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1.
Elife ; 102021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34636321

RESUMO

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.


Assuntos
Proteínas de Ligação a DNA/genética , Deleção de Genes , Neoplasias Pulmonares/genética , Neoplasias de Células Escamosas/genética , Fatores de Transcrição/genética , Ubiquitina Tiolesterase/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Fatores de Transcrição/metabolismo , Ubiquitina Tiolesterase/metabolismo
2.
J Med Chem ; 64(10): 6569-6580, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33719426

RESUMO

KRAS, the most common oncogenic driver in human cancers, is controlled and signals primarily through protein-protein interactions (PPIs). The interaction between KRAS and SOS1, crucial for the activation of KRAS, is a typical, challenging PPI with a large contact surface area and high affinity. Here, we report that the addition of only one atom placed between Y884SOS1 and A73KRAS is sufficient to convert SOS1 activators into SOS1 inhibitors. We also disclose the discovery of BI-3406. Combination with the upstream EGFR inhibitor afatinib shows in vivo efficacy against KRASG13D mutant colorectal tumor cells, demonstrating the utility of BI-3406 to probe SOS1 biology. These findings challenge the dogma that large molecules are required to disrupt challenging PPIs. Instead, a "foot in the door" approach, whereby single atoms or small functional groups placed between key PPI interactions, can lead to potent inhibitors even for challenging PPIs such as SOS1-KRAS.


Assuntos
Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SOS1/metabolismo , Afatinib/química , Afatinib/metabolismo , Afatinib/uso terapêutico , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Proteína SOS1/agonistas , Proteína SOS1/antagonistas & inibidores , Proteína SOS1/genética
4.
J Cell Biol ; 220(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33507233

RESUMO

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.


Assuntos
Ribossomos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Anticorpos/metabolismo , Biocatálise , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Estabilidade Proteica , Reprodutibilidade dos Testes , Ribonucleoproteínas/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores
5.
Cancer Discov ; 11(1): 142-157, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32816843

RESUMO

KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SIGNIFICANCE: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D.See related commentary by Zhao et al., p. 17.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Linhagem Celular Tumoral , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Nucleotídeos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética
6.
SLAS Discov ; 25(5): 515-522, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32107961

RESUMO

DNA-encoded library (DEL) technology has become a prominent screening platform in drug discovery owing to the capacity to screen billions or trillions of compounds in a single experiment. Although numerous successes with DEL technology have been reported, we are unaware of a rigorous examination of the many different variables that can influence a screen's success. Herein, we explore the impact of variable sample sequencing depth on the detection of tool compounds with known affinities toward a given target while simultaneously probing the effect of initial compound input. Our sequencing data confirm reports that high-affinity compounds can be discovered directly from a DEL screen, but we demonstrate that a mismatch between selection output and sequencing quantity can obscure useful ligands. Our results highlight the importance of selection coverage in grasping the entire picture of a DEL screen where the signal of a weak or underrepresented ligand may be suppressed by the inherent noise of a selection. These potential missed ligands may be critical to the success or failure of a drug discovery program.


Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/química , DNA/química , DNA/efeitos dos fármacos , Biblioteca Gênica , Humanos , Ligantes , Bibliotecas de Moléculas Pequenas/farmacologia
7.
Org Lett ; 22(4): 1290-1294, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31999466

RESUMO

2-Aminobenzimidazole cores are among the most common structural components in medicinal chemistry and can be found in many biologically active molecules. Herein, we report a mild protocol for the synthesis of multifunctional 2-aminobenzimidazoles on-DNA with broad substrate scopes. The reaction conditions expand our ability to design and synthesize 2-aminobenzimidazole core-focused DNA-encoded libraries.


