RESUMO
A novel, potent, and orally bioavailable inhibitor of the bromodomain of CBP, compound 35 (GNE-207), has been identified through SAR investigations focused on optimizing al bicyclic heteroarene to replace the aniline present in the published GNE-272 series. Compound 35â¯has excellent CBP potency (CBP IC50â¯=â¯1â¯nM, MYC EC50â¯=â¯18â¯nM), a selectively index ofâ¯>2500-fold against BRD4(1), and exhibits a good pharmacokinetic profile.
Assuntos
Compostos de Bifenilo/química , Desenho de Fármacos , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Animais , Sítios de Ligação , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/metabolismo , Proteínas de Ciclo Celular , Cristalografia por Raios X , Meia-Vida , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Camundongos , Microssomos Hepáticos/metabolismo , Simulação de Dinâmica Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Ratos , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
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
MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. In an effort to generate small molecule MAP4K4 inhibitors, a fragment-based screen was carried out and a pyrrolotriazine fragment with excellent ligand efficiency was identified. Further modification of this fragment guided by X-ray crystal structures and molecular modeling led to the discovery of a series of promising compounds with good structural diversity and physicochemical properties. These compounds exhibited single digit nanomolar potency and compounds 35 and 44 achieved good in vivo exposure.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Triazinas/farmacologia , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Camundongos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/química , Quinase Induzida por NF-kappaBRESUMO
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
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
USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017 , 550 , 534 - 538 ) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the "palm" region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Aminopiridinas/química , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Simulação por Computador , Cristalografia por Raios X , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Espectroscopia de Ressonância Magnética/métodos , Oxidiazóis/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismoRESUMO
The epigenetic regulator CBP/P300 presents a novel therapeutic target for oncology. Previously, we disclosed the development of potent and selective CBP bromodomain inhibitors by first identifying pharmacophores that bind the KAc region and then building into the LPF shelf. Herein, we report the "hybridization" of a variety of KAc-binding fragments with a tetrahydroquinoline scaffold that makes optimal interactions with the LPF shelf, imparting enhanced potency and selectivity to the hybridized ligand. To demonstrate the utility of our hybridization approach, two analogues containing unique Asn binders and the optimized tetrahydroquinoline moiety were rapidly optimized to yield single-digit nanomolar inhibitors of CBP with exquisite selectivity over BRD4(1) and the broader bromodomain family.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Animais , Asparagina/química , Asparagina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular , Cristalografia por Raios X , Feminino , Transferência Ressonante de Energia de Fluorescência/métodos , Camundongos Endogâmicos , Simulação de Acoplamento Molecular , Proteínas Nucleares/antagonistas & inibidores , Domínios Proteicos , Pirazóis/química , Piridinas/química , Quinolinas/química , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
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
Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.
Assuntos
Antineoplásicos/farmacologia , Proteína de Ligação a CREB/antagonistas & inibidores , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Proteína de Ligação a CREB/química , Cães , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacocinética , Piridinas/síntese química , Piridinas/química , Piridinas/farmacocinética , RNA/genética , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bromodomains, small protein modules that recognize acetylated lysine on histones, play a significant role in the epigenome, where they function as "readers" that ultimately determine the functional outcome of the post-translational modification. Because the initial discovery of selective BET inhibitors have helped define the role of that protein family in oncology and inflammation, BET bromodomains have continued to garner the most attention of any other bromodomain. More recently, non-BET bromodomain inhibitors that are potent and selective have been disclosed for ATAD2, CBP, BRD7/9, BRPF, BRPF/TRIM24, CECR2, SMARCA4, and BAZ2A/B. Such novel inhibitors can be used to probe the physiological function of these non-BET bromodomains and further understanding of their role in certain disease states. Here, we provide an update to the progress in identifying selective bromodomain inhibitors and their use as biological tools, as well as our perspective on the field.
Assuntos
Acetilação/efeitos dos fármacos , Descoberta de Drogas , Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Animais , Antígenos Nucleares/metabolismo , Proteína de Ligação a CREB/antagonistas & inibidores , Proteína de Ligação a CREB/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Descoberta de Drogas/métodos , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
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
CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.
RESUMO
The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Pirazóis/farmacologia , Piridonas/farmacologia , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Piridonas/síntese química , Piridonas/química , Relação Estrutura-AtividadeRESUMO
Diverse biological roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors in order to study its function in various disease contexts. In particular, compounds that can be used to carry out such studies in vivo would be critical for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compound 13 (GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were observed in the inducible Map4k4 knockout mice.
RESUMO
Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.