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Sample records for selective faah inhibitor

  1. Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor.

    PubMed

    Johnson, Douglas S; Stiff, Cory; Lazerwith, Scott E; Kesten, Suzanne R; Fay, Lorraine K; Morris, Mark; Beidler, David; Liimatta, Marya B; Smith, Sarah E; Dudley, David T; Sadagopan, Nalini; Bhattachar, Shobha N; Kesten, Stephen J; Nomanbhoy, Tyzoon K; Cravatt, Benjamin F; Ahn, Kay

    2011-02-10

    Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.

  2. FAAH inhibitors in the limelight, but regrettably

    PubMed Central

    Mallet, Christophe; Dubray, Claude; Dualé, Christian

    2016-01-01

    Abstract. This short review focuses on the recent drug development of FAAH inhibitors, as recent serious adverse events have been reported in a phase I study with a compound of this class. The authors overview the potential interest in targeting FAAH inhibition, the current programs, and the available information on the recent dramatic events. PMID:27191771

  3. Switching cannabinoid response from CB(2) agonists to FAAH inhibitors.

    PubMed

    Tourteau, Aurélien; Leleu-Chavain, Natascha; Body-Malapel, Mathilde; Andrzejak, Virginie; Barczyk, Amélie; Djouina, Madjid; Rigo, Benoit; Desreumaux, Pierre; Chavatte, Philippe; Millet, Régis

    2014-03-01

    A series of 3-carboxamido-5-aryl-isoxazoles designed as CB2 agonists were evaluated as FAAH inhibitors. The pharmacological results led to identify structure-activity relationships enabling to switch cannabinoid response from CB2 agonists to FAAH inhibitors. Two compounds were selected for their FAAH and/or CB2 activity, and evaluated in a colitis model for their anti-inflammatory activity. Results showed that compounds 10 and 11 inhibit the development of DSS-induced acute colitis in mice and then, are interesting leads to explore new drug candidates for IBD.

  4. Pyrazole phenylcyclohexylcarbamates as inhibitors of human fatty acid amide hydrolases (FAAH).

    PubMed

    Aghazadeh Tabrizi, Mojgan; Baraldi, Pier Giovanni; Ruggiero, Emanuela; Saponaro, Giulia; Baraldi, Stefania; Romagnoli, Romeo; Martinelli, Adriano; Tuccinardi, Tiziano

    2015-06-05

    Fatty acid amide hydrolase (FAAH) inhibitors have gained attention as potential therapeutic targets in the management of neuropathic pain. Here, we report a series of pyrazole phenylcyclohexylcarbamate derivatives standing on the known carbamoyl FAAH inhibitor URB597. Structural modifications led to the recognition of compound 22 that inhibited human recombinant FAAH (hrFAAH) in the low nanomolar range (IC50 = 11 nM). The most active compounds of this series showed significant selectivity toward monoacylglycerol lipase (MAGL) enzyme. In addition, molecular modeling and reversibility behavior of the new class of FAAH inhibitors are presented in this article.

  5. Systemic and spinal administration of FAAH, MAGL inhibitors and dual FAAH/MAGL inhibitors produce antipruritic effect in mice.

    PubMed

    Yesilyurt, Ozgur; Cayirli, Mutlu; Sakin, Yusuf Serdar; Seyrek, Melik; Akar, Ahmet; Dogrul, Ahmet

    2016-07-01

    The increase of endocannabinoid tonus by inhibiting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) represents a promising therapeutic approach in a variety of disease to overcome serious central side effects of exocannabinoids. Recent studies reported that systemic administration of FAAH and MAGL inhibitors produce antipruritic action. Dual FAAH/MAGL inhibitors have also been described to get enhanced endocannabinoid therapeutic effect. In this study, we examined and compared dose-related antipruritic effects of systemic (intraperitoneal; ip) or intrathecal (it) administration of selective FAAH inhibitor PF-3845 (5, 10, and 20 mg/kg, i.p.; 1, 5, and 10 µg, i.t.), MAGL inhibitor JZL184 (4, 20, and 40 mg/kg, i.p.; 1, 5, and 10 µg, i.t.) and dual FAAH/MAGL inhibitor JZL195 (2, 5, and 20 mg/kg, i.p.; 1, 5, and 10 µg, i.t.) on serotonin (5-HT)-induced scratching model. Serotonin (25 μg) was injected intradermally in a volume of 50 μl into the rostral part of skin on the back of male Balb-C mice. Both systemic or intrathecal administration of PF-3845, JZL184 or JZL195 produced similar dose-dependent antipruritic effects. Our results suggest that endocannabinoid-degrading enzymes FAAH and MAGL are involved in pruritic process at spinal level. FAAH, MAGL or dual FAAH/MAGL inhibitors have promising antipruritic effects, at least, in part through spinal site of action.

  6. Piperazine and piperidine carboxamides and carbamates as inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

    PubMed

    Korhonen, Jani; Kuusisto, Anne; van Bruchem, John; Patel, Jayendra Z; Laitinen, Tuomo; Navia-Paldanius, Dina; Laitinen, Jarmo T; Savinainen, Juha R; Parkkari, Teija; Nevalainen, Tapio J

    2014-12-01

    The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), are potential targets for various therapeutic applications. In this paper, we present more extensively the results of our previous work on piperazine and piperidine carboxamides and carbamates as FAAH and MAGL inhibitors. The best compounds of these series function as potent and selective MAGL/FAAH inhibitors or as dual FAAH/MAGL inhibitors at nanomolar concentrations. This study revealed that MAGL inhibitors should comprise leaving-groups with a conjugate acid pKa of 8-10, while diverse leaving groups are tolerated for FAAH inhibitors.

  7. Structure-guided inhibitor design for human FAAH by interspecies active site conversion.

    PubMed

    Mileni, Mauro; Johnson, Douglas S; Wang, Zhigang; Everdeen, Daniel S; Liimatta, Marya; Pabst, Brandon; Bhattacharya, Keshab; Nugent, Richard A; Kamtekar, Satwik; Cravatt, Benjamin F; Ahn, Kay; Stevens, Raymond C

    2008-09-02

    The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-A crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.

  8. Preclinical Characterization of the FAAH Inhibitor JNJ-42165279

    PubMed Central

    2015-01-01

    The pre-clinical characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concentrations of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compound was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good physical, ADME, and PD properties of JNJ-42165279 supported it entering the clinical portfolio. PMID:26713105

  9. Preclinical Characterization of the FAAH Inhibitor JNJ-42165279.

    PubMed

    Keith, John M; Jones, William M; Tichenor, Mark; Liu, Jing; Seierstad, Mark; Palmer, James A; Webb, Michael; Karbarz, Mark; Scott, Brian P; Wilson, Sandy J; Luo, Lin; Wennerholm, Michelle L; Chang, Leon; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra R; Breitenbucher, J Guy

    2015-12-10

    The pre-clinical characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concentrations of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compound was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good physical, ADME, and PD properties of JNJ-42165279 supported it entering the clinical portfolio.

  10. Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH).

    PubMed

    Min, Xiaoshan; Thibault, Stephen T; Porter, Amy C; Gustin, Darin J; Carlson, Timothy J; Xu, Haoda; Lindstrom, Michelle; Xu, Guifen; Uyeda, Craig; Ma, Zhihua; Li, Yihong; Kayser, Frank; Walker, Nigel P C; Wang, Zhulun

    2011-05-03

    Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.

  11. Design, synthesis and biological evaluation of potent FAAH inhibitors.

    PubMed

    Tuo, Wei; Leleu-Chavain, Natascha; Barczyk, Amélie; Renault, Nicolas; Lemaire, Lucas; Chavatte, Philippe; Millet, Régis

    2016-06-01

    A new series of 3-carboxamido-5-aryl-isoxazoles was designed, synthesized and evaluated for their biological activity. Different pharmacomodulations have been explored and the lipophilicity of these compounds was assessed. Investigation of the in vitro biological activity led to the identification of 5 compounds as potent FAAH inhibitors, their good FAAH inhibition capacity is probably correlated with their suitable lipophilicity. Specifically, compound 25 showed similar inhibition potency against FAAH in comparison with URB597, one of the most potent FAAH inhibitor known to date.

  12. FAAH selectively influences placebo effects

    PubMed Central

    Peciña, M.; Martínez-Jauand, M.; Hodgkinson, C.; Stohler, C.S.; Goldman, D.; Zubieta, J.K.

    2013-01-01

    Endogenous opioid and cannabinoid systems are thought to act synergistically regulating antinociceptive and reward mechanisms. To further understand the human implications of the interaction between these two systems, we investigated the role of the common, functional missense variant Pro129Thr of the gene coding fatty acid amide hydrolase (FAAH), the major degrading enzyme of endocannabinoids, on psychophysical and neurotransmitter (dopaminergic, opioid) responses to pain and placebo-induced analgesia in humans. FAAH Pro129/Pro129 homozygotes, who constitute nearly half of the population, reported higher placebo-analgesia and more positive affective states immediately and 24 hours after placebo administration; no effects on pain report in the absence of placebo were observed. Pro129/Pro129 homozygotes also showed greater placebo-induced μ-opioid, but not D2/3 dopaminergic, enhancements in neurotransmission in regions known involved in placebo effects. These results show that a common genetic variation affecting the function of the cannabinoid system is serving as a probe to demonstrate the involvement of cannabinoid and opioid transmitters on the formation of placebo effects. PMID:24042479

  13. Aryl Piperazinyl Ureas as Inhibitors of Fatty Acid Amide Hydrolase (FAAH) in Rat, Dog, and Primate.

    PubMed

    Keith, John M; Apodaca, Rich; Tichenor, Mark; Xiao, Wei; Jones, William; Pierce, Joan; Seierstad, Mark; Palmer, James; Webb, Michael; Karbarz, Mark; Scott, Brian; Wilson, Sandy; Luo, Lin; Wennerholm, Michelle; Chang, Leon; Brown, Sean; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra; Breitenbucher, J Guy

    2012-10-11

    A series of aryl piperazinyl ureas that act as covalent inhibitors of fatty acid amide hydrolase (FAAH) is described. A potent and selective (does not inhibit FAAH-2) member of this class, JNJ-40355003, was found to elevate the plasma levels of three fatty acid amides: anandamide, oleoyl ethanolamide, and palmitoyl ethanolamide, in the rat, dog, and cynomolgous monkey. The elevation of the levels of these lipids in the plasma of monkeys suggests that FAAH-2 may not play a significant role in regulating plasma levels of fatty acid ethanolamides in primates.

  14. Piperidinyl thiazole isoxazolines: A new series of highly potent, slowly reversible FAAH inhibitors with analgesic properties.

    PubMed

    Pember, Stephen O; Mejia, Galo L; Price, Theodore J; Pasteris, Robert J

    2016-06-15

    Fatty acid amide hydrolase (FAAH) is a membrane anchored serine hydrolase that has a principle role in the metabolism of the endogenous cannabinoid anandamide. Docking studies using representative FAAH crystal structures revealed that compounds containing a novel piperidinyl thiazole isoxazoline core fit within the ligand binding domains. New potential FAAH inhibitors were designed and synthesized incorporating urea, carbamate, alkyldione and thiourea reactive centers as potential pharmacophores. A small library of candidate compounds (75) was then screened against human FAAH leading to the identification of new carbamate and urea based inhibitors (Ki=pM and nM, respectively). Representative carbamate and urea based chemotypes displayed slow, time dependent inhibition kinetics leading to enzyme inactivation which was slowly reversible. However, evidence indicated that features of the mechanism of inactivation differ between the two pharmacophore types. Selected compounds were also evaluated for analgesic activity in the mouse-tail flick test.

  15. α-Ketoheterocycle-Based Inhibitors of Fatty Acid Amide Hydrolase (FAAH)

    PubMed Central

    2011-01-01

    A summary of the initial discovery and characterization of the enzyme fatty acid amide hydrolase (FAAH), and the subsequent advancement of an important class of competitive, reversible, potent, and selective inhibitors is presented. Initially explored using substrate-inspired inhibitors bearing electrophilic carbonyls, the examination of α-ketoheterocyle-based inhibitors of FAAH with the benefit of a unique activity-based protein-profiling (ABPP)-based proteome-wide selectivity assay, a powerful in vivo biomarker-based in vivo screen, and subsequent retrospective X-ray cocrystal structures with the enzyme, is summarized. These efforts defined the impact of the central activating heterocycle and its key substituents, provided key simplifications in the C2 acyl side chain and clear interpretations for the unique role and subsequent optimization of the central activating heterocycle, and established the basis for the recent further conformational constraints in the C2 acyl side chain, providing potent, long-acting, orally active FAAH inhibitors. PMID:22639704

  16. The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB1- and FAAH-independent mechanisms

    PubMed Central

    Bosier, Barbara; Muccioli, Giulio G; Lambert, Didier M

    2013-01-01

    Background Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between ‘on demand’ generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. Experimental Approach We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids. Key Results Anandamide decreased TH promoter activity in N1E115 cells through CB1 receptor activation. Unexpectedly, URB597 reduced TH expression (pEC50 = 8.7 ± 0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB1 receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain. Conclusions While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB1 and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when

  17. Actions of the dual FAAH/MAGL inhibitor JZL195 in a murine neuropathic pain model

    PubMed Central

    Adamson Barnes, Nicholas S.; Mitchell, Vanessa A.; Kazantzis, Nicholas P.

    2015-01-01

    Background and Purpose While cannabinoids have been proposed as a potential treatment for neuropathic pain, they have limitations. Cannabinoid receptor agonists have good efficacy in animal models of neuropathic pain; they have a poor therapeutic window. Conversely, selective fatty acid amide hydrolase (FAAH) inhibitors that enhance the endocannabinoid system have a better therapeutic window, but lesser efficacy. We examined whether JZL195, a dual inhibitor of FAAH and monacylglycerol lipase (MAGL), could overcome these limitations. Experimental Approach C57BL/6 mice underwent the chronic constriction injury (CCI) model of neuropathic pain. Mechanical and cold allodynia, plus cannabinoid side effects, were assessed in response to systemic drug application. Key Results JZL195 and the cannabinoid receptor agonist WIN55212 produced dose‐dependent reductions in CCI‐induced mechanical and cold allodynia, plus side effects including motor incoordination, catalepsy and sedation. JZL195 reduced allodynia with an ED50 at least four times less than that at which it produced side effects. By contrast, WIN55212 reduced allodynia and produce side effects with similar ED50s. The maximal anti‐allodynic effect of JZL195 was greater than that produced by selective FAAH, or MAGL inhibitors. The JZL195‐induced anti‐allodynia was maintained during repeated treatment. Conclusions and Implications These findings suggest that JZL195 has greater anti‐allodynic efficacy than selective FAAH, or MAGL inhibitors, plus a greater therapeutic window than a cannabinoid receptor agonist. Thus, dual FAAH/MAGL inhibition may have greater potential in alleviating neuropathic pain, compared with selective FAAH and MAGL inhibitors, or cannabinoid receptor agonists. PMID:26398331

  18. Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

    PubMed

    Nicolussi, Simon; Chicca, Andrea; Rau, Mark; Rihs, Sabine; Soeberdt, Michael; Abels, Christoph; Gertsch, Jürg

    2014-12-15

    Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

  19. Peripheral FAAH and soluble epoxide hydrolase inhibitors are synergistically antinociceptive.

    PubMed

    Sasso, Oscar; Wagner, Karen; Morisseau, Christophe; Inceoglu, Bora; Hammock, Bruce D; Piomelli, Daniele

    2015-07-01

    We need better medicines to control acute and chronic pain. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) catalyze the deactivating hydrolysis of two classes of bioactive lipid mediators--fatty acid ethanolamides (FAEs) and epoxidized fatty acids (EpFAs), respectively--which are biogenetically distinct but share the ability to attenuate pain responses and inflammation. In these experiments, we evaluated the antihyperalgesic activity of small-molecule inhibitors of FAAH and sEH, administered alone or in combination, in two pain models: carrageenan-induced hyperalgesia in mice and streptozocin-induced allodynia in rats. When administered separately, the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea (TPPU) and the peripherally restricted FAAH inhibitor URB937 were highly active in the two models. The combination TPPU plus URB937 was markedly synergistic, as assessed using isobolographic analyses. The results of these experiments reveal the existence of a possible functional crosstalk between FAEs and EpFAs in regulating pain responses. Additionally, the results suggest that combinations of sEH and FAAH inhibitors might be exploited therapeutically to achieve greater analgesic efficacy.

  20. Potent multitarget FAAH-COX inhibitors: Design and structure-activity relationship studies.

    PubMed

    Migliore, Marco; Habrant, Damien; Sasso, Oscar; Albani, Clara; Bertozzi, Sine Mandrup; Armirotti, Andrea; Piomelli, Daniele; Scarpelli, Rita

    2016-02-15

    Non-steroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological effects by inhibiting cyclooxygenase (COX)-1 and COX-2. Though widely prescribed for pain and inflammation, these agents have limited utility in chronic diseases due to serious mechanism-based adverse events such as gastrointestinal damage. Concomitant blockade of fatty acid amide hydrolase (FAAH) enhances the therapeutic effects of the NSAIDs while attenuating their propensity to cause gastrointestinal injury. This favorable interaction is attributed to the accumulation of protective FAAH substrates, such as the endocannabinoid anandamide, and suggests that agents simultaneously targeting COX and FAAH might provide an innovative strategy to combat pain and inflammation with reduced side effects. Here, we describe the rational design and structure-active relationship (SAR) properties of the first class of potent multitarget FAAH-COX inhibitors. A focused SAR exploration around the prototype 10r (ARN2508) led to the identification of achiral (18b) as well as racemic (29a-c and 29e) analogs. Absolute configurational assignment and pharmacological evaluation of single enantiomers of 10r are also presented. (S)-(+)-10r is the first highly potent and selective chiral inhibitor of FAAH-COX with marked in vivo activity, and represents a promising lead to discover novel analgesics and anti-inflammatory drugs.

  1. Development and characterization of endocannabinoid hydrolases FAAH and MAGL inhibitors bearing a benzotriazol-1-yl carboxamide scaffold.

    PubMed

    Morera, Ludovica; Labar, Geoffray; Ortar, Giorgio; Lambert, Didier M

    2012-11-01

    A series of (1H-benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-1-yl)methanones and of (1H-benzo[d][1,2,3]triazol-1-yl)(4-phenylpiperazin-1-yl)methanones has been prepared and tested on human fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). In the benzylpiperazinyl series, compound 29 (ML30) exhibited an IC(50) value of 0.54 nM on MAGL, combined with a 1000-fold selectivity versus FAAH, while compounds 11 and 16 acted as potent dual FAAH-MAGL inhibitors (IC(50)<10 nM). In the phenylpiperazinyl series, compounds 37, 38, 42, and 43 displayed IC(50) values against MAGL in the nanomolar range, whilst being between one and two orders of magnitude less potent on the FAAH, while compounds 31 and 32 were potent FAAH inhibitors (IC(50)<20 nM) and over 12-fold selective versus MAGL. The key structural determinants driving the structure-activity relationships were explored by the minimization of the inhibitors inside the active site of both enzymes.

  2. (4-Phenoxyphenyl)tetrazolecarboxamides and related compounds as dual inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

    PubMed

    Holtfrerich, Angela; Hanekamp, Walburga; Lehr, Matthias

    2013-05-01

    Inhibitors of the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principle enzymes involved in the degradation of endogenous cannabinoids like anandamide and 2-arachidonoylglycerol, have potential utility in the treatment of several disorders including pain, inflammation and anxiety. In the present study, the effectivity and selectivity of eight known FAAH and MAGL inhibitors for inhibition of the appropriate enzyme were measured applying in vitro assays, which work under comparable conditions. Because many of the known FAAH and MAGL inhibitors simply consist of a lipophilic scaffold to which a heterocyclic system is bound, furthermore, different heterocyclic structures were evaluated for their contribution to enzyme inhibition by attaching them to the same lipophilic backbone, namely 4-phenoxybenzene. One of the most active compound synthesized during this investigation was N,N-dimethyl-5-(4-phenoxyphenyl)-2H-tetrazole-2-carboxamide (16) (IC50 FAAH: 0.012 μM; IC50 MAGL: 0.028 μM). This inhibitor was systematically modified in the lipophilic 4-phenoxyphenyl region. Structure-activity relationship studies revealed that the inhibitory potency against FAAH and MAGL, respectively, could still be increased by replacement of the phenoxy residue of 16 by 3-chlorophenoxy (45) or pyrrol-1-yl groups (49). Finally, the tetrazolecarboxamide 16 and some related compounds were tested for metabolic stability with rat liver S9 fractions showing that these kind of FAAH/MAGL inhibitors are readily inactivated by cleavage of the bond between the tetrazole ring and its carboxamide substituent.

  3. N-aryl 2-aryloxyacetamides as a new class of fatty acid amide hydrolase (FAAH) inhibitors.

    PubMed

    Sunduru, Naresh; Svensson, Mona; Cipriano, Mariateresa; Marwaha, Sania; Andersson, C David; Svensson, Richard; Fowler, Christopher J; Elofsson, Mikael

    2017-12-01

    Fatty acid amide hydrolase (FAAH) is a promising target for the development of drugs to treat neurological diseases. In search of new FAAH inhibitors, we identified 2-(4-cyclohexylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4g, with an IC50 of 2.6 µM as a chemical starting point for the development of potent FAAH inhibitors. Preliminary hit-to-lead optimisation resulted in 2-(4-phenylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4i, with an IC50 of 0.35 µM.

  4. Design, synthesis and evaluation of polar head group containing 2-keto-oxazole inhibitors of FAAH.

    PubMed

    Rusch, Marion; Zahov, Stefan; Vetter, Ingrid R; Lehr, Matthias; Hedberg, Christian

    2012-01-15

    2-α-Keto oxazoles containing polar head groups in their C5-side chains were designed as fatty acid amide hydrolase (FAAH) inhibitors. Variation in the spacer length resulted in submicromolar α-keto-oxazole FAAH inhibitor (IC(50)=436 nM) presenting electrostatic stabilizing interactions between its polar head group contained in the C5-side chain and the hydrophilic pocket of the enzyme.

  5. Potential Therapeutic Value of a Novel FAAH Inhibitor for the Treatment of Anxiety

    PubMed Central

    Marco, Eva M.; Rapino, Cinzia; Caprioli, Antonio; Borsini, Franco; Laviola, Giovanni; Maccarrone, Mauro

    2015-01-01

    Anxiety disorders are among the most prevalent psychiatric diseases with high personal costs and a remarkable socio-economic burden. However, current treatment of anxiety is far from satisfactory. Novel pharmacological targets have emerged in the recent years, and attention has focused on the endocannabinoid (eCB) system, given the increasing evidence that supports its central role in emotion, coping with stress and anxiety. In the management of anxiety disorders, drug development strategies have left apart the direct activation of type-1 cannabinoid receptors to indirectly enhance eCB signalling through the inhibition of eCB deactivation, that is, the inhibition of the fatty acid amide hydrolase (FAAH) enzyme. In the present study, we provide evidence for the anxiolytic-like properties of a novel, potent and selective reversible inhibitor of FAAH, ST4070, orally administered to rodents. ST4070 (3 to 30 mg/kg per os) administered to CD1 male mice induced an increase of time spent in the exploration of the open arms of the elevated-plus maze. A partial reduction of anxiety-related behaviour by ST4070 was also obtained in Wistar male rats, which moderately intensified the time spent in the illuminated compartment of the light-dark box. ST4070 clearly inhibited FAAH activity and augmented the levels of two of its substrates, N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine, in anxiety-relevant brain regions. Altogether, ST4070 offers a promising anxiolytic-like profile in preclinical studies, although further studies are warranted to clearly demonstrate its efficacy in the clinic management of anxiety disorders. PMID:26360704

  6. Fatty acid amide hydrolase (FAAH) inhibitors exert pharmacological effects, but lack antinociceptive efficacy in rats with neuropathic spinal cord injury pain.

    PubMed

    Hama, Aldric T; Germano, Peter; Varghese, Matthew S; Cravatt, Benjamin F; Milne, G Todd; Pearson, James P; Sagen, Jacqueline

    2014-01-01

    Amelioration of neuropathic spinal cord injury (SCI) pain is a clinical challenge. Increasing the endocannabinoid anandamide and other fatty acid amides (FAA) by blocking fatty acid amide hydrolase (FAAH) has been shown to be antinociceptive in a number of animal models of chronic pain. However, an antinociceptive effect of blocking FAAH has yet to be demonstrated in a rat model of neuropathic SCI pain. Four weeks following a SCI, rats developed significantly decreased hind paw withdrawal thresholds, indicative of below-level cutaneous hypersensitivity. A group of SCI rats were systemically treated (i.p.) with either the selective FAAH inhibitor URB597 or vehicle twice daily for seven days. A separate group of SCI rats received a single dose (p.o.) of either the selective FAAH inhibitor PF-3845 or vehicle. Following behavioral testing, levels of the FAA N-arachidonoylethanolamide, N-oleoyl ethanolamide and N-palmitoyl ethanolamide were quantified in brain and spinal cord from SCI rats. Four weeks following SCI, FAA levels were markedly reduced in spinal cord tissue. Although systemic treatment with URB597 significantly increased CNS FAA levels, no antinociceptive effect was observed. A significant elevation of CNS FAA levels was also observed following oral PF-3845 treatment, but only a modest antinociceptive effect was observed. Increasing CNS FAA levels alone does not lead to robust amelioration of below-level neuropathic SCI pain. Perhaps utilizing FAAH inhibition in conjunction with other analgesic mechanisms could be an effective analgesic therapy.

  7. Actions of the dual FAAH/MAGL inhibitor JZL195 in a murine inflammatory pain model.

    PubMed

    Anderson, Wayne B; Gould, Michael J; Torres, Romeo D; Mitchell, Vanessa A; Vaughan, Christopher W

    2014-06-01

    The analgesic efficacy of cannabinoids in chronic pain models is limited by side-effects. It has been proposed that this might be overcome by using agents which indirectly activate the endocannabinoid system. We examined the analgesic and side-effect profile of the dual FAAH/MAGL inhibitor JZL195 in an inflammatory pain model. The effect of systemic injections of a range of doses of JZL195 and the pan-cannabinoid receptor agonist WIN55212 were performed 1 day following intraplantar injection of CFA in C57BL/6 mice. JZL195 and WIN55212 both reduced mechanical allodynia and thermal hyperalgesia, and produced catalepsy and sedation in a dose dependent manner. Unlike WIN55212, JZL195 reduced allodynia at doses below those at which side-effects were observed. The effects of JZL195 and WIN55212 were abolished by co-application with the CB1 antagonist AM251. The CB2 antagonist also reduced the JZL195 anti-allodynia, and reversed the WIN55212 anti-allodynia. The reduction in allodynia produced by JZL195 was greater than that produced individually by the FAAH and MAGL inhibitors, URB597 and JZL184. These findings suggest that JZL195 reduces inflammation induced allodynia at doses below those which produce side-effects, and displays greater efficacy that FAAH or MAGL inhibitors. Thus, dual FAAH/MAGL inhibition has the potential to alleviate inflammatory pain with reduced cannabinoid-like side-effects.

  8. The SAR of brain penetration for a series of heteroaryl urea FAAH inhibitors.

    PubMed

    Keith, John M; Tichenor, Mark S; Apodaca, Richard L; Xiao, Wei; Jones, William M; Seierstad, Mark; Pierce, Joan M; Palmer, James A; Webb, Michael; Karbarz, Mark J; Scott, Brian P; Wilson, Sandy J; Wennerholm, Michelle L; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra R; Breitenbucher, J Guy

    2016-07-01

    The SAR of brain penetration for a series of heteroaryl piperazinyl- and piperadinyl-urea fatty acid amide hydrolase (FAAH) inhibitors is described. Brain/plasma (B/P) ratios ranging from >4:1 to as low as 0.02:1 were obtained through relatively simple structural changes to various regions of the heteroaryl urea scaffold. It was not possible to predict the degree of central nervous system (CNS) penetration from the volumes of distribution (Vd) obtained from pharmacokinetic (PK) experiments as very high Vds did not correlate with high B/P ratios. Similarly, calculated topological polar surface areas (TPSAs) did not consistently correlate with the degree of brain penetration. The lowest B/P ratios were observed for those compounds that were significantly ionized at physiological pH. However, as this class of compounds inhibits the FAAH enzyme through covalent modification, low B/P ratios did not preclude effective central target engagement.

  9. 3-Heterocycle-phenyl N-alkylcarbamates as FAAH inhibitors: design, synthesis and 3D-QSAR studies.

    PubMed

    Käsnänen, Heikki; Myllymäki, Mikko J; Minkkilä, Anna; Kataja, Antti O; Saario, Susanna M; Nevalainen, Tapio; Koskinen, Ari M P; Poso, Antti

    2010-02-01

    Carbamates are a well-established class of fatty acid amide hydrolase (FAAH) inhibitors. Here we describe the synthesis of meta-substituted phenolic N-alkyl/aryl carbamates and their in vitro FAAH inhibitory activities. The most potent compound, 3-(oxazol-2yl)phenyl cyclohexylcarbamate (2 a), inhibited FAAH with a sub-nanomolar IC(50) value (IC(50)=0.74 nM). Additionally, we developed and validated three-dimensional quantitative structure-activity relationships (QSAR) models of FAAH inhibition combining the newly disclosed carbamates with our previously published inhibitors to give a total set of 99 compounds. Prior to 3D-QSAR modeling, the degree of correlation between FAAH inhibition and in silico reactivity was also established. Both 3D-QSAR methods used, CoMSIA and GRID/GOLPE, produced statistically significant models with coefficient of correlation for external prediction (R(2) (PRED)) values of 0.732 and 0.760, respectively. These models could be of high value in further FAAH inhibitor design.

  10. The macamide N-3-methoxybenzyl-linoleamide is a time-dependent fatty acid amide hydrolase (FAAH) inhibitor.

    PubMed

    Almukadi, Haifa; Wu, Hui; Böhlke, Mark; Kelley, Charles J; Maher, Timothy J; Pino-Figueroa, Alejandro

    2013-10-01

    The Peruvian plant Lepidium meyenii (Maca) has been shown to possess neuroprotective activity both in vitro and in vivo. Previous studies have also demonstrated the activity of the pentane extract and its macamides, the most representative lipophilic constituents of Maca, in the endocannabinoid system as fatty acid amide hydrolase (FAAH) inhibitors. One of the most active macamides, N-3-methoxybenzyl-linoleamide, was studied to determine its mechanism of interaction with FAAH and whether it has inhibitory activity on mono-acyl glycerol lipase (MAGL), the second enzyme responsible for endocannabinoid degradation. Macamide concentrations from 1 to 100 μM were tested using FAAH and MAGL inhibitor assay methods and showed no effect on MAGL. Tests with other conditions were performed in order to characterize the inhibitory mechanism of FAAH inhibition. N-3-methoxybenzyl-linoleamide displayed significant time-dependent and dose-dependent FAAH inhibitory activity. The mechanism of inhibition was most likely irreversible or slowly reversible. These results suggest the potential application of macamides isolated from Maca as FAAH inhibitors, as they might act on the central nervous system to provide analgesic, anti-inflammatory, or neuroprotective effects, by modulating the release of neurotransmitters.

  11. O-(triazolyl)methyl carbamates as a novel and potent class of fatty acid amide hydrolase (FAAH) inhibitors.

    PubMed

    Colombano, Giampiero; Albani, Clara; Ottonello, Giuliana; Ribeiro, Alison; Scarpelli, Rita; Tarozzo, Glauco; Daglian, Jennifer; Jung, Kwang-Mook; Piomelli, Daniele; Bandiera, Tiziano

    2015-02-01

    Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O-aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate derivatives were designed and synthesized exploiting a copper- catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure-activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC50 values in the single-digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug-like properties.

  12. The First Dual ChE/FAAH Inhibitors: New Perspectives for Alzheimer's Disease?

    PubMed Central

    2012-01-01

    The treatment of Alzheimer's disease (AD) still remains an area of significant unmet need, with drugs that only target the symptoms of the disease. Therefore, there is considerable need for disease-modifying therapies. The complex etiology of AD prompts scientists to develop multitarget strategies to combat causes and symptoms. To this aim, we designed, synthesized, and tested four new carbamates as dual cholinesterase-FAAH inhibitors. The dual activity of these compounds could lead to a potentially more effective treatment for the counteraction of AD progression, because they would allow regulation of both ACh and eCB signaling and improve neuronal transmission and/or counteract neuroinflammation. PMID:24900454

  13. The First Dual ChE/FAAH Inhibitors: New Perspectives for Alzheimer's Disease?

    PubMed

    Rampa, Angela; Bartolini, Manuela; Bisi, Alessandra; Belluti, Federica; Gobbi, Silvia; Andrisano, Vincenza; Ligresti, Alessia; Di Marzo, Vincenzo

    2012-03-08

    The treatment of Alzheimer's disease (AD) still remains an area of significant unmet need, with drugs that only target the symptoms of the disease. Therefore, there is considerable need for disease-modifying therapies. The complex etiology of AD prompts scientists to develop multitarget strategies to combat causes and symptoms. To this aim, we designed, synthesized, and tested four new carbamates as dual cholinesterase-FAAH inhibitors. The dual activity of these compounds could lead to a potentially more effective treatment for the counteraction of AD progression, because they would allow regulation of both ACh and eCB signaling and improve neuronal transmission and/or counteract neuroinflammation.

  14. Effects of Fatty Acid Amide Hydrolase (FAAH) Inhibitors in Non-Human Primate Models of Nicotine Reward and Relapse

    PubMed Central

    Justinova, Zuzana; Panlilio, Leigh V; Moreno-Sanz, Guillermo; Redhi, Godfrey H; Auber, Alessia; Secci, Maria E; Mascia, Paola; Bandiera, Tiziano; Armirotti, Andrea; Bertorelli, Rosalia; Chefer, Svetlana I; Barnes, Chanel; Yasar, Sevil; Piomelli, Daniele; Goldberg, Steven R

    2015-01-01

    Inhibition of the enzyme fatty acid amide hydrolase (FAAH) counteracts reward-related effects of nicotine in rats, but it has not been tested for this purpose in non-human primates. Therefore, we studied the effects of the first- and second-generation O-arylcarbamate-based FAAH inhibitors, URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) and URB694 (6-hydroxy-[1,1'-biphenyl]-3-yl-cyclohexylcarbamate), in squirrel monkeys. Both FAAH inhibitors: (1) blocked FAAH activity in brain and liver, increasing levels of endogenous ligands for cannabinoid and α-type peroxisome proliferator-activated (PPAR-α) receptors; (2) shifted nicotine self-administration dose–response functions in a manner consistent with reduced nicotine reward; (3) blocked reinstatement of nicotine seeking induced by reexposure to either nicotine priming or nicotine-associated cues; and (4) had no effect on cocaine or food self-administration. The effects of FAAH inhibition on nicotine self-administration and nicotine priming-induced reinstatement were reversed by the PPAR-α antagonist, MK886. Unlike URB597, which was not self-administered by monkeys in an earlier study, URB694 was self-administered at a moderate rate. URB694 self-administration was blocked by pretreatment with an antagonist for either PPAR-α (MK886) or cannabinoid CB1 receptors (rimonabant). In additional experiments in rats, URB694 was devoid of THC-like or nicotine-like interoceptive effects under drug-discrimination procedures, and neither of the FAAH inhibitors induced dopamine release in the nucleus accumbens shell—consistent with their lack of robust reinforcing effects in monkeys. Overall, both URB597 and URB694 show promise for the initialization and maintenance of smoking cessation because of their ability to block the rewarding effects of nicotine and prevent nicotine priming-induced and cue-induced reinstatement. PMID:25754762

  15. Effects of Fatty Acid Amide Hydrolase (FAAH) Inhibitors in Non-Human Primate Models of Nicotine Reward and Relapse.

    PubMed

    Justinova, Zuzana; Panlilio, Leigh V; Moreno-Sanz, Guillermo; Redhi, Godfrey H; Auber, Alessia; Secci, Maria E; Mascia, Paola; Bandiera, Tiziano; Armirotti, Andrea; Bertorelli, Rosalia; Chefer, Svetlana I; Barnes, Chanel; Yasar, Sevil; Piomelli, Daniele; Goldberg, Steven R

    2015-08-01

    Inhibition of the enzyme fatty acid amide hydrolase (FAAH) counteracts reward-related effects of nicotine in rats, but it has not been tested for this purpose in non-human primates. Therefore, we studied the effects of the first- and second-generation O-arylcarbamate-based FAAH inhibitors, URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) and URB694 (6-hydroxy-[1,1'-biphenyl]-3-yl-cyclohexylcarbamate), in squirrel monkeys. Both FAAH inhibitors: (1) blocked FAAH activity in brain and liver, increasing levels of endogenous ligands for cannabinoid and α-type peroxisome proliferator-activated (PPAR-α) receptors; (2) shifted nicotine self-administration dose-response functions in a manner consistent with reduced nicotine reward; (3) blocked reinstatement of nicotine seeking induced by reexposure to either nicotine priming or nicotine-associated cues; and (4) had no effect on cocaine or food self-administration. The effects of FAAH inhibition on nicotine self-administration and nicotine priming-induced reinstatement were reversed by the PPAR-α antagonist, MK886. Unlike URB597, which was not self-administered by monkeys in an earlier study, URB694 was self-administered at a moderate rate. URB694 self-administration was blocked by pretreatment with an antagonist for either PPAR-α (MK886) or cannabinoid CB1 receptors (rimonabant). In additional experiments in rats, URB694 was devoid of THC-like or nicotine-like interoceptive effects under drug-discrimination procedures, and neither of the FAAH inhibitors induced dopamine release in the nucleus accumbens shell--consistent with their lack of robust reinforcing effects in monkeys. Overall, both URB597 and URB694 show promise for the initialization and maintenance of smoking cessation because of their ability to block the rewarding effects of nicotine and prevent nicotine priming-induced and cue-induced reinstatement.

  16. Evaluation of the Safety and Efficacy of the FAAH Inhibitor URB597

    DTIC Science & Technology

    2016-01-01

    Evaluation2of2the2Safety2and2Efficacy2of2the2FAAH2Inhibitor2URB597 ! ! ! PRINCIPAL!INVESTIGATOR:!!!Alexander2Neumeister! ! ! ! CONTRACTING!ORGANIZATION:!New2York2University New York, NY 10016...nyumc.org! ! ! 5f.2WORK2UNIT2NUMBER 7.2PERFORMING2ORGANIZATION2NAME(S)2AND2ADDRESS(ES)2 New York University 550 First Avenue & One Park Avenue, Room 8...225 New York, New York 10016 8.2PERFORMING2ORGANIZATION2REPORT222 2222NUMBER 9.2SPONSORING2/2MONITORING2AGENCY2NAME(S)2AND2ADDRESS(ES

  17. The dual FAAH/MAGL inhibitor JZL195 has enhanced effects on endocannabinoid transmission and motor behavior in rats as compared to those of the MAGL inhibitor JZL184.

    PubMed

    Seillier, Alexandre; Dominguez Aguilar, David; Giuffrida, Andrea

    2014-09-01

    The biological actions of the endocannabinoids anandamide and 2-arachidonoyl glycerol (2-AG) are terminated by enzymatic hydrolysis of these lipids via fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. While several selective FAAH inhibitors have been developed and characterized in vitro and in vivo, none of the initial MAGL blockers have shown adequate potency and specificity for in vivo applications. More recently, a selective MAGL inhibitor, JZL184, has been shown to produce a long-lasting elevation of brain 2-AG, as well as cannabinoid-like behavioral responses in mice. However, its effectiveness in rats remains controversial. Indeed, although JZL184 can elicit behavioral responses that are mediated, at least in part, via activation of cannabinoid CB1 receptors, several reports indicate that this compound does not alter 2-AG levels in this species. In this study we compared the behavioral and neurochemical effects of JZL 184 with those of the dual FAAH/MAGL inhibitor JZL195, and showed that systemic administration of the former can selectively elevate brain 2-AG in rats and produce motor suppression through a CB1-independent mechanism. These findings indicate that, despite its lower potency against rat MAGL, JZL184 can be used to enhance 2-AG transmission and elicit behavioral responses in rodents.

  18. Chemistry around imidazopyrazine and ibuprofen: discovery of novel fatty acid amide hydrolase (FAAH) inhibitors.

    PubMed

    De Wael, Frédéric; Muccioli, Giulio G; Lambert, Didier M; Sergent, Thérèse; Schneider, Yves-Jacques; Rees, Jean-François; Marchand-Brynaert, Jacqueline

    2010-09-01

    Based on the imidazo-[1,2-a]-pyrazin-3-(7H)-one scaffold, a dual action prodrug has been designed for combining antioxidant and anti-inflammatory activities, possibly unmasked upon oxidation. The construction of the target-molecule requires two building blocks, namely a 2-amino-1,4-pyrazine and a 2-ketoaldehyde. Attempts to synthesize the 2-ketoaldehyde (5a) derived from ibuprofen failed, but led to the corresponding 2-ketoaldoxime (7a) which could not be condensed with the pyrazine synthons. However, a model compound, i.e. phenylglyoxal aldoxime, reacted well under microwave activation to furnish novel imidazo[1,2-a]-pyrazine-3-(7H)-imine derivatives (18a,b). These heterobicycles behave as antioxidants by inhibiting the lipid peroxidation, and one compound (18b) is endowed with a significant anti-inflammatory effect in a cellular test. Unexpectedly, all the synthetic intermediates derived from ibuprofen are good inhibitors of FAAH, the most active compound (4a) featuring the 1,3-dithian-2-yl motif.

  19. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

    PubMed

    Vandevoorde, Séverine; Fowler, Christopher J

    2005-08-01

    There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for

  20. Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain

    PubMed Central

    Moreno-Sanz, Guillermo; Barrera, Borja; Armirotti, Andrea; Bertozzi, Sine M.; Scarpelli, Rita; Bandiera, Tiziano; Prieto, Julio G.; Duranti, Andrea; Tarzia, Giorgio; Merino, Gracia

    2014-01-01

    The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family – Breast Cancer Resistance Protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) – play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested – even those able to enter the CNS in vivo – were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS. PMID:24993496

  1. Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain.

    PubMed

    Moreno-Sanz, Guillermo; Barrera, Borja; Armirotti, Andrea; Bertozzi, Sine M; Scarpelli, Rita; Bandiera, Tiziano; Prieto, Julio G; Duranti, Andrea; Tarzia, Giorgio; Merino, Gracia; Piomelli, Daniele

    2014-09-01

    The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family - breast cancer resistance protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) - play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested - even those able to enter the CNS in vivo - were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS.

  2. Attenuation of persistent pain-related behavior by fatty acid amide hydrolase (FAAH) inhibitors in a rat model of HIV sensory neuropathy

    PubMed Central

    Nasirinezhad, Farinaz; Jergova, Stanislava; Pearson, James P.; Sagen, Jacqueline

    2015-01-01

    Distal sensory neuropathies are a hallmark of HIV infections and can result in persistent and disabling pain despite advances in antiretroviral therapies. HIV-sensory neuropathic (HIV-SN) pain may be amenable to cannabinoid treatment, but currently available agonist treatments are limited by untoward side effects and potential for abuse in this patient population. Fatty acid amide hydrolase (FAAH) inhibitors may offer an alternative approach by inhibiting the degradation of endocannabinoids with purportedly fewer untoward CNS side effects. In order to evaluate this potential approach in the management of HIV-SN pain, the recombinant HIV envelope protein gp120 was applied epineurally to the rat sciatic nerve to induce an HIV-SN-like pain syndrome. Two distinct FAAH inhibitory compounds, URB597 and PF-3845 were tested, and contrasted with standard antinociceptive gabapentin or vehicle treatment, for attenuation of tactile allodynia, cold allodynia, and mechanical hyperalgesia. Both FAAH inhibitors markedly reduced cold and tactile allodynia with limited anti-hyperalgesic effects. Peak antinociceptive effects produced by both agents were more modest than gabapentin in reducing tactile allodynia with similar potency ranges. URB597 produced comparable cold anti-allodynic effects to gabapentin, and the effects of both FAAH inhibitors were longer lasting than gabapentin. To assess the contribution of cannabinoid receptors in these antinociceptive effects, CB1 antagonist AM251 or CB2 antagonist SR144528 were tested in conjunction with FAAH inhibitors. Results suggested a contribution of both CB1- and CB2-mediated effects, particularly in reducing tactile allodynia. In summary, these findings support inhibition of endocannabinoid degradation as a promising target for management of disabling persistent HIV-SN pain syndromes. PMID:25486617

  3. Attenuation of persistent pain-related behavior by fatty acid amide hydrolase (FAAH) inhibitors in a rat model of HIV sensory neuropathy.

    PubMed

    Nasirinezhad, Farinaz; Jergova, Stanislava; Pearson, James P; Sagen, Jacqueline

    2015-08-01

    Distal sensory neuropathies are a hallmark of HIV infections and can result in persistent and disabling pain despite advances in antiretroviral therapies. HIV-sensory neuropathic (HIV-SN) pain may be amenable to cannabinoid treatment, but currently available agonist treatments are limited by untoward side effects and potential for abuse in this patient population. Fatty acid amide hydrolase (FAAH) inhibitors may offer an alternative approach by inhibiting the degradation of endocannabinoids with purportedly fewer untoward CNS side effects. In order to evaluate this potential approach in the management of HIV-SN pain, the recombinant HIV envelope protein gp120 was applied epineurally to the rat sciatic nerve to induce an HIV-SN-like pain syndrome. Two distinct FAAH inhibitory compounds, URB597 and PF-3845 were tested, and contrasted with standard antinociceptive gabapentin or vehicle treatment, for attenuation of tactile allodynia, cold allodynia, and mechanical hyperalgesia. Both FAAH inhibitors markedly reduced cold and tactile allodynia with limited anti-hyperalgesic effects. Peak antinociceptive effects produced by both agents were more modest than gabapentin in reducing tactile allodynia with similar potency ranges. URB597 produced comparable cold anti-allodynic effects to gabapentin, and the effects of both FAAH inhibitors were longer lasting than gabapentin. To assess the contribution of cannabinoid receptors in these antinociceptive effects, CB1 antagonist AM251 or CB2 antagonist SR144528 were tested in conjunction with FAAH inhibitors. Results suggested a contribution of both CB1- and CB2-mediated effects, particularly in reducing tactile allodynia. In summary, these findings support inhibition of endocannabinoid degradation as a promising target for management of disabling persistent HIV-SN pain syndromes.

  4. The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice

    PubMed Central

    Booker, Lamont; Kinsey, Steven G; Abdullah, Rehab A; Blankman, Jacqueline L; Long, Jonathan Z; Ezzili, Cyrine; Boger, Dale L; Cravatt, Benjamin F; Lichtman, Aron H

    2012-01-01

    BACKGROUND AND PURPOSE Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB1 and CB2 cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ9-tetrahydrocannabinol (THC). EXPERIMENTAL APPROACH Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses. KEY RESULTS FAAH (−/−) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB1 and CB2 receptors, but other potential targets of FAA substrates (i.e. µ-opioid, TRPV1 and PPARα receptors) had no apparent role. CONCLUSIONS AND IMPLICATIONS AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To

  5. Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode

    PubMed Central

    Deplano, Alessandro; Smaldone, Giovanni; Pedone, Emilia; Luque, F. Javier; Svensson, Mona; Novellino, Ettore; Congiu, Cenzo; Onnis, Valentina; Catalanotti, Bruno; Fowler, Christopher J.

    2015-01-01

    Background Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here. Methodology/Principal Findings FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAHT488A-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 μM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 μM) was more potent than the (R)-enantiomer (IC50 5.7 μM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH. Conclusions/Significance The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors. PMID:26565710

  6. Inhibition of FAAH confers increased stem cell migration via PPARα.

    PubMed

    Wollank, Yvonne; Ramer, Robert; Ivanov, Igor; Salamon, Achim; Peters, Kirsten; Hinz, Burkhard

    2015-10-01

    Regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (MSCs). The present study focused on inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (N-oleoylethanolamine, N-palmitoylethanolamine). Boyden chamber assays, the FAAH inhibitors, URB597 and arachidonoyl serotonin (AA-5HT), were found to increase the migration of human adipose-derived MSCs. LC-MS analyses revealed increased levels of all four aforementioned FAAH substrates in MSCs incubated with either FAAH inhibitor. Following addition to MSCs, all FAAH substrates mimicked the promigratory action of FAAH inhibitors. Promigratory effects of FAAH inhibitors and substrates were causally linked to activation of p42/44 MAPKs, as well as to cytosol-to-nucleus translocation of the transcription factor, PPARα. Whereas PPARα activation by FAAH inhibitors and substrates became reversed upon inhibition of p42/44 MAPK activation, a blockade of PPARα left p42/44 MAPK phosphorylation unaltered. Collectively, these data demonstrate FAAH inhibitors and substrates to cause p42/44 MAPK phosphorylation, which subsequently activates PPARα to confer increased migration of MSCs. This novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by FAAH inhibitors.

  7. Inhibition of FAAH confers increased stem cell migration via PPARα

    PubMed Central

    Wollank, Yvonne; Ramer, Robert; Ivanov, Igor; Salamon, Achim; Peters, Kirsten; Hinz, Burkhard

    2015-01-01

    Regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (MSCs). The present study focused on inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (N-oleoylethanolamine, N-palmitoylethanolamine). Boyden chamber assays, the FAAH inhibitors, URB597 and arachidonoyl serotonin (AA-5HT), were found to increase the migration of human adipose-derived MSCs. LC-MS analyses revealed increased levels of all four aforementioned FAAH substrates in MSCs incubated with either FAAH inhibitor. Following addition to MSCs, all FAAH substrates mimicked the promigratory action of FAAH inhibitors. Promigratory effects of FAAH inhibitors and substrates were causally linked to activation of p42/44 MAPKs, as well as to cytosol-to-nucleus translocation of the transcription factor, PPARα. Whereas PPARα activation by FAAH inhibitors and substrates became reversed upon inhibition of p42/44 MAPK activation, a blockade of PPARα left p42/44 MAPK phosphorylation unaltered. Collectively, these data demonstrate FAAH inhibitors and substrates to cause p42/44 MAPK phosphorylation, which subsequently activates PPARα to confer increased migration of MSCs. This novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by FAAH inhibitors. PMID:26263913

  8. Synthesis and Preliminary PET Imaging Studies of a FAAH Radiotracer ([¹¹C]MPPO) Based on α-Ketoheterocyclic Scaffold.

    PubMed

    Wang, Lu; Yui, Joji; Wang, Qifan; Zhang, Yiding; Mori, Wakana; Shimoda, Yoko; Fujinaga, Masayuki; Kumata, Katsushi; Yamasaki, Tomoteru; Hatori, Akiko; Rotstein, Benjamin H; Collier, Thomas Lee; Ran, Chongzhao; Vasdev, Neil; Zhang, Ming-Rong; Liang, Steven H

    2016-01-20

    Fatty acid amide hydrolase (FAAH) is one of the principle enzymes for metabolizing endogenous cannabinoid neurotransmitters such as anandamide, and thus regulates endocannabinoid (eCB) signaling. Selective pharmacological blockade of FAAH has emerged as a potential therapy to discern the endogenous functions of anandamide-mediated eCB pathways in anxiety, pain, and addiction. Quantification of FAAH in the living brain by positron emission tomography (PET) would help our understanding of the endocannabinoid system in these conditions. While most FAAH radiotracers operate by an irreversible ("suicide") binding mechanism, a FAAH tracer with reversibility would facilitate quantitative analysis. We have identified and radiolabeled a reversible FAAH inhibitor, 7-(2-[(11)C]methoxyphenyl)-1-(5-(pyridin-2-yl)oxazol-2-yl)heptan-1-one ([(11)C]MPPO) in 13% radiochemical yield (nondecay corrected) with >99% radiochemical purity and 2 Ci/μmol (74 GBq/μmol) specific activity. The tracer showed moderate brain uptake (0.8 SUV) with heterogeneous brain distribution. However, blocking studies with a potent FAAH inhibitor URB597 demonstrated a low to modest specificity to the target. Measurement of lipophilicity, metabolite, and efflux pathway analysis were also performed to study the pharmacokinetic profile of [(11)C]MPPO. In all, we reported an efficient radiolabeling and preliminary evaluation of the first-in-class FAAH inhibitor [(11)C]MPPO with α-ketoheterocyclic scaffold.

  9. Just add water: cannabinoid discrimination in a water T-maze with FAAH(-/-) and FAAH(+/+) mice.

    PubMed

    Wiley, Jenny L; Lefever, Timothy W; Pulley, Nikita S; Marusich, Julie A; Cravatt, Benjamin F; Lichtman, Aron H

    2016-08-01

    Incomplete overlap in the discriminative stimulus effects of Δ-tetrahydrocannabinol (THC) and the endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol has been reported in food-reinforced tasks. The aim of this study was to examine cannabinoid discriminative stimulus effects in a nonappetitive procedure. Adult male mice lacking the gene for AEA's major metabolic enzyme, fatty acid amide hydrolase (FAAH), and FAAH mice were trained to discriminate THC or AEA in a water T-maze, in which the response was swimming to an escape platform on the injection-appropriate side. JZL184, a monoacylglycerol lipase inhibitor, was also tested. FAAH mice showed faster acquisition than FAAH mice. THC and AEA fully substituted, with only minor cross-procedure potency variations. Incomplete substitution of JZL184 was observed in THC-trained FAAH mice in the water-maze task, as contrasted with full substitution in a food-reinforced nose-poke procedure. Stress-induced changes in AEA and/or 2-arachidonoylglycerol concentrations in the brain may have mediated this attenuation. JZL184 also partially substituted in AEA-trained FAAH mice in the water maze, suggesting incomplete overlap in the stimulus effects of AEA and JZL184. Through the use of a novel water-maze procedure, the present study supports the work of previous behavioral pharmacologists in showing the robustness of the discrimination paradigm.

  10. Assay of FAAH Activity.

    PubMed

    Bari, Monica; Feole, Monica; Maccarrone, Mauro

    2016-01-01

    Fatty acid amide hydrolase (FAAH) is an intracellular enzyme responsible for the hydrolysis of endogenous anandamide (AEA), a reaction that terminates the biological effects of this lipid mediator. The final products of this reaction are arachidonic acid and ethanolamine. In the method described herein, FAAH activity is measured through the use of a radioactive substrate by quantification of reaction products, that is, [(14)C]-ethanolamine from [(14)C-ethanolamine]-AEA.

  11. FAAH genetic variation enhances fronto-amygdala function in mouse and human.

    PubMed

    Dincheva, Iva; Drysdale, Andrew T; Hartley, Catherine A; Johnson, David C; Jing, Deqiang; King, Elizabeth C; Ra, Stephen; Gray, J Megan; Yang, Ruirong; DeGruccio, Ann Marie; Huang, Chienchun; Cravatt, Benjamin F; Glatt, Charles E; Hill, Matthew N; Casey, B J; Lee, Francis S

    2015-03-03

    Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human.

  12. FAAH genetic variation enhances fronto-amygdala function in mouse and human

    PubMed Central

    Dincheva, Iva; Drysdale, Andrew T.; Hartley, Catherine A.; Johnson, David C.; Jing, Deqiang; King, Elizabeth C.; Ra, Stephen; Gray, Megan; Yang, Ruirong; DeGruccio, Ann Marie; Huang, Chienchun; Cravatt, Benjamin F.; Glatt, Charles E.; Hill, Matthew N.; Casey, B. J.; Lee, Francis S.

    2015-01-01

    Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry, and behavior. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviors. These results suggest a gain-of-function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human. PMID:25731744

  13. FAAH inhibitor, URB-597, promotes extinction and CB(1) antagonist, SR141716, inhibits extinction of conditioned aversion produced by naloxone-precipitated morphine withdrawal, but not extinction of conditioned preference produced by morphine in rats.

    PubMed

    Manwell, Laurie A; Satvat, Elham; Lang, Stefan T; Allen, Craig P; Leri, Francesco; Parker, Linda A

    2009-11-01

    Converging evidence suggests that the endogenous cannabinoid (eCB) system is involved in extinction of learned behaviours. Using operant and classical conditioning procedures, the potential of the fatty acid amide (FAAH) inhibitor, URB-597, and the CB(1) antagonist/inverse agonist, SR141716, to promote and inhibit (respectively) extinction of learned responses previously motivated by either rewarding or aversive stimuli was investigated. In the operant conditioning procedure (Expt. 1), rats previously trained to lever press for sucrose reward were administered URB-597 (0.3 mg/kg) or the CB(1) antagonist/inverse agonist SR141716 (2.5 mg/kg) prior to each of three extinction trials. In the conditioned floor preference procedure (Expts 2a-d), rats trained to associate morphine with one of two distinctive floors were administered one of several doses of the CB(1) antagonist/inverse agonist, AM-251 (Expt 2a) or URB-597 (Expt 2b and 2d) prior to each extinction/test trial wherein a choice of both floors was presented and prior to forced exposure to each floor (Expt 2c). In the conditioned floor aversion procedure (Expt. 3), rats trained to associate a naloxone-precipitated morphine withdrawal with a floor cue were administered URB-597 or SR141716 prior to each of 24 extinction/testing trials. URB-597 did not promote and SR141716 did not reduce extinction rates for sucrose reward-induced operant responding (Expt. 1) or morphine-induced conditioned floor preference (Expts. 2a-d). In contrast, URB-597 facilitated, whereas SR141716 impaired, extinction of the conditioned floor aversion (Expt. 3). These data support previous reports that the eCB system selectively facilitates extinction of aversive memories. URB-597 may prove useful in targeting extinction of aversively motivated behaviours.

  14. Supra-spinal FAAH is required for the analgesic action of paracetamol in an inflammatory context.

    PubMed

    Dalmann, Romain; Daulhac, Laurence; Antri, Myriam; Eschalier, Alain; Mallet, Christophe

    2015-04-01

    Paracetamol (acetaminophen) is the most commonly used analgesic in the world. Recently, a new view of its action has emerged: that paracetamol would be a pro-drug that should be metabolized by the FAAH enzyme into AM404, its active metabolite. However, this hypothesis has been demonstrated only in naive animals, a far cry from the clinical pathologic context of paracetamol use. Moreover, FAAH is a ubiquitous enzyme expressed both in the central nervous system and in the periphery. Thus, we explored: (i) the involvement of FAAH in the analgesic action of paracetamol in a mouse model of inflammatory pain; and (ii) the contributions of central versus peripheral FAAH in this action. The analgesic effect of paracetamol was evaluated in thermal hyperalgesia, mechanical allodynia and hyperalgesia induced by an intra-plantar injection of carrageenan (3%) in FAAH knock-out mice or their littermates. Moreover, the contribution of the central and peripheral enzymes was explored by comparing the effect of a global FAAH inhibitor (URB597) to that of a peripherally restricted FAAH inhibitor (URB937) on paracetamol action. Here, we show that in a model of inflammatory pain submitted to different stimuli, the analgesic action of paracetamol was abolished when FAAH was genetically or pharmacologically inhibited. Whereas a global FAAH inhibitor, URB597 (0.3 mg/kg), reduced the anti-hyperalgesic action of paracetamol, a brain-impermeant FAAH inhibitor, URB937 (0.3 mg/kg), had no influence. However, administered intracerebroventricularly, URB937 (5 μg/mouse) reduced the action of paracetamol. These results demonstrate that the supra-spinally-located FAAH enzyme is necessary for the analgesic action of paracetamol.

  15. High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker.

    PubMed

    Yapa, Udeni; Prusakiewicz, Jeffery J; Wrightstone, Ann D; Christine, Lori J; Palandra, Joe; Groeber, Elizabeth; Wittwer, Arthur J

    2012-02-15

    Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.

  16. Role of FAAH-like anandamide transporter in anandamide inactivation.

    PubMed

    Leung, Kwannok; Elmes, Matthew W; Glaser, Sherrye T; Deutsch, Dale G; Kaczocha, Martin

    2013-01-01

    The endocannabinoid system modulates numerous physiological processes including nociception and reproduction. Anandamide (AEA) is an endocannabinoid that is inactivated by cellular uptake followed by intracellular hydrolysis by fatty acid amide hydrolase (FAAH). Recently, FAAH-like anandamide transporter (FLAT), a truncated and catalytically-inactive variant of FAAH, was proposed to function as an intracellular AEA carrier and mediate its delivery to FAAH for hydrolysis. Pharmacological inhibition of FLAT potentiated AEA signaling and produced antinociceptive effects. Given that endocannabinoids produce analgesia through central and peripheral mechanisms, the goal of the current work was to examine the expression of FLAT in the central and peripheral nervous systems. In contrast to the original report characterizing FLAT, expression of FLAT was not observed in any of the tissues examined. To investigate the role of FLAT as a putative AEA binding protein, FLAT was generated from FAAH using polymerase chain reaction and further analyzed. Despite its low cellular expression, FLAT displayed residual catalytic activity that was sensitive to FAAH inhibitors and abolished following mutation of its catalytic serine. Overexpression of FLAT potentiated AEA cellular uptake and this appeared to be dependent upon its catalytic activity. Immunofluorescence revealed that FLAT localizes primarily to intracellular membranes and does not contact the plasma membrane, suggesting that its capability to potentiate AEA uptake may stem from its enzymatic rather than transport activity. Collectively, our data demonstrate that FLAT does not serve as a global intracellular AEA carrier, although a role in mediating localized AEA inactivation in mammalian tissues cannot be ruled out.

