RESUMO
Recurrent somatic mutations in the BRG1/BRM-associated factor (BAF) chromatin remodeling complex subunit ARID1A occur frequently in advanced urothelial, endometrial, and ovarian clear cell carcinomas, creating an alternative chromatin state that may be exploited therapeutically. The histone methyltransferase EZH2 has been previously identified as targetable vulnerability in the context of ARID1A mutations. In this study, we describe the discovery of tulmimetostat, an orally available, clinical stage EZH2 inhibitor, and it elucidates the aspects of its application potential in ARID1A mutant tumors. Tulmimetostat administration achieved efficacy in multiple ARID1A mutant bladder, ovarian, and endometrial tumor models and improved cisplatin response in chemotherapy-resistant models. Consistent with its comprehensive and durable level of target coverage, tulmimetostat demonstrated greater efficacy than other PRC2-targeted inhibitors at comparable or lower exposures in a bladder cancer xenograft mouse model. Tulmimetostat mediated extensive changes in gene expression, in addition to a profound reduction in global H3K27me3 levels in tumors. Phase I clinical pharmacokinetic and pharmacodynamic data indicated that tulmimetostat exhibits durable exposure and profound target engagement. Importantly, a tulmimetostat controlled gene expression signature identified in whole blood from a cohort of 32 patients with cancer correlated with tulmimetostat exposure, representing a pharmacodynamic marker for the assessment of target coverage for PRC2-targeted agents in the clinic. Collectively, these data suggest that tulmimetostat has the potential to achieve clinical benefit in solid tumors as a monotherapy but also in combination with chemotherapeutic agents, and may be beneficial in various indications with recurrent ARID1A mutations. Significance: The EZH2 inhibitor tulmimetostat achieves comprehensive target inhibition in ARID1A mutant solid tumor models and cancer patients that can be assessed with a pharmacodynamic gene signature in peripheral blood.
Assuntos
Proteínas de Ligação a DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Mutação , Fatores de Transcrição , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Feminino , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismoRESUMO
The histone methyltransferase EZH2 has been the target of numerous small-molecule inhibitor discovery efforts over the last 10+ years. Emerging clinical data have provided early evidence for single agent activity with acceptable safety profiles for first-generation inhibitors. We have developed kinetic methodologies for studying EZH2-inhibitor-binding kinetics that have allowed us to identify a unique structural modification that results in significant increases in the drug-target residence times of all EZH2 inhibitor scaffolds we have studied. The unexpected residence time enhancement bestowed by this modification has enabled us to create a series of second-generation EZH2 inhibitors with sub-pM binding affinities. We provide both biophysical evidence validating this sub-pM potency and biological evidence demonstrating the utility and relevance of such high-affinity interactions with EZH2.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Descoberta de Drogas , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Células HeLa , Humanos , Camundongos SCID , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Contemporary deep learning approaches still struggle to bring a useful improvement in the field of drug discovery because of the challenges of sparse, noisy, and heterogeneous data that are typically encountered in this context. We use a state-of-the-art deep learning method, Alchemite, to impute data from drug discovery projects, including multitarget biochemical activities, phenotypic activities in cell-based assays, and a variety of absorption, distribution, metabolism, and excretion (ADME) endpoints. The resulting model gives excellent predictions for activity and ADME endpoints, offering an average increase in R2 of 0.22 versus quantitative structure-activity relationship methods. The model accuracy is robust to combining data across uncorrelated endpoints and projects with different chemical spaces, enabling a single model to be trained for all compounds and endpoints. We demonstrate improvements in accuracy on the latest chemistry and data when updating models with new data as an ongoing medicinal chemistry project progresses.
Assuntos
Aprendizado Profundo , Descoberta de Drogas , Química Farmacêutica , Relação Quantitativa Estrutura-AtividadeRESUMO
Histone methyltransferase EZH2, which is the catalytic subunit of the PRC2 complex, catalyzes the methylation of histone H3K27-a transcriptionally repressive post-translational modification (PTM). EZH2 is commonly mutated in hematologic malignancies and frequently overexpressed in solid tumors, where its expression level often correlates with poor prognosis. First generation EZH2 inhibitors are beginning to show clinical benefit, and we believe that a second generation EZH2 inhibitor could further build upon this foundation to fully realize the therapeutic potential of EZH2 inhibition. During our medicinal chemistry campaign, we identified 4-thiomethyl pyridone as a key modification that led to significantly increased potency and prolonged residence time. Leveraging this finding, we optimized a series of EZH2 inhibitors, with enhanced antitumor activity and improved physiochemical properties, which have the potential to expand the clinical use of EZH2 inhibition.