Assuntos
Benzimidazóis/síntese química , DNA/química , Iodo/química , Benzimidazóis/química , Ciclização , Estrutura Molecular , Estereoisomerismo
8.
Methods Mol Biol ; 1683: 165-191, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29082493

RESUMO

Within the Drug Discovery industry, there is a growing recognition of the value of high content screening (HCS), particularly as researchers aim to screen compounds and identify hits using more physiologically relevant in vitro cell-based assays. Image-based high content screening, with its combined ability to yield multiparametric data, provide subcellular resolution, and enable cell population analysis, is well suited to this challenge. While HCS has been in routine use for over a decade, a number of hurdles have historically prohibited very large, miniaturized high-throughput screening efforts with this platform. Suitable hardware and consumables for conducting 1536-well HCS have only recently become available, and developing a reliable informatics framework to accommodate the scale of high-throughput HCS data remains a considerable challenge. Additionally, innovative approaches are needed to interpret the large volumes of content-rich information generated. Despite these hurdles, there has been a growing interest in screening large compound inventories using this platform. Here, we outline the infrastructure developed and applied at Bristol-Myers Squibb for 1536-well high content screening and discuss key lessons learned.


Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Análise por Conglomerados , Interpretação Estatística de Dados , Descoberta de Drogas/métodos , Células Hep G2 , Humanos , Processamento de Imagem Assistida por Computador , Microscopia , Imagem Molecular/métodos , Reprodutibilidade dos Testes
9.
J Pharmacol Exp Ther ; 356(2): 293-304, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26582730

RESUMO

The muscarinic acetylcholine receptor subtype 1 (M1) receptors play an important role in cognition and memory, and are considered to be attractive targets for the development of novel medications to treat cognitive impairments seen in schizophrenia and Alzheimer's disease. Indeed, the M1 agonist xanomeline has been shown to produce beneficial cognitive effects in both Alzheimer's disease and schizophrenia patients. Unfortunately, the therapeutic utility of xanomeline was limited by cholinergic side effects (sweating, salivation, gastrointestinal distress), which are believed to result from nonselective activation of other muscarinic receptor subtypes such as M2 and M3. Therefore, drug discovery efforts targeting the M1 receptor have focused on the discovery of compounds with improved selectivity profiles. Recently, allosteric M1 receptor ligands have been described, which exhibit excellent selectivity for M1 over other muscarinic receptor subtypes. In the current study, the following three compounds with mixed agonist/positive allosteric modulator activities that are highly functionally selective for the M1 receptor were tested in rats, dogs, and cynomologous monkeys: (3-((1S,2S)-2-hydrocyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one; 1-((4-cyano-4-(pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine-3-carboxylic acid; and (R)-ethyl 3-(2-methylbenzamido)-[1,4'-bipiperidine]-1'-carboxylate). Despite their selectivity for the M1 receptor, all three compounds elicited cholinergic side effects such as salivation, diarrhea, and emesis. These effects could not be explained by activity at other muscarinic receptor subtypes, or by activity at other receptors tested. Together, these results suggest that activation of M1 receptors alone is sufficient to produce unwanted cholinergic side effects such as those seen with xanomeline. This has important implications for the development of M1 receptor-targeted therapeutics since it suggests that dose-limiting cholinergic side effects still reside in M1 receptor selective activators.


Assuntos
Agonistas Muscarínicos/metabolismo , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Cães , Relação Dose-Resposta a Droga , Humanos , Macaca fascicularis , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley
10.
J Med Chem ; 58(10): 4220-9, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-25901762

RESUMO

Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, ß-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.


Assuntos
Receptores Opioides delta/metabolismo , Relação Estrutura-Atividade , Animais , Arrestinas/metabolismo , Benzamidas/farmacologia , Ligação Competitiva , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Técnicas de Química Sintética , Cricetulus , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Encefalina Leucina/farmacologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Estrutura Molecular , Terapia de Alvo Molecular , Piperazinas/farmacologia , Ligação Proteica , Quinolinas/farmacologia , beta-Arrestinas
11.
J Biomol Screen ; 19(9): 1255-65, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25047277

