RESUMO
Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins. We report here the discovery of a class of such inhibitors targeting the family VIII bromodomains of SMARCA2 (BRM) and SMARCA4 (BRG1), and PBRM1 (polybromo-1) bromodomain 5. We propose one example from this series, GNE-064, as a chemical probe for the bromodomains SMARCA2, SMARCA4, and PBRM1(5) with the potential for in vivo use.
Assuntos
DNA Helicases , Fatores de Transcrição , Proteínas de Ligação a DNA , Humanos , Proteínas Nucleares , Domínios ProteicosRESUMO
Leveraging the catalytic machinery of LSD1 (KDM1A), a series of covalent styrenylcyclopropane LSD1 inhibitors were identified. These inhibitors represent a new class of mechanism-based inhibitors that target and covalently label the FAD cofactor of LSD1. The series was rapidly progressed to potent biochemical and cellular LSD1 inhibitors with good physical properties. This effort resulted in the identification of 34, a highly potent (<4 nM biochemical, 2 nM cell, and 1 nM GI50), and selective LSD1 inhibitor. In-depth kinetic profiling of 34 confirmed its covalent mechanism of action, validated the styrenylcyclopropane as an FAD-directed warhead, and demonstrated that the potency of this inhibitor is driven by improved non-covalent binding (K I). 34 demonstrated robust cell-killing activity in a panel of AML cell lines and robust antitumor activity in a Kasumi-1 xenograft model of AML when dosed orally at 1.5 mg/kg once daily.
RESUMO
The biological functions of the dual bromodomains of human transcription-initiation-factor TFIID subunit 1 (TAF1(1,2)) remain unknown, although TAF1 has been identified as a potential target for oncology research. Here, we describe the discovery of a potent and selective in vitro tool compound for TAF1(2), starting from a previously reported lead. A cocrystal structure of lead compound 2 bound to TAF1(2) enabled structure-based design and structure-activity-relationship studies that ultimately led to our in vitro tool compound, 27 (GNE-371). Compound 27 binds TAF1(2) with an IC50 of 10 nM while maintaining excellent selectivity over other bromodomain-family members. Compound 27 is also active in a cellular-TAF1(2) target-engagement assay (IC50 = 38 nM) and exhibits antiproliferative synergy with the BET inhibitor JQ1, suggesting engagement of endogenous TAF1 by 27 and further supporting the use of 27 in mechanistic and target-validation studies.
Assuntos
Benzimidazóis/metabolismo , Desenho de Fármacos , Sondas Moleculares/metabolismo , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely unknown. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromodomain inhibitor (GNE-886) suitable for use as an in vitro tool compound.
RESUMO
Bromodomain-containing protein 9 (BRD9), an epigenetic "reader" of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell viability or gene expression assays, it showed remarkable potency in preventing the emergence of a drug tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance. BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.
Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Linhagem Celular Tumoral , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Piridonas/química , Piridonas/farmacologia , Retinal Desidrogenase , Fatores de Transcrição/metabolismoRESUMO
Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention. Here we report the optimization of our indole-based EZH2 inhibitor series that led to the identification of CPI-1205, a highly potent (biochemical IC50 = 0.002 µM, cellular EC50 = 0.032 µM) and selective inhibitor of EZH2. This compound demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the co-crystal structure of our inhibitor series bound to the human PRC2 complex.
Assuntos
Antineoplásicos/farmacologia , Ensaios Clínicos Fase I como Assunto , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Indóis/farmacologia , Linfoma de Células B/tratamento farmacológico , Piperidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Indóis/síntese química , Indóis/química , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Piperidinas/síntese química , Piperidinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2. The preference of distinct substituents for individual bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.
Assuntos
Histona Acetiltransferases/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Piridonas/farmacologia , Pirróis/farmacologia , Fatores Associados à Proteína de Ligação a TATA/antagonistas & inibidores , Fator de Transcrição TFIID/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Água/química , Sítios de Ligação/efeitos dos fármacos , Proteínas de Ciclo Celular , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Fluorometria , Histona Acetiltransferases/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Proteínas Nucleares/metabolismo , Piridonas/síntese química , Piridonas/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição/metabolismoRESUMO
A high-throughput screen based on a viral replication assay was used to identify inhibitors of the human cytomegalovirus. Using this approach, hit compound 1 was identified as a 4 µM inhibitor of HCMV that was specific and selective over other herpes viruses. Time of addition studies indicated compound 1 exerted its antiviral effect early in the viral life cycle. Mechanism of action studies also revealed that this series inhibited infection of MRC-5 and ARPE19 cells by free virus and via direct cell-to-cell spread from infected to uninfected cells. Preliminary structure-activity relationships demonstrated that the potency of compound 1 could be improved to a low nanomolar level, but metabolic stability was a key optimization parameter for this series. A strategy focused on minimizing metabolic hydrolysis of the N1-amide led to an alternative scaffold in this series with improved metabolic stability and good pharmacokinetic parameters in rat.