  17. Macamides and their synthetic analogs: evaluation of in vitro FAAH inhibition.

    PubMed

    Wu, Hui; Kelley, Charles J; Pino-Figueroa, Alejandro; Vu, Huyen D; Maher, Timothy J

    2013-09-01

    Maca (Lepidium meyenii), a traditional food crop of the Peruvian Andes is now widely touted as a dietary supplement. Among the various chemical constituents isolated from the plant are a unique series of non-polar, long-chain fatty acid N-benzylamides known as macamides. We have synthesized 11 of the 19 reported macamides and have tested each as potential inhibitors of the human enzyme, fatty acid amide hydrolase (FAAH). The five most potent macamides were FAAH inhibitors (IC50=10-17μM). These amides were derivatives of oleic, linoleic and linolenic acids and benzylamine or 3-methoxybenzylamine. Of the three compounds evaluated in a pre-incubation time study, two macamides were not irreversible inhibitors of FAAH. The third, a carbamate structurally related to macamides, was shown to be an irreversible inhibitor of FAAH (IC50=0.153μM).

  18. Selective Inhibitors of Protein Methyltransferases

    PubMed Central

    2015-01-01

    Mounting evidence suggests that protein methyltransferases (PMTs), which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and human diseases. In particular, PMTs have been recognized as major players in regulating gene expression and chromatin state. PMTs are divided into two categories: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs). There has been a steadily growing interest in these enzymes as potential therapeutic targets and therefore discovery of PMT inhibitors has also been pursued increasingly over the past decade. Here, we present a perspective on selective, small-molecule inhibitors of PMTs with an emphasis on their discovery, characterization, and applicability as chemical tools for deciphering the target PMTs’ physiological functions and involvement in human diseases. We highlight the current state of PMT inhibitors and discuss future directions and opportunities for PMT inhibitor discovery. PMID:25406853

  19. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants

    PubMed Central

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors’ therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic–pituitary–adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted. PMID:26630956

  20. Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Alapafuja, Shakiru O.; Nikas, Spyros P.; Bharatan, Indu; Shukla, Vidyanand G.; Nasr, Mahmoud L.; Bowman, Anna L.; Zvonok, Nikolai; Li, Jing; Shi, Xiaomeng; Engen, John R.; Makriyannis, Alexandros

    2013-01-01

    Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs. PMID:23083016

  1. Peripheral FAAH inhibition causes profound antinociception and protects against indomethacin-induced gastric lesions.

    PubMed

    Sasso, Oscar; Bertorelli, Rosalia; Bandiera, Tiziano; Scarpelli, Rita; Colombano, Giampiero; Armirotti, Andrea; Moreno-Sanz, Guillermo; Reggiani, Angelo; Piomelli, Daniele

    2012-05-01

    Fatty-acid amide hydrolase (FAAH) catalyzes the intracellular hydrolysis of the endocannabinoid anandamide and other bioactive lipid amides. In the present study, we conducted a comparative characterization of the effects of the newly identified brain-impermeant FAAH inhibitor, URB937 ([3-(3-carbamoylphenyl)-4-hydroxy-phenyl] N-cyclohexylcarbamate), in various rodent models of acute and persistent pain. When administered by the oral route in mice, URB937 was highly active (median effective dose, ED(50), to inhibit liver FAAH activity: 0.3mgkg(-1)) and had a bioavailability of 5.3%. The antinociceptive effects of oral URB937 were investigated in mouse models of acute inflammation (carrageenan), peripheral nerve injury (chronic sciatic nerve ligation) and arthritis (complete Freund's adjuvant). In all models, URB937 was as effective or more effective than standard analgesic and anti-inflammatory drugs (indomethacin, gabapentin, dexamethasone) and reversed pain-related responses (mechanical hyperalgesia, thermal hyperalgesia, and mechanical allodynia) in a dose-dependent manner. ED(50) values ranged from 0.2 to 10mgkg(-1), depending on model and readout. Importantly, URB937 was significantly more effective than two global FAAH inhibitors, URB597 and PF-04457845, in the complete Freund's adjuvant model. The effects of a combination of URB937 with the non-steroidal anti-inflammatory agent, indomethacin, were examined in the carrageenan and chronic sciatic nerve ligation models. Isobolographic analyses showed that the two compounds interacted synergistically to attenuate pain-related behaviors. Furthermore, URB937 reduced the number and severity of gastric lesions produced by indomethacin, while exerting no ulcerogenic effect when administered alone. The results indicate that the peripheral FAAH inhibitor URB937 is more effective than globally active FAAH inhibitors at inhibiting inflammatory pain. Our findings further suggest that FAAH and cyclooxygenase inhibitors

  2. The Molecular Basis for Dual Fatty Acid Amide Hydrolase (FAAH)/Cyclooxygenase (COX) Inhibition.

    PubMed

    Palermo, Giulia; Favia, Angelo D; Convertino, Marino; De Vivo, Marco

    2016-06-20

    The design of multitarget-directed ligands is a promising strategy for discovering innovative drugs. Here, we report a mechanistic study that clarifies key aspects of the dual inhibition of the fatty acid amide hydrolase (FAAH) and the cyclooxygenase (COX) enzymes by a new multitarget-directed ligand named ARN2508 (2-[3-fluoro-4-[3-(hexylcarbamoyloxy)phenyl]phenyl]propanoic acid). This potent dual inhibitor combines, in a single scaffold, the pharmacophoric elements often needed to block FAAH and COX, that is, a carbamate moiety and the 2-arylpropionic acid functionality, respectively. Molecular modeling and molecular dynamics simulations suggest that ARN2508 uses a noncovalent mechanism of inhibition to block COXs, while inhibiting FAAH via the acetylation of the catalytic Ser241, in line with previous experimental evidence for covalent FAAH inhibition. This study proposes the molecular basis for the dual FAAH/COX inhibition by this novel hybrid scaffold, stimulating further experimental studies and offering new insights for the rational design of novel anti-inflammatory agents that simultaneously act on FAAH and COX.

  3. Investigating the selectivity of metalloenzyme inhibitors.

    PubMed

    Day, Joshua A; Cohen, Seth M

    2013-10-24

    The inhibitory activity of a broad group of known metalloenzyme inhibitors against a panel of metalloenzymes was evaluated. Clinically approved inhibitors were selected as well as several other reported metalloprotein inhibitors in order to represent a broad range of metal binding groups (MBGs), including hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea functional groups. A panel of metalloenzymes, including carbonic anhydrase (hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme (ACE), histone deacetylase (HDAC-2), and tyrosinase (TY), was selected based on their clinical importance for a range of pathologies. In addition, each inhibitor was evaluated for its ability to remove Fe(3+) from holo-transferrin to gauge the ability of the inhibitors to access Fe(3+) from a primary transport protein. The results show that the metalloenzyme inhibitors are quite selective for their intended targets, suggesting that despite their ability to bind metal ions, metalloprotein inhibitors are not prone to widespread off-target enzyme inhibition activity.

  4. Fatty acid amide hydrolase (FAAH) blockade ameliorates experimental colitis by altering microRNA expression and suppressing inflammation.

    PubMed

    Shamran, Haidar; Singh, Narendra P; Zumbrun, Elizabeth E; Murphy, Angela; Taub, Dennis D; Mishra, Manoj K; Price, Robert L; Chatterjee, Saurabh; Nagarkatti, Mitzi; Nagarkatti, Prakash S; Singh, Udai P

    2017-01-01

    Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), which is thought to result from immune-mediated inflammatory disorders, leads to high morbidity and health care cost. Fatty acid amide hydrolase (FAAH) is an enzyme crucially involved in the modulation of intestinal physiology through anandamide (AEA) and other endocannabinoids. Here we examined the effects of an FAAH inhibitor (FAAH-II), on dextran sodium sulphate (DSS)-induced experimental colitis in mice. Treatments with FAAH-II improved overall clinical scores by reversing weight loss and colitis-associated pathogenesis. The frequencies of activated CD4(+) T cells in spleens, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and colon lamina propiria (LP) were reduced by FAAH inhibition. Similarly, the frequencies of macrophages, neutrophils, natural killer (NK), and NKT cells in the PPs and LP of mice with colitis declined after FAAH blockade, as did concentrations of systemic and colon inflammatory cytokines. Microarray analysis showed that 26 miRNAs from MLNs and 217 from PPs had a 1.5-fold greater difference in expression after FAAH inhibition. Among them, 8 miRNAs were determined by reverse-transcription polymerase chain reaction (RT-PCR) analysis to have anti-inflammatory properties. Pathway analysis demonstrated that differentially regulated miRNAs target mRNA associated with inflammation. Thus, FAAH-II ameliorates experimental colitis by reducing not only the number of activated T cells but also the frequency of macrophages, neutrophils, and NK/NKT cell, as well as inflammatory miRNAs and cytokine at effector sites in the colon. These studies demonstrate for the first time that FAAH-II inhibitor may suppress colitis through regulation of pro-inflammatory miRNAs expression.

  5. Selective serotonin reuptake inhibitor exposure.

    PubMed

    Fitzgerald, Kevin T; Bronstein, Alvin C

    2013-02-01

    Many antidepressants inhibit serotonin or norepinephrine reuptake or both to achieve their clinical effect. The selective serotonin reuptake inhibitor class of antidepressants (SSRIs) includes citalopram, escitalopram (active enantiomer of citalopram), fluoxetine, fluvoxamine, paroxetine, and sertraline. The SSRIs are as effective as tricyclic antidepressants in treatment of major depression with less significant side effects. As a result, they have become the largest class of medications prescribed to humans for depression. They are also used to treat obsessive-compulsive disorder, panic disorders, alcoholism, obesity, migraines, and chronic pain. An SSRI (fluoxetine) has been approved for veterinary use in treatment of canine separation anxiety. SSRIs act specifically on synaptic serotonin concentrations by blocking its reuptake in the presynapse and increasing levels in the presynaptic membrane. Clinical signs of SSRI overdose result from excessive amounts of serotonin in the central nervous system. These signs include nausea, vomiting, mydriasis, hypersalivation, and hyperthermia. Clinical signs are dose dependent and higher dosages may result in the serotonin syndrome that manifests itself as ataxia, tremors, muscle rigidity, hyperthermia, diarrhea, and seizures. Current studies reveal no increase in appearance of any specific clinical signs of serotonin toxicity with regard to any SSRI medication. In people, citalopram has been reported to have an increased risk of electrocardiographic abnormalities. Diagnosis of SSRI poisoning is based on history, clinical signs, and response to therapy. No single clinical test is currently available to confirm SSRI toxicosis. The goals of treatment in this intoxication are to support the animal, prevent further absorption of the drug, support the central nervous system, control hyperthermia, and halt any seizure activity. The relative safety of the SSRIs in overdose despite the occurrence of serotonin syndrome makes them

  6. Inhibition of fatty acid amide hydrolase (FAAH) reduces spinal nociceptive responses and expression of spinal long-term potentiation (LTP).

    PubMed

    Eriksen, Guro S; Jacobsen, Line Melå; Mahmood, Aqsa; Pedersen, Linda M; Gjerstad, Johannes

    2012-02-10

    Fatty acid amide hydrolase (FAAH) is an enzyme that metabolizes endocannabinoids and fatty acid amides possibly linked to activation of the opioid system. To examine how this enzyme affects spinal signalling, electrophysiological recordings in the dorsal horn and qPCR on dorsal horn tissue following systemic administration of the FAAH inhibitor URB597 (0.3 and 1.0mg/kg i.v.) and spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.), were performed. The present data showed that the suppressive effect of the FAAH inhibitor URB597 (1.0mg/kg i.v.) on the spinal nociceptive responses was prevented by spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.). Moreover, the present findings demonstrated that the FAAH inhibitor URB597 (1.0mg/kg i.v.) partly reversed expression of spinal long-term potentiation (LTP) and also attenuated the LTP-associated increased Zif expression. We conclude that pharmacological inactivation of FAAH may be a promising strategy to inhibit the development of central hyperalgesia; thereby reinforcing the role of FAAH as a potential therapeutic target.

  7. Inhibition of FAAH reduces nitroglycerin-induced migraine-like pain and trigeminal neuronal hyperactivity in mice.

    PubMed

    Nozaki, Chihiro; Markert, Astrid; Zimmer, Andreas

    2015-08-01

    There is evidence to suggest that a dysregulation of endocannabinoid signaling may contribute to the etiology and pathophysiology of migraine. Thus, patients suffering from chronic migraine or medication overuse headache showed alterations in the activity of the arachidonoylethanolamide (AEA) degrading enzyme fatty acid amide hydrolase (FAAH) and a specific AEA membrane transporter, alongside with changes in AEA levels. The precise role of different endocannabinoid system components is, however, not clear. We have therefore investigated mice with a genetic deletion of the two main cannabinoid receptors CB1 and CB2, or the main endocannabinoid degrading enzymes, FAAH and monoacylglycerol lipase (MAGL), which degrades 2-arachidonoylglycerol (2-AG), in a nitroglycerine-induced animal model of migraine. We found that nitroglycerin-induced mechanical allodynia and neuronal activation of the trigeminal nucleus were completely abolished in FAAH-deficient mice. To validate these results, we used two structurally different FAAH inhibitors, URB597 and PF3945. Both inhibitors also dose-dependently blocked nitroglycerin-induced hyperalgesia and the activation of trigeminal neurons. The effects of the genetic deletion of pharmacological blockade of FAAH are mediated by CB1 receptors, because they were completely disrupted with the CB1 antagonist rimonabant. These results identify FAAH as a target for migraine pharmacotherapy.

  8. Selective Phosphodiesterase 4B Inhibitors: A Review

    PubMed Central

    Azam, Mohammed Afzal; Tripuraneni, Naga Srinivas

    2014-01-01

    Abstract Phosphodiesterase 4B (PDE4B) is a member of the phosphodiesterase family of proteins that plays a critical role in regulating intracellular levels of cyclic adenosine monophosphate (cAMP) by controlling its rate of degradation. It has been demonstrated that this isoform is involved in the orchestra of events which includes inflammation, schizophrenia, cancers, chronic obstructive pulmonary disease, contractility of the myocardium, and psoriatic arthritis. Phosphodiesterase 4B has constituted an interesting target for drug development. In recent years, a number of PDE4B inhibitors have been developed for their use as therapeutic agents. In this review, an up-to-date status of the inhibitors investigated for the inhibition of PDE4B has been given so that this rich source of structural information of presently known PDE4B inhibitors could be helpful in generating a selective and potent inhibitor of PDE4B. PMID:25853062

  9. Inhibitors Selective for Mycobacterial Versus Human Proteasomes

    SciTech Connect

    Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

    2009-01-01

    Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

  10. Inhibition of FAAH and activation of PPAR: New approaches to the treatment of cognitive dysfunction and drug addiction

    PubMed Central

    Panlilio, Leigh V.; Justinova, Zuzana; Goldberg, Steven R.

    2013-01-01

    Enhancing the effects of endogenously-released cannabinoid ligands in the brain might provide therapeutic effects more safely and effectively than administering drugs that act directly at the cannabinoid receptor. Inhibitors of fatty acid amide hydrolase (FAAH) prevent the breakdown of endogenous ligands for cannabinoid receptors and peroxisome proliferator-activated receptors (PPAR), prolonging and enhancing the effects of these ligands when they are naturally released. This review considers recent research on the effects of FAAH inhibitors and PPAR activators in animal models of addiction and cognition (specifically learning and memory). These studies show that FAAH inhibitors can produce potentially therapeutic effects, some through cannabinoid receptors and some through PPAR. These effects include enhancing certain forms of learning, counteracting the rewarding effects of nicotine and alcohol, relieving symptoms of withdrawal from cannabis and other drugs, and protecting against relapse-like reinstatement of drug self-administration. Since FAAH inhibition might have a wide range of therapeutic actions but might also share some of the adverse effects of cannabis, it is noteworthy that at least one FAAH-inhibiting drug (URB597) has been found to have potentially beneficial effects but no indication of liability for abuse or dependence. Although these areas of research are new, the preliminary evidence indicates that they might lead to improved therapeutic interventions and a better understanding of the brain mechanisms underlying addiction and memory. PMID:23333350

  11. Inhibition of FAAH and activation of PPAR: new approaches to the treatment of cognitive dysfunction and drug addiction.

    PubMed

    Panlilio, Leigh V; Justinova, Zuzana; Goldberg, Steven R

    2013-04-01

    Enhancing the effects of endogenously-released cannabinoid ligands in the brain might provide therapeutic effects more safely and effectively than administering drugs that act directly at the cannabinoid receptor. Inhibitors of fatty acid amide hydrolase (FAAH) prevent the breakdown of endogenous ligands for cannabinoid receptors and peroxisome proliferator-activated receptors (PPAR), prolonging and enhancing the effects of these ligands when they are naturally released. This review considers recent research on the effects of FAAH inhibitors and PPAR activators in animal models of addiction and cognition (specifically learning and memory). These studies show that FAAH inhibitors can produce potentially therapeutic effects, some through cannabinoid receptors and some through PPAR. These effects include enhancing certain forms of learning, counteracting the rewarding effects of nicotine and alcohol, relieving symptoms of withdrawal from cannabis and other drugs, and protecting against relapse-like reinstatement of drug self-administration. Since FAAH inhibition might have a wide range of therapeutic actions but might also share some of the adverse effects of cannabis, it is noteworthy that at least one FAAH-inhibiting drug (URB597) has been found to have potentially beneficial effects but no indication of liability for abuse or dependence. Although these areas of research are new, the preliminary evidence indicates that they might lead to improved therapeutic interventions and a better understanding of the brain mechanisms underlying addiction and memory.

  12. Identification of potent, selective KDM5 inhibitors.

    PubMed

    Gehling, Victor S; Bellon, Steven F; Harmange, Jean-Christophe; LeBlanc, Yves; Poy, Florence; Odate, Shobu; Buker, Shane; Lan, Fei; Arora, Shilpi; Williamson, Kaylyn E; Sandy, Peter; Cummings, Richard T; Bailey, Christopher M; Bergeron, Louise; Mao, Weifeng; Gustafson, Amy; Liu, Yichin; VanderPorten, Erica; Audia, James E; Trojer, Patrick; Albrecht, Brian K

    2016-09-01

    This communication describes the identification and optimization of a series of pan-KDM5 inhibitors derived from compound 1, a hit initially identified against KDM4C. Compound 1 was optimized to afford compound 20, a 10nM inhibitor of KDM5A. Compound 20 is highly selective for the KDM5 enzymes versus other histone lysine demethylases and demonstrates activity in a cellular assay measuring the increase in global histone 3 lysine 4 tri-methylation (H3K4me3). In addition compound 20 has good ADME properties, excellent mouse PK, and is a suitable starting point for further optimization.

  13. Inhibition of fatty acid amide hydrolase (FAAH) as a novel therapeutic strategy in the treatment of pain and inflammatory diseases in the gastrointestinal tract.

    PubMed

    Sałaga, Maciej; Sobczak, Marta; Fichna, Jakub

    2014-02-14

    Fatty acid amide hydrolase (FAAH) is the enzyme crucially involved in the modulation of physiological processes mediated by anandamide (AEA), as well as other endocannabinoids and non-cannabinoid biolipids in the gastrointestinal (GI) tract. FAAH also plays a major role in the etiology and the course of GI diseases and the inhibition of the enzyme has recently become a potential target for their therapy. In this review we look at the pharmacology of FAAH and possible clinical application of FAAH inhibitors in the treatment of GI disorders. In particular, we focus on inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS), whose symptoms include abdominal pain and motility disturbances. We also discuss why the inhibitor-based drugs may replace in future conventional therapies for IBD and IBS.

  14. Biochemical and biological properties of 4-(3-phenyl-[1,2,4] thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide, a mechanism-based inhibitor of fatty acid amide hydrolase.

    PubMed

    Karbarz, Mark J; Luo, Lin; Chang, Leon; Tham, Chui-Se; Palmer, James A; Wilson, Sandy J; Wennerholm, Michelle L; Brown, Sean M; Scott, Brian P; Apodaca, Richard L; Keith, John M; Wu, Jiejun; Breitenbucher, James Guy; Chaplan, Sandra R; Webb, Michael

    2009-01-01

    Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme within the amidase-signature family. It catalyzes the hydrolysis of several endogenous biologically active lipids, including anandamide (arachidonoyl ethanolamide), oleoyl ethanolamide, and palmitoyl ethanolamide. These endogenous FAAH substrates have been shown to be involved in a variety of physiological and pathological processes, including synaptic regulation, regulation of sleep and feeding, locomotor activity, pain and inflammation. Here we describe the biochemical and biological properties of a potent and selective FAAH inhibitor, 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide (JNJ-1661010). The time-dependence of apparent IC(50) values at rat and human recombinant FAAH, dialysis and mass spectrometry data indicate that the acyl piperazinyl fragment of JNJ-1661010 forms a covalent bond with the enzyme. This bond is slowly hydrolyzed, with release of the piperazinyl fragment and recovery of enzyme activity. The lack of inhibition observed in a rat liver esterase assay suggests that JNJ-1661010 is not a general esterase inhibitor. JNJ-1661010 is >100-fold preferentially selective for FAAH-1 when compared to FAAH-2. JNJ-1661010 dose-dependently increases arachidonoyl ethanolamide, oleoyl ethanolamide, and palmitoyl ethanolamide in the rat brain. The compound attenuates tactile allodynia in the rat mild thermal injury model of acute tissue damage and in the rat spinal nerve ligation (Chung) model of neuropathic pain. JNJ-1661010 also diminishes thermal hyperalgesia in the inflammatory rat carrageenan paw model. These data suggest that FAAH inhibitors with modes of action similar to JNJ-1661010 may be useful clinically as broad-spectrum analgesics.

  15. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    ERIC Educational Resources Information Center

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  16. Binding and Inactivation Mechanism of a Humanized Fatty Acid Amide Hydrolase by [alpha]-Ketoheterocycle Inhibitors Revealed from Cocrystal Structures

    SciTech Connect

    Mileni, Mauro; Garfunkle, Joie; DeMartino, Jessica K.; Cravatt, Benjamin F.; Boger, Dale L.; Stevens, Raymond C.

    2010-08-17

    The cocrystal X-ray structures of two isomeric {alpha}-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional 'in-action' depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.

  17. [Selective serotonin reuptake inhibitors (SSRI) and osteoporosis].

    PubMed

    Fétique-Will, Anne-Catherine; Chevalley, Thierry; Rizzoli, René

    2011-06-15

    Selective serotonin reuptake inhibitors (SSRI) represent the first-line treatment of depression. Several studies demonstrate that use of therapeutical doses of SSRI is associated with a decreased bone mineral density (BMD) and an increased risk of fracture. Mechanisms of action of SSRI on bone tissue are not totally clarified. These treatments would be associated with an increased risk of falls and would also have a direct effect on bone metabolism. Regarding proofs existing of the implication of SSRI on osteoporosis, while waiting for larger-scale prospective studies, it appears reasonable that practitioners assess bone loss within risk groups of patients treated with SSRI.

  18. [Selective serotonin reuptake inhibitor and gastrointestinal hemorrhage].

    PubMed

    Yamamoto, Takatsugu; Abe, Koichiro; Kuyama, Yasushi

    2013-04-01

    Selective serotonin reuptake inhibitors (SSRI) are widely used antidepressants characterized by less-frequent adverse effects compared with classical anti-depressive agents. On the other hand, SSRI can cause hemorrhagic events more due to impaired platelet aggregation induced by a depletion of serotonin in the peripheral platelet. Epidemiological studies have indicated that patients taking SSRI are predisposed to gastrointestinal hemorrhage, especially in case that nonsteroidal anti-inflammatory drugs are prescribed concomitantly. Here we describe a risk of the gastrointestinal hemorrhage in patients taking SSRI.

  19. Genetically reduced FAAH activity may be a risk for the development of anxiety and depression in persons with repetitive childhood trauma.

    PubMed

    Lazary, Judit; Eszlari, Nora; Juhasz, Gabriella; Bagdy, Gyorgy

    2016-06-01

    Fatty acid amide hydrolase (FAAH) inhibitors are addressed for promising anxiolytics, but human studies on genetically reduced FAAH activity, stress and affective phenotypes are scarce. We investigated the effect of a functional polymorphism of FAAH (FAAH C385A or rs324420; low FAAH activity and high anandamide concentration are associated with the A allele) together with childhood adversity on the anxious and depressive phenotypes in 858 subjects from the general population. Phenotypes were measured by the Zung Self-Rating Depression Scale (ZSDS), the depression and anxiety subscales of the Brief Symptom Inventory (BSI-DEP, BSI-ANX) and the State-Trait Anxiety scales (STAI-S, STAI-T). Childhood Adversity Questionnaire (CHA) was used to assess early life traumas. Frequency of the A allele was greater among subjects with high ZSDS scores compared to the CC genotype. Furthermore, FAAH C385A and the CHA have shown a robust gene-environment interaction, namely, significantly higher anxiety and depression scores were exhibited by individuals carrying the A allele if they had high CHA scores compared to CC carriers. These data provided preliminary evidence that genetically reduced FAAH activity and repetitive stress in the childhood are associated with increased vulnerability for anxiety and depression in later life. Our results together with earlier experimental data suggest that permanently elevated anandamide level together with early life stress may cause a lifelong damage on stress response probably via the downregulation of CB1R during the neurodevelopment in the brain. It may also point to pharmacogenomic consequences, namely ineffectiveness or adverse effects of FAAH inhibitors in this subpopulation.

  20. Knipholone, a selective inhibitor of leukotriene metabolism.

    PubMed

    Wube, A A; Bucar, F; Asres, K; Gibbons, S; Adams, M; Streit, B; Bodensieck, A; Bauer, R

    2006-06-01

    Inhibition of leukotriene formation is one of the approaches to the treatment of asthma and other inflammatory diseases. We have investigated knipholone, isolated from the roots of Kniphofia foliosa, Hochst (Asphodelaceae), for inhibition of leukotriene biosynthesis in an ex vivo bioassay using activated human neutrophile granulocytes. Moreover, activities on 12-lipoxygenase from human platelets and cycloxygenase (COX)-1 and -2 from sheep cotyledons and seminal vesicles, respectively, have been evaluated. Knipholone was found to be a selective inhibitor of leukotriene metabolism in a human blood assay with an IC(50) value of 4.2microM. However, at a concentration of 10microg/ml, the compound showed weak inhibition of 12(S)-HETE production in human platelets and at a concentration of 50microM it produced no inhibition of COX-1 and -2. In our attempt to explain the mechanism of inhibition, we examined the antioxidant activity of knipholone using various in vitro assay systems including free radical scavenging, non-enzymatic lipid peroxidation, and metal chelation. Knipholone was found to be a weak dose-independent free radical scavenger and lipid peroxidation inhibitor, but not a metal chelator. Therefore, the leukotriene biosynthesis inhibitory effect of knipholone was evident by its ability either to inhibit the 5-lipoxygenase activating protein (FLAP) or as a competitive (non-redox) inhibitor of the enzyme. Cytotoxicity results also provided evidence that knipholone exhibits less toxicity for a mammalian host cell.

  1. Diacerein is a potent and selective inhibitor of palmitoylethanolamide inactivation with analgesic activity in a rat model of acute inflammatory pain.

    PubMed

    Petrosino, Stefania; Ahmad, Akbar; Marcolongo, Gabriele; Esposito, Emanuela; Allarà, Marco; Verde, Roberta; Cuzzocrea, Salvatore; Di Marzo, Vincenzo

    2015-01-01

    Palmitoylethanolamide (PEA) is produced by mammalian cells from its biosynthetic precursor, N-palmitoyl-phosphatidyl-ethanolamine, and inactivated by enzymatic hydrolysis to palmitic acid and ethanolamine. Apart from fatty acid amide hydrolase (FAAH), the N-acylethanolamine-hydrolyzing acid amidase (NAAA), a lysosomal enzyme, was also shown to catalyze the hydrolysis of PEA and to limit its analgesic and anti-inflammatory action. Here we report the finding of a new potential inhibitor of NAAA, EPT4900 (4,5-diacetyloxy-9,10-dioxo-anthracene-2-carboxylic acid, diacerein). EPT4900 exhibited a high inhibitory activity on human recombinant NAAA over-expressed in HEK293 cells (HEK-NAAA cells). EPT4900 selectively increased the levels of PEA in intact HEK-NAAA cells, and inhibited inflammation as well as hyperalgesia in rats treated with an intraplantar injection of carrageenan. This latter effect was accompanied by elevation of PEA endogenous levels in the paw skin.

  2. Variants in the CNR1 and the FAAH genes and adiposity traits in the community

    PubMed Central

    Lieb, Wolfgang; Manning, Alisa K.; Florez, Jose C.; Dupuis, Josée; Cupples, L. Adrienne; McAteer, Jarred B.; Vasan, Ramachandran S.; Hoffmann, Udo; O'Donnell, Christopher J.; Meigs, James B.; Fox, Caroline S.

    2011-01-01

    Pharmacologic blockade of the endocannabinoid receptor 1 leads to weight loss and an improved metabolic risk profile in overweight and obese individuals. We hypothesize that common genetic variants in the CNR1 (encoding endocannabinoid receptor 1) and FAAH genes (encoding fatty acid amide hydrolase, a key enzyme hydrolyzing endocannabinoids) are associated with adiposity traits. We genotyped 18 single nucleotide polymorphisms (SNPs) in the CNR1 and 9 SNPs in the FAAH gene in 2,415 Framingham Offspring Study participants (mean age 61±10 years; 52.6% women; mean BMI 28.2±5.4 kg/m2; 30.3% obese) and studied them for association with cross-sectional and longitudinal measures of adiposity (body mass index [BMI], waist circumference, change over time in BMI and waist circumference, visceral and subcutaneous adipose tissue) using linear mixed-effect models. The selected SNPs captured 85% (r2=0.8) of the common variation (minor allele frequency >5%) at the CNR1 locus and 96% (r2=0.8) of the common variation at the FAAH locus (defined as the genomic segment containing the gene +20 kb upstream and +10 kb downstream). After correction for multiple testing, none of the SNPs in the CNR1 gene or in the FAAH gene displayed statistical evidence for association with BMI, waist circumference and visceral adipose tissue or subcutaneous adipose tissue (all P>0.18). Despite comprehensive SNP mapping across the genes and their regulatory regions in a large unselected sample, we failed to find evidence for an association of common variants in the CNR1 and FAAH genes with measures of adiposity in our community-based sample. PMID:19165169

  3. Chemical origins of isoform selectivity in histone deacetylase inhibitors.

    PubMed

    Butler, Kyle V; Kozikowski, Alan P

    2008-01-01

    Histones undergo extensive posttranslational modifications that affect gene expression. Acetylation is a key histone modification that is primarily regulated by two enzymes, one of which is histone deacetylase (HDAC). The activity of HDAC causes transcriptional silencing of DNA. Eleven distinct zinc-dependent histone deacetylase isoforms have been identified in humans. Each isoform has a unique structure and function, and regulates a unique set of genes. HDAC is responsible for the regulation of many genes involved in cancer cell proliferation, and it has been implicated in the pathogenesis of many neurological conditions. HDAC inhibitors are known to be very effective anti-cancer agents, and research has shown them to be potential treatments for many other conditions. Histone deacetylase inhibitors modify the expression of many genes, and it is possible that inhibition of one isoform could cause epigenetic changes that are beneficial to treatment of a disease, while inhibition of another isoform could cause contradictory changes. Selective HDAC inhibitors will be better able to avoid these types of situations than non-specific inhibitors, and may also be less toxic than pan-HDAC inhibitors. Many potent pan-HDAC inhibitors have already been developed, leaving the development of selective inhibitors at the forefront of HDAC drug development. Certain structural moieties may be added to HDAC inhibitors to give isoform selectivity, and these will be discussed in this review. This review will focus on the applications of selective HDAC inhibitors, inhibitors reported to show selectivity, and the relationship between inhibitor structure and selectivity.

  4. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs)

    PubMed Central

    Deutsch, Dale G.

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that “solubilize” anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992

  5. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

    PubMed

    Deutsch, Dale G

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to

  6. Fatty acid amide hydrolase inhibitors confer anti-invasive and antimetastatic effects on lung cancer cells

    PubMed Central

    Winkler, Katrin; Ramer, Robert; Dithmer, Sophie; Ivanov, Igor; Merkord, Jutta; Hinz, Burkhard

    2016-01-01

    Inhibition of endocannabinoid degradation has been suggested as tool for activation of endogenous tumor defense. One of these strategies lies in blockade of fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA], 2-arachidonoylglycerol [2-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA], N-palmitoylethanolamine [PEA]). This study addressed the impact of two FAAH inhibitors (arachidonoyl serotonin [AA-5HT], URB597) on A549 lung cancer cell metastasis and invasion. LC-MS analyses revealed increased levels of FAAH substrates (AEA, 2-AG, OEA, PEA) in cells incubated with either FAAH inhibitor. In athymic nude mice FAAH inhibitors were shown to elicit a dose-dependent antimetastatic action yielding a 67% and 62% inhibition of metastatic lung nodules following repeated administration of 15 mg/kg AA-5HT and 5 mg/kg URB597, respectively. In vitro, a concentration-dependent anti-invasive action of either FAAH inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Using siRNA approaches, a causal link between the TIMP-1-upregulating and anti-invasive action of FAAH inhibitors was confirmed. Moreover, knockdown of FAAH by siRNA was shown to confer decreased cancer cell invasiveness and increased TIMP-1 expression. Inhibitor experiments point toward a role of CB2 and transient receptor potential vanilloid 1 in conferring anti-invasive effects of FAAH inhibitors and FAAH siRNA. Finally, antimetastatic and anti-invasive effects were confirmed for all FAAH substrates with AEA and OEA causing a TIMP-1-dependent anti-invasive action. Collectively, the present study provides first-time proof for an antimetastatic action of FAAH inhibitors. As mechanism of its anti-invasive properties an upregulation of TIMP-1 was identified. PMID:26930716

  7. Chronic FAAH inhibition during nicotine abstinence alters habenular CB1 receptor activity and precipitates depressive-like behaviors.

    PubMed

    Simonnet, A; Zamberletti, E; Cador, M; Rubino, T; Caillé, S

    2017-02-01

    The role of the endocannabinoid system in nicotine addiction is being increasingly acknowledged. Acute inhibition of anandamide (AEA) degradation efficiently reduces nicotine withdrawal-induced affective symptoms in rats and fatty acid amide hydrolase (FAAH), the degradation enzyme of AEA, has been proposed as a possible treatment against nicotine addiction. However, it is unclear whether chronic inhibition of AEA during nicotine abstinence will have beneficial or deleterious affective side-effects. Using a rat model of nicotine addiction, we found that, during abstinence, rats injected daily with a FAAH inhibitor (URB597) developed a depressive-like phenotype. Our results show that in the nicotine abstinent rats, URB597 induced low saccharin consumption, persistent immobility in the forced swim test and increased corticosterone levels in response to stress. In addition, URB597decreased CB1 receptor binding and activity in the habenula, a key structure in the control of nicotine-related emotional states. In contrast, non-treated abstinent rats showed increased CB1 receptor activity and behaviors comparable to controls. No FAAH inhibition-induced alterations were observed in animals that had a previous history of saline self-administration. Taken together, our results suggest that chronic FAAH inhibition prevents the homeostatic adaptations of habenular CB1 receptor function that are necessary for the recovery from nicotine dependence.

  8. New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis.

    PubMed

    Ligresti, Alessia; Cascio, Maria Grazia; Pryce, Gareth; Kulasegram, Sanjitha; Beletskaya, Irina; De Petrocellis, Luciano; Saha, Bijali; Mahadevan, Anu; Visintin, Cristina; Wiley, Jenny L; Baker, David; Martin, Billy R; Razdan, Raj K; Di Marzo, Vincenzo

    2006-01-01

    We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo and synthesized four structural analogs (O-2247, O-2248, O-3246, and O-3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB(1) and CB(2) receptors; (b) assays of transient receptor potential of the vanilloid type-1 (TRPV1) channel functional activity (measurement of [Ca(2+)](i)); (c) [(14)C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL-2H3) cells; (d) the mouse 'tetrad' tests (analgesia on a hot plate, immobility on a 'ring', rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O-2093, either synthesized by us or commercially available, was inactive in the 'tetrad' up to a 20 mg kg(-1) dose (i.v.). Like O-2093, the other four compounds exhibited low affinity in CB(1) (K(i) from 1.3 to >10 microM) and CB(2) binding assays (1.310 microM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC(50)>25 microM) and were inactive in the 'tetrad' up to a 30 mg kg(-1) dose (i.v.). While O-2247 and O-2248 were poor inhibitors of [(14)C]AEA cellular uptake (IC(50)>40 microM), O-3246 and O-3262 were quite potent in this assay. O-3246, which exhibits only a very subtle structural difference with O-2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC(50)=1.4 microM) and is 12-fold more potent than O-2093. When injected intravenously O-3246 and O-3262, again like O-2093 and unlike O-2247 and O-2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective

  9. New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis

    PubMed Central

    Ligresti, Alessia; Cascio, Maria Grazia; Pryce, Gareth; Kulasegram, Sanjitha; Beletskaya, Irina; De Petrocellis, Luciano; Saha, Bijali; Mahadevan, Anu; Visintin, Cristina; Wiley, Jenny L; Baker, David; Martin, Billy R; Razdan, Raj K; Di Marzo, Vincenzo

    2005-01-01

    We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo and synthesized four structural analogs (O-2247, O-2248, O-3246, and O-3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB1 and CB2 receptors; (b) assays of transient receptor potential of the vanilloid type-1 (TRPV1) channel functional activity (measurement of [Ca2+]i); (c) [14C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL-2H3) cells; (d) the mouse ‘tetrad' tests (analgesia on a hot plate, immobility on a ‘ring', rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O-2093, either synthesized by us or commercially available, was inactive in the ‘tetrad' up to a 20 mg kg−1 dose (i.v.). Like O-2093, the other four compounds exhibited low affinity in CB1 (Ki from 1.3 to >10 μM) and CB2 binding assays (1.310 μM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC50>25 μM) and were inactive in the ‘tetrad' up to a 30 mg kg−1 dose (i.v.). While O-2247 and O-2248 were poor inhibitors of [14C]AEA cellular uptake (IC50>40 μM), O-3246 and O-3262 were quite potent in this assay. O-3246, which exhibits only a very subtle structural difference with O-2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC50=1.4 μM) and is 12-fold more potent than O-2093. When injected intravenously O-3246 and O-3262, again like O-2093 and unlike O-2247 and O-2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective AEA uptake

  10. Estrogenic/antiestrogenic activity of selected selective serotonin reuptake inhibitors

    PubMed Central

    POP, ANCA; LUPU, DIANA IOANA; CHERFAN, JULIEN; KISS, BELA; LOGHIN, FELICIA

    2015-01-01

    Background and aims Selective serotonin reuptake inhibitors (SSRIs) are one of the most prescribed classes of psychotropics. Even though the SSRI class consists of 6 molecules (citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline), only fluoxetine was intensively studied for endocrine disruptive effects, while the other SSRIs received less attention. This study was designed to evaluate the estrogenic/antiestrogenic effect of fluoxetine, sertraline and paroxetine. Methods The in vitro (anti)estrogenic activity was assessed using a firefly luciferase reporter construct in the T47D-KBluc breast cancer cell line. These cells express nuclear estrogen receptors that can activate the transcription of the luciferase reporter gene upon binding of estrogen receptor agonists. Results All three compounds were found to interact with the estrogen receptor. Fluoxetine had dual properties, weak estrogenic at lower concentrations and antiestrogenic effect at higher concentrations. Sertraline shared the same properties with fluoxetine, but also increased the estradiol-mediated transcriptional activity. Paroxetine presented only one type of effect, the ability to increase the estradiol-mediated transcriptional activity. Conclusions Overall, our results indicate a possible interaction of SSRIs with the estrogen receptor. As SSRIs are being used by all categories of population, including pregnant women or children, establishing whether they can affect the endocrine mediated mechanisms should be a priority. PMID:26609273

  11. Selective serotonin reuptake inhibitors for fibromyalgia syndrome

    PubMed Central

    Walitt, Brian; Urrútia, Gerard; Nishishinya, María Betina; Cantrell, Sarah E; Häuser, Winfried

    2016-01-01

    Background Fibromyalgia is a clinically well-defined chronic condition with a biopsychosocial aetiology. Fibromyalgia is characterized by chronic widespread musculoskeletal pain, sleep problems, cognitive dysfunction, and fatigue. Patients often report high disability levels and poor quality of life. Since there is no specific treatment that alters the pathogenesis of fibromyalgia, drug therapy focuses on pain reduction and improvement of other aversive symptoms. Objectives The objective was to assess the benefits and harms of selective serotonin reuptake inhibitors (SSRIs) in the treatment of fibromyalgia. Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL; 2014, Issue 5), MEDLINE (1966 to June 2014), EMBASE (1946 to June 2014), and the reference lists of reviewed articles. Selection criteria We selected all randomized, double-blind trials of SSRIs used for the treatment of fibromyalgia symptoms in adult participants. We considered the following SSRIs in this review: citalopram, fluoxetine, escitalopram, fluvoxamine, paroxetine, and sertraline. Data collection and analysis Three authors extracted the data of all included studies and assessed the risks of bias of the studies. We resolved discrepancies by discussion. Main results The quality of evidence was very low for each outcome. We downgraded the quality of evidence to very low due to concerns about risk of bias and studies with few participants. We included seven placebo-controlled studies, two with citalopram, three with fluoxetine and two with paroxetine, with a median study duration of eight weeks (4 to 16 weeks) and 383 participants, who were pooled together. All studies had one or more sources of potential major bias. There was a small (10%) difference in patients who reported a 30% pain reduction between SSRIs (56/172 (32.6%)) and placebo (39/171 (22.8%)) risk difference (RD) 0.10, 95% confidence interval (CI) 0.01 to 0.20; number needed to treat for an additional

  12. Development of a potent and selective cell penetrant Legumain inhibitor.

    PubMed

    Ness, Kerry A; Eddie, Sharon L; Higgins, Catherine A; Templeman, Amy; D'Costa, Zenobia; Gaddale, Kishore K D; Bouzzaoui, Samira; Jordan, Linda; Janssen, Dominic; Harrison, Timothy; Burkamp, Frank; Young, Andrew; Burden, Roberta; Scott, Christopher J; Mullan, Paul B; Williams, Rich

    2015-12-01

    This Letter describes the continued SAR exploration of small molecule Legumain inhibitors with the aim of developing a potent and selective in vitro tool compound. Work continued in this Letter explores the use of alternative P2-P3 linker units and the P3 group SAR which led to the identification of 10t, a potent, selective and cellularly active Legumain inhibitor. We also demonstrate that 10t has activity in both cancer cell viability and colony formation assays.

  13. Acalabrutinib (ACP-196): a selective second-generation BTK inhibitor.

    PubMed

    Wu, Jingjing; Zhang, Mingzhi; Liu, Delong

    2016-03-09

    More and more targeted agents become available for B cell malignancies with increasing precision and potency. The first-in-class Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, has been in clinical use for the treatment of chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom's macroglobulinemia. More selective BTK inhibitors (ACP-196, ONO/GS-4059, BGB-3111, CC-292) are being explored. Acalabrutinib (ACP-196) is a novel irreversible second-generation BTK inhibitor that was shown to be more potent and selective than ibrutinib. This review summarized the preclinical research and clinical data of acalabrutinib.

  14. Screening of selective histone deacetylase inhibitors by proteochemometric modeling

    PubMed Central

    2012-01-01

    Background Histone deacetylase (HDAC) is a novel target for the treatment of cancer and it can be classified into three classes, i.e., classes I, II, and IV. The inhibitors selectively targeting individual HDAC have been proved to be the better candidate antitumor drugs. To screen selective HDAC inhibitors, several proteochemometric (PCM) models based on different combinations of three kinds of protein descriptors, two kinds of ligand descriptors and multiplication cross-terms were constructed in our study. Results The results show that structure similarity descriptors are better than sequence similarity descriptors and geometry descriptors in the leftacterization of HDACs. Furthermore, the predictive ability was not improved by introducing the cross-terms in our models. Finally, a best PCM model based on protein structure similarity descriptors and 32-dimensional general descriptors was derived (R2 = 0.9897, Qtest2 = 0.7542), which shows a powerful ability to screen selective HDAC inhibitors. Conclusions Our best model not only predict the activities of inhibitors for each HDAC isoform, but also screen and distinguish class-selective inhibitors and even more isoform-selective inhibitors, thus it provides a potential way to discover or design novel candidate antitumor drugs with reduced side effect. PMID:22913517

  15. Identification of new peptide amides as selective cathepsin L inhibitors: the first step towards selective irreversible inhibitors?

    PubMed

    Torkar, Ana; Lenarčič, Brigita; Lah, Tamara; Dive, Vincent; Devel, Laurent

    2013-05-15

    A small library of peptide amides was designed to profile the cathepsin L active site. Within the cathepsin family of cysteine proteases, the first round of selection was on cathepsin L and cathepsin B, and then selected hits were further evaluated for binding to cathepsin K and cathepsin S. Five highly selective sequences with submicromolar affinities towards cathepsin L were identified. An acyloxymethyl ketone warhead was then attached to these sequences. Although these original irreversible inhibitors inactivate cathepsin L, it appears that the nature of the warhead drastically impact the selectivity profile of the resulting covalent inhibitors.

  16. Triazaspirodimethoxybenzoyls as Selective Inhibitors of Mycobacterial Lipoamide Dehydrogenase

    SciTech Connect

    Bryk, Ruslana; Arango, Nancy; Venugopal, Aditya; Warren, J. David; Park, Yun-Hee; Patel, Mulchand S.; Lima, Christopher D.; Nathan, Carl

    2010-06-25

    Mycobacterium tuberculosis (Mtb) remains the leading single cause of death from bacterial infection. Here we explored the possibility of species-selective inhibition of lipoamide dehydrogenase (Lpd), an enzyme central to Mtb's intermediary metabolism and antioxidant defense. High-throughput screening of combinatorial chemical libraries identified triazaspirodimethoxybenzoyls as high-nanomolar inhibitors of Mtb's Lpd that were noncompetitive versus NADH, NAD{sup +}, and lipoamide and >100-fold selective compared to human Lpd. Efficacy required the dimethoxy and dichlorophenyl groups. The structure of an Lpd-inhibitor complex was resolved to 2.42 {angstrom} by X-ray crystallography, revealing that the inhibitor occupied a pocket adjacent to the Lpd NADH/NAD{sup +} binding site. The inhibitor did not overlap with the adenosine moiety of NADH/NAD{sup +} but did overlap with positions predicted to bind the nicotinamide rings in NADH and NAD{sup +} complexes. The dimethoxy ring occupied a deep pocket adjacent to the FAD flavin ring where it would block coordination of the NADH nicotinamide ring, while the dichlorophenyl group occupied a more exposed pocket predicted to coordinate the NAD{sup +} nicotinamide. Several residues that are not conserved between the bacterial enzyme and its human homologue were predicted to contribute both to inhibitor binding and to species selectivity, as confirmed for three residues by analysis of the corresponding mutant Mtb Lpd proteins. Thus, nonconservation of residues lining the electron-transfer tunnel in Mtb Lpd can be exploited for development of species-selective Lpd inhibitors.

  17. Triazaspirodimethoxybenzoyls as selective inhibitors of mycobacterial lipoamide dehydrogenase .

    PubMed

    Bryk, Ruslana; Arango, Nancy; Venugopal, Aditya; Warren, J David; Park, Yun-Hee; Patel, Mulchand S; Lima, Christopher D; Nathan, Carl

    2010-03-02

    Mycobacterium tuberculosis (Mtb) remains the leading single cause of death from bacterial infection. Here we explored the possibility of species-selective inhibition of lipoamide dehydrogenase (Lpd), an enzyme central to Mtb's intermediary metabolism and antioxidant defense. High-throughput screening of combinatorial chemical libraries identified triazaspirodimethoxybenzoyls as high-nanomolar inhibitors of Mtb's Lpd that were noncompetitive versus NADH, NAD(+), and lipoamide and >100-fold selective compared to human Lpd. Efficacy required the dimethoxy and dichlorophenyl groups. The structure of an Lpd-inhibitor complex was resolved to 2.42 A by X-ray crystallography, revealing that the inhibitor occupied a pocket adjacent to the Lpd NADH/NAD(+) binding site. The inhibitor did not overlap with the adenosine moiety of NADH/NAD(+) but did overlap with positions predicted to bind the nicotinamide rings in NADH and NAD(+) complexes. The dimethoxy ring occupied a deep pocket adjacent to the FAD flavin ring where it would block coordination of the NADH nicotinamide ring, while the dichlorophenyl group occupied a more exposed pocket predicted to coordinate the NAD(+) nicotinamide. Several residues that are not conserved between the bacterial enzyme and its human homologue were predicted to contribute both to inhibitor binding and to species selectivity, as confirmed for three residues by analysis of the corresponding mutant Mtb Lpd proteins. Thus, nonconservation of residues lining the electron-transfer tunnel in Mtb Lpd can be exploited for development of species-selective Lpd inhibitors.

  18. FAAH inhibition produces antidepressant-like efforts of mice to acute stress via synaptic long-term depression.

    PubMed

    Wang, Ying; Zhang, Xia

    2017-05-01

    Recent studies have shown that inhibition of fatty acid amide hydrolase (FAAH), the major degradative enzyme of the endocannabinoid N-arachidonoylethanolamine (AEA), produced antidepressant behavioral responses, but its underlying mechanism is not clear. Here we find that a systemic administration of the FAAH inhibitor PF3845 or an intra-CA1 application of AEA elicits an in vivo long-term depression (LTD) at excitatory glutamatergic CA3-CA1 synapses of the hippocampus. The PF3845- and/or AEA-elicited LTD are abolished by the LTD-blocking peptide Tat-GluR2. PF3845 significantly decreases passive behavioral coping of naïve mice to acute inescapable stress, which is also abolished by Tat-GluR2 peptide. However, PF3845 does not significantly affect sucrose assumption ratio of mice receiving chronic administration of corticosterone. These results suggest that FAAH inhibitors are able to produce antidepressant effects in naïve animals in response to acute stress through LTD at hippocampal glutamatergic CA3-CA1 synapses.

  19. Refined homology model of monoacylglycerol lipase: toward a selective inhibitor

    NASA Astrophysics Data System (ADS)

    Bowman, Anna L.; Makriyannis, Alexandros

    2009-11-01

    Monoacylglycerol lipase (MGL) is primarily responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both cannabinoid receptors. Increased tissue 2-AG levels consequent to MGL inhibition are considered therapeutic against pain, inflammation, and neurodegenerative disorders. However, the lack of MGL structural information has hindered the development of MGL-selective inhibitors. Here, we detail a fully refined homology model of MGL which preferentially identifies MGL inhibitors over druglike noninhibitors. We include for the first time insight into the active-site geometry and potential hydrogen-bonding interactions along with molecular dynamics simulations describing the opening and closing of the MGL helical-domain lid. Docked poses of both the natural substrate and known inhibitors are detailed. A comparison of the MGL active-site to that of the other principal endocannabinoid metabolizing enzyme, fatty acid amide hydrolase, demonstrates key differences which provide crucial insight toward the design of selective MGL inhibitors as potential drugs.

  20. Amygdala FAAH and anandamide: mediating protection and recovery from stress.

    PubMed

    Gunduz-Cinar, Ozge; Hill, Matthew N; McEwen, Bruce S; Holmes, Andrew

    2013-11-01

    A long-standing literature linking endocannabinoids (ECBs) to stress, fear, and anxiety has led to growing interest in developing novel anxiolytics targeting the ECB system. Following rapid on-demand biosynthesis and degradation upon neuronal activation, the ECB N-arachidonoylethanolamide (anandamide, AEA) is actively degraded by the serine hydrolase enzyme, fatty acid amide hydrolase (FAAH). Exposure to stress rapidly mobilizes FAAH to deplete the signaling pool of AEA and increase neuronal excitability in a key anxiety-mediating region--the basolateral amygdala (BLA). Gene deletion or pharmacological inhibition of FAAH prevents stress-induced reductions in AEA and associated increases in BLA dendritic hypertrophy and anxiety-like behavior. Additionally, inhibition of FAAH facilitates long-term fear extinction and rescues deficient fear extinction in rodent models by enhancing AEA-CB1 (cannabinoid type 1) receptor signaling and synaptic plasticity in the BLA. These preclinical findings propose restoring deficient BLA AEA levels by pharmacologically inhibiting FAAH as a mechanism to therapeutically mitigate the effects of traumatic stress.

  1. 1-(3-biaryloxy-2-oxopropyl)indole-5-carboxylic acids and related compounds as dual inhibitors of human cytosolic phospholipase A2α and fatty acid amide hydrolase.

    PubMed

    Zahov, Stefan; Drews, Andreas; Hess, Mark; Schulze Elfringhoff, Alwine; Lehr, Matthias

    2011-03-07

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes that have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (5) is a dual inhibitor of cPLA2α and FAAH. Structure-activity relationship studies revealed that substituents at the indole 3- and 5-positions and replacement of the indole scaffold of this compound by other heterocycles strongly influences the inhibitory potency against cPLA2α and FAAH, respectively. Herein we report the effect of variation of the 4-octyl residue of 5 and an exchange of its carboxylic acid moiety by some bioisosteric functional groups. Several of the compounds assayed were favorably active against both enzymes, and could therefore represent agents with improved analgesic and anti-inflammatory qualities in comparison with selective cPLA2 α and FAAH inhibitors.

  2. SAR156497, an exquisitely selective inhibitor of aurora kinases.

    PubMed

    Carry, Jean-Christophe; Clerc, François; Minoux, Hervé; Schio, Laurent; Mauger, Jacques; Nair, Anil; Parmantier, Eric; Le Moigne, Ronan; Delorme, Cécile; Nicolas, Jean-Paul; Krick, Alain; Abécassis, Pierre-Yves; Crocq-Stuerga, Véronique; Pouzieux, Stéphanie; Delarbre, Laure; Maignan, Sébastien; Bertrand, Thomas; Bjergarde, Kirsten; Ma, Nina; Lachaud, Sylvette; Guizani, Houlfa; Lebel, Rémi; Doerflinger, Gilles; Monget, Sylvie; Perron, Sébastien; Gasse, Francis; Angouillant-Boniface, Odile; Filoche-Rommé, Bruno; Murer, Michel; Gontier, Sylvie; Prévost, Céline; Monteiro, Marie-Line; Combeau, Cécile

    2015-01-08

    The Aurora family of serine/threonine kinases is essential for mitosis. Their crucial role in cell cycle regulation and aberrant expression in a broad range of malignancies have been demonstrated and have prompted intensive search for small molecule Aurora inhibitors. Indeed, over 10 of them have reached the clinic as potential anticancer therapies. We report herein the discovery and optimization of a novel series of tricyclic molecules that has led to SAR156497, an exquisitely selective Aurora A, B, and C inhibitor with in vitro and in vivo efficacy. We also provide insights into its mode of binding to its target proteins, which could explain its selectivity.

  3. The fatty-acid amide hydrolase inhibitor URB597 does not affect triacylglycerol hydrolysis in rat tissues.

    PubMed

    Clapper, Jason R; Duranti, Andrea; Tontini, Andrea; Mor, Marco; Tarzia, Giorgio; Piomelli, Daniele

    2006-11-01

    The O-arylcarbamate URB597 (cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester; also referred to as KDS-4103) is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the inactivation of the endogenous cannabinoid anandamide. URB597 demonstrates a remarkable degree of selectivity for FAAH over other serine hydrolases (e.g. cholinesterases) or other components of the endocannabinoid system (e.g. cannabinoid receptors). However, in a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain proteins, it was recently shown that URB597 prevents FPR binding to triacylglycerol hydrolase (TGH) with a median inhibitory concentration of 192nM. To determine whether this effect correlates with inhibition of TGH activity, we investigated the ability of URB597 to inhibit triolein hydrolysis in rat liver and heart tissues, which are rich in TGH, as well as white adipose tissue (WAT), which is rich in adipose triacylglycerol lipase (TGL) and hormone-sensitive lipase. The results show that URB597 does not affect triolein hydrolysis in any of these tissues at concentrations as high as 10microM, whereas it inhibits FAAH activity at low nanomolar concentrations. Moreover, intraperitoneal (i.p.) administration of URB597 at doses that maximally inhibit FAAH in vivo (0.3-3mgkg(-1)) exerts no effect on triolein hydrolysis and tissue triacylglycerol (TAG) levels in rat liver, heart or WAT. The results indicate that URB597, while potent at inhibiting FAAH, does not affect TGH and TGL activities in rat tissues.

  4. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

    PubMed Central

    Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  5. Anilino-monoindolylmaleimides as potent and selective JAK3 inhibitors.

    PubMed

    McDonnell, Mark E; Bian, Haiyan; Wrobel, Jay; Smith, Garry R; Liang, Shuguang; Ma, Haiching; Reitz, Allen B

    2014-02-15

    We designed a series of anilino-indoylmaleimides based on structural elements from literature JAK3 inhibitors 3 and 4, and our lead 5. These new compounds were tested as inhibitors of JAKs 1, 2 and 3 and TYK2 for therapeutic intervention in rheumatoid arthritis (RA). Our requirements, based on current scientific rationale for optimum efficacy against RA with reduced side effects, was for potent, mixed JAK1 and 3 inhibition, and selectivity over JAK2. Our efforts yielded a potent JAK3 inhibitor 11d and its eutomer 11e. These compounds were highly selective for inhibition of JAK3 over JAK2 and TYK. The compounds displayed only modest JAK1 inhibition.

  6. Discovery of potent, selective chymase inhibitors via fragment linking strategies.

    PubMed

    Taylor, Steven J; Padyana, Anil K; Abeywardane, Asitha; Liang, Shuang; Hao, Ming-Hong; De Lombaert, Stéphane; Proudfoot, John; Farmer, Bennett S; Li, Xiang; Collins, Brandon; Martin, Leslie; Albaugh, Daniel R; Hill-Drzewi, Melissa; Pullen, Steven S; Takahashi, Hidenori

    2013-06-13

    Chymase plays an important and diverse role in the homeostasis of a number of cardiovascular processes. Herein, we describe the identification of potent, selective chymase inhibitors, developed using fragment-based, structure-guided linking and optimization techniques. High-concentration biophysical screening methods followed by high-throughput crystallography identified an oxindole fragment bound to the S1 pocket of the protein exhibiting a novel interaction pattern hitherto not observed in chymase inhibitors. X-ray crystallographic structures were used to guide the elaboration/linking of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over cathepsin G and that mitigated a number of liabilities associated with poor physicochemical properties of the series it was derived from.

  7. Fatty Acid Amide Hydrolase (FAAH), Acetylcholinesterase (AChE), and Butyrylcholinesterase (BuChE): Networked Targets for the Development of Carbamates as Potential Anti-Alzheimer's Disease Agents.

    PubMed

    Montanari, Serena; Scalvini, Laura; Bartolini, Manuela; Belluti, Federica; Gobbi, Silvia; Andrisano, Vincenza; Ligresti, Alessia; Di Marzo, Vincenzo; Rivara, Silvia; Mor, Marco; Bisi, Alessandra; Rampa, Angela

    2016-07-14

    The modulation of the endocannabinoid system is emerging as a viable avenue for the treatment of neurodegeneration, being involved in neuroprotective and anti-inflammatory processes. In particular, indirectly enhancing endocannabinoid signaling to therapeutic levels through FAAH inhibition might be beneficial for neurodegenerative disorders such as Alzheimer's disease, effectively preventing or slowing the progression of the disease. Hence, in the search for a more effective treatment for Alzheimer's disease, in this paper, the multitarget-directed ligand paradigm was applied to the design of carbamates able to simultaneously target the recently proposed endocannabinoid system and the classic cholinesterase system, and achieve effective dual FAAH/cholinesterase inhibitors. Among the two series of synthesized compounds, while some derivatives proved to be extremely potent on a single target, compounds 9 and 19 were identified as effective dual FAAH/ChE inhibitors, with well-balanced nanomolar activities. Thus, 9 and 19 might be considered as new promising candidates for Alzheimer's disease treatment.

  8. Cardiac Toxicity in Selective Serotonin Reuptake Inhibitor Users.

    PubMed

    Lusetti, Monia; Licata, Manuela; Silingardi, Enrico; Reggiani Bonetti, Luca; Palmiere, Cristian

    2015-12-01

    Several classes of recreational and prescription drugs have been associated with an increased risk of cardiovascular disease and the occurrence of arrhythmias, which may be involved in sudden deaths in chronic users even at therapeutic doses. The study presented herein focuses on pathological changes involving the heart, which may be caused by selective serotonin reuptake inhibitor use and their possible role in the occurrence of sudden cardiac death. A total of 40 cases were included in the study and were divided evenly into 2 groups: 20 cases of patients treated with selective serotonin reuptake inhibitors and 20 cases of sudden deaths involving patients void of any drug treatment. The first group included 16 patients treated with citalopram and 4 with sertraline. Autopsies, histology, biochemistry, and toxicology were performed in all cases. Pathological changes in selective serotonin reuptake inhibitor users consisted of various degrees of interstitial and perivascular fibrosis as well as a small degree of perineural fibrosis within the myocardium of the left ventricle. Within the limits of the small number of investigated cases, the results of this study seem to confirm former observations on this topic, suggesting that selective serotonin reuptake inhibitors may play a potential, causative role in the pathogenesis of sudden deaths in chronic users even at therapeutic concentrations.

  9. Selective serotonin reuptake inhibitors for depression in pregnancy.

    PubMed

    Susser, Leah C; Sansone, Stephanie A; Hermann, Alison D

    2016-12-01

    Perinatal depression is associated with a high risk of morbidity and mortality and may have long-term consequences on child development. The US Preventive Services Task Force has recently recognized the importance of identifying and treating women with depression in the perinatal period. However, screening and accessing appropriate treatment come with logistical challenges. In many areas, there may not be sufficient access to psychiatric care, and, until these resources develop, the burden may inadvertently fall on obstetricians. As a result, understanding the risks of perinatal depression in comparison with the risks of treatment is important. Many studies of selective serotonin reuptake inhibitors in pregnancy fail to control for underlying depressive illness, which can lead to misinterpretation of selective serotonin reuptake inhibitor risk by clinicians. This review discusses the risks and benefits of selective serotonin reuptake inhibitor treatment in pregnancy within the context of perinatal depression. Whereas selective serotonin reuptake inhibitors may be associated with certain risks, the absolute risks are low and may be outweighed by the risks of untreated depression for many women and their offspring.