RESUMO

Hetero-oligomeric complexes of G protein-coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a ß-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Ensaios de Triagem em Larga Escala , Receptores Opioides delta/agonistas , Receptores Opioides delta/química , Receptores Opioides mu/agonistas , Receptores Opioides mu/química , Animais , Arrestinas/metabolismo , Células CHO , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Antagonistas de Entorpecentes/farmacologia , Multimerização Proteica , beta-Arrestinas
12.
ACS Med Chem Lett ; 5(7): 760-5, 2014 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25050161

RESUMO

A series of 4-bicyclic heteroaryl 1,2,3,4-tetrahydroisoquinoline inhibitors of the serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT) was discovered. The synthesis and structure-activity relationship (SAR) of these triple reuptake inhibitors (TRIs) will be discussed. Compound 10i (AMR-2), a very potent inhibitor of SERT, NET, and DAT, showed efficacy in the rat forced-swim and mouse tail suspension models with minimum effective doses of 0.3 and 1 mg/kg (po), respectively. At efficacious doses in these assays, 10i exhibited substantial occupancy levels at the three transporters in both rat and mouse brain. The study of the metabolism of 10i revealed the formation of a significant active metabolite, compound 13.

13.
J Biomol Screen ; 19(7): 1079-89, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24789006

RESUMO

G protein-coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment. Focusing on the sphingosine-1-phosphate (S1P1) receptor, we evaluated the utility of high-content approaches in hit identification efforts by developing and applying assays to monitor ß-arrestin translocation, GPCR internalization, and GPCR recycling kinetics. Using these approaches in combination with more traditional GPCR screening assays, we identified compounds whose unique pharmacological profiles would have gone unnoticed if using a single platform. In addition, we identified a compound that induces an atypical pattern of ß-arrestin translocation and GPCR recycling kinetics. Our results highlight the value of high-content imaging in GPCR drug discovery efforts and emphasize the value of a multiassay approach to study pharmacological properties of compounds of interest.


Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/química , Sítio Alostérico , Animais , Bioensaio/métodos , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , AMP Cíclico/química , Descoberta de Drogas , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Ligação Proteica , Transporte Proteico , Ratos , Reprodutibilidade dos Testes , beta-Arrestinas/metabolismo
14.
J Biomol Screen ; 19(4): 595-605, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24241710

RESUMO

Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.


Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Lipase Lipoproteica/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Lipase Lipoproteica/metabolismo , Reprodutibilidade dos Testes , Especificidade por Substrato
15.
J Biomol Screen ; 18(9): 1072-83, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24062352

RESUMO

Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.


Assuntos
Descoberta de Drogas , Drogas em Investigação/farmacologia , Ensaios de Triagem em Larga Escala , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Drogas em Investigação/química , Drogas em Investigação/metabolismo , Corantes Fluorescentes , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteínas de Membrana Transportadoras/química , Relação Estrutura-Atividade
16.
Proc Natl Acad Sci U S A ; 110(26): 10830-5, 2013 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-23754417

RESUMO

µ-Opioid receptors are among the most studied G protein-coupled receptors because of the therapeutic value of agonists, such as morphine, that are used to treat chronic pain. However, these drugs have significant side effects, such as respiratory suppression, constipation, allodynia, tolerance, and dependence, as well as abuse potential. Efforts to fine tune pain control while alleviating the side effects of drugs, both physiological and psychological, have led to the development of a wide variety of structurally diverse agonist ligands for the µ-opioid receptor, as well as compounds that target κ- and δ-opioid receptors. In recent years, the identification of allosteric ligands for some G protein-coupled receptors has provided breakthroughs in obtaining receptor subtype-selectivity that can reduce the overall side effect profiles of a potential drug. However, positive allosteric modulators (PAMs) can also have the specific advantage of only modulating the activity of the receptor when the orthosteric agonist occupies the receptor, thus maintaining spatial and temporal control of receptor signaling in vivo. This second advantage of allosteric modulators may yield breakthroughs in opioid receptor research and could lead to drugs with improved side-effect profiles or fewer tolerance and dependence issues compared with orthosteric opioid receptor agonists. Here, we describe the discovery and characterization of µ-opioid receptor PAMs and silent allosteric modulators, identified from high-throughput screening using a ß-arrestin-recruitment assay.


