potentials. Using neurotransmission modulators, the simultaneous monitoring of drug responses from neuronal micro-circuit arrays was also demonstrated. The proposed approach will be powerful for neurobiological functional assay studies or neuron-based biosensor fields which require repeated trials to obtain a single data point due to biological variations. PMID:19865730

  6. Multilayered, Hyaluronic Acid-Based Hydrogel Formulations Suitable for Automated 3D High Throughput Drug Screening of Cancer-Stromal Cell Cocultures.

    PubMed

    Engel, Brian J; Constantinou, Pamela E; Sablatura, Lindsey K; Doty, Nathaniel J; Carson, Daniel D; Farach-Carson, Mary C; Harrington, Daniel A; Zarembinski, Thomas I

    2015-08-01

    Validation of a high-throughput compatible 3D hyaluronic acid hydrogel coculture of cancer cells with stromal cells. The multilayered hyaluronic acid hydrogels improve drug screening predictability as evaluated with a panel of clinically relevant chemotherapeutics in both prostate and endometrial cancer cell lines compared to 2D culture. PMID:26059746

  7. Selection of a trioxaquine as an antimalarial drug candidate

    PubMed Central

    Coslédan, Frédéric; Fraisse, Laurent; Pellet, Alain; Guillou, François; Mordmüller, Benjamin; Kremsner, Peter G.; Moreno, Alicia; Mazier, Dominique; Maffrand, Jean-Pierre; Meunier, Bernard

    2008-01-01

    Trioxaquines are antimalarial agents based on hybrid structures with a dual mode of action. One of these molecules, PA1103/SAR116242, is highly active in vitro on several sensitive and resistant strains of Plasmodium falciparum at nanomolar concentrations (e.g., IC50 value = 10 nM with FcM29, a chloroquine-resistant strain) and also on multidrug-resistant strains obtained from fresh patient isolates in Gabon. This molecule is very efficient by oral route with a complete cure of mice infected with chloroquine-sensitive or chloroquine-resistant strains of Plasmodia at 26–32 mg/kg. This compound is also highly effective in humanized mice infected with P. falciparum. Combined with a good drug profile (preliminary absorption, metabolism, and safety parameters), these data were favorable for the selection of this particular trioxaquine for development as drug candidate among 120 other active hybrid molecules. PMID:18987321

  8. A high-throughput lab-on-a-chip interface for zebrafish embryo tests in drug discovery and ecotoxicology

    NASA Astrophysics Data System (ADS)

    Zhu, Feng; Akagi, Jin; Hall, Chris J.; Crosier, Kathryn E.; Crosier, Philip S.; Delaage, Pierre; Wlodkowic, Donald

    2013-12-01

    Drug discovery screenings performed on zebrafish embryos mirror with a high level of accuracy. The tests usually performed on mammalian animal models, and the fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, conventional methods utilising 96-well microtiter plates and manual dispensing of fish embryos are very time-consuming. They rely on laborious and iterative manual pipetting that is a main source of analytical errors and low throughput. In this work, we present development of a miniaturised and high-throughput Lab-on-a-Chip (LOC) platform for automation of FET assays. The 3D high-density LOC array was fabricated in poly-methyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining while the off-chip interfaces were fabricated using additive manufacturing processes (FDM and SLA). The system's design facilitates rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It has been conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. We also present proof-of-concept interfacing with a high-speed imaging cytometer Plate RUNNER HD® capable of multispectral image acquisition with resolution of up to 8192 x 8192 pixels and depth of field of about 40 μm. Furthermore, we developed a miniaturized and self-contained analytical device interfaced with a miniaturized USB microscope. This system modification is capable of performing rapid imaging of multiple embryos at a low resolution for drug toxicity analysis.

  9. A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine

    PubMed Central

    Tian, H; Ip, L; Luo, H; Chang, D C; Luo, K Q

    2006-01-01

    Background and purpose: We report the development of a very efficient cell-based high throughput screening (HTS) method, which utilizes a novel bio-sensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique. Experimental approach: We generated a stable HeLa cell line expressing a FRET-based bio-sensor protein. When cells undergo apoptosis, they activate a protease called ‘caspase-3'. Activation of this enzyme will cleave our sensor protein and cause its fluorescence emission to shift from a wavelength of 535 nm (green) to 486 nm (blue). A decrease in the green/blue emission ratio thus gives a direct indication of apoptosis. The sensor cells are grown in 96-well plates. After addition of different chemical compounds to each well, a fluorescence profile can be measured at various time-points using a fluorescent plate reader. Compounds that can trigger apoptosis are potential candidates as anti-cancer drugs. Key results: This novel cell-based HTS method is highly effective in identifying anti-cancer compounds. It was very sensitive in detecting apoptosis induced by various known anti-cancer drugs. Further, this system detects apoptosis, but not necrosis, and is thus more useful than the conventional cell viability assays, such as those using MTT. Finally, we used this system to screen compounds, isolated from two plants used in Chinese medicine, and identified several effective compounds for inducing apoptosis. Conclusions and Implications: This FRET-based HTS method is a powerful tool for identifying anti-cancer compounds and can serve as a highly efficient platform for drug discovery. PMID:17179946

  10. Novel Arenavirus Entry Inhibitors Discovered by Using a Minigenome Rescue System for High-Throughput Drug Screening

    PubMed Central

    Rathbun, Jessica Y.; Droniou, Magali E.; Damoiseaux, Robert; Haworth, Kevin G.; Henley, Jill E.; Exline, Colin M.; Choe, Hyeryun

    2015-01-01

    ABSTRACT Certain members of the Arenaviridae family are category A agents capable of causing severe hemorrhagic fevers in humans. Specific antiviral treatments do not exist, and the only commonly used drug, ribavirin, has limited efficacy and can cause severe side effects. The discovery and development of new antivirals are inhibited by the biohazardous nature of the viruses, making them a relatively poorly understood group of human pathogens. We therefore adapted a reverse-genetics minigenome (MG) rescue system based on Junin virus, the causative agent of Argentine hemorrhagic fever, for high-throughput screening (HTS). The MG rescue system recapitulates all stages of the virus life cycle and enables screening of small-molecule libraries under biosafety containment level 2 (BSL2) conditions. The HTS resulted in the identification of four candidate compounds with potent activity against a broad panel of arenaviruses, three of which were completely novel. The target for all 4 compounds was the stage of viral entry, which positions the compounds as potentially important leads for future development. IMPORTANCE The arenavirus family includes several members that are highly pathogenic, causing acute viral hemorrhagic fevers with high mortality rates. No specific effective treatments exist, and although a vaccine is available for Junin virus, the causative agent of Argentine hemorrhagic fever, it is licensed for use only in areas where Argentine hemorrhagic fever is endemic. For these reasons, it is important to identify specific compounds that could be developed as antivirals against these deadly viruses. PMID:26041296

  11. An efficient high-throughput screening method for MYST family acetyltransferases, a new class of epigenetic drug targets.

    PubMed

    Falk, Hendrik; Connor, Theresa; Yang, Hong; Loft, Karen J; Alcindor, Joanne L; Nikolakopoulos, George; Surjadi, Regina N; Bentley, John D; Hattarki, Meghan K; Dolezal, Olan; Murphy, James M; Monahan, Brendon J; Peat, Thomas S; Thomas, Tim; Baell, Jonathan B; Parisot, John P; Street, Ian P

    2011-12-01

    Epigenetic aberrations are increasingly regarded as key factors in cancer progression. Recently, deregulation of histone acetyltransferases (HATs) has been linked to several types of cancer. Monocytic leukemia zinc finger protein (MOZ) is a member of the MYST family of HATs, which regulate gene expression in cell proliferation and differentiation. Deregulation of these processes through constitutively active MOZ fusion proteins gives rise to the formation of leukemic stem cells, rendering MOZ an excellent target for treating myeloid leukemia. The authors implemented a hit discovery campaign to identify small-molecule inhibitors of MOZ-HAT activity. They developed a robust, homogeneous assay measuring the acetylation of synthetic histone peptides. In a primary screening campaign testing 243 000 lead-like compounds, they identified inhibitors from several chemical classes. Secondary assays were used to eliminate assay-interfering compounds and prioritize confirmed hits. This study establishes a new high-throughput assay for HAT activity and could provide the foundation for the development of a new class of drugs for the treatment of leukemias. PMID:22086725

  12. Limitations in a frataxin knockdown cell model for Friedreich ataxia in a high-throughput drug screen

    PubMed Central

    Calmels, Nadège; Seznec, Hervé; Villa, Pascal; Reutenauer, Laurence; Hibert, Marcel; Haiech, Jacques; Rustin, Pierre; Koenig, Michel; Puccio, Hélène

    2009-01-01

    Background Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay. Methods Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA. Results The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model. Conclusion Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future. PMID

  13. Coartemether (artemether and lumefantrine): an oral antimalarial drug.

    PubMed

    Wernsdorfer, Walther H

    2004-04-01

    Coartemether (Riamet, Coartem, Novartis), a tablet formulation of artemether and lumefantrine, is a well-tolerated, fast-acting and effective blood schizontocidal drug that serves primarily in the treatment of uncomplicated falciparum malaria that is resistant to other antimalarials. Initial clinical-parasitological response relies mainly on the artemether component, while lumefantrine effects radical cure. The absorption of lumefantrine is poor during the fasting state, the normal condition in acutely ill malaria patients, but with return to normal diet it becomes adequate. This highlights the need for an appropriate adjustment of the dose regimen. In the area where Plasmodium falciparum shows the highest degree of multidrug resistance worldwide, the best results (99% cure) were obtained with a six-dose regimen given over 5 days. Extensive cardiological investigations have demonstrated the high cardiac safety of coartemether. PMID:15482185

  14. Modulation of PF10_0355 (MSPDBL2) Alters Plasmodium falciparum Response to Antimalarial Drugs

    PubMed Central

    Van Tyne, Daria; Uboldi, Alessandro D.; Healer, Julie; Cowman, Alan F.

    2013-01-01

    Malaria's ability to rapidly adapt to new drugs has allowed it to remain one of the most devastating infectious diseases of humans. Understanding and tracking the genetic basis of these adaptations are critical to the success of treatment and intervention strategies. The novel antimalarial resistance locus PF10_0355 (Pfmspdbl2) was previously associated with the parasite response to halofantrine, and functional validation confirmed that overexpression of this gene lowered parasite sensitivity to both halofantrine and the structurally related antimalarials mefloquine and lumefantrine, predominantly through copy number variation. Here we further characterize the role of Pfmspdbl2 in mediating the antimalarial drug response of Plasmodium falciparum. Knockout of Pfmspdbl2 increased parasite sensitivity to halofantrine, mefloquine, and lumefantrine but not to unrelated antimalarials, further suggesting that this gene mediates the parasite response to a specific class of antimalarial drugs. A single nucleotide polymorphism encoding a C591S mutation within Pfmspdbl2 had the strongest association with halofantrine sensitivity and showed a high derived allele frequency among Senegalese parasites. Transgenic parasites expressing the ancestral Pfmspdbl2 allele were more sensitive to halofantrine and structurally related antimalarials than were parasites expressing the derived allele, revealing an allele-specific effect on drug sensitivity in the absence of copy number effects. Finally, growth competition experiments showed that under drug pressure, parasites expressing the derived allele of Pfmspdbl2 outcompeted parasites expressing the ancestral allele within a few generations. Together, these experiments demonstrate that modulation of Pfmspdbl2 affects malaria parasite responses to antimalarial drugs. PMID:23587962

  15. Modulation of PF10_0355 (MSPDBL2) alters Plasmodium falciparum response to antimalarial drugs.

    PubMed

    Van Tyne, Daria; Uboldi, Alessandro D; Healer, Julie; Cowman, Alan F; Wirth, Dyann F

    2013-07-01

    Malaria's ability to rapidly adapt to new drugs has allowed it to remain one of the most devastating infectious diseases of humans. Understanding and tracking the genetic basis of these adaptations are critical to the success of treatment and intervention strategies. The novel antimalarial resistance locus PF10_0355 (Pfmspdbl2) was previously associated with the parasite response to halofantrine, and functional validation confirmed that overexpression of this gene lowered parasite sensitivity to both halofantrine and the structurally related antimalarials mefloquine and lumefantrine, predominantly through copy number variation. Here we further characterize the role of Pfmspdbl2 in mediating the antimalarial drug response of Plasmodium falciparum. Knockout of Pfmspdbl2 increased parasite sensitivity to halofantrine, mefloquine, and lumefantrine but not to unrelated antimalarials, further suggesting that this gene mediates the parasite response to a specific class of antimalarial drugs. A single nucleotide polymorphism encoding a C591S mutation within Pfmspdbl2 had the strongest association with halofantrine sensitivity and showed a high derived allele frequency among Senegalese parasites. Transgenic parasites expressing the ancestral Pfmspdbl2 allele were more sensitive to halofantrine and structurally related antimalarials than were parasites expressing the derived allele, revealing an allele-specific effect on drug sensitivity in the absence of copy number effects. Finally, growth competition experiments showed that under drug pressure, parasites expressing the derived allele of Pfmspdbl2 outcompeted parasites expressing the ancestral allele within a few generations. Together, these experiments demonstrate that modulation of Pfmspdbl2 affects malaria parasite responses to antimalarial drugs. PMID:23587962

  16. Antimalarial Drug Resistance: Literature Review and Activities and Findings of the ICEMR Network.

    PubMed

    Cui, Liwang; Mharakurwa, Sungano; Ndiaye, Daouda; Rathod, Pradipsinh K; Rosenthal, Philip J

    2015-09-01

    Antimalarial drugs are key tools for the control and elimination of malaria. Recent decreases in the global malaria burden are likely due, in part, to the deployment of artemisinin-based combination therapies. Therefore, the emergence and potential spread of artemisinin-resistant parasites in southeast Asia and changes in sensitivities to artemisinin partner drugs have raised concerns. In recognition of this urgent threat, the International Centers of Excellence for Malaria Research (ICEMRs) are closely monitoring antimalarial drug efficacy and studying the mechanisms underlying drug resistance. At multiple sentinel sites of the global ICEMR network, research activities include clinical studies to track the efficacies of antimalarial drugs, ex vivo/in vitro assays to measure drug susceptibilities of parasite isolates, and characterization of resistance-mediating parasite polymorphisms. Taken together, these efforts offer an increasingly comprehensive assessment of the efficacies of antimalarial therapies, and enable us to predict the emergence of drug resistance and to guide local antimalarial drug policies. Here we briefly review worldwide antimalarial drug resistance concerns, summarize research activities of the ICEMRs related to drug resistance, and assess the global impacts of the ICEMR programs. PMID:26259943

  17. Antimalarial Drug Resistance: Literature Review and Activities and Findings of the ICEMR Network

    PubMed Central

    Cui, Liwang; Mharakurwa, Sungano; Ndiaye, Daouda; Rathod, Pradipsinh K.; Rosenthal, Philip J.