  10. Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context

    PubMed Central

    Rivera, Patricia; Bindila, Laura; Pastor, Antoni; Pérez-Martín, Margarita; Pavón, Francisco J.; Serrano, Antonia; de la Torre, Rafael; Lutz, Beat; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3+ or BrdU+ cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3+), astroglia (GFAP+), and microglia (Iba1+ cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3+ and BrdU+ subgranular cells as well as GFAP+, Iba1+ and cleaved caspase-3+ cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3+, GFAP+ and 3-weeks-old BrdU+ cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context. PMID:25870539

  11. FAAH inhibition enhances anandamide mediated anti-tumorigenic effects in non-small cell lung cancer by downregulating the EGF/EGFR pathway.

    PubMed

    Ravi, Janani; Sneh, Amita; Shilo, Konstantin; Nasser, Mohd W; Ganju, Ramesh K

    2014-05-15

    The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anti-cancer effects. Fatty acid amide hydrolase (FAAH) metabolizes AEA and decreases its anti-tumorigenic activity. In this study, we have analyzed the role of FAAH inhibition in non-small cell lung cancer (NSCLC). We have shown that FAAH and CB1 receptor which is activated by AEA are expressed in lung adenocarcinoma patient samples and NSCLC cell lines A549 and H460. Since the synthetic analogue of anandamide (Met-F-AEA) did not possess significant anti-tumorigenic effects, we used Met-F-AEA in combination with FAAH inhibitor URB597 which significantly reduced EGF (epidermal growth factor)-induced proliferative and chemotactic activities in vitro when compared to anti-tumorigenic activity of Met-F-AEA alone. Further analysis of signaling mechanisms revealed that Met-F-AEA in combination with URB597 inhibits activation of EGFR and its downstream signaling ERK, AKT and NF-kB. In addition, it inhibited MMP2 secretion and stress fiber formation. We have also shown that the Met-F-AEA in combination with URB597 induces G0/G1 cell cycle arrest by downregulating cyclin D1 and CDK4 expressions, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, the combination treatment inhibited tumor growth in a xenograft nude mouse model system. Tumors derived from Met-F-AEA and URB597 combination treated mice showed reduced EGFR, AKT and ERK activation and MMP2/MMP9 expressions when compared to Met-F-AEA or URB597 alone. Taken together, these data suggest in EGFR overexpressing NSCLC that the combination of Met-F-AEA with FAAH inhibitor resulted in superior therapeutic response compared to individual compound activity alone.

  12. Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context.

    PubMed

    Rivera, Patricia; Bindila, Laura; Pastor, Antoni; Pérez-Martín, Margarita; Pavón, Francisco J; Serrano, Antonia; de la Torre, Rafael; Lutz, Beat; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context.

  13. Pharmacological inhibition of FAAH modulates TLR-induced neuroinflammation, but not sickness behaviour: An effect partially mediated by central TRPV1.

    PubMed

    Henry, Rebecca J; Kerr, Daniel M; Flannery, Lisa E; Killilea, Marykate; Hughes, Edel M; Corcoran, Louise; Finn, David P; Roche, Michelle

    2017-05-01

    Aberrant activation of toll-like receptors (TLRs), key components of the innate immune system, has been proposed to underlie and exacerbate a range of central nervous system disorders. Increasing evidence supports a role for the endocannabinoid system in modulating inflammatory responses including those mediated by TLRs, and thus this system may provide an important treatment target for neuroinflammatory disorders. However, the effect of modulating endocannabinoid tone on TLR-induced neuroinflammation in vivo and associated behavioural changes is largely unknown. The present study examined the effect of inhibiting fatty acid amide hydrolyase (FAAH), the primary enzyme responsible for the metabolism of anandamide (AEA), in vivo on TLR4-induced neuroimmune and behavioural responses, and evaluated sites and mechanisms of action. Systemic administration of the FAAH inhibitor PF3845 increased levels of AEA, and related FAAH substrates N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA), in the frontal cortex and hippocampus of rats, an effect associated with an attenuation in the expression of pro- and anti-inflammatory cytokines and mediators measured 2hrs following systemic administration of the TLR4 agonist, lipopolysaccharide (LPS). These effects were mimicked by central i.c.v. administration of PF3845, but not systemic administration of the peripherally-restricted FAAH inhibitor URB937. Central antagonism of TRPV1 significantly attenuated the PF3845-induced decrease in IL-6 expression, effects not observed following antagonism of CB1, CB2, PPARα, PPARγ or GPR55. LPS-induced a robust sickness-like behavioural response and increased the expression of markers of glial activity and pro-inflammatory cytokines over 24hrs. Systemic administration of PF3845 modulated the TLR4-induced expression of neuroimmune mediators and anhedonia without altering acute sickness behaviour. Overall, these findings support an important role for FAAH substrates directly within

  14. DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening

    PubMed Central

    Navrátil, Václav; Schimer, Jiří; Tykvart, Jan; Knedlík, Tomáš; Vik, Viktor; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2017-01-01

    Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery. PMID:27679479

  15. Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

    PubMed Central

    Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

    2015-01-01

    Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

  16. The fatty acid amide hydrolase inhibitor PF-3845 promotes neuronal survival, attenuates inflammation and improves functional recovery in mice with traumatic brain injury.

    PubMed

    Tchantchou, Flaubert; Tucker, Laura B; Fu, Amanda H; Bluett, Rebecca J; McCabe, Joseph T; Patel, Sachin; Zhang, Yumin

    2014-10-01

    Traumatic brain injury (TBI) is the leading cause of death in young adults in the United States, but there is still no effective agent for treatment. N-arachidonoylethanolamine (anandamide, AEA) is a major endocannabinoid in the brain. Its increase after brain injury is believed to be protective. However, the compensatory role of AEA is transient due to its rapid hydrolysis by the fatty acid amide hydrolase (FAAH). Thus, inhibition of FAAH can boost the endogenous levels of AEA and prolong its protective effect. Using a TBI mouse model, we found that post-injury chronic treatment with PF3845, a selective and potent FAAH inhibitor, reversed TBI-induced impairments in fine motor movement, hippocampus dependent working memory and anxiety-like behavior. Treatment with PF3845 inactivated FAAH activity and enhanced the AEA levels in the brain. It reduced neurodegeneration in the dentate gyrus, and up-regulated the expression of Bcl-2 and Hsp70/72 in both cortex and hippocampus. PF3845 also suppressed the increased production of amyloid precursor protein, prevented dendritic loss and restored the levels of synaptophysin in the ipsilateral dentate gyrus. Furthermore, PF3845 suppressed the expression of inducible nitric oxide synthase and cyclooxygenase-2 and enhanced the expression of arginase-1 post-TBI, suggesting a shift of microglia/macrophages from M1 to M2 phenotype. The effects of PF3845 on TBI-induced behavioral deficits and neurodegeneration were mediated by activation of cannabinoid type 1 and 2 receptors and might be attributable to the phosphorylation of ERK1/2 and AKT. These results suggest that selective inhibition of FAAH is likely to be beneficial for TBI treatment.

  17. The exploration of thienothiazines as selective butyrylcholinesterase inhibitors.

    PubMed

    Karlsson, Daniela; Fallarero, Adyary; Brunhofer, Gerda; Mayer, Claudia; Prakash, Ohm; Mohan, C Gopi; Vuorela, Pia; Erker, Thomas

    2012-08-30

    The role of butyrylcholinesterase (BChE) in the progression of Alzheimer's disease (AD) has recently become more crucial. In the AD brain, selective BChE inhibitors have been demonstrated to have a beneficial effect in vivo, probably by recovering cholinergic activity and/or by restoring AChE:BChE activity ratios to the levels observed in the healthy brain. Thienothiazines are compounds sharing some structural features with phenothiazines, which are known to be potent BChE inhibitors. Thus, in this contribution 45 thienothiazines were investigated for their BChE inhibitory activity. Six of them were proven to be potent and selective inhibitors of equine BChE's hydrolase activity. Structure-activity relationships were laid out, and a tentative pharmacophore model for BChE inhibitors of the thienothiazine type was proposed. The most active compound, 3f, displayed a mixed type of inhibition and was also active against the human BChE (huBChE) with an IC(50) huBChE of 0.51 ± 0.07 μM. Computational studies suggested that 3f likely binds to the catalytic site and nearby to the peripheral site of the huBChE in an extended form. In addition, the chemical space occupied by the active thienothiazines, as opposed to phenothiazines and other representative chemical classes of BChE inhibitors, was explored with the aid of ChemGPS-NP, and the relevant chemical space regions were identified. This study shows for the first time that thienothiazines represent a new group of BChE inhibitors that can be used as molecular probes for studying the role of BChE in the brain or for developing newer drug leads for AD therapy.

  18. Development of Selective Covalent Janus Kinase 3 Inhibitors.

    PubMed

    Tan, Li; Akahane, Koshi; McNally, Randall; Reyskens, Kathleen M S E; Ficarro, Scott B; Liu, Suhu; Herter-Sprie, Grit S; Koyama, Shohei; Pattison, Michael J; Labella, Katherine; Johannessen, Liv; Akbay, Esra A; Wong, Kwok-Kin; Frank, David A; Marto, Jarrod A; Look, Thomas A; Arthur, J Simon C; Eck, Michael J; Gray, Nathanael S

    2015-08-27

    The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å cocrystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology.

  19. Selectivity of IMAC columns in trypsin inhibitor purification.

    PubMed

    Yeomans-Reina, H; Ruiz-Manriquez, A; Wong, B R; Mansir, A T

    2001-01-01

    The properties of an adsorbent and the parameters in an adsorption process affect the resolution of chromatographic purifications. This is reflected in the elution profile, which shows the relative affinity of different proteins for a specific adsorbent. In the work presented here, elution profiles for trypsin inhibitor were used to study the effects of the concentration of trypsin inhibitor, ionic strength of the protein solution, slope of the elution gradient, and the regeneration treatment of the chromatography column on the selectivity of the adsorbent Cellufine Chelate-Cu(II)(ida). Cytochrome c was used as a reference protein. Variations in the concentrations of trypsin inhibitor and in the ionic strength of the buffered solution did not have any effects on the elution profile. On the other hand, changes in the slope of the pH gradient used for elution caused shifting of the elution peaks toward lower values of the elution volume, resulting in the best strategy to modify the elution profile of the system. Finally, using a constant slope pH gradient of elution, the variation of the selectivity of the adsorbent for trypsin inhibitor when subjected to cleaning treatments with 0.5 N NaOH was studied. Appropriate cleaning practices used in industry were followed. The adsorbent showed only a slight tendency for resolution loss in the order of 2 x 10(-4) days(-1). The results presented here show a good stability of the adsorbent when compared to other biospecific adsorbents commonly used.

  20. Synthesis of a selective HDAC6 inhibitor active in neuroblasts.

    PubMed

    Zwick, Vincent; Simões-Pires, Claudia A; Nurisso, Alessandra; Petit, Charlotte; Dos Santos Passos, Carolina; Randazzo, Giuseppe Marco; Martinet, Nadine; Bertrand, Philippe; Cuendet, Muriel

    2016-10-15

    In recent years, the role of HDAC6 in neurodegeneration has been partially elucidated, which led some authors to propose HDAC6 inhibitors as a therapeutic strategy to treat neurodegenerative diseases. In an effort to develop a selective HDAC6 inhibitor which can cross the blood brain barrier (BBB), a modified hydroxamate derivative (compound 3) was designed and synthetized. This compound was predicted to have potential for BBB penetration based on in silico and in vitro evaluation of passive permeability. When tested for its HDAC inhibitory activity, the IC50 value of compound 3 towards HDAC6 was in the nM range in both enzymatic and cell-based assays. Compound 3 showed a cell-based selectivity profile close to that of tubastatin A in SH-SY5Y human neuroblastoma cells, and a good BBB permeability profile.

  1. Loratadine analogues as MAGL inhibitors.

    PubMed

    Patel, Jayendra Z; Ahenkorah, Stephen; Vaara, Miia; Staszewski, Marek; Adams, Yahaya; Laitinen, Tuomo; Navia-Paldanius, Dina; Parkkari, Teija; Savinainen, Juha R; Walczyński, Krzysztof; Laitinen, Jarmo T; Nevalainen, Tapio J

    2015-04-01

    Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAGL, IC50=46 nM), and was found to have almost 150-fold higher selectivity over human recombinant fatty acid amide hydrolase (hFAAH, IC50=7.24 μM) and 35-fold higher selectivity over human α/β-hydrolase-6 (hABHD6, IC50=1.79 μM). Additionally, compound 12a retained H1 antagonistic affinity (pA2=6.81) but did not show cannabinoid receptor activity, when tested at concentrations ⩽ 10 μM. Hence, compound 12a represents a novel dual-acting pharmacological tool possessing both MAGL-inhibitory and antihistaminergic activities.

  2. Selective Inhibitors of 17beta-Hydroxysteroid Dehydrogenase

    DTIC Science & Technology

    2003-07-01

    this project is to develop selective inhibitors of human Type I 17 Beta-HSD as "lead compounds" for stmcture-based drug design . The crystal structure...of human Type I 17 Beta-HSD is available to aid in structure-based drug design . The concept that the Rossmann fold may represent a useful drug target is a new concept in drug design .

  3. Selective indole-based ECE inhibitors: synthesis and pharmacological evaluation.

    PubMed

    Brands, Michael; Ergüden, Jens-Kerim; Hashimoto, Kentaro; Heimbach, Dirk; Krahn, Thomas; Schröder, Christian; Siegel, Stephan; Stasch, Johannes-Peter; Tsujishita, Hideki; Weigand, Stefan; Yoshida, Nagahiro H

    2006-01-01

    Inhibition of the metalloprotease ECE-1 may be beneficial for the treatment of coronary heart disease, cancer, renal failure, and urological disorders. A novel class of indole-based ECE inhibitors was identified by high throughput screening. Optimization of the original screening lead structure 6 led to highly potent inhibitors such as 11, which bears a bisaryl amide moiety linked to the indole C2 position through an amide group. Docking of 11 into a model structure of ECE revealed a unique binding mode in which the Zn center of the enzyme is not directly addressed by the inhibitor, but key interactions are suggested for the central amide group. Testing of the lead compound 6 in hypertensive Dahl S rats resulted in a decrease in blood pressure after an initial period in which the blood pressure remained unchanged, most probably the result of ET-1 already present. Indole derivative 6 also displays a cardio-protective effect in a mouse model of acute myocardial infarction after oral administration. The more potent chloropyridine derivative 9 antagonizes big-ET-1-induced increase in blood pressure in rats at intravenous administration of 3 mg kg-1. All ECE inhibitors of the indole class showed high selectivity for ECE over related metalloproteases such as NEP and ACE. Therefore, these compounds might have further potential as drugs for the treatment of coronary heart diseases.

  4. Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH.

    PubMed

    Dainese, Enrico; De Fabritiis, Gianni; Sabatucci, Annalaura; Oddi, Sergio; Angelucci, Clotilde Beatrice; Di Pancrazio, Chiara; Giorgino, Toni; Stanley, Nathaniel; Del Carlo, Michele; Cravatt, Benjamin F; Maccarrone, Mauro

    2014-02-01

    Lipid composition is expected to play an important role in modulating membrane enzyme activity, in particular if the substrates are themselves lipid molecules. A paradigmatic case is FAAH (fatty acid amide hydrolase), an enzyme critical in terminating endocannabinoid signalling and an important therapeutic target. In the present study, using a combined experimental and computational approach, we show that membrane lipids modulate the structure, subcellular localization and activity of FAAH. We report that the FAAH dimer is stabilized by the lipid bilayer and shows a higher membrane-binding affinity and enzymatic activity within membranes containing both cholesterol and the natural FAAH substrate AEA (anandamide). Additionally, co-localization of cholesterol, AEA and FAAH in mouse neuroblastoma cells suggests a mechanism through which cholesterol increases the substrate accessibility of FAAH.

  5. Arachidonate 12-lipoxygenases with reference to their selective inhibitors

    SciTech Connect

    Yamamoto, Shozo . E-mail: yamamosh@kyoto-wu.ac.jp; Katsukawa, Michiko; Nakano, Ayumi; Hiraki, Emi; Nishimura, Kohji; Jisaka, Mitsuo; Yokota, Kazushige; Ueda, Natsuo

    2005-12-09

    Lipoxygenase is a dioxygenase recognizing a 1-cis,4-cis-pentadiene of polyunsaturated fatty acids. The enzyme oxygenates various carbon atoms of arachidonic acid as a substrate and produces 5-, 8-, 12- or 15-hydroperoxy eicosatetraenoic acid with a conjugated diene chromophore. The enzyme is referred to as 5-, 8-, 12- or 15-lipoxygenase, respectively. Earlier we found two isoforms of 12-lipoxygenase, leukocyte- and platelet-type enzymes, which were distinguished by substrate specificity, catalytic activity, primary structure, gene intron size, and antigenicity. Recently, the epidermis-type enzyme was found as the third isoform. Attempts have been made to find isozyme-specific inhibitors of 12-lipoxygenase, and earlier we found hinokitol, a tropolone, as a potent inhibitor selective for the platelet-type 12-lipoxygenase. More recently, we tested various catechins of tea leaves and found that (-)-geotechnical gallate was a potent and selective inhibitor of human platelet 12-lipoxygenase with an IC{sub 5} of 0.14 {mu}M. The compound was much less active with 12-lipoxygenase of leukocyte-type, 15-, 8-, and 5-lipoxygenases, and cyclo oxygenases-1 and -2.

  6. Identification of an Adamantyl Azaquinolone JNK Selective Inhibitor

    PubMed Central

    2012-01-01

    3-[4-((1S,2S,3R,5S,7S)-5-Hydroxyadamantan-2-ylcarbamoyl)benzyl]-4-oxo-1-phenyl-1,4-dihydro-[1,8]naphthyridine-2-carboxylic acid methyl ester (4) was identified as a novel, druglike and selective quinolone pan JNK inhibitor. In this communication, some of the structure–activity relationship of the azaquinolone analogues leading to 4 is discussed. The focus is on how changes at the amide functionality affected the biochemical potency, cellular potency, metabolic properties, and solubility of this class of JNK inhibitors. Optimization of these properties led to the identification of the adamantyl analogue, 4. 4 achieved proof of mechanism in both rat and mouse TNF-α challenge models. PMID:24900545

  7. The Selectivity of CK2 Inhibitor Quinalizarin: A Reevaluation

    PubMed Central

    Cozza, Giorgio; Venerando, Andrea; Sarno, Stefania; Pinna, Lorenzo A.

    2015-01-01

    Many polyphenolic compounds have been reported to inhibit protein kinases, with special reference to CK2, a pleiotropic serine/threonine kinase, implicated in neoplasia, neurodegenerative disease, and viral infections. In general however these compounds are not endowed with stringent selectivity. Among them quinalizarin (1,2,5,8-tetrahydroxyanthraquinone) turned out to be particularly potent (Ki = 0.058 μM) and quite selective as judged by profiling it on a small panel of 70 protein kinases. Here, by profiling quinalizarin on a larger panel of 140 kinases we reach the conclusion that quinalizarin is one of the most selective inhibitors of CK2, superior to the first-in-class CK2 inhibitor, CX-4945, now in clinical trials for the treatment of cancer. Moreover here we show that quinalizarin is able to discriminate between the isolated CK2 catalytic subunit (CK2α) and CK2 holoenzyme (CK2α2β2), consistent with in silico and in vitro analyses. PMID:26558278

  8. Development of a selective inhibitor of Protein Arginine Deiminase 2.

    PubMed

    Muth, Aaron; Subramanian, Venkataraman; Beaumont, Edward; Nagar, Mitesh; Kerry, Philip; McEwan, Paul; Srinath, Hema; Clancy, Kathleen Wanda; Parelkar, Sangram S; Thompson, Paul R

    2017-03-22

    Protein arginine deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and selectivity (30a, 41a, and 49a) as well as cellular efficacy 30a. Notably, these compounds use the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated.

  9. Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors.

    PubMed

    Zaro, Balyn W; Whitby, Landon R; Lum, Kenneth M; Cravatt, Benjamin F

    2016-12-14

    Electrophilic small molecules are an important class of chemical probes and drugs that produce biological effects by irreversibly modifying proteins. Examples of electrophilic drugs include covalent kinase inhibitors that are used to treat cancer and the multiple sclerosis drug dimethyl fumarate. Optimized covalent drugs typically inactivate their protein targets rapidly in cells, but ensuing time-dependent, off-target protein modification can erode selectivity and diminish the utility of reactive small molecules as chemical probes and therapeutics. Here, we describe an approach to confer kinetic selectivity to electrophilic drugs. We show that an analogue of the covalent Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib bearing a fumarate ester electrophile is vulnerable to enzymatic metabolism on a time-scale that preserves rapid and sustained BTK inhibition, while thwarting more slowly accumulating off-target reactivity in cell and animal models. These findings demonstrate that metabolically labile electrophilic groups can endow covalent drugs with kinetic selectivity to enable perturbation of proteins and biochemical pathways with greater precision.

  10. N-methyl-citalopram: A quaternary selective serotonin reuptake inhibitor.

    PubMed

    Bismuth-Evenzal, Yona; Roz, Netta; Gurwitz, David; Rehavi, Moshe

    2010-11-15

    We describe the synthesis and the pharmacological characterization of a new quaternary selective serotonin reuptake inhibitor (SSRI) N-methyl-citalopram (NMC) with periphery restricted action due to its inability to cross the blood brain barrier. NMC recognized and blocked the human platelet serotonin transporter (SERT) with similar affinity to that of citalopram as was evident from competition binding studies with [(3)H]citalopram and uptake studies with [(3)H]5-HT. In contrast, the affinity of NMC to rat brain SERT was 10-fold lower than its parent compound citalopram. Similarly to citalopram, NMC did not inhibit dopamine and noradrenaline uptake in rat brain synaptosomes at 10(-7)M as well as [(3)H]ketanserin binding to rat brain membranes at 10(-5)M, demonstrating its SSRI profile. A comparison of radioactivity retained in perfused mice brain following in vivo intraperitoneal injections of tritium-labeled NMC or citalopram showed that unlike citalopram, NMC did not penetrate the brain. Taken together, our observations suggest that N-methyl-citalopram is a selective serotonin reuptake inhibitor that does not penetrate the mouse brain. Epidemiological studies have suggested that chronic use of SSRI drugs may confer a protective effect against myocardial infarction (MI) apparently reflecting reduced platelet aggregation secondary to reduced platelet serotonin levels. N-methyl-citalopram may therefore have a potential as a new anti-platelet drug that does not cross the blood brain barrier and is thus devoid of the adverse CNS effects of SSRI drugs.

  11. Synthesis and evaluation of heteroarylalanine diacids as potent and selective neutral endopeptidase inhibitors.

    PubMed

    Glossop, Melanie S; Bazin, Richard J; Dack, Kevin N; Fox, David N A; MacDonald, Graeme A; Mills, Mark; Owen, Dafydd R; Phillips, Chris; Reeves, Keith A; Ringer, Tracy J; Strang, Ross S; Watson, Christine A L

    2011-06-01

    Heteroarylalanine derivatives 4 were designed as potential inhibitors of neutral endopeptidase (NEP EC 3.4.24.11). Selectivity over other zinc metalloproteinases was explored through occupation of the S2' subsite within NEP. Structural optimisation led to the identification of 5-phenyl oxazole 4f, a potent and selective NEP inhibitor. A crystal structure of the inhibitor bound complex is reported.

  12. Full Inhibition of Spinal FAAH Leads to TRPV1-Mediated Analgesic Effects in Neuropathic Rats and Possible Lipoxygenase-Mediated Remodeling of Anandamide Metabolism

    PubMed Central

    Starowicz, Katarzyna; Makuch, Wioletta; Korostynski, Michal; Malek, Natalia; Slezak, Michal; Zychowska, Magdalena; Petrosino, Stefania; De Petrocellis, Luciano; Cristino, Luigia; Przewlocka, Barbara; Di Marzo, Vincenzo

    2013-01-01

    Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats. PMID:23573230

  13. Full inhibition of spinal FAAH leads to TRPV1-mediated analgesic effects in neuropathic rats and possible lipoxygenase-mediated remodeling of anandamide metabolism.

    PubMed

    Starowicz, Katarzyna; Makuch, Wioletta; Korostynski, Michal; Malek, Natalia; Slezak, Michal; Zychowska, Magdalena; Petrosino, Stefania; De Petrocellis, Luciano; Cristino, Luigia; Przewlocka, Barbara; Di Marzo, Vincenzo

    2013-01-01

    Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.

  14. Selective Serotonin Reuptake Inhibitor (SSRI) Antidepressants, Prolactin and Breast Cancer.

    PubMed

    Ashbury, Janet E; Lévesque, Linda E; Beck, Patricia A; Aronson, Kristan J

    2012-01-01

    Selective serotonin reuptake inhibitors (SSRIs) are a widely prescribed class of antidepressants. Laboratory and epidemiologic evidence suggests that a prolactin-mediated mechanism secondary to increased serotonin levels at neuronal synapses could lead to a potentially carcinogenic effect of SSRIs. In this population-based case-control study, we evaluated the association between SSRI use and breast cancer risk as a function of their relative degree of inhibition of serotonin reuptake as a proxy for their impact on prolactin levels. Cases were 2,129 women with primary invasive breast cancer diagnosed from 2003 to 2007, and controls were 21,297 women randomly selected from the population registry. Detailed information for each SSRI prescription dispensed was compiled using the Saskatchewan prescription database. Logistic regression was used to evaluate the impact of use of high and lower inhibitors of serotonin reuptake and duration of use, as well as to assess the effect of individual high inhibitors on the risk of breast cancer. Exclusive users of high or lower inhibitors of serotonin reuptake were not at increased risk for breast cancer compared with non-users of SSRIs (OR = 1.01, CI = 0.88-1.17 and OR = 0.91, CI = 0.67-1.25 respectively), regardless of their duration of use or menopausal status. While we cannot rule out the possibility of a clinically important risk increase (OR = 1.83, CI = 0.99-3.40) for long-term users of sertraline (≥24 prescriptions), given the small number of exposed cases (n = 12), the borderline statistical significance, and the wide confidence interval, these results need to be interpreted cautiously. In this large population-based case-control study, we found no conclusive evidence of breast cancer risk associated with the use of SSRIs even after assessing the degree of serotonin reuptake inhibition and duration of use. Our results do not support the serotonin-mediated pathway for the prolactin

  15. A Selective Phenelzine Analogue Inhibitor of Histone Demethylase LSD1

    PubMed Central

    2015-01-01

    Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1–/– cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease. PMID:24707965

  16. A selective phenelzine analogue inhibitor of histone demethylase LSD1.

    PubMed

    Prusevich, Polina; Kalin, Jay H; Ming, Shonoi A; Basso, Manuela; Givens, Jeffrey; Li, Xin; Hu, Jianfei; Taylor, Martin S; Cieniewicz, Anne M; Hsiao, Po-Yuan; Huang, Rong; Roberson, Heather; Adejola, Nkosi; Avery, Lindsay B; Casero, Robert A; Taverna, Sean D; Qian, Jiang; Tackett, Alan J; Ratan, Rajiv R; McDonald, Oliver G; Feinberg, Andrew P; Cole, Philip A

    2014-06-20

    Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.

  17. Quinazoline derivatives as selective CYP1B1 inhibitors.

    PubMed

    Mohd Siddique, Mohd Usman; McCann, Glen J P; Sonawane, Vinay R; Horley, Neill; Gatchie, Linda; Joshi, Prashant; Bharate, Sandip B; Jayaprakash, Venkatesan; Sinha, Barij N; Chaudhuri, Bhabatosh

    2017-04-21

    CYP1B1 is implicated to have a role in the development of breast, ovarian, renal, skin and lung carcinomas. It has been suggested that identification of potent and specific CYP1B1 inhibitors can lead to a novel treatment of cancer. Flavonoids have a compact rigid skeleton which fit precisely within the binding cavity of CYP1B1. Systematic isosteric replacement of flavonoid 'O' atom with 'N' atom led to the prediction that a 'quinazoline' scaffold could be the basis for designing potential CYP1B1 inhibitors. A total of 20 quinazoline analogs were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes™. IC50 determinations of six compounds with capability of inhibiting CYP1B1 identified quinazolines 5c and 5h as the best candidates for CYP1B1 inhibition, with IC50 values in the nM range. Further selectivity studies with homologous CYPs, belonging to the CYP1, CYP2 and CYP3 family of enzymes, showed that the compounds are likely to be free from critical drug-drug interaction liability. Molecular modelling studies were performed to rationalize the observed enzymatic inhibitions. Further biological studies in live yeast and human cells, harboring CYP1A1 and CYP1B1 enzymes, have illustrated the most potent compounds' cellular permeability and capability of potently inhibiting CYP1B1 enzyme expressed within live cells.

  18. Integrated ligand based pharmacophore model derived from diverse FAAH covalent ligand classes.

    PubMed

    Shen, Lingling; Huang, Hongwei; Makriyannis, Alexandros; Fisher, Luke S

    2012-12-01

    3D pharmacophore modeling is an important computational methodology for ligand-enzyme binding interactions in drug discovery. More specifically, a consensus pharmacophore model derived from diverse ligands is a key determinant upon which the prediction power of computational models is based for designing novel ligands. In this work, by merging the important pharmacophore features based on four classes of covalent FAAH ligands, and then integrating the exclusion volume spheres derived from the crystal structure, we created for the first time an integrated FAAH pharmacophore model to describe the ligand-enzyme binding interactions. This new integrated FAAH pharmacophore model can correctly predict the covalent ligand binding mode, which correlates with the SAR data. The study is expected to provide insights into novel covalent ligand-FAAH binding interactions, and facilitate the design of covalent ligands against FAAH.

  19. A selective inhibitor of cell proliferation from normal serum.

    PubMed Central

    Harrington, W N; Godman, G C

    1980-01-01

    A factor in normal serum that selectively and reversibly inhibits proliferation of cells in culture has been enriched 160-fold from calf serum by sequential ammonium sulfate precipitation, gel filtration, and lectin-affinity chromatography. DNA synthesis of normal (but not transformed) rat hepatocytes, human lymphoblast lines, and mitogen-stimulated murine spleen cells is inhibited by greater than 90%, and Vero, murine myeloma, MELC, and a human colon carcinoma cell line to a lesser extent. Growth of other cell lines tested was not affected. Responsive cells are arrested apparently in G1 by this inhibitor, the effect of which is maximal by 24 hr and is spontaneously reversible thereafter unless it is renewed. The active fraction is a protein that migrates with the alpha 2-globulins; it is not a lipoprotein, and it is of high apparent molecular weight. PMID:6928635

  20. Serotonin syndrome: fentanyl and selective serotonin reuptake inhibitor interactions.

    PubMed

    Greenier, Ewa; Lukyanova, Valentina; Reede, Lynn

    2014-10-01

    Serotonin syndrome is a rare but potentially fatal adverse drug reaction associated with increased serotonergic activity in the central nervous system. It is characterized by a triad of symptoms, which include altered mental status, neuromuscular hyperactivity, and autonomic instability or hyperactivity. Due to the potential of rapid onset, it is important for clinicians to recognize the signs and symptoms of serotonin syndrome. Serotonin syndrome symptoms may resemble other conditions. Although this article focuses on serotonin syndrome as a result of an adverse interaction of selective serotonin reuptake inhibitors (SSRI) and fentanyl, it is important for not only anesthesia professionals, but all clinicians--such as those in emergency medicine and critical care--to be aware of this syndrome and its management. This article discusses the clinical manifestations of the serotonin syndrome and highlights reported cases of serotonin syndrome specifically related to an interaction between SSRIs and fentanyl, a commonly used opioid in anesthesia practice.

  1. Selective Monoacylglycerol Lipase Inhibitors: Antinociceptive versus Cannabimimetic Effects in Mice

    PubMed Central

    Wilkerson, Jenny L.; Mustafa, Mohammed; Abdullah, Rehab; Niphakis, Micah; Wiley, Jenny L.; Cravatt, Benjamin F.; Lichtman, Aron H.

    2015-01-01

    The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) plays an important role in a variety of physiologic processes, but its rapid breakdown by monoacylglycerol lipase (MAGL) results in short-lived actions. Initial MAGL inhibitors were limited by poor selectivity and low potency. In this study, we tested JZL184 [4-nitrophenyl 4-[bis(2H-1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate] and MJN110 [2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate], MAGL inhibitors that possess increased selectivity and potency, in mouse behavioral assays of neuropathic pain [chronic constriction injury (CCI) of the sciatic nerve], interoceptive cannabimimetic effects (drug-discrimination paradigm), and locomotor activity in an open field test. MJN110 (1.25 and 2.5 mg/kg) and JZL184 (16 and 40 mg/kg) significantly elevated 2-AG and decreased arachidonic acid but did not affect anandamide in whole brains. Both MAGL inhibitors significantly reduced CCI-induced mechanical allodynia with the following potencies [ED50 (95% confidence limit [CL]) values in mg/kg: MJN110 (0.43 [0.30–0.63]) > JZL184 (17.8 [11.6–27.4])] and also substituted for the potent cannabinoid receptor agonist CP55,940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol] in the drug-discrimination paradigm [ED50 (95% CL) values in mg/kg: MJN110 (0.84 [0.69–1.02]) > JZL184 (24.9 [14.6–42.5])]; however, these compounds elicited differential effects on locomotor behavior. Similar to cannabinoid 1 (CB1) receptor agonists, JZL184 produced hypomotility, whereas MJN110 increased locomotor behavior and did not produce catalepsy or hypothermia. Although both drugs substituted for CP55,940 in the drug discrimination assay, MJN110 was more potent in reversing allodynia in the CCI model than in producing CP55,940-like effects. Overall, these results suggest that MAGL inhibition may alleviate neuropathic pain, while displaying limited

  2. Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase.

    PubMed

    Keung, W M; Vallee, B L

    1993-02-15

    Human mitochondrial aldehyde dehydrogenase (ALDH-I) is potently, reversibly, and selectively inhibited by an isoflavone isolated from Radix puerariae and identified as daidzin, the 7-glucoside of 4',7-dihydroxyisoflavone. Kinetic analysis with formaldehyde as substrate reveals that daidzin inhibits ALDH-I competitively with respect to formaldehyde with a Ki of 40 nM, and uncompetitively with respect to the coenzyme NAD+. The human cytosolic aldehyde dehydrogenase isozyme (ALDH-II) is nearly 3 orders of magnitude less sensitive to daidzin inhibition. Daidzin does not inhibit human class I, II, or III alcohol dehydrogenases, nor does it have any significant effect on biological systems that are known to be affected by other isoflavones. Among more than 40 structurally related compounds surveyed, 12 inhibit ALDH-I, but only prunetin and 5-hydroxydaidzin (genistin) combine high selectivity and potency, although they are 7- to 15-fold less potent than daidzin. Structure-function relationships have established a basis for the design and synthesis of additional ALDH inhibitors that could both be yet more potent and specific.

  3. UPREGULATION OF FATTY ACID AMIDE HYDROLASE (FAAH) IN THE DORSAL PERIAQUEDUCTAL GRAY IS ASSOCIATED WITH NEUROPATHIC PAIN AND REDUCED HEART RATE IN RATS.

    PubMed

    Dean, Caron; Hillard, Cecilia J; Seagard, Jeanne L; Hopp, Francis A; Hogan, Quinn H

    2017-02-01

    Nerve damage can induce a heightened pain response to noxious stimulation, which is termed hyperalgesia. Pain itself acts as a stressor, initiating autonomic and sensory effects through the dorsal periaqueductal gray (dPAG) to induce both sympathoexcitation and analgesia, which prior studies have shown to be affected by endocannabinoid signaling. The present study addressed the hypothesis that neuropathic pain disrupts autonomic and analgesic regulation by endocannabinoid signaling in the dPAG. Endocannabinoid contents, transcript levels of endocannabinoid signaling components, and catabolic enzyme activity were analyzed in the dPAG of rats at 21 days after painful nerve injury. The responses to two nerve injury models were similar, with two-thirds of animals developing hyperalgesia that was maintained throughout the post injury period, while no sustained change in sensory function was observed in the remaining rats. Anandamide content was lower in the dPAG of rats that developed sustained hyperalgesia and activity of the catabolic enzyme fatty acid amide hydrolase (FAAH) was higher. Intensity of hyperalgesia was correlated to transcript levels of FAAH and negatively correlated to heart rate and sympatho-vagal balance. These data suggest that maladaptive endocannabinoid signaling in the dPAG after nerve injury could contribute to chronic neuropathic pain and associated autonomic dysregulation. This study demonstrates that reduced anandamide content and upregulation of FAAH in the dPAG are associated with hyperalgesia and reduced heart rate sustained weeks after nerve injury. These data provide support for the evaluation of FAAH inhibitors for the treatment of chronic neuropathic pain.

  4. Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance.

    PubMed

    Willemsen-Seegers, Nicole; Uitdehaag, Joost C M; Prinsen, Martine B W; de Vetter, Judith R F; de Man, Jos; Sawa, Masaaki; Kawase, Yusuke; Buijsman, Rogier C; Zaman, Guido J R

    2017-02-17

    Target residence time (τ) has been suggested to be a better predictor of the biological activity of kinase inhibitors than inhibitory potency (IC50) in enzyme assays. Surface plasmon resonance binding assays for 46 human protein and lipid kinases were developed. The association and dissociation constants of 80 kinase inhibitor interactions were determined. τ and equilibrium affinity constants (KD) were calculated to determine kinetic selectivity. Comparison of τ and KD or IC50 values revealed a strikingly different view on the selectivity of several kinase inhibitors, including the multi-kinase inhibitor ponatinib, which was tested on 10 different kinases. In addition, known pan-Aurora inhibitors resided much longer on Aurora B than on Aurora A, despite having comparable affinity for Aurora A and B. Furthermore, the γ/δ-selective PI3K inhibitor duvelisib and the δ-selective drug idelalisib had similar 20-fold selectivity for δ- over γ-isoform but duvelisib resided much longer on both targets.

  5. Identification of potential ACAT-2 selective inhibitors using pharmacophore, SVM and SVR from Chinese herbs.

    PubMed

    Qiao, Lian-Sheng; Zhang, Xian-Bao; Jiang, Lu-di; Zhang, Yan-Ling; Li, Gong-Yu

    2016-11-01

    Acyl-coenzyme A cholesterol acyltransferase (ACAT) plays an important role in maintaining cellular and organismal cholesterol homeostasis. Two types of ACAT isozymes with different functions exist in mammals, named ACAT-1 and ACAT-2. Numerous studies showed that ACAT-2 selective inhibitors are effective for the treatment of hypercholesterolemia and atherosclerosis. However, as a typical endoplasmic reticulum protein, ACAT-2 protein has not been purified and revealed, so combinatorial ligand-based methods might be the optimal strategy for discovering the ACAT-2 selective inhibitors. In this study, selective pharmacophore models of ACAT-1 inhibitors and ACAT-2 inhibitors were built, respectively. The optimal pharmacophore model for each subtype was identified and utilized as queries for the Traditional Chinese Medicine Database screening. A total of 180 potential ACAT-2 selective inhibitors were obtained, which were identified using an ACAT-2 pharmacophore and not by our ACAT-1 model. Selective SVM model and bioactive SVR model were generated for further identification of the obtained ACAT-2 inhibitors. Ten compounds were finally obtained with predicted inhibitory activities toward ACAT-2. Hydrogen bond acceptor, 2D autocorrelations, GETAWAY descriptors, and BCUT descriptors were identified as key structural features for selectivity and activity of ACAT-2 inhibitors. This study provides a reasonable ligand-based approach to discover potential ACAT-2 selective inhibitors from Chinese herbs, which could help in further screening and development of ACAT-2 selective inhibitors.

  6. Candidate genes for antidepressant response to selective serotonin reuptake inhibitors

    PubMed Central

    Lotrich, Francis E; Pollock, Bruce G

    2005-01-01

    Selective serotonin reuptake inhibitors (SSRIs) can safely and successfully treat major depression, although a substantial number of patients benefit only partially or not at all from treatment. Genetic polymorphisms may play a major role in determining the response to SSRI treatment. Nonetheless, it is likely that efficacy is determined by multiple genes, with individual genetic polymorphisms having a limited effect size. Initial studies have identified the promoter polymorphism in the gene coding for the serotonin reuptake transporter as moderating efficacy for several SSRIs. The goal of this review is to suggest additional plausible polymorphisms that may be involved in antidepressant efficacy. These include genes affecting intracellular transductional cascades; neuronal growth factors; stress-related hormones, such as corticotropin-releasing hormone and glucocorticoid receptors; ion channels and synaptic efficacy; and adaptations of monoaminergic pathways. Association analyses to examine these candidate genes may facilitate identification of patients for targeted alternative therapies. Determining which genes are involved may also assist in identifying future, novel treatments. PMID:18568127

  7. Selective serotonin reuptake inhibitors and cardiovascular events: A systematic review

    PubMed Central

    Nezafati, Mohammad Hassan; Eshraghi, Ali; Vojdanparast, Mohammad; Abtahi, Saeed; Nezafati, Pouya

    2016-01-01

    Background: Given the importance of the role of depression in predicting the outcome of cardiovascular disorders, current medications for treating depression, particularly selective serotonin reuptake inhibitors (SSRIs), are taken into consideration. This study aimed to systematically review the published findings in the use of SSRIs and the risk for cardiac events. Materials and Methods: An independent review of the Web of Science, PubMed, Scopus, Cochrane, CINAHL, index Copernicus, and Google Scholar, up to 2014, was performed. We identified studies evaluating the effect of SSRIs, on cardiovascular events. Articles in English with full text availability, review articles, and experimental studies were included in the study. Among 150 studies reviewed based on the included keywords, 17 met the study criteria and were finally reviewed. Results: The use of some types of SSRIs may prevent platelet adhesion and aggregation; control the cardiovascular risk profile including hypertension, insulin resistance, and body weight; and also inhibit inflammatory processes. The appearance of adverse cardiac events, including cardiac arrhythmias (torsade de pointes and QT prolongation), syncope, increased systolic and diastolic right ventricular volume, and the production of pro-inflammatory cytokines leading atherosclerosis development, has also been expected with the chronic use of some types of SSRIs. Conclusion: According to our systematic review, both beneficial and adverse cardiovascular events can be established following the chronic use of various types of SSRIs. Therefore, when taking SSRIs, the cardiovascular effect of each SSRI has to be carefully considered, based on patients’ cardiovascular risk profiles. PMID:27904611

  8. Selective serotonin reuptake inhibitor drug interactions in patients receiving statins.

    PubMed

    Andrade, Chittaranjan

    2014-02-01

    Elderly patients commonly receive statin drugs for the primary or secondary prevention of cardiovascular and cerebrovascular events. Elderly patients also commonly receive antidepressant drugs, usually selective serotonin reuptake inhibitors (SSRIs), for the treatment of depression, anxiety, or other conditions. SSRIs are associated with many pharmacokinetic drug interactions related to the inhibition of the cytochrome P450 (CYP) metabolic pathways. There is concern that drugs that inhibit statin metabolism can trigger statin adverse effects, especially myopathy (which can be potentially serious, if rhabdomyolysis occurs). However, a detailed literature review of statin metabolism and of SSRI effects on CYP enzymes suggests that escitalopram, citalopram, and paroxetine are almost certain to be safe with all statins, and rosuvastatin, pitavastatin, and pravastatin are almost certain to be safe with all SSRIs. Even though other SSRI-statin combinations may theoretically be associated with risks, the magnitude of the pharmacokinetic interaction is likely to be below the threshold for clinical significance. Risk, if at all, lies in combining fluvoxamine with atorvastatin, simvastatin, or lovastatin, and even this risk can be minimized by using lower statin doses and monitoring the patient.

  9. Arylboronic acids as dual-action FAAH and TRPV1 ligands.

    PubMed

    Morera, Enrico; Di Marzo, Vincenzo; Monti, Ludovica; Allarà, Marco; Schiano Moriello, Aniello; Nalli, Marianna; Ortar, Giorgio; De Petrocellis, Luciano

    2016-03-01

    A series of 31 arylboronic acids designed on the basis of the pharmacophore model for a variety of TRPV1 antagonists was prepared and tested on FAAH and TRPV1 channel. Four of them, that is, compounds 3c, 4a, 5a,b acted as dual FAAH/TRPV1 blockers with IC50 values between 0.56 and 8.11μM whereas ten others (compounds 1c,f-i, 2c-f, 4b) inhibited FAAH and activated/desensitized TRPV1.

  10. Design of highly potent urea-based, exosite-binding inhibitors selective for glutamate carboxypeptidase II.

    PubMed

    Tykvart, Jan; Schimer, Jiří; Jančařík, Andrej; Bařinková, Jitka; Navrátil, Václav; Starková, Jana; Šrámková, Karolína; Konvalinka, Jan; Majer, Pavel; Šácha, Pavel

    2015-05-28

    We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.

  11. Selective serotonin reuptake inhibitors for premature ejaculation: review of erectile and ejaculatory side effects.

    PubMed

    Lasker, George F; Halis, Fikret; Gokce, Ahmet

    2014-01-01

    Premature ejaculation is one of the most prevalent sexual disorders affecting men today. The lack of approved therapies has resulted in the prescription of many 'off-label' treatments to manage the condition. Selective serotonin reuptake inhibitors have an interesting side effect of prolonging ejaculatory latency. Consequently, these agents are often considered a first line treatment for patients suffering from premature ejaculation. Erectile dysfunction is another common side effect reported by men treated with selective serotonin reuptake inhibitors. Nitric oxide is the primary mediator of erectile function. Preclinical studies have provided evidence that selective serotonin reuptake inhibitors decrease nitric oxide bioavailability. This invited mini-review aims to examine the physiology of the erectile and ejaculatory responses, discuss the indicated and 'off-label' clinical utility of selective serotonin reuptake inhibitors, and to summarize evidence from basic science and clinical studies pertaining to mechanisms of how selective serotonin reuptake inhibitor therapy modifies ejaculatory and erectile function.

  12. Piperazinyl carbamate fatty acid amide hydrolase inhibitors and transient receptor potential channel modulators as "dual-target" analgesics.

    PubMed

    Maione, Sabatino; Costa, Barbara; Piscitelli, Fabiana; Morera, Enrico; De Chiaro, Maria; Comelli, Francesca; Boccella, Serena; Guida, Francesca; Verde, Roberta; Ortar, Giorgio; Di Marzo, Vincenzo

    2013-10-01

    We showed previously that inhibiting fatty acid amide hydrolase (FAAH), an endocannabinoid degrading enzyme, and transient receptor potential vanilloid type-1 (TRPV1) channels with the same molecule, the naturally occurring N-arachidonoyl-serotonin (AA-5-HT), produces more efficacious anti-nociceptive and anti-hyperalgesic actions than the targeting of FAAH or TRPV1 alone. We also reported the synthesis of some piperazinyl carbamates as "dual" FAAH inhibitors and either antagonists at TRPV1 or agonists/desensitizers of the transient receptor potential ankyrin type-1 (TRPA1) cannel, another target for analgesic drugs. We investigated here if two such compounds, the FAAH/TRPV1 blocker OMDM198 and the FAAH inhibitor/TRPA1 agonist, OMDM202, exert anti-nociceptive actions in the formalin test of pain in mice, and through what mechanism. Both compounds inhibited the second phase of the response to formalin, the effect being maximal at 3 mg/kg, i.p. Antagonism of CB1 or CB2 receptors with AM251 or AM630 (1 mg/kg, i.p.), respectively, reversed this effect. A TRPV1 agonist, palvanil (0.1 mg/kg, i.p.), also reversed the analgesic effect of OMDM198. OMDM202 action was also antagonized by a per se inactive dose of the selective TRPA1 blocker, AP-18 (0.05 mg/kg, i.p.), but not by a TRPV1 antagonist. AP-18 at higher doses (0.1-0.2 mg/kg) inhibited both the first and second phase of the formalin response. The effects of OMDM198 and OMDM202 were accompanied by elevation of anandamide levels in the spinal cord. OMDM198 (0.1-5.0 mg/kg, i.p.) also reversed carrageenan-induced oedema and thermal hyperalgesia in mice with efficacy similar to that of AA-5-HT. These data suggest that "dual" fatty acid amide hydrolase and transient receptor potential channel modulators should be clinically evaluated as novel analgesics.

  13. Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors.

    PubMed

    Bertrand, J A; Thieffine, S; Vulpetti, A; Cristiani, C; Valsasina, B; Knapp, S; Kalisz, H M; Flocco, M

    2003-10-17

    GSK-3beta is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3beta may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3beta, we determined crystal structures of unphosphorylated GSK-3beta in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3beta in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3beta inhibitors.

  14. Optimization of the Central Heterocycle of α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Garfunkle, Joie; Ezzili, Cyrine; Rayl, Thomas J.; Hochstatter, Dustin G.; Hwang, Inkyu; Boger, Dale L.

    2008-01-01

    The synthesis and evaluation of a refined series of α-ketoheterocycles based on the oxazole 2 (OL-135) incorporating systematic changes in the central heterocycle bearing a key set of added substituents are described. The nature of the central heterocycle, even within the systematic and minor perturbations explored herein, significantly influenced the inhibitor activity: 1,3,4-oxadiazoles and 1,2,4-oxadiazoles 9 > tetrazoles, the isomeric 1,2,4-oxadiazoles 10, 1,3,4-thiadiazoles > oxazoles including 2 > 1,2-diazines > thiazoles > 1,3,4-triazoles. Most evident in these trends is the observation that introduction of an additional heteroatom at position 4 (oxazole numbering, N > O > CH) substantially increases activity that may be attributed to a reduced destabilizing steric interaction at the FAAH active site. Added heterocycle substituents displaying well defined trends may be utilized to enhance the inhibitor potency and, more significantly, to enhance the inhibitor selectivity. These trends, exemplified herein, emerge from both enhancements in the FAAH activity and simultaneous disruption of binding affinity for competitive off-target enzymes. PMID:18630870

  15. A Systems Pharmacology Perspective on the Clinical Development of Fatty Acid Amide Hydrolase Inhibitors for Pain

    PubMed Central

    Benson, N; Metelkin, E; Demin, O; Li, G L; Nichols, D; van der Graaf, P H

    2014-01-01

    The level of the endocannabinoid anandamide is controlled by fatty acid amide hydrolase (FAAH). In 2011, PF-04457845, an irreversible inhibitor of FAAH, was progressed to phase II clinical trials for osteoarthritic pain. This article discusses a prospective, integrated systems pharmacology model evaluation of FAAH as a target for pain in humans, using physiologically based pharmacokinetic and systems biology approaches. The model integrated physiological compartments; endocannabinoid production, degradation, and disposition data; PF-04457845 pharmacokinetics and pharmacodynamics, and cannabinoid receptor CB1-binding kinetics. The modeling identified clear gaps in our understanding and highlighted key risks going forward, in particular relating to whether methods are in place to demonstrate target engagement and pharmacological effect. The value of this modeling exercise will be discussed in detail and in the context of the clinical phase II data, together with recommendations to enable optimal future evaluation of FAAH inhibitors. PMID:24429592

  16. Enhanced Responsiveness to Selective Serotonin Reuptake Inhibitors during Lactation

    PubMed Central

    Jury, Nicholas J.; McCormick, Betsy A.; Horseman, Nelson D.; Benoit, Stephen C.; Gregerson, Karen A.

    2015-01-01

    The physiology of mood regulation in the postpartum is poorly understood despite the fact that postpartum depression (PPD) is a common pathology. Serotonergic mechanisms and their dysfunction are widely presumed to be involved, which has led us to investigate whether lactation induces changes in central or peripheral serotonin (5-HT) systems and related affective behaviors. Brain sections from lactating (day 10 postpartum) and age-matched nulliparous (non-pregnant) C57BL/6J mice were processed for 5-HT immunohistochemistry. The total number of 5-HT immunostained cells and optical density were measured. Lactating mice exhibited lower immunoreactive 5-HT and intensity in the dorsal raphe nucleus when compared with nulliparous controls. Serum 5-HT was quantified from lactating and nulliparous mice using radioimmunoassay. Serum 5-HT concentrations were higher in lactating mice than in nulliparous controls. Affective behavior was assessed in lactating and non-lactating females ten days postpartum, as well as in nulliparous controls using the forced swim test (FST) and marble burying task (MBT). Animals were treated for the preceding five days with a selective serotonin reuptake inhibitor (SSRI, citalopram, 5mg/kg/day) or vehicle. Lactating mice exhibited a lower baseline immobility time during the FST and buried fewer marbles during the MBT as compared to nulliparous controls. Citalopram treatment changed these behaviors in lactating mice with further reductions in immobility during the FST and decreased marble burying. In contrast, the same regimen of citalopram treatment had no effect on these behaviors in either non-lactating postpartum or nulliparous females. Our findings demonstrate changes in both central and peripheral 5-HT systems associated with lactation, independent of pregnancy. They also demonstrate a significant interaction of lactation and responsiveness to SSRI treatment, which has important implications in the treatment of PPD. Although recent evidence

  17. Enhanced responsiveness to selective serotonin reuptake inhibitors during lactation.

    PubMed

    Jury, Nicholas J; McCormick, Betsy A; Horseman, Nelson D; Benoit, Stephen C; Gregerson, Karen A

    2015-01-01

    The physiology of mood regulation in the postpartum is poorly understood despite the fact that postpartum depression (PPD) is a common pathology. Serotonergic mechanisms and their dysfunction are widely presumed to be involved, which has led us to investigate whether lactation induces changes in central or peripheral serotonin (5-HT) systems and related affective behaviors. Brain sections from lactating (day 10 postpartum) and age-matched nulliparous (non-pregnant) C57BL/6J mice were processed for 5-HT immunohistochemistry. The total number of 5-HT immunostained cells and optical density were measured. Lactating mice exhibited lower immunoreactive 5-HT and intensity in the dorsal raphe nucleus when compared with nulliparous controls. Serum 5-HT was quantified from lactating and nulliparous mice using radioimmunoassay. Serum 5-HT concentrations were higher in lactating mice than in nulliparous controls. Affective behavior was assessed in lactating and non-lactating females ten days postpartum, as well as in nulliparous controls using the forced swim test (FST) and marble burying task (MBT). Animals were treated for the preceding five days with a selective serotonin reuptake inhibitor (SSRI, citalopram, 5mg/kg/day) or vehicle. Lactating mice exhibited a lower baseline immobility time during the FST and buried fewer marbles during the MBT as compared to nulliparous controls. Citalopram treatment changed these behaviors in lactating mice with further reductions in immobility during the FST and decreased marble burying. In contrast, the same regimen of citalopram treatment had no effect on these behaviors in either non-lactating postpartum or nulliparous females. Our findings demonstrate changes in both central and peripheral 5-HT systems associated with lactation, independent of pregnancy. They also demonstrate a significant interaction of lactation and responsiveness to SSRI treatment, which has important implications in the treatment of PPD. Although recent evidence

  18. Intracranial haemorrhage and use of selective serotonin reuptake inhibitors

    PubMed Central

    de Abajo, Francisco J; Jick, Hershel; Derby, Laura; Jick, Susan; Schmitz, Stephen

    2000-01-01

    Aims In the past few years an increasing number of bleeding disorders have been reported in association with the use of selective serotonin reuptake inhibitors (SSRIs), including serious cases of intracranial haemorrhage, raising concerns about the safety of this class of drugs. The present study was performed to test the hypothesis of an increased risk of intracranial haemorrhage associated with the use of SSRIs. Methods We carried out a case-control study nested in a cohort of antidepressants users with the UK-based General Practice Research Database (GPRD) as the primary source of information. The study cohort encompassed subjects aged between 18 and 79 years who received a first-time prescription for any antidepressant from January, 1990 to October, 1997. Patients with presenting conditions or treatments that could be associated with an increased risk of intracranial haemorrhage were excluded from the cohort. Patients were followed-up until the occurrence of an idiopathic intracranial haemorrhage. Up to four controls per case, matched on age, sex, calendar time and practice were randomly selected from the study cohort. We estimated adjusted odds ratios and 95% confidence intervals of intracranial haemorrhage with current use of SSRIs and other antidepressants as compared with nonuse using conditional logistic regression. Results We identified 65 cases of idiopathic intracranial haemorrhage and 254 matched controls. Current exposure to SSRIs was ascertained in 7 cases (10.8%) and 24 controls (9.7%) resulting in an adjusted OR (95%CI) of 0.8 (0.3,2.3). The estimate for ‘other antidepressants’ was 0.7 (0.3,1.6). The effect measures were not modified by gender or age. No effect related to dose or treatment duration was detected. The risk estimates did not change according to the location of bleeding (intracerebral or subarachnoid). Conclusions Our results are not compatible with a major increased risk of intracranial haemorrhage among users of SSRIs or other

  19. Structural Biology Insight for the Design of Sub-type Selective Aurora Kinase Inhibitors.

    PubMed

    Sarvagalla, Sailu; Coumar, Mohane Selvaraj

    2015-01-01

    Aurora kinase A, B and C, are key regulators of mitosis and are over expressed in many of the human cancers, making them an ideal drug target for cancer chemotherapy. Currently, over a dozen of Aurora kinase inhibitors are in various phases of clinical development. The majority of the inhibitors (VX-680/MK-0457, PHA-739358, CYC116, SNS-314, AMG 900, AT-9283, SCH- 1473759, ABT-348, PF-03814735, R-763/AS-703569, KW-2449 and TAK-901) are pan-selective (isoform non-selective) and few are Aurora A (MLN8054, MLN8237, VX-689/MK5108 and ENMD 2076) and Aurora B (AZD1152 and GSK1070916) sub-type selective. Despite the intensive research efforts in the past decade, no Aurora kinase inhibitor has reached the market. Recent evidence suggests that the sub-type selective Aurora kinase A inhibitor could possess advantages over pan-selective Aurora inhibitors, by avoiding Aurora B mediated neutropenia. However, sub-type selective Aurora kinase A inhibitor design is very challenging due to the similarity in the active site among the isoforms. Structural biology and computational aspects pertaining to the design of Aurora kinase inhibitors were analyzed and found that a possible means to develop sub-type selective inhibitor is by targeting Aurora A specific residues (Leu215, Thr217 and Arg220) or Aurora B specific residues (Arg159, Glu161 and Lys164), near the solvent exposed region of the protein. Particularly, a useful strategy for the design of sub-type selective Aurora A inhibitor could be by targeting Thr217 residue as in the case of MLN8054. Further preclinical and clinical studies with the sub-type selective Aurora inhibitors could help bring them to the market for the treatment of cancer.

  20. Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors.

    PubMed

    Brasca, Maria Gabriella; Nesi, Marcella; Avanzi, Nilla; Ballinari, Dario; Bandiera, Tiziano; Bertrand, Jay; Bindi, Simona; Canevari, Giulia; Carenzi, Davide; Casero, Daniele; Ceriani, Lucio; Ciomei, Marina; Cirla, Alessandra; Colombo, Maristella; Cribioli, Sabrina; Cristiani, Cinzia; Della Vedova, Franco; Fachin, Gabriele; Fasolini, Marina; Felder, Eduard R; Galvani, Arturo; Isacchi, Antonella; Mirizzi, Danilo; Motto, Ilaria; Panzeri, Achille; Pesenti, Enrico; Vianello, Paola; Gnocchi, Paola; Donati, Daniele

    2014-09-01

    We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.

  1. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

    SciTech Connect

    Zhou, Wenjun; Ercan, Dalia; Chen, Liang; Yun, Cai-Hong; Li, Danan; Capelletti, Marzia; Cortot, Alexis B.; Chirieac, Lucian; Iacob, Roxana E.; Padera, Robert; Engen, John R.; Wong, Kwok-Kin; Eck, Michael J.; Gray, Nathanael S.; Jänne, Pasi A.

    2010-01-12

    The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.

  2. Progesterone reverts LPS-reduced FAAH activity in murine peripheral blood mononuclear cells by a receptor-mediated fashion.

    PubMed

    Wolfson, Manuel L; Aisemberg, Julieta; Salazar, Ana I; Domínguez Rubio, Ana P; Vercelli, Claudia A; Franchi, Ana M

    2013-12-05

    Increased anandamide concentrations are associated with pregnancy failure. Anandamide levels are regulated by the fatty acid amide hydrolase (FAAH). The aim of the study was to investigate the role of progesterone (P) on FAAH modulation in murine peripheral blood mononuclear cells (PBMC) under septic conditions. We observed that in vivo administration of LPS to non-pregnant (NP) mice decreased FAAH activity of PBMC while in pregnant mice no changes in FAAH activity were observed. NP animals administered with P had a similar response to LPS as the pregnant animals. Also, NP mice injected with P antagonist and P showed that the effect of P on LPS-reduced FAAH activity was impaired. Furthermore, LPS produced a decrease in the ratio of PR-B/PR-A in NP animals. Our results showed that, in our model the endotoxin decreased PBMC's FAAH activity and this condition was reverted by P in a receptor-mediated fashion.

  3. Biochemical And Structural Evaluation of Highly Selective 2-Arylbenzoxazole-Based Transthyretin Amyloidogenesis Inhibitors

    SciTech Connect

    Johnson, S.M.; Connelly, S.; Wilson, I.A.; Kelly, J.W.

