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1.
Cell ; 150(3): 575-89, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22863010

RESUMO

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.


Assuntos
Azepinas/farmacologia , Descoberta de Drogas , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Megacariócitos/metabolismo , Poliploidia , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Aurora Quinase A , Aurora Quinases , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Megacarioblástica Aguda/genética , Megacariócitos/citologia , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Associadas a rho/metabolismo
2.
Proc Natl Acad Sci U S A ; 113(4): 830-7, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26699492

RESUMO

Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K(+) efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K(+) efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K(+) and subsequent induction of stress-activated protein kinase pathways.


Assuntos
Apolipoproteínas/genética , Transporte de Íons/genética , Nefropatias/genética , Lipoproteínas HDL/genética , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Mutação de Sentido Incorreto , Potássio/metabolismo , Substituição de Aminoácidos , Apolipoproteína L1 , Apolipoproteínas/fisiologia , População Negra/genética , Morte Celular , Tamanho Celular , Receptor gp130 de Citocina/biossíntese , Receptor gp130 de Citocina/genética , Progressão da Doença , Ativação Enzimática , Frequência do Gene , Predisposição Genética para Doença , Células HEK293 , Humanos , Nefropatias/etnologia , Lipoproteínas HDL/fisiologia , Sistema de Sinalização das MAP Quinases , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Risco , Fator de Transcrição STAT3/metabolismo , Transfecção
3.
J Biol Chem ; 291(13): 7029-44, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26839313

RESUMO

Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages. A new diagnostic marker product for ONOO(-)is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO(-)formed from Nox2-derived O2 (̇̄)and nitric oxide synthase-derived nitric oxide.


Assuntos
Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Glicoproteínas de Membrana/antagonistas & inibidores , Sondas Moleculares/química , NADPH Oxidases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Cromatografia Líquida de Alta Pressão , Cumarínicos/química , Inibidores Enzimáticos/química , Fluorometria , Expressão Gênica , Células HL-60 , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Oxazinas/química , Oxirredução , Ácido Peroxinitroso/antagonistas & inibidores , Ácido Peroxinitroso/biossíntese , Ácido Peroxinitroso/química , Fenantridinas/química , Compostos de Amônio Quaternário/química , Bibliotecas de Moléculas Pequenas/química , Superóxidos/antagonistas & inibidores , Superóxidos/química , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
4.
Bioorg Med Chem Lett ; 23(6): 1834-8, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23403082

RESUMO

A high-throughput screen (HTS) was conducted against stably propagated cancer stem cell (CSC)-enriched populations using a library of 300,718 compounds from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR). A cinnamide analog displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control cell line (HMLE_sh_eGFP). Herein, we report structure-activity relationships of this class of cinnamides for selective lethality towards CSC-enriched populations.


Assuntos
Amidas/química , Bibliotecas de Moléculas Pequenas/química , Amidas/toxicidade , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/toxicidade , Relação Estrutura-Atividade
5.
Bioorg Med Chem Lett ; 22(10): 3571-4, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22503247

RESUMO

A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP).


Assuntos
Neoplasias da Mama/tratamento farmacológico , Hidrazonas/farmacologia , Células-Tronco Neoplásicas/citologia , Pirróis/farmacologia , Neoplasias da Mama/patologia , Feminino , Humanos
6.
Mol Biol Cell ; 16(5): 2529-43, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15772160

RESUMO

Cytokinesis requires the coordination of many cellular complexes, particularly those involved in the constriction and reconstruction of the plasma membrane in the cleavage furrow. We have investigated the regulation and function of vesicle transport and fusion during cytokinesis in budding yeast. By using time-lapse confocal microscopy, we show that post-Golgi vesicles, as well as the exocyst, a complex required for the tethering and fusion of these vesicles, localize to the bud neck at a precise time just before spindle disassembly and actomyosin ring contraction. Using mutants affecting cyclin degradation and the mitotic exit network, we found that targeted secretion, in contrast to contractile ring activation, requires cyclin degradation but not the mitotic exit network. Analysis of cells in late anaphase bearing exocyst and myosin V mutations show that both vesicle transport and fusion machineries are required for the completion of cytokinesis, but this is not due to a delay in mitotic exit or assembly of the contractile ring. Further investigation of the dynamics of contractile rings in exocyst mutants shows these cells may be able to initiate contraction but often fail to complete the contraction due to premature disassembly during the contraction phase. This phenotype led us to identify Chs2, a transmembrane protein targeted to the bud neck through the exocytic pathway, as necessary for actomyosin ring stability during contraction. Chs2, as the chitin synthase that produces the primary septum, thus couples the assembly of the extracellular matrix with the dynamics of the contractile ring during cytokinesis.