    2015-01-01

    Antimalarial drugs are key tools for the control and elimination of malaria. Recent decreases in the global malaria burden are likely due, in part, to the deployment of artemisinin-based combination therapies. Therefore, the emergence and potential spread of artemisinin-resistant parasites in southeast Asia and changes in sensitivities to artemisinin partner drugs have raised concerns. In recognition of this urgent threat, the International Centers of Excellence for Malaria Research (ICEMRs) are closely monitoring antimalarial drug efficacy and studying the mechanisms underlying drug resistance. At multiple sentinel sites of the global ICEMR network, research activities include clinical studies to track the efficacies of antimalarial drugs, ex vivo/in vitro assays to measure drug susceptibilities of parasite isolates, and characterization of resistance-mediating parasite polymorphisms. Taken together, these efforts offer an increasingly comprehensive assessment of the efficacies of antimalarial therapies, and enable us to predict the emergence of drug resistance and to guide local antimalarial drug policies. Here we briefly review worldwide antimalarial drug resistance concerns, summarize research activities of the ICEMRs related to drug resistance, and assess the global impacts of the ICEMR programs. PMID:26259943

  18. Assessment of urinary excretion of antimalarial drugs in large-scale chemotherapeutic eradication projects.

    PubMed

    BRUCE-CHWATT, L J

    1959-01-01

    Assessment of the urinary excretion of an antimalarial drug is a useful means of checking the amount of drug administered and the regularity of intake. The author describes the various methods available for the qualitative and quantitative estimation of antimalarial drugs in urine and discusses their relative merits, with special reference to their suitability for use in the field. He points out the difficulties involved in estimating the urinary excretion of antimalarials in large-scale chemotherapeutic eradication projects and stress the importance of simplifying testing techniques as far as possible. PMID:13805135

  19. Superinfection and the evolution of resistance to antimalarial drugs

    PubMed Central

    Klein, Eili Y.; Smith, David L.; Laxminarayan, Ramanan; Levin, Simon

    2012-01-01

    A major issue in the control of malaria is the evolution of drug resistance. Ecological theory has demonstrated that pathogen superinfection and the resulting within-host competition influences the evolution of specific traits. Individuals infected with Plasmodium falciparum are consistently infected by multiple parasites; however, while this probably alters the dynamics of resistance evolution, there are few robust mathematical models examining this issue. We developed a general theory for modelling the evolution of resistance with host superinfection and examine: (i) the effect of transmission intensity on the rate of resistance evolution; (ii) the importance of different biological costs of resistance; and (iii) the best measure of the frequency of resistance. We find that within-host competition retards the ability and slows the rate at which drug-resistant parasites invade, particularly as the transmission rate increases. We also find that biological costs of resistance that reduce transmission are less important than reductions in the duration of drug-resistant infections. Lastly, we find that random sampling of the population for resistant parasites is likely to significantly underestimate the frequency of resistance. Considering superinfection in mathematical models of antimalarial drug resistance may thus be important for generating accurate predictions of interventions to contain resistance. PMID:22787024

  20. A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair

    PubMed Central

    Shahar, Or David; Kalousi, Alkmini; Eini, Lital; Fisher, Benoit; Weiss, Amelie; Darr, Jonatan; Mazina, Olga; Bramson, Shay; Kupiec, Martin; Eden, Amir; Meshorer, Eran; Mazin, Alexander V.; Brino, Laurent; Goldberg, Michal; Soutoglou, Evi

    2014-01-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy. PMID:24682826

  1. Targeting Plasmodium Metabolism to Improve Antimalarial Drug Design.

    PubMed

    Avitia-Domínguez, Claudia; Sierra-Campos, Erick; Betancourt-Conde, Irene; Aguirre-Raudry, Miriam; Vázquez-Raygoza, Alejandra; Luevano-De la Cruz, Artemisa; Favela-Candia, Alejandro; Sarabia-Sanchez, Marie; Ríos-Soto, Lluvia; Méndez-Hernández, Edna; Cisneros-Martínez, Jorge; Palacio-Gastélum, Marcelo Gómez; Valdez-Solana, Mónica; Hernández-Rivera, Jessica; De Lira-Sánchez, Jaime; Campos-Almazán, Mara; Téllez-Valencia, Alfredo

    2016-01-01

    Malaria is one of the main infectious diseases in tropical developing countries and represents high morbidity and mortality rates nowadays. The principal etiological agent P. falciparum is transmitted through the bite of the female Anopheles mosquito. The issue has escalated due to the emergence of resistant strains to most of the antimalarials used for the treatment including Chloroquine, Sulfadoxine-Pyrimethamine, and recently Artemisinin derivatives, which has led to diminished effectiveness and by consequence increased the severity of epidemic outbreaks. Due to the lack of effective compounds to treat these drug-resistant strains, the discovery or development of novel anti-malaria drugs is important. In this context, one strategy has been to find inhibitors of enzymes, which play an important role for parasite survival. Today, promising results have been obtained in this regard, involving the entire P. falciparum metabolism. These inhibitors could serve as leads in the search of a new chemotherapy against malaria. This review focuses on the achievements in recent years with regard to inhibition of enzymes used as targets for drug design against malaria. PMID:26983887

  2. Detecting metabolic activities of bacteria using a simple carbon nanotube device for high-throughput screening of anti-bacterial drugs.

    PubMed

    Huang, Yinxi; Sudibya, Herry Gunadi; Chen, Peng

    2011-06-15

    In this contribution, we demonstrate a simple carbon nanotube device to electrically detect the presence of bacteria (Escherichai coli) with high sensitivity (<100 cfu/mL) and the glucose triggered metabolic activities of bacteria in real-time. As proof-of-concept demonstration, we also show that this nanoelectronic approach can be employed for high-throughput screening of anti-bacterial drugs. PMID:21570819

  3. A concentration gradient generator on a paper-based microfluidic chip coupled with cell culture microarray for high-throughput drug screening.

    PubMed

    Hong, Bo; Xue, Peng; Wu, Yafeng; Bao, Jingnan; Chuah, Yon Jin; Kang, Yuejun

    2016-02-01

    Inspired by the paper platforms for 3-D cell culture, a paper-based microfluidic device containing drug concentration gradient was designed and constructed for investigating cell response to drugs based on high throughput analysis. This drug gradient generator was applied to generate concentration gradients of doxorubicin (DOX) as the model drug. HeLa cells encapsulated in collagen hydrogel were incubated in the device reservoirs to evaluate the cell viability based on the controlled release of DOX spatially. It was demonstrated that drug diffusion through the paper fibers created a gradient of drug concentration, which influenced cell viability. This drug screening platform has a great opportunity to be applied for drug discovery and diagnostic studies with simultaneous and parallel tests of drugs under various gradient concentrations. PMID:26864970

  4. Use and quality of antimalarial drugs in the private sector in Viet Nam.

    PubMed

    Cong, L D; Yen, P T; Nhu, T V; Binh, L N

    1998-01-01

    This study examines the use and quality of antimalarial drugs in the growing private sector of Viet Nam. The practices of drug vendors (called alternative treatment providers (ATPs)) as well as their stocks and the quality of drugs sold by them, and the local production and distribution of antimalarials were investigated. Antimalarials were sold by the vast majority of ATPs, almost all the common antimalarials being available for sale. The practices and indications for sale, however, varied. Underdosing for malaria was frequent in all three provinces studied, and lack of knowledge of the appropriate regimen for cure was common among the drug-sellers. Samples of antimalarials were collected from ATP outlets in the three provinces, and the drugs were assessed for their contents and expiry date by the Institute of Drug Quality Control in Hanoi. Of the 218 samples of drugs examined by the Institute, over 96% met the quality requirements. However, a 10% sample of these drugs were independently assessed by WHO and revealed a different picture: 70% of them failed to meet the standard specifications required. There is therefore an urgent need to improve the capability and monitoring procedures of bodies involved in assessing and regulating drugs in Viet Nam. PMID:9763723

  5. High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.

    PubMed

    Mathews Griner, Lesley A; Guha, Rajarshi; Shinn, Paul; Young, Ryan M; Keller, Jonathan M; Liu, Dongbo; Goldlust, Ian S; Yasgar, Adam; McKnight, Crystal; Boxer, Matthew B; Duveau, Damien Y; Jiang, Jian-Kang; Michael, Sam; Mierzwa, Tim; Huang, Wenwei; Walsh, Martin J; Mott, Bryan T; Patel, Paresma; Leister, William; Maloney, David J; Leclair, Christopher A; Rai, Ganesha; Jadhav, Ajit; Peyser, Brian D; Austin, Christopher P; Martin, Scott E; Simeonov, Anton; Ferrer, Marc; Staudt, Louis M; Thomas, Craig J

    2014-02-11

    The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL. PMID:24469833

  6. Targeting protein kinases in the malaria parasite: update of an antimalarial drug target.

    PubMed

    Zhang, Veronica M; Chavchich, Marina; Waters, Norman C

    2012-01-01

    Millions of deaths each year are attributed to malaria worldwide. Transmitted through the bite of an Anopheles mosquito, infection and subsequent death from the Plasmodium species, most notably P. falciparum, can readily spread through a susceptible population. A malaria vaccine does not exist and resistance to virtually every antimalarial drug predicts that mortality and morbidity associated with this disease will increase. With only a few antimalarial drugs currently in the pipeline, new therapeutic options and novel chemotypes are desperately needed. Hit-to-Lead diversity may successfully provide novel inhibitory scaffolds when essential enzymes are targeted, for example, the plasmodial protein kinases. Throughout the entire life cycle of the malaria parasite, protein kinases are essential for growth and development. Ongoing efforts continue to characterize these kinases, while simultaneously pursuing them as antimalarial drug targets. A collection of structural data, inhibitory profiles and target validation has set the foundation and support for targeting the malarial kinome. Pursuing protein kinases as cancer drug targets has generated a wealth of information on the inhibitory strategies that can be useful for antimalarial drug discovery. In this review, progress on selected protein kinases is described. As the search for novel antimalarials continues, an understanding of the phosphor-regulatory pathways will not only validate protein kinase targets, but also will identify novel chemotypes to thwart malaria drug resistance. PMID:22242850

  7. High-throughput transfection and engineering of primary cells and cultured cell lines - an invaluable tool for research as well as drug development.

    PubMed

    Müller-Hartmann, Herbert; Faust, Nicole; Kazinski, Michael; Kretzschmar, Titus

    2007-11-01

    The manipulation of eukaryotic cells by introducing nucleic acids and other substrates using chemical, physical or viral methods is one of the ground-breaking tools in the life sciences. Changes in the molecular equipment of a cell induced by introducing different molecules not only enable the dissection of signal transduction and metabolic pathways, but also allow the exploitation of engineered cells as bio-factories for the production of proteins in the processes of target research and drug development. In addition to the application of engineered cells for modern cell-based assays, medically relevant engineered cells can be used in clinical settings for adoptive immunotherapy or gene therapy. With the advent of methods exploiting RNA interference (RNAi), gene identification and functional validation in eukaryotic cells have clearly become one of the most exciting methods in life sciences during the past few years. To accelerate research and development in these areas, high-quality, high-throughput approaches (i.e., using sample formats of at least 96 wells) for cell engineering are needed with increasing demand. Recent developments, especially in the field of electroporation, now allow the efficient, high-throughput engineering of virtually any cell type, including primary cells, many of which were previously considered difficult or even impossible to transfect. Primary cells freshly isolated from native tissues are gaining more and more interest, as data obtained with these cells are considered to be of higher physiological relevance than data obtained with immortalized cell lines that have been cultured for extensive periods. In this review, the various methods for cell engineering (with focus on higher eukaryotic cells) are summarized and their impact for high-throughput applications in research and drug development is discussed. PMID:23484597

  8. Inclusion of gametocyte parameters in anti-malarial drug efficacy studies: filling a neglected gap needed for malaria elimination.

    PubMed

    Abdul-Ghani, Rashad; Basco, Leonardo K; Beier, John C; Mahdy, Mohammed A K

    2015-01-01

    Standard anti-malarial drug efficacy and drug resistance assessments neglect the gametocyte parameters in their protocols. With the spread of drug resistance and the absence of clinically proven vaccines, the use of gametocytocidal drugs or drug combinations with transmission-blocking activity is a high priority for malaria control and elimination. However, the limited repertoire of gametocytocidal drugs and induction of gametocytogenesis after treatment with certain anti-malarial drugs necessitate both regular monitoring of gametocytocidal activities of anti-malarial drugs in clinical use and the effectiveness of candidate gametocytocidal agents. Therefore, updating current protocols of anti-malarial drug efficacy is needed to reflect the effects of anti-malarial drugs or drug combinations on gametocyte carriage and gametocyte density along with asexual parasite density. Developing protocols of anti-malarial drug efficacy that include gametocyte parameters related to both microscopic and submicroscopic gametocytaemias is important if drugs or drug combinations are to be strategically used in transmission-blocking interventions in the context of malaria elimination. The present piece of opinion highlights the challenges in gametocyte detection and follow-up and discuss the need for including the gametocyte parameter in anti-malarial efficacy studies. PMID:26481312

  9. Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.

    PubMed

    Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen

    2016-02-25

    The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s(-1) for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug. PMID:26851087

  10. Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

    PubMed Central

    Zhang, Haili; Zhu, Guan

    2015-01-01

    Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds. PMID:26441920

  11. Retinal toxicity induced by antimalarial drugs: literature review and case report.

    PubMed

    Garza-Leon, Manuel; Flores-Alvarado, Diana Elsa; Muñoz-Bravo, Juan Manuel

    2016-01-01

    Antimalarial drugs are widely used in several countries for control of rheumatologic diseases such as systemic lupus erythematosus and rheumatoid arthritis. They are still used in Mexico because of their low cost and few secondary effects, most of which are mild and reversible. Even so, at an ophthalmological level, they could produce irreversible visual damage, which is why it is important to have ophthalmological evaluation and proper follow up. We present a clinical case as an example of characteristic ophthalmological findings as well as risk factors for retinal toxicity. We then discuss guidelines for diagnosis and follow up of patients who use antimalarial drugs for the treatment of rheumatologic illnesses. PMID:27391903

  12. A High-Throughput Fluorescence-Based Assay System for Appetite-Regulating Gene and Drug Screening

    PubMed Central

    Shimada, Yasuhito; Hirano, Minoru; Nishimura, Yuhei; Tanaka, Toshio

    2012-01-01

    The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish). This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf), knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1), and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant) revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers. PMID:23300705

  13. The University of Kansas High-Throughput Screening laboratory. Part I: meeting drug-discovery needs in the heartland of America with entrepreneurial flair.

    PubMed

    McDonald, Peter R; Roy, Anuradha; Chaguturu, Rathnam

    2011-05-01

    The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core applies pharmaceutical industry project-management principles in an academic setting by bringing together multidisciplinary teams to fill critical scientific and technology gaps, using an experienced team of industry-trained researchers and project managers. The KU HTS proactively engages in supporting grant applications for extramural funding, intellectual-property management and technology transfer. The KU HTS staff further provides educational opportunities for the KU faculty and students to learn cutting-edge technologies in drug-discovery platforms through seminars, workshops, internships and course teaching. This is the first instalment of a two-part contribution from the KU HTS laboratory. PMID:21644824

  14. Rational Design of Proteasome Inhibitors as Antimalarial Drugs.

    PubMed

    Le Chapelain, Camille; Groll, Michael

    2016-05-23

    One life, two strategies: Crucial structural differences between the human and the Plasmodium falciparum proteasomes were recently identified. A combination of cryo-EM and functional characterization enabled the design of a selective antimalarial proteasome inhibitor that shows low toxicity in the host. When used with artemisinin, this ligand offers a new approach for the efficient treatment of malaria at all stages of the parasite lifecycle. PMID:27079849

  15. A new high-throughput method utilizing porous silica-based nano-composites for the determination of partition coefficients of drug candidates.

    PubMed

    Yu, Chih H; Tam, Kin; Tsang, Shik C

    2011-09-01

    We show that highly porous silica-based nanoparticles prepared via micro-emulsion and sol-gel techniques are stable colloids in aqueous solution. By incorporating a magnetic core into the porous silica nano-composite, it is found that the material can be rapidly separated (precipitated) upon exposure to an external magnetic field. Alternatively, the porous silica nanoparticles without magnetic cores can be equally separated from solution by applying a high-speed centrifugation. Using these silica-based nanostructures a new high-throughput method for the determination of partition coefficient for water/n-octanol is hereby described. First, a tiny quantity of n-octanol phase is pre-absorbed in the porous silica nano-composite colloids, which allows an establishment of interface at nano-scale between the adsorbed n-octanol with the bulk aqueous phase. Organic compounds added to the mixture can therefore undergo a rapid partition between the two phases. The concentration of drug compound in the supernatant in a small vial can be determined by UV-visible absorption spectroscopy. With the adaptation of a robotic liquid handler, a high-throughput technology for the determination of partition coefficients of drug candidates can be employed for drug screening in the industry based on these nano-separation skills. The experimental results clearly suggest that this new method can provide partition coefficient values of potential drug candidates comparable to the conventional shake-flask method but requires much shorter analytical time and lesser quantity of chemicals. PMID:21780284

  16. Model for high-throughput screening of drug immunotoxicity--study of the anti-microbial G1 over peritoneal macrophages using flow cytometry.