    2009-05-18

    To develop potent transthyretin (TTR) amyloidogenesis inhibitors that also display high binding selectivity in blood, it proves useful to systematically optimize each of the three substructural elements that comprise a typical inhibitor: the two aryl rings and the linker joining them. In the first study, described herein, structural modifications to one aryl ring were evaluated by screening a library of 2-arylbenzoxazoles bearing thyroid hormone-like aryl substituents on the 2-aryl ring. Several potent and highly selective amyloidogenesis inhibitors were identified that exhibit minimal thyroid hormone nuclear receptor and COX-1 binding. High resolution crystal structures (1.3-1.5 A) of three inhibitors (2f, 4f, and 4d) in complex with TTR were obtained to characterize their binding orientation. Collectively, the results demonstrate that thyroid hormone-like substitution patterns on one aryl ring lead to potent and highly selective TTR amyloidogenesis inhibitors that lack undesirable thyroid hormone receptor or COX-1 binding.

  4. Chemical parsing: Dissecting cell dependencies with a toolkit of selective BCL-2 family inhibitors.

    PubMed

    Leverson, Joel D

    2016-01-01

    The BCL-2/BCL-XL inhibitor navitoclax has shown promise for the treatment of cancer but on-target toxicities have limited its utility. Recently, the generation of selective BCL-2 family inhibitors has enabled a careful dissection of BCL-2 biology, and early work indicates that these molecules have improved therapeutic profiles for the treatment of cancer.

  5. Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors.

    PubMed

    Golkowski, Martin; Vidadala, Rama Subba Rao; Lombard, Chloe K; Suh, Hyong Won; Maly, Dustin J; Ong, Shao-En

    2017-03-03

    ATP-competitive protein kinase inhibitors are important research tools and therapeutic agents. Because there are >500 human kinases that contain highly conserved active sites, the development of selective inhibitors is extremely challenging. Methods to rapidly and efficiently profile kinase inhibitor targets in cell lysates are urgently needed to discover selective compounds and to elucidate the mechanisms of action for polypharmacological inhibitors. Here, we describe a protocol for microgram-scale chemoproteomic profiling of ATP-competitive kinase inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography-mass spectrometry to profile ∼200 kinases in single analytical runs using as little as 5 μL of kinobeads and 300 μg of protein. With our kinobead reagents, we obtained broad coverage of the kinome, monitoring the relative expression levels of 312 kinases in a diverse panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors in competition-binding experiments with label-free quantification, leading to the discovery of a novel selective and potent inhibitor of protein kinase D (PKD) 1, 2, and 3. Our protocol is useful for rapid and sensitive profiling of kinase expression levels and ATP-competitive kinase inhibitor selectivity in native proteomes.

  6. Potent and selective inhibitors of Helicobacter pylori glutamate racemase (MurI): pyridodiazepine amines.

    PubMed

    Geng, Bolin; Basarab, Gregory; Comita-Prevoir, Janelle; Gowravaram, Madhusudhan; Hill, Pamela; Kiely, Andrew; Loch, James; MacPherson, Lawrence; Morningstar, Marshall; Mullen, George; Osimboni, Ekundayo; Satz, Alexander; Eyermann, Charles; Lundqvist, Tomas

    2009-02-01

    An SAR study of an HTS screening hit generated a series of pyridodiazepine amines as potent inhibitors of Helicobacter pylori glutamate racemase (MurI) showing highly selective anti-H. pylori activity, marked improved solubility, and reduced plasma protein binding. X-ray co-crystal E-I structures were obtained. These uncompetitive inhibitors bind at the MurI dimer interface.

  7. ERK Signal Suppression and Sensitivity to CH5183284/Debio 1347, a Selective FGFR Inhibitor.

    PubMed

    Nakanishi, Yoshito; Mizuno, Hideaki; Sase, Hitoshi; Fujii, Toshihiko; Sakata, Kiyoaki; Akiyama, Nukinori; Aoki, Yuko; Aoki, Masahiro; Ishii, Nobuya

    2015-12-01

    Drugs that target specific gene alterations have proven beneficial in the treatment of cancer. Because cancer cells have multiple resistance mechanisms, it is important to understand the downstream pathways of the target genes and monitor the pharmacodynamic markers associated with therapeutic efficacy. We performed a transcriptome analysis to characterize the response of various cancer cell lines to a selective fibroblast growth factor receptor (FGFR) inhibitor (CH5183284/Debio 1347), a mitogen-activated protein kinase kinase (MEK) inhibitor, or a phosphoinositide 3-kinase (PI3K) inhibitor. FGFR and MEK inhibition produced similar expression patterns, and the extracellular signal-regulated kinase (ERK) gene signature was altered in several FGFR inhibitor-sensitive cell lines. Consistent with these findings, CH5183284/Debio 1347 suppressed phospho-ERK in every tested FGFR inhibitor-sensitive cell line. Because the mitogen-activated protein kinase (MAPK) pathway functions downstream of FGFR, we searched for a pharmacodynamic marker of FGFR inhibitor efficacy in a collection of cell lines with the ERK signature and identified dual-specificity phosphatase 6 (DUSP6) as a candidate marker. Although a MEK inhibitor suppressed the MAPK pathway, most FGFR inhibitor-sensitive cell lines are insensitive to MEK inhibitors and we found potent feedback activation of several pathways via FGFR. We therefore suggest that FGFR inhibitors exert their effect by suppressing ERK signaling without feedback activation. In addition, DUSP6 may be a pharmacodynamic marker of FGFR inhibitor efficacy in FGFR-addicted cancers.

  8. Mechanism-Based Inhibitors of Serine Proteases with High Selectivity Through Optimization of S’ Subsite Binding

    PubMed Central

    Li, Yi; Dou, Dengfeng; He, Guijia; Lushington, Gerald H.; Groutas, William C.

    2009-01-01

    A series of mechanism-based inhibitors designed to interact with the S’ subsites of serine proteases was synthesized and their inhibitory activity toward the closely-related serine proteases human neutrophil elastase (HNE) and proteinase 3 (PR 3) was investigated. The compounds were found to be time-dependent inhibitors of HNE and were devoid of any inhibitory activity toward PR 3. The results suggest that highly selective inhibitors of serine proteases whose primary substrate specificity and active sites are similar can be identified by exploiting differences in their S’ subsites. The best inhibitor (compound 16) had a kinact/KI value of 4580 M−1 s−1. PMID:19394830

  9. Selective Serotonin Reuptake Inhibitors and Violent Crime: A Cohort Study

    PubMed Central

    Molero, Yasmina; Lichtenstein, Paul; Zetterqvist, Johan; Gumpert, Clara Hellner; Fazel, Seena

    2015-01-01

    Background Although selective serotonin reuptake inhibitors (SSRIs) are widely prescribed, associations with violence are uncertain. Methods and Findings From Swedish national registers we extracted information on 856,493 individuals who were prescribed SSRIs, and subsequent violent crimes during 2006 through 2009. We used stratified Cox regression analyses to compare the rate of violent crime while individuals were prescribed these medications with the rate in the same individuals while not receiving medication. Adjustments were made for other psychotropic medications. Information on all medications was extracted from the Swedish Prescribed Drug Register, with complete national data on all dispensed medications. Information on violent crime convictions was extracted from the Swedish national crime register. Using within-individual models, there was an overall association between SSRIs and violent crime convictions (hazard ratio [HR] = 1.19, 95% CI 1.08–1.32, p < 0.001, absolute risk = 1.0%). With age stratification, there was a significant association between SSRIs and violent crime convictions for individuals aged 15 to 24 y (HR = 1.43, 95% CI 1.19–1.73, p < 0.001, absolute risk = 3.0%). However, there were no significant associations in those aged 25–34 y (HR = 1.20, 95% CI 0.95–1.52, p = 0.125, absolute risk = 1.6%), in those aged 35–44 y (HR = 1.06, 95% CI 0.83–1.35, p = 0.666, absolute risk = 1.2%), or in those aged 45 y or older (HR = 1.07, 95% CI 0.84–1.35, p = 0.594, absolute risk = 0.3%). Associations in those aged 15 to 24 y were also found for violent crime arrests with preliminary investigations (HR = 1.28, 95% CI 1.16–1.41, p < 0.001), non-violent crime convictions (HR = 1.22, 95% CI 1.10–1.34, p < 0.001), non-violent crime arrests (HR = 1.13, 95% CI 1.07–1.20, p < 0.001), non-fatal injuries from accidents (HR = 1.29, 95% CI 1.22–1.36, p < 0.001), and emergency inpatient or outpatient treatment for alcohol intoxication or misuse

  10. Novel Associations between FAAH Genetic Variants and Postoperative Central Opioid related Adverse Effects

    PubMed Central

    Sadhasivam, Senthilkumar; Zhang, Xue; Chidambaran, Vidya; Mavi, Jagroop; Pilipenko, Valentina; Mersha, Tesfaye B.; Meller, Jaroslaw; Kaufman, Kenneth M.; Martin, Lisa J.; McAuliffe, John

    2014-01-01

    Opioid effects are potentiated by cannabinoid agonists including anandamide, an endocannabinoid. Inter-individual variability in responses to opioids is a major clinical problem. Multiple deaths and anoxic brain injuries occur every year in due to opioid induced respiratory depression in surgical patients and drug abusers of opioids and cannabinoids. This study aimed to determine specific associations between genetic variants of fatty acid amide hydrolase (FAAH) and postoperative central opioid adverse effects in children undergoing tonsillectomy. This is a prospective genotype blinded observational study 259 healthy children between 6 and 15 years that received standard perioperative care with a standard anesthetic and an intraoperative dose of morphine were enrolled. Associations between frequent polymorphisms of FAAH and central postoperative opioid adverse effects including, respiratory depression (RD), postoperative nausea and vomiting (PONV) and prolonged stay in Post Anesthesia Recovery Room (PACU) due to RD and PONV were analyzed. Five specific FAAH SNPs had significant associations with more than 2 fold increased risk for refractory PONV (adjusted p<0.0018), and nominal associations (p<0.05) with RD and prolonged PACU stay in white children undergoing tonsillectomy. FAAH SNP, rs324420 is a missense mutation with altered FAAH function and it is linked with other FAAH SNPs associated with PONV and RD in our cohort; association between PONV and rs324420 was confirmed in our extended cohort with additional 66 white children. Specific FAAH polymorphisms are associated with refractory PONV, opioid-related respiratory depression, and prolonged PACU stay due to opioid adverse effects in white children undergoing tonsillectomy. PMID:25558980

  11. Novel associations between FAAH genetic variants and postoperative central opioid-related adverse effects.

    PubMed

    Sadhasivam, S; Zhang, X; Chidambaran, V; Mavi, J; Pilipenko, V; Mersha, T B; Meller, J; Kaufman, K M; Martin, L J; McAuliffe, J

    2015-10-01

    Opioid effects are potentiated by cannabinoid agonists including anandamide, an endocannabinoid. Inter-individual variability in responses to opioids is a major clinical problem. Multiple deaths and anoxic brain injuries occur every year because of opioid-induced respiratory depression (RD) in surgical patients and drug abusers of opioids and cannabinoids. This study aimed to determine specific associations between genetic variants of fatty acid amide hydrolase (FAAH) and postoperative central opioid adverse effects in children undergoing tonsillectomy. This is a prospective genotype-blinded observational study in which 259 healthy children between 6 and 15 years of age who received standard perioperative care with a standard anesthetic and an intraoperative dose of morphine were enrolled. Associations between frequent polymorphisms of FAAH and central postoperative opioid adverse effects including, RD, postoperative nausea and vomiting (PONV) and prolonged stay in Post Anesthesia Recovery Room (postoperative anesthesia care unit, PACU) due to RD and PONV were analyzed. Five specific FAAH single nucleotide polymorphisms (SNPs) had significant associations with more than twofold increased risk for refractory PONV (adjusted P<0.0018), and nominal associations (P<0.05) with RD and prolonged PACU stay in white children undergoing tonsillectomy. The FAAH SNP, rs324420, is a missense mutation with altered FAAH function and it is linked with other FAAH SNPs associated with PONV and RD in our cohort; association between PONV and rs324420 was confirmed in our extended cohort with additional 66 white children. Specific FAAH polymorphisms are associated with refractory PONV, opioid-related RD, and prolonged PACU stay due to opioid adverse effects in white children undergoing tonsillectomy.

  12. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth

    PubMed Central

    Bergman, Joel A.; Woan, Karrune; Perez-Villarroel, Patricio; Villagra, Alejandro; Sotomayor, Eduardo M.; Kozikowski, Alan P.

    2012-01-01

    The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet non-selective inhibitor. Structure activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ~600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6 selective inhibitors that possess antiproliferative effects against melanoma cells. PMID:23009203

  13. Evaluation of NHS Carbamates as a Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors

    PubMed Central

    2013-01-01

    Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) 2-arachidonoylglycerol (2-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme’s function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. N-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate 1a (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors. PMID:23731016

  14. Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

    PubMed Central

    Finzel, B. C.; Baldwin, E. T.; Bryant, G. L.; Hess, G. F.; Wilks, J. W.; Trepod, C. M.; Mott, J. E.; Marshall, V. P.; Petzold, G. L.; Poorman, R. A.; O'Sullivan, T. J.; Schostarez, H. J.; Mitchell, M. A.

    1998-01-01

    The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity. PMID:9792098

  15. Hyperglycemia induces apoptosis via CB1 activation through the decrease of FAAH 1 in retinal pigment epithelial cells.

    PubMed

    Lim, Seul Ki; Park, Min Jung; Lim, Jae Cheong; Kim, Jong Choon; Han, Ho Jae; Kim, Gye-Yeop; Cravatt, Benjamin F; Woo, Chang Hoon; Ma, Seung Jin; Yoon, Kyung Cheol; Park, Soo Hyun

    2012-02-01

    Fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the main endocannabinoid, anandamide, and related fatty acid amides, has emerged as a regulator of endocannabinoid signaling. Retinal pigment epithelial (RPE) cells are believed to be important cells in the pathogenesis of diabetic retinopathy. However, the pathophysiology of FAAH in diabetic retinopathy has not been determined. Thus, we examined the effect of high glucose (HG) on the expression of FAAH and CB(1)R in the ARPE-19 human RPE cells. We found that HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, it upregulated the expression of CB(1)R mRNA and protein. HG-induced internalization of CB(1)R in HEK 293 cells and ARPE-19 cells was blocked by overexpression of FAAH 1 and treatment with the CB(1)R blocker, AM 251. HG-induced generation of reactive oxygen species and lipid peroxide formation were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced expression of CB(1)R in the cytosolic fraction. We also investigated whether the overexpression of FAAH 1 protected against HG-induced apoptosis. High glucose increased the Bax/Bcl-2 ratio and levels of cleaved PARP, cleaved caspase-9 and caspase-3, and reduced cell viability. HG-induced apoptotic effects were reduced by the overexpression of FAAH 1, treatment with the CB(1)R-specific antagonist AM 251 and CB(1)R siRNA transfection. In conclusion, HG-induced apoptosis in ARPE-19 cells by inducing CB(1)R expression through the downregulation of FAAH 1 expression. Our results provide evidence that CB(1)R blockade through the recovery of FAAH 1 expression may be a potential anti-diabetic therapy for the treatment of diabetic retinopathy.

  16. Selective inhibitors of the Janus kinase Jak3--Are they effective?

    PubMed

    Thoma, Gebhard; Drückes, Peter; Zerwes, Hans-Günter

    2014-10-01

    Jak3, together with Jak1, is involved in signal transduction initiated by cytokines signaling through the common gamma chain which are important in immune homeostasis and immune pathologies. Based on genetic evidence Jak3 has been considered to be an attractive target for immunosuppression. The Jak inhibitor tofacitinib (CP-690,550) which is an approved drug for rheumatoid arthritis was originally introduced as a selective Jak3 inhibitor, however, it also inhibits Jak1 and Jak2. The search for new selective Jak3 inhibitors has yielded several compounds whose profiles will be reviewed here. Implications on Jak3 as a therapeutic target are also discussed.

  17. Palladium-Catalyzed Arylation of Carbasugars Enables the Discovery of Potent and Selective SGLT2 Inhibitors.

    PubMed

    Ng, Wai-Lung; Lau, Kit-Man; Lau, Clara B-S; Shing, Tony K M

    2016-10-24

    Selective inhibition of the transporter protein sodium-glucose cotransporter 2 (SGLT2) has emerged as a promising way to control blood glucose level in diabetes patients. Reported herein is a short and convergent synthetic route towards some small-molecule SGLT2 inhibitors by a chemo- and diastereospecific palladium-catalyzed arylation reaction. This synthetic strategy enabled the discovery of two highly selective and potent SGLT2 inhibitors, thereby paving the way towards the development of carbasugar SGLT2 inhibitors as potential antidiabetic/antitumor agents.

  18. Discovery of GSK2656157: An Optimized PERK Inhibitor Selected for Preclinical Development.

    PubMed

    Axten, Jeffrey M; Romeril, Stuart P; Shu, Arthur; Ralph, Jeffrey; Medina, Jesús R; Feng, Yanhong; Li, William Hoi Hong; Grant, Seth W; Heerding, Dirk A; Minthorn, Elisabeth; Mencken, Thomas; Gaul, Nathan; Goetz, Aaron; Stanley, Thomas; Hassell, Annie M; Gampe, Robert T; Atkins, Charity; Kumar, Rakesh

    2013-10-10

    We recently reported the discovery of GSK2606414 (1), a selective first in class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), which inhibited PERK activation in cells and demonstrated tumor growth inhibition in a human tumor xenograft in mice. In continuation of our drug discovery program, we applied a strategy to decrease inhibitor lipophilicity as a means to improve physical properties and pharmacokinetics. This report describes our medicinal chemistry optimization culminating in the discovery of the PERK inhibitor GSK2656157 (6), which was selected for advancement to preclinical development.

  19. Use of selective-serotonin reuptake inhibitors and platelet aggregation inhibitors among individuals with co-occurring atherosclerotic cardiovascular disease and depression or anxiety

    PubMed Central

    Thornton, J Douglas; Agarwal, Parul; Sambamoorthi, Usha

    2016-01-01

    Objective: Medications commonly used to treat heart disease, anxiety, and depression can interact resulting in an increased risk of bleeding, warranting a cautious approach in medical decision making. This retrospective, descriptive study examined the prevalence and the factors associated with the use of both selective-serotonin reuptake inhibitor and platelet aggregation inhibitor among individuals with co-occurring atherosclerotic cardiovascular disease and anxiety or depression. Methods: Respondents aged 22 years and older, alive throughout the study period, and diagnosed with co-occurring atherosclerotic cardiovascular disease and anxiety or depression (n = 1507) in years 2007 through 2013 of the Medical Expenditures Panel Survey were included. The use of treatment was grouped as follows: selective-serotonin reuptake inhibitor and platelet aggregation inhibitor, selective-serotonin reuptake inhibitor or platelet aggregation inhibitor, and neither selective-serotonin reuptake inhibitor nor platelet aggregation inhibitor. Results: Overall, 16.5% used both selective-serotonin reuptake inhibitor and platelet aggregation inhibitor, 61.2% used selective-serotonin reuptake inhibitor or platelet aggregation inhibitor, and 22.3% used neither selective-serotonin reuptake inhibitor nor platelet aggregation inhibitor. Respondents aged over 65 years (adjusted odds ratio = 1.93 (95% confidence interval = 1.08–3.45)) and having a diagnosis of diabetes (adjusted odds ratio = 1.63 (95% confidence interval = 1.15–2.31)) and hypertension (adjusted odds ratio = 1.84 (95% confidence interval = 1.04–3.27)) were more likely to be prescribed the combination. Conclusion: The drug interaction was prevalent in patients who are already at higher risk of health disparities and worse outcomes thus requiring vigilant evaluation. PMID:28348738

  20. Selective and potent furin inhibitors protect cells from anthrax without significant toxicity.

    PubMed

    Remacle, Albert G; Gawlik, Katarzyna; Golubkov, Vladislav S; Cadwell, Gregory W; Liddington, Robert C; Cieplak, Piotr; Millis, Sherri Z; Desjardins, Roxane; Routhier, Sophie; Yuan, Xue Wen; Neugebauer, Witold A; Day, Robert; Strongin, Alex Y

    2010-06-01

    Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions.

  1. Structural Basis for Binding and Selectivity of Antimalarial and Anticancer Ethylenediamine Inhibitors to Protein Farnesyltransferase

    SciTech Connect

    Hast, Michael A.; Fletcher, Steven; Cummings, Christopher G.; Pusateri, Erin E.; Blaskovich, Michelle A.; Rivas, Kasey; Gelb, Michael H.; Voorhis, Wesley C.Van; Sebti, Said M.; Hamilton, Andrew D.; Beese, Lorena S. ); ); ); )

    2009-03-20

    Protein farnesyltransferase (FTase) catalyzes an essential posttranslational lipid modification of more than 60 proteins involved in intracellular signal transduction networks. FTase inhibitors have emerged as a significant target for development of anticancer therapeutics and, more recently, for the treatment of parasitic diseases caused by protozoan pathogens, including malaria (Plasmodium falciparum). We present the X-ray crystallographic structures of complexes of mammalian FTase with five inhibitors based on an ethylenediamine scaffold, two of which exhibit over 1000-fold selective inhibition of P. falciparum FTase. These structures reveal the dominant determinants in both the inhibitor and enzyme that control binding and selectivity. Comparison to a homology model constructed for the P. falciparum FTase suggests opportunities for further improving selectivity of a new generation of antimalarial inhibitors.

  2. Ligand-protein interactions of selective casein kinase 1δ inhibitors.

    PubMed

    Mente, Scot; Arnold, Eric; Butler, Todd; Chakrapani, Subramanyam; Chandrasekaran, Ramalakshmi; Cherry, Kevin; DiRico, Ken; Doran, Angela; Fisher, Katherine; Galatsis, Paul; Green, Michael; Hayward, Matthew; Humphrey, John; Knafels, John; Li, Jianke; Liu, Shenping; Marconi, Michael; McDonald, Scott; Ohren, Jeff; Paradis, Vanessa; Sneed, Blossom; Walton, Kevin; Wager, Travis

    2013-09-12

    Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.

  3. In search of potent and selective inhibitors of neuronal nitric oxide synthase with more simple structures

    PubMed Central

    Jing, Qing; Li, Huiying; Fang, Jianguo; Roman, Linda J.; Martásek, Pavel; Poulos, Thomas L.; Silverman, Richard B.

    2013-01-01

    In certain neurodegenerative diseases damaging levels of nitric oxide (NO) are produced by neuronal nitric oxide synthase (nNOS). It, therefore, is important to develop inhibitors selective for nNOS that do not interfere with other NOS isoforms, especially endothelial NOS (eNOS), which is critical for proper functioning of the cardiovascular system. While we have been successful in developing potent and isoform-selective inhibitors, such as lead compounds 1 and 2, the ease of synthesis and bioavailability have been problematic. Here we describe a new series of compounds including crystal structures of NOS-inhibitor complexes that integrate the advantages of easy synthesis and good biological properties compared to the lead compounds. These results provide the basis for additional structure–activity relationship (SAR) studies to guide further improvement of isozyme selective inhibitors. PMID:23867386

  4. A Rational Approach for the Identification of Non-Hydroxamate HDAC6-Selective Inhibitors

    PubMed Central

    Goracci, Laura; Deschamps, Nathalie; Randazzo, Giuseppe Marco; Petit, Charlotte; Dos Santos Passos, Carolina; Carrupt, Pierre-Alain; Simões-Pires, Claudia; Nurisso, Alessandra

    2016-01-01

    The human histone deacetylase isoform 6 (HDAC6) has been demonstrated to play a major role in cell motility and aggresome formation, being interesting for the treatment of multiple tumour types and neurodegenerative conditions. Currently, most HDAC inhibitors in preclinical or clinical evaluations are non-selective inhibitors, characterised by a hydroxamate zinc-binding group (ZBG) showing off-target effects and mutagenicity. The identification of selective HDAC6 inhibitors with novel chemical properties has not been successful yet, also because of the absence of crystallographic information that makes the rational design of HDAC6 selective inhibitors difficult. Using HDAC inhibitory data retrieved from the ChEMBL database and ligand-based computational strategies, we identified 8 original new non-hydroxamate HDAC6 inhibitors from the SPECS database, with activity in the low μM range. The most potent and selective compound, bearing a hydrazide ZBG, was shown to increase tubulin acetylation in human cells. No effects on histone H4 acetylation were observed. To the best of our knowledge, this is the first report of an HDAC6 selective inhibitor bearing a hydrazide ZBG. Its capability to passively cross the blood-brain barrier (BBB), as observed through PAMPA assays, and its low cytotoxicity in vitro, suggested its potential for drug development. PMID:27404291

  5. A Rational Approach for the Identification of Non-Hydroxamate HDAC6-Selective Inhibitors

    NASA Astrophysics Data System (ADS)

    Goracci, Laura; Deschamps, Nathalie; Randazzo, Giuseppe Marco; Petit, Charlotte; Dos Santos Passos, Carolina; Carrupt, Pierre-Alain; Simões-Pires, Claudia; Nurisso, Alessandra

    2016-07-01

    The human histone deacetylase isoform 6 (HDAC6) has been demonstrated to play a major role in cell motility and aggresome formation, being interesting for the treatment of multiple tumour types and neurodegenerative conditions. Currently, most HDAC inhibitors in preclinical or clinical evaluations are non-selective inhibitors, characterised by a hydroxamate zinc-binding group (ZBG) showing off-target effects and mutagenicity. The identification of selective HDAC6 inhibitors with novel chemical properties has not been successful yet, also because of the absence of crystallographic information that makes the rational design of HDAC6 selective inhibitors difficult. Using HDAC inhibitory data retrieved from the ChEMBL database and ligand-based computational strategies, we identified 8 original new non-hydroxamate HDAC6 inhibitors from the SPECS database, with activity in the low μM range. The most potent and selective compound, bearing a hydrazide ZBG, was shown to increase tubulin acetylation in human cells. No effects on histone H4 acetylation were observed. To the best of our knowledge, this is the first report of an HDAC6 selective inhibitor bearing a hydrazide ZBG. Its capability to passively cross the blood-brain barrier (BBB), as observed through PAMPA assays, and its low cytotoxicity in vitro, suggested its potential for drug development.

  6. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth.

    PubMed

    Bergman, Joel A; Woan, Karrune; Perez-Villarroel, Patricio; Villagra, Alejandro; Sotomayor, Eduardo M; Kozikowski, Alan P

    2012-11-26

    The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet nonselective inhibitor. Structure-activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5 g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ∼600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.

  7. High-throughput screening to identify selective inhibitors of microbial sulfate reduction (and beyond)

    NASA Astrophysics Data System (ADS)

    Carlson, H. K.; Coates, J. D.; Deutschbauer, A. M.

    2015-12-01

    The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive and corrosive. Current strategies to selectively inhibit sulfidogenesis are based on non-specific biocide treatments, bio-competitive exclusion by alternative electron acceptors or sulfate-analogs which are competitive inhibitors or futile/alternative substrates of the sulfate reduction pathway. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to target SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Once inhibitor selectivity is defined, high-throughput characterization of microbial community structure across compound gradients and identification of fitness determinants using isolate bar-coded transposon mutant libraries can give insights into the genetic mechanisms whereby compounds structure microbial communities. The high-throughput (HT) approach we present can be readily applied to target SRM in diverse environments and more broadly, could be used to identify and quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant for engineering environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

  8. Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease

    PubMed Central

    2010-01-01

    Introduction Inhibition of gamma-secretase presents a direct target for lowering Aβ production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. Methods In vitro assays monitoring inhibitor potencies at APP γ-site cleavage (equivalent to Aβ40), and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aβ production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aβ was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aβ reduction vs. Notch signaling endpoints in periphery. Results The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aβ production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aβ in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aβ was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post

  9. Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance.

    PubMed

    Mhlongo, Sizwe I; den Haan, Riaan; Viljoen-Bloom, Marinda; van Zyl, Willem H

    2015-12-01

    In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme-inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.

  10. The faah gene is the first direct target of estrogen in the testis: role of histone demethylase LSD1.

    PubMed

    Grimaldi, Paola; Pucci, Mariangela; Di Siena, Sara; Di Giacomo, Daniele; Pirazzi, Valentina; Geremia, Raffaele; Maccarrone, Mauro

    2012-12-01

    Estrogen (E(2)) regulates spermatogenesis, yet its direct target genes have not been identified in the testis. Here, we cloned the proximal 5' flanking region of the mouse fatty acid amide hydrolase (faah) gene upstream of the luciferase reporter gene, and demonstrated its promoter activity and E(2) inducibility in primary mouse Sertoli cells. Specific mutations in the E(2) response elements (ERE) of the faah gene showed that two proximal ERE sequences (ERE2/3) are essential for E(2)-induced transcription, and chromatin immunoprecipitation experiments showed that E(2) induced estrogen receptor β binding at ERE2/3 sites in the faah promoter in vivo. Moreover, the histone demethylase LSD1 was found to be associated with ERE2/3 sites and to play a role in mediating E(2) induction of FAAH expression. E(2) induced epigenetic modifications at the faah proximal promoter compatible with transcriptional activation by remarkably decreasing methylation of both DNA at CpG site and histone H3 at lysine 9. Finally, FAAH silencing abolished E(2) protection against apoptosis induced by the FAAH substrate anandamide. Taken together, our results identify FAAH as the first direct target of E(2).

  11. Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement

    PubMed Central

    Altenhöfer, Sebastian; Radermacher, Kim A.; Kleikers, Pamela W.M.; Wingler, Kirstin

    2015-01-01

    Abstract Significance: Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Recent Advances: Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Critical Issues: Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. Future Directions: The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition. Antioxid. Redox Signal. 23, 406–427. PMID:24383718

  12. Zaprinast, a phosphodiesterase 5 inhibitor, overcomes sexual dysfunction produced by fluoxetine, a selective serotonin reuptake inhibitor in hamsters.

    PubMed

    Frye, Cheryl A; Rhodes, Madeline E

    2003-02-01

    A high incidence of sexual dysfunction among women is reported in the clinical literature. Little experimental investigation has been initiated on the ability of phosphodiesterase (PDE) inhibitors to overcome deficits in sexual functioning because of selective serotonin reuptake inhibitors (SSRIs). The effects of fluoxetine, an SSRI, and zaprinast, a PDE-5 inhibitor, on the lateral displacement response (used as a measure of sensitivity to reproductively relevant stimuli) of hamsters in behavioral estrus were investigated. In Experiment 1, hamsters that were maximally sensitive to reproductively relevant stimuli because they were at the peak of behavioral estrus were administered fluoxetine (10 mg/kg, i.p.); they had significantly decreased lateral displacement responses compared to vehicle-administered hamsters. In Experiment 2, hamsters that were relatively less sensitive to sexual stimuli because they were at the termination of behavioral estrus were administered zaprinast (3 mg/kg; i.p.); they had significantly enhanced lateral displacement responses compared to responses seen following vehicle administration. In Experiment 3, fluoxetine-induced deficits in the lateral displacement of hamsters at the peak of behavioral estrus were overcome by the coadministration of zaprinast. These data confirm previous findings that sexual dysfunction can be induced by SSRIs and extend the current knowledge to suggest that administration of a PDE-5 inhibitor can override SSRI-induced deficits in sexual functioning.

  13. Novel, selective CDK9 inhibitors for the treatment of HIV infection.

    PubMed

    Németh, G; Varga, Z; Greff, Z; Bencze, G; Sipos, A; Szántai-Kis, C; Baska, F; Gyuris, A; Kelemenics, K; Szathmáry, Z; Minárovits, J; Kéri, G; Orfi, L

    2011-01-01

    Cyclin Dependent Kinases (CDKs) are important regulators of cell cycle and gene expression. Since an up-to-date review about the pharmacological inhibitors of CDK family (CDK1-10) is not available; therefore in the present paper we briefly summarize the most relevant inhibitors and point out the low number of selective inhibitors. Among CDKs, CDK9 is a validated pathological target in HIV infection, inflammation and cardiac hypertrophy; however selective CDK9 inhibitors are still not available. We present a selective inhibitor family of CDK9 based on the 4-phenylamino-6- phenylpyrimidine nucleus. We show a convenient synthetic method to prepare a useful intermediate and its derivatisation resulting in novel compounds. The CDK9 inhibitory activity of the derivatives was measured in specific kinase assay and the CDK inhibitory profile of the best ones (IC(50) < 100 nM) was determined. The most selective compounds had high selectivity over CDK1, 2, 3, 5, 6, 7 and showed at least one order of magnitude higher inhibitory activity over CDK4 inhibition. The most selective molecules were examined in cytotoxicity assays and their ability to inhibit HIV-1 replication was determined in cellular assays.

  14. Identification of a suitable and selective inhibitor towards aldehyde oxidase catalyzed reactions.

    PubMed

    Nirogi, Ramakrishna; Kandikere, Vishwottam; Palacharla, Raghava Choudary; Bhyrapuneni, Gopinadh; Kanamarlapudi, Vijaya Bhargava; Ponnamaneni, Ranjith Kumar; Manoharan, Arun Kumar

    2014-03-01

    1. Aldehyde oxidase (AO) is a liver cytosolic molybdoflavoprotein enzyme whose importance in drug metabolism is gaining in the recent. The objective of this work is to find a potent and selective inhibitor for AO activity using phthalazine oxidation as a marker reaction. 2. Among organic solvents tested, it was identified that methanol was not a suitable choice for AO activity even at concentrations less than 0.2% v/v. Acetonitrile and DMSO did not show any effect till 0.5% v/v but thereafter activites tend to decrease. 3. For selectivity, 23 compounds were selected and evaluated for their effects on AO and nine CYP450 enzymes. Among the tested compounds chlorpromazine, estradiol, hydralazine, quetiapine and raloxifene were selected based on their potency of inhibition towards AO activity. 4. Raloxifene was found to be a non-specific inhibitor of all major tested CYP450 enzymes and was excluded as a selective inhibitor for AO. Quetiapine also showed a degree of inhibition towards the major CYP450 tested. Hydralazine used as a specific inhibitor during the past for AO activity demonstrated a stimulation of AO activity at high and low concentrations respectively and the inhibition noted to be time dependent while inhibiting other enzymes like monoamine oxidase. 5. Estradiol showed no inhibition towards the tested CYP450 enzymes and thus proved to be a selective and specific inhibitor for AO activity with an uncompetitive mode of inhibition.

  15. Differential regulation of Bdnf expression in cortical neurons by class-selective histone deacetylase inhibitors.

    PubMed

    Koppel, Indrek; Timmusk, Tõnis

    2013-12-01

    Histone deactylase (HDAC) inhibitors show promise as therapeutics for neurodegenerative and psychiatric diseases. Increased expression of brain-derived neurotrophic factor (BDNF) has been associated with memory-enhancing and neuroprotective properties of these drugs, but the mechanism of BDNF induction is not well understood. Here, we compared the effects of a class I/IIb selective HDAC inhibitor SAHA, a class I selective inhibitor MS-275, a class II selective inhibitor MC1568 and a HDAC6 selective inhibitor tubacin on Bdnf mRNA expression in rat primary neurons. We show that inhibition of class II HDACs resulted in rapid upregulation of Bdnf mRNA levels, whereas class I HDAC inhibition produced a markedly delayed Bdnf induction. In contrast to relatively slow upregulation of Bdnf transcripts, histone acetylation at BDNF promoters I and IV was rapidly induced by SAHA. Bdnf induction by SAHA and MS-275 at 24 h was sensitive to protein synthesis inhibition, suggesting that delayed Bdnf induction by HDAC inhibitors is secondary to changed expression of its regulators. HDAC4 and HDAC5 repressed Bdnf promoter IV activity, supporting the role of class II HDACs in regulation of Bdnf expression. In addition, we show a critical role for the cAMP/Ca2+ response element (CRE) in induction of Bdnf promoter IV by MS-275, MC1568, SAHA and sodium valproate. In contrast, MEF2-binding CaRE1 element was not necessary for promoter IV induction by HDAC inhibition. Finally, we show that similarly to Bdnf, the studied HDAC inhibitors differentially induced expression of neuronal activity-regulated genes c-fos and Arc. Together, our findings implicate class II HDACs in transcriptional regulation of Bdnf and indicate that class II selective HDAC inhibitors may have potential as therapeutics for nervous system disorders.

  16. Tricyclic covalent inhibitors selectively target Jak3 through an active site thiol.

    PubMed

    Goedken, Eric R; Argiriadi, Maria A; Banach, David L; Fiamengo, Bryan A; Foley, Sage E; Frank, Kristine E; George, Jonathan S; Harris, Christopher M; Hobson, Adrian D; Ihle, David C; Marcotte, Douglas; Merta, Philip J; Michalak, Mark E; Murdock, Sara E; Tomlinson, Medha J; Voss, Jeffrey W

    2015-02-20

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.

  17. Selective COX-2 Inhibitors: A Review of Their Structure-Activity Relationships

    PubMed Central

    Zarghi, Afshin; Arfaei, Sara

    2011-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are the competitive inhibitors of cyclooxygenase (COX), the enzyme which mediates the bioconversion of arachidonic acid to inflammatory prostaglandins (PGs). Their use is associated with the side effects such as gastrointestinal and renal toxicity. The therapeutic anti-inflammatory action of NSAIDs is produced by the inhibition of COX-2, while the undesired side effects arise from inhibition of COX-1 activity. Thus, it was though that more selective COX-2 inhibitors would have reduced side effects. Based upon a number of selective COX-2 inhibitors (rofecoxib, celecoxib, valdecoxibetc.) were developed as safer NSAIDs with improved gastric safety profile. However, the recent market removal of some COXIBs such as rofecoxib due to its adverse cardiovascular side effects clearly encourages the researchers to explore and evaluate alternative templates with COX-2 inhibitory activity. Recognition of new avenues for selective COX-2 inhibitors in cancer chemotherapy and neurological diseases such as Parkinson and Alzheimer’s diseases still continues to attract investigations on the development of COX-2 inhibitors. This review highlights the various structural classes of selective COX-2 inhibitors with special emphasis on their structure-activity relationships. PMID:24250402

  18. Structures of the four subfamilies of phosphodiesterase-4 provide insight into the selectivity of their inhibitors.

    PubMed

    Wang, Huanchen; Peng, Ming-Sheng; Chen, Yi; Geng, Jie; Robinson, Howard; Houslay, Miles D; Cai, Jiwen; Ke, Hengming

    2007-12-01

    PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP {4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid} as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.

  19. Structures of the Four Subfamilies of Phosphodiesterase-4 Provide Insight into the Selectivity of Their Inhibitors

    SciTech Connect

    Wang, H.; Peng, M; Chen , Y; Geng, J; Robinson, H; Houslay , M; Cai, J; Ke, H

    2007-01-01

    PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP 4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.

  20. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  1. Selective Serotonin–norepinephrine Reuptake Inhibitors-induced Takotsubo Cardiomyopathy

    PubMed Central

    Vasudev, Rahul; Rampal, Upamanyu; Patel, Hiten; Patel, Kunal; Bikkina, Mahesh; Shamoon, Fayez

    2016-01-01

    Context: Takotsubo translates to “octopus pot” in Japanese. Takotsubo cardiomyopathy (TTC) is characterized by a transient regional systolic dysfunction of the left ventricle. Catecholamine excess is the one most studied and favored theories explaining the pathophysiology of TTC. Case Report: We present the case of a 52-year-old Hispanic female admitted for venlafaxine-induced TTC with a review literature on all the cases of Serotonin–norepinephrine reuptake inhibitors (SNRI)-associated TTC published so far. Conclusion: SNRI inhibit the reuptake of catecholamines into the presynaptic neuron, resulting in a net gain in the concentration of epinephrine and serotonin in the neuronal synapses and causing iatrogenic catecholamine excess, ultimately leading to TTC. PMID:27583240

  2. Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

    PubMed Central

    Gouveia-Figueira, Sandra; Karlsson, Jessica; Deplano, Alessandro; Hashemian, Sanaz; Svensson, Mona; Fredriksson Sundbom, Marcus; Congiu, Cenzo; Onnis, Valentina; Fowler, Christopher J.

    2015-01-01

    Background Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known. Methodology/Principal Findings COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM). Conclusions/Significance Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the

  3. Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT.

    PubMed

    Wang, Chao; Abegg, Daniel; Hoch, Dominic G; Adibekian, Alexander

    2016-02-18

    We present a novel chemical scaffold for cysteine-reactive covalent inhibitors. Chloromethyl triazoles (CMTs) are readily accessed in only two chemical steps, thus enabling the rapid optimization of the pharmacological properties of these inhibitors. We demonstrate the tunability of the CMTs towards a specific biological target by synthesizing AA-CW236 as the first potent non-pseudosubstrate inhibitor of the O(6) -alkylguanine DNA methyltransferase (MGMT), a protein of major clinical significance for the treatment of several severe cancer forms. Using quantitative proteomics profiling techniques, we show that AA-CW236 exhibits a high degree of selectivity towards MGMT. Finally, we validate the effectiveness of our MGMT inhibitor in combination with the DNA alkylating drug temozolomide in breast and colon cancer cells by fluorescence imaging and a cell-viability assay. Our results may open a new avenue towards the development of a clinically approved MGMT inhibitor.

  4. Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable γ-Secretase Inhibitor

    PubMed Central

    2010-01-01

    During the course of our research efforts to develop a potent and selective γ-secretase inhibitor for the treatment of Alzheimer's disease, we investigated a series of carboxamide-substituted sulfonamides. Optimization based on potency, Notch/amyloid-β precursor protein selectivity, and brain efficacy after oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent inhibitor of γ-secretase (Aβ40 IC50 = 0.30 nM), demonstrating a 193-fold selectivity against Notch. Oral administration of 4 significantly reduced Aβ40 levels for sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs. PMID:24900185

  5. Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor

    PubMed Central

    2015-01-01

    The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase. PMID:26191365

  6. Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT).

    PubMed

    Ding, Yun; O'Keefe, Heather; DeLorey, Jennifer L; Israel, David I; Messer, Jeffrey A; Chiu, Cynthia H; Skinner, Steven R; Matico, Rosalie E; Murray-Thompson, Monique F; Li, Fan; Clark, Matthew A; Cuozzo, John W; Arico-Muendel, Christopher; Morgan, Barry A

    2015-08-13

    The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure-activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality.

  7. Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT)

    PubMed Central

    2015-01-01

    The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure–activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality. PMID:26288689

  8. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    SciTech Connect

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G.

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  9. Usefulness of selective COX-2 inhibitors as therapeutic agents against canine mammary tumors.

    PubMed

    Saito, Teruyoshi; Tamura, Dai; Asano, Ryuji

    2014-04-01

    Cyclooxygenase-2 (COX-2) is a key enzyme for converting arachidonic acids to prostanoids, which are known to be induced during inflammation and cancer initiation. Previously, it has been reported that COX inhibitors, such as aspirin, reduce the incidence of human colorectal cancer; therefore, it is widely believed that COX-2 is a potential therapeutic and chemoprevention target for several types of human cancer. However, whether selective COX-2 inhibitors have antitumor effects against canine mammary tumor cells remains unclear. In the present study, to elucidate the antitumor effect of selective COX-2 inhibitors against canine mammary tumors, we investigated the antitumor effects of meloxicam, etodolac and celecoxib using COX-2-expressing canine mammary tumor (CF33) cells. We analyzed the effects of selective COX-2 inhibitors on COX-2 protein expression levels in CF33 cells. Celecoxib (100 µM) was found to induce downregulation of COX-2 protein expression. We examined the effect of selective COX-2 inhibitors on CF33 cell proliferation. All the selective COX-2 inhibitors suppressed CF33 cell growth. Specifically, etodolac and celecoxib inhibited cell proliferation via a decrease in S-phase cells and an increase in G0/G1 arrest. We examined the apoptotic effect of selective COX-2 inhibitors on CF33 cells. Our data suggested that etodolac and celecoxib induced apoptosis in CF33 cells. In particular, celecoxib led to apoptosis mediated by the activation of the mitochondrial apoptosis pathway, including the upregulation of BAX expression, downregulation of Bcl-2 expression and activation of caspase-3/7. Furthermore, celecoxib increased the percentages of cells in both early apoptosis and late apoptosis. Our results revealed that celecoxib induced apoptosis and cell cycle arrest in CF33 cells. The data suggested that celecoxib is the most viable candidate as a therapeutic agent for the treatment of canine mammary tumors. Furthermore, our findings provide the first

  10. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    PubMed Central

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy. PMID:26713267

  11. Discovery of GS-9973, a selective and orally efficacious inhibitor of spleen tyrosine kinase.

    PubMed

    Currie, Kevin S; Kropf, Jeffrey E; Lee, Tony; Blomgren, Peter; Xu, Jianjun; Zhao, Zhongdong; Gallion, Steve; Whitney, J Andrew; Maclin, Deborah; Lansdon, Eric B; Maciejewski, Patricia; Rossi, Ann Marie; Rong, Hong; Macaluso, Jennifer; Barbosa, James; Di Paolo, Julie A; Mitchell, Scott A

    2014-05-08

    Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

  12. Sulfated Pentagalloylglucoside is a Potent, Allosteric, and Selective Inhibitor of Factor XIa

    PubMed Central

    Al-Horani, Rami A.; Ponnusamy, Pooja; Mehta, Akul Y.; Gailani, David; Desai, Umesh R.

    2013-01-01

    Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor 6 reduced VMAX of FXIa hydrolysis of chromogenic substrate without affecting the KM suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance. PMID:23316863

  13. Selective inhibitors of Cyclin-G associated kinase (GAK) as anti-HCV agents

    PubMed Central

    Kovackova, Sona; Chang, Lei; Bekerman, Elena; Neveu, Gregory; Barouch-Bentov, Rina; Chaikuad, Apirat; Heroven, Christina; Šála, Michal; De Jonghe, Steven; Knapp, Stefan; Einav, Shirit; Herdewijn, Piet

    2015-01-01

    Cyclin-G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Co-crystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV lifecycle (i.e. viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer and Parkinson's disease). PMID:25822739

  14. Novel inhibitors of human leukocyte elastase and cathepsin G. Sequence variants of squash seed protease inhibitor with altered protease selectivity

    SciTech Connect

    McWherter, C.A.; Walkenhorst, W.F.; Glover, G.I. ); Campbell, E.J. )

    1989-07-11

    Novel peptide inhibitors of human leukocyte elastase (HLE) and cathepsin G (CG) were prepared by solid-phase peptide synthesis of P1 amino acid sequence variants of Curcurbita maxima trypsin inhibitor III (CMTI-III), a 29-residue peptide found in squash seed. A systematic study of P1 variants indicated that P1, Arg, Lys, Leu, Ala, Phe, and Met inhibit trypsin; P1, Val, Ile, Gly, Leu, Ala, Phe, and Met inhibit HLE; P1 Leu, Ala, Phe, and Met inhibit CG and chymotrypsin. Variants with P1, Val, Ile, or Gly were selective inhibitors of HLE, while inhibition of trypsin required P1 amino acids with an unbranched {beta} carbon. Studies of Val-5-CMTI-III (P1 Val) inhibition of HLE demonstrated a 1:1 binding stoichiometry with a (K{sub i}){sub app} of 8.7 nM. Inhibition of HLE by Gly-5-CMTI-III indicated a significant role for reactive-site structural moieties other than the P1 side chain. Val-5-CMTI-III inhibited both HLE and human polymorphonuclear leukocyte (PMN) proteolysis of surface-bound {sup 125}I-labeled fibronectin. Val-5-CMTI-III was more effective at preventing turnover of a peptide p-nitroanilide substrate than halting dissolution of {sup 125}I-labeled fibronectin. It was about as effective as human serum {alpha}{sub 1}-proteinase inhibitor in preventing PMN degradation of the connective tissue substrate. In addition to providing interesting candidates for controlling inflammatory cell proteolytic injury, the CMTI-based inhibitors are ideal for studying molecular recognition because of their small size, their ease of preparation, and the availability of sensitive and quantitative assays for intermolecular interactions.

  15. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    SciTech Connect

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G.

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  16. Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors

    PubMed Central

    Davare, Monika A.; Vellore, Nadeem A.; Wagner, Jacob P.; Eide, Christopher A.; Goodman, James R.; Drilon, Alexander; Deininger, Michael W.; O’Hare, Thomas; Druker, Brian J.

    2015-01-01

    Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib’s dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1G2032R mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure–function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies. PMID:26372962

  17. Natural and synthetic geiparvarins are strong and selective MAO-B inhibitors. Synthesis and SAR studies.

    PubMed

    Carotti, Angelo; Carrieri, Antonio; Chimichi, Stefano; Boccalini, Marco; Cosimelli, Barbara; Gnerre, Carmela; Carotti, Andrea; Carrupt, Pierre Alain; Testa, Bernard

    2002-12-16

    Natural geiparvarin 1 and a number of its analogues were prepared and tested as inhibitors of both monoamine oxidase isoforms, MAO-B and MAO-A. The desmethyl congener 6 of geiparvarin, proved potent and selective MAO-B inhibitor (pIC(50)=7.55 vs 4.62). X-ray crystallography and molecular modelling studies helped the understanding of the observed structure-activity relationships.

  18. Carbonic anhydrase II-induced selection of inhibitors from a dynamic combinatorial library of Schiff's bases.

    PubMed

    Nasr, Gihane; Petit, Eddy; Supuran, Claudiu T; Winum, Jean-Yves; Barboiu, Mihail

    2009-11-01

    A dynamic combinatorial library (DCL) has been generated under thermodynamic control by using the aminocarbonyl/imine interconversion as reversible chemistry, combined with non-covalent binding within the active site of the metalloenzyme human carbonic anhydrase II (hCA II, EC 4.2.1.1). The high affinity of hCA II isozyme towards some sulfonamide inhibitors obtained here was used to select from the dynamic library specific inhibitors of this isoform. These results point out to the possibility of identifying sulfonamide amplified compounds presenting potent inhibition and high yield of formation in the presence of the isoform(s) towards which the inhibitors were designed.

  19. Novel selective inhibitor of Leishmania (Leishmania) amazonensis arginase.

    PubMed

    da Silva, Edson R; Boechat, Nubia; Pinheiro, Luiz C S; Bastos, Monica M; Costa, Carolina C P; Bartholomeu, Juliana C; da Costa, Talita H

    2015-11-01

    Arginase is a glycosomal enzyme in Leishmania that is involved in polyamine and trypanothione biosynthesis. The central role of arginase in Leishmania (Leishmania) amazonensis was demonstrated by the generation of two mutants: one with an arginase lacking the glycosomal addressing signal and one in which the arginase-coding gene was knocked out. Both of these mutants exhibited decreased infectivity. Thus, arginase seems to be a potential drug target for Leishmania treatment. In an attempt to search for arginase inhibitors, 29 derivatives of the [1,2,4]triazolo[1,5-a]pyrimidine system were tested against Leishmania (Leishmania) amazonensis arginase in vitro. The [1,2,4]triazolo[1,5-a]pyrimidine scaffold containing R1  = CF3 exhibited greater activity against the arginase rather than when the substituent R1  = CH3 in the 2-position. The novel compound 2-(5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)hydrazinecarbothioamide (30) was the most potent, inhibiting arginase by a non-competitive mechanism, with the Ki and IC50 values for arginase inhibition estimated to be 17 ± 1 μm and 16.5 ± 0.5 μm, respectively. These results can guide the development of new drugs against leishmaniasis based on [1,2,4]triazolo[1,5-a]pyrimidine derivatives targeting the arginase enzyme.

  20. Drosotoxin, a selective inhibitor of tetrodotoxin-resistant sodium channels.

    PubMed

    Zhu, Shunyi; Gao, Bin; Deng, Meichun; Yuan, Yuzhe; Luo, Lan; Peigneur, Steve; Xiao, Yucheng; Liang, Songping; Tytgat, Jan

    2010-10-15

    The design of animal toxins with high target selectivity has long been a goal in protein engineering. Based on evolutionary relationship between the Drosophila antifungal defensin (drosomycin) and scorpion depressant Na(+) channel toxins, we exploited a strategy to create a novel chimeric molecule (named drosotoxin) with high selectivity for channel subtypes, which was achieved by using drosomycin to substitute the structural core of BmKITc, a depressant toxin acting on both insect and mammalian Na(+) channels. Recombinant drosotoxin selectively inhibited tetrodotoxin-resistant (TTX-R) Na(+) channels in rat dorsal root ganglion (DRG) neurons with a 50% inhibitory concentration (IC(50)) of 2.6+/-0.5muM. This chimeric peptide showed no activity on K(+), Ca(2+) and TTX-sensitive (TTX-S) Na(+) channels in rat DRG neurons and Drosophila para/tipE channels at micromolar concentrations. Drosotoxin represents the first chimeric toxin and example of a non-toxic core scaffold with high selectivity on mammalian TTX-R Na(+) channels.

  1. Structure-activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps.

    PubMed

    Sundermann, Tom; Hanekamp, Walburga; Lehr, Matthias

    2016-08-01

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA2α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA2α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA2α and FAAH, respectively.

  2. Synthesis and QSAR of Fatty Acid Amide Hydrolase Inhibitors: Modulation at the N-Portion of Biphenyl-3-yl Alkylcarbamates

    PubMed Central

    Mor, Marco; Lodola, Alessio; Rivara, Silvia; Vacondio, Federica; Duranti, Andrea; Tontini, Andrea; Sanchini, Silvano; Piersanti, Giovanni; Clapper, Jason R.; King, Alvin R.; Tarzia, Giorgio; Piomelli, Daniele

    2013-01-01

    Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (IC50, for FAAH = 63 nM) with a selected set of substituents of different size, shape, flexibility and lipophilicity. Docking experiments and Linear Interaction Energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the β-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3′-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors. PMID:18507372

  3. Selection of Protease Inhibitors to Prevent or Attenuate Inflammatory Processes

    DTIC Science & Technology

    2007-08-01

    activation of phospholipase CO3 and to the production of inositol 1,4,5-triphosphate, with consequent increase of intracellular calcium concentration [ Campos ...selective kinin BI-receptor antagonists would not produce undesirable side effects [ Campos et al., 2006]. The constitutive expression of B2-receptors on...infection [ Campos et al., 2006]. In a study done by Monteiro et al. (2006) an infection model (mouse model of subcutaneous infection by Trypanosoma cruzi

  4. Virtual screening of selective multitarget kinase inhibitors by combinatorial support vector machines.

    PubMed

    Ma, X H; Wang, R; Tan, C Y; Jiang, Y Y; Lu, T; Rao, H B; Li, X Y; Go, M L; Low, B C; Chen, Y Z

    2010-10-04

    Multitarget agents have been increasingly explored for enhancing efficacy and reducing countertarget activities and toxicities. Efficient virtual screening (VS) tools for searching selective multitarget agents are desired. Combinatorial support vector machines (C-SVM) were tested as VS tools for searching dual-inhibitors of 11 combinations of 9 anticancer kinase targets (EGFR, VEGFR, PDGFR, Src, FGFR, Lck, CDK1, CDK2, GSK3). C-SVM trained on 233-1,316 non-dual-inhibitors correctly identified 26.8%-57.3% (majority >36%) of the 56-230 intra-kinase-group dual-inhibitors (equivalent to the 50-70% yields of two independent individual target VS tools), and 12.2% of the 41 inter-kinase-group dual-inhibitors. C-SVM were fairly selective in misidentifying as dual-inhibitors 3.7%-48.1% (majority <20%) of the 233-1,316 non-dual-inhibitors of the same kinase pairs and 0.98%-4.77% of the 3,971-5,180 inhibitors of other kinases. C-SVM produced low false-hit rates in misidentifying as dual-inhibitors 1,746-4,817 (0.013%-0.036%) of the 13.56 M PubChem compounds, 12-175 (0.007%-0.104%) of the 168 K MDDR compounds, and 0-84 (0.0%-2.9%) of the 19,495-38,483 MDDR compounds similar to the known dual-inhibitors. C-SVM was compared to other VS methods Surflex-Dock, DOCK Blaster, kNN and PNN against the same sets of kinase inhibitors and the full set or subset of the 1.02 M Zinc clean-leads data set. C-SVM produced comparable dual-inhibitor yields, slightly better false-hit rates for kinase inhibitors, and significantly lower false-hit rates for the Zinc clean-leads data set. Combinatorial SVM showed promising potential for searching selective multitarget agents against intra-kinase-group kinases without explicit knowledge of multitarget agents.

  5. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors

    PubMed Central

    Ojo, Kayode K; Larson, Eric T; Keyloun, Katelyn R; Castaneda, Lisa J; DeRocher, Amy E; Inampudi, Krishna K; Kim, Jessica E; Arakaki, Tracy L; Murphy, Ryan C; Zhang, Li; Napuli, Alberto J; Maly, Dustin J; Verlinde, Christophe LMJ; Buckner, Frederick S; Parsons, Marilyn; Hol, Wim GJ; Merritt, Ethan A; Van Voorhis, Wesley C

    2010-01-01

    New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable. PMID:20436472

  6. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

    SciTech Connect

    Yu, Wenyu; Chory, Emma J.; Wernimont, Amy K.; Tempel, Wolfram; Scopton, Alex; Federation, Alexander; Marineau, Jason J.; Qi, Jun; Barsyte-Lovejoy, Dalia; Yi, Joanna; Marcellus, Richard; Iacob, Roxana E.; Engen, John R.; Griffin, Carly; Aman, Ahmed; Wienholds, Erno; Li, Fengling; Pineda, Javier; Estiu, Guillermina; Shatseva, Tatiana; Hajian, Taraneh; Al-awar, Rima; Dick, John E.; Vedadi, Masoud; Brown, Peter J.; Arrowsmith, Cheryl H.; Bradner, James E.; Schapira, Matthieu

    2012-12-18

    Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound toEPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

  7. Identification of Selective Nanomolar Inhibitors of the Human Neuraminidase, NEU4

    PubMed Central

    2013-01-01

    The human neuraminidase enzymes (hNEU) play important roles in human physiology and pathology. The lack of potent and selective inhibitors toward these enzymes has limited our understanding of their function and the development of therapeutic applications. Here we report the evaluation of a panel of compounds against the four human neuraminidase isoenzymes. Among the compounds tested, we identified the first selective, nanomolar inhibitors of the human neuraminidase 4 enzyme (NEU4). The most potent NEU4 inhibitor (5-acetamido-9-[4-hydroxymethyl[1,2,3]triazol-1-yl]-2,3,5,9-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosonic acid) was found to have an inhibitory constant (Ki) of 30 ± 19 nM and was 500-fold selective for its target over the other hNEU isoenzymes tested in vitro (NEU1, NEU2, and NEU3). This is the first report of any inhibitor of hNEU with nanomolar potency, and this confirms that the 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) scaffold can be exploited to develop new, potent, and selective inhibitors that target this important family of human enzymes. PMID:24900705

  8. Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness

    PubMed Central

    Scott, Sarah A; Selvy, Paige E; Buck, Jason R; Cho, Hyekyung P; Criswell, Tracy L; Thomas, Ashley L; Armstrong, Michelle D; Arteaga, Carlos L; Lindsley, Craig W; Brown, H Alex

    2013-01-01

    Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. The lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. We used a diversity-oriented synthetic approach and developed a library of PLD inhibitors with considerable pharmacological characterization. Here we report the rigorous evaluation of that library, which contains highly potent inhibitors, including the first isoform-selective PLD inhibitors. Specific members of this series inhibit isoforms with > 100-fold selectivity both in vitro and in cells. A subset of inhibitors was shown to block invasiveness in metastatic breast cancer models. These findings demonstrate the power of diversity-oriented synthesis combined with biochemical assays and mass spectrometric lipid profiling of cellular responses to develop the first isoform-selective PLD inhibitors—a new class of antimetastatic agents. PMID:19136975

  9. Conformationally-Restricted Dipeptide Amides as Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors

    PubMed Central

    Ji, Haitao; Gómez-Vidal, José A.; Martásek, Pavel; Roman, Linda J.; Silverman, Richard B.

    2008-01-01

    Four new conformationally-restricted analogues of the potent and selective neuronal nitric oxide synthase inhibitor, L-nitroargininyl-L-2,4-diaminobutyramide (1), have been synthesized. Nα-Methyl and Nα-benzyl derivatives (3 and 4, respectively) of 4N-(L-ArgNO2)-trans-4-amino-L-prolineamide (2) are also selective inhibitors, but the potency and selectivity of 3 are weak. Analogue 4 has only one-third the potency and one-half to one-third the selectivity of 2 against iNOS and eNOS, respectively. 3-N-(L-ArgNO2)-trans-3-amino-L-prolineamide (6) is as potent an inhibitor of nNOS as is 2; selectivity for nNOS over iNOS is half of that for 2 but the selectivity for nNOS over eNOS is almost double that for 2. The corresponding cis-isomer (5) is a weak inhibitor of nNOS. These results are supported by computer modeling. PMID:17034131

  10. Free energy calculation provides insight into the action mechanism of selective PARP-1 inhibitor.

    PubMed

    Cao, Ran

    2016-04-01

    Selective poly (ADP-ribose) polymerase (PARP)-1 inhibitor represents promising therapy against cancers with a good balance between efficacy and safety. Owing to the conserved structure between PARP-1 and PARP-2, most of the clinical and experimental drugs show equivalent inhibition against both targets. Most recently, it's disclosed a highly selective PARP-1 inhibitor (NMS-P118) with promising pharmacokinetic properties. Herein, we combined molecular simulation with free energy calculation to gain insights into the selective mechanism of NMS-P118. Our results suggest the reduction of binding affinity for PARP-2 is attributed to the unfavorable conformational change of protein, which is accompanied by a significant energy penalty. Alanine-scanning mutagenesis study further reveals the important role for a tyrosine residue of donor loop (Tyr889(PARP-1) and Tyr455(PARP-2)) in contributing to the ligand selectivity. Retrospective structural analysis indicates the ligand-induced movement of Tyr455(PARP-2) disrupts the intra-molecule hydrogen bonding network, which partially accounts for the "high-energy" protein conformation in the presence of NMS-P118. Interestingly, such effect isn't observed in other non-selective PARP inhibitors including BMN673 and A861695, which validates the computational prediction. Our work provides energetic insight into the subtle variations in the crystal structures and could facilitate rational design of new selective PARP inhibitor.