Assuntos
Actomiosina/química , Actomiosina/metabolismo , Quitina Sintase/metabolismo , Citocinese/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Actomiosina/genética , Ciclo Celular/fisiologia , Divisão Celular , Quitina Sintase/genética , Estabilidade de Medicamentos , Exocitose , Genes Fúngicos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fusão de Membrana , Mitose , Modelos Biológicos , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Curr Biol ; 12(21): 1864-70, 2002 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-12419188

RESUMO

In eukaryotic cells, dynamic rearrangement of the actin cytoskeleton is critical for cell division. In the yeast Saccharomyces cerevisiae, three main structures constitute the actin cytoskeleton: cortical actin patches, cytoplasmic actin cables, and the actin-based cytokinetic ring. The conserved Arp2/3 complex and a WASP-family protein mediate actin patch formation, whereas the yeast formins (Bni1 and Bnr1) promote assembly of actin cables. However, the mechanism of actin ring formation is currently unclear. Here, we show that actin filaments are required for cytokinesis in S. cerevisiae, and that the actin ring is a highly dynamic structure that undergoes constant turnover. Assembly of the actin ring requires the formin-like proteins and profilin, but is not Arp2/3-mediated. Furthermore, the formin-dependent actin ring assembly pathway is regulated by the Rho-type GTPase Rho1 but not Cdc42. Finally, we show that the formins are not required for localization of Cyk1/Iqg1, an IQGAP-like protein previously shown to be required for actin ring formation, suggesting that formin-like proteins and Cyk1 act synergistically but independently in assembly of the actin ring.


Assuntos
Actinas/fisiologia , Ciclo Celular/fisiologia , Saccharomyces cerevisiae/citologia , Proteínas rho de Ligação ao GTP/fisiologia , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae
8.
PLoS One ; 11(2): e0149659, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26901445

RESUMO

The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors.


Assuntos
Antibacterianos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/metabolismo , Animais , Antibacterianos/química , Bovinos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico
9.
Antiviral Res ; 131: 19-25, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27059228

RESUMO

Respiratory syncytial virus (RSV) infections affect millions of children and adults every year. Despite the significant disease burden, there are currently no safe and effective vaccines or therapeutics. We employed a replicon-based high throughput screen combined with live-virus triaging assays to identify three novel diversity-oriented synthesis-derived scaffolds with activity against RSV. One of these small molecules is shown to target the RSV polymerase (L protein) to inhibit viral replication and transcription; the mechanisms of action of the other small molecules are currently unknown. The compounds described herein may provide attractive inhibitors for lead optimization campaigns.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Testes de Sensibilidade Microbiana , Replicon/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Antivirais/química , Antivirais/isolamento & purificação , Células Hep G2 , Humanos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Infecções por Vírus Respiratório Sincicial/terapia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/enzimologia , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos
10.
ACS Chem Biol ; 9(7): 1536-44, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24824984

RESUMO

Pseudomonas aeruginosa produces the peptide siderophore pyoverdine, which is used to acquire essential Fe(3+) ions from the environment. PvdQ, an Ntn hydrolase, is required for the biosynthesis of pyoverdine. PvdQ knockout strains are not infectious in model systems, suggesting that disruption of siderophore production via PvdQ inhibition could be exploited as a target for novel antibacterial agents, by preventing cells from acquiring iron in the low iron environments of most biological settings. We have previously described a high-throughput screen to identify inhibitors of PvdQ that identified inhibitors with IC50 values of ∼100 µM. Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.


Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oligopeptídeos/metabolismo , Pseudomonas aeruginosa/enzimologia , Amidoidrolases/química , Amidoidrolases/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo
11.
J Clin Invest ; 124(2): 644-55, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24401270

RESUMO

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug's antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.


Assuntos
Apoptose , Fenotiazinas/química , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteína Fosfatase 2/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatografia de Afinidade , Modelos Animais de Doenças , Antagonistas de Dopamina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectrometria de Massas , Camundongos , Perfenazina/química , Fosforilação , Pigmentação , Proteômica , Receptores Notch/metabolismo , Fatores de Tempo , Peixe-Zebra
12.
Chem Biol ; 20(5): 713-25, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23706636

RESUMO

While cytotoxic chemotherapy remains the hallmark of cancer treatment, intensive regimens fall short in many malignancies, including high-risk neuroblastoma. One alternative strategy is to therapeutically promote tumor differentiation. We created a gene expression signature to measure neuroblast maturation, adapted it to a high-throughput platform, and screened a diversity oriented synthesis-generated small-molecule library for differentiation inducers. We identified BRD8430, containing a nine-membered lactam, an ortho-amino anilide functionality, and three chiral centers, as a selective class I histone deacetylase (HDAC) inhibitor (HDAC1 > 2 > 3). Further investigation demonstrated that selective HDAC1/HDAC2 inhibition using compounds or RNA interference induced differentiation and decreased viability in neuroblastoma cell lines. Combined treatment with 13-cis retinoic acid augmented these effects and enhanced activation of retinoic acid signaling. Therefore, by applying a chemical genomic screening approach, we identified selective HDAC1/HDAC2 inhibition as a strategy to induce neuroblastoma differentiation.


Assuntos
Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 2/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Lactamas/farmacologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/enzimologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/química , Humanos , Lactamas/química , Neuroblastoma/genética , Neuroblastoma/patologia , Tretinoína/metabolismo
13.
J Biomol Screen ; 17(9): 1204-10, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22941295

RESUMO

Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).


Assuntos
Neoplasias da Mama/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Hidrazonas/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Pirróis/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Hidrazonas/química , Concentração Inibidora 50 , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/citologia
14.
ACS Med Chem Lett ; 3(3): 232-237, 2012 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-22408714

RESUMO

A high-throughput screen of the NIH-MLSMR compound collection, along with a series of secondary assays to identify potential targets of hit compounds, previously identified a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We now report additional structure-activity relationship (SAR) studies that delineate the requirements for activity at PAR1 and identify plasma-stable analogues with nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may be important for the discovery of antithrombotics with an improved safety profile.

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