    PubMed

    Tenorio-Borroto, Esvieta; Peñuelas-Rivas, Claudia G; Vásquez-Chagoyán, Juan C; Castañedo, Nilo; Prado-Prado, Francisco J; García-Mera, Xerardo; González-Díaz, Humberto

    2014-01-24

    Quantitative Structure-Activity (mt-QSAR) techniques may become an important tool for prediction of cytotoxicity and High-throughput Screening (HTS) of drugs to rationalize drug discovery process. In this work, we train and validate by the first time mt-QSAR model using TOPS-MODE approach to calculate drug molecular descriptors and Linear Discriminant Analysis (LDA) function. This model correctly classifies 8258 out of 9000 (Accuracy = 91.76%) multiplexing assay endpoints of 7903 drugs (including both train and validation series). Each endpoint correspond to one out of 1418 assays, 36 molecular and cellular targets, 46 standard type measures, in two possible organisms (human and mouse). After that, we determined experimentally, by the first time, the values of EC50 = 21.58 μg/mL and Cytotoxicity = 23.6% for the anti-microbial/anti-parasite drug G1 over Balb/C mouse peritoneal macrophages using flow cytometry. In addition, the model predicts for G1 only 7 positive endpoints out 1251 cytotoxicity assays (0.56% of probability of cytotoxicity in multiple assays). The results obtained complement the toxicological studies of this important drug. This work adds a new tool to the existing pool of few methods useful for multi-target HTS of ChEMBL and other libraries of compounds towards drug discovery. PMID:24445280

  17. Development of antimalarial drugs and their application in China: a historical review

    PubMed Central

    2014-01-01

    This historical review covers antimalarials developed in China, which include artemisinin, artemether, artesunate, and dihydroartemisinin, as well as other synthetic drugs such as piperaquine, pyronaridine, benflumetol (lumefantrine), and naphthoquine. The curative effects of these antimalarials in the treatment of falciparum malaria, including chloroquine-resistant strain, are especially discussed. Following the World Health Organization (WHO) recommended artemisinin-based combination therapy (ACT), different combinations of artemisinin, or its derivative, along with another antimalarial drug were orally used to treat Plasmodium falciparum infections. The recrudescence rates were low, gametocyte carriers lessened, and the curative rate increased remarkably. The combination therapy effectively deferred the emergence of drug resistance in the parasite. The regulation “The guidelines and regimens for the use of antimalarial drugs in China” was issued to guide rational application and standardize malaria treatment in the country. As the recommended first-line drug to treat falciparum malaria in the world, ACT was adopted in the regulation. In response to the global initiative of malaria eradication proposed by the UN Millennium Development Goals (MDGs), the Chinese government has set a target to eliminate malaria by 2020. PMID:24650735

  18. High-throughput screening of FDA-approved drugs using oxygen biosensor plates reveals secondary mitofunctional effects

    PubMed Central

    Sahdeo, Sunil; Tomilov, Alexey; Komachi, Kelly; Iwahashi, Christine; Datta, Sandipan; Hughes, Owen; Hagerman, Paul; Cortopassi, Gino

    2014-01-01

    Repurposing of FDA-approved drugs with effects on mitochondrial function might shorten the critical path to mitochondrial disease drug development. We improved a biosensor-based assay of mitochondrial O2 consumption, and identified mitofunctional defects in cell models of LHON and FXTAS. Using this platform, we screened a 1600-compound library of clinically used drugs. The assay identified drugs known to affect mitochondrial function, such as metformin and decoquinate. We also identified several drugs not previously known to affect mitochondrial respiration including acarbose, metaraminol, gallamine triethiodide, and acamprosate. These previously unknown ‘mitoactives’ represent novel links to targets for mitochondrial regulation and potentially therapy, for mitochondrial disease. PMID:25034306

  19. Microfluidic System Based High Throughput Drug Screening System for Curcumin/TRAIL Combinational Chemotherapy in Human Prostate Cancer PC3 Cells

    PubMed Central

    An, Dami; Kim, Kwangmi; Kim, Jeongyun

    2014-01-01

    We have developed a fully automated high throughput drug screening (HTDS) system based on the microfluidic cell culture array to perform combinational chemotherapy. This system has 64 individually addressable cell culture chambers where the sequential combinatorial concentrations of two different drugs can be generated by two microfluidic diffusive mixers. Each diffusive mixer has two integrated micropumps connected to the media and the drug reservoirs respectively for generating the desired combination without the need for any extra equipment to perfuse the solution such as syringe pumps. The cell array is periodically exposed to the drug combination with the programmed LabVIEW system during a couple of days without extra handling after seeding the cells into the microfluidic device and also, this device does not require the continuous generation of solutions compared to the previous systems. Therefore, the total amount of drug being consumed per experiment is less than a few hundred micro liters in each reservoir. The utility of this system is demonstrated through investigating the viability of the prostate cancer PC3 cell line with the combinational treatments of curcumin and tumor necrosis factor-alpha related apoptosis inducing ligand (TRAIL). Our results suggest that the system can be used for screening and optimizing drug combination with a small amount of reagent for combinatorial chemotherapy against cancer cells. PMID:25143816

  20. Peroxide bond strength of antimalarial drugs containing an endoperoxide cycle. Relation with biological activity.

    PubMed

    Fernández, Israel; Robert, Anne

    2011-06-01

    Several endoperoxide compounds are very efficient antimalarial analogues of the natural drug artemisinin. Quantum chemical calculations have been used to correlate the computed free energies of the O-O bond with respect to the total number of oxygen atoms contained in the cycle, and with the size/strain of the cycle (5- or 6-membered cycles). The gas-phase homolysis of the O-O bond has been studied for five- and six-membered oxygenated cycles which are models of the "real" drugs. Our results indicate that, in 6-membered cycles, the stability order is the following: 1,2-dioxane > 1,2,4-trioxane > 1,2,4,5-tetraoxane. In cycles containing 3 oxygen atoms, the 5-membered cycle 1,2,4-trioxolane was found much less stable than its 6-membered counterpart 1,2,4-trioxane. This feature indicates the possible role of the cycle strain for the O-O bond stability, and may also explain the high antimalarial activity of some trioxolane derivatives. Similar trends in the O-O bond strength have been found for the real antimalarial drugs. However, the O-O bond stability is not in itself a decisive argument to anticipate the antimalarial activity of drugs. PMID:21487624

  1. Synthesis of the antimalarial drug FR900098 utilizing the nitroso-ene reaction.

    PubMed

    Fokin, Andrey A; Yurchenko, Alexander G; Rodionov, Vladimir N; Gunchenko, Pavel A; Yurchenko, Raisa I; Reichenberg, Armin; Wiesner, Jochen; Hintz, Martin; Jomaa, Hassan; Schreiner, Peter R

    2007-10-11

    The antimalarial drug FR900098 was prepared from diethyl allylphosphonate involving the nitroso-ene reaction with nitrosocarbonyl methane as the key step followed by hydrogenation and dealkylation. The utilization of dibenzyl allylphosphonate as the starting compound allows one-step hydrogenation with dealkylation, which simplifies the preparative scheme further. PMID:17887769

  2. Generation of SNCA Cell Models Using Zinc Finger Nuclease (ZFN) Technology for Efficient High-Throughput Drug Screening

    PubMed Central

    Dansithong, Warunee; Paul, Sharan; Scoles, Daniel R.; Pulst, Stefan M.; Huynh, Duong P.

    2015-01-01

    Parkinson’s disease (PD) is a progressive neurodegenerative disorder caused by loss of dopaminergic neurons of the substantia nigra. The hallmark of PD is the appearance of neuronal protein aggregations known as Lewy bodies and Lewy neurites, of which α-synuclein forms a major component. Familial PD is rare and is associated with missense mutations of the SNCA gene or increases in gene copy number resulting in SNCA overexpression. This suggests that lowering SNCA expression could be therapeutic for PD. Supporting this hypothesis, SNCA reduction was neuroprotective in cell line and rodent PD models. We developed novel cell lines expressing SNCA fused to the reporter genes luciferase (luc) or GFP with the objective to enable high-throughput compound screening (HTS) for small molecules that can lower SNCA expression. Because SNCA expression is likely regulated by far-upstream elements (including the NACP-REP1 located at 8852 bp upstream of the transcription site), we employed zinc finger nuclease (ZFN) genome editing to insert reporter genes in-frame downstream of the SNCA gene in order to retain native SNCA expression control. This ensured full retention of known and unknown up- and downstream genetic elements controlling SNCA expression. Treatment of cells with the histone deacetylase inhibitor valproic acid (VPA) resulted in significantly increased SNCA-luc and SNCA-GFP expression supporting the use of our cell lines for identifying small molecules altering complex modes of expression control. Cells expressing SNCA-luc treated with a luciferase inhibitor or SNCA siRNA resulted in Z’-scores ≥ 0.75, suggesting the suitability of these cell lines for use in HTS. This study presents a novel use of genome editing for the creation of cell lines expressing α-synuclein fusion constructs entirely under native expression control. These cell lines are well suited for HTS for compounds that lower SNCA expression directly or by acting at long-range sites to the SNCA

  3. Serine Proteases of Malaria Parasite Plasmodium falciparum: Potential as Antimalarial Drug Targets

    PubMed Central

    2014-01-01

    Malaria is a major global parasitic disease and a cause of enormous mortality and morbidity. Widespread drug resistance against currently available antimalarials warrants the identification of novel drug targets and development of new drugs. Malarial proteases are a group of molecules that serve as potential drug targets because of their essentiality for parasite life cycle stages and feasibility of designing specific inhibitors against them. Proteases belonging to various mechanistic classes are found in P. falciparum, of which serine proteases are of particular interest due to their involvement in parasite-specific processes of egress and invasion. In P. falciparum, a number of serine proteases belonging to chymotrypsin, subtilisin, and rhomboid clans are found. This review focuses on the potential of P. falciparum serine proteases as antimalarial drug targets. PMID:24799897

  4. Critical analysis of 3-D organoid in vitro cell culture models for high-throughput drug candidate toxicity assessments.

    PubMed

    Astashkina, Anna; Grainger, David W

    2014-04-01

    Drug failure due to toxicity indicators remains among the primary reasons for staggering drug attrition rates during clinical studies and post-marketing surveillance. Broader validation and use of next-generation 3-D improved cell culture models are expected to improve predictive power and effectiveness of drug toxicological predictions. However, after decades of promising research significant gaps remain in our collective ability to extract quality human toxicity information from in vitro data using 3-D cell and tissue models. Issues, challenges and future directions for the field to improve drug assay predictive power and reliability of 3-D models are reviewed. PMID:24613390

  5. The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology

    PubMed Central

    McDonald, Peter R; Roy, Anuradha; Chaguturu, Rathnam

    2011-01-01

    The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory. PMID:21806374

  6. The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology.

    PubMed

    McDonald, Peter R; Roy, Anuradha; Chaguturu, Rathnam

    2011-07-01

    The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory. PMID:21806374

  7. Amphiphilic dendritic derivatives as nanocarriers for the targeted delivery of antimalarial drugs.

    PubMed

    Movellan, Julie; Urbán, Patricia; Moles, Ernest; de la Fuente, Jesús M; Sierra, Teresa; Serrano, José Luis; Fernàndez-Busquets, Xavier

    2014-09-01

    It can be foreseen that in a future scenario of malaria eradication, a varied armamentarium will be required, including strategies for the targeted administration of antimalarial compounds. The development of nanovectors capable of encapsulating drugs and of delivering them to Plasmodium-infected cells with high specificity and efficacy and at an affordable cost is of particular interest. With this objective, dendritic derivatives based on 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) and Pluronic(®) polymers have been herein explored. Four different dendritic derivatives have been tested for their capacity to encapsulate the antimalarial drugs chloroquine (CQ) and primaquine (PQ), their specific targeting to Plasmodium-infected red blood cells (pRBCs), and their antimalarial activity in vitro against the human pathogen Plasmodium falciparum and in vivo against the rodent malaria species Plasmodium yoelii. The results obtained have allowed the identification of two dendritic derivatives exhibiting specific targeting to pRBCs vs. non-infected RBCs, which reduce the in vitro IC50 of CQ and PQ by ca. 3- and 4-fold down to 4.0 nm and 1.1 μm, respectively. This work on the application of dendritic derivatives to antimalarial targeted drug delivery opens the way for the use of this new type of chemicals in future malaria eradication programs. PMID:24930847

  8. Discovery of New Compounds Active against Plasmodium falciparum by High Throughput Screening of Microbial Natural Products

    PubMed Central

    Pérez-Moreno, Guiomar; Cantizani, Juan; Sánchez-Carrasco, Paula; Ruiz-Pérez, Luis Miguel; Martín, Jesús; el Aouad, Noureddine; Pérez-Victoria, Ignacio; Tormo, José Rubén; González-Menendez, Víctor; González, Ignacio; de Pedro, Nuria; Reyes, Fernando; Genilloud, Olga; Vicente, Francisca; González-Pacanowska, Dolores

    2016-01-01

    Due to the low structural diversity within the set of antimalarial drugs currently available in the clinic and the increasing number of cases of resistance, there is an urgent need to find new compounds with novel modes of action to treat the disease. Microbial natural products are characterized by their large diversity provided in terms of the chemical complexity of the compounds and the novelty of structures. Microbial natural products extracts have been underexplored in the search for new antiparasitic drugs and even more so in the discovery of new antimalarials. Our objective was to find new druggable natural products with antimalarial properties from the MEDINA natural products collection, one of the largest natural product libraries harboring more than 130,000 microbial extracts. In this work, we describe the optimization process and the results of a phenotypic high throughput screen (HTS) based on measurements of Plasmodium lactate dehydrogenase. A subset of more than 20,000 extracts from the MEDINA microbial products collection has been explored, leading to the discovery of 3 new compounds with antimalarial activity. In addition, we report on the novel antiplasmodial activity of 4 previously described natural products. PMID:26735308

  9. Discovery of New Compounds Active against Plasmodium falciparum by High Throughput Screening of Microbial Natural Products.

    PubMed

    Pérez-Moreno, Guiomar; Cantizani, Juan; Sánchez-Carrasco, Paula; Ruiz-Pérez, Luis Miguel; Martín, Jesús; El Aouad, Noureddine; Pérez-Victoria, Ignacio; Tormo, José Rubén; González-Menendez, Víctor; González, Ignacio; de Pedro, Nuria; Reyes, Fernando; Genilloud, Olga; Vicente, Francisca; González-Pacanowska, Dolores

    2016-01-01

    Due to the low structural diversity within the set of antimalarial drugs currently available in the clinic and the increasing number of cases of resistance, there is an urgent need to find new compounds with novel modes of action to treat the disease. Microbial natural products are characterized by their large diversity provided in terms of the chemical complexity of the compounds and the novelty of structures. Microbial natural products extracts have been underexplored in the search for new antiparasitic drugs and even more so in the discovery of new antimalarials. Our objective was to find new druggable natural products with antimalarial properties from the MEDINA natural products collection, one of the largest natural product libraries harboring more than 130,000 microbial extracts. In this work, we describe the optimization process and the results of a phenotypic high throughput screen (HTS) based on measurements of Plasmodium lactate dehydrogenase. A subset of more than 20,000 extracts from the MEDINA microbial products collection has been explored, leading to the discovery of 3 new compounds with antimalarial activity. In addition, we report on the novel antiplasmodial activity of 4 previously described natural products. PMID:26735308

  10. Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania.

    PubMed

    Gazanion, Élodie; Fernández-Prada, Christopher; Papadopoulou, Barbara; Leprohon, Philippe; Ouellette, Marc

    2016-05-24

    Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms. PMID:27162331

  11. Irrational use of antimalarial drugs in rural areas of eastern Pakistan: a random field study

    PubMed Central

    2012-01-01

    Background Prescription of antimalarial drugs in the absence of malarial disease is a common practice in countries where malaria is endemic. However, unwarranted use of such drugs can cause side effects in some people and is a financial drain on local economies. In this study, we surveyed the prevalence of malaria parasites in humans, and the prevalence of the malaria transmitting mosquito vectors in the study area. We also investigated the use of antimalarial drugs in the local people. We focused on randomly selected rural areas of eastern Pakistan where no malaria cases had been reported since May 2004. Methods Mass blood surveys, active case detection, passive case detection, and vector density surveys were carried out in selected areas of Sargodha district from September 2008 to August 2009. Data pertaining to the quantities and types of antimalarial drugs used in these areas were collected from health centers, pharmacies, and the district CDC program of the Health Department of the Government of the Punjab. Results Seven hundred and forty four blood samples were examined, resulting in a Blood Examination Rate (BER) of 3.18; microscopic analysis of blood smears showed that none of the samples were positive for malaria parasites. Investigation of the mosquito vector density in 43 living rooms (bedrooms or rooms used for sleeping), 23 stores, and 32 animal sheds, revealed no vectors capable of transmitting malaria in these locations. In contrast, the density of Culex mosquitoes was high. Substantial consumption of a variety of antimalarial tablets, syrups, capsules and injections costing around 1000 US$, was documented for the region. Conclusion Use of antimalarial drugs in the absence of malarial infection or the vectors that transmit the disease was common in the study area. Continuous use of such drugs, not only in Pakistan, but in other parts of the world, may lead to drug-induced side effects amongst users. Better training of health care professionals is

  12. Metabolic Dysregulation Induced in Plasmodium falciparum by Dihydroartemisinin and Other Front-Line Antimalarial Drugs.

    PubMed

    Cobbold, Simon A; Chua, Hwa H; Nijagal, Brunda; Creek, Darren J; Ralph, Stuart A; McConville, Malcolm J

    2016-01-15

    Detailed information on the mode of action of antimalarial drugs can be used to improve existing drugs, identify new drug targets, and understand the basis of drug resistance. In this study we describe the use of a time-resolved, mass spectrometry (MS)-based metabolite profiling approach to map the metabolic perturbations induced by a panel of clinical antimalarial drugs and inhibitors on Plasmodium falciparum asexual blood stages. Drug-induced changes in metabolite levels in P. falciparum-infected erythrocytes were monitored over time using gas chromatography-MS and liquid chromatography-MS and changes in specific metabolic fluxes confirmed by nonstationary [(13)C]-glucose labeling. Dihydroartemisinin (DHA) was found to disrupt hemoglobin catabolism within 1 hour of exposure, resulting in a transient decrease in hemoglobin-derived peptides. Unexpectedly, it also disrupted pyrimidine biosynthesis, resulting in increased [(13)C]-glucose flux toward malate production, potentially explaining the susceptibility of P. falciparum to DHA during early blood-stage development. Unique metabolic signatures were also found for atovaquone, chloroquine, proguanil, cycloguanil and methylene blue. We also show that this approach can be used to identify the mode of action of novel antimalarials, such as the compound Torin 2, which inhibits hemoglobin catabolism. PMID:26150544

  13. Lead Clinical and Preclinical Antimalarial Drugs Can Significantly Reduce Sporozoite Transmission to Vertebrate Populations

    PubMed Central

    Upton, L. M.; Brock, P. M.; Churcher, T. S.; Ghani, A. C.; Gething, P. W.; Delves, M. J.; Sala, K. A.; Leroy, D.; Sinden, R. E.