  11. Selective Inhibitors of Histone Deacetylases 1 and 2 Synergize with Azacitidine in Acute Myeloid Leukemia

    PubMed Central

    Shearstone, Jeffrey R.; Quayle, Steven N.; Huang, Pengyu; van Duzer, John H.; Jarpe, Matthew B.; Jones, Simon S.; Yang, Min

    2017-01-01

    Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. Outcomes for patients with AML remain poor, highlighting the need for novel treatment options. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and inhibitors of DNA methyltransferase or histone deacetylase (HDAC) enzymes have exhibited activity in preclinical AML models. Combination studies with HDAC inhibitors plus DNA methyltransferase inhibitors have potential beneficial clinical activity in AML, however the toxicity profiles of non-selective HDAC inhibitors in the combination setting limit their clinical utility. In this work, we describe the preclinical development of selective inhibitors of HDAC1 and HDAC2, which are hypothesized to have improved safety profiles, for combination therapy in AML. We demonstrate that selective inhibition of HDAC1 and HDAC2 is sufficient to achieve efficacy both as a single agent and in combination with azacitidine in preclinical models of AML, including established AML cell lines, primary leukemia cells from AML patient bone marrow samples and in vivo xenograft models of human AML. Gene expression profiling of AML cells treated with either an HDAC1/2 inhibitor, azacitidine, or the combination of both have identified a list of genes involved in transcription and cell cycle regulation as potential mediators of the combinatorial effects of HDAC1/2 inhibition with azacitidine. Together, these findings support the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML patients. PMID:28060870

  12. Chemical inhibitors of CYP450 enzymes in liver microsomes: combining selectivity and unbound fractions to guide selection of appropriate concentration in phenotyping assays.

    PubMed

    Nirogi, Ramakrishna; Palacharla, Raghava Choudary; Uthukam, Venkatesham; Manoharan, Arunkumar; Srikakolapu, Surya Rao; Kalaikadhiban, Ilayaraja; Boggavarapu, Rajesh Kumar; Ponnamaneni, Ranjith Kumar; Ajjala, Devender Reddy; Bhyrapuneni, Gopinadh

    2015-02-01

    1. Chemical inhibition is the widely used method in reaction phenotyping assays for estimation of specific enzyme contribution to a given metabolic pathway. The results from phenotyping assays depend on the selectivity of chemical inhibitor and the concentration of inhibitor used in the incubation. 2. The higher protein concentrations used in the in vitro phenotyping assays will impact the inhibitory potency of chemical inhibitors. The objective of the study is to evaluate comprehensively the selectivity of chemical inhibitors and to guide in selecting appropriate concentration of the chemical inhibitors to be used in the phenotyping assays based on unbound fractions. 3. Selectivity of chemical inhibitors against nine major CYP450 isoforms was determined in liver microsomes using standard probe substrates. The unbound fractions of the selective inhibitors were determined in human liver microsomes using high-throughput equilibrium dialysis. Combining unbound inhibitor concentrations that are required to inhibit the CYP450 activities by 90% and unbound fractions of the chemical inhibitors in liver microsomes appropriate total concentrations of the inhibitors to be used in the phenotyping assays were reported. 4. The findings suggest that non-specific binding of the chemical inhibitors need to be taken into account while selecting concentrations for phenotyping assays.

  13. Structural basis for isoform selectivity in a class of benzothiazole inhibitors of phosphoinositide 3-kinase γ.

    PubMed

    Collier, Philip N; Martinez-Botella, Gabriel; Cornebise, Mark; Cottrell, Kevin M; Doran, John D; Griffith, James P; Mahajan, Sudipta; Maltais, François; Moody, Cameron S; Huck, Emilie Porter; Wang, Tiansheng; Aronov, Alex M

    2015-01-08

    Phosphoinositide 3-kinase γ (PI3Kγ) is an attractive target to potentially treat a range of disease states. Herein, we describe the evolution of a reported phenylthiazole pan-PI3K inhibitor into a family of potent and selective benzothiazole inhibitors. Using X-ray crystallography, we discovered that compound 22 occupies a previously unreported hydrophobic binding cleft adjacent to the ATP binding site of PI3Kγ, and achieves its selectivity by exploiting natural sequence differences among PI3K isoforms in this region.

  14. The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

    SciTech Connect

    MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Riera, Thomas V.; D’Aquino, J. Alejandro; Zhang, Minjia; Cuny, Gregory D.; Hedstrom, Lizbeth

    2010-03-29

    Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

  15. Association between selective serotonin reuptake inhibitors and upper gastrointestinal bleeding: population based case-control study

    PubMed Central

    de Abajo, Francisco José; Rodríguez, Luis Alberto García; Montero, Dolores

    1999-01-01

    Objective To examine the association between selective serotonin reuptake inhibitors and risk of upper gastrointestinal bleeding. Design Population based case-control study. Setting General practices included in the UK general practice research database. Subjects 1651 incident cases of upper gastrointestinal bleeding and 248 cases of ulcer perforation among patients aged 40 to 79 years between April 1993 and September 1997, and 10 000 controls matched for age, sex, and year that the case was identified. Interventions Review of computer profiles for all potential cases, and an internal validation study to confirm the accuracy of the diagnosis on the basis of the computerised information. Main outcome measures Current use of selective serotonin reuptake inhibitors or other antidepressants within 30 days before the index date. Results Current exposure to selective serotonin reuptake inhibitors was identified in 3.1% (52 of 1651) of patients with upper gastrointestinal bleeding but only 1.0% (95 of 10 000) of controls, giving an adjusted rate ratio of 3.0 (95% confidence interval 2.1 to 4.4). This effect measure was not modified by sex, age, dose, or treatment duration. A crude incidence of 1 case per 8000 prescriptions was estimated. A small association was found with non-selective serotonin reuptake inhibitors (relative risk 1.4, 1.1 to 1.9) but not with antidepressants lacking this inhibitory effect. None of the groups of antidepressants was associated with ulcer perforation. The concurrent use of selective serotonin reuptake inhibitors with non-steroidal anti-inflammatory drugs increased the risk of upper gastrointestinal bleeding beyond the sum of their independent effects (15.6, 6.6 to 36.6). A smaller interaction was also found between selective serotonin reuptake inhibitors and low dose aspirin (7.2, 3.1 to 17.1). Conclusions Selective serotonin reuptake inhibitors increase the risk of upper gastrointestinal bleeding. The absolute effect is, however

  16. Structure-activity relationship study of selective benzimidazole-based inhibitors of Cryptosporidium parvum IMPDH

    PubMed Central

    Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Sharling, Lisa; Zhang, Minjia; Liu, Xiaoping; Ray, Soumya S.; MacPherson, Iain S.; Striepen, Boris; Hedstrom, Lizbeth; Cuny, Gregory D.

    2012-01-01

    Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. parvum and C. hominis genomes indicate that the only route to guanine nucleotides is via inosine 5'-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro. PMID:22310229

  17. Computational selection of inhibitors of Abeta aggregation and neuronal toxicity.

    PubMed

    Chen, Deliang; Martin, Zane S; Soto, Claudio; Schein, Catherine H

    2009-07-15

    Alzheimer's disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-beta protein (Abeta). Disease symptoms can be alleviated, in vitro and in vivo, by 'beta-sheet breaker' pentapeptides that reduce plaque load. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related beta-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Abeta. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features and a conformation similar to the active peptides were selected, ranked by docking and biochemical parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Abeta aggregation at 2-3microM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Abeta on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD.

  18. Discovery of potent and selective sirtuin 2 (SIRT2) inhibitors using a fragment-based approach.

    PubMed

    Cui, Huaqing; Kamal, Zeeshan; Ai, Teng; Xu, Yanli; More, Swati S; Wilson, Daniel J; Chen, Liqiang

    2014-10-23

    Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD(+)-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the discovery and development of SIRT2 inhibitors. Herein we report our discovery of novel SIRT2 inhibitors using a fragment-based approach. Inspired by the purported close binding proximity of suramin and nicotinamide, we prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene-benzamides and -nicotinamides. Biochemical evaluation of these two series provided structure-activity relationship (SAR) information, which led to the design of (5-benzamidonaphthalen-1/2-yloxy)nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2 activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-tubulin, a well-established SIRT2 substrate, in both concentration- and time-dependent manners. Further kinetic studies revealed that this compound behaves as a competitive inhibitor against the peptide substrate and most likely as a noncompetitive inhibitor against NAD(+). Taken together, these results indicate that we have discovered a potent and selective SIRT2 inhibitor whose novel structure merits further exploration.

  19. Discovery of a Highly Selective, Brain-Penetrant Aminopyrazole LRRK2 Inhibitor.

    PubMed

    Chan, Bryan K; Estrada, Anthony A; Chen, Huifen; Atherall, John; Baker-Glenn, Charles; Beresford, Alan; Burdick, Daniel J; Chambers, Mark; Dominguez, Sara L; Drummond, Jason; Gill, Andrew; Kleinheinz, Tracy; Le Pichon, Claire E; Medhurst, Andrew D; Liu, Xingrong; Moffat, John G; Nash, Kevin; Scearce-Levie, Kimberly; Sheng, Zejuan; Shore, Daniel G; Van de Poël, Hervé; Zhang, Shuo; Zhu, Haitao; Sweeney, Zachary K

    2013-01-10

    The modulation of LRRK2 kinase activity by a selective small molecule inhibitor has been proposed as a potentially viable treatment for Parkinson's disease. By using aminopyrazoles as aniline bioisosteres, we discovered a novel series of LRRK2 inhibitors. Herein, we describe our optimization effort that resulted in the identification of a highly potent, brain-penetrant aminopyrazole LRRK2 inhibitor (18) that addressed the liabilities (e.g., poor solubility and metabolic soft spots) of our previously disclosed anilino-aminopyrimidine inhibitors. In in vivo rodent PKPD studies, 18 demonstrated good brain exposure and engendered significant reduction in brain pLRRK2 levels post-ip administration. The strategies of bioisosteric substitution of aminopyrazoles for anilines and attenuation of CYP1A2 inhibition described herein have potential applications to other drug discovery programs.

  20. Selective ACAT inhibitors as promising antihyperlipidemic, antiathero-sclerotic and anti-Alzheimer drugs.

    PubMed

    Giovannoni, M P; Piaz, V Dal; Vergelli, C; Barlocco, D

    2003-09-01

    Inhibition of ACAT, the enzyme which catalyses the intracellular formation of cholesteryl esters, is a very attractive target for the treatment of hypercholesterolaemia and atherosclerosis. However, in the past years many ACAT inhibitors gave disappointing results in clinical trials showing very low efficacy. In addition, their development was affected by the adrenotoxicity observed in many compounds. The discovery of two isoforms of the enzyme, namely ACAT1 and ACAT2, with different substrate specificity and different potential function, offers a precious information for planning selective inhibitors with reduced secondary effects. Today some potent, bioavailable and non adrenotoxic ACAT inhibitors are under clinical evaluation. Amongst others, a very promising compound is Avasimibe, presently in phase III clinical trials as anti-hyperlipidemic and anti-atherosclerotic agent. Finally, ACAT inhibitors have recently been proposed for the treatment of Alzheimer's disease.

  1. Field trials for corrosion inhibitor selection and optimization, using a new generation of electrical resistance probes

    SciTech Connect

    Ridd, B.; Blakset, T.J.; Queen, D.

    1998-12-31

    Even with today`s availability of corrosion resistant alloys, carbon steels protected by corrosion inhibitors still dominate the material selection for pipework in the oil and gas production. Even though laboratory screening tests of corrosion inhibitor performance provides valuable data, the real performance of the chemical can only be studied through field trials which provide the ultimate test to evaluate the effectiveness of an inhibitor under actual operating conditions. A new generation of electrical resistance probe has been developed, allowing highly sensitive and immediate response to changes in corrosion rates on the internal environment of production pipework. Because of the high sensitivity, the probe responds to small changes in the corrosion rate, and it provides the corrosion engineer with a highly effective method of optimizing the use of inhibitor chemicals resulting in confidence in corrosion control and minimizing detrimental environmental effects.

  2. Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

    PubMed

    Liu, Tao; Nair, Somarajan J; Lescarbeau, André; Belani, Jitendra; Peluso, Stéphane; Conley, James; Tillotson, Bonnie; O'Hearn, Patrick; Smith, Sherri; Slocum, Kelly; West, Kip; Helble, Joseph; Douglas, Mark; Bahadoor, Adilah; Ali, Janid; McGovern, Karen; Fritz, Christian; Palombella, Vito J; Wylie, Andrew; Castro, Alfredo C; Tremblay, Martin R

    2012-10-25

    Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

  3. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity.

    PubMed

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A

    2017-03-07

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1(R132H) mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  4. Identification of HDAC6-Selective Inhibitors of Low Cancer Cell Cytotoxicity.

    PubMed

    Gaisina, Irina N; Tueckmantel, Werner; Ugolkov, Andrey; Shen, Sida; Hoffen, Jessica; Dubrovskyi, Oleksii; Mazar, Andrew; Schoon, Renee A; Billadeau, Daniel; Kozikowski, Alan P

    2016-01-05

    The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform-selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first-generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell-cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole-based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1-3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.

  5. Crystal Structure of Checkpoint Kinase 2 in Complex with Nsc 109555, a Potent and Selective Inhibitor

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Zhang, Di; Jobson, Andrew G.; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2009-03-05

    Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA-damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC{sub 50} = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATP-binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.

  6. Novel Selective Calpain 1 Inhibitors as Potential Therapeutics in Alzheimer's Disease.

    PubMed

    Fà, Mauro; Zhang, Hong; Staniszewski, Agnieszka; Saeed, Faisal; Shen, Li W; Schiefer, Isaac T; Siklos, Marton I; Tapadar, Subhasish; Litosh, Vladislav A; Libien, Jenny; Petukhov, Pavel A; Teich, Andrew F; Thatcher, Gregory R J; Arancio, Ottavio

    2016-01-01

    Alzheimer's disease, one of the most important brain pathologies associated with neurodegenerative processes, is related to overactivation of calpain-mediated proteolysis. Previous data showed a compelling efficacy of calpain inhibition against abnormal synaptic plasticity and memory produced by the excess of amyloid-β, a distinctive marker of the disease. Moreover, a beneficial effect of calpain inhibitors in Alzheimer's disease is predictable by the occurrence of calpain hyperactivation leading to impairment of memory-related pathways following abnormal calcium influxes that might ensue independently of amyloid-β elevation. However, molecules currently available as effective calpain inhibitors lack adequate selectivity. This work is aimed at characterizing the efficacy of a novel class of epoxide-based inhibitors, synthesized to display improved selectivity and potency towards calpain 1 compared to the prototype epoxide-based generic calpain inhibitor E64. Both functional and preliminary toxicological investigations proved the efficacy, potency, and safety of the novel and selective calpain inhibitors NYC438 and NYC488 as possible therapeutics against the disease.

  7. Potent, Highly Selective, and Orally Bioavailable Gem-Difluorinated Monocationic Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    Xue, Fengtian; Li, Huiying; Delker, Silvia L.; Fang, Jianguo; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2010-01-01

    In our efforts to discover neuronal isoform selective nitric oxide synthase (NOS) inhibitors we have developed a series of compounds containing a pyrrolidine ring with two stereogenic centers. The enantiomerically pure compounds, (S,S) vs. (R,R), exhibited two different binding orientations, with (R,R) inhibitors showing much better potency and selectivity. To improve the bioavailability of these inhibitors we have introduced a CF2 moiety geminal to an amino group in the long tail of one of these inhibitors, which reduced its basicity, resulting in compounds with monocationic character under physiological pH conditions. Biological evaluations have led to a nNOS inhibitor with a Ki of 36 nM and high selectivity for nNOS over eNOS (3800-fold) and iNOS (1400-fold). MM-PBSA calculations indicated that the low pKa NH is, at least, partially protonated when bound to the active site. A comparison of rat oral bioavailability of the difluorinated compound to the parent molecule shows 22% for the difluorinated compound versus essentially no oral bioavailability for the parent compound. This indicates that the goal of this research to make compounds with only one protonated nitrogen atom at physiological pH to allow for membrane permeability, but which can become protonated when bound to NOS, has been accomplished. PMID:20843082

  8. Selective inhibitors of digestive enzymes from Aedes aegypti larvae identified by phage display.

    PubMed

    Soares, Tatiane Sanches; Soares Torquato, Ricardo Jose; Alves Lemos, Francisco Jose; Tanaka, Aparecida Sadae

    2013-01-01

    Dengue is a serious disease transmitted by the mosquito Aedes aegypti during blood meal feeding. It is estimated that the dengue virus is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies have been based on controlling the vector, Ae. aegypti, using insecticide, but the emergence of resistance poses new challenges. The aim of this study was the identification of specific protease inhibitors of the digestive enzymes from Ae. aegypti larvae, which may serve as a prospective alternative biocontrol method. High affinity protein inhibitors were selected by all of the digestive serine proteases of the 4th instar larval midgut, and the specificity of these inhibitors was characterized. These inhibitors were obtained from a phage library displaying variants of HiTI, a trypsin inhibitor from Haematobia irritans, that are mutated in the reactive loop (P1-P4'). Based on the selected amino acid sequence pattern, seven HiTI inhibitor variants were cloned, expressed and purified. The results indicate that the HiTI variants named T6 (RGGAV) and T128 (WNEGL) were selected by larval trypsin-like (IC(50) of 1.1 nM) and chymotrypsin-like enzymes (IC(50) of 11.6 nM), respectively. The variants T23 (LLGGL) and T149 (GGVWR) inhibited both larval chymotrypsin-like (IC(50) of 4.2 nM and 29.0 nM, respectively) and elastase-like enzymes (IC(50) of 1.2 nM for both). Specific inhibitors were successfully obtained for the digestive enzymes of Ae. aegypti larvae by phage display. Our data also strongly suggest the presence of elastase-like enzymes in Ae. aegypti larvae. The HiTI variants T6 and T23 are good candidates for the development as a larvicide to control the vector.

  9. Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors.

    PubMed Central

    Tisdale, M; Myers, R E; Maschera, B; Parry, N R; Oliver, N M; Blair, E D

    1995-01-01

    Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy. PMID:7486905

  10. Evaluation of JAK3 biology in autoimmune disease using a highly selective, irreversible JAK3 inhibitor.

    PubMed

    Elwood, Fiona; Witter, David; Piesvaux, Jennifer; Kraybill, Brian; Bays, Nathan; Alpert, Carla; Goldenblatt, Peter; Qu, Yujie; Ivanovska, Irena; Lee, Hyun-Hee; Chiu, Chi-Sung; Tang, Hao; Scott, Mark E; Deshmukh, Sujal; Zielstorff, Mark; Byford, Alan; Chakravarty, Kalyan; Dorosh, Lauren; Rivkin, Alexy; Klappenbach, Joel; Pan, Bo-Sheng; Kariv, Ilona; Dinsmore, Christopher; Slipetz, Deborah; Dandliker, Peter

    2017-02-13

    Reversible Janus kinase (JAK) inhibitors such as Tofacitinib(Changelian, et al., 2003;Flanagan, et al., 2010) and Decernotinib(Farmer, et al., 2015;Mahajan, et al., 2015) block cytokine signaling and are efficacious in treating autoimmune diseases (Kremer, et al., 2009;Fleischmann, et al., 2015;Fleischmann, et al., 2015;Krueger, et al., 2016;Sandborn, et al., 2012). However therapeutic doses are limited due to inhibition of other JAK/STAT pathways associated with hematopoiesis, lipid biogenesis, infection and immune responses(Kahn C, 2012). A selective JAK3 inhibitor may have a better therapeutic index, however, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km for each JAK isozyme ranged from 31.8 μM for JAK1 to 2.9 μM for JAK3. To confirm compound activity in cells, we developed a novel enzyme complimentation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro, in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold (IL-2 vs. IL-6) or 140-fold (IL-2 vs. EPO or GMCSF) selective in cellular reporter assays and >35-fold selective in human PBMC assays (IL-7 vs. IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of Rheumatoid Arthritis, while sparing hematopoiesis.

  11. Indazole- and indole-5-carboxamides: selective and reversible monoamine oxidase B inhibitors with subnanomolar potency.

    PubMed

    Tzvetkov, Nikolay T; Hinz, Sonja; Küppers, Petra; Gastreich, Marcus; Müller, Christa E

    2014-08-14

    Indazole- and indole-carboxamides were discovered as highly potent, selective, competitive, and reversible inhibitors of monoamine oxidase B (MAO-B). The compounds are easily accessible by standard synthetic procedures with high overall yields. The most potent derivatives were N-(3,4-dichlorophenyl)-1-methyl-1H-indazole-5-carboxamide (38a, PSB-1491, IC50 human MAO-B 0.386 nM, >25000-fold selective versus MAO-A) and N-(3,4-dichlorophenyl)-1H-indole-5-carboxamide (53, PSB-1410, IC50 human MAO-B 0.227 nM, >5700-fold selective versus MAO-A). Replacement of the carboxamide linker with a methanimine spacer leading to (E)-N-(3,4-dichlorophenyl)-1-(1H-indazol-5-yl)methanimine (58) represents a further novel class of highly potent and selective MAO-B inhibitors (IC50 human MAO-B 0.612 nM, >16000-fold selective versus MAO-A). In N-(3,4-difluorophenyl-1H-indazole-5-carboxamide (30, PSB-1434, IC50 human MAO-B 1.59 nM, selectivity versus MAO-A>6000-fold), high potency and selectivity are optimally combined with superior physicochemical properties. Computational docking studies provided insights into the inhibitors' interaction with the enzyme binding site and a rationale for their high potency despite their small molecular size.

  12. Short-term cardiovascular effects of selective phosphodiesterase 3 inhibitor olprinone versus non-selective phosphodiesterase inhibitor aminophylline in a meconium-induced acute lung injury.

    PubMed

    Mokra, D; Tonhajzerova, I; Pistekova, H; Visnovcova, Z; Mokry, J; Drgova, A; Repcakova, M; Calkovska, A

    2013-12-01

    Various anti-inflammatory drugs have been used for treatment of neonatal meconium aspiration syndrome (MAS). As their adverse effects are poorly described, this study compared effects of selective phosphodiesterase (PDE) 3 inhibitor olprinone and non-selective PDE inhibitor aminophylline on cardiovascular parameters in animal model of MAS. Oxygen-ventilated rabbits were intratracheally instilled 4 mL/kg of meconium (25 mg/mL) or saline. Thirty minutes later, meconium-instilled animals were intravenously given olprinone (0.2 mg/kg) at a single dose at 0.5 h after meconium instillation, or aminophylline (2.0 mg/kg) at two doses at 0.5 and 2.5 h after meconium instillation, or were left without treatment. Cardiovascular changes were evaluated within 5 min of administration and 5 min after finishing the administration. Furthermore, respiratory and cardiovascular parameters were measured within 5 hours following treatment delivery. Oxidation markers (thiobarbituric acid-reactive substances (TBARS), and total antioxidant status) and markers of cardiovascular injury (aldosterone, gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT)) were determined in the plasma. Meconium instillation induced acute lung injury associated with oxidative stress, elevated aldosterone, and slightly increased GGT and AST levels. Both aminophylline and olprinone improved lung functions and reduced oxidation stress. However, the PDE inhibitors acutely increased blood pressure and heart rate, whereas heart rate variability remained higher till the end of experiment and correlated well with markers of cardiovascular injury. Considering that systemic administration of olprinone and aminophylline was accompanied by acute cardiovascular changes in the meconium-instilled animals, use of PDE inhibitors in the newborns with MAS should be carefully monitored.

  13. Synthesis of selective inhibitors against V. cholerae sialidase and human cytosolic sialidase NEU2.

    PubMed

    Khedri, Zahra; Li, Yanhong; Cao, Hongzhi; Qu, Jingyao; Yu, Hai; Muthana, Musleh M; Chen, Xi

    2012-08-14

    Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized. Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that Neu5Gc9N(3)2en and Neu5AcN(3)9N(3)2en are selective inhibitors against V. cholerae sialidase and human NEU2, respectively.

  14. Studies on pyrrolopyrimidines as selective inhibitors of multidrug-resistance-associated protein in multidrug resistance.

    PubMed

    Wang, Shouming; Folkes, Adrian; Chuckowree, Irina; Cockcroft, Xiaoling; Sohal, Sukhjit; Miller, Warren; Milton, John; Wren, Stephen P; Vicker, Nigel; Depledge, Paul; Scott, John; Smith, Lyndsay; Jones, Hazel; Mistry, Prakash; Faint, Richard; Thompson, Deanne; Cocks, Simon

    2004-03-11

    Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.

  15. 6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site

    PubMed Central

    Ghizzoni, Massimo; Wu, Jiang; Gao, Tielong; Haisma, Hidde J.; Dekker, Frank J.; Zheng, Y. George

    2011-01-01

    Histone acetyltransferases are important enzymes that regulate various cellular functions, such as epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency will provide powerful mechanistic tools for the elucidation of the biological functions of HATs and may also have pharmacological value for potential new therapies. In this work, analogs of the known HAT inhibitor anacardic acid were synthesized and evaluated for inhibition of HAT activity. Biochemical assays revealed novel anacardic acid analogs that inhibited the human recombinant enzyme Tip60 selectively compared to PCAF and p300. Enzyme kinetics studies demonstrated that inhibition of Tip60 by one such novel anacardic acid derive, 20, was essentially competitive with Ac-CoA and noncompetitive with the histone substrate. In addition, these HAT inhibitors effectively inhibited acetyltransferase activity of nuclear extracts on the histone H3 and H4 at micromolar concentrations. PMID:22100137

  16. Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitors.

    PubMed

    Brullo, Chiara; Massa, Matteo; Villa, Carla; Ricciarelli, Roberta; Rivera, Daniela; Pronzato, Maria Adelaide; Fedele, Ernesto; Barocelli, Elisabetta; Bertoni, Simona; Flammini, Lisa; Bruno, Olga

    2015-07-01

    A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All compounds showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 isoforms (PDE4A4, PDE4B2 and PDE4C2). Compound 3b, chosen among the synthesized compounds as the most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacokinetic profile compared to its non fluorinated analogue. Spontaneous locomotor activity, assessed in an open field apparatus, showed that, differently from rolipram and diazepam, selective PDE4D inhibitors, such as compounds 3b, 5b and 7b, did not affect locomotion, whereas compound 1b showed a tendency to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity.

  17. Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides

    NASA Astrophysics Data System (ADS)

    Nguyen, G. T. T.; Erlenkamp, G.; Jäck, O.; Küberl, A.; Bott, M.; Fiorani, F.; Gohlke, H.; Groth, G.

    2016-06-01

    Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2‧,3‧,4‧,3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2‧,3‧,4‧-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15–45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme.

  18. Effect of selective serotonin reuptake inhibitors on in vitro fertilization outcome.

    PubMed

    Friedman, Brooke E; Rogers, Jayna L; Shahine, Lora K; Westphal, Lynn M; Lathi, Ruth B

    2009-10-01

    A review of 950 patients was performed to investigate the impact of selective serotonin reuptake inhibitors (SSRIs) on in vitro fertilization outcome. The 41 patients (4.3%) taking an SSRI had a higher cycle cancellation rate but no statistically significant difference in pregnancy rate and live birth rate per cycle started.

  19. Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors

    PubMed Central

    Morgan, Cynthia A.; Hurley, Thomas D.

    2015-01-01

    Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson’s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit sub-micromolar inhibition constants, but have different mechanisms of inhibition. The crystal structures of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique Glycine residue to achieve their selectivity. These two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states. PMID:25634381

  20. Novel, potent, selective, and orally bioavailable human betaII-tryptase inhibitors.

    PubMed

    Sperandio, David; Tai, Vincent W-F; Lohman, Julia; Hirschbein, Bernie; Mendonca, Rohan; Lee, Chang-Sun; Spencer, Jeffrey R; Janc, James; Nguyen, Margaret; Beltman, Jerlyn; Sprengeler, Paul; Scheerens, Heleen; Lin, Tong; Liu, Liang; Gadre, Ashwini; Kellogg, Alisha; Green, Michael J; McGrath, Mary E

    2006-08-01

    The synthesis of novel [1,2,4]oxadiazoles and their structure-activity relationship (SAR) for the inhibition of tryptase and related serine proteases is presented. Elaboration of the P'-side afforded potent, selective, and orally bioavailable tryptase inhibitors.

  1. Potent, Selective, and CNS-Penetrant Tetrasubstituted Cyclopropane Class IIa Histone Deacetylase (HDAC) Inhibitors

    PubMed Central

    2015-01-01

    Potent and selective class IIa HDAC tetrasubstituted cyclopropane hydroxamic acid inhibitors were identified with high oral bioavailability that exhibited good brain and muscle exposure. Compound 14 displayed suitable properties for assessment of the impact of class IIa HDAC catalytic site inhibition in preclinical disease models. PMID:26819662

  2. Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides

    PubMed Central

    Nguyen, G. T. T.; Erlenkamp, G.; Jäck, O.; Küberl, A.; Bott, M.; Fiorani, F.; Gohlke, H.; Groth, G.

    2016-01-01

    Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2′,3′,4′,3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2′,3′,4′-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15–45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme. PMID:27263468

  3. Selective Serotonin Reuptake Inhibitor-Induced Sexual Dysfunction in Adolescents: A Review.

    ERIC Educational Resources Information Center

    Scharko, Alexander M.

    2004-01-01

    Objective: To review the existing literature on selective serotonin reuptake inhibitor (SSRI)-induced sexual dysfunction in adolescents. Method: A literature review of SSRI-induced adverse effects in adolescents focusing on sexual dysfunction was done. Nonsexual SSRI-induced adverse effects were compared in adult and pediatric populations.…

  4. In Utero Exposure to Selective Serotonin Reuptake Inhibitors and Risk for Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Gidaya, Nicole B.; Lee, Brian K.; Burstyn, Igor; Yudell, Michael; Mortensen, Erik L.; Newschaffer, Craig J.

    2014-01-01

    We investigated whether there is an association between increased risk for autism spectrum disorders (ASD) and selective serotonin reuptake inhibitors (SSRIs) used during pregnancy. This study used Denmark's health and population registers to obtain information regarding prescription drugs, ASD diagnosis, and health and socioeconomic status. There…

  5. Anhedonia Predicts Poorer Recovery among Youth with Selective Serotonin Reuptake Inhibitor Treatment-Resistant Depression

    ERIC Educational Resources Information Center

    McMakin, Dana L.; Olino, Thomas M.; Porta, Giovanna; Dietz, Laura J.; Emslie, Graham; Clarke, Gregory; Wagner, Karen Dineen; Asarnow, Joan R.; Ryan, Neal D.; Birmaher, Boris; Shamseddeen, Wael; Mayes, Taryn; Kennard, Betsy; Spirito, Anthony; Keller, Martin; Lynch, Frances L.; Dickerson, John F.; Brent, David A.

    2012-01-01

    Objective: To identify symptom dimensions of depression that predict recovery among selective serotonin reuptake inhibitor (SSRI) treatment-resistant adolescents undergoing second-step treatment. Method: The Treatment of Resistant Depression in Adolescents (TORDIA) trial included 334 SSRI treatment-resistant youth randomized to a medication…

  6. Selective Monocationic Inhibitors of Neuronal Nitric Oxide Synthase. Binding Mode Insights from Molecular Dynamics Simulations

    PubMed Central

    Huang, He; Ji, Haitao; Li, Huiying; Jing, Qing; Labby, Kristin Jansen; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2012-01-01

    The reduction of pathophysiologic levels of nitric oxide through inhibition of neuronal nitric oxide synthase (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. We have developed a series of pyrrolidine-based nNOS inhibitors that exhibit excellent potencies and isoform selectivities (J. Am. Chem. Soc. 2010, 132, 5437). However, there are still important challenges, such as how to decrease the multiple positive charges derived from basic amino groups, which contribute to poor bioavailability, without losing potency and/or selectivity. Here we present an interdisciplinary study combining molecular docking, crystallography, molecular dynamics simulations, synthesis, and enzymology to explore potential pharmacophoric features of nNOS inhibitors and to design potent and selective monocationic nNOS inhibitors. The simulation results indicate that different hydrogen bond patterns, electrostatic interactions, hydrophobic interactions, and a water molecule bridge are key factors for stabilizing ligands and controlling ligand orientation. We find that a heteroatom in the aromatic head or linker chain of the ligand provides additional stability and blocks the substrate binding pocket. Finally, the computational insights are experimentally validated with double-headed pyridine analogs. The compounds reported here are among the most potent and selective monocationic pyrrolidine-based nNOS inhibitors reported to date, and 10 shows improved membrane permeability. PMID:22731813

  7. "Addition" and "Subtraction": Selectivity Design for Type II Maternal Embryonic Leucine Zipper Kinase Inhibitors.

    PubMed

    Chen, Xin; Giraldes, John; Sprague, Elizabeth R; Shakya, Subarna; Chen, Zhuoliang; Wang, Yaping; Joud, Carol; Mathieu, Simon; Chen, Christine Hiu-Tung; Straub, Christopher; Duca, Jose; Hurov, Kristen; Yuan, Yanqiu; Shao, Wenlin; Touré, B Barry

    2017-03-09

    While adding the structural features that are more favored by on-target activity is the more common strategy in selectivity optimization, the opposite strategy of subtracting the structural features that contribute more to off-target activity can also be very effective. Reported here is our successful effort of improving the kinase selectivity of type II maternal embryonic leucine zipper kinase inhibitors by applying these two complementary approaches together, which clearly demonstrates the powerful synergy between them.

  8. A Novel Selective Prostaglandin E2 Synthesis Inhibitor Relieves Pyrexia and Chronic Inflammation in Rats.

    PubMed

    Sugita, Ryusuke; Kuwabara, Harumi; Sugimoto, Kotaro; Kubota, Kazufumi; Imamura, Yuichiro; Kiho, Toshihiro; Tengeiji, Atsushi; Kawakami, Katsuhiro; Shimada, Kohei

    2016-04-01

    Prostaglandin E2 (PGE2) is a terminal prostaglandin in the cyclooxygenase (COX) pathway. Inhibition of PGE2 production may relieve inflammatory symptoms such as fever, arthritis, and inflammatory pain. We report here the profile of a novel selective PGE2 synthesis inhibitor, compound A [N-[(1S,3S)-3-carbamoylcyclohexyl]-1-(6-methyl-3-phenylquinolin-2-yl)piperidine-4-carboxamide], in animal models of pyrexia and inflammation. The compound selectively suppressed the synthesis of PGE2 in human alveolar adenocarcinoma cell line A549 cells and rat macrophages. In the lipopolysaccharide-induced pyrexia model, this compound selectively reduced PGE2 production in cerebrospinal fluid and showed an anti-pyretic effect. In the adjuvant-induced arthritis model, compound A therapeutically decreased foot swelling in the established arthritis. Our data demonstrates that selective suppression of PGE2 synthesis shows anti-pyretic and anti-inflammatory effects, suggesting that selective PGE2 synthesis inhibitors can be applied as an alternative treatment to nonsteroidal, anti-inflammatory drugs (NSAIDs) or COX-2-selective inhibitors.

  9. Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

    PubMed

    Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

    2017-02-28

    Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.

  10. Structure based lead optimization approach in discovery of selective DPP4 inhibitors.

    PubMed

    Ghate, Manjunath; Jain, Shailesh V

    2013-05-01

    Diabetes mellitus is a chronic progressive metabolic disorder that has profound consequences for individuals, families, and society. To date, main available oral antidiabetic medications target either insulin resistance (metformin, glitazones), or insulin deficiency (sulfonylureas, glinides), but leading to shortfalls in medication. Advancement in modern oral hypoglycemic agents may be encouraged with or in place of traditional therapies. The lower risk for hypoglycemic events as compared with other insulinotropic or insulin-sensitizing agents make DPP-4 inhibitors very promising candidates for a more physiological treatment of type-2 diabetes. Only some DPP-4 inhibitors are currently used for the treatment of type 2 diabetes (T2DM) and various inhibitors currently undergoing animal and human testing. A number of catalytically active DPPs distinct from DPP-4 (DPP II, FAP, DPP-8, and DPP-9) have been described that is associated with side-effect and toxicity. To discover potent and selective and safer drugs in a shorter time frame and with reduced cost it requires using an innovative approach for designing novel inhibitors. This review article focuses on the status of advanced lead candidates of DPP group and their binding affinity with the active site residue of target structure which help in discovery of potent and selective DPP-4 inhibitors by lead optimization approach.

  11. Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

    PubMed

    Abdul-Hay, Samer O; Bannister, Thomas D; Wang, Hui; Cameron, Michael D; Caulfield, Thomas R; Masson, Amandine; Bertrand, Juliette; Howard, Erin A; McGuire, Michael P; Crisafulli, Umberto; Rosenberry, Terrone R; Topper, Caitlyn L; Thompson, Caroline R; Schürer, Stephan C; Madoux, Franck; Hodder, Peter; Leissring, Malcolm A

    2015-12-18

    Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 μM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

  12. A roadmap to evaluate the proteome-wide selectivity of covalent kinase inhibitors

    PubMed Central

    Dix, Melissa M.; Douhan, John; Gilbert, Adam M.; Hett, Erik C.; Johnson, Theodore O.; Joslyn, Chris; Kath, John C.; Niessen, Sherry; Roberts, Lee R.; Schnute, Mark E.; Wang, Chu; Hulce, Jonathan J.; Wei, Baoxian; Whiteley, Laurence O.; Hayward, Matthew M.; Cravatt, Benjamin F.

    2014-01-01

    Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active-sites have emerged as valuable probes and approved drugs. Many protein classes, however, possess functional cysteines and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative mass spectrometry to globally map the targets, both specific and non-specific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent non-kinase proteins that, interestingly, possess conserved, active-site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental roadmap to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors. PMID:25038787

  13. Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A2

    PubMed Central

    Nagano, Joseph M. G.; Hsu, Ku-Lung; Whitby, Landon R.; Niphakis, Micah J.; Speers, Anna E.; Brown, Steven J.; Spicer, Timothy; Fernandez-Vega, Virneliz; Ferguson, Jill; Hodder, Peter; Srinivasan, Prabhavathi; Gonzalez, Tara D.; Rosen, Hugh; Bahnson, Brian J.

    2013-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2 or PLA2G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA2 has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA2 have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA2 inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA2 and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA2 in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA2 function in biological systems. PMID:23260346

  14. Use of Selective Cyclooxygenase-2 Inhibitors, Other Analgesics, and Risk of Glioma

    PubMed Central

    Seliger, Corinna; Meier, Christoph R.; Becker, Claudia; Jick, Susan S.; Bogdahn, Ulrich; Hau, Peter; Leitzmann, Michael F.

    2016-01-01

    Background Selective cyclooxygenase-2 (COX-2) inhibitors are analgesic, antipyretic, and anti-inflammatory drugs. They have been found to inhibit the development of glioma in laboratory investigations. Whether these drugs reduce the risk of glioma incidence in humans is unknown. Methods We conducted a matched case-control analysis using the U.K.-based Clinical Practice Research Datalink (CPRD). We identified 2,469 cases matched to 24,690 controls on age, sex, calendar time, general practice, and number of years of active history in the CPRD prior to the index date. We conducted conditional logistic regression analyses to determine relative risks, estimated as odds ratios (ORs) with 95% confidence intervals (CIs) of glioma in relation to use of selective COX-2 inhibitors, adjusted for several confounding variables. Results Use of selective COX-2 inhibitors was unrelated to risk of glioma (adjusted OR for 1–9 versus 0 prescriptions = 1.02; 95% CI = 0.92–1.13, 10–29 versus 0 prescriptions = 1.01; 95% CI = 0.80–1.28, ≥30 versus 0 prescriptions = 1.16; 95% CI = 0.86–1.55). Trends for increasing numbers of prescriptions for other non-steroidal anti-inflammatory drugs (NSAIDs), and non-NSAID analgesics were also not associated with glioma risk. Conclusion Further epidemiologic studies are needed to confirm the null relation of use of selective COX-2 inhibitors to glioma risk and to explain the discrepancy between laboratory investigations and our observational study. Impact: Use of selective COX-2 inhibitors is unrelated to glioma risk. PMID:26871579

  15. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket.

    PubMed

    Forster, Michael; Chaikuad, Apirat; Bauer, Silke M; Holstein, Julia; Robers, Matthew B; Corona, Cesear R; Gehringer, Matthias; Pfaffenrot, Ellen; Ghoreschi, Kamran; Knapp, Stefan; Laufer, Stefan A

    2016-11-17

    Janus kinases (JAKs) are a family of cytoplasmatic tyrosine kinases that are attractive targets for the development of anti-inflammatory drugs given their roles in cytokine signaling. One question regarding JAKs and their inhibitors that remains under intensive debate is whether JAK inhibitors should be isoform selective. Since JAK3 functions are restricted to immune cells, an isoform-selective inhibitor for JAK3 could be especially valuable to achieve clinically more useful and precise effects. However, the high degree of structural conservation makes isoform-selective targeting a challenging task. Here, we present picomolar inhibitors with unprecedented kinome-wide selectivity for JAK3. Selectivity was achieved by concurrent covalent reversible targeting of a JAK3-specific cysteine residue and a ligand-induced binding pocket. We confirmed that in vitro activity and selectivity translate well into the cellular environment and suggest that our inhibitors are powerful tools to elucidate JAK3-specific functions.

  16. Enhancement of pomalidomide anti-tumor response with ACY-241, a selective HDAC6 inhibitor

    PubMed Central

    Tamang, David; Yang, Min; Jones, Simon S.; Quayle, Steven N.

    2017-01-01

    Thalidomide-based Immunomodulatory Drugs (IMiDs®), including lenalidomide and pomalidomide, are effective therapeutics for multiple myeloma. These agents have been approved with, or are under clinical development with, other targeted therapies including proteasome inhibitors, αCD38 monoclonal antibodies, as well as histone deacetylase (HDAC) inhibitors for combination therapy. HDAC inhibitors broadly targeting Class I and IIb HDACs have shown potent preclinical efficacy but have frequently demonstrated an undesirable safety profile in combination therapy approaches in clinical studies. Therefore, development of more selective HDAC inhibitors could provide enhanced efficacy with reduced side effects in combination with IMiDs® for the treatment of B-cell malignancies, including multiple myeloma. Here, the second generation selective HDAC6 inhibitor citarinostat (ACY-241), with a more favorable safety profile than non-selective pan-HDAC inhibitors, is shown to synergize with pomalidomide in in vitro assays through promoting greater apoptosis and cell cycle arrest. Furthermore, utilizing a multiple myeloma in vivo murine xenograft model, combination treatment with pomalidomide and ACY-241 leads to increased tumor growth inhibition. At the molecular level, combination treatment with ACY-241 and pomalidomide leads to greater suppression of the pro-survival factors survivin, Myc, and IRF4. The results presented here demonstrate synergy between pomalidomide and ACY-241 in both in vitro and in vivo preclinical models, providing further impetus for clinical development of ACY-241 for use in combination with IMiDs for patients with multiple myeloma and potentially other B-cell malignancies. PMID:28264055

  17. Structure- and function-based design of Plasmodium-selective proteasome inhibitors.

    PubMed

    Li, Hao; O'Donoghue, Anthony J; van der Linden, Wouter A; Xie, Stanley C; Yoo, Euna; Foe, Ian T; Tilley, Leann; Craik, Charles S; da Fonseca, Paula C A; Bogyo, Matthew

    2016-02-11

    The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a

  18. Structure and function based design of Plasmodium-selective proteasome inhibitors

    PubMed Central

    Li, Hao; O'Donoghue, Anthony J.; van der Linden, Wouter A.; Xie, Stanley C.; Yoo, Euna; Foe, Ian T.; Tilley, Leann; Craik, Charles S.; da Fonseca, Paula C. A.; Bogyo, Matthew

    2016-01-01

    The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation1. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle2-5. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome resulting in toxicity that precludes their use as therapeutic agents2,6. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, we used a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We designed inhibitors based on amino acid preferences specific to the parasite proteasome, and found that they preferentially inhibit the β 2 subunit. We determined the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy (cryo-EM) and single particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information regarding active site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin (ART) family anti-malarials7,8, we observed growth inhibition synergism with low doses of this β 2 selective inhibitor in ART sensitive and resistant parasites. Finally, we demonstrated that a parasite selective inhibitor could be used to attenuate parasite growth in vivo without significant toxicity to the host. Thus, the

  19. Acute neural effects of selective serotonin reuptake inhibitors versus noradrenaline reuptake inhibitors on emotion processing: Implications for differential treatment efficacy.

    PubMed

    Outhred, Tim; Hawkshead, Brittany E; Wager, Tor D; Das, Pritha; Malhi, Gin S; Kemp, Andrew H

    2013-09-01

    Clinical research has demonstrated differential efficacy of selective serotonin reuptake inhibitors (SSRIs) and norepinephrine reuptake inhibitors (NRIs), which may relate to differential acute effects these medications have on emotional brain processes. Here we present findings from a Multi-Level Kernel Density Analysis meta-analysis that integrates and contrasts activations from disparate fMRI studies in order to examine whether single dose SSRIs and NRIs have different effects on emotion processing tasks in healthy participants. Seven SSRI and four NRI studies were eligible for inclusion. SSRIs decreased amygdala responses, suggesting reduced emotional reactivity to emotional stimuli, whereas NRIs increased frontal and medial activation, suggesting increased emotion regulation. As hypothesised, an interaction of antidepressant and task type was found, such that SSRIs modulated amygdaloid-hippocampal, medial and frontal activity during both the presentation of faces and pictures, whereas NRIs only modulated the activation in medial and frontal regions during the presentation of pictures. Findings are interpreted within a novel model of the differential effects of SSRIs and NRIs on emotion processing.

  20. Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

    PubMed

    Tao, Zhi-Fu; Hasvold, Lisa; Wang, Le; Wang, Xilu; Petros, Andrew M; Park, Chang H; Boghaert, Erwin R; Catron, Nathaniel D; Chen, Jun; Colman, Peter M; Czabotar, Peter E; Deshayes, Kurt; Fairbrother, Wayne J; Flygare, John A; Hymowitz, Sarah G; Jin, Sha; Judge, Russell A; Koehler, Michael F T; Kovar, Peter J; Lessene, Guillaume; Mitten, Michael J; Ndubaku, Chudi O; Nimmer, Paul; Purkey, Hans E; Oleksijew, Anatol; Phillips, Darren C; Sleebs, Brad E; Smith, Brian J; Smith, Morey L; Tahir, Stephen K; Watson, Keith G; Xiao, Yu; Xue, John; Zhang, Haichao; Zobel, Kerry; Rosenberg, Saul H; Tse, Chris; Leverson, Joel D; Elmore, Steven W; Souers, Andrew J

    2014-10-09

    A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.

  1. 5-Amino-pyrazoles as potent and selective p38[alpha] inhibitors

    SciTech Connect

    Das, Jagabandhu; Moquin, Robert V.; Dyckman, Alaric J.; Li, Tianle; Pitt, Sidney; Zhang, Rosemary; Shen, Ding Ren; McIntyre, Kim W.; Gillooly, Kathleen; Doweyko, Arthur M.; Newitt, John A.; Sack, John S.; Zhang, Hongjian; Kiefer, Susan E.; Kish, Kevin; McKinnon, Murray; Barrish, Joel C.; Dodd, John H.; Schieven, Gary L.; Leftheris, Katerina

    2012-02-07

    The synthesis and structure-activity relationships (SAR) of p38{alpha} MAP kinase inhibitors based on a 5-amino-pyrazole scaffold are described. These studies led to the identification of compound 2j as a potent and selective inhibitor of p38{alpha} MAP kinase with excellent cellular potency toward the inhibition of TNF{alpha} production. Compound 2j was highly efficacious in vivo in inhibiting TNF{alpha} production in an acute murine model of TNF{alpha} production. X-ray co-crystallography of a 5-amino-pyrazole analog 2f bound to unphosphorylated p38{alpha} is also disclosed.

  2. Structural Characterization of Inhibitors with Selectivity against Members of a Homologous Enzyme Family

    SciTech Connect

    Pavlovsky, Alexander G.; Liu, Xuying; Faehnle, Christopher R.; Potente, Nina; Viola, Ronald E.

    2013-01-31

    The aspartate biosynthetic pathway provides essential metabolites for many important biological functions, including the production of four essential amino acids. As this critical pathway is only present in plants and microbes, any disruptions will be fatal to these organisms. An early pathway enzyme, L-aspartate-{beta}-semialdehyde dehydrogenase, produces a key intermediate at the first branch point of this pathway. Developing potent and selective inhibitors against several orthologs in the L-aspartate-{beta}-semialdehyde dehydrogenase family can serve as lead compounds for antibiotic development. Kinetic studies of two small molecule fragment libraries have identified inhibitors that show good selectivity against L-aspartate-{beta}-semialdehyde dehydrogenases from two different bacterial species, Streptococcus pneumoniae and Vibrio cholerae, despite the presence of an identical constellation of active site amino acids in this homologous enzyme family. Structural characterization of enzyme-inhibitor complexes have elucidated different modes of binding between these structurally related enzymes. This information provides the basis for a structure-guided approach to the development of more potent and more selective inhibitors.

  3. Novel Selective Butyrylcholinesterase Inhibitors Incorporating Antioxidant Functionalities as Potential Bimodal Therapeutics for Alzheimer's Disease.

    PubMed

    Jones, Mike; Wang, Jun; Harmon, Shona; Kling, Beata; Heilmann, Jörg; Gilmer, John F

    2016-04-01

    Isosorbide-2-carbamates-5-aryl esters are highly potent and very selective butyrylcholinesterase inhibitors. The objective of the present work was to address the hypothesis that the isosorbide-aryl-5-ester group could be replaced with an antioxidant functionality while maintaining inhibitor effects and selectivity. We successfully incorporated ferulic acid or lipoic acid groups producing potent selective inhibitors of butyrylcholinesterase (BuChE). The hybrid compounds were non-toxic to the murine hippocampal cell line HT-22 and lipoate esters were neuroprotective at 10 and 25 µM when the cells were challenged with glutamate (5 mM) in a similar manner to the positive control quercetin. The benzyl carbamate 7a was a potent inhibitor of BuChE (IC50 150 nM) and it was effective in reducing glutamate toxicity to neuronal cells at >5 µM. Representative compounds exhibited an antioxidant effect in the oxygen radical absorbance capacity assay as the lipoate 7d was not active, whereas the ferulate 8a showed a weak, but significant, activity with 0.635 ± 0.020 Trolox Equivalent.

  4. Preferential and non-selective cyclooxygenase inhibitors reduce inflammation during lipopolysaccharide-induced synovitis.

    PubMed

    Morton, Alison J; Campbell, Nigel B; Gayle, J'mai M; Redding, W Rich; Blikslager, Anthony T

    2005-04-01

    Synovitis in horses is frequently treated by administration of non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenase isoforms (COX-1 and COX-2). Constitutively expressed COX-1 is involved in physiologic functions such as maintenance of gastric mucosal integrity, whereas COX-2 is up-regulated at sites of inflammation. Thus, COX-2 inhibitors reduce inflammation with reduced gastrointestinal side effects as compared to non-selective COX inhibitors. The objective of the present study was to compare the anti-inflammatory effects of the preferential COX-2 inhibitor etodolac with the non-selective COX inhibitor phenylbutazone in horses with lipopolysaccharide (LPS)-induced synovitis. Three groups of horses (n=6) received no treatment, phenylbutazone (4.4 mg/kg, IV, q12h), or etodolac (23 mg/kg, IV, q12h), respectively, 2-h following injection of LPS into one middle carpal joint. Synovial fluid was analyzed for white blood cell (WBC) count, and TXB2 and PGE2 levels. Phenylbutazone and etodolac significantly reduced WBC count 6 and 24-h following injection of LPS compared to untreated horses. In addition, both drugs significantly reduced PGE2 levels (P<0.05) 6-h following LPS injection, whereas the probable COX-1 prostanoid TXB2 was significantly reduced by phenylbutazone (P<0.05), but not etodolac. Etodolac may serve as a more selective anti-inflammatory agent than phenylbutazone for treatment of equine synovitis.

  5. Structural characterization of inhibitors with selectivity against members of a homologous enzyme family.

    PubMed

    Pavlovsky, Alexander G; Liu, Xuying; Faehnle, Christopher R; Potente, Nina; Viola, Ronald E

    2012-01-01

    The aspartate biosynthetic pathway provides essential metabolites for many important biological functions, including the production of four essential amino acids. As this critical pathway is only present in plants and microbes, any disruptions will be fatal to these organisms. An early pathway enzyme, l-aspartate-β-semialdehyde dehydrogenase, produces a key intermediate at the first branch point of this pathway. Developing potent and selective inhibitors against several orthologs in the l-aspartate-β-semialdehyde dehydrogenase family can serve as lead compounds for antibiotic development. Kinetic studies of two small molecule fragment libraries have identified inhibitors that show good selectivity against l-aspartate-β-semialdehyde dehydrogenases from two different bacterial species, Streptococcus pneumoniae and Vibrio cholerae, despite the presence of an identical constellation of active site amino acids in this homologous enzyme family. Structural characterization of enzyme-inhibitor complexes have elucidated different modes of binding between these structurally related enzymes. This information provides the basis for a structure-guided approach to the development of more potent and more selective inhibitors.

  6. Amino Acid Derivatives as New Zinc Binding Groups for the Design of Selective Matrix Metalloproteinase Inhibitors

    PubMed Central

    Giustiniano, Mariateresa; Agamennone, Mariangela; Rossello, Armando; Gomez-Monterrey, Isabel; Novellino, Ettore; Campiglia, Pietro; Vernieri, Ermelinda; Bertamino, Alessia; Carotenuto, Alfonso

    2013-01-01

    A number of matrix metalloproteinases (MMPs) are important medicinal targets for conditions ranging from rheumatoid arthritis to cardiomyopathy, periodontal disease, liver cirrhosis, multiple sclerosis, and cancer invasion and metastasis, where they showed to have a dual role, inhibiting or promoting important processes involved in the pathology. MMPs contain a zinc (II) ion in the protein active site. Small-molecule inhibitors of these metalloproteins are designed to bind directly to the active site metal ions. In an effort to devise new approaches to selective inhibitors, in this paper, we describe the synthesis and preliminary biological evaluation of amino acid derivatives as new zinc binding groups (ZBGs). The incorporation of selected metal-binding functions in more complex biphenyl sulfonamide moieties allowed the identification of one compound able to interact selectively with different MMP enzymatic isoforms. PMID:23555050

  7. Discovery and antiplatelet activity of a selective PI3Kβ inhibitor (MIPS-9922).

    PubMed

    Zheng, Zhaohua; Pinson, Jo-Anne; Mountford, Simon J; Orive, Stephanie; Schoenwaelder, Simone M; Shackleford, David; Powell, Andrew; Nelson, Erin M; Hamilton, Justin R; Jackson, Shaun P; Jennings, Ian G; Thompson, Philip E

    2016-10-21

    A series of amino-substituted triazines were developed and examined for PI3Kβ inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kβ selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kβ selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbβ3 activation and αIIbβ3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss.

  8. Novel, potent and selective inhibitors of protein kinase C show oral anti-inflammatory activity.

    PubMed

    Nixon, J S; Bishop, J; Bradshaw, D; Davis, P D; Hill, C H; Elliott, L H; Kumar, H; Lawton, G; Lewis, E J; Mulqueen, M

    1991-01-01

    Clarification of the precise role of protein kinase C (PKC) in cellular functional responses has been hampered by a lack of potent, selective inhibitors. The structural lead provided by staurosporine, a potent but non-selective protein kinase (PK) inhibitor, was used to derive a series of bis(indolyl)maleimides of which the most potent, Ro 31-8425 (I50: PKC = 8 nM) showed 350-fold selectivity for PKC over cAMP-dependent protein kinase. Ro 31-8425 antagonised cellular processes triggered by phorbol esters (potent, specific PKC activators) and inhibited the allogeneic mixed lymphocyte reaction, suggesting a role for PKC in T-cell activation. Methylation of the primary amine in Ro 31-8425 produced an analogue. Ro 31-8830 which, when administered orally, produced a dose-dependent inhibition of a phorbol ester-induced paw oedema in mice (minimum effective dose = 15 mg/kg). Ro 31-8830 also selectively inhibited the secondary inflammation in a developing adjuvant arthritis model in the rat. The results presented here suggest that these selective inhibitors of PKC may have therapeutic value in the treatment of T-cell-mediated autoimmune diseases.

  9. Antidepressant-Like Properties of Novel HDAC6-Selective Inhibitors with Improved Brain Bioavailability

    PubMed Central

    Jochems, Jeanine; Boulden, Janette; Lee, Bridgin G; Blendy, Julie A; Jarpe, Matthew; Mazitschek, Ralph; Van Duzer, John H; Jones, Simon; Berton, Olivier

    2014-01-01

    HDAC inhibitors have been reported to produce antidepressant and pro-cognitive effects in animal models, however, poor brain bioavailability or lack of isoform selectivity of current probes has limited our understanding of their mode of action. We report the characterization of novel pyrimidine hydroxyl amide small molecule inhibitors of HDAC6, brain bioavailable upon systemic administration. We show that two compounds in this family, ACY-738 and ACY-775, inhibit HDAC6 with low nanomolar potency and a selectivity of 60- to 1500-fold over class I HDACs. In contrast to tubastatin A, a reference HDAC6 inhibitor with similar potency and peripheral activity, but more limited brain bioavailability, ACY-738 and ACY-775 induce dramatic increases in α-tubulin acetylation in brain and stimulate mouse exploratory behaviors in novel, but not familiar environments. Interestingly, despite a lack of detectable effect on histone acetylation, we show that ACY-738 and ACY-775 share the antidepressant-like properties of other HDAC inhibitors, such as SAHA and MS-275, in the tail suspension test and social defeat paradigm. These effects of ACY-738 and ACY-775 are directly attributable to the inhibition of HDAC6 expressed centrally, as they are fully abrogated in mice with a neural-specific loss of function of HDAC6. Furthermore, administered in combination, a behaviorally inactive dose of ACY-738 markedly potentiates the anti-immobility activity of a subactive dose of the selective serotonin reuptake inhibitor citalopram. Our results validate new isoform-selective probes for in vivo pharmacological studies of HDAC6 in the CNS and reinforce the viability of this HDAC isoform as a potential target for antidepressant development. PMID:23954848

  10. Discovery of MK-1832, a Kv1.5 inhibitor with improved selectivity and pharmacokinetics.

    PubMed

    Wolkenberg, Scott E; Nolt, M Brad; Bilodeau, Mark T; Trotter, B Wesley; Manley, Peter J; Kett, Nathan R; Nanda, Kausik K; Wu, Zhicai; Cato, Matthew J; Kane, Stefanie A; Kiss, Laszlo; Spencer, Robert H; Wang, Jixin; Lynch, Joseph J; Regan, Christopher P; Stump, Gary L; Li, Bing; White, Rebecca; Yeh, Suzie; Dinsmore, Christopher J; Lindsley, Craig W; Hartman, George D

    2017-02-15

    Selective inhibition of Kv1.5, which underlies the ultra-rapid delayed rectifier current, IKur, has been pursued as a treatment for atrial fibrillation. Here we describe the discovery of MK-1832, a Kv1.5 inhibitor with improved selectivity versus the off-target current IKs, whose inhibition has been associated with ventricular proarrhythmia. MK-1832 exhibits improved selectivity for IKur over IKs (>3000-fold versus 70-fold for MK-0448), consistent with an observed larger window between atrial and ventricular effects in vivo (>1800-fold versus 210-fold for MK-0448). MK-1832 also exhibits an improved preclinical pharmacokinetic profile consistent with projected once daily dosing in humans.

  11. Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox.

    PubMed

    Guimarães, Ana P; de Souza, Felipe R; Oliveira, Aline A; Gonçalves, Arlan S; de Alencastro, Ricardo B; Ramalho, Teodorico C; França, Tanos C C

    2015-02-16

    Recently we constructed a homology model of the enzyme thymidylate kinase from Variola virus (VarTMPK) and proposed it as a new target to the drug design against smallpox. In the present work, we used the antivirals cidofovir and acyclovir as reference compounds to choose eleven compounds as leads to the drug design of inhibitors for VarTMPK. Docking and molecular dynamics (MD) studies of the interactions of these compounds inside VarTMPK and human TMPK (HssTMPK) suggest that they compete for the binding region of the substrate and were used to propose the structures of ten new inhibitors for VarTMPK. Further docking and MD simulations of these compounds, inside VarTMPK and HssTMPK, suggest that nine among ten are potential selective inhibitors of VarTMPK.