Assuntos
Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Sulfonas/farmacologia , Tiazóis/farmacologia , Regulação Alostérica , Sítio Alostérico , Animais , Arrestinas/metabolismo , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Ratos , Sulfonas/química , Tiazóis/química , beta-Arrestinas
17.
Assay Drug Dev Technol ; 10(5): 457-67, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22746835

RESUMO

In recent years, the increased use of cell-based functional assays for G protein-coupled receptors in high-throughput screening has enabled the design of robust assays to identify allosteric modulators (AMs) in addition to the more traditional orthosteric agonists and antagonists. In this article, the authors describe a screening format able to identify all ligand types using a triple-add assay that measures changes in cytosolic calcium concentration with three separate additions and reads in the same assay plate. This triple-add assay captures more small molecule ligand types than previously described assay formats without a significant increase in screening cost. Finally, the customizability of the triple-add assay to suit the needs of various AM screening programs is demonstrated.


Assuntos
Sinalização do Cálcio/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/fisiologia , Regulação Alostérica/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Ligantes , Ligação Proteica/fisiologia
18.
J Med Chem ; 54(19): 6548-62, 2011 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-21882820

RESUMO

Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of tyrosine residues, a process that involves a conserved tryptophan-proline-aspartate (WPD) loop in catalysis. In previously determined structures of PTPs, the WPD-loop has been observed in either an "open" conformation or a "closed" conformation. In the current work, X-ray structures of the catalytic domain of receptor-like protein tyrosine phosphatase γ (RPTPγ) revealed a ligand-induced "superopen" conformation not previously reported for PTPs. In the superopen conformation, the ligand acts as an apparent competitive inhibitor and binds in a small hydrophobic pocket adjacent to, but distinct from, the active site. In the open and closed WPD-loop conformations of RPTPγ, the side chain of Trp1026 partially occupies this pocket. In the superopen conformation, Trp1026 is displaced allowing a 3,4-dichlorobenzyl substituent to occupy this site. The bound ligand prevents closure of the WPD-loop over the active site and disrupts the catalytic cycle of the enzyme.


Assuntos
Modelos Moleculares , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/antagonistas & inibidores , Tiofenos/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/química , Relação Estrutura-Atividade , Tiofenos/síntese química
19.
J Biomol Screen ; 16(5): 476-85, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21406618

RESUMO

Protein tyrosine phosphatase-γ (PTP-γ) is a receptor-like PTP whose biological function is poorly understood. A recent mouse PTP-γ genetic deletion model associated the loss of PTP-γ gene expression with a potential antidepressant phenotype. This led the authors to screen a subset of the Bristol-Myers Squibb (BMS) compound collection to identify selective small-molecule inhibitors of receptor-like PTP-γ (RPTP-γ) for use in evaluating enzyme function in vivo. Here, they report the design of a high-throughput fluorescence resonance energy transfer (FRET) assay based on the Z'-LYTE technology to screen for inhibitors of RPTP-γ. A subset of the BMS diverse compound collection was screened and several compounds identified as RPTP-γ inhibitors in the assay. After chemical triage and clustering, compounds were assessed for potency and selectivity by IC(50) determination with RPTP-γ and two other phosphatases, PTP-1B and CD45. One hundred twenty-nine RPTP-γ selective (defined as IC(50) value greater than 5- to 10-fold over PTP-1B and CD45) inhibitors were identified and prioritized for evaluation. One of these hits, 3-(3, 4-dichlorobenzylthio) thiophene-2-carboxylic acid, was the primary chemotype for the initiation of a medicinal chemistry program.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/metabolismo , Ensaios de Triagem em Larga Escala , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/antagonistas & inibidores , Dimetil Sulfóxido/farmacologia , Inibidores Enzimáticos/química , Estabilidade Enzimática/efeitos dos fármacos , Reprodutibilidade dos Testes , Projetos de Pesquisa , Sensibilidade e Especificidade , Solventes/farmacologia
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