    2014-01-01

    To achieve malarial elimination, we must employ interventions that reduce the exposure of human populations to infectious mosquitoes. To this end, numerous antimalarial drugs are under assessment in a variety of transmission-blocking assays which fail to measure the single crucial criteria of a successful intervention, namely impact on case incidence within a vertebrate population (reduction in reproductive number/effect size). Consequently, any reduction in new infections due to drug treatment (and how this may be influenced by differing transmission settings) is not currently examined, limiting the translation of any findings. We describe the use of a laboratory population model to assess how individual antimalarial drugs can impact the number of secondary Plasmodium berghei infections over a cycle of transmission. We examine the impact of multiple clinical and preclinical drugs on both insect and vertebrate populations at multiple transmission settings. Both primaquine (>6 mg/kg of body weight) and NITD609 (8.1 mg/kg) have significant impacts across multiple transmission settings, but artemether and lumefantrine (57 and 11.8 mg/kg), OZ439 (6.5 mg/kg), and primaquine (<1.25 mg/kg) demonstrated potent efficacy only at lower-transmission settings. While directly demonstrating the impact of antimalarial drug treatment on vertebrate populations, we additionally calculate effect size for each treatment, allowing for head-to-head comparison of the potential impact of individual drugs within epidemiologically relevant settings, supporting their usage within elimination campaigns. PMID:25385107

  14. Analysis of antimicrobial resistance mechanisms in multi-drug resistant (MDR) Salmonella enterica by high-throughput DNA sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Multi drug resistant (MDR) Salmonella enterica is found in food animals and may consequently pose a risk to humans through food borne transmission. To understand the mechanisms that drive this problem, the genetic elements associated with MDR need to be determined. These MDR elements in ...

  15. High-Throughput Screening for Identification of Blood-Brain Barrier Integrity Enhancers: A Drug Repurposing Opportunity to Rectify Vascular Amyloid Toxicity.

    PubMed

    Qosa, Hisham; Mohamed, Loqman A; Al Rihani, Sweilem B; Batarseh, Yazan S; Duong, Quoc-Viet; Keller, Jeffrey N; Kaddoumi, Amal

    2016-07-01

    The blood-brain barrier (BBB) is a dynamic interface that maintains brain homeostasis and protects it from free entry of chemicals, toxins, and drugs. The barrier function of the BBB is maintained mainly by capillary endothelial cells that physically separate brain from blood. Several neurological diseases, such as Alzheimer's disease (AD), are known to disrupt BBB integrity. In this study, a high-throughput screening (HTS) was developed to identify drugs that rectify/protect BBB integrity from vascular amyloid toxicity associated with AD progression. Assessing Lucifer Yellow permeation across in-vitro BBB model composed from mouse brain endothelial cells (bEnd3) grown on 96-well plate inserts was used to screen 1280 compounds of Sigma LOPAC®1280 library for modulators of bEnd3 monolayer integrity. HTS identified 62 compounds as disruptors, and 50 compounds as enhancers of the endothelial barrier integrity. From these 50 enhancers, 7 FDA approved drugs were identified with EC50 values ranging from 0.76-4.56 μM. Of these 7 drugs, 5 were able to protect bEnd3-based BBB model integrity against amyloid toxicity. Furthermore, to test the translational potential to humans, the 7 drugs were tested for their ability to rectify the disruptive effect of Aβ in the human endothelial cell line hCMEC/D3. Only 3 (etodolac, granisetron, and beclomethasone) out of the 5 effective drugs in the bEnd3-based BBB model demonstrated a promising effect to protect the hCMEC/D3-based BBB model integrity. These drugs are compelling candidates for repurposing as therapeutic agents that could rectify dysfunctional BBB associated with AD. PMID:27392852

  16. Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine.

    PubMed

    Olafson, Katy N; Ketchum, Megan A; Rimer, Jeffrey D; Vekilov, Peter G

    2015-04-21

    Hematin crystallization is the primary mechanism of heme detoxification in malaria parasites and the target of the quinoline class of antimalarials. Despite numerous studies of malaria pathophysiology, fundamental questions regarding hematin growth and inhibition remain. Among them are the identity of the crystallization medium in vivo, aqueous or organic; the mechanism of crystallization, classical or nonclassical; and whether quinoline antimalarials inhibit crystallization by sequestering hematin in the solution, or by blocking surface sites crucial for growth. Here we use time-resolved in situ atomic force microscopy (AFM) and show that the lipid subphase in the parasite may be a preferred growth medium. We provide, to our knowledge, the first evidence of the molecular mechanisms of hematin crystallization and inhibition by chloroquine, a common quinoline antimalarial drug. AFM observations demonstrate that crystallization strictly follows a classical mechanism wherein new crystal layers are generated by 2D nucleation and grow by the attachment of solute molecules. We identify four classes of surface sites available for binding of potential drugs and propose respective mechanisms of drug action. Further studies reveal that chloroquine inhibits hematin crystallization by binding to molecularly flat {100} surfaces. A 2-μM concentration of chloroquine fully arrests layer generation and step advancement, which is ∼10(4)× less than hematin's physiological concentration. Our results suggest that adsorption at specific growth sites may be a general mode of hemozoin growth inhibition for the quinoline antimalarials. Because the atomic structures of the identified sites are known, this insight could advance the future design and/or optimization of new antimalarials. PMID:25831526

  17. A Model of Excitotoxic Brain Injury in Larval Zebrafish: Potential Application for High-Throughput Drug Evaluation to Treat Traumatic Brain Injury.

    PubMed

    McCutcheon, Victoria; Park, Eugene; Liu, Elaine; Wang, Youdong; Wen, Xiao-Yan; Baker, Andrew J

    2016-06-01

    Traumatic brain injury (TBI) is a leading cause of death and morbidity with no effective therapeutic treatments for secondary injury. Preclinical drug evaluation in rodent models of TBI is a lengthy process. In this regard, the zebrafish has numerous advantages to address the technical and time-dependent obstacles associated with drug evaluation. We developed a reproducible brain injury using glutamate excitoxicity in zebrafish larvae, a known initiator of delayed cell death in TBI. Glutamate challenge resulted in dose-dependent lethality over an 84-h observation period. We report significant decrease in locomotion (p < 0.0001) and mean velocity (p < 0.001) with 10 μM glutamate application as measured through automated 96-well plate behavioral analysis. Application of the NMDA receptor antagonist MK-801 (400 nM) or the calpain inhibitor, MDL-28170 (20 μM), resulted in significant recovery of locomotor function. A secA5-YFP transgenic line was used to visualize the localization of cell death due to glutamate exposure in vivo using confocal fluorescence microscopy. Our results indicate that zebrafish larvae exhibit responses to excitotoxic injury and pharmacotherapeutic intervention with pathophysiological relevance to mammalian excitotoxic brain injury. This system has potential to be applied as a high-throughput drug screening model to quickly identify candidate lead compounds for further evaluation. PMID:27028704

  18. Impact of prepackaging antimalarial drugs on cost to patients and compliance with treatment.

    PubMed Central

    Yeboah-Antwi, K.; Gyapong, J. O.; Asare, I. K.; Barnish, G.; Evans, D. B.; Adjei, S.

    2001-01-01

    OBJECTIVE: To examine the extent to which district health teams could reduce the burden of malaria, a continuing major cause of mortality and morbidity, in a situation where severe resource constraints existed and integrated care was provided. METHODS: Antimalarial drugs were prepackaged into unit doses in an attempt to improve compliance with full courses of chemotherapy. FINDINGS: Compliance improved by approximately 20% in both adults and children. There were 50% reductions in cost to patients, waiting time at dispensaries and drug wastage at facilities. The intervention, which tended to improve both case and drug management at facilities, was well accepted by health staff and did not involve them in additional working time. CONCLUSION: The prepackaging of antimalarials at the district level offers the prospect of improved compliance and a reduction in the spread of resistance. PMID:11417034

  19. High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes.

    PubMed

    Bedut, Stephane; Seminatore-Nole, Christine; Lamamy, Veronique; Caignard, Sarah; Boutin, Jean A; Nosjean, Olivier; Stephan, Jean-Philippe; Coge, Francis

    2016-07-01

    Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety. PMID:27199128

  20. To kill or not to kill, that is the question: cytocidal antimalarial drug resistance

    PubMed Central

    Roepe, Paul D.

    2014-01-01

    Elucidating mechanisms of antimalarial drug resistance accelerates development of improved diagnostics and the design of new, effective malaria therapy. Recently, several studies have emphasized that chloroquine (CQ) resistance (CQR) can be quantified in two very distinct ways, depending on whether sensitivity to the growth inhibitory effects or parasite – kill effects of the drug are being measured. It is now clear that these cytostatic and cytocidal CQR phenotypes are not equivalent, and recent genetic, cell biological, and biophysical evidence suggests how the molecular mechanisms may overlap. These conclusions have important implications for elucidating other drug resistance phenomena and emphasize new concepts that are essential for the development of new drug therapy. PMID:24530127

  1. A High-Throughput In Vitro Drug Screen in a Genetically Engineered Mouse Model of Diffuse Intrinsic Pontine Glioma Identifies BMS-754807 as a Promising Therapeutic Agent

    PubMed Central

    Halvorson, Kyle G.; Barton, Kelly L.; Schroeder, Kristin; Misuraca, Katherine L.; Hoeman, Christine; Chung, Alex; Crabtree, Donna M.; Cordero, Francisco J.; Singh, Raj; Spasojevic, Ivan; Berlow, Noah; Pal, Ranadip; Becher, Oren J.

    2015-01-01

    Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system, including a model driven by PDGF-B, H3.3K27M, and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study, an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R, IR, MET, TRKA, TRKB, AURKA, AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation. Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo, systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50, suggesting that inadequate drug delivery may limit in vivo efficacy. In summary, an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG, but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice. PMID:25748921

  2. Interspecies Allometric Scaling of Antimalarial Drugs and Potential Application to Pediatric Dosing

    PubMed Central

    Senarathna, S. M. D. K. Ganga

    2014-01-01

    Pharmacopeial recommendations for administration of antimalarial drugs are the same weight-based (mg/kg of body weight) doses for children and adults. However, linear calculations are known to underestimate pediatric doses; therefore, interspecies allometric scaling data may have a role in predicting doses in children. We investigated the allometric scaling relationships of antimalarial drugs using data from pharmacokinetic studies in mammalian species. Simple allometry (Y = a × Wb) was utilized and compared to maximum life span potential (MLP) correction. All drugs showed a strong correlation with clearance (CL) in healthy controls. Insufficient data from malaria-infected species other than humans were available for allometric scaling. The allometric exponents (b) for CL of artesunate, dihydroartemisinin (from intravenous artesunate), artemether, artemisinin, clindamycin, piperaquine, mefloquine, and quinine were 0.71, 0.85, 0.66, 0.83, 0.62, 0.96, 0.52, and 0.40, respectively. Clearance was significantly lower in malaria infection than in healthy (adult) humans for quinine (0.07 versus 0.17 liter/h/kg; P = 0.0002) and dihydroartemisinin (0.81 versus 1.11 liters/h/kg; P = 0.04; power = 0.6). Interpolation of simple allometry provided better estimates of CL for children than MLP correction, which generally underestimated CL values. Pediatric dose calculations based on simple allometric exponents were 10 to 70% higher than pharmacopeial (mg/kg) recommendations. Interpolation of interspecies allometric scaling could provide better estimates than linear scaling of adult to pediatric doses of antimalarial drugs; however, the use of a fixed exponent for CL was not supported in the present study. The variability in allometric exponents for antimalarial drugs also has implications for scaling of fixed-dose combinations. PMID:25092696

  3. High-throughput behavioral phenotyping of drug and alcohol susceptibility traits in the expanded panel of BXD recombinant inbred strains

    SciTech Connect

    Philip, Vivek M; Ansah, T; Blaha, C,; Cook, Melloni N.; Hamre, Kristin M.; Lariviere, William R; Matthews, Douglas B; Goldowitz, Daniel; Chesler, Elissa J

    2010-01-01

    Genetic reference populations, particularly the BXD recombinant inbred strains, are a valuable resource for the discovery of the bio-molecular substrates and genetic drivers responsible for trait variation and co- ariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic co-regulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium have obtained behavioral phenotype data from 260 measures related to multiple behavioral assays across several domains: self-administration, response to, and withdrawal from cocaine, MDMA, morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity; and sleep/wake cycles. All traits have been measured in both sexes and the recently expanded panel of 69 additional BXD recombinant inbred strains (N=69). Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent BXD RI lines was performed. Primary data is publicly available for heritability, sex difference and genetic analyses using www.GeneNetwork.org. These analyses include QTL detection and genetic analysis of gene expression. Stored results from these analyses are available at http://ontologicaldiscovery.org for comparison to other genomic analysis results. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits.

  4. Molecular surveillance of antimalarial drug resistance related genes in Plasmodium falciparum isolates from Eritrea.

    PubMed

    Menegon, Michela; Nurahmed, Abduselam M; Talha, Albadawi A; Nour, Bakri Y M; Severini, Carlo

    2016-05-01

    The introduction of artemisinin-based combination therapy has led to extraordinary results in malaria control, however the recent emergence of partial resistance to artemisinin therapy in Southeast Asia jeopardizes these successes. This study aimed at investigating resistance to the antimalarial drugs by evaluating the polymorphisms in the PfK13, Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolates obtained from patients in Eritrea. PMID:26875763

  5. Evaluating the Pharmacodynamic Effect of Antimalarial Drugs in Clinical Trials by Quantitative PCR

    PubMed Central

    Marquart, Louise; Baker, Mark; O'Rourke, Peter

    2015-01-01

    The ongoing development of new antimalarial drugs and the increasing use of controlled human malaria infection (CHMI) studies to investigate their activity in early-stage clinical trials require the development of methods to analyze their pharmacodynamic effect. This is especially so for studies where quantitative PCR (qPCR) is becoming the preferred method for assessing parasite clearance as the study endpoint. We report the development and validation of an analytic approach for qPCR-determined parasite clearance data. First, in a clinical trial with the licensed antimalarial combination sulfadoxine-pyrimethamine (S/P), qPCR data were collected from 12 subjects and used to determine qPCR replicate variability and to identify outliers. Then, an iterative analytic approach based on modeling the log-linear decay of parasitemia following drug treatment was developed to determine the parasite reduction ratio (PRR) and parasite clearance half-life, both measures of parasite clearance. This analytic approach was then validated with data from 8 subjects enrolled in a second study with the licensed antimalarial drug mefloquine. By this method, the PRR and parasite clearance half-lives for S/P and Mefloquine were determined to be 38,878 (95% confidence interval [95% CI], 17,396 to 86,889) at 3.15 (95% CI, 2.93 to 3.41) days and 157 (95% CI, 130 to 189) at 6.58 (95% CI, 6.35 to 6.83) days for the respective studies. No serious adverse events occurred in the two trials, and pharmacokinetic values were within expected ranges for sulfadoxine and pyrimethamine. The robust statistical method that we have developed to analyze qPCR-derived pharmacodynamic data from CHMI studies will facilitate the assessment of the activity of a range of experimental antimalarial drugs now entering clinical trials. (This trial was registered with the Australian New Zealand Clinical Trials Registry under registration numbers ACTRN12611001203943 and ACTRN12612000323820.) PMID:25963983

  6. High-Throughput Proteomics

    NASA Astrophysics Data System (ADS)

    Zhang, Zhaorui; Wu, Si; Stenoien, David L.; Paša-Tolić, Ljiljana

    2014-06-01

    Mass spectrometry (MS)-based high-throughput proteomics is the core technique for large-scale protein characterization. Due to the extreme complexity of proteomes, sophisticated separation techniques and advanced MS instrumentation have been developed to extend coverage and enhance dynamic range and sensitivity. In this review, we discuss the separation and prefractionation techniques applied for large-scale analysis in both bottom-up (i.e., peptide-level) and top-down (i.e., protein-level) proteomics. Different approaches for quantifying peptides or intact proteins, including label-free and stable-isotope-labeling strategies, are also discussed. In addition, we present a brief overview of different types of mass analyzers and fragmentation techniques as well as selected emerging techniques.

  7. High throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

    A cryocondensation pump with a unique regeneration mechanism that allows continuous operation has been constructed and tested. The pump features a device referred to as the ''Snail'' which removes the cryofrost layer as it is moved over the pumping surfaces. A forepump pumps the sublimed gas generated inside the Snail. The compression ratio of the pump is the ratio of the cryopump speed to the leakage conductance of the Snail. Deuterium had been pumped continuously at 30 torr.L/s at a speed of 2000 L/s and a compression ratio of 100. The pump, being all metal sealed and free of lubricating fluids, has many potential applications where untraclean high throughput pumping is desirable. Since the pump regenerates on a time scale of 60 seconds, the inventory in the pump is minimized - an important consideration when pumping radioactive materials such as tritium. Test data and a videotape of the Snail removing the cryofrost will be shown.