RESUMO
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 histone acetyltransferases, CREB binding protein (CBP) and EP300, are master transcriptional co-regulators that have been implicated in numerous diseases, such as cancer, inflammatory disorders, and neurodegeneration. A novel, highly potent, orally bioavailable EP300/CBP histone acetyltransferase (HAT) inhibitor, CPI-1612 or 17, was developed from the lead compound 3. Replacement of the indole scaffold of 3 with the aminopyridine scaffold of 17 led to improvements in potency, solubility, and bioavailability. These characteristics resulted in a 20-fold lower efficacious dose for 17 relative to lead 3 in a JEKO-1 tumor mouse xenograft study.
RESUMO
EP300 and CBP (KAT3A/3B) are two highly homologous, multidomain, epigenetic coregulators that play central roles in transcription through the acetylation of lysine residues on histones and other proteins. Both enzymes have been implicated in human diseases, especially cancer. From a high-throughput screen of 191 000 compounds searching for EP300/CBP histone acetyltransferase (HAT) inhibitors, 18 compounds were characterized by a suite of biochemical enzymatic assays and biophysical methods, including X-ray crystallography and native mass spectrometry. This work resulted in the discovery of three distinct mechanistic classes of EP300/CBP HAT inhibitors, including two classes not previously described. The profiles of an example of each class of inhibitor are described in detail. A subsequent medicinal chemistry effort led to the development of a novel class of orally bioavailable AcCoA-competitive EP300/CBP HAT inhibitors with inâ vivo activity. We believe that this work will prove to be a useful guide for other groups interested in the development of HAT inhibitors.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1R132H. This Letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker 2-HG.
RESUMO
Inhibition of mutant IDH1 is being evaluated clinically as a promising treatment option for various cancers with hotspot mutation at Arg132. Having identified an allosteric, induced pocket of IDH1R132H, we have explored 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors for in vivo modulation of 2-HG production and potential brain penetration. We report here optimization efforts toward the identification of clinical candidate IDH305 (13), a potent and selective mutant IDH1 inhibitor that has demonstrated brain exposure in rodents. Preclinical characterization of this compound exhibited in vivo correlation of 2-HG reduction and efficacy in a patient-derived IDH1 mutant xenograft tumor model. IDH305 (13) has progressed into human clinical trials for the treatment of cancers with IDH1 mutation.
RESUMO
The observed structure-activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.
Assuntos
Anti-Hipertensivos/farmacologia , Hipertensão/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Regulação Alostérica , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacocinética , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Simulação de Acoplamento Molecular , Piperazinas/síntese química , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacocinética , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
High throughput screening and subsequent hit validation identified 4-isopropyl-3-(2-((1-phenylethyl)amino)pyrimidin-4-yl)oxazolidin-2-one as a potent inhibitor of IDH1R132H. Synthesis of the four separate stereoisomers identified the (S,S)-diastereomer (IDH125, 1f) as the most potent isomer. This also showed reasonable cellular activity and excellent selectivity vs IDH1wt. Initial structure-activity relationship exploration identified the key tolerances and potential for optimization. X-ray crystallography identified a functionally relevant allosteric binding site amenable to inhibitors, which can penetrate the blood-brain barrier, and aided rational optimization. Potency improvement and modulation of the physicochemical properties identified (S,S)-oxazolidinone IDH889 (5x) with good exposure and 2-HG inhibitory activity in a mutant IDH1 xenograft mouse model.
RESUMO
Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.
Assuntos
Inibidores Enzimáticos/farmacologia , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Neoplasias/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica , Sítio Alostérico , Cristalografia por Raios X , Glutaratos/metabolismo , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genéticaRESUMO
Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH and 2-HG alter signaling pathways to promote cancer, however, remains unclear. Additionally, there exist relatively few cell lines with IDH mutations. To examine the effect of endogenous IDH mutations and 2-HG, we created a panel of isogenic epithelial cell lines with either wild-type IDH1/2 or clinically relevant IDH1/2 mutations. Differences were noted in the ability of IDH mutations to cause robust 2-HG accumulation. IDH1/2 mutants that produce high levels of 2-HG cause an epithelial-mesenchymal transition (EMT)-like phenotype, characterized by changes in EMT-related gene expression and cellular morphology. 2-HG is sufficient to recapitulate aspects of this phenotype in the absence of an IDH mutation. In the cells types examined, mutant IDH-induced EMT is dependent on up-regulation of the transcription factor ZEB1 and down-regulation of the miR-200 family of microRNAs. Furthermore, sustained knockdown of IDH1 in IDH1 R132H mutant cells is sufficient to reverse many characteristics of EMT, demonstrating that continued expression of mutant IDH is required to maintain this phenotype. These results suggest mutant IDH proteins can reversibly deregulate discrete signaling pathways that contribute to tumorigenesis.