  12. (R)-2-Phenylpyrrolidine Substituted Imidazopyridazines: A New Class of Potent and Selective Pan-TRK Inhibitors

    PubMed Central

    2015-01-01

    Deregulated kinase activities of tropomyosin receptor kinase (TRK) family members have been shown to be associated with tumorigenesis and poor prognosis in a variety of cancer types. In particular, several chromosomal rearrangements involving TRKA have been reported in colorectal, papillary thyroid, glioblastoma, melanoma, and lung tissue that are believed to be the key oncogenic driver in these tumors. By screening the Novartis compound collection, a novel imidazopyridazine TRK inhibitor was identified that served as a launching point for drug optimization. Structure guided drug design led to the identification of (R)-2-phenylpyrrolidine substituted imidazopyridazines as a series of potent, selective, orally bioavailable pan-TRK inhibitors achieving tumor regression in rats bearing KM12 xenografts. From this work the (R)-2-phenylpyrrolidine has emerged as an ideal moiety to incorporate in bicyclic TRK inhibitors by virtue of its shape complementarity to the hydrophobic pocket of TRKs. PMID:26005534

  13. A SIRT2-selective inhibitor promotes c-Myc oncoprotein degradation and exhibits broad anticancer activity

    PubMed Central

    Jing, Hui; Hu, Jing; He, Bin; Negron Abril, Yashira L.; Stupinski, Jack; Weiser, Keren; Carbonaro, Marisa; Chiang, Ying-Ling; Southard, Teresa; Giannakakou, Paraskevi; Weiss, Robert S.; Lin, Hening

    2016-01-01

    Summary Targeting sirtuins for cancer treatment has been a topic of debate due to conflicting reports and lack of potent and specific inhibitors. We have developed a thiomyristoyl lysine compound, TM, as a potent SIRT2-specific inhibitor with broad anticancer effect in various human cancer cells and mouse models of breast cancer. Mechanistically, SIRT2 inhibition promotes c-Myc ubiquitination and degradation. The anticancer effect of TM correlates with its ability to decrease c-Myc level. TM had limited effects on non-cancerous cells and tumor-free mice, suggesting that cancer cells have an increased dependency on SIRT2 that can be exploited for therapeutic benefit. Our studies demonstrate that SIRT2-selective inhibitors are promising anticancer agents and may represent a general strategy to target certain c-Myc-driven cancers. PMID:26977881

  14. Design and Synthesis of Phenylpyrrolidine Phenylglycinamides As Highly Potent and Selective TF-FVIIa Inhibitors

    PubMed Central

    2013-01-01

    Inhibitors of the Tissue Factor/Factor VIIa (TF-FVIIa) complex are promising novel anticoagulants that show excellent efficacy and minimal bleeding in preclinical models. On the basis of a zwitterionic phenylglycine acylsulfonamide 1, a phenylglycine benzylamide 2 was shown to possess improved permeability and oral bioavailability. Optimization of the benzylamide, guided by X-ray crystallography, led to a potent TF-FVIIa inhibitor 18i with promising oral bioavailability, but promiscuous activity in an in vitro safety panel of receptors and enzymes. Introducing an acid on the pyrrolidine ring, guided by molecular modeling, resulted in highly potent, selective, and efficacious TF-FVIIa inhibitors with clean in vitro safety profile. The pyrrolidine acid 20 showed a moderate clearance, low volume of distribution, and a short t1/2 in dog PK studies. PMID:24900796

  15. Yeast as a tool to select inhibitors of the cullin deneddylating enzyme Csn5.

    PubMed

    Cirigliano, Angela; Stirpe, Alessandro; Menta, Sergio; Mori, Mattia; Dell'Edera, Domenico; Pick, Elah; Negri, Rodolfo; Botta, Bruno; Rinaldi, Teresa

    2016-12-01

    The CSN complex plays a key role in various cellular pathways: through a metalloprotease activity of its Csn5 deneddylating enzyme, it regulates the activity of Cullin-RING ligases (CRLs). Indeed, Csn5 has been found amplified in many tumors, but, due to its pleiotropic effects, it is difficult to dissect its function and the involvement in cancer progression. Moreover, while growing evidences point to the neddylation function as a good target for drug development; specific inhibitors have not yet been developed for the CSN. Here, we propose the yeast Saccharomyces cerevisiae as a model system to screen libraries of small molecules as inhibitors of cullins deneddylation, taking advantage of the unique feature of this organism to survive without a functional CSN5 gene and to accumulate a fully neddylated cullin substrate. By combining molecular modeling and simple genetic tools, we were able to identify two small molecular fragments as selective inhibitors of Csn5 deneddylation function.

  16. Potent and Selective Inhibition of Plasma Membrane Monoamine Transporter by HIV Protease Inhibitors

    PubMed Central

    Duan, Haichuan; Hu, Tao; Foti, Robert S.; Pan, Yongmei; Swaan, Peter W.

    2015-01-01

    Plasma membrane monoamine transporter (PMAT) is a major uptake-2 monoamine transporter that shares extensive substrate and inhibitor overlap with organic cation transporters 1–3 (OCT1–3). Currently, there are no PMAT-specific inhibitors available that can be used in in vitro and in vivo studies to differentiate between PMAT and OCT activities. In this study, we showed that IDT307 (4-(4-(dimethylamino)phenyl)-1-methylpyridinium iodide), a fluorescent analog of 1-methyl-4-phenylpyridinium (MPP+), is a transportable substrate for PMAT and that IDT307-based fluorescence assay can be used to rapidly identify and characterize PMAT inhibitors. Using the fluorescent substrate-based assays, we analyzed the interactions of eight human immunodeficiency virus (HIV) protease inhibitors (PIs) with human PMAT and OCT1–3 in human embryonic kidney 293 (HEK293) cells stably transfected with individual transporters. Our data revealed that PMAT and OCTs exhibit distinct sensitivity and inhibition patterns toward HIV PIs. PMAT is most sensitive to PI inhibition whereas OCT2 and OCT3 are resistant. OCT1 showed an intermediate sensitivity and a distinct inhibition profile from PMAT. Importantly, lopinavir is a potent PMAT inhibitor and exhibited >120 fold selectivity toward PMAT (IC50 = 1.4 ± 0.2 µM) over OCT1 (IC50 = 174 ± 40 µM). Lopinavir has no inhibitory effect on OCT2 or OCT3 at maximal tested concentrations. Lopinavir also exhibited no or much weaker interactions with uptake-1 monoamine transporters. Together, our results reveal that PMAT and OCTs have distinct specificity exemplified by their differential interaction with HIV PIs. Further, we demonstrate that lopinavir can be used as a selective PMAT inhibitor to differentiate PMAT-mediated monoamine and organic cation transport from those mediated by OCT1–3. PMID:26285765

  17. Preclinical testing of selective Aurora kinase inhibitors on a medullary thyroid carcinoma-derived cell line.

    PubMed

    Tuccilli, Chiara; Baldini, Enke; Prinzi, Natalie; Morrone, Stefania; Sorrenti, Salvatore; Filippini, Angelo; Catania, Antonio; Alessandrini, Stefania; Rendina, Roberta; Coccaro, Carmela; D'Armiento, Massimino; Ulisse, Salvatore

    2016-05-01

    Deregulated expression of the Aurora kinases (Aurora-A, B, and C) is thought to be involved in cell malignant transformation and genomic instability in several cancer types. Over the last decade, a number of small-molecule inhibitors of Aurora kinases have been developed, which have proved to efficiently restrain malignant cell growth and tumorigenicity. Regarding medullary thyroid carcinoma (MTC), we previously showed the efficacy of a pan-Aurora kinase inhibitor (MK-0457) in impairing growth and survival of the MTC-derived cell line TT. In the present study, we sought to establish if one of the Aurora kinases might represent a preferential target for MTC therapy. The effects of selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) were analyzed on TT cell proliferation, apoptosis, cell cycle, and ploidy. The two inhibitors reduced TT cell proliferation in a time- and dose-dependent manner, with IC50 of 19.0 ± 2.4 nM for MLN8237 and 401.6 ± 44.1 nM for AZD1152. Immunofluorescence experiments confirmed that AZD1152 inhibited phosphorylation of histone H3 (Ser10) by Aurora-B, while it did not affect Aurora-A autophosphorylation. MLN8237 inhibited Aurora-A autophosphorylation as expected, but at concentrations required to achieve the maximum antiproliferative effects it also abolished H3 (Ser10) phosphorylation. Cytofluorimetry experiments showed that both inhibitors induced accumulation of cells in G2/M phase and increased the subG0/G1 fraction and polyploidy. Finally, both inhibitors triggered apoptosis. We demonstrated that inhibition of either Aurora-A or Aurora-B has antiproliferative effects on TT cells, and thus it would be worthwhile to further investigate the therapeutical potential of Aurora kinase inhibitors in MTC treatment.

  18. Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes.

    PubMed

    Forsberg, Kevin J; Patel, Sanket; Witt, Evan; Wang, Bin; Ellison, Tyler D; Dantas, Gautam

    2015-11-06

    The production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications.

  19. Metabolic interactions of central nervous system medications and selective serotonin reuptake inhibitors.

    PubMed

    Naranjo, C A; Sproule, B A; Knoke, D M

    1999-05-01

    Selective serotonin reuptake inhibitors (SSRIs) are prescribed alone and in combination with other psychotropic medications in the treatment of a variety of psychiatric disorders. Such combinations create the potential for pharmacokinetic interactions by affecting the activity of the cytochromes P450 (CYP450), drug metabolizing oxidative enzymes. SSRIs are not equivalent in their potential for interactions when combined with other central nervous system (CNS) medication. Generally citalopram and sertraline are characterized by weaker inhibition of CYP450 enzymes and, therefore, hold less potential for interaction than the other SSRIs. Paroxetine potently inhibits CYP2D6, which can result in increased neuroleptic serum concentrations, accompanied by increased CNS side-effects. Similarly, as a potent inhibitor of CYP2D6, fluoxetine can increase serum concentrations of neuroleptics and antidepressants and numerous case reports have documented concomitant adverse events. Fluoxetine also inhibits CYP3A and CYP2C19, increasing serum concentrations of some benzodiazepines. Fluvoxamine is a potent inhibitor of CYP1A2, a moderate inhibitor of CYP3A and a mild inhibitor of CYP2D6. Therefore, interactions with clozapine and benzodiazepines are evident.

  20. Some selective serotonin reuptake inhibitors inhibit dynamin I guanosine triphosphatase (GTPase).

    PubMed

    Otomo, Masahiro; Takahashi, Kiyofumi; Miyoshi, Hiroshi; Osada, Kenichi; Nakashima, Hideki; Yamaguchi, Noboru

    2008-08-01

    Neuronal dynamin I plays a critical role in the recycling of synaptic vesicles, and thus in nervous system function. We expressed and purified dynamin I to explore potentially clinically useful endocytosis inhibitors and to examine the mechanism of their action. We estimated the IC(50) of nineteen psychotropic drugs for dynamin I. The IC(50) values of two selective serotonin reuptake inhibitors (sertraline and fluvoxamine) were 7.3+/-1.0 and 14.7+/-1.6 microM, respectively. Kinetic analyses revealed that fluvoxamine is a noncompetitive inhibitor of dynamin I guanosine triphosphatase (GTPase) with respect to guanosine 5'-triphosphate (GTP) and a competitive inhibitor with respect to L-phosphatidylserine (PS). Fluvoxamine may compete with PS for binding to the pleckstrin homology domain of dynamin I. On the other hand, sertraline was a mixed type inhibitor with respect to both GTP and PS. Our results indicate that sertraline and fluvoxamine may regulate the transportation of neurotransmitters by modulating synaptic vesicle endocytosis via the inhibition of dynamin I GTPase.

  1. Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes

    PubMed Central

    Forsberg, Kevin J.; Patel, Sanket; Witt, Evan; Wang, Bin; Ellison, Tyler D.

    2015-01-01

    The production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications. PMID:26546427

  2. Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors.

    PubMed

    Thangavelu, Bharani; Bhansali, Pravin; Viola, Ronald E

    2015-10-15

    Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme.

  3. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response.

    PubMed

    Kruidenier, Laurens; Chung, Chun-wa; Cheng, Zhongjun; Liddle, John; Che, KaHing; Joberty, Gerard; Bantscheff, Marcus; Bountra, Chas; Bridges, Angela; Diallo, Hawa; Eberhard, Dirk; Hutchinson, Sue; Jones, Emma; Katso, Roy; Leveridge, Melanie; Mander, Palwinder K; Mosley, Julie; Ramirez-Molina, Cesar; Rowland, Paul; Schofield, Christopher J; Sheppard, Robert J; Smith, Julia E; Swales, Catherine; Tanner, Robert; Thomas, Pamela; Tumber, Anthony; Drewes, Gerard; Oppermann, Udo; Patel, Dinshaw J; Lee, Kevin; Wilson, David M

    2012-08-16

    The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

  4. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

    SciTech Connect

    Kruidenier, Laurens; Chung, Chun-wa; Cheng, Zhongjun; Liddle, John; Che, KaHing; Joberty, Gerard; Bantscheff, Marcus; Bountra, Chas; Bridges, Angela; Diallo, Hawa; Eberhard, Dirk; Hutchinson, Sue; Jones, Emma; Katso, Roy; Leveridge, Melanie; Mander, Palwinder K.; Mosley, Julie; Ramirez-Molina, Cesar; Rowland, Paul; Schofield, Christopher J.; Sheppard, Robert J.; Smith, Julia E.; Swales, Catherine; Tanner, Robert; Thomas, Pamela; Tumber, Anthony; Drewes, Gerard; Oppermann, Udo; Patel, Dinshaw J.; Lee, Kevin; Wilson, David M.

    2012-10-11

    The jumonji (JMJ) family of histone demethylases are Fe{sup 2+}- and {alpha}-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

  5. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

    PubMed Central

    Kruidenier, Laurens; Chung, Chun-wa; Cheng, Zhongjun; Liddle, John; Che, KaHing; Joberty, Gerard; Bantscheff, Marcus; Bountra, Chas; Bridges, Angela; Diallo, Hawa; Eberhard, Dirk; Hutchinson, Sue; Jones, Emma; Katso, Roy; Leveridge, Melanie; Mander, Palwinder K.; Mosley, Julie; Ramirez-Molina, Cesar; Rowland, Paul; Schofield, Christopher J.; Sheppard, Robert J.; Smith, Julia E.; Swales, Catherine; Tanner, Robert; Thomas, Pamela; Tumber, Anthony; Drewes, Gerard; Oppermann, Udo; Patel, Dinshaw J.; Lee, Kevin; Wilson, David M.

    2015-01-01

    The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes1–4. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context5. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance2,4,6, as well as in development, physiology and disease3,7. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX)8. The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family. PMID:22842901

  6. Functional proteomics-aided selection of protease inhibitors for herbivore insect control

    PubMed Central

    Rasoolizadeh, Asieh; Munger, Aurélie; Goulet, Marie-Claire; Sainsbury, Frank; Cloutier, Conrad; Michaud, Dominique

    2016-01-01

    Studies have reported the potential of protease inhibitors to engineer insect resistance in transgenic plants but the general usefulness of this approach in crop protection still remains to be established. Insects have evolved strategies to cope with dietary protease inhibitors, such as the use of proteases recalcitrant to inhibition, that often make the selection of effective inhibitors very challenging. Here, we used a functional proteomics approach for the ‘capture’ of Cys protease targets in crude protein extracts as a tool to identify promising cystatins for plant improvement. Two cystatins found to differ in their efficiency to capture Cys proteases of the coleopteran pest Leptinotarsa decemlineata also differed in their usefulness to produce transgenic potato lines resistant to this insect. Plants expressing the most potent cystatin at high level had a strong repressing effect on larval growth and leaf intake, while plants expressing the weakest cystatin showed no effect on both two parameters compared to untransformed parental line used for genetic transformation. Our data underline the relevance of considering the whole range of possible protease targets when selecting an inhibitor for plant pest control. They also confirm the feasibility of developing cystatin-expressing transgenics resistant to a major pest of potato. PMID:27958307

  7. Discovery of Selective Small Molecule ROMK Inhibitors as Potential New Mechanism Diuretics

    PubMed Central

    2012-01-01

    The renal outer medullary potassium channel (ROMK or Kir1.1) is a putative drug target for a novel class of diuretics that could be used for the treatment of hypertension and edematous states such as heart failure. An internal high-throughput screening campaign identified 1,4-bis(4-nitrophenethyl)piperazine (5) as a potent ROMK inhibitor. It is worth noting that this compound was identified as a minor impurity in a screening hit that was responsible for all of the initially observed ROMK activity. Structure–activity studies resulted in analogues with improved rat pharmacokinetic properties and selectivity over the hERG channel, providing tool compounds that can be used for in vivo pharmacological assessment. The featured ROMK inhibitors were also selective against other members of the inward rectifier family of potassium channels. PMID:24900480

  8. Discovery of highly selective and potent p38 inhibitors based on a phthalazine scaffold.

    PubMed

    Herberich, Brad; Cao, Guo-Qiang; Chakrabarti, Partha P; Falsey, James R; Pettus, Liping; Rzasa, Robert M; Reed, Anthony B; Reichelt, Andreas; Sham, Kelvin; Thaman, Maya; Wurz, Ryan P; Xu, Shimin; Zhang, Dawei; Hsieh, Faye; Lee, Matthew R; Syed, Rashid; Li, Vivian; Grosfeld, David; Plant, Matthew H; Henkle, Bradley; Sherman, Lisa; Middleton, Scot; Wong, Lu Min; Tasker, Andrew S

    2008-10-23

    Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of p38 are described. These efforts originated from quinazoline 1 and through rational design led to the development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved by exploiting a collection of interactions with p38alpha including close contact to Ala157, occupation of the hydrophobic gatekeeper pocket, and a residue flip with Gly110. Substitutions on the phthalazine influenced the pharmacokinetic properties, of which compound 16 displayed the most desirable profile. Oral dosing (0.03 mg/kg) of 16 in rats 1 h prior to LPS challenge gave a >50% decrease in TNFalpha production.

  9. Discovery of selective ATP-competitive eIF4A3 inhibitors.

    PubMed

    Ito, Masahiro; Iwatani, Misa; Kamada, Yusuke; Sogabe, Satoshi; Nakao, Shoichi; Tanaka, Toshio; Kawamoto, Tomohiro; Aparicio, Samuel; Nakanishi, Atsushi; Imaeda, Yasuhiro

    2017-04-01

    Eukaryotic initiation factor 4A3 (eIF4A3), an ATP-dependent RNA helicase, is a core component of exon junction complex (EJC). EJC has a variety of roles in RNA metabolism such as translation, surveillance, and localization of spliced RNA. It is worthwhile to identify selective eIF4A3 inhibitors with a view to investigating the functions of eIF4A3 and EJC further to clarify the roles of the ATPase and helicase activities in cells. Our chemical optimization of hit compound 2 culminated in the discovery of ATP-competitive eIF4A3 inhibitor 18 with submicromolar ATPase inhibitory activity and excellent selectivity over other helicases. Hence, compound 18 could be a valuable chemical probe to elucidate the detailed functions of eIF4A3 and EJC.

  10. Combination therapy with selective serotonin reuptake inhibitors and phosphodiesterase-5 inhibitors in the treatment of premature ejaculation.

    PubMed

    Polat, E C; Ozbek, E; Otunctemur, A; Ozcan, L; Simsek, A

    2015-06-01

    We aimed to evaluate the effectiveness of paroxetine and tadalafil combination in the treatment of premature ejaculation (PE). A total of 150 primary (lifelong)PE patients were randomly distributed into three groups of 50 patients each. Group 1 received 20 mg paroxetine every day for 1 month, Group 2 received 20 mg tadalafil on demand 2 h before intercourse, and Group 3 received paroxetine and tadalafil on demand 2 h before intercourse. Intravaginal ejaculatory latency times (IELT) scores were evaluated at baseline, at the end of the first month of therapy and 1 month after discontinuation of the treatment, while International Index of Erectile Function (IIEF) questionnaire scores were evaluated both prior to and after the treatment. At the end of the first month of therapy, IELT scores were compared with the basal values and statistically significant changes were detected (60.6 ± 30.2-117.3 ± 67.3, 68.5 ± 21.4-110.2 ± 37.3, 71.56 ± 40.23-175.2 ± 60.2)(P < 0.01). IELT scores after discontinuation of treatment were found to be close to the baseline IELT scores (P > 0.05). IIEF scores were evaluated both prior to and after the treatment, and no statistically significant difference was detected (P > 0.05). It is concluded that utilisation of selective serotonin reuptake inhibitors (SSRI) and phosphodiesterase-5 inhibitors (PDE5i) combination before intercourse seems to provide significantly longer ejaculatory latency times as compared with SSRI alone for a long time in patients with PE.

  11. Structural Basis for the Design of Selective Phosphodiesterase 4B Inhibitors

    PubMed Central

    Fox, David; Burgin, Alex B.; Gurney, Mark E.

    2014-01-01

    Phosphodiesterase-4B (PDE4B) regulates the pro-inflammatory Toll Receptor –Tumor Necrosis Factor α (TNFα) pathway in monocytes, macrophages and microglial cells. As such, it is an important, although under-exploited molecular target for anti-inflammatory drugs. This is due in part to the difficulty of developing selective PDE4B inhibitors as the amino acid sequence of the PDE4 active site is identical in all PDE4 subtypes (PDE4A-D). We show that highly selective PDE4B inhibitors can be designed by exploiting sequence differences outside the active site. Specifically, PDE4B selectivity can be achieved by capture of a C-terminal regulatory helix, now termed CR3 (Control Region 3), across the active site in a conformation that closes access by cAMP. PDE4B selectivity is driven by a single amino acid polymorphism in CR3 (Leu674 in PDE4B1 versus Gln594 in PDE4D). The reciprocal mutations in PDE4B and PDE4D cause a 70-80 fold shift in selectivity. Our structural studies show that CR3 is flexible and can adopt multiple orientations and multiple registries in the closed conformation. The new co-crystal structure with bound ligand provides a guide map for the design of PDE4B selective anti-inflammatory drugs. PMID:24361374

  12. Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms

    PubMed Central

    2015-01-01

    JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile. PMID:26288683

  13. Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY‐876

    PubMed Central

    Siebeneicher, Holger; Cleve, Arwed; Rehwinkel, Hartmut; Neuhaus, Roland; Heisler, Iring; Müller, Thomas; Bauser, Marcus

    2016-01-01

    Abstract Despite the long‐known fact that the facilitative glucose transporter GLUT1 is one of the key players safeguarding the increase in glucose consumption of many tumor entities even under conditions of normal oxygen supply (known as the Warburg effect), only few endeavors have been undertaken to find a GLUT1‐selective small‐molecule inhibitor. Because other transporters of the GLUT1 family are involved in crucial processes, these transporters should not be addressed by such an inhibitor. A high‐throughput screen against a library of ∼3 million compounds was performed to find a small molecule with this challenging potency and selectivity profile. The N‐(1H‐pyrazol‐4‐yl)quinoline‐4‐carboxamides were identified as an excellent starting point for further compound optimization. After extensive structure–activity relationship explorations, single‐digit nanomolar inhibitors with a selectivity factor of >100 against GLUT2, GLUT3, and GLUT4 were obtained. The most promising compound, BAY‐876 [N 4‐[1‐(4‐cyanobenzyl)‐5‐methyl‐3‐(trifluoromethyl)‐1H‐pyrazol‐4‐yl]‐7‐fluoroquinoline‐2,4‐dicarboxamide], showed good metabolic stability in vitro and high oral bioavailability in vivo. PMID:27552707

  14. Development of Selective Inhibitors for Aldehyde Dehydrogenases Based on Substituted Indole-2,3-diones

    PubMed Central

    2015-01-01

    Aldehyde dehydrogenases (ALDH) participate in multiple metabolic pathways and have been indicated to play a role in several cancerous disease states. Our laboratory is interested in developing novel and selective ALDH inhibitors. We looked to further work recently published by developing a class of isoenzyme-selective inhibitors using similar indole-2,3-diones that exhibit differential inhibition of ALDH1A1, ALDH2, and ALDH3A1. Kinetic and X-ray crystallography data suggest that these inhibitors are competitive against aldehyde binding, forming direct interactions with active-site cysteine residues. The selectivity is precise in that these compounds appear to interact directly with the catalytic nucleophile, Cys243, in ALDH3A1 but not in ALDH2. In ALDH2, the 3-keto group is surrounded by the adjacent Cys301/303. Surprisingly, the orientation of the interaction changes depending on the nature of the substitutions on the basic indole ring structure and correlates well with the observed structure–activity relationships for each ALDH isoenzyme. PMID:24444054

  15. Bis-Aryl Urea Derivatives as Potent and Selective LIM Kinase (Limk) Inhibitors

    PubMed Central

    Yin, Yan; Zheng, Ke; Eid, Nibal; Howard, Shannon; Jeong, Ji-Hak; Yi, Fei; Guo, Jia; Park, Chul M; Bibian, Mathieu; Wu, Weilin; Hernandez, Pamela; Park, HaJeung; Wu, Yuntao; Luo, Jun-Li; LoGrasso, Philip V.; Feng, Yangbo

    2015-01-01

    The discovery/optimization of bis-aryl ureas as Limk inhibitors to obtain high potency and selectivity, and appropriate pharmacokinetic properties through systematic SAR studies is reported. Docking studies supported the observed SAR. Optimized Limk inhibitors had high biochemical potency (IC50 < 25 nM), excellent selectivity against ROCK and JNK kinases (> 400-fold), potent inhibition of cofilin phosphorylation in A7r5,PC-3, and CEM-SS T cells (IC50 < 1 μM), and good in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61 kinases, compound 18b at 1 μM inhibited only Limk1 and STK16 with ≥ 80% inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class of bis-aryl urea derived potent and selective Limk inhibitors. PMID:25621531

  16. Discovery of Potent and Selective Inhibitors of Human Reticulocyte 15- Lipoxygenase-1

    PubMed Central

    Rai, Ganesha; Kenyon, Victor; Jadhav, Ajit; Schultz, Lena; Armstrong, Michelle; Jameson, J Brian; Hoobler, Eric; Leister, William; Simeonov, Anton; Holman, Theodore R.; Maloney, David J.

    2010-01-01

    There are a variety of lipoxygenases in the human body (hLO), each having a distinct role in cellular biology. Human reticulocyte 15-Lipoxygenase-1 (15-hLO-1), which catalyzes the dioxygenation of 1,4-cis,cis-pentadiene-containing polyunsaturated fatty acids, is implicated in a number of diseases including cancer, atherosclerosis, and neurodegenerative conditions. Despite the potential therapeutic relevance of this target, few inhibitors have been reported that are both potent and selective. To this end, we have employed a quantitative high-throughput (qHTS) screen against ~74,000 small molecules in search of reticulocyte 15-hLO-1 selective inhibitors. This screen led to the discovery of a novel chemotype for 15-hLO-1 inhibition, which displays nM potency and is >7,500-fold selective against the related isozymes, 5-hLO, platelet 12-hLO, epithelial 15-hLO-2, ovine cyclooxygenase-1 and human cyclooxygenase-2. In addition, kinetic experiments were performed which indicate that this class of inhibitor is tight binding, reversible, and appears not to reduce the active-site ferric ion. PMID:20866075

  17. A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3)**

    PubMed Central

    Kaniskan, H. Ümit; Szewczyk, Magdalena M.; Yu, Zhengtian; Eram, Mohammad S.; Yang, Xiaobao; Schmidt, Keith; Luo, Xiao; Dai, Miao; He, Feng; Zang, Irene; Lin, Ying; Kennedy, Steven; Li, Fengling; Dobrovetsky, Elena; Dong, Aiping; Smil, David; Min, Sun-Joon; Landon, Melissa; Lin-Jones, Jennifer; Huang, Xi-Ping; Roth, Bryan L.; Schapira, Matthieu; Atadja, Peter; Barsyte-Lovejoy, Dalia; Arrowsmith, Cheryl H.; Brown, Peter J.; Zhao, Kehao; Jin, Jian; Vedadi, Masoud

    2015-01-01

    PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell- active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 = 31 ± 2 nm, KD = 53 ± 2 nm) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well- characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease. PMID:25728001

  18. Selective, tight-binding inhibitors of integrin alpha4beta1 that inhibit allergic airway responses.

    PubMed

    Lin, K c; Ateeq, H S; Hsiung, S H; Chong, L T; Zimmerman, C N; Castro, A; Lee, W C; Hammond, C E; Kalkunte, S; Chen, L L; Pepinsky, R B; Leone, D R; Sprague, A G; Abraham, W M; Gill, A; Lobb, R R; Adams, S P

    1999-03-11

    Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.

  19. Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies.

    PubMed

    Li, Yanhong; Cao, Hongzhi; Yu, Hai; Chen, Yi; Lau, Kam; Qu, Jingyao; Thon, Vireak; Sugiarto, Go; Chen, Xi

    2011-04-01

    Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.

  20. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

    PubMed

    Miller, J Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H James; Huband, Michael D; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y; Mehrens, Shawn; Mueller, W Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J V N Vara; Shelly, John A; Skerlos, Laura; Sulavik, Mark; Thomas, V Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C Kendall

    2009-02-10

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  1. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

    SciTech Connect

    Miller, J. Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H. James; Huband, Michael D.; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y.; Mehrens, Shawn; Mueller, W. Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J.V.N. Vara; Shelly, John A.; Skerlos, Laura; Sulavik, Mark; Thomas, V. Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C. Kendall

    2009-06-25

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  2. Structure-Based Design of Potent and Selective CK1γ Inhibitors

    PubMed Central

    2012-01-01

    Aberrant activation of the Wnt pathway is believed to drive the development and growth of some cancers. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We describe a structure-based approach that led to the discovery of a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms. A single oral dose of compound 13 resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model. PMID:24900428

  3. Synthesis and Biological Investigation of Oxazole Hydroxamates as Highly Selective Histone Deacetylase 6 (HDAC6) Inhibitors.

    PubMed

    Senger, Johanna; Melesina, Jelena; Marek, Martin; Romier, Christophe; Oehme, Ina; Witt, Olaf; Sippl, Wolfgang; Jung, Manfred

    2016-02-25

    Histone deacetylase 6 (HDAC6) catalyzes the removal of an acetyl group from lysine residues of several non-histone proteins. Here we report the preparation of thiazole-, oxazole-, and oxadiazole-containing biarylhydroxamic acids by a short synthetic procedure. We identified them as selective HDAC6 inhibitors by investigating the inhibition of recombinant HDAC enzymes and the protein acetylation in cells by Western blotting (tubulin vs histone acetylation). The most active compounds exhibited nanomolar potency and high selectivity for HDAC6. For example, an oxazole hydroxamate inhibits HDAC6 with an IC50 of 59 nM and has a selectivity index of >200 against HDAC1 and HDAC8. This is the first report showing that the nature of a heterocycle directly connected to a zinc binding group (ZBG) can be used to modulate subtype selectivity and potency for HDAC6 inhibitors to such an extent. We rationalize the high potency and selectivity of the oxazoles by molecular modeling and docking.

  4. Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library.

    PubMed

    Naimuddin, Mohammed; Kitamura, Koichirou; Kinoshita, Yasunori; Honda-Takahashi, Yoko; Murakami, Marina; Ito, Masato; Yamamoto, Kenji; Hanada, Kazunori; Husimi, Yuzuru; Nishigaki, Koichi

    2007-01-01

    A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.

  5. Development of fragment-based n-FABS NMR screening applied to the membrane enzyme FAAH.

    PubMed

    Lambruschini, Chiara; Veronesi, Marina; Romeo, Elisa; Garau, Gianpiero; Bandiera, Tiziano; Piomelli, Daniele; Scarpelli, Rita; Dalvit, Claudio

    2013-09-02

    Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹⁹F NMR spectroscopic methods, n-fluorine atoms for biochemical screening (n-FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n-FABS to the discovery of novel fragment hits, targeting the membrane-bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP-FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n-FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC₅₀) in the low mM-μM range. To the best of our knowledge, these results represent the first application of a ¹⁹F NMR fragment-based functional assay to a membrane protein.

  6. α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase: Exploration of Conformational Constraints in the Acyl Side Chain

    PubMed Central

    Duncan, Katharine K.; Otrubova, Katerina; Boger, Dale L.

    2014-01-01

    A series of α-ketooxazoles containing heteroatoms embedded within conformational constraints in the C2 acyl side chain of 2 (OL-135) were synthesized and evaluated as inhibitors of fatty acid amide hydrolase (FAAH). The studies reveal that the installation of a heteroatom (O) in the conformational constraint is achievable, although the potency of these novel derivatives is reduced slightly relative to 2 and the analogous 1,2,3,4-tetrahydronaphthalene series. Interestingly, both enantiomers (R and S) of the candidate inhibitors bearing a chiral center adjacent to the electrophilic carbonyl were found to effectively inhibit FAAH. PMID:24690529

  7. Kinetic and Structural Studies of Phosphodiesterase-8A and Implication on the Inhibitor Selectivity

    SciTech Connect

    Wang, H.; Yan, Z; Yang, S; Cai, J; Robinson, H; Ke, H

    2008-01-01

    Cyclic nucleotide phosphodiesterase-8 (PDE8) is a family of cAMP-specific enzymes and plays important roles in many biological processes, including T-cell activation, testosterone production, adrenocortical hyperplasia, and thyroid function. However, no PDE8 selective inhibitors are available for trial treatment of human diseases. Here we report kinetic properties of the highly active PDE8A1 catalytic domain prepared from refolding and its crystal structures in the unliganded and 3-isobutyl-1-methylxanthine (IBMX) bound forms at 1.9 and 2.1 Angstroms resolutions, respectively. The PDE8A1 catalytic domain has a KM of 1.8 eM, Vmax of 6.1 emol/min/mg, a kcat of 4.0 s-1 for cAMP, and a KM of 1.6 mM, Vmax of 2.5 emol/min/mg, a kcat of 1.6 s-1 for cGMP, thus indicating that the substrate specificity of PDE8 is dominated by KM. The structure of the PDE8A1 catalytic domain has similar topology as those of other PDE families but contains two extra helices around Asn685-Thr710. Since this fragment is distant from the active site of the enzyme, its impact on the catalysis is unclear. The PDE8A1 catalytic domain is insensitive to the IBMX inhibition (IC50 = 700 eM). The unfavorable interaction of IBMX in the PDE8A1-IBMX structure suggests an important role of Tyr748 in the inhibitor binding. Indeed, the mutation of Tyr748 to phenylalanine increases the PDE8A1 sensitivity to several nonselective or family selective PDE inhibitors. Thus, the structural and mutagenesis studies provide not only insight into the enzymatic properties but also guidelines for design of PDE8 selective inhibitors.

  8. SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV.

    PubMed

    Greco, George E; Matsumoto, Yoshihiro; Brooks, Rhys C; Lu, Zhengfei; Lieber, Michael R; Tomkinson, Alan E

    2016-07-01

    DNA ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA ligase inhibitors with differing selectivity for the three human DNA ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA ligase IV, inhibit the repair of DSBs by the DNA ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA ligases I and III than DNA ligase IV. Furthermore, SCR7-R failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA ligase IV.

  9. Discovery of a Selective Inhibitor of Oncogenic B-Raf Kinase With Potent Antimelanoma Activity

    SciTech Connect

    Tsai, J.; Lee, J.T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.; Bremer, R.; Gillette, S.; Kong, J.; Haass, N.K.; Sproesser, K.; Li, L.; Smalley, K.S.M.; Fong, D.; Zhu, Y.-L.; Marimuthu, A.; Nguyen, H.; Lam, B.; Liu, J.; Cheung, I.; Rice, J.

    2009-05-26

    BRAF{sup V600E} is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting 'active' protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf{sup V600E} with an IC{sub 50} of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf{sup V600E} kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf{sup V600E}-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf{sup V600E}-positive cells. In B-Raf{sup V600E}-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf{sup V600E}-driven tumors.

  10. Overcoming EGFR T790M and C797S resistance with mutant-selective allosteric inhibitors

    PubMed Central

    Jia, Yong; Yun, Cai-Hong; Park, Eunyoung; Ercan, Dalia; Manuia, Mari; Juarez, Jose; Xu, Chunxiao; Rhee, Kevin; Chen, Ting; Zhang, Haikuo; Palakurthi, Sangeetha; Jang, Jaebong; Lelais, Gerald; DiDonato, Michael; Bursulaya, Badry; Michellys, Pierre-Yves; Epple, Robert; Marsilje, Thomas H.; McNeill, Matthew; Lu, Wenshuo; Harris, Jennifer; Bender, Steven; Wong, Kwok-Kin; Jänne, Pasi A.; Eck, Michael J.

    2016-01-01

    EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.3,4 Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors. PMID:27251290

  11. Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors.

    PubMed

    Basile, Kevin J; Le, Kaitlyn; Hartsough, Edward J; Aplin, Andrew E

    2014-05-01

    Vemurafenib and dabrafenib block MEK-ERK1/2 signaling and cause tumor regression in the majority of advanced-stage BRAF(V600E) melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next-generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF(V600E) melanoma cells without paradoxical effects in wild-type cells. Furthermore, PLX7904 blocks the growth of vemurafenib-resistant BRAF(V600E) cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF(V600E) splice variant-mediated vemurafenib-resistant cells. We show that paradox-breaker RAF inhibitors potently block MEK-ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720-resistant cells harboring distinct BRAF(V600E) splice variants. These data support the further investigation of paradox-breaker RAF inhibitors as a second-line treatment option for patients failing on vemurafenib or dabrafenib.

  12. Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ.

    PubMed

    Rutaganira, Florentine U; Fowler, Melissa L; McPhail, Jacob A; Gelman, Michael A; Nguyen, Khanh; Xiong, Anming; Dornan, Gillian L; Tavshanjian, Brandon; Glenn, Jeffrey S; Shokat, Kevan M; Burke, John E

    2016-03-10

    Type III phosphatidylinositol 4-kinase (PI4KIIIβ) is an essential enzyme in mediating membrane trafficking and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles and may lead to novel antiviral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design, we have designed novel potent and selective (>1000-fold over class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed antiviral activity against hepatitis C virus. The co-crystal structure of PI4KIIIβ bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIβ inhibitors, which may play significant roles as antiviral therapeutics.

  13. A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity

    DOE PAGES

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; ...

    2015-01-09

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes withmore » different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. As a result, these findings offer a potential strategy for further ligand optimization.« less

  14. A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

    PubMed Central

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-01-01

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

  15. Design, Synthesis and Evaluation of Marinopyrrole Derivatives as Selective Inhibitors of Mcl-1 Binding to Pro-apoptotic Bim and Dual Mcl-1/Bcl-xL Inhibitors

    PubMed Central

    Li, Rongshi; Daniel, Kenyon G.; Li, Jerry; Qin, Yong; Gavathiotis, Evripidis; Sebti, Said M.

    2015-01-01

    Inhibition of anti-apoptotic Mcl-1 is a promising anticancer strategy to overcome the survival and chemoresistance of a broad spectrum of human cancers. We previously reported on the identification of a natural product marinopyrrole A (1) that induces apoptosis in Mcl-1-dependent cells through Mcl-1 degradation. Here, we report the design and synthesis of novel marinopyrrole-based analogues and their evaluation as selective inhibitors of Mcl-1 as well as dual Mcl-1/Bcl-xL inhibitors. The most selective Mcl-1 antagonists were 34, 36 and 37 with 16-, 13- and 9-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding, respectively. Among the most potent dual inhibitors is 42 which inhibited Mcl-1/Bim and Bcl-xL/Bim binding 15-fold (IC50 = 600 nM) and 33-fold (500 nM) more potently than (±)-marinopyrrole A (1), respectively. Fluorescence quenching, NMR analysis and molecular docking indicated binding of marinopyrroles to the BH3 binding site of Mcl-1. Several marinopyrroles potently decreased Mcl-1 cellular levels and induced caspase 3 activation in human breast cancer cells. Our studies provide novel “lead” marinopyrroles for further optimization as selective Mcl-1 inhibitors and dual Mcl-1 and Bcl-xL inhibitors. PMID:25437618

  16. Individual and Additive Effects of the CNR1 and FAAH Genes on Brain Response to Marijuana Cues

    PubMed Central

    Filbey, Francesca M; Schacht, Joseph P; Myers, Ursula S; Chavez, Robert S; Hutchison, Kent E

    2010-01-01

    As previous work has highlighted the significance of the cannabinoid receptor 1 (CNR1) and fatty acid amide hydrolase (FAAH) genes with respect to cannabis dependence (CD), this study sought to characterize the neural mechanisms that underlie these genetic effects. To this end, we collected DNA samples and fMRI data using a cue-elicited craving paradigm in thirty-seven 3-day-abstinent regular marijuana users. The participants were grouped according to their genotype on two single-nucleotide polymorphisms (SNPs) earlier associated with CD phenotypes: rs2023239 in CNR1 and rs324420 in FAAH. Between-group comparisons showed that carriers of the CNR1 rs2023239 G allele had significantly greater activity in reward-related areas of the brain, such as the orbitofrontal cortex (OFC), inferior frontal gyrus (IFG), and anterior cingulate gyrus (ACG), during exposure to marijuana cues, as compared with those with the A/A genotype for this SNP. The FAAH group contrasts showed that FAAH rs324420 C homozygotes also had greater activation in widespread areas within the reward circuit, specifically in the OFC, ACG, and nucleus accumbens (NAc), as compared with the FAAH A-allele carriers. Moreover, there was a positive correlation between neural response in OFC and NAc and the total number of risk alleles (cluster-corrected p<0.05). These findings are in accord with earlier reported associations between CNR1 and FAAH and CD intermediate phenotypes, and suggest that the underlying mechanism of these genetic effects may be enhanced neural response in reward areas of the brain in carriers of the CNR1 G allele and FAAH C/C genotype in response to marijuana cues. PMID:20010552

  17. Individual and additive effects of the CNR1 and FAAH genes on brain response to marijuana cues.

    PubMed

    Filbey, Francesca M; Schacht, Joseph P; Myers, Ursula S; Chavez, Robert S; Hutchison, Kent E

    2010-03-01

    As previous work has highlighted the significance of the cannabinoid receptor 1 (CNR1) and fatty acid amide hydrolase (FAAH) genes with respect to cannabis dependence (CD), this study sought to characterize the neural mechanisms that underlie these genetic effects. To this end, we collected DNA samples and fMRI data using a cue-elicited craving paradigm in thirty-seven 3-day-abstinent regular marijuana users. The participants were grouped according to their genotype on two single-nucleotide polymorphisms (SNPs) earlier associated with CD phenotypes: rs2023239 in CNR1 and rs324420 in FAAH. Between-group comparisons showed that carriers of the CNR1 rs2023239 G allele had significantly greater activity in reward-related areas of the brain, such as the orbitofrontal cortex (OFC), inferior frontal gyrus (IFG), and anterior cingulate gyrus (ACG), during exposure to marijuana cues, as compared with those with the A/A genotype for this SNP. The FAAH group contrasts showed that FAAH rs324420 C homozygotes also had greater activation in widespread areas within the reward circuit, specifically in the OFC, ACG, and nucleus accumbens (NAc), as compared with the FAAH A-allele carriers. Moreover, there was a positive correlation between neural response in OFC and NAc and the total number of risk alleles (cluster-corrected p<0.05). These findings are in accord with earlier reported associations between CNR1 and FAAH and CD intermediate phenotypes, and suggest that the underlying mechanism of these genetic effects may be enhanced neural response in reward areas of the brain in carriers of the CNR1 G allele and FAAH C/C genotype in response to marijuana cues.

  18. Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

    PubMed Central

    Kasbekar, Monica; Fischer, Gerhard; Mott, Bryan T.; Yasgar, Adam; Hyvönen, Marko; Boshoff, Helena I. M.; Abell, Chris; Barry, Clifton E.; Thomas, Craig J.

    2016-01-01

    Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen. PMID:27325754

  19. Cheminformatics-driven discovery of selective, nanomolar inhibitors for staphylococcal pyruvate kinase.

    PubMed

    Axerio-Cilies, Peter; See, Raymond H; Zoraghi, Roya; Worral, Liam; Lian, Tian; Stoynov, Nikolay; Jiang, Jihong; Kaur, Sukhbir; Jackson, Linda; Gong, Huansheng; Swayze, Rick; Amandoron, Emily; Kumar, Nag S; Moreau, Anne; Hsing, Michael; Strynadka, Natalie C; McMaster, William R; Finlay, B Brett; Foster, Leonard J; Young, Robert N; Reiner, Neil E; Cherkasov, Artem

    2012-02-17

    We have recently mapped the protein interaction network of methicillin-resistant Staphylococcus aureus (MRSA), which revealed its scale-free organization with characteristic presence of highly connected hub proteins that are critical for bacterial survival. Here we report the discovery of inhibitors that are highly potent against one such hub target, staphylococcal pyruvate kinase (PK). Importantly, the developed compounds demonstrate complete selectivity for the bacterial enzyme compared to all human orthologues. The lead 91nM inhibitor IS-130 has been identified through ligand-based cheminformatic exploration of a chemical space around micromolar hits initially generated by experimental screening. The following crystallographic study resulted in identification of a tetrameric MRSA PK structure where IS-130 is bound to the interface between the protein's subunits. This newly described binding pocket is not present in otherwise highly similar human orthologues and can be effectively utilized for selective inhibition of bacterial PK. The following synthetic modifications of IS-130, guided by structure-based molecular modeling, resulted in the development of MRSA PK inhibitors with much improved antimicrobial properties. Considering a notable lack of recent reports on novel antibacterial targets and cognate antibacterial compounds, this study provides a valuable perspective on the development of a new generation of antimicrobials. Equally noteworthy, the results of the current work highlight the importance of rigorous cheminformatics-based exploration of the results of high-throughput experiments.

  20. Selective and potent urea inhibitors of Cryptosporidium parvum inosine 5′ monophosphate dehydrogenase

    PubMed Central

    Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Liu, Xiaoping; Sharling, Lisa; Gollapalli, Deviprasad R.; Striepen, Boris; Hedstrom, Lizbeth; Cuny, Gregory D.

    2012-01-01

    Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC50 < 2 nM) against CpIMPDH, excellent selectivity > 1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model. PMID:22950983

  1. Potent and Selective Inhibitors of Human Reticulocyte 12/15-Lipoxygenase as Anti-Stroke Therapies

    PubMed Central

    2015-01-01

    A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition that has nanomolar potency (IC50 = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Lastly, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke. PMID:24684213

  2. The 2′-Trifluoromethyl Analogue of Indomethacin Is a Potent and Selective COX-2 Inhibitor

    PubMed Central

    2013-01-01

    Indomethacin is a potent, time-dependent, nonselective inhibitor of the cyclooxygenase enzymes (COX-1 and COX-2). Deletion of the 2′-methyl group of indomethacin produces a weak, reversible COX inhibitor, leading us to explore functionality at that position. Here, we report that substitution of the 2′-methyl group of indomethacin with trifluoromethyl produces CF3–indomethacin, a tight-binding inhibitor with kinetic properties similar to those of indomethacin and unexpected COX-2 selectivity (IC50 mCOX-2 = 267 nM; IC50 oCOX-1 > 100 μM). Studies with site-directed mutants reveal that COX-2 selectivity results from insertion of the CF3 group into a small hydrophobic pocket formed by Ala-527, Val-349, Ser-530, and Leu-531 and projection of the methoxy group toward a side pocket bordered by Val-523. CF3–indomethacin inhibited COX-2 activity in human head and neck squamous cell carcinoma cells and exhibited in vivo anti-inflammatory activity in the carrageenan-induced rat paw edema model with similar potency to that of indomethacin. PMID:23687559

  3. The 2'-Trifluoromethyl Analogue of Indomethacin Is a Potent and Selective COX-2 Inhibitor.

    PubMed

    Blobaum, Anna L; Uddin, Md Jashim; Felts, Andrew S; Crews, Brenda C; Rouzer, Carol A; Marnett, Lawrence J

    2013-05-09

    Indomethacin is a potent, time-dependent, nonselective inhibitor of the cyclooxygenase enzymes (COX-1 and COX-2). Deletion of the 2'-methyl group of indomethacin produces a weak, reversible COX inhibitor, leading us to explore functionality at that position. Here, we report that substitution of the 2'-methyl group of indomethacin with trifluoromethyl produces CF3-indomethacin, a tight-binding inhibitor with kinetic properties similar to those of indomethacin and unexpected COX-2 selectivity (IC50 mCOX-2 = 267 nM; IC50 oCOX-1 > 100 μM). Studies with site-directed mutants reveal that COX-2 selectivity results from insertion of the CF3 group into a small hydrophobic pocket formed by Ala-527, Val-349, Ser-530, and Leu-531 and projection of the methoxy group toward a side pocket bordered by Val-523. CF3-indomethacin inhibited COX-2 activity in human head and neck squamous cell carcinoma cells and exhibited in vivo anti-inflammatory activity in the carrageenan-induced rat paw edema model with similar potency to that of indomethacin.

  4. Structure-Based Design of an in Vivo Active Selective BRD9 Inhibitor

    PubMed Central

    2016-01-01

    Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings. PMID:26914985

  5. Discovery of benzoylisoindolines as a novel class of potent, selective and orally active GlyT1 inhibitors.

    PubMed

    Pinard, Emmanuel; Alberati, Daniela; Bender, Markus; Borroni, Edilio; Brom, Virginie; Burner, Serge; Fischer, Holger; Hainzl, Dominik; Halm, Remy; Hauser, Nicole; Jolidon, Synèse; Lengyel, Judith; Marty, Hans-Peter; Meyer, Thierry; Moreau, Jean-Luc; Mory, Roland; Narquizian, Robert; Norcross, Roger D; Schmid, Philipp; Wermuth, Roger; Zimmerli, Daniel

    2010-12-01

    Benzoylisoindolines were discovered as a novel structural class of GlyT1 inhibitors. SAR studies and subsequent lead optimization efforts focused primarily on addressing hERG liability and on improving in vivo efficacy resulted in the identification of potent GlyT1 inhibitors displaying excellent selectivity and in vivo PD and PK profiles.

  6. The design and synthesis of 5- and 6-isoxazolylbenzimidazoles as selective inhibitors of the BET bromodomains†‡

    PubMed Central

    Hay, Duncan; Fedorov, Oleg; Filippakopoulos, Panagis; Martin, Sarah; Philpott, Martin; Picaud, Sarah; Hewings, David S.; Uttakar, Sagar; Heightman, Tom D.; Conway, Stuart J.; Knapp, Stefan; Brennan, Paul E.

    2015-01-01

    Simple 1-substituted 5- and 6-isoxazolyl-benzimidazoles have been shown to be potent inhibitors of the BET bromodomains with selectivity over the related bromodomain of CBP. The reported inhibitors were prepared from simple starting materials in two steps followed by separation of the regioisomers or regioselectively in three steps. PMID:26682033

  7. Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).

    PubMed

    Bahudhanapati, Harinath; Zhang, Yingnan; Sidhu, Sachdev S; Brew, Keith

    2011-09-09

    Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.

  8. Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines.

    PubMed

    Baldini, Enke; Tuccilli, Chiara; Prinzi, Natalie; Sorrenti, Salvatore; Antonelli, Alessandro; Gnessi, Lucio; Morrone, Stefania; Moretti, Costanzo; Bononi, Marco; Arlot-Bonnemains, Yannick; D'Armiento, Massimino; Ulisse, Salvatore

    2014-10-01

    Aurora kinases are serine/threonine kinases that play an essential role in cell division. Their aberrant expression and/or function induce severe mitotic abnormalities, resulting in either cell death or aneuploidy. Overexpression of Aurora kinases is often found in several malignancies, among which is anaplastic thyroid carcinoma (ATC). We have previously demonstrated the in vitro efficacy of Aurora kinase inhibitors in restraining cell growth and survival of different ATC cell lines. In this study, we sought to establish which Aurora might represent the preferential drug target for ATC. To this end, the effects of two selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) on four human ATC cell lines (CAL-62, BHT-101, 8305C, and 8505C) were analysed. Both inhibitors reduced cell proliferation in a time- and dose-dependent manner, with IC50 ranges of 44.3-134.2 nM for MLN8237 and of 9.2-461.3 nM for AZD1152. Immunofluorescence experiments and time-lapse videomicroscopy yielded evidence that each inhibitor induced distinct mitotic phenotypes, but both of them prevented the completion of cytokinesis. As a result, poliploidy increased in all AZD1152-treated cells, and in two out of four cell lines treated with MLN8237. Apoptosis was induced in all the cells by MLN8237, and in BHT-101, 8305C, and 8505C by AZD1152, while CAL-62 exposed to AZD1152 died through necrosis after multiple rounds of endoreplication. Both inhibitors were capable of blocking anchorage-independent cell growth. In conclusion, we demonstrated that either Aurora-A or Aurora-B might represent therapeutic targets for the ATC treatment, but inhibition of Aurora-A appears more effective for suppressing ATC cell proliferation and for inducing the apoptotic pathway.

  9. Design, synthesis, and 3D QSAR of novel potent and selective aromatase inhibitors.

    PubMed

    Leonetti, Francesco; Favia, Angelo; Rao, Angela; Aliano, Rosaria; Paluszcak, Anja; Hartmann, Rolf W; Carotti, Angelo

    2004-12-30

    The design, synthesis, and biological evaluation of a series of new aromatase inhibitors bearing an imidazole or triazole ring linked to a fluorene (A), indenodiazine (B), or coumarin scaffold (C) are reported. Properly substituted coumarin derivatives displayed the highest aromatase inhibitory potency and selectivity over 17-alpha-hydroxylase/17-20 lyase. The modeling of the aromatase inhibition data by Comparative Molecular Field Analysis (CoMFA/GOLPE 3D QSAR approach) led to the development of a PLS model with good fitting and predictive powers (n = 22, ONC = 3, r(2) = 0.949, s = 0.216, and q(2) = 0.715). The relationship between aromatase inhibition and the steric and electrostatic fields generated by the examined azole inhibitors enables a clear understanding of the nature and spatial location of the main interactions modulating the aromatase inhibitory potency.

  10. Discovery of selective and orally available spiro-3-piperidyl ATP-competitive MK2 inhibitors.

    PubMed

    Kaptein, Allard; Oubrie, Arthur; de Zwart, Edwin; Hoogenboom, Niels; de Wit, Joeri; van de Kar, Bas; van Hoek, Maaike; Vogel, Gerard; de Kimpe, Vera; Schultz-Fademrecht, Carsten; Borsboom, Judith; van Zeeland, Mario; Versteegh, Judith; Kazemier, Bert; de Roos, Jeroen; Wijnands, Frank; Dulos, John; Jaeger, Martin; Leandro-Garcia, Paula; Barf, Tjeerd

    2011-06-15

    The identification of a potent, selective, and orally available MK2 inhibitor series is described. The initial absence of oral bioavailability was successfully tackled by moving the basic nitrogen of the spiro-4-piperidyl moiety towards the electron-deficient pyrrolepyridinedione core, thereby reducing the pK(a) and improving Caco-2 permeability. The resulting racemic spiro-3-piperidyl analogues were separated by chiral preparative HPLC, and the activity towards MK2 inhibition was shown to reside mostly in the first eluting stereoisomer. This led to the identification of new MK2 inhibitors, such as (S)-23, with low nanomolar biochemical inhibition (EC(50) 7.4 nM) and submicromolar cellular target engagement activity (EC(50) 0.5 μM).

  11. Synthesis of stable and selective inhibitors of human galectins-1 and -3.

    PubMed

    Giguère, Denis; Bonin, Marc-André; Cloutier, Philipe; Patnam, Ramesh; St-Pierre, Christian; Sato, Sachiko; Roy, René

    2008-08-15

    The syntheses of glycolytically stable galactosides and lactosides have been made toward the selective inhibition of human galectins-1 and -3. Transition metal-catalyzed cross-coupling reactions were used to create carbon-carbon bond formation (Sonogashira, Suzuki, Heck, Glaser). Additionally, Hantzsch condensation was used to create novel 2-aminothiazoles which reacted with a panel of acylating and sulfonylating reagents. Moreover, dimeric galactosides and lactosides bearing triazoles, regiospecifically prepared using copper-catalyzed Huisgen azide-alkyne [1,3]-dipolar cycloaddition, provided efficient galectins-1 and -3 inhibitors. Best monovalent inhibitor among the tested series was (E)-methyl 2-phenyl-4-(beta-D-galactopyranosyl)-but-2-enoate 15 with inhibitory potency of 313 microM against galectin-1 and best dimers were bis-lactoside 68 and 75 having both inhibitory properties of 160 microM against Galectin-3.

  12. Tetrahydroisoquinoline-7-carboxamide Derivatives as New Selective Discoidin Domain Receptor 1 (DDR1) Inhibitors.

    PubMed

    Wang, Zhen; Zhang, Yali; Bartual, Sergio G; Luo, Jinfeng; Xu, Tingting; Du, Wenting; Xun, Qiuju; Tu, Zhengchao; Brekken, Rolf A; Ren, Xiaomei; Bullock, Alex N; Liang, Guang; Lu, Xiaoyun; Ding, Ke

    2017-03-09

    Acute lung injury (ALI) is a deadly symptom for serious lung inflammation. Discoidin Domain Receptor 1 (DDR1) is a new potential target for anti-inflammatory drug discovery. A new selective tetrahydroisoquinoline-7-carboxamide based DDR1 inhibitor 7ae was discovered to tightly bind the DDR1 protein and potently inhibit its kinase function with a Kd value of 2.2 nM and an IC50 value of 6.6 nM, respectively. The compound dose-dependently inhibited lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) release in mouse primary peritoneal macrophages (MPMs). In addition, 7ae also exhibited promising in vivo anti-inflammatory effects in a LPS-induced mouse ALI model. To the best of our knowledge, this is the first "proof of concept" investigation on the potential application of a small molecule DDR1 inhibitor to treat ALI.

  13. Relation between flexibility and positively selected HIV-1 protease mutants against inhibitors.

    PubMed

    Braz, Antônio S K; Tufanetto, Patrícia; Perahia, David; Scott, Luis P B

    2012-12-01

    The antiretroviral chemotherapy helps to reduce the mortality of HIVs infected patients. However, RNA dependant virus replication has a high mutation rate. Human immunodeficiency virus Type 1 protease plays an essential role in viral replication cycle. This protein is an important target for therapy with viral protein inhibitors. There are few works using normal mode analysis to investigate this problem from the structural changes viewpoint. The investigation of protein flexibility may be important for the study of processes associated with conformational changes and state transitions. The normal mode analysis allowed us to investigate structural changes in the protease (such as flexibility) in a straightforward way and try to associate these changes with the increase of fitness for each positively selected HIV-1 mutant protease of patients treated with several protease inhibitors (saquinavir, indinavir, ritonavir, nelfinavir, lopinavir, fosamprenavir, atazanavir, darunavir, and tripanavir) in combination or separately. These positively selected mutations introduce significant flexibility in important regions such as the active site cavity and flaps. These mutations were also able to cause changes in accessible solvent area. This study showed that the majority of HIV-1 protease mutants can be grouped into two main classes of protein flexibility behavior. We presented a new approach to study structural changes caused by positively selected mutations in a pathogen protein, for instance the HIV-1 protease and their relationship with their resistance mechanism against known inhibitors. The method can be applied to any pharmaceutically relevant pathogen proteins and could be very useful to understand the effects of positively selected mutations in the context of structural changes.

  14. Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

    SciTech Connect

    Lin, Yen -Lin; Meng, Yilin; Huang, Lei; Roux, Benoît

    2014-10-22

    Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to the DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.

  15. Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

    DOE PAGES

    Lin, Yen -Lin; Meng, Yilin; Huang, Lei; ...

    2014-10-22

    Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to themore » DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.« less

  16. Diverse modes of binding in structures of Leishmania major N-myristoyltransferase with selective inhibitors

    PubMed Central

    Brannigan, James A.; Roberts, Shirley M.; Bell, Andrew S.; Hutton, Jennie A.; Hodgkinson, Michael R.; Tate, Edward W.; Leatherbarrow, Robin J.; Smith, Deborah F.; Wilkinson, Anthony J.

    2014-01-01

    The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed. PMID:25075346

  17. Selective non-steroidal inhibitors of 5 alpha-reductase type 1.

    PubMed

    Occhiato, Ernesto G; Guarna, Antonio; Danza, Giovanna; Serio, Mario

    2004-01-01

    The enzyme 5 alpha-reductase (5 alpha R) catalyses the reduction of testosterone (T) into the more potent androgen dihydrotestosterone (DHT). The abnormal production of DHT is associated to pathologies of the main target organs of this hormone: the prostate and the skin. Benign prostatic hyperplasia (BPH), prostate cancer, acne, androgenetic alopecia in men, and hirsutism in women appear related to the DHT production. Two isozymes of 5 alpha-reductase have been cloned, expressed and characterized (5 alpha R-1 and 5 alpha R-2). They share a poor homology, have different chromosomal localization, enzyme kinetic parameters, and tissue expression patterns. Since 5 alpha R-1 and 5 alpha R-2 are differently distributed in the androgen target organs, a different involvement of the two isozymes in the pathogenesis of prostate and skin disorders can be hypothesized. High interest has been paid to the synthesis of inhibitors of 5 alpha-reductase for the treatment of DHT related pathologies, and the selective inhibition of any single isozyme represents a great challenge for medical and pharmaceutical research in order to have more specific drugs. At present, no 5 alpha R-1 inhibitor is marketed for the treatment of 5 alpha R-1 related pathologies but pharmaceutical research is very active in this field. This paper will review the major classes of 5 alpha R inhibitors focusing in particular on non-steroidal inhibitors and on structural features that enhance the selectivity versus the type 1 isozyme. Biological tests to assess the inhibitory activity towards the two 5 alpha R isozymes will be also discussed.

  18. The selective norepinephrine reuptake inhibitor antidepressant reboxetine: pharmacological and clinical profile.

    PubMed

    Hajós, Mihály; Fleishaker, Joseph C; Filipiak-Reisner, Jacqueline K; Brown, Mark T; Wong, Erik H F

    2004-01-01

    Reboxetine is the first commercially available norepinephrine reuptake inhibitor developed specifically as a first line therapy for major depressive disorder. In vitro and in vivo pharmacological studies indicated that reboxetine methanesulphonate has high affinity and selectivity for the human norepinephrine transporter over the serotonin and dopamine transporters. Pharmacological specificity is further demonstrated by the absence of affinity for 45 transmitter receptors and CNS targets. Pharmacokinetic studies demonstrated that reboxetine is suitable for twice daily administration (8-10 mg/day) and that it exhibits minimal drug-drug interactions. The starting dose of reboxetine should be reduced in the elderly, in patients with renal or hepatic impairment, or in patients receiving potent CYP3A inhibitors. A total of 20 phase II/III clinical studies comprising placebo-controlled, active comparator-controlled and open-label uncontrolled studies in both short-term and long-term treatment of major depression have been conducted. In the treatment of major depression, reboxetine was superior to placebo in 5 of 12 short- or long-term placebo-controlled studies and was comparable in efficacy to active comparators in 3 out of 3 active-controlled studies. Unlike conventional tricyclic antidepressants (TCAs), reboxetine had only minimal sedative and cardiovascular liabilities, probably due to increased pharmacological specificity of reboxetine as compared with TCAs. Unlike serotonin reuptake inhibitors, this selective and specific norepinephrine reuptake inhibitor demonstrated a distinct side-effect profile with diminishing sexual dysfunction and GI side effects. The availability of this agent has afforded patients suffering from major depressive disorder a new class of agents to combat the debilitating consequence of this psychiatric disease. The demonstrated pharmacological specificity of this compound has provided the psychopharmacology community with a tool to elucidate

  19. Discovery of potent and selective inhibitors of 11beta-HSD1 for the treatment of metabolic syndrome.

    PubMed

    Richards, Steven; Sorensen, Bryan; Jae, Hwan-Soo; Winn, Marty; Chen, Yixian; Wang, Jiahong; Fung, Steven; Monzon, Katina; Frevert, Ernst U; Jacobson, Peer; Sham, Hing; Link, J T

    2006-12-15

    High throughput screening efforts have identified a novel class of dichloroaniline amide 11beta-HSD1 inhibitors. SAR studies initiated from dichloroaniline 4 focused on retaining the potency and selectivity profile of the lead.