  8. Linking Murine and Human Plasmodium falciparum Challenge Models in a Translational Path for Antimalarial Drug Development

    PubMed Central

    McCarthy, James S.; Marquart, Louise; Sekuloski, Silvana; Trenholme, Katharine; Elliott, Suzanne; Griffin, Paul; Rockett, Rebecca; O'Rourke, Peter; Sloots, Theo; Angulo-Barturen, Iñigo; Ferrer, Santiago; Jiménez-Díaz, María Belén; Martínez, María-Santos; Duparc, Stephan; Leroy, Didier; Wells, Timothy N. C.; Baker, Mark

    2016-01-01

    Effective progression of candidate antimalarials is dependent on optimal dosing in clinical studies, which is determined by a sound understanding of pharmacokinetics and pharmacodynamics (PK/PD). Recently, two important translational models for antimalarials have been developed: the NOD/SCID/IL2Rγ−/− (NSG) model, whereby mice are engrafted with noninfected and Plasmodium falciparum-infected human erythrocytes, and the induced blood-stage malaria (IBSM) model in human volunteers. The antimalarial mefloquine was used to directly measure the PK/PD in both models, which were compared to previously published trial data for malaria patients. The clinical part was a single-center, controlled study using a blood-stage Plasmodium falciparum challenge inoculum in volunteers to characterize the effectiveness of mefloquine against early malaria. The study was conducted in three cohorts (n = 8 each) using different doses of mefloquine. The characteristic delay in onset of action of about 24 h was seen in both NSG and IBSM systems. In vivo 50% inhibitory concentrations (IC50s) were estimated at 2.0 μg/ml and 1.8 μg/ml in the NSG and IBSM models, respectively, aligning with 1.8 μg/ml reported previously for patients. In the IBSM model, the parasite reduction ratios were 157 and 195 for the 10- and 15-mg/kg doses, within the range of previously reported clinical data for patients but significantly lower than observed in the mouse model. Linking mouse and human challenge models to clinical trial data can accelerate the accrual of critical data on antimalarial drug activity. Such data can guide large clinical trials required for development of urgently needed novel antimalarial combinations. (This trial was registered at the Australian New Zealand Clinical Trials Registry [http://anzctr.org.au] under registration number ACTRN12612000323820.) PMID:27044554

  9. Linking Murine and Human Plasmodium falciparum Challenge Models in a Translational Path for Antimalarial Drug Development.

    PubMed

    McCarthy, James S; Marquart, Louise; Sekuloski, Silvana; Trenholme, Katharine; Elliott, Suzanne; Griffin, Paul; Rockett, Rebecca; O'Rourke, Peter; Sloots, Theo; Angulo-Barturen, Iñigo; Ferrer, Santiago; Jiménez-Díaz, María Belén; Martínez, María-Santos; Hooft van Huijsduijnen, Rob; Duparc, Stephan; Leroy, Didier; Wells, Timothy N C; Baker, Mark; Möhrle, Jörg J

    2016-06-01

    Effective progression of candidate antimalarials is dependent on optimal dosing in clinical studies, which is determined by a sound understanding of pharmacokinetics and pharmacodynamics (PK/PD). Recently, two important translational models for antimalarials have been developed: the NOD/SCID/IL2Rγ(-/-) (NSG) model, whereby mice are engrafted with noninfected and Plasmodium falciparum-infected human erythrocytes, and the induced blood-stage malaria (IBSM) model in human volunteers. The antimalarial mefloquine was used to directly measure the PK/PD in both models, which were compared to previously published trial data for malaria patients. The clinical part was a single-center, controlled study using a blood-stage Plasmodium falciparum challenge inoculum in volunteers to characterize the effectiveness of mefloquine against early malaria. The study was conducted in three cohorts (n = 8 each) using different doses of mefloquine. The characteristic delay in onset of action of about 24 h was seen in both NSG and IBSM systems. In vivo 50% inhibitory concentrations (IC50s) were estimated at 2.0 μg/ml and 1.8 μg/ml in the NSG and IBSM models, respectively, aligning with 1.8 μg/ml reported previously for patients. In the IBSM model, the parasite reduction ratios were 157 and 195 for the 10- and 15-mg/kg doses, within the range of previously reported clinical data for patients but significantly lower than observed in the mouse model. Linking mouse and human challenge models to clinical trial data can accelerate the accrual of critical data on antimalarial drug activity. Such data can guide large clinical trials required for development of urgently needed novel antimalarial combinations. (This trial was registered at the Australian New Zealand Clinical Trials Registry [http://anzctr.org.au] under registration number ACTRN12612000323820.). PMID:27044554

  10. Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells.

    PubMed

    Johnson, Paul H; Walker, Roger P; Jones, Steven W; Stephens, Kathy; Meurer, Janet; Zajchowski, Deborah A; Luke, May M; Eeckman, Frank; Tan, Yuping; Wong, Linda; Parry, Gordon; Morgan, Thomas K; McCarrick, Meg A; Monforte, Joseph

    2002-12-01

    Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as cancer. We report a new cell-based high-throughput technology for screening chemical libraries against several potential cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in cancer cells was used to screen 9000 chemicals and known drugs in the human prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind DNA, cell surface receptors, and components of intracellular signaling pathways. A class of steroids related to the cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-ATPase resulting in the inhibition of four of the prostate target genes including transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis, survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA methyltransferase, and cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis. PMID:12516962

  11. Development of high-throughput multi-residue method for non-steroidal anti-inflammatory drugs monitoring in swine muscle by LC-MS/MS.

    PubMed

    Castilhos, Tamara S; Barreto, Fabiano; Meneghini, Leonardo; Bergold, Ana Maria

    2016-07-01

    A reliable and simple method for the detection and quantification of residues of 14 non-steroidal anti-inflammatory drugs and a metamizole metabolite in swine muscle was developed using liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The samples were extracted with acetonitrile (ACN) in solid-liquid extraction followed by a low-temperature partitioning (LLE-LTP) process at -20 ± 2°C. After evaporation to dryness, the residue was reconstituted with hexane and a mixture of water:acetonitrile (1:1). LC separation was achieved on a reversed-phase (RP18) column with gradient elution using water (phase A) and ACN (phase B) both containing 1 mmol l(-)(1) ammonium acetate (NH4COO) with 0.025% acetic acid. Analysis was carried out on a triple-quadrupole tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode using an electrospray interface in negative and positive mode in a single run. Method validation was performed according to the criteria of Commission Decision No. 2002/657/EC. The matrix effect and linearity were evaluated. Decision limit (CCα), detection capability (CCβ), accuracy and repeatability of the method are also reported. The proposed method proved to be simple, easy and adequate for high-throughput analysis and was applied to routine analysis by the Brazilian Ministry of Agriculture, Livestock and Food Supply. PMID:27268755

  12. High-throughput quantification of drugs and their metabolites in biosamples by LC-MS/MS and CE-MS/MS: possibilities and limitations.

    PubMed

    Hopfgartner, G; Husser, C; Zell, M

    2002-02-01

    Off-line solid phase extraction with C18 disk plates and turbulent flow chromatography were evaluated versus on-line solid phase extraction using column-switching HPLC as sample preparation techniques for high-throughput analysis of pharmaceutical compounds and their metabolites by LC-MS/MS. Turbulent flow chromatography was found to be very straightforward in its applicaton, but the LOQs were more than fivefold higher compared with off-line or other on-line solid phase extraction methods. Solid phase extraction (SPE) on disk was found to be fast and sufficient efficient to minimize matrix effects and therefore an apprach to provide sensitive and reliable LC-MS/MS methods. Column-switching HPLC with microbore columns (0.5 mm i.d.) were used for fast analysis of a parent drug and four of its metabolites utilizing steep gradients in 1 minute. The application of CZE-MS/MS for bionalysis of pharamaceutical compounds is also discussed. PMID:11805734

  13. The development and assessment of high-throughput mass spectrometry-based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate.

    PubMed

    Buse, Joshua; Purves, Randy W; Verrall, Ronald E; Badea, Ildiko; Zhang, Haixia; Mulligan, Christopher C; Peru, Kerry M; Bailey, Jonathan; Headley, John V; El-Aneed, Anas

    2014-11-01

    The safe use of lipid-based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high-throughput MS-based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3). Analysis utilized MS instruments that detect analytes using low-resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high-resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)-QTRAP-MS/MS, FC- LTQ-Orbitrap-MS, desorption electrospray ionization-collision-induced dissociation (CID)-MS/MS and matrix assisted laser desorption ionization-ToF/ToF-MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)-CID-MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS-based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC-CID-MS/MS, the new MS-based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high-throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC-CID-MS/MS for the quantification of the G16-3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16-3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells. PMID:25395133

  14. Altering Antimalarial Drug Regimens May Dramatically Enhance and Restore Drug Effectiveness.

    PubMed

    Kay, Katherine; Hodel, Eva Maria; Hastings, Ian M

    2015-10-01

    There is considerable concern that malaria parasites are starting to evolve resistance to the current generation of antimalarial drugs, the artemisinin-based combination therapies (ACTs). We use pharmacological modeling to investigate changes in ACT effectiveness likely to occur if current regimens are extended from 3 to 5 days or, alternatively, given twice daily over 3 days. We show that the pharmacology of artemisinins allows both regimen changes to substantially increase the artemisinin killing rate. Malaria patients rarely contain more than 10(12) parasites, while the standard dosing regimens allow approximately 1 in 10(10) parasites to survive artemisinin treatment. Parasite survival falls dramatically, to around 1 in 10(17) parasites if the dose is extended or split; theoretically, this increase in drug killing appears to be more than sufficient to restore failing ACT efficacy. One of the most widely used dosing regimens, artemether-lumefantrine, already successfully employs a twice-daily dosing regimen, and we argue that twice-daily dosing should be incorporated into all ACT regimen design considerations as a simple and effective way of ensuring the continued long-term effectiveness of ACTs. PMID:26239993

  15. Altering Antimalarial Drug Regimens May Dramatically Enhance and Restore Drug Effectiveness

    PubMed Central

    Hodel, Eva Maria; Hastings, Ian M.

    2015-01-01

    There is considerable concern that malaria parasites are starting to evolve resistance to the current generation of antimalarial drugs, the artemisinin-based combination therapies (ACTs). We use pharmacological modeling to investigate changes in ACT effectiveness likely to occur if current regimens are extended from 3 to 5 days or, alternatively, given twice daily over 3 days. We show that the pharmacology of artemisinins allows both regimen changes to substantially increase the artemisinin killing rate. Malaria patients rarely contain more than 1012 parasites, while the standard dosing regimens allow approximately 1 in 1010 parasites to survive artemisinin treatment. Parasite survival falls dramatically, to around 1 in 1017 parasites if the dose is extended or split; theoretically, this increase in drug killing appears to be more than sufficient to restore failing ACT efficacy. One of the most widely used dosing regimens, artemether-lumefantrine, already successfully employs a twice-daily dosing regimen, and we argue that twice-daily dosing should be incorporated into all ACT regimen design considerations as a simple and effective way of ensuring the continued long-term effectiveness of ACTs. PMID:26239993

  16. Investigation of some medicinal plants traditionally used for treatment of malaria in Kenya as potential sources of antimalarial drugs.

    PubMed

    Muthaura, C N; Keriko, J M; Derese, S; Yenesew, A; Rukunga, G M

    2011-03-01

    Malaria is a major public health problem in many tropical and subtropical countries and the burden of this disease is getting worse, mainly due to the increasing resistance of Plasmodium falciparum against the widely available antimalarial drugs. There is an urgent need for discovery of new antimalarial agents. Herbal medicines for the treatment of various diseases including malaria are an important part of the cultural diversity and traditions of which Kenya's biodiversity has been an integral part. Two major antimalarial drugs widely used today came originally from indigenous medical systems, that is quinine and artemisinin, from Peruvian and Chinese ancestral treatments, respectively. Thus ethnopharmacology is a very important resource in which new therapies may be discovered. The present review is an analysis of ethnopharmacological publications on antimalarial therapies from some Kenyan medicinal plants. PMID:21095187

  17. Factors determining anti-malarial drug use in a peri-urban population from malaria holoendemic region of western kenya

    PubMed Central

    2010-01-01

    Background Interventions to reverse trends in malaria-related morbidity and mortality in Kenya focus on preventive strategies and drug efficacy. However, the pattern of use of anti-malarials in malaria-endemic populations, such as in western Kenya, is still poorly understood. It is critical to understand the patterns of anti-malarial drug use to ascertain that the currently applied new combination therapy to malaria treatment, will achieve sustained cure rates and protection against parasite resistance. Therefore, this cross-sectional study was designed to determine the patterns of use of anti-malarial drugs in households (n = 397) in peri-urban location of Manyatta-B sub-location in Kisumu in western Kenya. Methods Household factors, associated with the pattern of anti-malarials use, were evaluated. Using clusters, questionnaire was administered to a particular household member who had the most recent malaria episode (within <2 weeks) and used an anti-malarial for cure. Mothers/caretakers provided information for children aged <13 years. Results Stratification of the type of anti-malarial drugs taken revealed that 37.0% used sulphadoxine/pyrimethamine (SP), 32.0% artemisinin-based combined therapy (ACT), 11.1% anti-pyretics, 7.3% chloroquine (CQ), 7.1% quinine, 2.5% amodiaquine (AQ), while 3.0% used others which were perceived as anti-malarials (cough syrups and antibiotics). In a regression model, it was demonstrated that age (P = 0.050), household size (P = 0.047), household head (P = 0.049), household source of income (P = 0.015), monthly income (P = 0.020), duration of use (P = 0.029), dosage of drugs taken (P = 0.036), and source of drugs (P = 0.005) significantly influenced anti-malarial drug use. Overall, 38.8% of respondents used drugs as recommended by the Ministry of Health. Conclusion This study demonstrates that consumers require access to correct and comprehensible information associated with use of drugs, including self-prescription. There is

  18. How Patients Take Malaria Treatment: A Systematic Review of the Literature on Adherence to Antimalarial Drugs

    PubMed Central

    Bruxvoort, Katia; Goodman, Catherine; Kachur, S. Patrick; Schellenberg, David

    2014-01-01

    Background High levels of patient adherence to antimalarial treatment are important in ensuring drug effectiveness. To achieve this goal, it is important to understand levels of patient adherence, and the range of study designs and methodological challenges involved in measuring adherence and interpreting results. Since antimalarial adherence was reviewed in 2004, there has been a major expansion in the use of artemisinin-based combination therapies (ACTs) in the public sector, as well as initiatives to make them more widely accessible through community health workers and private retailers. These changes and the large number of recent adherence studies raise the need for an updated review on this topic. Objective We conducted a systematic review of studies reporting quantitative results on patient adherence to antimalarials obtained for treatment. Results The 55 studies identified reported extensive variation in patient adherence to antimalarials, with many studies reporting very high adherence (90–100%) and others finding adherence of less than 50%. We identified five overarching approaches to assessing adherence based on the definition of adherence and the methods used to measure it. Overall, there was no clear pattern in adherence results by approach. However, adherence tended to be higher among studies where informed consent was collected at the time of obtaining the drug, where patient consultations were directly observed by research staff, and where a diagnostic test was obtained. Conclusion Variations in reported adherence may reflect factors related to patient characteristics and the nature of their consultation with the provider, as well as methodological variations such as interaction between the research team and patients before and during the treatment. Future studies can benefit from an awareness of the impact of study procedures on adherence outcomes, and the identification of improved measurement methods less dependent on self-report. PMID:24465418

  19. Stated preferences for anti-malarial drug characteristics in Zomba, a malaria endemic area of Malawi

    PubMed Central

    2014-01-01

    Background The evidence on determinants of individuals’ choices for anti-malarial drug treatments is scarce. This study sought to measure the strength of preference for adult antimalarial drug treatment attributes of heads of urban, rural and peri-urban households in a resource-limited malaria-endemic area of sub-Saharan Africa. Methods Discrete choice experiments were conducted with 508 heads of household interviewed face-to-face for a household population survey of health-seeking behavior in Zomba District, Malawi. The interviews were held in Chichewa and the choice experiment questions were presented with cartoon aids. The anti-malarial drug attributes included in the stated preference experiment were: speed of fever resolution, side effects (pruritus) risk, protection (duration of prophylactic effect), price, duration of treatment course and recommendation by a health professional. Sixteen treatment profiles from a fractional factorial design by orthogonal array were paired into choice scenarios, and scenarios were randomly assigned to participants so that each participant was presented with a series of eight pairwise choice scenarios. Respondents had the option to state indifference between the two profiles or decline to choose. Data were analysed in a mixed logit model, with normally distributed coefficients for all six attributes. Results The sex ratio was balanced in urban areas, whereas 63% of participants in rural areas were male. The proportion of individuals with no education was considerably higher in the rural group (25%) than in the urban (5%) and peri-urban (6%) groups. All attributes investigated had the expected influence, and traded-off in most respondents’ choices. There were heterogeneous effects of price, pruritus risk, treatment recommendation by a professional, and duration of prophylaxis across respondents, only partly explained by their differences in education, household per capita expenditure, sex and age. Individuals´ demand

  20. Measuring topology of low-intensity DNA methylation sites for high-throughput assessment of epigenetic drug-induced effects in cancer cells

    SciTech Connect

    Gertych, Arkadiusz; Farkas, Daniel L.; Tajbakhsh, Jian

    2010-11-15

    potentially be a valuable component in the high-throughput assessment of demethylation and risk of chromatin reorganization in epigenetic-drug screening tasks.

  1. Current issues for anti-malarial drugs to control P. falciparum malaria.

    PubMed

    Schellenberg, D; Abdulla, S; Roper, C

    2006-03-01

    Successful malaria control depends heavily on efficacious anti-malarial drugs for the treatment of malaria. Artesunate-containing Combination Treatments (ACT) are increasingly recommended as first line malaria treatment in endemic countries, but implementation of this recommendation is limited by the small number of available and affordable co-formulated anti-malarial drugs. In recent years Intermittent Preventive Treatment has been recommended for malaria control in pregnancy and has been shown to be of potential public health importance in the prevention of malaria and anaemia in children. The use of drugs for malaria treatment or prevention is associated with the development of resistance and recent advances in molecular biology facilitate the evaluation of the impact on drug resistance of new drug-based strategies. This review concentrates on the challenges surrounding the use of ACT, the current understanding of IPT in infants and the use of molecular approaches to enhance our understanding of the effects of interventions on the spread of drug resistance. PMID:16515515

  2. High throughput optical scanner

    DOEpatents

    Basiji, David A.; van den Engh, Gerrit J.