RESUMO
The discovery and optimization of a series of small molecule EZH2 inhibitors is described. Starting from dimethylpyridone HTS hit (2), a series of indole-based EZH2 inhibitors were identified. Biochemical potency and microsomal stability were optimized during these studies and afforded compound 22. This compound demonstrates nanomolar levels of biochemical potency (IC50=0.002 µM), cellular potency (EC50=0.080 µM), and afforded tumor regression when dosed (200 mpk SC BID) in an EZH2 dependent tumor xenograft model.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Indóis/química , Complexo Repressor Polycomb 2/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Técnicas de Química Sintética , Desenho de Fármacos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Proteína Potenciadora do Homólogo 2 de Zeste , Células HeLa/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Terapia de Alvo Molecular/métodos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We describe our efforts to identify analogs of thumb pocket 1 HCV NS5B inhibitor 1 (aza-analog of BI 207524) with improved plasma to liver partitioning and a predicted human half-life consistent with achieving a strong antiviral effect at a reasonable dose in HCV-infected patients. Compounds 3 and 7 were identified that met these criteria but exhibited off-target promiscuity in an in vitro pharmacology screen and in vivo toxicity in rats. High lipophilicity in this class was found to correlate with increased probability for promiscuous behavior and toxicity. The synthesis of an 8×11 matrix of analogs allowed the identification of C3, an inhibitor that displayed comparable potency to 1, improved partitioning to the liver and reduced lipophilicity. Although C3 displayed reduced propensity for in vitro off-target inhibition and the toxicity profile in rats was improved, the predicted human half-life of this compound was short, resulting in unacceptable dosing requirements to maintain a strong antiviral effect in patients.
Assuntos
Acrilatos/química , Acrilatos/farmacologia , Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Indóis/química , Indóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Acrilatos/farmacocinética , Acrilatos/toxicidade , Animais , Antivirais/farmacocinética , Antivirais/toxicidade , Cães , Haplorrinos , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Indóis/farmacocinética , Indóis/toxicidade , Lipídeos/química , Fígado/metabolismo , Fígado/virologia , Ratos , Proteínas não Estruturais Virais/metabolismoRESUMO
A series of heterocyclic aza-analogs of BI 207524 (2), a potent HCV NS5B polymerase thumb pocket 1 inhibitor, was investigated with the goal to reduce the liability associated with the release of a genotoxic aniline metabolite in vivo. Analog 4, containing a 2-aminopyridine aniline isostere that is negative in the Ames test was identified, and was found to provide comparable GT1a/1b potency to 2. Although the cross-species PK profile, poor predicted human liver distribution of analog 4 and allometry principles projected high doses to achieve a strong antiviral response in patients, this work has provided a path forward toward the design of novel thumb pocket 1 NS5B polymerase inhibitors with improved safety profiles.
Assuntos
Acrilatos/metabolismo , Acrilatos/farmacologia , Compostos de Anilina/metabolismo , Antivirais/metabolismo , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Indóis/metabolismo , Indóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Acrilatos/química , Acrilatos/farmacocinética , Animais , Antivirais/química , Antivirais/farmacocinética , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Haplorrinos , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Indóis/química , Indóis/farmacocinética , Ratos , Proteínas não Estruturais Virais/metabolismoRESUMO
A method for the synthesis of N-functionalized C2-/C3-substituted indoles via Pd-catalyzed C-N bond coupling of halo-aryl enamines is described. The general strategy utilizes a variety of amines and ß-keto esters which are elaborated into halo-aryl enamines as latent precursors to indoles. The preferred conditions comprising the RuPhos precatalyst and RuPhos in the presence of NaOMe in 1,4-dioxane tolerate a variety of substituents and are scalable for the construction of indoles in multigram quantities.
Assuntos
Indóis/síntese química , Paládio/química , Aminas/química , Catálise , Ciclização , Indóis/química , Estrutura MolecularRESUMO
Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1 and 2 resulted in the identification of multiple structural elements that contributed to improved cell culture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic (IC50) and cell culture (EC50) potencies of less than 100 nanomolar.
Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , ortoaminobenzoatos/química , Antivirais/síntese química , Antivirais/farmacologia , Sítios de Ligação , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , ortoaminobenzoatos/síntese química , ortoaminobenzoatos/farmacologiaRESUMO
Recently, a new class of HIV reverse transcriptase (HIV-RT) inhibitors has been reported. The novel mechanism of inhibition by this class involves competitive binding to the active site of the RT enzyme and has been termed Nucleotide-Competing Reverse Transcriptase Inhibitors (NcRTIs). In this publication we describe the optimization of a novel benzofurano[3,2-d]pyrimidin-2-one series of NcRTIs. The starting point for the current study was inhibitor 2, which had high biochemical and antiviral potency but only moderate permeability in a Caco-2 assay and high B-to-A efflux, resulting in moderate rat bioavailability and low Cmax. We present herein the results and strategies we employed to optimize both the potency as well as the permeability, metabolic stability and pharmacokinetic profile of this series. One of the key observations of the present study was the importance of shielding polar functionality, at least in the context of the current chemotype, to enhance permeability. These studies led to the identification of inhibitors 39 and 45, which display sub-nanomolar antiviral potency in a p24 ELISA assay with significantly reduced efflux ratios (ratios <1.5). These inhibitors also display excellent rat pharmacokinetic profiles with high bioavailabilities and low clearance.
Assuntos
Antivirais/farmacologia , Benzofuranos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV/efeitos dos fármacos , Pirimidinonas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/química , Benzofuranos/química , Disponibilidade Biológica , Células CACO-2 , Relação Dose-Resposta a Droga , Transcriptase Reversa do HIV/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Pirimidinonas/administração & dosagem , Pirimidinonas/química , Ratos , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/química , Relação Estrutura-AtividadeRESUMO
A HTS screen led to the identification of a benzofurano[3,2-d]pyrimidin-2-one core structure which upon further optimization resulted in 1 as a potent HIV-1 nucleotide competing reverse transcriptase inhibitor (NcRTI). Investigation of the SAR at N-1 allowed significant improvements in potency and when combined with the incorporation of heterocycles at C-8 resulted in potent analogues not requiring a basic amine to achieve antiviral activity. Additional modifications at N-1 resulted in 33 which demonstrated excellent antiviral potency and improved physicochemical properties.
Assuntos
Benzofuranos/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Nucleotídeos/química , Pirimidinonas/química , Inibidores da Transcriptase Reversa/química , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Microssomos Hepáticos/metabolismo , Nucleotídeos/metabolismo , Ligação Proteica , Pirimidinonas/síntese química , Pirimidinonas/farmacologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.
Assuntos
Benzofuranos/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Nucleotídeos/química , Pirimidinonas/química , Inibidores da Transcriptase Reversa/química , Animais , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Microssomos Hepáticos/metabolismo , Nucleotídeos/metabolismo , Ligação Proteica , Pirimidinonas/síntese química , Pirimidinonas/farmacologia , Ratos , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-AtividadeRESUMO
A uHTS campaign led to the discovery of a 5-(5-furan-2-ylpyrazol-1-yl)-1H-benzimidazole series that inhibits assembly of HIV-1 capsid. Synthetic manipulations at N1, C2 and C16 positions improved the antiviral potency by a . The X-ray structure of 33 complexed with the capsid N-terminal domain allowed identification of major interactions between the inhibitor and the protein.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Benzimidazóis/síntese química , Benzimidazóis/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A novel strategy for the stereoselective construction of all-carbon quaternary centers on acyclic molecules using a two-step tandem process is reported. The first step involves an intramolecular and stereoselective atom transfer radical cyclization reaction from an allyl or vinyl subunit attached on a silyloxy, serving as a tether, to a tertiary radical alpha to an ester. A subsequent mild acidic elimination leads stereoselectively to a quaternary center bearing an allyl or a vinyl in high yield.
Assuntos
Compostos Alílicos/síntese química , Silício/química , Compostos de Vinila/síntese química , Compostos Alílicos/química , Ciclização , Estrutura Molecular , Estereoisomerismo , Compostos de Vinila/químicaRESUMO
The palladium-catalyzed cross-coupling reaction of tricyclopropylbismuth with aryl and heterocyclic halides and triflates is reported. The reaction tolerates numerous functional groups and does not require anhydrous conditions. The method was successfully extended to the cross-coupling of triethylbismuth.