  20. Potent, Selective, and Orally Bioavailable Inhibitors of VPS34 Provide Chemical Tools to Modulate Autophagy in Vivo

    PubMed Central

    2015-01-01

    Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools, but selective inhibitors are now available to modulate the PI 3-kinase VPS34, which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models. PMID:26819669

  1. The Potential of Inhibitors of Endocannabinoid Metabolism for Drug Development: A Critical Review.

    PubMed

    Fowler, Christopher J

    2015-01-01

    The endocannabinoids anandamide and 2-arachidonoylglycerol are metabolised by both hydrolytic enzymes (primarily fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL)) and oxygenating enzymes (e.g. cyclooxygenase-2, COX-2). In the present article, the in vivo data for compounds inhibiting endocannabinoid metabolism have been reviewed, focussing on inflammation and pain. Potential reasons for the failure of an FAAH inhibitor in a clinical trial in patients with osteoarthritic pain are discussed. It is concluded that there is a continued potential for compounds inhibiting endocannabinoid metabolism in terms of drug development, but that it is wise not to be unrealistic in terms of expectations of success.

  2. Discovery of Highly Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase by Fragment Hopping

    PubMed Central

    Ji, Haitao; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2009-01-01

    Selective inhibition of neuronal nitric oxide synthase (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. This study shows that not only greater inhibitory potency and isozyme selectivity, but more drug-like properties can be achieved by fragment hopping. Based on the structure of lead molecule 6, fragment hopping effectively extracted the minimal pharmacophoric elements in the active site of nNOS for ligand hydrophobic and steric interactions and generated appropriate lipophilic fragments for lead optimization. More potent and selective inhibitors with better drug-like properties were obtained within the design of 20 derivatives (compounds 7-26). Our structure-based inhibitor design for nNOS and SAR analysis reveal the robustness and efficiency of fragment hopping in lead discovery and structural optimization, which implicates a broad application of this approach to many other therapeutic targets for which known drug-like small-molecule modulators are still limited. PMID:19125620

  3. Fluorinated indazoles as novel selective inhibitors of nitric oxide synthase (NOS): synthesis and biological evaluation.

    PubMed

    Claramunt, Rosa M; López, Concepción; Pérez-Medina, Carlos; Pérez-Torralba, Marta; Elguero, José; Escames, Germaine; Acuña-Castroviejo, Darío

    2009-09-01

    In order to find new compounds with neuroprotective activity and NOS-I/NOS-II selectivity, we have designed, synthesized, and characterized 14 new NOS inhibitors with an indazole structure. The first group corresponds to 4,5,6,7-tetrahydroindazoles (4-8), the second to the N-methyl derivatives (9-12) of 7-nitro-1H-indazole (1) and 3-bromo-7-nitro-1H-indazole (2), and the latter to 4,5,6,7-tetrafluoroindazoles (13-17). Compound 13 (4,5,6,7-tetrafluoro-3-methyl-1H-indazole) inhibited NOS-I by 63% and NOS-II by 83%. Interestingly, compound 16 (4,5,6,7-tetrafluoro-3-perfluorophenyl-1H-indazole) inhibited NOS-II activity by 80%, but it did not affect to NOS-I activity. Structural comparison between these new indazoles further supports the importance of the aromatic indazole skeleton for NOS inhibition and indicate that bulky groups or N-methylation of 1 and 2 diminish their effect on NOS activity. The fluorination of the aromatic ring increased the inhibitory potency and NOS-II selectivity, suggesting that this is a promising strategy for NOS selective inhibitors.

  4. 2,5-Disubstituted tetrahydrofurans as selective serotonin re-uptake inhibitors.

    PubMed

    Voelker, Troy; Xia, Haiji; Fandrick, Keith; Johnson, Robert; Janowsky, Aaron; Cashman, John R

    2009-03-01

    Enhancement of 5-hydroxytryptamine (5-HT, serotonin) neurotransmission is a viable means of treating depression. On the basis of this observation, agents that inhibit re-uptake of 5-HT were prepared based on (-)-cocaine and aryltropanes as lead compounds because they are reasonably potent 5-HT re-uptake inhibitors. Molecular dissection of an aryltropane provided a series of 5- and 6-membered ring compounds. From among this library of compounds a series of disubstituted tetrahydrofurans bearing 2-alkyl aryl and 5-alkyl amino groups were identified as having highly potent and selective 5-HT re-uptake inhibition. The compounds were evaluated for their ability to compete with radiolabeled RTI-55 binding and to inhibit re-uptake of neurotransmitters at the human dopamine, serotonin and norepinephrine transporters. Based on potency (e.g., K(i)=800 pM) and significant functional selectivity (e.g., IC(50) ratios for human dopamine:serotonin or norepinephrine:serotonin, >or=1397) highly potent and selective serotonin re-uptake inhibitors were identified. Optimal features playing a dominant role in binding affinity and re-uptake inhibition included lipophilic substitution on the aromatic moiety, trans relative stereochemistry of the 2,5-disubstituted tetrahydrofuran ring, and a total of four or five methylene groups between the alkyl amine and the alkyl aryl moiety and the tetrahydrofuran group. A number of the most potent serotonin re-uptake inhibitors were tested in Balb/c mice in the forced-swim test (FST), a behavioral test used to measure the effects of antidepressant agents. Acute administration of 32c (10mg/kg), or 32d (10mg/kg) ip tended to decrease the duration of mouse immobility in the FST although the effect was not statistically significant.

  5. Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter.

    PubMed

    Andersen, Jacob; Ringsted, Kristoffer B; Bang-Andersen, Benny; Strømgaard, Kristian; Kristensen, Anders S

    2015-10-27

    The transporters for norepinephrine and dopamine (NET and DAT, respectively) constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity binding site in NET and DAT was performed to examine their role for selective inhibitor recognition. Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor selectivity in NET and DAT, and provide important new insight into the molecular basis for NET/DAT selectivity of therapeutic and recreational drugs.

  6. Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter

    PubMed Central

    Andersen, Jacob; Ringsted, Kristoffer B.; Bang-Andersen, Benny; Strømgaard, Kristian; Kristensen, Anders S.

    2015-01-01

    The transporters for norepinephrine and dopamine (NET and DAT, respectively) constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity binding site in NET and DAT was performed to examine their role for selective inhibitor recognition. Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor selectivity in NET and DAT, and provide important new insight into the molecular basis for NET/DAT selectivity of therapeutic and recreational drugs. PMID:26503701

  7. Discovery and optimization of indazoles as potent and selective interleukin-2 inducible T cell kinase (ITK) inhibitors.

    PubMed

    Pastor, Richard M; Burch, Jason D; Magnuson, Steven; Ortwine, Daniel F; Chen, Yuan; De La Torre, Kelly; Ding, Xiao; Eigenbrot, Charles; Johnson, Adam; Liimatta, Marya; Liu, Yichin; Shia, Steven; Wang, Xiaolu; Wu, Lawren C; Pei, Zhonghua

    2014-06-01

    There is evidence that small molecule inhibitors of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signaling cascade, could represent a novel asthma therapeutic class. Moreover, given the expected chronic dosing regimen of any asthma treatment, highly selective as well as potent inhibitors would be strongly preferred in any potential therapeutic. Here we report hit-to-lead optimization of a series of indazoles that demonstrate sub-nanomolar inhibitory potency against ITK with strong cellular activity and good kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of the complexes.

  8. Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1

    PubMed Central

    Jacques, Sylvain A.; Johansson, Lars; Calderón-Montaño, José Manuel; Claesson, Magnus; Loseva, Olga; Valerie, Nicholas C. K.; Lundbäck, Thomas; Piedrafita, Javier; Maga, Giovanni; Crespan, Emmanuele; Meijer, Laurent; Morón, Estefanía Burgos; Baranczewski, Pawel; Hagbjörk, Ann-Louise; Svensson, Richard; Wiita, Elisee; Almlöf, Ingrid; Visnes, Torkild; Jeppsson, Fredrik; Sigmundsson, Kristmundur; Jensen, Annika Jenmalm; Artursson, Per; Jemth, Ann-Sofie; Stenmark, Pål; Berglund, Ulrika Warpman; Scobie, Martin; Helleday, Thomas

    2016-01-01

    The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies. PMID:26771665

  9. A potent and selective inhibitor targeting human and murine 12/15-LOX.

    PubMed

    Armstrong, Michelle M; Freedman, Cody J; Jung, Joo Eun; Zheng, Yi; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Simeonov, Anton; Maloney, David J; van Leyen, Klaus; Jadhav, Ajit; Holman, Theodore R

    2016-03-15

    Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4±0.5 μM against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 μM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 μM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a Kic of 1.0±0.08 μM and a Kiu of 6.0±3.3 μM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.

  10. Selective serotonin re-uptake inhibitors (SSRIs) in the aquatic environment: an ecopharmacovigilance approach.

    PubMed

    Silva, Liliana J G; Lino, Celeste M; Meisel, Leonor M; Pena, Angelina

    2012-10-15

    Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are among the most prescribed pharmaceutical active substances throughout the world. The occurrence of these widely used compounds in different environmental compartments (wastewaters, surface, ground and drinking waters, and sediments), justify the growing concern about these emerging environmental pollutants. Viewing an ecopharmacovigilance approach, a comprehensive discussion of the state of the art regarding different contamination sources, fate, degradation and occurrence is presented. Information on the current distribution levels and fate in different environmental matrices continues to be sparse and measures are imperative to improve awareness and encourage precautionary actions to minimize SSRIs' environmental impact.

  11. Selection, Preparation, and Evaluation of Small-Molecule Inhibitors of Toll-Like Receptor 4

    PubMed Central

    2010-01-01

    Toll-like receptor 4 (TLR4), a membrane-spanning receptor protein that functions in complex with its accessory protein MD-2, is an intriguing target for therapeutic development. Herein, we report the identification of a series of novel TLR4 inhibitors and the development of a robust, enantioselective synthesis using an unprecedented Mannich type reaction to functionalize a pyrazole ring. In silico and cellular assay results demonstrated that compound 1 and its analogues selectively block TLR4 activation in live cells. Animal model tests showed that 1 and its derivatives could potentiate morphine-induced analgesia in vivo, presumably by attenuating the opioid-induced TLR4 activation. PMID:20824192

  12. Discovery of azabenzimidazole derivatives as potent, selective inhibitors of TBK1/IKKε kinases.

    PubMed

    Wang, Tao; Block, Michael A; Cowen, Scott; Davies, Audrey M; Devereaux, Erik; Gingipalli, Lakshmaiah; Johannes, Jeffrey; Larsen, Nicholas A; Su, Qibin; Tucker, Julie A; Whitston, David; Wu, Jiaquan; Zhang, Hai-Jun; Zinda, Michael; Chuaqui, Claudio

    2012-03-01

    The design, synthesis and biological evaluation of a series of azabenzimidazole derivatives as TBK1/IKKε kinase inhibitors are described. Starting from a lead compound 1a, iterative design and SAR exploitation of the scaffold led to analogues with nM enzyme potencies against TBK1/IKKε. These compounds also exhibited excellent cellular activity against TBK1. Further structure-based design to improve selectivity over CDK2 and Aurora B resulted in compounds such as 5b-e. These probe compounds will facilitate study of the complex cancer biology of TBK1 and IKKε.

  13. Syntheses of Mycobactin Analogs as Potent and Selective Inhibitors of Mycobacterium tuberculosis

    PubMed Central

    Juárez-Hernández, Raúl E.; Franzblau, Scott G.

    2012-01-01

    Three analogs of mycobactin T, the siderophore secreted by Mycobacterium tuberculosis (Mtb) were synthesized and screened for their antibiotic activity against Mtb H37Rv and a broad panel of Gram-positive and Gram-negative bacteria. The synthetic mycobactins were potent (MIC90 0.02–0.88 μM in 7H12 media) and selective Mtb inhibitors, with no inhibitory activity observed against any other of the microorganisms tested. The maleimide-containing analog 40 represents a versatile platform for the development of mycobactin-drug conjugates, as well as other applications. PMID:22895786

  14. [HYPERPROLACTINEMIA AND SELECTIVE SEROTONIN REUPTAKE INHIBITORS. A NARRATIVE REVIEW OF THE LITERATURE].

    PubMed

    Rosón-González, Mirian; Tajima-Pozo, Kazuhiro; Montañés-Rada, Francisco

    2015-01-01

    A large number of scientific papers have reported the relationship between the development of hyperprolactinemia and the use of psychotropic drugs, especially the role of antipsychotics which are antidopaminergic drugs. However, less information is known about the role of antidepressants in the development of hyperprolactinemia, specially the selective reuptake inhibitors (SSRIs). The prevalence of hyperprolactinemia as a pharmacological side effect of SSRIs is still unknown, despite the widespread use over the last decade. The aim of this review is to explore the relationship between hyperprolactinemia and SSRIs.

  15. Exacerbation of migraine attacks during treatment with the selective serotonin reuptake inhibitor sertraline. A case report.

    PubMed

    Bickel, A; Kornhuber, J; Maihöfner, C; Ropohl, A

    2005-11-01

    Abnormal signal transmission in central serotonergic pathways is supposed to play an important role in the pathogenesis of migraine and major depression. We report on a patient, who was treated during an episode of depression with the selective serotonin reuptake inhibitor (SSRI) sertraline and developed frequent migraine attacks under this therapeutical regime. Single migraine attacks were treated successfully with triptanes. Although SSRIs may be beneficial for migraine prophylaxis at long term administration, this case suggests that acute administration of SSRIs in migraineurs may include the risk of worsening migraine.

  16. Selective Serotonin Reuptake Inhibitor Use during Pregnancy and the Risk of Autism Spectrum Disorders: A Review

    PubMed Central

    Boukhris, Takoua; Bérard, Anick

    2015-01-01

    Antidepressants are widely used during pregnancy. Several studies have shown that the use of antidepressants during pregnancy is linked to adverse outcomes, including congenital malformations, prematurity, and low birth weight. However, there is a knowledge gap regarding the potential association between gestational exposure to antidepressants and the risk of autism spectrum disorders (ASD). The etiology of ASD remains unclear, although studies have implicated genetic predispositions and environmental risk factors in the development of ASD in children. In this review, we describe the association between gestational use of antidepressants, specifically selective serotonin reuptake inhibitors, and the risk of ASD. PMID:27617119

  17. Selective Serotonin Reuptake Inhibitors for the Treatment of Obsessive-Compulsive Disorder in Children and Adolescents

    PubMed Central

    Williams, Jennifer Schoelles; Moore, Thea; Collins, Candace L.; Thomas, Kerry-Ann E.

    2003-01-01

    The introduction of the selective serotonin reuptake inhibitors (SSRIs) has been a major advance in pediatric psychiatry, while contemporary advances in the understanding of obsessive-compulsive disorder (OCD) phenomenon in children have facilitated its identification and treatment. Currently, fluvoxamine and sertraline are the only SSRIs that have received FDA approval for the treatment of childhood OCD. The purpose of this article is to review the safety and efficacy of SSRIs in the treatment of obsessive-compulsive disorder (OCD) in children and adolescents. PMID:23118677

  18. Fragment-based drug discovery of potent and selective MKK3/6 inhibitors.

    PubMed

    Adams, Mark; Kobayashi, Toshitake; Lawson, J David; Saitoh, Morihisa; Shimokawa, Kenichiro; Bigi, Simone V; Hixon, Mark S; Smith, Christopher R; Tatamiya, Takayuki; Goto, Masayuki; Russo, Joseph; Grimshaw, Charles E; Swann, Steven

    2016-02-01

    The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38.

  19. Novel Triazole-Quinoline Derivatives as Selective Dual Binding Site Acetylcholinesterase Inhibitors.

    PubMed

    Mantoani, Susimaire P; Chierrito, Talita P C; Vilela, Adriana F L; Cardoso, Carmen L; Martínez, Ana; Carvalho, Ivone

    2016-02-05

    Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder worldwide. Currently, the only strategy for palliative treatment of AD is to inhibit acetylcholinesterase (AChE) in order to increase the concentration of acetylcholine in the synaptic cleft. Evidence indicates that AChE also interacts with the β-amyloid (Aβ) protein, acting as a chaperone and increasing the number and neurotoxicity of Aβ fibrils. It is known that AChE has two binding sites: the peripheral site, responsible for the interactions with Aβ, and the catalytic site, related with acetylcholine hydrolysis. In this work, we reported the synthesis and biological evaluation of a library of new tacrine-donepezil hybrids, as a potential dual binding site AChE inhibitor, containing a triazole-quinoline system. The synthesis of hybrids was performed in four steps using the click chemistry strategy. These compounds were evaluated as hAChE and hBChE inhibitors, and some derivatives showed IC50 values in the micro-molar range and were remarkably selective towards hAChE. Kinetic assays and molecular modeling studies confirm that these compounds block both catalytic and peripheral AChE sites. These results are quite interesting since the triazole-quinoline system is a new structural scaffold for AChE inhibitors. Furthermore, the synthetic approach is very efficient for the preparation of target compounds, allowing a further fruitful new chemical library optimization.

  20. A Selective Small Molecule DNA2 Inhibitor for Sensitization of Human Cancer Cells to Chemotherapy

    PubMed Central

    Liu, Wenpeng; Zhou, Mian; Li, Zhengke; Li, Hongzhi; Polaczek, Piotr; Dai, Huifang; Wu, Qiong; Liu, Changwei; Karanja, Kenneth K.; Popuri, Vencat; Shan, Shu-ou; Schlacher, Katharina; Zheng, Li; Campbell, Judith L.; Shen, Binghui

    2016-01-01

    Cancer cells frequently up-regulate DNA replication and repair proteins such as the multifunctional DNA2 nuclease/helicase, counteracting DNA damage due to replication stress and promoting survival. Therefore, we hypothesized that blocking both DNA replication and repair by inhibiting the bifunctional DNA2 could be a potent strategy to sensitize cancer cells to stresses from radiation or chemotherapeutic agents. We show that homozygous deletion of DNA2 sensitizes cells to ionizing radiation and camptothecin (CPT). Using a virtual high throughput screen, we identify 4-hydroxy-8-nitroquinoline-3-carboxylic acid (C5) as an effective and selective inhibitor of DNA2. Mutagenesis and biochemical analysis define the C5 binding pocket at a DNA-binding motif that is shared by the nuclease and helicase activities, consistent with structural studies that suggest that DNA binding to the helicase domain is necessary for nuclease activity. C5 targets the known functions of DNA2 in vivo: C5 inhibits resection at stalled forks as well as reducing recombination. C5 is an even more potent inhibitor of restart of stalled DNA replication forks and over-resection of nascent DNA in cells defective in replication fork protection, including BRCA2 and BOD1L. C5 sensitizes cells to CPT and synergizes with PARP inhibitors. PMID:27211550

  1. Charge optimization increases the potency and selectivity of a chorismate mutase inhibitor.

    PubMed

    Mandal, Ajay; Hilvert, Donald

    2003-05-14

    The highest affinity inhibitor for chorismate mutases, a conformationally constrained oxabicyclic dicarboxylate transition state analogue, was modified as suggested by computational charge optimization methods. As predicted, replacement of the C10 carboxylate in this molecule with a nitro group yields an even more potent inhibitor of a chorismate mutase from Bacillus subtilis (BsCM), but the magnitude of the improvement (roughly 3-fold, corresponding to a DeltaDeltaG of -0.7 kcal/mol) is substantially lower than the gain of 2-3 kcal/mol binding free energy anticipated for the reduced desolvation penalty upon binding. Experiments with a truncated version of the enzyme show that the flexible C terminus, which was only partially resolved in the crystal structure and hence omitted from the calculations, provides favorable interactions with the C10 group that partially compensate for its desolvation. Although truncation diminishes the affinity of the enzyme for both inhibitors, the nitro derivative binds 1.7 kcal/mol more tightly than the dicarboxylate, in reasonable agreement with the calculations. Significantly, substitution of the C10 carboxylate with a nitro group also enhances the selectivity of inhibition of BsCM relative to a chorismate mutase from Escherichia coli (EcCM), which has a completely different fold and binding pocket, by 10-fold. These results experimentally verify the utility of charge optimization methods for improving interactions between proteins and low-molecular weight ligands.

  2. The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation

    PubMed Central

    Evans, Margery L.; Chorell, Erik; Taylor, Jonathan D.; Åden, Jörgen; Göteson, Anna; Li, Fei; Koch, Marion; Sefer, Lea; Matthews, Steve J.; Wittung-Stafshede, Pernilla; Almqvist, Fredrik; Chapman, Matthew R.

    2014-01-01

    Summary Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids. PMID:25620560

  3. Selective serotonin reuptake inhibitor antidepressants potentiate methylphenidate (Ritalin)-induced gene regulation in the adolescent striatum.

    PubMed

    Van Waes, Vincent; Beverley, Joel; Marinelli, Michela; Steiner, Heinz

    2010-08-01

    The psychostimulant methylphenidate (Ritalin) is used in conjunction with selective serotonin reuptake inhibitors (SSRIs) in the treatment of medical conditions such as attention-deficit hyperactivity disorder with anxiety/depression comorbidity and major depression. Co-exposure also occurs in patients on SSRIs who use psychostimulant 'cognitive enhancers'. Methylphenidate is a dopamine/norepinephrine reuptake inhibitor that produces altered gene expression in the forebrain; these effects partly mimic gene regulation by cocaine (dopamine/norepinephrine/serotonin reuptake inhibitor). We investigated whether the addition of SSRIs (fluoxetine or citalopram; 5 mg/kg) modified gene regulation by methylphenidate (2-5 mg/kg) in the striatum and cortex of adolescent rats. Our results show that SSRIs potentiate methylphenidate-induced expression of the transcription factor genes zif268 and c-fos in the striatum, rendering these molecular changes more cocaine-like. Present throughout most of the striatum, this potentiation was most robust in its sensorimotor parts. The methylphenidate + SSRI combination also enhanced behavioral stereotypies, consistent with dysfunction in sensorimotor striatal circuits. In so far as such gene regulation is implicated in psychostimulant addiction, our findings suggest that SSRIs may enhance the addiction potential of methylphenidate.

  4. [Tubulointerstitial nephritis associated with treatment with selective Cox-2 inhibitors, celecoxib and rofecoxib].

    PubMed

    Ortiz, M; Mon, C; Fernández, M J; Sánchez, R; Mampaso, F; Alvarez Ude, F

    2005-01-01

    The nephrotoxic effect of nonselective nonsteroidal anti-inflamatory drugs (NSAIDS) has been widely described. The main benefit of the Cox-2 inhibitors in relation to the NSAIDS is the production of a very similar analgesic effect, but with fewer gastrointestinal side effects. However, their effects on renal function are little known as yet and their long-term safety is still pending definition. The use of selective Cox-2 inhibitors as anti-inflamatory analgesic is becoming more and more common in our environment. We report two cases of tubulointersticial nephritis confirmed by renal biopsy, associated with administration of the two Cox-2 inhibitors currently available on the market, celecoxib and rofecoxib. In both cases, we were talking about elderly women, with deterioration of the general condition and acute renal failure. In the former case, renal biopsy showed an acute tubulo-intersticial nephritis (TIN) so highly "variegated" in its histologic expression. In the second case, was associated with strong indications of chronicity. Treatment with steroid was initiated in both patients and improvement of renal function was observed.

  5. Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

    PubMed

    Kitamura, Seiya; Hvorecny, Kelli L; Niu, Jun; Hammock, Bruce D; Madden, Dean R; Morisseau, Christophe

    2016-05-26

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.

  6. CGX1037 is a novel PKC isoform delta selective inhibitor in platelets.

    PubMed

    Bhavanasi, Dheeraj; Kostyak, John C; Swindle, John; Kilpatrick, Laurie E; Kunapuli, Satya P

    2015-01-01

    Platelets upon activation change their shape, aggregate and secrete alpha and dense granule contents among which ADP acts as a feedback activator. Different Protein Kinase C (PKC) isoforms have specific non-redundant roles in mediating platelet responses including secretion and thrombus formation. Murine platelets lacking specific PKC isoforms have been used to evaluate the isoform specific functions. Novel PKC isoform δ has been shown to play an important role in some pathological processes. Lack of specific inhibitors for PKCδ has restricted analysis of its role in various cells. The current study was carried out to evaluate a novel small molecule PKCδ inhibitor, CGX1037 in platelets. Platelet aggregation, dense granule secretion and western blotting experiments were performed to evaluate CGX1037. In human platelets, CGX1037 inhibited PAR4-mediated phosphorylation on PKD2, a PKCδ-specific substrate. Pre-treatment of human or murine platelets with CGX1037 inhibited PAR4-mediated dense granule secretion whereas it potentiated GPVI-mediated dense granule secretion similar to the responses observed in murine platelets lacking PKCδ· Furthermore, pre-treatment of platelets from PKCδ(-/-) mice with CGX1037 had no significant additive effect on platelet responses suggesting the specificity of CGX1037. Hence, we show that CGX1037 is a selective small molecule inhibitor of PKCδ in platelets.

  7. The effects of celecoxib, a COX-2 selective inhibitor, on acute inflammation induced in irradiated rats.

    PubMed

    Khayyal, M T; El-Ghazaly, Mona A; El-Hazek, R M; Nada, A S

    2009-10-01

    The potential value of selective and non-selective COX-2 inhibitors in preventing some of the biochemical changes induced by ionizing radiation was studied in rats exposed to carrageenan-induced paw edema and 6-day-old air pouch models. The animals were exposed to different exposure levels of gamma-radiation, namely either to single doses of 2 and 7.5 Gy or a fractionated dose level of 7.5 Gy delivered as 0.5 Gy twice weekly for 7.5 weeks. The inflammatory response produced by carrageenan in irradiated rats was markedly higher than that induced in non-irradiated animals, and depended on the extent of irradiation. Celecoxib, a selective COX-2 inhibitor, in doses of 3, 5, 10, and 15 mg/kg was effective in reducing paw edema in irradiated and non-irradiated rats in a dose-dependent manner as well as diclofenac (3 mg/kg), a non-selective COX inhibitor. Irradiation of animals before the induction of the air pouch by an acute dose of 2 Gy led to a significant increase in leukocytic count, as well as in the level of interleukin-6 (IL-6), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), LTB(4), PGE(2) (as an index of COX-2 activity), TXB(2) (as an index of COX-1 activity), and the plasma level of MDA. This increase in level of these parameters was more marked than that observed in the non-irradiated animals subjected to the inflammagen. The blood GSH level was not affected by the dose of irradiation used, whereas superoxide dismutase (SOD) activity was suppressed. In many respects, celecoxib (5 mg/kg) was as potent as diclofenac in decreasing the elevated levels of IL-6, IL-1beta, TNF-alpha, LTB(4), PGE(2), but lacked any significant effect on TXB(2) level. Since it is mostly selective for COX-2 with a rare effect on COX-1 enzyme, both drugs at the selected dose levels showed no effect on level of MDA, GSH, and SOD activity.

  8. Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor

    PubMed Central

    Riendeau, D; Percival, M D; Boyce, S; Brideau, C; Charleson, S; Cromlish, W; Ethier, D; Evans, J; Falgueyret, J -P; Ford-Hutchinson, A W; Gordon, R; Greig, G; Gresser, M; Guay, J; Kargman, S; Léger, S; Mancini, J A; O'Neill, G; Ouellet, M; Rodger, I W; Thérien, M; Wang, Z; Webb, J K; Wong, E; Xu, L; Young, R N; Zamboni, R; Prasit, P; Chan, C -C

    1997-01-01

    DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor.In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50=41±14 nM) over COX-1 (IC50>50 μM). Indomethacin was a potent inhibitor of both COX-1 (IC50=18±3 nM) and COX-2 (IC50=26±6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50>50 μM and 4.1±1.7 nM, respectively).DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki value of 140±68 μM for the initial reversible binding to enzyme and a k2 value of 0.11±0.06 s−1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62±26 μM and 0.06±0.01 s−1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t1/2=1–3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site.Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50=63±5 μM at 0.1 μM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1.DFU inhibited lipopolysaccharide (LPS)-induced PGE2 production (COX-2) in a human whole blood assay with a potency (IC50=0.28±0.04 μM) similar

  9. CRM1 Blockade by Selective Inhibitors of Nuclear Export (SINE) attenuates Kidney Cancer Growth

    PubMed Central

    Inoue, Hiromi; Kauffman, Michael; Shacham, Sharon; Landesman, Yosef; Yang, Joy; Evans, Christopher P.; Weiss, Robert H.

    2015-01-01

    Since renal cell carcinoma (RCC) often presents asymptomatically, patients are commonly diagnosed at the metastatic stage when treatment options are limited and survival is poor. Given that progression-free survival with current therapies for metastatic RCC is only one to two years and existing drugs are associated with a high rate of resistance, new pharmacological targets are desperately needed. We identified and evaluated the nuclear exporter protein, chromosome region maintenance protein 1 (CRM1), as a novel potential therapeutic for RCC. Purpose To evaluate novel, selective inhibitors of nuclear export as potential RCC therapeutics. Materials and Methods Efficacy of the CRM1 inhibitors, KPT-185 and -251, was tested in several RCC cell lines and in a RCC xenograft model. Apoptosis and cell cycle arrest were quantified, and localization of p53 family proteins was assessed using standard techniques. Results KPT-185 attenuated CRM1 and showed increased cytotoxicity in RCC cells in vitro, with evidence of increased apoptosis as well as cell cycle arrest. KPT-185 caused both p53 and p21 to remain primarily in the nucleus in all RCC cell lines, suggesting a mechanism of action of these compounds dependent upon tumor-suppressor protein localization. Furthermore, when administered orally in a high-grade RCC xenograft model, the bioavailable CRM1 inhibitor KPT-251 significantly inhibited tumor growth in vivo with the expected on-target effects and with no obvious toxicity. Conclusions The CRM1 inhibitor family of proteins are novel therapeutic targets RCC and deserve further intensive investigation in this and other urologic malignancies. PMID:23079374

  10. Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L

    PubMed Central

    Parker, Erica N.; Song, Jiangli; Kumar, G. D. Kishore; Odutola, Samuel O.; Chavarria, Gustavo E.; Charlton-Sevcik, Amanda K.; Strecker, Tracy E.; Barnes, Ashleigh L.; Sudhan, Dhivya R.; Wittenborn, Thomas R.; Siemann, Dietmar W.; Horsman, Michael R.; Chaplin, David J.; Trawick, Mary Lynn; Pinney, Kevin G.

    2016-01-01

    Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 μM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre

  11. A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity

    SciTech Connect

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-01-09

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. As a result, these findings offer a potential strategy for further ligand optimization.

  12. Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L.

    PubMed

    Parker, Erica N; Song, Jiangli; Kishore Kumar, G D; Odutola, Samuel O; Chavarria, Gustavo E; Charlton-Sevcik, Amanda K; Strecker, Tracy E; Barnes, Ashleigh L; Sudhan, Dhivya R; Wittenborn, Thomas R; Siemann, Dietmar W; Horsman, Michael R; Chaplin, David J; Trawick, Mary Lynn; Pinney, Kevin G

    2015-11-01

    Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 μM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre

  13. Moguntinones--new selective inhibitors for the treatment of human colorectal cancer.

    PubMed

    Maderer, Annett; Plutizki, Stanislav; Kramb, Jan-Peter; Göpfert, Katrin; Linnig, Monika; Khillimberger, Katrin; Ganser, Christopher; Lauermann, Eva; Dannhardt, Gerd; Galle, Peter R; Moehler, Markus

    2014-06-01

    3-Indolyl and 3-azaindolyl-4-aryl maleimide derivatives, called moguntinones (MOG), have been selected for their ability to inhibit protein kinases associated with angiogenesis and induce apoptosis. Here, we characterize their mode of action and their potential clinical value in human colorectal cancer in vitro and in vivo. MOG-19 and MOG-13 were characterized in vitro using kinase, viability, and apoptosis assays in different human colon cancer (HT-29, HCT-116, Caco-2, and SW480) and normal colon cell lines (CCD-18Co, FHC, and HCoEpiC) alone or in combination with topoisomerase I inhibitors. Intracellular signaling pathways were analyzed by Western blotting. To determine their potential to inhibit tumor growth in vivo, the human HT-29 tumor xenograft model was used. Moguntinones prominently inhibit several protein kinases associated with tumor growth and metastasis. Specific signaling pathways such as GSK3β and mTOR downstream targets were inhibited with IC(50) values in the nanomolar range. GSK3β signaling inhibition was independent of KRAS, BRAF, and PI3KCA mutation status. While moguntinones alone induced apoptosis only in concentrations >10 μmol/L, MOG-19 in combination with topoisomerase I inhibitors induced apoptosis synergistically at lower concentrations. Consistent with in vitro data, MOG-19 significantly reduced tumor volume and weight in combination with a topoisomerase I inhibitor in vivo. Our in vitro and in vivo data present significant proapoptotic, antiangiogenic, and antiproliferative effects of MOG-19 in different human colon cancer cells. Combination with clinically relevant topoisomerase I inhibitors in vitro and xenograft mouse model demonstrate a high potency of moguntinones to complement and improve standard chemotherapy options in human colorectal cancer.

  14. Discovery of pyrazole as C-terminus of selective BACE1 inhibitors.

    PubMed

    Zou, Yiquan; Xu, Lei; Chen, Wuyan; Zhu, Yiping; Chen, Tiantian; Fu, Yan; Li, Li; Ma, Lanping; Xiong, Bing; Wang, Xin; Li, Jian; He, Jianhua; Zhang, Haiyan; Xu, Yechun; Li, Jia; Shen, Jingkang

    2013-10-01

    We recently discovered and reported dual inhibitor 5 of AChE and BACE1 with N-benzylpiperidine ethyl as C-terminus. Compound 5 showed potent inhibitory activities for BACE1, and could reduce endogenous Aβ1-40 production in APP transgenic mice. In present work, we rapidly identified substituted triazole as the C-terminus of compound 5 by replacing the benzylpiperidine ethyl group with click chemistry and tested these synthesized compounds by in situ screening assay. As revealed by the crystal structures of BACE1 in complex with our triazole compound 12, we found that Pro70 and Thr72 located in the flap region were the critical components for binding with these inhibitors. With the aid of the crystal structure, a new series of five-membered heterocyclic compounds was prepared in order to explore the structure-activity relationship (SAR) of this class of molecules. From these efforts, pyrazole was discovered as a novel C-terminus of BACE1 inhibitors. After further modification of pyrazole with variable substituents, compound 37 exhibited good potency in enzyme inhibition assay (IC50=0.025 μM) and compound 33 showed moderate inhibition effects on Aβ production of APP transfected HEK293 cells. Moreover, these pyrazole derivatives demonstrated good selectivity versus cathepsin D. Our results indicated that the vicinity of Pro70 and Thr72 might be utilized as a subsite, and the discovered pyrazole derivatives might provide useful hints for developing novel BACE1 inhibitors as anti-AD drugs.

  15. [Conformation of adenosine deaminase in complexes with inhibitors: application of selective quenching of fluorescence emission].

    PubMed

    Vermishian, I G; Sharoian, S G; Antonian, A A; Grigorian, N A; Mardanian, S S; Khoetsian, A V; Markarian, Sh A

    2008-01-01

    The effect of inhibitors, 1-deazaadenosine (1-dAdo) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), on the conformation of adenosine deaminase was studied using the method of selective quenching of fluorescence emission by acrylamide, I- and Cs+. Both in free adenosine deaminase and in its complexes with the inhibitors, the wavelength maxima and half-width of the emission characterize the environment of fluorescing tryptophan residues in adenosine deaminase as weak polar with limited access to solvent. The formation of complexes with the ground state inhibitors used did not quench or change the main emission characteristics of tryptophan fluorescence in adenosine deaminase. Small blue shifts of emission maxima were observed upon quenching in all three samples. The Stern-Volmer parameters of tryptophan fluorescence quenching by acrylamide were not essentially influenced by complex formation of the enzyme with the inhibitors: in general, the folding of the enzyme molecule in the complexes is not perturbed. On the contrary, the emission quenching by charged heavy ions, I- and Cs+, in the complexes was hindered in comparison with free adenosine deaminase. In the complex with 1-deazaadenosine, the parameters for quenching by both ions evidence the essential worsening of their interaction with tryptophans. In the complex with erythro-9-(2-hydroxy-3-nonyl)adenine, along with the worse quenching by I-, complete prohibition of quenching by Cs+ was observed. These data indicate that the local environments of fluorescing tryptophan residues is substantially distorted compared with free adenosine deaminase, which leads to their screening from charged heavy ions.

  16. Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I-Inhibitor Design and Validation

    SciTech Connect

    Zhu, Zhaoning; Sun, Zhong-Yue; Ye, Yuanzan; Voigt, Johannes; Strickland, Corey; Smith, Elizabeth M; Cumming, Jared; Wang, Lingyan; Wong, Jesse; Wang, Yu-Sen; Wyss, Daniel F; Chen, Xia; Kuvelkar, Reshma; Kennedy, Matthew E; Favreau, Leonard; Parker, Eric; McKittrick, Brian A; Stamford, Andrew; Czarniecki, Michael; Greenlee, William; Hunter, John C

    2010-10-18

    A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180º in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

  17. Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I–Inhibitor Design and Validation

    SciTech Connect

    Zhu, Zhaoning; Sun, Zhong-Yue; Ye, Yuanzan; Voigt, Johannes; Strickland, Corey; Smith, Elizabeth M.; Cumming, Jared; Wang, Lingyan; Wong, Jesse; Wang, Yu-Sen; Wyss, Daniel F.; Chen, Xia; Kuvelkar, Reshma; Kennedy, Matthew E.; Favreau, Leonard; Parker, Eric; McKittrick, Brian A.; Stamford, Andrew; Czarniecki, Michael; Greenlee, William; Hunter, John C.

    2013-11-20

    A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit, with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180° in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

  18. Using self-organizing map (SOM) and support vector machine (SVM) for classification of selectivity of ACAT inhibitors.

    PubMed

    Wang, Ling; Wang, Maolin; Yan, Aixia; Dai, Bin

    2013-02-01

    Using a self-organizing map (SOM) and support vector machine, two classification models were built to predict whether a compound is a selective inhibitor toward the two Acyl-coenzyme A: cholesterol acyltransferase (ACAT) isozymes, ACAT-1 and ACAT-2. A dataset of 97 ACAT inhibitors was collected. For each molecule, the global descriptors, 2D and 3D property autocorrelation descriptors and autocorrelation of surface properties were calculated from the program ADRIANA.Code. The prediction accuracies of the models (based on the training/ test set splitting by SOM method) for the test sets are 88.9 % for SOM1, 92.6 % for SVM1 model. In addition, the extended connectivity fingerprints (ECFP_4) for all the molecules were calculated and the structure-activity relationship of selective ACAT inhibitors was summarized, which may help find important structural features of inhibitors relating to the selectivity of ACAT isozymes.

  19. Identification of the molecular basis of inhibitor selectivity between the human and streptococcal type I methionine aminopeptidases.

    PubMed

    Arya, Tarun; Reddi, Ravikumar; Kishor, Chandan; Ganji, Roopa Jones; Bhukya, Supriya; Gumpena, Rajesh; McGowan, Sheena; Drag, Marcin; Addlagatta, Anthony

    2015-03-12

    The methionine aminopeptidase (MetAP) family is responsible for the cleavage of the initiator methionine from newly synthesized proteins. Currently, there are no small molecule inhibitors that show selectivity toward the bacterial MetAPs compared to the human enzyme. In our current study, we have screened 20 α-aminophosphonate derivatives and identified a molecule (compound 15) that selectively inhibits the S. pneumonia MetAP in low micromolar range but not the human enzyme. Further bioinformatics, biochemical, and structural analyses suggested that phenylalanine (F309) in the human enzyme and methionine (M205) in the S. pneumonia MetAP at the analogous position render them with different susceptibilities against the identified inhibitor. X-ray crystal structures of various inhibitors in complex with wild type and F309M enzyme further established the molecular basis for the inhibitor selectivity.

  20. Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

    SciTech Connect

    Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M.

    2014-10-02

    Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

  1. A Potent, Selective and Cell-active Inhibitor of Human Type I Protein Arginine Methyltransferases

    PubMed Central

    Wu, Hong; Senisterra, Guillermo; Li, Fengling; Butler, Kyle V.; Kaniskan, H. Ümit; Speed, Brandon A.; dela Seña, Carlo; Dong, Aiping; Zeng, Hong; Schapira, Matthieu; Brown, Peter J.; Arrowsmith, Cheryl H.; Barsyte-Lovejoy, Dalia; Liu, Jing; Vedadi, Masoud; Jin, Jian

    2015-01-01

    Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, 3, 4, 6 and 8, but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease. PMID:26598975

  2. Clinical effects of pharmacological variations in selective serotonin reuptake inhibitors: an overview.

    PubMed

    Carrasco, J L; Sandner, C

    2005-12-01

    Although the selective serotonin reuptake inhibitor (SSRI) class of antidepressants shares a common primary pharmacology, namely the inhibition of serotonin reuptake, their secondary pharmacology is remarkably heterogeneous. Inhibition of serotonin reuptake and the consequent increase in serotonin availability are responsible for the relief of depressive symptoms and for some of the adverse effects of this class of drugs. Transsynaptic effects such as modulation of signalling cascades, gene expression processes and neuroplasticity are also important in the mechanism of action of antidepressants. However, this review shows that secondary properties of the SSRIs may contribute to the differences in efficacy and tolerability between members of the class. For example, fluvoxamine has affinity for sigma(1)-receptors -- a property likely to be responsible for its particular efficacy in delusional depression. By understanding the properties of SSRIs and employing careful selection of agents for individual patients, physicians are more able to tailor antidepressant treatments to their patients' particular circumstances.

  3. Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

    PubMed

    Luo, Guanglin; Chen, Ling; Burton, Catherine R; Xiao, Hong; Sivaprakasam, Prasanna; Krause, Carol M; Cao, Yang; Liu, Nengyin; Lippy, Jonathan; Clarke, Wendy J; Snow, Kimberly; Raybon, Joseph; Arora, Vinod; Pokross, Matt; Kish, Kevin; Lewis, Hal A; Langley, David R; Macor, John E; Dubowchik, Gene M

    2016-02-11

    GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics.

  4. Modulating the selectivity of matriptase-2 inhibitors with unnatural amino acids.

    PubMed

    St-Georges, Catherine; Désilets, Antoine; Béliveau, François; Ghinet, Mariana; Dion, Sébastien P; Colombo, Éloic; Boudreault, Pierre-Luc; Najmanovich, Rafael J; Leduc, Richard; Marsault, Éric

    2017-03-31

    Matriptase-2, a type II transmembrane serine protease (TTSP), is expressed in the liver and regulates iron homeostasis via the cleavage of hemojuvelin. Matriptase-2 emerges as an attractive target for the treatment of conditions associated with iron overload, such as hemochromatosis or beta-thalassemia. Starting from the crystal structure of its closest homolog matriptase, we constructed a homology model of matriptase-2 in order to further optimize the selectivity of serine trap peptidomimetic inhibitors for matriptase-2 vs matriptase. Careful modifications of the P4, P3 and P2 positions with the help of unnatural amino acids led to a thorough understanding of Structure-Activity Relationship and a >60-fold increase in selectivity for matriptase-2 vs matriptase. Additionally, the introduction of unnatural amino acids led to significant increases in plasma stability. Such compounds represent useful pharmacological tools to test matriptase-2 inhibition in a context of iron overload.

  5. Synthesis and biological evaluation of 2,4-diaminopyrimidines as selective Aurora A kinase inhibitors.

    PubMed

    Qin, Wen-Wen; Sang, Chun-Yan; Zhang, Lin-Lin; Wei, Wei; Tian, Heng-Zhi; Liu, Huan-Xiang; Chen, Shi-Wu; Hui, Ling

    2015-05-05

    The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic apparatus and serve as potential therapeutic targets in oncology. Here we synthesized 15 2,4-diaminopyrimidines and evaluated their biological activities, including antiproliferation, inhibition against Aurora kinases and cell cycle effects. These compounds generally exhibited more potent cytotoxicity against tumor cell lines compared with the VX-680 control, especially compound 11c, which showed the highest cytotoxicities, with IC50 values of 0.5-4.0 μM. Compound 11c had more than 35-fold more selectivity for Aurora A over Aurora B, and molecular docking analysis indicated that compound 11c form better interaction with Aurora A both from the perspective of structure and energy. Furthermore, compound 11c induced G2/M cell cycle arrest in HeLa cells. This series of compounds has the potential for further development as selective Aurora A inhibitors for anticancer activity.

  6. N-(3-Ethynyl-2,4-difluorophenyl)sulfonamide Derivatives as Selective Raf Inhibitors

    PubMed Central

    2015-01-01

    A series of N-(3-ethynyl-2,4-difluorophenyl)sulfonamides were identified as new selective Raf inhibitors. The compounds potently inhibit B-RafV600E with low nanomolar IC50 values and exhibit excellent target specificity in a selectivity profiling investigation against 468 kinases. They strongly suppress proliferation of a panel of human cancer cell lines and patient-derived melanoma cells with B-RafV600E mutation while being significantly less potent to the cells with B-RafWT. The compounds also display favorable pharmacokinetic properties with a preferred example (3s) demonstrating significant in vivo antitumor efficacy in a xenograft mouse model of B-RafV600E mutated Colo205 human colorectal cancer cells, supporting it as a promising lead compound for further anticancer drug discovery. PMID:26005530

  7. Selective serotonin reuptake inhibitors for children and adolescents with major depression: current controversies and recommendations.

    PubMed

    Hamrin, Vanya; Scahill, Lawrence

    2005-05-01

    Recent warnings about potential serious adverse effects with the selective serotonin reuptake inhibitors in children and adolescents with depression raises questions about the risk-benefit ratio of these drugs in this population. Published safety and efficacy trials of SSRIs for the treatment of youth with depression are critically reviewed. These data were augmented by information from regulatory hearings in 2003-2004 and selected open-label reports. Based on this review, recommendations for medication treatment and monitoring of children and adolescents with major depression on SSRIs are provided. Emerging data from several clinical trials show that the SSRIs provide moderate benefits for youth with depression. In addition, SSRI treatment may be associated with increased risk of behavioral activation, self-harm, and suicidal ideation. Appropriate use of the SSRIs in children and adolescents requires careful diagnostic assessment, evaluation of comorbidity, and close monitoring, especially early in treatment.

  8. A Highly Potent and Selective Caspase 1 Inhibitor that Utilizes a Key 3-Cyanopropanoic Acid Moiety

    PubMed Central

    Boxer, Matthew B.; Quinn, Amy M.; Shen, Min; Jadhav, Ajit; Leister, William; Simeonov, Anton; Auld, Douglas S.; Thomas, Craig J.

    2011-01-01

    Herein we examine the potential of a nitrile-containing proprionic acid moiety as an electrophile for covalent attack by the active site cysteine residue of caspase 1. The syntheses of several cyanopropanate containing small molecules based upon the optimized peptidic scaffold of the prodrug VX-765 were accomplished and found to be potent inhibitors of caspase 1 (IC50s ≤ 1 nM). Examination of these novel small molecules versus a caspase panel demonstrated an impressive degree of selectivity for caspase 1 inhibition. Assessment of hydrolytic stability and selected ADME properties highlighted these agents as potentially useful tools for studying caspase 1 down-regulation in various settings including in vivo analyses. PMID:20229566

  9. An update on the pharmacokinetics and pharmacodynamics of alisertib, a selective Aurora kinase A inhibitor.

    PubMed

    Durlacher, Cameron T; Li, Zhi-Ling; Chen, Xiao-Wu; He, Zhi-Xu; Zhou, Shu-Feng

    2016-06-01

    Human Aurora kinases, including Aurora kinase A (AURKA), B (AURKB), and C (AURKC), play an essential role in mitotic events such as monitoring of the mitotic checkpoint, creation of bipolar mitotic spindle and alignment of centrosomes on it, also regulating centrosome separation, bio-orientation of chromosomes and cytokinesis. AURKA and AURKB are key regulators of mitosis and centrosome via polymerizing microfilaments and controlling chromatid segregation. In particular, AURKA plays critical roles in the regulation of mitotic entry, centrosome function, bipolar spindle assembly, and chromosome segregation. AURKA has been found to be overexpressed in various solid and haematological cancers and has been linked with poor prognosis. Its important role in cancer initiation, growth, and metastasis has brought the focus to search for potent and selective AURKA inhibitors for cancer treatment. MLN8237, also known as alisertib, is one selective AURKA inhibitor that has shown remarkable anticancer effects in preclinical studies. Alisertib exhibits favourable pharmacokinetic properties. Alisertib has generally showed good partial response rates of 4-52% and good safety profiles in Phase I and II trials when it is solely administered as well as combined with cytotoxic chemotherapeutic drugs. Recently, the multicentre, randomized Phase III study of alisertib in patients with relapsed or refractory peripheral T-cell lymphoma has been discontinued due to unsatisfactory efficacy. The low risk of side effects, accessibility, and effectiveness of alisertib makes it a new promising anticancer therapy and further mechanistic and clinical studies are warranted.

  10. SD0006: A Potent, Selective and Orally Available Inhibitor of p38 Kinase

    PubMed Central

    Burnette, Barry L.; Selness, Shaun; Devraj, Raj; Jungbluth, Gail; Kurumbail, Ravi; Stillwell, Loreen; Anderson, Gary; Mnich, Stephen; Hirsch, Jeffrey; Compton, Robert; De Ciechi, Pamela; Hope, Heidi; Hepperle, Michael; Keith, Robert H.; Naing, Win; Shieh, Huey; Portanova, Joseph; Zhang, Yan; Zhang, Jian; Leimgruber, Richard M.; Monahan, Joseph

    2009-01-01

    SD0006 is a diarylpyrazole that was prepared as an inhibitor of p38 kinase-α (p38α). In vitro, SD0006 was selective for p38α kinase over 50 other kinases screened (including p38γ and p38δ with modest selectivity over p38β). Crystal structures with p38α show binding at the ATP site with additional residue interactions outside the ATP pocket unique to p38α that can confer advantages over other ATP competitive inhibitors. Direct correlation between inhibition of p38α activity and that of lipopolysaccharide-stimulated TNFα release was established in cellular models and in vivo, including a phase 1 clinical trial. Potency (IC50) for inhibiting tumor necrosis factor-α (TNFα) release, in vitro and in vivo, was <200 nmol/l. In vivo, SD0006 was effective in the rat streptococcal-cell-wall-induced arthritis model, with dramatic protective effects on paw joint integrity and bone density as shown by radiographic analysis. In the murine collagen-induced arthritis model, equivalence was demonstrated to anti-TNFα treatment. SD0006 also demonstrated good oral anti-inflammatory efficacy with excellent cross-species correlation between the rat, cynomolgus monkey, and human. SD0006 suppressed expression of multiple proinflammatory proteins at both the transcriptional and translational levels. These properties suggest SD0006 could provide broader therapeutic efficacy than cytokine-targeted monotherapeutics. PMID:19590255

  11. Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor.

    PubMed

    Flinspach, M; Xu, Q; Piekarz, A D; Fellows, R; Hagan, R; Gibbs, A; Liu, Y; Neff, R A; Freedman, J; Eckert, W A; Zhou, M; Bonesteel, R; Pennington, M W; Eddinger, K A; Yaksh, T L; Hunter, M; Swanson, R V; Wickenden, A D

    2017-01-03

    Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

  12. Cyclooxygenase-1-Selective Inhibitors Based on the (E)-2′-Des-methyl-sulindac Sulfide Scaffold

    PubMed Central

    2012-01-01

    Prostaglandins (PGs) are powerful lipid mediators in many physiological and pathophysiological responses. They are produced by oxidation of arachidonic acid (AA) by cyclooxygenases (COX-1 and COX-2) followed by metabolism of endoperoxide intermediates by terminal PG synthases. PG biosynthesis is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). Specific inhibition of COX-2 has been extensively investigated, but relatively few COX-1-selective inhibitors have been described. Recent reports of a possible contribution of COX-1 in analgesia, neuroinflammation, or carcinogenesis suggest that COX-1 is a potential therapeutic target. We designed, synthesized, and evaluated a series of (E)-2′-des-methyl-sulindac sulfide (E-DMSS) analogues for inhibition of COX-1. Several potent and selective inhibitors were discovered, and the most promising compounds were active against COX-1 in intact ovarian carcinoma cells (OVCAR-3). The compounds inhibited tumor cell proliferation but only at concentrations >100-fold higher than the concentrations that inhibit COX-1 activity. E-DMSS analogues may be useful probes of COX-1 biology in vivo and promising leads for COX-1-targeted therapeutic agents. PMID:22263894

  13. Discovery of a potent, selective, and orally bioavailable pyridinyl-pyrimidine phthalazine aurora kinase inhibitor.

    PubMed

    Cee, Victor J; Schenkel, Laurie B; Hodous, Brian L; Deak, Holly L; Nguyen, Hanh N; Olivieri, Philip R; Romero, Karina; Bak, Annette; Be, Xuhai; Bellon, Steve; Bush, Tammy L; Cheng, Alan C; Chung, Grace; Coats, Steve; Eden, Patrick M; Hanestad, Kelly; Gallant, Paul L; Gu, Yan; Huang, Xin; Kendall, Richard L; Lin, Min-Hwa Jasmine; Morrison, Michael J; Patel, Vinod F; Radinsky, Robert; Rose, Paul E; Ross, Sandra; Sun, Ji-Rong; Tang, Jin; Zhao, Huilin; Payton, Marc; Geuns-Meyer, Stephanie D

    2010-09-09

    The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.

  14. A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

    PubMed

    Chan-Penebre, Elayne; Kuplast, Kristy G; Majer, Christina R; Boriack-Sjodin, P Ann; Wigle, Tim J; Johnston, L Danielle; Rioux, Nathalie; Munchhof, Michael J; Jin, Lei; Jacques, Suzanne L; West, Kip A; Lingaraj, Trupti; Stickland, Kimberly; Ribich, Scott A; Raimondi, Alejandra; Scott, Margaret Porter; Waters, Nigel J; Pollock, Roy M; Smith, Jesse J; Barbash, Olena; Pappalardi, Melissa; Ho, Thau F; Nurse, Kelvin; Oza, Khyati P; Gallagher, Kathleen T; Kruger, Ryan; Moyer, Mikel P; Copeland, Robert A; Chesworth, Richard; Duncan, Kenneth W

    2015-06-01

    Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.

  15. Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor

    PubMed Central

    Flinspach, M.; Xu, Q.; Piekarz, A. D.; Fellows, R.; Hagan, R.; Gibbs, A.; Liu, Y.; Neff, R. A.; Freedman, J.; Eckert, W. A.; Zhou, M.; Bonesteel, R.; Pennington, M. W.; Eddinger, K. A.; Yaksh, T. L.; Hunter, M.; Swanson, R. V.; Wickenden, A. D.

    2017-01-01

    Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain. PMID:28045073

  16. The Marine Cyanobacterial Metabolite Gallinamide A is a Potent and Selective Inhibitor of Human Cathepsin L

    PubMed Central

    Miller, Bailey; Friedman, Aaron J; Choi, Hyukjae; Hogan, James; McCammon, J. Andrew; Hook, Vivian; Gerwick, William H.

    2014-01-01

    A number of marine natural products are potent inhibitors of proteases, an important drug target class in human diseases. Hence, marine cyanobacterial extracts were assessed for inhibitory activity to human cathepsin L. Herein, we have shown that gallinamide A potently and selectively inhibits the human cysteine protease, cathepsin L. With 30 min of preincubation, gallinamide A displayed an IC50 of 5.0 nM, and kinetic analysis demonstrated an inhibition constant of ki = 9000 ± 260 M−1 s−1. Preincubation-dilution and activity-probe experiments revealed an irreversible mode of inhibition, and comparative IC50 values display a 28- to 320- fold greater selectivity toward cathepsin L than closely related human cysteine cathepsins V or B. Molecular docking and molecular dynamics simulations were used to determine the pose of gallinamide in the active site of cathepsin L. These data resulted in the identification of a pose characterized by high stability, a consistent hydrogen bond network, and the reactive Michael acceptor enamide of gallinamide A positioned near the active site cysteine of the protease, leading to a proposed mechanism of covalent inhibition. These data reveal and characterize the novel activity of gallinamide A as a potent inhibitor of human cathepsin L. PMID:24364476

  17. Peripheral Blood Mononuclear Cells Infiltration Downregulates Decidual FAAH Activity in an LPS-Induced Embryo Resorption Model.

    PubMed

    Wolfson, Manuel Luis; Aisemberg, Julieta; Correa, Fernando; Franchi, Ana María

    2017-06-01

    Maternal infections with gram-negative bacteria are associated with miscarriage and are one of the most common complications during pregnancy. Previous studies from our group have shown that lipopolysaccharide (LPS)-activated infiltrating peripheral blood mononuclear cells (PBMC) into decidual tissue plays an important role in the establishment of a local inflammatory process that results in embryo cytotoxicity and early embryo resorption. Moreover, we have also shown that an increased endocannabinoid tone mediates LPS-induced deleterious effects during early pregnancy loss. Here, we sought to investigate whether the infiltrating PBMC modulates the decidual endocannabinoid tone and the molecular mechanisms involved. PBMC isolated from 7-day pregnant mice subjected to different treatments were co-cultured in a transwell system with decidual tissue from control 7-day pregnant mice. Decidual fatty acid amide hydrolase (FAAH) activity was measured by radioconvertion, total decidual protein nitration by Western blot (WB), and decidual FAAH nitration by immunoprecipitation followed by WB. We found that co-culture of PBMC obtained from LPS-treated mice increased the level of nitration of decidual FAAH, which resulted in a negative modulation of decidual FAAH activity. Interestingly, co-treatment with progesterone or aminoguanidine prevented this effect. We found that LPS-treated PBMC release high amounts of nitric oxide (NO) which causes tyrosine nitration of decidual FAAH, diminishing its enzymatic activity. Inactivation of FAAH, the main degrading enzyme of anandamide and similar endocannabinoids, could lead to an increased decidual endocannabinoid tone with embryotoxic effects. J. Cell. Physiol. 232: 1441-1447, 2017. © 2016 Wiley Periodicals, Inc.

  18. Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

    PubMed

    Shi, Z; Ma, X H; Qin, C; Jia, J; Jiang, Y Y; Tan, C Y; Chen, Y Z

    2012-02-01

    Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority <15.4%) of the individual target inhibitors of the same target pair and 0.58-7.1% of the other 6 targets outside the target pair. COMBI-SVMs showed low dual inhibitor false hit rates (0.006-0.056%, 0.042-0.21%, 0.2-4%) in screening 17 million PubChem compounds, 168,000 MDDR compounds, and 7-8181 MDDR compounds similar to the dual inhibitors. Compared with similarity searching, k-NN and PNN methods, COMBI-SVM produced comparable dual inhibitor yields, similar target selectivity, and lower false hit rate in screening 168,000 MDDR compounds. The annotated classes of many COMBI-SVMs identified MDDR virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents.

  19. Intermediate cannabis dependence phenotypes and the FAAH C385A variant: an exploratory analysis

    PubMed Central

    Selling, Rebecca E.; Hutchison, Kent E.

    2010-01-01

    Rationale Cannabis dependence is a growing problem among individuals who use marijuana frequently, and genetic differences make some users more liable to progress to dependence. The identification of intermediate phenotypes of cannabis dependence may aid candidate genetic analysis. Promising intermediate phenotypes include craving for marijuana, withdrawal symptoms after abstinence, and sensitivity to its acute effects. A single nucleotide polymorphism (SNP) in the gene encoding for fatty acid amide hydrolase (FAAH) has demonstrated association with substance use disorder diagnoses, but has not been studied with respect to these narrower phenotypes. FAAH is an enzyme that inactivates anandamide, an endogenous agonist for CB1 receptors (to which Δ9-tetrahydrocannabinol binds). CB1 binding modulates mesocorticolimbic dopamine release, which underlies many facets of addiction. Objectives The SNP, FAAH C385A (rs324420), was examined to determine whether its variance was associated with changes in craving and withdrawal after marijuana abstinence, craving after cue exposure, or sensitivity to the acute effects of marijuana. Materials and methods Forty daily marijuana users abstained for 24 h, were presented with a cue-elicited craving paradigm and smoked a marijuana cigarette in the laboratory. Results C385A variance was significantly associated with changes in withdrawal after abstinence, and happiness after smoking marijuana in the predicted directions, was associated with changes in heart rate after smoking in the opposite of the predicted direction, and was not associated with changes in craving or other acute effects. Conclusions These data lend support to some previous association studies of C385A, but suggest that further refinement of these intermediate phenotypes is necessary. PMID:19002671

  20. Potent and Selective Small Molecule Inhibitors of Specific Isoforms of Cdc2-like Kinases (Clk) and Dual Specificity Tyrosine-Phosphorylation-Regulated Kinases (Dyrk)

    PubMed Central

    Rosenthal, Andrew S.; Tanega, Cordelle; Shen, Min; Mott, Bryan T.; Bougie, James M.; Nguyen, Dac-Trung; Misteli, Tom; Auld, Douglas S.; Maloney, David J.; Thomas, Craig J.