    2001-01-01

    A scanning apparatus is provided to obtain automated, rapid and sensitive scanning of substrate fluorescence, optical density or phosphorescence. The scanner uses a constant path length optical train, which enables the combination of a moving beam for high speed scanning with phase-sensitive detection for noise reduction, comprising a light source, a scanning mirror to receive light from the light source and sweep it across a steering mirror, a steering mirror to receive light from the scanning mirror and reflect it to the substrate, whereby it is swept across the substrate along a scan arc, and a photodetector to receive emitted or scattered light from the substrate, wherein the optical path length from the light source to the photodetector is substantially constant throughout the sweep across the substrate. The optical train can further include a waveguide or mirror to collect emitted or scattered light from the substrate and direct it to the photodetector. For phase-sensitive detection the light source is intensity modulated and the detector is connected to phase-sensitive detection electronics. A scanner using a substrate translator is also provided. For two dimensional imaging the substrate is translated in one dimension while the scanning mirror scans the beam in a second dimension. For a high throughput scanner, stacks of substrates are loaded onto a conveyor belt from a tray feeder.

  3. Study of the efficacy of antimalarial drugs delivered inside targeted immunoliposomal nanovectors

    NASA Astrophysics Data System (ADS)

    Urbán, Patricia; Estelrich, Joan; Adeva, Alberto; Cortés, Alfred; Fernàndez-Busquets, Xavier

    2011-12-01

    Paul Ehrlich's dream of a 'magic bullet' that would specifically destroy invading microbes is now a major aspect of clinical medicine. However, a century later, the implementation of this medical holy grail continues being a challenge in three main fronts: identifying the right molecular or cellular targets for a particular disease, having a drug that is effective against it, and finding a strategy for the efficient delivery of sufficient amounts of the drug in an active state exclusively to the selected targets. In a previous work, we engineered an immunoliposomal nanovector for the targeted delivery of its contents exclusively to Plasmodium falciparum-infected red blood cells [pRBCs]. In preliminary assays, the antimalarial drug chloroquine showed improved efficacy when delivered inside immunoliposomes targeted with the pRBC-specific monoclonal antibody BM1234. Because difficulties in determining the exact concentration of the drug due to its low amounts prevented an accurate estimation of the nanovector performance, here, we have developed an HPLC-based method for the precise determination of the concentrations in the liposomal preparations of chloroquine and of a second antimalarial drug, fosmidomycin. The results obtained indicate that immunoliposome encapsulation of chloroquine and fosmidomycin improves by tenfold the efficacy of antimalarial drugs. The targeting antibody used binds preferentially to pRBCs containing late maturation stages of the parasite. In accordance with this observation, the best performing immunoliposomes are those added to Plasmodium cultures having a larger number of late form-containing pRBCs. An average of five antibody molecules per liposome significantly improves in cell cultures the performance of immunoliposomes over non-functionalized liposomes as drug delivery vessels. Increasing the number of antibodies on the liposome surface correspondingly increases performance, with a reduction of 50% parasitemia achieved with

  4. Antimalarial Drug Discovery: Approaches and Progress towards New Medicines

    PubMed Central

    Flannery, Erika L.; Chatterjee, Arnab K.; Winzeler, Elizabeth A.

    2014-01-01

    Malaria elimination has recently been reinstated as a global health priority but current therapies seem to be insufficient for the task. Elimination efforts require new drug classes that alleviate symptoms, prevent transmission and provide a radical cure. To develop these next generation medicines, public-private partnerships are funding innovative approaches to identify compounds that target multiple parasite species at multiple stages of the parasite lifecycle. Here, we review the cell-, chemistry- and target-based approaches used to discover new drug candidates that are currently in clinical trials or undergoing preclinical testing. PMID:24217412

  5. Heme as trigger and target for trioxane-containing antimalarial drugs.

    PubMed

    Meunier, Bernard; Robert, Anne

    2010-11-16

    Heme is not only just the binding site responsible for oxygen transport by hemoglobin, but it is also the prosthetic group of many different heme-containing enzymes, such as cytochromes P450, peroxidases, catalase, and several proteins involved in electron transfer. Heme plays a key role in the mechanism of action of many different antimalarial drugs. In degrading the host's hemoglobin, the malaria parasite Plasmodium and several other heme-eating parasites are faced with this redox-active metal complex. Heme is able to induce the toxic reductive cascade of molecular oxygen, which leads to the production of destructive hydroxyl radicals. Plasmodium detoxifies heme by converting it into a redox-inactive iron(III) polymer called hemozoin. Artemisinin, a natural drug containing a biologically important 1,2,4-trioxane structure, is now the first-line treatment for multidrug-resistant malaria. The peroxide moiety in artemisinin reacts in the presence of the flat, achiral iron(II)-heme; the mechanism does not reflect the classical "key and lock" paradigm for drugs. Instead, the reductive activation of the peroxide function generates a short-lived alkoxy radical, which quickly rearranges to a C-centered primary radical. This radical alkylates heme via an intramolecular process to produce covalent heme-drug adducts. The accumulation of non-polymerizable redox-active heme derivatives, a consequence of heme alkylation, is thought to be toxic for the parasite. The alkylation of heme by artemisinin has been demonstrated in malaria-infected mice, indicating that heme is acting as the trigger and target of artemisinin. The alkylation of heme by artemisinin is not limited to this natural compound: the mechanism is invoked for a large number of antimalarial semisynthetic derivatives. Synthetic trioxanes or trioxolanes also alkylate heme, and their alkylation ability correlates well with their antimalarial efficacy. In addition, several reports have demonstrated the cytotoxicity of

  6. Atorvastatin Is a Promising Partner for Antimalarial Drugs in Treatment of Plasmodium falciparum Malaria▿

    PubMed Central

    Parquet, Véronique; Briolant, Sébastien; Torrentino-Madamet, Marylin; Henry, Maud; Almeras, Lionel; Amalvict, Rémy; Baret, Eric; Fusaï, Thierry; Rogier, Christophe; Pradines, Bruno

    2009-01-01

    Atorvastatin (AVA) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. AVA exposure resulted in the reduced in vitro growth of 22 Plasmodium falciparum strains, with the 50% inhibitory concentrations (IC50s) ranging from 2.5 μM to 10.8 μM. A significant positive correlation was found between the strains’ responses to AVA and mefloquine (r = 0.553; P = 0.008). We found no correlation between the responses to AVA and to chloroquine, quinine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, or doxycycline. These data could suggest that the mechanism of AVA uptake and/or the mode of action of AVA is different from those for other antimalarial drugs. The IC50s for AVA were unrelated to the occurrence of mutations in the transport protein genes involved in quinoline antimalarial drug resistance, such as the P. falciparum crt, mdr1, mrp, and nhe-1 genes. Therefore, AVA can be ruled out as a substrate for the transport proteins (CRT, Pgh1, and MRP) and is not subject to the pH modification induced by the P. falciparum NHE-1 protein. The absence of in vitro cross-resistance between AVA and chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone, and doxycycline argues that these antimalarial drugs could potentially be paired with AVA as a treatment for malaria. In conclusion, the present observations suggest that AVA is a good candidate for further studies on the use of statins in association with drugs known to have activities against the malaria parasite. PMID:19307369

  7. Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island, China

    PubMed Central

    Wang, Shan-Qing; Wang, Guang-Ze; Li, Yu-Chun; Meng, Feng; Lin, Shi-Gan; Zhu, Zhen-Hu; Sun, Ding-Wei; He, Chang-Hua; Hu, Xi-Min; Du, Jian-Wei

    2015-01-01

    Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5±10.6 hr) and the mean parasite clearance time (27.3±12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (P<0.0001). The resistance intensities decreased as follows: chloroquine>piperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC50) was 3.77×10-6 mol/L, 2.09×10-6 mol/L, 0.09×10-6 mol/L, and 0.05×10-6 mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60×10-6 mol/L, 9.26×10-6 mol/L, 0.55×10-6 mol/L, and 0.07×10-6 mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity. PMID:25748707

  8. The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation.

    PubMed

    Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

    2016-01-01

    The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10(-6) cm/sec, followed by amodiaquine around 20 x 10(-6) cm/sec; both mefloquine and artesunate were around 10 x 10(-6) cm/sec. Methylene blue was between 2 and 6 x 10(-6) cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine. PMID:27045516

  9. The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation

    PubMed Central

    Senarathna, S M D K Ganga; Page-Sharp, Madhu; Crowe, Andrew

    2016-01-01

    The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10−6 cm/sec, followed by amodiaquine around 20 x 10−6 cm/sec; both mefloquine and artesunate were around 10 x 10−6 cm/sec. Methylene blue was between 2 and 6 x 10−6 cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine. PMID:27045516

  10. Policy options for deploying anti-malarial drugs in endemic countries: a population genetics approach

    PubMed Central

    2012-01-01

    Background Anti-malarial drugs are constantly exposed to the threat of evolving drug resistance so good stewardship of existing therapy is an essential component of public health policy. However, the widespread availability of numerous different drugs through informal providers could undermine official drug deployment policies. A policy of multiple first-line therapy (MFT) is compared with the conventional policy of sequential drug deployment, i.e., where one drug is used until resistance evolves and then replaced by the next drug in the sequence. Methods Population genetic models of drug resistance are used to make the comparison; this methodology explicitly tracks the genetics of drug resistance (including, importantly, recombination in the sexual stage, intrahost dynamics, and direction of linkage disequilibrium). Results A policy of MFT outlasts sequential application providing drug usages are low to moderate, and appears not to drive widespread multi-drug resistance. Inadequate dosing is an even more potent driver of drug resistance than the MFT/sequential policy decision. Conclusions The provision of MFT as a deliberate policy can be encouraged provided overall treatment rates are low or moderate (less than around half of malaria infections are treated) and the ad hoc provision of MFT through the private sector may be tolerated. This must be fully supported by education to ensure people take adequate doses of each of the drugs. PMID:23244624

  11. Coupling laser ablation/desorption electrospray ionization to atmospheric pressure drift tube ion mobility spectrometry for the screening of antimalarial drug quality.

    PubMed

    Harris, Glenn A; Graf, Stephan; Knochenmuss, Richard; Fernández, Facundo M

    2012-07-01

    Significant developments in the field of ambient desorption/ionization mass spectrometry (MS) have led to high-throughput direct analysis and imaging capabilities. However, advances in coupling ambient ionization techniques with standalone drift tube ion mobility spectrometry (DTIMS) have been comparatively slower, despite the attractive ruggedness and simplicity of IMS. In this study, we have developed and characterized a laser ablation/desorption electrospray ionization (LADESI) DTIMS platform, and applied it to the detection of active pharmaceutical ingredients (APIs) in antimalarial tablets collected in developing countries. The overarching goal of this work was to perform an initial evaluation of LADESI DTIMS as a technique with the potential for constituting the core of a portable drug quality-testing platform. The set-up consisted of an IR laser for desorption and an electrospray ionizer for capturing the ablated plume coupled to a high-resolution monolithic resistive glass drift tube ion mobility spectrometer. For more confident API identification, tablet extracts were also investigated via electrospray IM MS to correlate LADESI DTIMS reduced mobility (K(0)) values to m/z values. Overall, it was found that the IR LADESI DTIMS platform provided distinct ion mobility spectral fingerprints that could be used to detect the presence of the expected APIs, helping to distinguish counterfeit drugs from their genuine counterparts. PMID:22606690

  12. Antimalarial drug resistance: a review of the biology and strategies to delay emergence and spread

    PubMed Central

    Klein, E.Y.

    2013-01-01

    The emergence of resistance to former first-line antimalarial drugs has been an unmitigated disaster. In recent years, artemisinin class drugs have become standard and they are considered an essential tool for helping to eradicate the disease. However, their ability to reduce morbidity and mortality and to slow transmission requires the maintenance of effectiveness. Recently, an artemisinin delayed-clearance phenotype was described. This is believed to be the precursor to resistance and threatens local elimination and global eradication plans. Understanding how resistance emerges and spreads is important for developing strategies to contain its spread. Resistance is the result of two processes: (i) drug selection of resistant parasites; and (ii) the spread of resistance. In this review, we examine the factors that lead to both drug selection and the spread of resistance. We then examine strategies for controlling the spread of resistance, pointing out the complexities and deficiencies in predicting how resistance will spread. PMID:23394809

  13. Resistance of Plasmodium falciparum to antimalarial drugs in Equatorial Guinea.

    PubMed

    Roche, J; Benito, A; Ayecaba, S; Amela, C; Molina, R; Alvar, J

    1993-10-01

    One hundred and sixty-six children from Equatorial Guinea, all under 10 years of age and with acute uncomplicated falciparum malaria, were randomly allocated to four groups and treated with one of the following regimens: chloroquine or amodiaquine (25 mg base/kg body weight over 3 days), quinine (8 mg/kg every 8 h for 3 or 5 days), and sulphadoxine-pyrimethamine (25-1.25 mg/kg, in one dose). The parasite clearance rates up to day 14 were 28% with chloroquine, 74% with amodiaquine, and 95% with quinine or sulphadoxine-pyrimethamine. The times required to clear asexual blood forms of Plasmodium falciparum in sensitive cases were 64, 70, 73 and 65 h, respectively. Although quinine and sulphadoxine-pyrimethamine are equally effective, quinine is recommended for treatment of multidrug-resistant malaria in paediatric patients, essentially because of the risk of serious reactions to sulpha drugs. Health providers are, however, encouraged to keep supplies of sulphadoxine-pyrimethamine as an option and to refer patients quickly, if required. PMID:8311568

  14. Antimalarial drug targets in Plasmodium falciparum predicted by stage-specific metabolic network analysis

    PubMed Central

    2010-01-01

    Background Despite enormous efforts to combat malaria the disease still afflicts up to half a billion people each year of which more than one million die. Currently no approved vaccine is available and resistances to antimalarials are widely spread. Hence, new antimalarial drugs are urgently needed. Results Here, we present a computational analysis of the metabolism of Plasmodium falciparum, the deadliest malaria pathogen. We assembled a compartmentalized metabolic model and predicted life cycle stage specific metabolism with the help of a flux balance approach that integrates gene expression data. Predicted metabolite exchanges between parasite and host were found to be in good accordance with experimental findings when the parasite's metabolic network was embedded into that of its host (erythrocyte). Knock-out simulations identified 307 indispensable metabolic reactions within the parasite. 35 out of 57 experimentally demonstrated essential enzymes were recovered and another 16 enzymes, if additionally the assumption was made that nutrient uptake from the host cell is limited and all reactions catalyzed by the inhibited enzyme are blocked. This predicted set of putative drug targets, shown to be enriched with true targets by a factor of at least 2.75, was further analyzed with respect to homology to human enzymes, functional similarity to therapeutic targets in other organisms and their predicted potency for prophylaxis and disease treatment. Conclusions The results suggest that the set of essential enzymes predicted by our flux balance approach represents a promising starting point for further drug development. PMID:20807400

  15. Chemogenomic profiling of Plasmodium falciparum as a tool to aid antimalarial drug discovery

    PubMed Central

    Pradhan, Anupam; Siwo, Geoffrey H.; Singh, Naresh; Martens, Brian; Balu, Bharath; Button-Simons, Katrina A.; Tan, Asako; Zhang, Min; Udenze, Kenneth O.; Jiang, Rays H.Y.; Ferdig, Michael T.; Adams, John H.; Kyle, Dennis E.

    2015-01-01

    The spread of Plasmodium falciparum multidrug resistance highlights the urgency to discover new targets and chemical scaffolds. Unfortunately, lack of experimentally validated functional information about most P. falciparum genes remains a strategic hurdle. Chemogenomic profiling is an established tool for classification of drugs with similar mechanisms of action by comparing drug fitness profiles in a collection of mutants. Inferences of drug mechanisms of action and targets can be obtained by associations between shifts in drug fitness and specific genetic changes in the mutants. In this screen, P. falciparum, piggyBac single insertion mutants were profiled for altered responses to antimalarial drugs and metabolic inhibitors to create chemogenomic profiles. Drugs targeting the same pathway shared similar response profiles and multiple pairwise correlations of the chemogenomic profiles revealed novel insights into drugs’ mechanisms of action. A mutant of the artemisinin resistance candidate gene - “K13-propeller” gene (PF3D7_1343700) exhibited increased susceptibility to artemisinin drugs and identified a cluster of 7 mutants based on similar enhanced responses to the drugs tested. Our approach of chemogenomic profiling reveals artemisinin functional activity, linked by the unexpected drug-gene relationships of these mutants, to signal transduction and cell cycle regulation pathways. PMID:26541648

  16. Purine import into malaria parasites as a target for antimalarial drug development.

    PubMed

    Frame, I J; Deniskin, Roman; Arora, Avish; Akabas, Myles H

    2015-04-01

    Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage pathway enzymes to form the required purine nucleotides. The Plasmodium falciparum genome encodes four putative ENTs (PfENT1-4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (<10 μM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target. PMID:25424653

  17. 2D Inorganic-Antimalarial Drug-Polymer Hybrid with pH-Responsive Solubility.

    PubMed

    Kim, Ji-Yeong; Yang, Jae-Hun; Lee, Ji-Hee; Choi, Goeun; Park, Dae-Hwan; Jo, Mi-Rea; Choi, Soo-Jin; Choy, Jin-Ho

    2015-10-01

    Artesunic acid (ASH), an antimalarial drug, has low oral bioavailability due to its low aqueous solubility. To overcome this problem, artesunate (AS) was intercalated into zinc basic salt (ZBS) via co-precipitation. AS was immobilized with a tilted double layer arrangement, which was also confirmed by XRD and 1-D electron density mapping. In order to decrease the release rate of AS under gastrointestinal conditions and to simultaneously increase the release rate of AS under intestinal conditions, ZBS-AS was coated with EUDRAGIT L100 (ZBS-AS-L100). Finally, we performed an in-vivo pharmacokinetic study to compare the oral bioavailability of AS of ZBS-AS-L100 with that of ASH. Surprisingly, it was found that the former is 5.5 times greater than the latter due to an enhanced solubility of AS thanks to the ternary hybridization with ZBS and EUDRAGIT L100. Therefore, the present ZBS-AS-L100 system has a great potential as a novel antimalarial drug formulation with pH selectivity and enhanced bioavailability. PMID:25965188

  18. Purine import into malaria parasites as a target for antimalarial drug development

    PubMed Central

    Frame, I.J.; Deniskin, Roman; Arora, Avish; Akabas, Myles H.

    2014-01-01

    Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage–pathway enzymes to form the required purine nucleotides. The P. falciparum genome encodes four putative ENTs (PfENT1–4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine-salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (< 10 µM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target. PMID:25424653

  19. CRIMALDDI: a co-ordinated, rational, and integrated effort to set logical priorities in anti-malarial drug discovery initiatives

    PubMed Central

    2010-01-01

    Despite increasing efforts and support for anti-malarial drug R&D, globally anti-malarial drug discovery and development remains largely uncoordinated and fragmented. The current window of opportunity for large scale funding of R&D into malaria is likely to narrow in the coming decade due to a contraction in available resources caused by the current economic difficulties and new priorities (e.g. climate change). It is, therefore, essential that stakeholders are given well-articulated action plans and priorities to guide judgments on where resources can be best targeted. The CRIMALDDI Consortium (a European Union funded initiative) has been set up to develop, through a process of stakeholder and expert consultations, such priorities and recommendations to address them. It is hoped that the recommendations will help to guide the priorities of the European anti-malarial research as well as the wider global discovery agenda in the coming decade. PMID:20626844

  20. A novel asymmetric-loop molecular beacon-based two-phase hybridization assay for accurate and high-throughput detection of multiple drug resistance-conferring point mutations in Mycobacterium tuberculosis

    PubMed Central

    Chen, Qinghai; Wu, Nan; Xie, Meng; Zhang, Bo; Chen, Ming; Li, Jianjun; Zhuo, Lisha; Kuang, Hong; Fu, Weiling

    2012-01-01

    Summary The accurate and high-throughput detection of drug resistance-related multiple point mutations remains a challenge. Although the combination of molecular beacons with bio-immobilization technology, such as microarray, is promising, its application is difficult due to the ineffective immobilization of molecular beacons on the chip surface. Here, we propose a novel asymmetric-loop molecular beacon in which the loop consists of 2 parts. One is complementary to a target, while the other is complementary to an oligonucleotide probe immobilized on the chip surface. With this novel probe, a two-phase hybridization assay can be used for simultaneously detecting multiple point mutations. This assay will have advantages, such as easy probe availability, multiplex detection, low background, and high-efficiency hybridization, and may provide a new avenue for the immobilization of molecular beacons and high-throughput detection of point mutations. PMID:22460100

  1. Molecular Farming in Artemisia annua, a Promising Approach to Improve Anti-malarial Drug Production

    PubMed Central

    Pulice, Giuseppe; Pelaz, Soraya; Matías-Hernández, Luis

    2016-01-01

    Malaria is a parasite infection affecting millions of people worldwide. Even though progress has been made in prevention and treatment of the disease; an estimated 214 million cases of malaria occurred in 2015, resulting in 438,000 estimated deaths; most of them occurring in Africa among children under the age of five. This article aims to review the epidemiology, future risk factors and current treatments of malaria, with particular focus on the promising potential of molecular farming that uses metabolic engineering in plants as an effective anti-malarial solution. Malaria represents an example of how a health problem may, on one hand, influence the proper development of a country, due to its burden of the disease. On the other hand, it constitutes an opportunity for lucrative business of diverse stakeholders. In contrast, plant biofarming is proposed here as a sustainable, promising, alternative for the production, not only of natural herbal repellents for malaria prevention but also for the production of sustainable anti-malarial drugs, like artemisinin (AN), used for primary parasite infection treatments. AN, a sesquiterpene lactone, is a natural anti-malarial compound that can be found in Artemisia annua. However, the low concentration of AN in the plant makes this molecule relatively expensive and difficult to produce in order to meet the current worldwide demand of Artemisinin Combination Therapies (ACTs), especially for economically disadvantaged people in developing countries. The biosynthetic pathway of AN, a process that takes place only in glandular secretory trichomes of A. annua, is relatively well elucidated. Significant efforts have been made using plant genetic engineering to increase production of this compound. These include diverse genetic manipulation approaches, such as studies on diverse transcription factors which have been shown to regulate the AN genetic pathway and other biological processes. Results look promising; however, further

  2. Psychiatric effects of malaria and anti-malarial drugs: historical and modern perspectives.

    PubMed

    Nevin, Remington L; Croft, Ashley M

    2016-01-01

    The modern medical literature implicates malaria, and particularly the potentially fatal form of cerebral malaria, with a risk of neurocognitive impairment. Yet historically, even milder forms of malaria were associated in the literature with a broad range of psychiatric effects, including disorders of personality, mood, memory, attention, thought, and behaviour. In this article, the history of psychiatric effects attributed to malaria and post-malaria syndromes is reviewed, and insights from the historical practice of malariotherapy in contributing to understanding of these effects are considered. This review concludes with a discussion of the potentially confounding role of the adverse effects of anti-malarial drugs, particularly of the quinoline class, in the unique attribution of certain psychiatric effects to malaria, and of the need for a critical reevaluation of the literature in light of emerging evidence of the chronic nature of these adverse drug effects. PMID:27335053

  3. Holographic analysis on deformation and restoration of malaria-infected red blood cells by antimalarial drug

    NASA Astrophysics Data System (ADS)

    Byeon, Hyeokjun; Ha, Young-Ran; Lee, Sang Joon

    2015-11-01

    Malaria parasites induce morphological, biochemical, and mechanical changes in red blood cells (RBCs). Mechanical variations are closely related to the deformability of individual RBCs. The deformation of various RBCs, including healthy and malaria-infected RBCs (iRBCs), can be directly observed through quantitative phase imaging (QPI). The effects of chloroquine treatment on the mechanical property variation of iRBCs were investigated using time-resolved holographic QPI of single live cells on a millisecond time scale. The deformabilities of healthy RBCs, iRBCs, and drug-treated iRBCs were compared, and the effect of chloroquine on iRBC restoration was experimentally examined. The present results are beneficial to elucidate the dynamic characteristics of iRBCs and the effect of the antimalarial drug on iRBCs.

  4. MIPHENO: Data normalization for high throughput metabolic analysis.

    EPA Science Inventory

    High throughput methodologies such as microarrays, mass spectrometry and plate-based small molecule screens are increasingly used to facilitate discoveries from gene function to drug candidate identification. These large-scale experiments are typically carried out over the course...

  5. Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

    NASA Astrophysics Data System (ADS)

    Wright, Megan H.; Clough, Barbara; Rackham, Mark D.; Rangachari, Kaveri; Brannigan, James A.; Grainger, Munira; Moss, David K.; Bottrill, Andrew R.; Heal, William P.; Broncel, Malgorzata; Serwa, Remigiusz A.; Brady, Declan; Mann, David J.; Leatherbarrow, Robin J.; Tewari, Rita; Wilkinson, Anthony J.; Holder, Anthony A.; Tate, Edward W.

    2014-02-01

    Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase.

  6. Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach.

    PubMed

    Wright, Megan H; Clough, Barbara; Rackham, Mark D; Rangachari, Kaveri; Brannigan, James A; Grainger, Munira; Moss, David K; Bottrill, Andrew R; Heal, William P; Broncel, Malgorzata; Serwa, Remigiusz A; Brady, Declan; Mann, David J; Leatherbarrow, Robin J; Tewari, Rita; Wilkinson, Anthony J; Holder, Anthony A; Tate, Edward W

    2014-02-01

    Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase. PMID:24451586

  7. Understanding the biology of the Plasmodium falciparum apicoplast; an excellent target for antimalarial drug development.

    PubMed

    Chakraborty, Arnish

    2016-08-01

    Malaria is a life-threatening tropical disease, caused by the intracellular parasite Plasmodium falciparum. The World Health Organization counts malaria as one of the top ten causes of worldwide death. The unavailability of a successful malaria vaccine and the ever-increasing instances of drug resistance in the malaria parasite demand the discovery of new targets within P. falciparum for the development of next generation antimalarials. Fortunately, all apicomplexan parasites, including P. falciparum harbor a relict, non-photosynthetic plastid known as the apicoplast. The apicoplast is a semi-autonomous organelle within P. falciparum containing a 35kb circular genome. Despite a genome of its own, majority of the apicoplast proteins are encoded by the parasite nucleus and imported into the apicoplast. The organelle has been shown to be essential to P. falciparum survival and the loss the apicoplast manifests as a 'delayed death' response in the parasite. The apicoplast has evolved out of cyanobacteria in a complex, two step endosymbiotic event. As a result the architecture and the gene expression machinery of the apicoplast is quite bacteria-like and is susceptible to a wide range of antibiotics such as fosmidomycin, tetracycline, azithromycin, clindamycin and triclosan. The biosynthetic pathways for isoprenoids, fatty acids and heme operate within the malaria apicoplast, making the organelle an excellent target for drug development. The review focuses on the evolution, biology and the essentiality of the apicoplast within the malaria parasite and discusses some of the recent achievements towards the design and discovery of apicoplast targeted antimalarial compounds. PMID:27381078

  8. Evaluation of bioluminescence-based assays of anti-malarial drug activity

    PubMed Central

    2013-01-01

    Background Transgenic Plasmodium falciparum expressing luciferase offers an attractive bioluminescence-based assay platform for the investigation of the pharmacological properties of anti-malarial drugs. Here a side-by-side comparison of bioluminescence and fluorescence-based assays, utilizing a luciferase reporter cassette that confers a strong temporal pattern of luciferase expression during the S-phase of intraerythrocytic development, is reported. Methods Key assay parameters for a range of commercially available luminogenic substrates are determined and compared to those measured using a Malaria Sybr Green I fluorescence assay. In addition, the short-term temporal effects of anti-malarial compounds are evaluated using both bioluminescent and fluorescent assay platforms. Results The Z’, % coefficient of variation and 50% inhibition concentrations are essentially the same for bioluminescent and fluorescent assays in transgenic parasites generated in both chloroquine-sensitive and -resistant genetic backgrounds. Bioluminescent assays, irrespective of the luminogenic agent employed, do, however, offer significantly enhanced signal-to-noise ratios. Moreover, the bioluminescent assay is more dynamic in terms of determining temporal effects immediately following drug perturbation. Conclusion This study suggests that opportunities for bioluminescence-based assays lie not in the measurement of 50% inhibition concentrations, where the cheaper fluorescence assay performs excellently and is not restricted by the need to genetically modify the parasite clone under investigation. Instead, assays that use the dynamic response of the luciferase reporter for semi-automated screening of additional pharmacological properties, such as relative rate-of-kill and lethal dose estimation, are a more attractive development opportunity. PMID:23394077

  9. Stability of the Antimalarial Drug Dihydroartemisinin under Physiologically Relevant Conditions: Implications for Clinical Treatment and Pharmacokinetic and In Vitro Assays

    PubMed Central

    Parapini, Silvia; Olliaro, Piero; Navaratnam, Visweswaran; Taramelli, Donatella

    2015-01-01

    Artemisinins are peroxidic antimalarial drugs known to be very potent but highly chemically unstable; they degrade in the presence of ferrous iron, Fe(II)-heme, or biological reductants. Less documented is how this translates into chemical stability and antimalarial activity across a range of conditions applying to in vitro testing and clinical situations. Dihydroartemisinin (DHA) is studied here because it is an antimalarial drug on its own and the main metabolite of other artemisinins. The behaviors of DHA in phosphate-buffered saline, plasma, or erythrocyte lysate at different temperatures and pH ranges were examined. The antimalarial activity of the residual drug was evaluated using the chemosensitivity assay on Plasmodium falciparum, and the extent of decomposition of DHA was established through use of high-performance liquid chromatography with electrochemical detection analysis. The role of the Fe(II)-heme was investigated by blocking its reactivity using carbon monoxide (CO). A significant reduction in the antimalarial activity of DHA was seen after incubation in plasma and to a lesser extent in erythrocyte lysate. Activity was reduced by half after 3 h and almost completely abolished after 24 h. Serum-enriched media also affected DHA activity. Effects were temperature and pH dependent and paralleled the increased rate of decomposition of DHA from pH 7 upwards and in plasma. These results suggest that particular care should be taken in conducting and interpreting in vitro studies, prone as their results are to experimental and drug storage conditions. Disorders such as fever, hemolysis, or acidosis associated with malaria severity may contribute to artemisinin instability and reduce their clinical efficacy. PMID:25918150

  10. Practical aspects of in vivo antimalarial drug efficacy testing in the Americas.

    PubMed

    Ruebush, Trenton K; Marquiño, Wilmer; Zegarra, Jorge; Neyra, Daniel; Villaroel, Rodolfo; Avila, Juan Carlos; Díaz, César; Beltrán, Efraín

    2003-04-01

    The World Health Organization (WHO) has developed guidelines for in vivo antimalarial drug efficacy testing for Plasmodium falciparum and Plasmodium vivax in areas with low-to-moderate transmission, such as the Americas. These guidelines are used widely by ministries of health and national malaria control programs to assess the efficacy of their first-line and second-line drugs for the treatment of malaria and to provide the information necessary to update national malaria treatment policies. Following the WHO guidelines, we have conducted in vivo efficacy trials with a variety of drugs and drug combinations against P. falciparum and P. vivax at 13 sites in Peru, Bolivia, and Ecuador. Based on these experiences, we have identified several modifications that we believe should be made in the WHO recommendations to make them more suitable to the relatively low levels of P. falciparum transmission in the Americas and to the logistic challenges of carrying out such studies in sparsely populated areas, such as the Amazon Basin. These include changes in inclusion and exclusion criteria, in enrollment and follow-up procedures, and in the measurement of study outcomes. PMID:12875285

  11. Mass administration of the antimalarial drug mefloquine to Guantánamo detainees: a critical analysis.

    PubMed

    Nevin, Remington L

    2012-10-01

    Recently, evidence has emerged from an unusual form of mass drug administration practised among detainees held at US Naval Station Guantánamo Bay, Cuba ('Guantánamo'), ostensibly as a public health measure. Mefloquine, an antimalarial drug originally developed by the US military, whose use is associated with a range of severe neuropsychiatric adverse effects, was administered at treatment doses to detainees immediately upon their arrival at Guantánamo, prior to laboratory testing for malaria and irrespective of symptoms of disease. In this analysis, the history of mefloquine's development is reviewed and the indications for its administration at treatment doses are discussed. The stated rationale for the use of mefloquine among Guantánamo detainees is then evaluated in the context of accepted forms of population-based malaria control. It is concluded that there was no plausible public health indication for the use of mefloquine at Guantánamo and that based on prevailing standards of care, the clinical indications for its use are decidedly unclear. This analysis suggests the troubling possibility that the use of mefloquine at Guantánamo may have been motivated in part by knowledge of the drug's adverse effects, and points to a critical need for further investigation to resolve unanswered questions regarding the drug's potentially inappropriate use. PMID:22882560

  12. High throughput screening technologies for ion channels

    PubMed Central

    Yu, Hai-bo; Li, Min; Wang, Wei-ping; Wang, Xiao-liang

    2016-01-01

    Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications. PMID:26657056

  13. High-throughput tetrad analysis.

    PubMed

    Ludlow, Catherine L; Scott, Adrian C; Cromie, Gareth A; Jeffery, Eric W; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L; Hays, Michelle; Dudley, Aimée M

    2013-07-01

    Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious. PMID:23666411

  14. High-throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

    A cryopump with a unique method of regeneration which allows continuous operation at high throughput has been constructed and tested. Deuterium was pumped continuously at a throughput of 30 Torr.L/s at a speed of 2000 L/s and a compression ratio of 200. Argon was pumped at a throughput of 60 Torr.L/s at a speed of 1275 L/s. To produce continuous operation of the pump, a method of regeneration that does not thermally cycle the pump is employed. A small chamber (the ''snail'') passes over the pumping surface and removes the frost from it either by mechanical action with a scraper or by local heating. The material removed is topologically in a secondary vacuum system with low conductance into the primary vacuum; thus, the exhaust can be pumped at pressures up to an effective compression ratio determined by the ratio of the pumping speed to the leakage conductance of the snail. The pump, which is all-metal-sealed and dry and which regenerates every 60 s, would be an ideal system for pumping tritium. Potential fusion applications are for mpmp limiters, for repeating pneumatic pellet injection lines, and for the centrifuge pellet injector spin tank, all of which will require pumping tritium at high throughput. Industrial applications requiring ultraclean pumping of corrosive gases at high throughput, such as the reactive ion etch semiconductor process, may also be feasible.