    2011-01-01

    Continued examination of substituted 6-arylquinazolin-4-amines as Clk4 inhibitors resulted in selective inhibitors of Clk1, Clk4, Dyrk1A and Dyrk1B. Several of the most potent inhibitors were validated as being highly selective within a comprehensive kinome scan. PMID:21450467

  1. Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy.

    PubMed

    Leverson, Joel D; Phillips, Darren C; Mitten, Michael J; Boghaert, Erwin R; Diaz, Dolores; Tahir, Stephen K; Belmont, Lisa D; Nimmer, Paul; Xiao, Yu; Ma, Xiaoju Max; Lowes, Kym N; Kovar, Peter; Chen, Jun; Jin, Sha; Smith, Morey; Xue, John; Zhang, Haichao; Oleksijew, Anatol; Magoc, Terrance J; Vaidya, Kedar S; Albert, Daniel H; Tarrant, Jacqueline M; La, Nghi; Wang, Le; Tao, Zhi-Fu; Wendt, Michael D; Sampath, Deepak; Rosenberg, Saul H; Tse, Chris; Huang, David C S; Fairbrother, Wayne J; Elmore, Steven W; Souers, Andrew J

    2015-03-18

    The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.

  2. Mutation screen and association studies for the fatty acid amide hydrolase (FAAH) gene and early onset and adult obesity

    PubMed Central

    2010-01-01

    Background The orexigenic effects of cannabinoids are limited by activation of the endocannabinoid degrading enzyme fatty acid amide hydrolase (FAAH). The aim of this study was to analyse whether FAAH alleles are associated with early and late onset obesity. Methods We initially assessed association of five single nucleotide polymorphisms (SNPs) in FAAH with early onset extreme obesity in up to 521 German obese children and both parents. SNPs with nominal p-values ≤ 0.1 were subsequently analysed in 235 independent German obesity families. SNPs associated with childhood obesity (p-values ≤ 0.05) were further analysed in 8,491 adult individuals of a population-based cohort (KORA) for association with adult obesity. One SNP was further analysed in 985 German obese adults and 588 normal and underweight controls. In parallel, we screened the FAAH coding region for novel sequence variants in 92 extremely obese children using single-stranded-conformation-polymorphism-analysis and denaturing HPLC and assessed the implication of the identified new variants for childhood obesity. Results The trio analysis revealed some evidence for an association of three SNPs in FAAH (rs324420 rs324419 and rs873978) with childhood obesity (two-sided p-values between 0.06 and 0.10). Although analyses of these variants in 235 independent obesity families did not result in statistically significant effects (two-sided p-values between 0.14 and 0.75), the combined analysis of all 603 obesity families supported the idea of an association of two SNPs in FAAH (rs324420 and rs2295632) with early onset extreme obesity (p-values between 0.02 and 0.03). No association was, however, found between these variants and adult obesity. The mutation screen revealed four novel variants, which were not associated with early onset obesity (p > 0.05). Conclusions As we observed some evidence for an association of the FAAH variants rs2295632 rs324420 with early onset but not adult obesity, we conclude that the

  3. Rational Design of Fatty Acid Amide Hydrolase Inhibitors that Act by Covalently Bonding to Two Active Site Residues

    PubMed Central

    Otrubova, Katerina; Brown, Monica; McCormick, Michael S.; Han, Gye W.; O’Neal, Scott T.; Cravatt, Benjamin F.; Stevens, Raymond C.; Lichtman, Aron H.; Boger, Dale L.

    2013-01-01

    The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent non-competitive inhibition, the co-crystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrate that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1–6 h time course monitored. PMID:23581831

  4. Evaluation of substituted 6-arylquinazolin-4-amines as potent and selective inhibitors of cdc2-like kinases (Clk)

    PubMed Central

    Mott, Bryan T.; Tanega, Cordelle; Shen, Min; Maloney, David J.; Shinn, Paul; Leister, William; Marugan, Juan J.; Inglese, James; Austin, Christopher P.; Misteli, Tom; Auld, Douglas S.; Thomas, Craig J.

    2010-01-01

    A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure activity-relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4 and Dyrk1A. PMID:19837585

  5. Alpha-amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold.

    PubMed

    Lehtiö, J; Teeri, T T; Nygren, P A

    2000-11-15

    A disulfide bridge-constrained cellulose binding domain (CBD(WT)) derived from the cellobiohydrolase Cel7A from Trichoderma reesei has been investigated for use in scaffold engineering to obtain novel binding proteins. The gene encoding the wild-type 36 aa CBD(WT) domain was first inserted into a phagemid vector and shown to be functionally displayed on M13 filamentous phage as a protein III fusion protein with retained cellulose binding activity. A combinatorial library comprising 46 million variants of the CBD domain was constructed through randomization of 11 positions located at the domain surface and distributed over three separate beta-sheets of the domain. Using the enzyme porcine alpha-amylase (PPA) as target in biopannings, two CBD variants showing selective binding to the enzyme were characterized. Reduction and iodoacetamide blocking of cysteine residues in selected CBD variants resulted in a loss of binding activity, indicating a conformation dependent binding. Interestingly, further studies showed that the selected CBD variants were capable of competing with the binding of the amylase inhibitor acarbose to the enzyme. In addition, the enzyme activity could be partially inhibited by addition of soluble protein, suggesting that the selected CBD variants bind to the active site of the enzyme.

  6. Discovery of a novel proteasome inhibitor selective for cancer cells over non-transformed cells.

    PubMed

    Kazi, Aslamuzzaman; Lawrence, Harshani; Guida, Wayne C; McLaughlin, Mark L; Springett, Gregory M; Berndt, Norbert; Yip, Richard M L; Sebti, Saïd M

    2009-06-15

    Numerous proteins controlling cell cycle progression, apoptosis and angiogenesis are degraded by the ubiquitin/proteasome system, which has become the subject for intense investigations for cancer therapeutics. Therefore, we used in silico and experimental approaches to screen compounds from the NCI chemical libraries for inhibitors against the chymotrypsin-like (CT-L) activity of the proteasome and discovered PI-083. Molecular docking indicates that PI-083 interacts with the Thr21, Gly47 and Ala49 residues of the beta5 subunit and Asp114 of the beta6 subunit of the proteasome. PI-083 inhibits CT-L activity and cell proliferation and induces apoptosis selectively in cancer cells (ovarian T80-Hras, pancreatic C7-Kras and breast MCF-7) as compared to their normal/immortalized counterparts (T80, C7 and MCF-10A, respectively). In contrast, Bortezomib, the only proteasome inhibitor approved by the Food and Drug Administration (FDA), did not exhibit this selectivity for cancer over non-transformed cells. In addition, in all cancer cells tested, including Multiple Myeloma (MM), breast, pancreatic, ovarian, lung, prostate cancer cell lines as well as fresh MM cells from patients, PI-083 required less time than Bortezomib to induce its antitumor effects. Furthermore, in nude mouse xenografts in vivo, PI-083, but not Bortezomib, suppressed the growth of human breast and lung tumors. Finally, following in vivo treatment of mice, PI-083 inhibited tumor, but not hepatic liver CT-L activity, whereas Bortezomib inhibited both tumor and liver CT-L activities. These results suggest that PI-083 is more selective for cancer cells and may have broader antitumor activity and therefore warrants further advanced preclinical studies.

  7. Evaluation of a tyrosine kinase peptide microarray for tyrosine kinase inhibitor therapy selection in cancer

    PubMed Central

    Labots, Mariette; Gotink, Kristy J; Dekker, Henk; Azijli, Kaamar; van der Mijn, Johannes C; Huijts, Charlotte M; Piersma, Sander R; Jiménez, Connie R; Verheul, Henk M W

    2016-01-01

    Personalized cancer medicine aims to accurately predict the response of individual patients to targeted therapies, including tyrosine kinase inhibitors (TKIs). Clinical implementation of this concept requires a robust selection tool. Here, using both cancer cell lines and tumor tissue from patients, we evaluated a high-throughput tyrosine kinase peptide substrate array to determine its readiness as a selection tool for TKI therapy. We found linearly increasing phosphorylation signal intensities of peptides representing kinase activity along the kinetic curve of the assay with 7.5–10 μg of lysate protein and up to 400 μM adenosine triphosphate (ATP). Basal kinase activity profiles were reproducible with intra- and inter-experiment coefficients of variation of <15% and <20%, respectively. Evaluation of 14 tumor cell lines and tissues showed similar consistently high phosphorylated peptides in their basal profiles. Incubation of four patient-derived tumor lysates with the TKIs dasatinib, sunitinib, sorafenib and erlotinib primarily caused inhibition of substrates that were highly phosphorylated in the basal profile analyses. Using recombinant Src and Axl kinase, relative substrate specificity was demonstrated for a subset of peptides, as their phosphorylation was reverted by co-incubation with a specific inhibitor. In conclusion, we demonstrated robust technical specifications of this high-throughput tyrosine kinase peptide microarray. These features required as little as 5–7 μg of protein per sample, facilitating clinical implementation as a TKI selection tool. However, currently available peptide substrates can benefit from an enhancement of the differential potential for complex samples such as tumor lysates. We propose that mass spectrometry-based phosphoproteomics may provide such an enhancement by identifying more discriminative peptides. PMID:27980342

  8. Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition.

    PubMed

    Telliez, Jean-Baptiste; Dowty, Martin E; Wang, Lu; Jussif, Jason; Lin, Tsung; Li, Li; Moy, Erick; Balbo, Paul; Li, Wei; Zhao, Yajuan; Crouse, Kimberly; Dickinson, Caitlyn; Symanowicz, Peter; Hegen, Martin; Banker, Mary Ellen; Vincent, Fabien; Unwalla, Ray; Liang, Sidney; Gilbert, Adam M; Brown, Matthew F; Hayward, Matthew; Montgomery, Justin; Yang, Xin; Bauman, Jonathan; Trujillo, John I; Casimiro-Garcia, Agustin; Vajdos, Felix F; Leung, Louis; Geoghegan, Kieran F; Quazi, Amira; Xuan, Dejun; Jones, Lyn; Hett, Erik; Wright, Katherine; Clark, James D; Thorarensen, Atli

    2016-12-16

    PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. In vitro, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and in vivo it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1β production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases.

  9. The role of chemoprevention by selective cyclooxygenase-2 inhibitors in colorectal cancer patients - a population-based study

    PubMed Central

    2012-01-01

    Background There are limited population-based studies focusing on the chemopreventive effects of selective cyclooxygenase-2 (COX-2) inhibitors against colorectal cancer. The purpose of this study is to assess the trends and dose–response effects of various medication possession ratios (MPR) of selective COX-2 inhibitor used for chemoprevention of colorectal cancer. Methods A population-based case–control study was conducted using the Taiwan Health Insurance Research Database (NHIRD). The study comprised 21,460 colorectal cancer patients and 79,331 controls. The conditional logistic regression was applied to estimate the odds ratios (ORs) for COX-2 inhibitors used for several durations (5 years, 3 years, 1 year, 6 months and 3 months) prior to the index date. Results In patients receiving selective COX-2 inhibitors, the OR was 0.51 (95% CI=0.29~0.90, p=0.021) for an estimated 5-year period in developing colorectal cancer. ORs showing significant protection effects were found in 10% of MPRs for 5-year, 3-year, and 1-year usage. Risk reduction against colorectal cancer by selective COX-2 inhibitors was observed as early as 6 months after usage. Conclusion Our results indicate that selective COX-2 inhibitors may reduce the development of colorectal cancer by at least 10% based on the MPRs evaluated. Given the limited number of clinical reports from general populations, our results add to the knowledge of chemopreventive effects of selective COX-2 inhibitors against cancer in individuals at no increased risk of colorectal cancer. PMID:23217168

  10. Rational Design Synthesis and Evaluation of New Selective Inhibitors of Microbial Class II (Zinc Dependent) Fructose Bis-phosphate Aldolases

    SciTech Connect

    R Daher; M Coincon; M Fonvielle; P Gest; M Guerin; M Jackson; J Sygusch; M Therisod

    2011-12-31

    We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows Ki against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 105). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.

  11. Discovery of 3-Alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitriles as Selective, Orally Bioavailable CHK1 Inhibitors

    PubMed Central

    2012-01-01

    Inhibitors of checkpoint kinase 1 (CHK1) are of current interest as potential antitumor agents, but the most advanced inhibitor series reported to date are not orally bioavailable. A novel series of potent and orally bioavailable 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitrile CHK1 inhibitors was generated by hybridization of two lead scaffolds derived from fragment-based drug design and optimized for CHK1 potency and high selectivity using a cell-based assay cascade. Efficient in vivo pharmacokinetic assessment was used to identify compounds with prolonged exposure following oral dosing. The optimized compound (CCT244747) was a potent and highly selective CHK1 inhibitor, which modulated the DNA damage response pathway in human tumor xenografts and showed antitumor activity in combination with genotoxic chemotherapies and as a single agent. PMID:23082860

  12. Structure-based de novo design and identification of D816V mutant-selective c-KIT inhibitors.

    PubMed

    Park, Hwangseo; Lee, Soyoung; Lee, Suhyun; Hong, Sungwoo

    2014-07-14

    To identify potent and selective inhibitors of D816V, the most common gain-of-function c-KIT mutant, we carried out structure-based de novo design using 7-azaindole as the core and the scoring function improved by implementing an accurate solvation free energy term. This approach led to the identification of new c-KIT inhibitors specific for the D816V mutant. The 3-(3,4-dimethoxyphenyl)-7-azaindole scaffold was optimized and represents a lead structure for the design of the potent and specific inhibitors of the D816V mutant. The results of molecular dynamics simulations indicate that hydrogen bonding interactions between the 7-azadindole moiety and the backbone groups of Cys673 are the most significant determinant for the potency and selectivity of c-KIT inhibitors.

  13. Aza-peptidyl Michael acceptor and epoxide inhibitors--potent and selective inhibitors of Schistosoma mansoni and Ixodes ricinus legumains (asparaginyl endopeptidases).

    PubMed

    Ovat, Asli; Muindi, Fanuel; Fagan, Crystal; Brouner, Michelle; Hansell, Elizabeth; Dvorák, Jan; Sojka, Daniel; Kopácek, Petr; McKerrow, James H; Caffrey, Conor R; Powers, James C

    2009-11-26

    Aza-peptide Michael acceptors and epoxides with the general structure of YCO-Ala-Ala-AAsn-trans-CH horizontal lineCHCOR and YCO-Ala-Ala-AAsn-EP-COR, respectively, are shown to be potent inhibitors of asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE), and the hard tick, Ixodes ricinus (IrAE). Structure-activity relationships (SARs) were determined for a set of 41 aza-peptide Michael acceptors and eight aza-peptide epoxides. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position, and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues at P2. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors, and for some of these compounds, second-order inhibiton rate constants are the fastest yet discovered. Given the central functions of these enzymes in both parasites, the data presented here may facilitate the eventual design of selective antiparasitic drugs.

  14. Identifying a Selective Substrate and Inhibitor Pair for the Evaluation of CYP2J2 Activity

    PubMed Central

    Lee, Caroline A.; Jones, J. P.; Katayama, Jonathan; Kaspera, Rüdiger; Jiang, Ying; Freiwald, Sascha; Smith, Evan; Walker, Gregory S.

    2012-01-01

    CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC50 = 77 nM) and O-demethylation of astemizole (Ki = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC50 values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues. PMID:22328583

  15. Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity.

    PubMed

    Lee, Caroline A; Jones, J P; Katayama, Jonathan; Kaspera, Rüdiger; Jiang, Ying; Freiwald, Sascha; Smith, Evan; Walker, Gregory S; Totah, Rheem A

    2012-05-01

    CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC(50) = 77 nM) and O-demethylation of astemizole (K(i) = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC(50) values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues.

  16. A NMR and MD study of the active site of factor Xa by selective inhibitors

    NASA Astrophysics Data System (ADS)

    Doan, B. T.; Fraternali, F.; Do, Q. T.; Atkinson, R. A.; Palmas, P.; Sklenar, V.; Wildgoose, P.; Strop, P.; Saudek, V.

    1998-02-01

    The structure of two selective inhibitors obtained by the screening of a vast combinatorial library, Ac-Tyr-Ile-Arg-Ile-NH2 and Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, in the active site of the blood clotting enzyme factor Xa was determined using transferred NOE NMR and simulated annealing (SA) under NMR constraints. The refined structures of the inhibitors were docked in the active site and SA was performed inside the enzyme which has been kept as a rigid charged template. The final structures were optimised by molecular dynamics simulation of the complexes in water. The inhibitors assume a compact, very well defined conformation embedded in the binding site without blocking the catalysis. The model allows to explain the mode of action, affinity and specificity. L'étude structurale d'inhibiteurs du facteur Xa, une enzyme de coagulation, obtenus par chimie combinatoire : Ac-Tyr-Ile-Arg-Ile-NH2, Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, a été réalisée par RMN NOE de transfert et modélisation moléculaire. Les structures ont été calculées sous contraintes RMN : géométrie de distance, recuit simulé et minimisation, affinées par une recherche conformationnelle et recuit de l'inhibiteur placé dans le site actif et optimisées par simulation de dynamique moléculaire du complexe dans l'eau. L'inhibiteur présente une structure compacte positionnée dans le site d'interaction hors d'accès du site catalytique. Ce modèle permet d'expliquer le mode d'action, l'affinité et la spécificité des peptides.

  17. The dual blocker of FAAH/TRPV1 N-arachidonoylserotonin reverses the behavioral despair induced by stress in rats and modulates the HPA-axis.

    PubMed

    Navarria, Andrea; Tamburella, Alessandra; Iannotti, Fabio A; Micale, Vincenzo; Camillieri, Giovanni; Gozzo, Lucia; Verde, Roberta; Imperatore, Roberta; Leggio, Gian Marco; Drago, Filippo; Di Marzo, Vincenzo

    2014-09-01

    In recent years, several studies have explored the involvement of the deregulation of the hypothalamus-pituitary-adrenal (HPA) axis in the pathophysiology of stress-related disorders. HPA hyper-activation as a consequence of acute/chronic stress has been found to play a major role in the neurobiological changes that are responsible for the onset of such states. Currently available medications for depression, one of the most relevant stress-related disorders, present several limitations, including a time lag for treatment response and low rates of efficacy. N-Arachidonoylserotonin (AA-5-HT), a dual blocker at fatty acid amide hydrolase (FAAH, the enzyme responsible for the inactivation of the endocannabinoid anandamide) and transient receptor potential vanilloid type-1 channel (TRPV1), produces anxiolytic-like effects in mice. The present study was designed to assess the capability of AA-5-HT to reverse the behavioral despair following exposure to stress in rats and the role of the HPA-axis. Behavioral tasks were performed, and corticosterone and endocannabinoid (anandamide and 2-arachidonoylglycerol) levels were measured in selected brain areas critically involved in the pathophysiology of stress-related disorders (medial PFC and hippocampus) under basal and stress conditions, and in response to treatment with AA-5-HT. Our data show that AA-5-HT reverses the rat behavioral despair in the forced swim test under stress conditions, and this effect is associated with the normalization of the HPA-axis deregulation that follows stress application and only in part with elevation of anandamide levels. Blockade of FAAH and TRPV1 may thus represent a novel target to design novel therapeutic strategies for the treatment of stress-related disorders.

  18. ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines

    SciTech Connect

    Zask, Arie; Verheijen, Jeroen C.; Curran, Kevin; Kaplan, Joshua; Richard, David J.; Nowak, Pawel; Malwitz, David J.; Brooijmans, Natasja; Bard, Joel; Svenson, Kristine; Lucas, Judy; Toral-Barza, Lourdes; Zhang, Wei-Guo; Hollander, Irwin; Gibbons, James J.; Abraham, Robert T.; Ayral-Kaloustian, Semiramis; Mansour, Tarek S.; Yu, Ker

    2009-09-18

    The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

  19. Synthesis and evaluation of pyrazolo[1,5-b]pyridazines as selective cyclin dependent kinase inhibitors

    SciTech Connect

    Stevens, Kirk L.; Reno, Michael J.; Alberti, Jennifer B.; Price, Daniel J.; Kane-Carson, Laurie S.; Knick, Victoria B.; Shewchuk, Lisa M.; Hassell, Anne M.; Veal, James M.; Davis, Stephen T.; Griffin, Robert J.; Peel, Michael R.

    2010-10-01

    A novel series of pyrazolo[1,5-b]pyridazines have been synthesized and identified as cyclin dependant kinase inhibitors potentially useful for the treatment of solid tumors. Modification of the hinge-binding amine or the C(2)- and C(6)-substitutions on the pyrazolopyridazine core provided potent inhibitors of CDK4 and demonstrated enzyme selectivity against VEGFR-2 and GSK3{beta}.

  20. SAR Exploration Guided by LE and Fsp3: Discovery of a Selective and Orally Efficacious RORγ Inhibitor

    PubMed Central

    2015-01-01

    A novel series of RORγ inhibitors was identified starting with the HTS hit 1. After SAR investigation based on a prospective consideration of two drug-likeness metrics, ligand efficiency (LE) and fraction of sp3 carbon atoms (Fsp3), significant improvement of metabolic stability as well as reduction of CYP inhibition was observed, which finally led to discovery of a selective and orally efficacious RORγ inhibitor 3z. PMID:26819660

  1. Selinexor, a Selective Inhibitor of Nuclear Export (SINE) compound, acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death

    PubMed Central

    Kashyap, Trinayan; Argueta, Christian; Aboukameel, Amro; Unger, Thaddeus John; Klebanov, Boris; Mohammad, Ramzi M.; Muqbil, Irfana; Azmi, Asfar S.; Drolen, Claire; Senapedis, William; Lee, Margaret; Kauffman, Michael; Shacham, Sharon; Landesman, Yosef

    2016-01-01

    The nuclear export protein, exportin-1 (XPO1/CRM1), is overexpressed in many cancers and correlates with poor prognosis. Selinexor, a first-in-class Selective Inhibitor of Nuclear Export (SINE) compound, binds covalently to XPO1 and blocks its function. Treatment of cancer cells with selinexor results in nuclear retention of major tumor suppressor proteins and cell cycle regulators, leading to growth arrest and apoptosis. Recently, we described the selection of SINE compound resistant cells and reported elevated expression of inflammation-related genes in these cells. Here, we demonstrated that NF-κB transcriptional activity is up-regulated in cells that are naturally resistant or have acquired resistance to SINE compounds. Resistance to SINE compounds was created by knockdown of the cellular NF-κB inhibitor, IκB-α. Combination treatment of selinexor with proteasome inhibitors decreased NF-κB activity, sensitized SINE compound resistant cells and showed synergistic cytotoxicity in vitro and in vivo. Furthermore, we showed that selinexor inhibited NF-κB activity by blocking phosphorylation of the IκB-α and the NF-κB p65 subunits, protecting IκB-α from proteasome degradation and trapping IκB-α in the nucleus to suppress NF-κB activity. Therefore, combination treatment of selinexor with a proteasome inhibitor may be beneficial to patients with resistance to either single-agent. PMID:27713151

  2. Effects of tyramine administration in Parkinson's disease patients treated with selective MAO-B inhibitor rasagiline.

    PubMed

    deMarcaida, J Antonelle; Schwid, Steven R; White, William B; Blindauer, Karen; Fahn, Stanley; Kieburtz, Karl; Stern, Matthew; Shoulson, Ira

    2006-10-01

    Rasagiline is a novel, potent, and selective MAO-B inhibitor shown to be effective for Parkinson's disease. Traditional nonselective MAO inhibitors have been associated with dietary tyramine interactions that can induce hypertensive reactions. To test safety, tyramine challenges (50-75 mg) were performed in 72 rasagiline-treated and 38 placebo-treated Parkinson's disease (PD) patients at the end of two double-blind placebo-controlled trials of rasagiline. An abnormal pressor response was prespecified as three consecutive measurements of systolic blood pressure (BP) increases of >or= 30 mm Hg and/or bradycardia of < 40 beats/min. In the first study involving 55 patients with early PD on rasagiline monotherapy, no patients randomized to rasagiline (1 mg/2 mg; n = 38) or placebo (n = 17) developed systolic BP (SBP) or heart rate changes indicative of a tyramine reaction. In the second trial involving 55 levodopa-treated patients, 3 of 22 subjects on rasagiline 0.5 mg/day and 1 of 21 subjects on placebo developed asymptomatic, self-limiting SBP elevations >or= 30 mm Hg on three measurements. No subject on 1 mg/day rasagiline (0/12) experienced significant BP or heart rate changes following tyramine ingestion. These data demonstrate that rasagiline 0.5 to 2 mg daily is not associated with clinically significant tyramine reactions and can be used as monotherapy or adjunct to levodopa in PD patients without specific dietary tyramine restriction.

  3. Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases

    PubMed Central

    Schonbrunn, Ernst; Betzi, Stephane; Alam, Riazul; Martin, Mathew P.; Becker, Andreas; Han, Huijong; Francis, Rawle; Chakrasali, Ramappa; Jakkaraj, Sudhakar; Kazi, Aslamuzzaman; Sebti, Said M.; Cubitt, Christopher L.; Gebhard, Anthony W.; Hazlehurst, Lori A.; Tash, Joseph S.; Georg, Gunda I.

    2013-01-01

    Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009 – 0.0015 µM) from a single hit compound with weak inhibitory activity (IC50 = 15 µM), discovered by high-throughput screening. Structure-based design was performed using 35 co-crystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4 and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 µM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics. PMID:23600925

  4. Possible role of selective, irreversible, proteasome inhibitor (carfilzomib) in the treatment of rat hepatocellular carcinoma.

    PubMed

    Mansour, Mahmoud A; Aljoufi, Mohammed A; Al-Hosaini, Khaled; Al-Rikabi, Ammar C; Nagi, Mahmoud N

    2014-05-25

    We investigated the possible therapeutic effect of irreversible proteasome inhibitor, carfilzomib against hepatocellular carcinoma induced chemically by chronic administration of diethylnitrosoamines (DENA). Hepatocellular carcinoma induced by DENA in male Wistar rats was manifested biochemically by significant elevation of serum α-feto protein (AFP) and carcinoembryonic antigen (CEA). In addition, hepatic cancer was further confirmed by a significant increase in hepatic tissue growth factors; vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (FGF). Moreover a marked increase in matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) content were also observed, along with a profound decrease in hepatic endostatin and metallothionein level. Treatment of rats with the selected doses of carfilzomib produced a significant protection against hepatic cancer. The present results claimed that chosen doses of carfilzomib succeeded in suppressing serum tumor markers level AFP and CEA. Furthermore, the drug reduced the elevated level of hepatic growth factors, MMP-2 and TIMP-1 induced by the carcinogen. The antitumor effect of carfilzomib was also accompanied by augmentation of hepatic content of endostatin and metallothionein. Histopathological examination of liver tissues also correlated with the biochemical observations. It could be concluded that treatment with carfilzomib confers a possible antitumor effect against hepatocellular carcinoma induced by DENA model in rats.

  5. Resveratrol-salicylate derivatives as selective DNMT3 inhibitors and anticancer agents.

    PubMed

    Aldawsari, Fahad S; Aguayo-Ortiz, Rodrigo; Kapilashrami, Kanishk; Yoo, Jakyung; Luo, Minkui; Medina-Franco, José L; Velázquez-Martínez, Carlos A

    2016-10-01

    Resveratrol is a natural polyphenol with plethora of biological activities. Resveratrol has previously shown to decrease DNA-methyltransferase (DNMT) enzymes expression and to reactivate silenced tumor suppressor genes. Currently, it seems that no resveratrol analogs have been developed as DNMT inhibitors. Recently, we reported the synthesis of resveratrol-salicylate derivatives and by examining the chemical structure of these analogs, we proposed that these compounds could exhibit DNMT inhibition especially that they resembled NSC 14778, a compound we previously identified as a DNMT inhibitor by virtual screening. Indeed, using in vitro DNMT inhibition assay, some of the resveratrol-salicylate analogs we screened in this work that showed selective inhibition against DNMT3 enzymes which were greater than resveratrol. A molecular docking study revealed key binding interactions with DNMT3A and DNMT3B enzymes. In addition, the most active analog, 10 showed considerable cytotoxicity against three human cancer cells; HT-29, HepG2 and SK-BR-3, which was greater than resveratrol. Further studies are needed to understand the anticancer mechanisms of these derivatives.

  6. Selective Covalent Targeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors.

    PubMed

    Huhn, Annissa J; Guerra, Rachel M; Harvey, Edward P; Bird, Gregory H; Walensky, Loren D

    2016-09-22

    Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical α-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of ABT-199, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. BFL-1 is a BCL-2 homolog implicated in melanoma, lymphoma, and other cancers, and remains undrugged. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1 by covalent targeting. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting pathologic protein interactions.

  7. Combining structure-based pharmacophore and in silico approaches to discover novel selective serotonin reuptake inhibitors.

    PubMed

    Zhou, Zheng-Li; Liu, Hsuan-Liang; Wu, Josephine W; Tsao, Cheng-Wen; Chen, Wei-Hsi; Liu, Kung-Tien; Ho, Yih

    2013-12-01

    Inhibition of human serotonin transporter (hSERT) has been reported to be a potent strategy for the treatment for depression. To discover novel selective serotonin reuptake inhibitors (SSRIs), a structure-based pharmacophore model (SBPM) was developed using the docked conformations of six highly active SSRIs. The best SBPM, consisting of four chemical features: two ring aromatics (RAs), one hydrophobic (HY), and one positive ionizable (PI), was further validated using Gunner-Henry (GH) scoring and receiver operating characteristic (ROC) curve methods. This well-validated SBPM was then used as a 3D-query in virtual screening to identify potential hits from National Cancer Institute (NCI) database. These hits were subsequently filtered by absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction and molecular docking, and their binding stabilities were validated by 20-ns MD simulations. Finally, only two compounds (NSC175176 and NSC705841) were identified as potential leads, which exhibited higher binding affinities in comparison with the paroxetine. Our results also suggest that cation-π interaction plays a crucial role in stabilizing the hSERT-inhibitor complex. To our knowledge, the present work is the first structure-based virtual screening study for new SSRI discovery, which should be a useful guide for the rapid identification of novel therapeutic agents from chemical database.

  8. NO-SSRIs: Nitric Oxide Chimera Drugs Incorporating a Selective Serotonin Reuptake Inhibitor.

    PubMed

    Abdul-Hay, Samer; Schiefer, Isaac T; Chandrasena, R Esala P; Li, Min; Abdelhamid, Ramy; Wang, Yue-Ting; Tavassoli, Ehsan; Michalsen, Bradley; Asghodom, Rezene T; Luo, Jia; Thatcher, Gregory R J

    2011-09-08

    Hybrid nitrate drugs have been reported to provide NO bioactivity to ameliorate side effects or to provide ancillary therapeutic activity. Hybrid nitrate selective serotonin reuptake inhibitors (NO-SSRIs) were prepared to improve the therapeutic profile of this drug class. A synthetic strategy for use of a thiocarbamate linker was developed, which in the case of NO-fluoxetine facilitated hydrolysis to fluoxetine at pH 7.4 within 7 hours. In cell culture, NO-SSRIs were weak inhibitors of the serotonin transporter, however, in the forced swimming task (FST) in rats, NO-fluoxetine demonstrated classical antidepressant activity. Comparison of NO-fluoxetine, with fluoxetine, and an NO-chimera nitrate developed for Alzheimer's disease (GT-1061), was made in the step through passive avoidance (STPA) test of learning and memory in rats treated with scopolamine as an amnesic agent. Fluoxetine was inactive, whereas NO-fluoxetine and GT-1061 both restored long-term memory. GT-1061 also produced antidepressant behavior in FST. These data support the potential for NO-SSRIs to overcome the lag in onset of therapeutic action and provide co-therapy of neuropathologies concomitant with depression.

  9. PFI-1, a highly selective protein interaction inhibitor, targeting BET Bromodomains.

    PubMed

    Picaud, Sarah; Da Costa, David; Thanasopoulou, Angeliki; Filippakopoulos, Panagis; Fish, Paul V; Philpott, Martin; Fedorov, Oleg; Brennan, Paul; Bunnage, Mark E; Owen, Dafydd R; Bradner, James E; Taniere, Philippe; O'Sullivan, Brendan; Müller, Susanne; Schwaller, Juerg; Stankovic, Tatjana; Knapp, Stefan

    2013-06-01

    Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycle progression. BET proteins are recruited on transcriptionally active chromatin via their two N-terminal bromodomains (BRD), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional downregulation of a number of oncogenes, providing a novel pharmacologic strategy for the treatment of cancer. Here, we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1, which efficiently blocks the interaction of BET BRDs with acetylated histone tails. Cocrystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell-cycle arrest, downregulation of MYC expression, as well as induction of apoptosis and induces differentiation of primary leukemic blasts. Intriguingly, cells exposed to PFI-1 showed significant downregulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10, providing an alternative strategy for the specific inhibition of this well-established oncology target.

  10. PFI-1 – A highly Selective Protein Interaction Inhibitor Targeting BET Bromodomains

    PubMed Central

    Picaud, Sarah; Costa, David Da; Thanasopoulou, Angeliki; Filippakopoulos, Panagis; Fish, Paul V.; Philpott, Martin; Fedorov, Oleg; Brennan, Paul; Bunnage, Mark E.; Owen, Dafydd R.; Bradner, James E.; Taniere, Philippe; O’Sullivan, Brendan; Müller, Susanne; Schwaller, Juerg; Stankovic, Tatjana; Knapp, Stefan

    2013-01-01

    Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and anti-apoptotic genes as well as for cell cycle progression. BET proteins are recruited to transcriptionally active chromatin via their two N-terminal bromodomains (BRDs), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional down-regulation of a number of oncogenes providing a novel pharmacological strategy for the treatment of cancer. Here we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1 that efficiently blocks the interaction of BET BRDs with acetylated histone tails. Co-crystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukaemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell cycle arrest, down-regulation of MYC expression as well as induction of apoptosis and induces differentiation of primary leukaemic blasts. Intriguingly, cells exposed to PFI-1 showed significant down-regulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10 providing an alternative strategy for the specific inhibition of this well established oncology target. PMID:23576556

  11. Kukoamine A and other hydrophobic acylpolyamines: potent and selective inhibitors of Crithidia fasciculata trypanothione reductase.

    PubMed Central

    Ponasik, J A; Strickland, C; Faerman, C; Savvides, S; Karplus, P A; Ganem, B

    1995-01-01

    The enzyme trypanothione reductase (TR), together with its substrate, the glutathione-spermidine conjugate trypanothione, plays an essential role in protecting parasitic trypanosomatids against oxidative stress and is a target for drug design. Here we show that a naturally occurring spermine derivative, the antihypertensive agent kukoamine A [N1N12-bis(dihydrocaffeoyl)-spermine] inhibits TR as a mixed inhibitor (Ki = 1.8 microM, Kii = 13 microM). Kukoamine shows no significant inhibition of human glutathione reductase (Ki > 10 mM) and thus provides a novel selective drug lead. The corresponding N1N8-bis(dihydrocaffeoyl)spermidine derivative was synthesized and acted as a purely competitive inhibitor with Ki = 7.5 microM. A series of mono- and di-acylated spermines and spermidines were synthesized to gain an insight into the effect of polyamine chain length, the nature and position of the acyl substituent and the importance of conformational mobility. These compounds inhibited TR with Ki values ranging from 11 to 607 microM. PMID:7487870

  12. Insight into selectivity of peptidomimetic inhibitors with modified statine core for plasmepsin II of Plasmodium falciparum over human cathepsin D.

    PubMed

    Dali, Brice; Keita, Melalie; Megnassan, Eugene; Frecer, Vladimir; Miertus, Stanislav

    2012-04-01

    Plasmepsin II (PlmII), an aspartic protease expressed in the food vacuole of Plasmodium falciparum (pf), cleaves the hemoglobin of the host during the erythrocytic stage of the parasite life cycle. Various peptidomimetic inhibitors of PlmII reported so far discriminate poorly between the drug target and aspartic proteases of the host organism, e.g., human cathepsinD (hCatD). hCatD is a protein digestion enzyme and signaling molecule involved in a variety of physiological processes; therefore, inhibition of hCatD by PlmII inhibitors may lead to pathophysiological conditions. In this study, binding of PlmII inhibitors has been modeled using the crystal structures of pfPlmII and hCatD complexes to gain insight into structural requirements underlying the target selectivity. A series of 26 inhibitors were modeled in the binding clefts of the pfPlmII and hCatD to establish QSAR models of the protease inhibition. In addition, 3D-QSAR pharmacophore models were generated for each enzyme. It was concluded that the contributions of the P(2) and P(3') residues to the inhibitor's binding affinity are responsible for the target selectivity. Based on these findings, new inhibitor candidates were designed with predicted inhibition constants K (pre)(i PlmII) reaching 0.2nm and selectivity index (S.I.)=K(pre)(i PlmII) >1200.

  13. Selective CNS Uptake of the GCP-II Inhibitor 2-PMPA following Intranasal Administration

    PubMed Central

    Rais, Rana; Wozniak, Krystyna; Wu, Ying; Niwa, Minae; Stathis, Marigo; Alt, Jesse; Giroux, Marc; Sawa, Akira; Rojas, Camilo; Slusher, Barbara S.

    2015-01-01

    Glutamate carboxypeptidase II (GCP-II) is a brain metallopeptidase that hydrolyzes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to NAA and glutamate. Small molecule GCP-II inhibitors increase brain NAAG, which activates mGluR3, decreases glutamate, and provide therapeutic utility in a variety of preclinical models of neurodegenerative diseases wherein excess glutamate is presumed pathogenic. Unfortunately no GCP-II inhibitor has advanced clinically, largely due to their highly polar nature resulting in insufficient oral bioavailability and limited brain penetration. Herein we report a non-invasive route for delivery of GCP-II inhibitors to the brain via intranasal (i.n.) administration. Three structurally distinct classes of GCP-II inhibitors were evaluated including DCMC (urea-based), 2-MPPA (thiol-based) and 2-PMPA (phosphonate-based). While all showed some brain penetration following i.n. administration, 2-PMPA exhibited the highest levels and was chosen for further evaluation. Compared to intraperitoneal (i.p.) administration, equivalent doses of i.n. administered 2-PMPA resulted in similar plasma exposures (AUC0-t, i.n./AUC0-t, i.p. = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC0-t, i.n./AUC0-t, i.p. = 67), cortex (AUC0-t, i.n./AUC0-t, i.p. = 46) and cerebellum (AUC0-t, i.n./AUC0-t, i.p. = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC0-t in the olfactory bulb, cortex, and cerebellum were 1.49, 0.71 and 0.10, respectively, compared to an i.p. brain tissue to plasma ratio of less than 0.02 in all areas. Furthermore, i.n. administration of 2-PMPA resulted in complete inhibition of brain GCP-II enzymatic activity ex-vivo confirming target engagement. Lastly, because the rodent nasal system is not similar to humans, we evaluated i.n. 2-PMPA also in a non-human primate. We report that i.n. 2-PMPA provides selective brain delivery with micromolar concentrations. These studies support

  14. Pharmacogenetics of selective serotonin reuptake inhibitors in pediatric depression and anxiety.

    PubMed

    Kronenberg, Sefi; Frisch, Amos; Rotberg, Beni; Carmel, Miri; Apter, Alan; Weizman, Abraham

    2008-11-01

    Selective serotonin reuptake inhibitors (SSRIs) are now an accepted and widely used first-line treatment for pediatric depression and anxiety. However, the data indicate that SSRI treatment achieves a clinical response in only 55-60% of children, and some may develop drug-induced suicidal behavior. Clinicians have no reliable tools to help them identify in advance those youths who are not likely to respond to an SSRI, or who are likely to develop SSRI-induced suicidality. Pharmacogenetic research attempts to identify genetic markers that are associated with response and side-effect profile. This review covers all the pharmacogenetic studies conducted as yet on pediatric samples and compares them with available data on adult samples. An emphasis is put on serotonergic genes such as the serotonin transporter (5-HTT) and additional genes known to be active in the CNS.

  15. Synthesis and structural characterisation of selective non-carbohydrate-based inhibitors of bacterial sialidases.

    PubMed

    Brear, Paul; Telford, Judith; Taylor, Garry L; Westwood, Nicholas J

    2012-11-05

    The major human pathogen Streptococcus pneumoniae plays a key role in several disease states including septicaemia, meningitis and community-acquired pneumonia. Although vaccines against S. pneumoniae are available as prophylactics, there remains a need to identify and characterise novel chemical entities that can treat the diseases caused by this pathogen. S. pneumoniae expresses three sialidases, enzymes that cleave sialic acid from carbohydrate-based surface molecules. Two of these enzymes, NanA and NanB, have been implicated in the pathogenesis of S. pneumoniae and are considered to be validated drug targets. Here we report our studies on the synthesis and structural characterisation of novel NanB-selective inhibitors that are inspired by the β-amino-sulfonic acid family of buffers.

  16. Augmenting selective serotonin reuptake inhibitors with clomipramine in obsessive-compulsive disorder: benefits and risks.

    PubMed

    Andrade, Chittaranjan

    2013-12-01

    A small body of literature suggests that clomipramine may usefully augment selective serotonin reuptake inhibitor (SSRI) treatment in obsessive-compulsive disorder (OCD) patients who do not respond to SSRI monotherapy. The combination, however, is associated with the risk of clinically significant drug interactions. Clomipramine can raise the blood levels and hence the adverse effects of most SSRIs, and many SSRIs can raise the blood levels and hence the adverse effects of clomipramine. The latter situation is more important because certain dose-dependent adverse effects of clomipramine, such as seizures, can be life-threatening. This article presents an evidence-based discussion of the pharmacodynamic and pharmacokinetic adverse effects of the SSRI-clomipramine combination along with suggestions for dosing and monitoring when the combination is used in OCD.

  17. Dapoxetine, a novel selective serotonin transport inhibitor for the treatment of premature ejaculation

    PubMed Central

    Kendirci, Muammer; Salem, Emad; Hellstrom, Wayne JG

    2007-01-01

    Premature ejaculation (PE) is the most common male sexual disorder, estimated to affect up to 30% of men. Over the past one or two decades, clinical investigators have participated in an increasing number of studies that are helping in our understanding of PE, which will undoubtedly facilitate future treatments. Apart from a number of behavioral approaches, the treatment of PE consists of primarily off-label use of oral selective serotonin reuptake inhibitors (SSRIs) via either on-demand or daily delivery. However, various undesirable side-effects of these medications have led researchers to search for and develop new therapeutic approaches for PE. Dapoxetine is a short-acting SSRI developed specifically for the treatment of PE. Early trials with dapoxetine have documented successful outcomes without serious short- or long-term side-effects. This review addresses the definition, classification, diagnosis, physiology, and neurobiopathology of PE, and evaluates therapeutic strategies with novel treatments for PE. PMID:18360636

  18. Delayed pressure urticaria treated with the selective serotonin reuptake inhibitor escitalopram.

    PubMed

    Eskeland, S; Tanum, L; Halvorsen, J A

    2016-07-01

    There is increasing evidence of platelet activation and systemic inflammation in chronic spontaneous urticaria and delayed pressure urticaria (DPU). Inflammation may be central to understanding the high comorbidity of depression and anxiety in patients with chronic urticaria (CU). We report a case of DPU and depression in a patient, which responded favourably to treatment with the selective serotonin reuptake inhibitor (SSRI) escitalopram. Sustained administration of SSRIs is associated with downregulation of serotonin transporters/receptors and depletion of platelet stored serotonin, which may reduce the ability of platelets to aggregate after thrombotic triggers. SSRIs are easier to manage and have significantly less disturbing adverse effects and cardiotoxicity than the tricyclic antidepressants (TCAs). SSRIs may represent an alternative to the traditional use of TCAs in treatment of CU.

  19. Selective serotonin reuptake inhibitor exposure constricts the mouse ductus arteriosus in utero.

    PubMed

    Hooper, Christopher W; Delaney, Cassidy; Streeter, Taylor; Yarboro, Michael T; Poole, Stanley; Brown, Naoko; Slaughter, James C; Cotton, Robert B; Reese, Jeff; Shelton, Elaine L

    2016-09-01

    Use of selective serotonin reuptake inhibitors (SSRIs) is common during pregnancy. Fetal exposure to SSRIs is associated with persistent pulmonary hypertension of the newborn (PPHN); however, a direct link between the two has yet to be established. Conversely, it is well known that PPHN can be caused by premature constriction of the ductus arteriosus (DA), a fetal vessel connecting the pulmonary and systemic circulations. We hypothesized that SSRIs could induce in utero DA constriction. Using isolated vessels and whole-animal models, we sought to determine the effects of two commonly prescribed SSRIs, fluoxetine and sertraline, on the fetal mouse DA. Cannulated vessel myography studies demonstrated that SSRIs caused concentration-dependent DA constriction and made vessels less sensitive to prostaglandin-induced dilation. Moreover, in vivo studies showed that SSRI-exposed mice had inappropriate DA constriction in utero. Taken together, these findings establish that SSRIs promote fetal DA constriction and provide a potential mechanism by which SSRIs could contribute to PPHN.

  20. Perinatal selective serotonin reuptake inhibitor exposure: impact on brain development and neural plasticity.

    PubMed

    Pawluski, Jodi L

    2012-01-01

    Selective serotonin reuptake inhibitor (SSRI) medications are the most common antidepressant treatment used during pregnancy and the postpartum period. Up to 10% of pregnant women are prescribed SSRIs. Serotonin plays an integral part in neurodevelopment, and questions have been raised about the placental transfer of SSRIs and the effects of preventing reuptake of presynaptic serotonin on fetal neurodevelopment. Preclinical data is beginning to document a role of early exposure to SSRIs in long-term developmental outcomes related to a number of brain regions, such as the hippocampus, cortex and cerebellum. To date, the majority of preclinical work has investigated the developmental effects of SSRIs in the offspring of healthy mothers; however, more research is needed on the effects of these medications in the face of maternal adversity. This minireview will highlight emerging evidence from clinical and preclinical studies investigating the impact of perinatal SSRI exposure on brain development and neural plasticity.

  1. The use of selective serotonin reuptake inhibitors during pregnancy and lactation: current knowledge.

    PubMed

    Misri, S; Kostaras, D; Kostaras, X

    2000-04-01

    This article reviews the effects of the selective serotonin reuptake inhibitor (SSRI) class of antidepressants in pregnant and lactating women for the treatment of depression and anxiety disorders. An examination of the literature was conducted using Medline (1966 to present). Despite methodological concerns and the scarcity of data on this important subject, the majority of recent investigations demonstrate safety of the fetus exposed to SSRIs during pregnancy. All of the SSRIs reported in the studies are excreted into breast milk, and low levels have been found in infant serum. The implications of this for practice include identifying the effects of treatment versus nontreatment on the mother-infant dyad. Further research must examine long-term neurobehavioural teratogenicity in exposed infants.

  2. Selective serotonin reuptake inhibitor use during pregnancy and possible neonatal complications.

    PubMed

    Cantor Sackett, Jillan; Weller, Ronald A; Weller, Elizabeth B

    2009-06-01

    This paper reviews the risks and benefits of using selective serotonin reuptake inhibitors (SSRIs) in pregnant women. The effects of SSRI use on pregnant women and fetuses are discussed, and the need for SSRI treatment is explained. Persistent pulmonary hypertension of the newborn, teratogenic risks of SSRI treatment during pregnancy, neonatal adaptations, and long-term outcomes for children whose mothers used SSRIs during pregnancy are specifically considered. Due to conflicting results from current studies, there are no clear guidelines for SSRI treatment in pregnancy. Patients and doctors must discuss together the risks and benefits of SSRI use during pregnancy and decide on a course of treatment. Potential risks must be balanced against the effects of untreated maternal depression.

  3. Fatal serotonin syndrome precipitated by oxcarbazepine in a patient using an selective serotonin reuptake inhibitor.

    PubMed

    Dardis, Christopher; Omoregie, Eghosa; Ly, Vanthanh

    2012-07-01

    Oxcarbazepine, a metabolite of carbamazepine, is used as an antiepileptic, analgesic for neuropathic pain and in the treatment of affective disorders. It has been approved by the Food and Drug Administration for partial seizures in adults as both adjunctive and monotherapy, and as adjunctive therapy in children aged from 2 to 16 years (http://www.fda.gov/ohrms/dockets/ac/06/briefing/2006-4254b_07_05_KP%20OxcarbazepineFDAlabel102005.pdf). We present a case of serotonin syndrome, which was precipitated by this medicine in a patient who had been predisposed by long-term treatment with sertraline, a selective serotonin reuptake inhibitor. This is the first reported fatality due to this drug interaction and only the second case of serotonin syndrome reported with oxcarbazepine. Physicians should consider this risk when prescribing the above combination.

  4. Down-regulation of the rat serotonin transporter upon exposure to a selective serotonin reuptake inhibitor.

    PubMed

    Horschitz, S; Hummerich, R; Schloss, P

    2001-07-20

    The serotonin transporter (SERT) terminates serotonergic neurotransmission by rapid reuptake of 5-hydroxytryptamine (5-HT) into the nerve terminal or axonal varicosities. SERT represents the target of various antidepressants which inhibit 5-HT transport and are widely used for the pharmacotherapy of depression. Here, we have analyzed the function of SERT stably expressed in HEK 293 cells upon exposure to citalopram, a selective serotonin reuptake inhibitor (SSRI), with respect to 5-HT transport activity and protein expression as estimated by ligand binding experiments. Our results show that long-term exposure to an SSRI causes a down-regulation of transport activity as revealed by a reduction of the maximal transport rate, without affecting substrate affinity, accompanied by a decrease in ligand binding sites.

  5. Dapoxetine, a novel selective serotonin transport inhibitor for the treatment of premature ejaculation.

    PubMed

    Kendirci, Muammer; Salem, Emad; Hellstrom, Wayne Jg

    2007-06-01

    Premature ejaculation (PE) is the most common male sexual disorder, estimated to affect up to 30% of men. Over the past one or two decades, clinical investigators have participated in an increasing number of studies that are helping in our understanding of PE, which will undoubtedly facilitate future treatments. Apart from a number of behavioral approaches, the treatment of PE consists of primarily off-label use of oral selective serotonin reuptake inhibitors (SSRIs) via either on-demand or daily delivery. However, various undesirable side-effects of these medications have led researchers to search for and develop new therapeutic approaches for PE. Dapoxetine is a short-acting SSRI developed specifically for the treatment of PE. Early trials with dapoxetine have documented successful outcomes without serious short- or long-term side-effects. This review addresses the definition, classification, diagnosis, physiology, and neurobiopathology of PE, and evaluates therapeutic strategies with novel treatments for PE.

  6. Selective serotonin reuptake inhibitors and hyponatraemia: review and proposed mechanisms in the elderly.

    PubMed

    Kirchner, V; Silver, L E; Kelly, C A

    1998-01-01

    The association between selective serotonin reuptake inhibitors (SSRIs) and hyponatraemia has been well documented, the elderly appearing to be at greatest risk. An analysis of data of hyponatraemia in the elderly using SSRIs from all published cases and from the Committee on Safety of Medicines found that the mean time to detection was about 3 weeks after commencing SSRIs. A wide range of time to detection (1-253 days) and non-specific symptoms suggest hyponatraemia is detected by chance rather than being specifically looked for. In the elderly there are physiological changes, a high prevalence of medical illnesses and concomitant drug use, which may precipitate hyponatraemia. Together with a risk of altered water regulation in psychiatric illness this may account for the particular susceptibility of the elderly to hyponatraemia whilst using SSRIs.

  7. [Susceptibility of the elderly patient to hyponatremia induced by selective serotonin reuptake inhibitors].

    PubMed

    Fonzo-Christe, C; Vogt, N

    2000-01-01

    Numerous spontaneous reports of the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) have followed the increased use of selective serotonin reuptake inhibitors (SSRI). It has been estimated that 1 in 200 patients treated per year developed SIADH, age and low body weight being particular risk factors. No clear gender effect has been detected when confounding factors such as body weight or antidepressant consumption are taken into account. Age-related susceptibility to hyponatraemia may be explained by physiological changes in renal and endocrine function. The high prevalence of polymedication and pluripathology in the elderly may be a contributing factor as well. To date, no study has demonstrated how SSRIs affect the regulation of fluid/sodium balance nor whether they have an independent effect on this regulation in depressed subjects.

  8. Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9.

    PubMed

    Appleby, Todd C; Greenstein, Andrew E; Hung, Magdeleine; Liclican, Albert; Velasquez, Maile; Villaseñor, Armando G; Wang, Ruth; Wong, Melanie H; Liu, Xiaohong; Papalia, Giuseppe A; Schultz, Brian E; Sakowicz, Roman; Smith, Victoria; Kwon, Hyock Joo

    2017-02-24

    Matrix metalloproteinase 9 (MMP9) is a key regulator of the extracellular matrix (ECM), involved in the degradation of various ECM proteins. MMP9 is a member of a large family of proteases that are secreted as inactive zymogens. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745 is a potent, highly selective humanized monoclonal antibody inhibitor of MMP9 that has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745:MMP9 complex and biochemical studies to elucidate the mechanism of GS-5745 inhibition of MMP9. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain. GS-5745 inhibits MMP9 by two mechanisms: binding to active MMP9 allosterically inhibits MMP9 activity and binding to pro-MMP9 prevents MMP9 activation.

  9. Cefsulodin Inspired Potent and Selective Inhibitors of mPTPB, a Virulent Phosphatase from Mycobacterium tuberculosis

    PubMed Central

    2015-01-01

    mPTPB is a virulent phosphatase from Mycobacterium tuberculosis and a promising therapeutic target for tuberculosis. To facilitate mPTPB-based drug discovery, we identified α-sulfophenylacetic amide (SPAA) from cefsulodin, a third generation β-lactam cephalosporin antibiotic, as a novel pTyr pharmacophore for mPTPB. Structure-guided and fragment-based optimization of SPAA led to the most potent and selective mPTPB inhibitor 9, with a Ki of 7.9 nM and more than 10,000-fold preference for mPTPB over a large panel of 25 phosphatases. Compound 9 also exhibited excellent cellular activity and specificity in blocking mPTPB function in macrophage. Given its novel structure, modest molecular mass, and extremely high ligand efficiency (0.46), compound 9 represents an outstanding lead compound for anti-TB drug discovery targeting mPTPB. PMID:26713110

  10. Discovery of Potent, Selective, and Structurally Novel Dot1L Inhibitors by a Fragment Linking Approach.

    PubMed

    Möbitz, Henrik; Machauer, Rainer; Holzer, Philipp; Vaupel, Andrea; Stauffer, Frédéric; Ragot, Christian; Caravatti, Giorgio; Scheufler, Clemens; Fernandez, Cesar; Hommel, Ulrich; Tiedt, Ralph; Beyer, Kim S; Chen, Chao; Zhu, Hugh; Gaul, Christoph

    2017-03-09

    Misdirected catalytic activity of histone methyltransferase Dot1L is believed to be causative for a subset of highly aggressive acute leukemias. Targeting the catalytic domain of Dot1L represents a potential therapeutic approach for these leukemias. In the context of a comprehensive Dot1L hit finding strategy, a knowledge-based virtual screen of the Dot1L SAM binding pocket led to the discovery of 2, a non-nucleoside fragment mimicking key interactions of SAM bound to Dot1L. Fragment linking of 2 and 3, an induced back pocket binder identified in earlier studies, followed by careful ligand optimization led to the identification of 7, a highly potent, selective and structurally novel Dot1L inhibitor.

  11. Δ9-tetrahydrocannabinol and endocannabinoid degradative enzyme inhibitors attenuate intracranial self-stimulation in mice.

    PubMed

    Wiebelhaus, Jason M; Grim, Travis W; Owens, Robert A; Lazenka, Matthew F; Sim-Selley, Laura J; Abdullah, Rehab A; Niphakis, Micah J; Vann, Robert E; Cravatt, Benjamin F; Wiley, Jenny L; Negus, S Stevens; Lichtman, Aron H

    2015-02-01

    A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and 2-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type 2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on

  12. Crystal Structure of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation

    SciTech Connect

    Mileni, Mauro; Kamtekar, Satwik; Wood, David C.; Benson, Timothy E.; Cravatt, Benjamin F.; Stevens, Raymond C.

    2010-08-12

    The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 {angstrom} resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH's catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 {angstrom}) than previously reported. The higher-resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggests a functional convergence between the amidase signature enzymes and serine proteases.

  13. Pharmacological inhibition of FAAH activity in rodents: A promising pharmacological approach for psychological-cardiac comorbidity?

    PubMed

    Carnevali, Luca; Rivara, Silvia; Nalivaiko, Eugene; Thayer, Julian F; Vacondio, Federica; Mor, Marco; Sgoifo, Andrea

    2017-03-01

    Numerous studies have documented a link between psychological disorders and cardiac disease. Yet, no systematic attempts have been made to develop pharmacological approaches for mood and anxiety disorders that could also be beneficial for cardiac health. The endocannabinoid system has been implicated in the regulation of stress, emotional behavior and cardiovascular function. General preclinical findings indicate that the endocannabinoid anandamide modulates physiological and behavioral stress responses and may also protect the heart from arrhythmias. Moreover, recent experimental studies suggest that pharmacological enhancement of anandamide signaling via inhibition of its degrading enzyme fatty acid amide hydrolase (FAAH) exerts anxiolytic- and antidepressive-like effects and improves cardiac autonomic function and the electrical stability of the myocardium in rodent models that reproduce aspects of human psychological/cardiac comorbidity. Here we summarize and discuss such experimental findings, which might guide future preclinical studies towards a systematic evaluation of the therapeutic potential of pharmacological approaches that target FAAH activity for the treatment of the comorbidity between psychological disorders and cardiac disease.

  14. Selective overproduction of the proteasome inhibitor salinosporamide A via precursor pathway regulation

    PubMed Central

    Lechner, Anna; Eustáquio, Alessandra S.; Gulder, Tobias A. M.; Hafner, Mathias; Moore, Bradley S.

    2011-01-01

    The chlorinated natural product salinosporamide A is a potent 20S proteasome inhibitor currently in clinical trials as an anticancer agent. To deepen our understanding of salinosporamide biosynthesis, we investigated the function of a LuxR-type pathway-specific regulatory gene, salR2, and observed a selective effect on the production of salinosporamide A over its less active aliphatic analogs. SalR2 was shown to specifically activate genes involved in the biosynthesis of the halogenated precursor chloroethylmalonyl-CoA, which is a dedicated precursor of salinosporamide A. Specifically, SalR2 activates transcription of two divergent operons – one of which contains the unique S-adenosyl-L-methionine-dependent chlorinase encoding gene salL. By applying this knowledge towards rational engineering, we were able to selectively double salinosporamide A production. This study exemplifies the specialized regulation of a polyketide precursor pathway and its application to the selective overproduction of a specific natural product congener. PMID:22195555

  15. Design, synthesis and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitor.

    PubMed

    Fujimori, Ikuo; Yukawa, Tomoya; Kamei, Taku; Nakada, Yoshihisa; Sakauchi, Nobuki; Yamada, Masami; Ohba, Yusuke; Takiguchi, Maiko; Kuno, Masako; Kamo, Izumi; Nakagawa, Hideyuki; Hamada, Teruki; Igari, Tomoko; Okuda, Teruaki; Yamamoto, Satoshi; Tsukamoto, Tetsuya; Ishichi, Yuji; Ueno, Hiroyuki

    2015-08-01

    Centrally acting noradrenaline reuptake inhibitor (NRI) is reportedly effective for patients with stress urinary incontinence (SUI) by increasing urethral closure in the clinical Phase IIa study with esreboxetine. Noradrenaline transporters are expressed in both central and peripheral nervous systems and the contribution of each site to efficacy has not been clarified. This report describes the development of a series of peripheral-selective 7-phenyl-1,4-oxazepane NRIs to investigate the contribution of the peripheral site to increasing urethral resistance in rats. (6S,7R)-1,4-Oxazepane derivative 7 exhibited noradrenaline transporter inhibition with high selectivity against inhibitions of serotonin and dopamine transporters. A replacement of hydroxyl with acetamide group contributed to enhancement of peripheral selectivity by increasing molecular polarity. Compound 12, N-{[(6S,7R)-7-(3,4-dichlorophenyl)-1,4-oxazepan-6-yl]methyl}acetamide 0.5 fumarate, which showed effectively no brain penetration in rats, increased urethral resistance in a dose-dependent manner and exhibited a maximal effect on par with esreboxetine. These results demonstrate that the urethral resistance-increasing effects of NRI in rats are mainly caused by the inhibition of noradrenaline transporters in the peripheral sites.

  16. Design and synthesis of benzodiazepine analogs as isoform-selective human lysine deacetylase inhibitors.

    PubMed

    Reddy, D Rajasekhar; Ballante, Flavio; Zhou, Nancy J; Marshall, Garland R

    2017-02-15

    A comprehensive investigation was performed to identify new benzodiazepine (BZD) derivatives as potent and selective human lysine deacetylase inhibitors (hKDACis). A total of 108 BZD compounds were designed, synthesized and from that 104 compounds were biologically evaluated against human lysine deacetylases (hKDACs) 1, 3 and 8 (class I) and 6 (class IIb). The most active compounds showed mid-nanomolar potencies against hKDACs 1, 3 and 6 and micromolar activity against hKDAC8, while a promising compound (6q) showed selectivity towards hKDAC3 among the different enzyme isoforms. An hKDAC6 homology model, refined by molecular dynamics simulation was generated, and molecular docking studies performed to rational