  15. Interactions between artemisinins and other antimalarial drugs in relation to the cofactor model--a unifying proposal for drug action.

    PubMed

    Haynes, Richard K; Cheu, Kwan-Wing; Chan, Ho-Wai; Wong, Ho-Ning; Li, Ka-Yan; Tang, Maggie Mei-Ki; Chen, Min-Jiao; Guo, Zu-Feng; Guo, Zhi-Hong; Sinniah, Kumar; Witte, Amanda B; Coghi, Paolo; Monti, Diego

    2012-12-01

    Artemisinins are proposed to act in the malaria parasite cytosol by oxidizing dihydroflavin cofactors of redox-active flavoenzymes, and under aerobic conditions by inducing their autoxidation. Perturbation of redox homeostasis coupled with the generation of reactive oxygen species (ROS) ensues. Ascorbic acid-methylene blue (MB), N-benzyl-1,4-dihydronicotinamide (BNAH)-MB, BNAH-lumiflavine, BNAH-riboflavin (RF), and NADPH-FAD-E. coli flavin reductase (Fre) systems at pH 7.4 generate leucomethylene blue (LMB) and reduced flavins that are rapidly oxidized in situ by artemisinins. These oxidations are inhibited by the 4-aminoquinolines piperaquine (PPQ), chloroquine (CQ), and others. In contrast, the arylmethanols lumefantrine, mefloquine (MFQ), and quinine (QN) have little or no effect. Inhibition correlates with the antagonism exerted by 4-aminoquinolines on the antimalarial activities of MB, RF, and artemisinins. Lack of inhibition correlates with the additivity/synergism between the arylmethanols and artemisinins. We propose association via π complex formation between the 4-aminoquinolines and LMB or the dihydroflavins; this hinders hydride transfer from the reduced conjugates to the artemisinins. The arylmethanols have a decreased tendency to form π complexes, and so exert no effect. The parallel between chemical reactivity and antagonism or additivity/synergism draws attention to the mechanism of action of all drugs described herein. CQ and QN inhibit the formation of hemozoin in the parasite digestive vacuole (DV). The buildup of heme-Fe(III) results in an enhanced efflux from the DV into the cytosol. In addition, the lipophilic heme-Fe(III) complexes of CQ and QN that form in the DV are proposed to diffuse across the DV membrane. At the higher pH of the cytosol, the complexes decompose to liberate heme-Fe(III) . The quinoline or arylmethanol reenters the DV, and so transfers more heme-Fe(III) out of the DV. In this way, the 4-aminoquinolines and arylmethanols

  16. Validation of ELISA for Quantitation of Artemisinin-Based Antimalarial Drugs

    PubMed Central

    Wang, Min; Cui, Yongliang; Zhou, Guofa; Yan, Guiyun; Cui, Liwang; Wang, Baomin

    2013-01-01

    The circulation of counterfeit or substandard artemisinins (ARTs) in malaria-endemic areas poses a serious threat to the long-term use of these drugs. Here, we validated an indirect competitive enzyme-linked immunosorbent assay (icELISA) for quantification of ARTs and found that 50% of inhibitory concentrations of dihydroartemisinin, artemether, and artesunate were 8.1, 207.0, and 4.7 ng/mL, respectively. We compared the icELISA with high-performance liquid chromatography (HPLC) for quantifying ART and its derivatives in 22 convenience samples of commercial antimalarial drugs. Paired t tests showed a borderline significant difference between the two methods (mean = 0.03, 95% confidence interval [CI] 0.00–0.07, P = 0.074) and the icELISA results were more variable than those of the HPLC analysis (P < 0.001), suggesting that further improvement is needed to enhance the performance of the icELISA. Our results showed that the icELISA has the potential to be improved for quality assurance of ARTs at the point of care in endemic settings. PMID:24080636

  17. Fast Gradient Elution Reversed-Phase HPLC with Diode-Array Detection as a High Throughput Screening Method for Drugs of Abuse

    SciTech Connect

    Peter W. Carr; K.M. Fuller; D.R. Stoll; L.D. Steinkraus; M.S. Pasha; Glenn G. Hardin

    2005-12-30

    A new approach has been developed by modifying a conventional gradient elution liquid chromatograph for the high throughput screening of biological samples to detect the presence of regulated intoxicants. The goal of this work was to improve the speed of a gradient elution screening method over current approaches by optimizing the operational parameters of both the column and the instrument without compromising the reproducibility of the retention times, which are the basis for the identification. Most importantly, the novel instrument configuration substantially reduces the time needed to re-equilibrate the column between gradient runs, thereby reducing the total time for each analysis. The total analysis time for each gradient elution run is only 2.8 minutes, including 0.3 minutes for column reequilibration between analyses. Retention times standard calibration solutes are reproducible to better than 0.002 minutes in consecutive runs. A corrected retention index was adopted to account for day-to-day and column-to-column variations in retention time. The discriminating power and mean list length were calculated for a library of 47 intoxicants and compared with previous work from other laboratories to evaluate fast gradient elution HPLC as a screening tool.

  18. High-Throughput Cell Toxicity Assays.

    PubMed

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

    Understanding compound-driven cell toxicity is vitally important for all drug discovery approaches. With high-throughput screening (HTS) being the key strategy to find hit and lead compounds for drug discovery projects in the pharmaceutical industry [1], an understanding of the cell toxicity profile of hit molecules from HTS activities is fundamentally important. Recently, there has been a resurgence of interest in phenotypic drug discovery and these cell-based assays are now being run in HTS labs on ever increasing numbers of compounds. As the use of cell assays increases the ability to measure toxicity of compounds on a large scale becomes increasingly important to ensure that false hits are not progressed and that compounds do not carry forward a toxic liability that may cause them to fail at later stages of a project. Here we describe methods employed in the AstraZeneca HTS laboratory to carry out very large scale cell toxicity screening. PMID:27317000

  19. Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

    PubMed

    Ro, Dae-Kyun; Paradise, Eric M; Ouellet, Mario; Fisher, Karl J; Newman, Karyn L; Ndungu, John M; Ho, Kimberly A; Eachus, Rachel A; Ham, Timothy S; Kirby, James; Chang, Michelle C Y; Withers, Sydnor T; Shiba, Yoichiro; Sarpong, Richmond; Keasling, Jay D

    2006-04-13

    Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices. PMID:16612385

  20. Salinomycin and Other Ionophores as a New Class of Antimalarial Drugs with Transmission-Blocking Activity

    PubMed Central

    D'Alessandro, Sarah; Corbett, Yolanda; Ilboudo, Denise P.; Misiano, Paola; Dahiya, Nisha; Abay, Solomon M.; Habluetzel, Annette; Grande, Romualdo; Gismondo, Maria R.; Dechering, Koen J.; Koolen, Karin M. J.; Sauerwein, Robert W.; Taramelli, Donatella; Parapini, Silvia

    2015-01-01

    The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads. PMID:26055362

  1. High throughput protein production screening

    DOEpatents

    Beernink, Peter T.; Coleman, Matthew A.; Segelke, Brent W.

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

  2. High-Throughput Sequencing Technologies

    PubMed Central

    Reuter, Jason A.; Spacek, Damek; Snyder, Michael P.

    2015-01-01

    Summary The human genome sequence has profoundly altered our understanding of biology, human diversity and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past ten years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them as well as the challenges facing current sequencing platforms and their clinical application. PMID:26000844

  3. Antimalarial drug utilization by women in Ethiopia: a knowledge-attitudes-practice study.

    PubMed Central

    Yeneneh, H.; Gyorkos, T. W.; Joseph, L.; Pickering, J.; Tedla, S.

    1993-01-01

    A survey was undertaken between December 1991 and February 1992 to assess the knowledge, attitudes, and practices with respect to malaria of 300 women from six randomly selected rural communities in central Ethiopia. A total of 85% were able to recognize one or more of the common symptoms of the disease; however, the modes of transmission were generally misunderstood and only 23% believed that transmission could be prevented. More women preferred to obtain antimalarials from government clinics rather than from private drug shops, mission clinics, unofficial suppliers of injections, open markets, or from leftover sources. Under-5-year-olds were identified as the most malaria-vulnerable group and given priority for treatment; severity of illness was the principal determinant in seeking treatment. Decisions about treatment were generally made jointly by both parents. Knowledge about the transmissibility of malaria decreased with increasing distance from a health unit (odds ratio: 0.48; 95% confidence interval: 0.27, 0.86). A logistic regression analysis indicated that literacy and village were the most important variables associated with knowledge about preventing malaria. PMID:8313494

  4. Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

    SciTech Connect

    Paeshuyse, Jan; Coelmont, Lotte; Vliegen, Inge; Hemel, Johan van; Vandenkerckhove, Jan; Peys, Eric; Sas, Benedikt; Clercq, Erik De; Neyts, Johan . E-mail: johan.neyts@rega.kuleuven.be

    2006-09-15

    We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC{sub 5}) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78 {+-} 21 {mu}M. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 {mu}M) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.

  5. High-throughput screening for modulators of cellular contractile force†

    PubMed Central

    Park, Chan Young; Zhou, Enhua H.; Tambe, Dhananjay; Chen, Bohao; Lavoie, Tera; Dowell, Maria; Simeonov, Anton; Maloney, David J.; Marinkovic, Aleksandar; Tschumperlin, Daniel J.; Burger, Stephanie; Frykenberg, Matthew; Butler, James P.; Stamer, W. Daniel; Johnson, Mark; Solway, Julian; Fredberg, Jeffrey J.

    2015-01-01

    When cellular contractile forces are central to pathophysiology, these forces comprise a logical target of therapy. Nevertheless, existing high-throughput screens are limited to upstream signalling intermediates with poorly defined relationships to such a physiological endpoint. Using cellular force as the target, here we report a new screening technology and demonstrate its applications using human airway smooth muscle cells in the context of asthma and Schlemm's canal endothelial cells in the context of glaucoma. This approach identified several drug candidates for both asthma and glaucoma. We attained rates of 1000 compounds per screening day, thus establishing a force-based cellular platform for high-throughput drug discovery. PMID:25953078

  6. Assessing the utility of an anti-malarial pharmacokinetic-pharmacodynamic model for aiding drug clinical development

    PubMed Central

    2012-01-01

    Background Mechanistic within-host models relating blood anti-malarial drug concentrations with the parasite-time profile help in assessing dosing schedules and partner drugs for new anti-malarial treatments. A comprehensive simulation study to assess the utility of a stage-specific pharmacokinetic-pharmacodynamic (PK-PD) model for predicting within-host parasite response was performed. Methods Three anti-malarial combination therapies were selected: artesunate-mefloquine, dihydroartemisinin-piperaquine, and artemether-lumefantrine. The PK-PD model included parameters to represent the concentration-time profiles of both drugs, the initial parasite burden and distribution across the parasite life cycle, and the parasite multiplication factor due to asexual reproduction. The model also included the maximal killing rate of each drug, and the blood drug concentration associated with half of that killing effect (in vivo EC50), derived from the in vitro IC50, the extent of binding to 0.5% Albumax present in the in vitro testing media, and the drugs plasma protein binding and whole blood to plasma partitioning ratio. All stochastic simulations were performed using a Latin-Hypercube-Sampling approach. Results The simulations demonstrated that the proportion of patients cured was highly sensitive to the in vivo EC50 and the maximal killing rate of the partner drug co-administered with the artemisinin derivative. The in vivo EC50 values that corresponded to on average 95% of patients cured were much higher than the adjusted values derived from the in vitro IC50. The proportion clinically cured was not strongly influenced by changes in the parameters defining the age distribution of the initial parasite burden (mean age of 4 to 16 hours) and the parasite multiplication factor every life cycle (ranging from 8 to 12 fold/cycle). The median parasite clearance times, however, lengthened as the standard deviation of the initial parasite burden increased (i.e. the infection became

  7. High-throughput Tetrad Analysis

    PubMed Central

    Ludlow, Catherine L.; Scott, Adrian C.; Cromie, Gareth A.; Jeffery, Eric W.; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L.; Hays, Michelle; Dudley, Aimée M.

    2013-01-01

    Tetrad analysis has been a gold standard genetic technique for several decades. Unfortunately, the manual nature of the process has relegated its application to small-scale studies and limited its integration with rapidly evolving DNA sequencing technologies. We have developed a rapid, high-throughput method, called Barcode Enabled Sequencing of Tetrads (BEST), that replaces the manual processes of isolating, disrupting and spacing tetrads. BEST uses a meiosis-specific GFP fusion protein to isolate tetrads by fluorescence-activated cell sorting and molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. We demonstrate the approach in Saccharomyces cerevisiae, but BEST is readily transferable to microorganisms in which meiotic mapping is significantly more laborious. PMID:23666411

  8. Incorporating Stage-Specific Drug Action into Pharmacological Modeling of Antimalarial Drug Treatment

    PubMed Central

    2016-01-01

    Pharmacological modeling of antiparasitic treatment based on a drug's pharmacokinetic and pharmacodynamic properties plays an increasingly important role in identifying optimal drug dosing regimens and predicting their potential impact on control and elimination programs. Conventional modeling of treatment relies on methods that do not distinguish between parasites at different developmental stages. This is problematic for malaria parasites, as their sensitivity to drugs varies substantially during their 48-h developmental cycle. We investigated four drug types (short or long half-lives with or without stage-specific killing) to quantify the accuracy of the standard methodology. The treatment dynamics of three drug types were well characterized with standard modeling. The exception were short-half-life drugs with stage-specific killing (i.e., artemisinins) because, depending on time of treatment, parasites might be in highly drug-sensitive stages or in much less sensitive stages. We describe how to bring such drugs into pharmacological modeling by including additional variation into the drug's maximal killing rate. Finally, we show that artemisinin kill rates may have been substantially overestimated in previous modeling studies because (i) the parasite reduction ratio (PRR) (generally estimated to be 104) is based on observed changes in circulating parasite numbers, which generally overestimate the “true” PRR, which should include both circulating and sequestered parasites, and (ii) the third dose of artemisinin at 48 h targets exactly those stages initially hit at time zero, so it is incorrect to extrapolate the PRR measured over 48 h to predict the impact of doses at 48 h and later. PMID:26902760

  9. Incorporating Stage-Specific Drug Action into Pharmacological Modeling of Antimalarial Drug Treatment.

    PubMed

    Hodel, Eva Maria; Kay, Katherine; Hastings, Ian M

    2016-05-01

    Pharmacological modeling of antiparasitic treatment based on a drug's pharmacokinetic and pharmacodynamic properties plays an increasingly important role in identifying optimal drug dosing regimens and predicting their potential impact on control and elimination programs. Conventional modeling of treatment relies on methods that do not distinguish between parasites at different developmental stages. This is problematic for malaria parasites, as their sensitivity to drugs varies substantially during their 48-h developmental cycle. We investigated four drug types (short or long half-lives with or without stage-specific killing) to quantify the accuracy of the standard methodology. The treatment dynamics of three drug types were well characterized with standard modeling. The exception were short-half-life drugs with stage-specific killing (i.e., artemisinins) because, depending on time of treatment, parasites might be in highly drug-sensitive stages or in much less sensitive stages. We describe how to bring such drugs into pharmacological modeling by including additional variation into the drug's maximal killing rate. Finally, we show that artemisinin kill rates may have been substantially overestimated in previous modeling studies because (i) the parasite reduction ratio (PRR) (generally estimated to be 10(4)) is based on observed changes in circulating parasite numbers, which generally overestimate the "true" PRR, which should include both circulating and sequestered parasites, and (ii) the third dose of artemisinin at 48 h targets exactly those stages initially hit at time zero, so it is incorrect to extrapolate the PRR measured over 48 h to predict the impact of doses at 48 h and later. PMID:26902760

  10. Blocking Plasmodium falciparum Malaria Transmission with Drugs: The Gametocytocidal and Sporontocidal Properties of Current and Prospective Antimalarials

    PubMed Central

    Kiszewski, Anthony E.

    2011-01-01

    Drugs that kill or inhibit the sexual stages of Plasmodium could potentially amplify or synergize the impact of other interventions by blocking transmission to mosquitoes. Primaquine and other 8-aminoquinolines have long offered such potential, but safety and other concerns have limited their use. Although transmission-blocking properties are not often a priority of drug discovery efforts, a number of interesting gametocytocidal and/or sporontocidal drug candidates have emerged in recent years. Some still bear significant technical and safety concerns, while others have passed clinical trials and are on the verge of entering the antimalarial armamentarium. Recent advances in our knowledge of gametocyte differentiation, gametogenesis and sporogony have also led to the identification of a large array of potential new targets for drugs that might interfere with malaria transmission. This review examines the properties of existing and prospective drugs, mechanisms of action, counter-indications and their potential role in regional malaria elimination efforts.