Assuntos
Transição Epitelial-Mesenquimal , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Isocitrato Desidrogenase/biossíntese , MicroRNAs/biossíntese , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , RNA Neoplásico/biossíntese , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Linhagem Celular Tumoral , Glutaratos/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Isocitrato Desidrogenase/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , RNA Neoplásico/genética , Fatores de Transcrição/genética , Regulação para Cima/genética , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
A solid phase combinatorial library was designed based on X-ray structures and in-silico models to explore an inducible S4+ pocket, which is formed by a simple side-chain rotation of Tyr95. This inducible S4+ pocket is unique to ß-tryptase and does not exist for other trypsin-like serine proteases of interest. Therefore, inhibitors utilizing this pocket have inherent advantages for being selective against other proteases in the same family. A member of this library was found to be a potent and selective ß-tryptase inhibitor with a suitable pharmacokinetic profile for further clinical evaluation.
Assuntos
Inibidores Enzimáticos/farmacologia , Mastócitos/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Triptases/antagonistas & inibidores , Administração Oral , Animais , Técnicas de Química Combinatória , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , Humanos , Modelos Moleculares , Estrutura Molecular , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-AtividadeRESUMO
LDL cholesterol (LDL-C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low-density lipoprotein cholesterol receptor (LDL-R). LDL-R is targeted for lysosomal degradation by association with proprotein convertase subtilisin-kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor-sparing phenomena, including loss-of-function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate-determining steps for protein-ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild-type versus gain-of-function mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins ("hydration sites"). We compared these results with binding kinetics data for PCSK9, full-length LDL-R ectodomain, and isolated EGF-A repeat. We propose that the fast k(on) and entropically driven thermodynamics observed for PCSK9-EGF-A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H-bonds from water to polar EGF-A groups). We further propose that the relatively fast k(off) observed for EGF-A unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower k(off) observed for EGF-A and LDL-R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex.
Assuntos
Simulação por Computador , Fator de Crescimento Epidérmico/química , Conformação Proteica , Serina Endopeptidases/química , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Fator de Crescimento Epidérmico/genética , Humanos , Modelos Moleculares , Mutação , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Receptores de LDL/química , Serina Endopeptidases/genética , Solventes/química , Relação Estrutura-Atividade , Termodinâmica , Água/químicaRESUMO
A new series of novel mast cell tryptase inhibitors is reported, which features the use of an indole structure as the hydrophobic substituent on a m-benzylaminepiperidine template. The best members of this series display good in vitro activity and excellent selectivity against other serine proteases.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Mastócitos/enzimologia , Serina Endopeptidases/efeitos dos fármacos , Inibidores Enzimáticos/química , Modelos Moleculares , Relação Estrutura-Atividade , TriptasesRESUMO
Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests tryptase beta is the most important form released during mast cell activation in allergic diseases. It is shown to have numerous pro-inflammatory cellular activities in vitro, and in animal models tryptase provokes broncho-constriction and induces a cellular inflammatory infiltrate characteristic of human asthma. Screening of in-house inhibitors of factor Xa (a closely related serine protease) identified beta-amidoester benzamidines as potent inhibitors of recombinant human betaII tryptase. X-ray structure driven template modification and exchange of the benzamidine to optimize potency and pharmacokinetic properties gave selective, potent and orally bioavailable 4-(3-aminomethyl phenyl)piperidinyl-1-amides.
Assuntos
Amidas , Piperidinas , Serina Endopeptidases/efeitos dos fármacos , Administração Oral , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Animais , Disponibilidade Biológica , Células CACO-2 , Cristalografia por Raios X , Desenho de Fármacos , Inibidores do Fator Xa , Humanos , Fígado/enzimologia , Modelos Moleculares , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Piperidinas/farmacologia , Conformação Proteica , Ratos , Proteínas Recombinantes/efeitos dos fármacos , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , TriptasesRESUMO
We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human beta-tryptase.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos , Serina Endopeptidases/efeitos dos fármacos , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Relação Estrutura-Atividade , TriptasesRESUMO
4H-Substituted pyrylium 9, benzo[b]pyrylium 15, and xanthylium salts 22 react with benzotriazole to give the corresponding 4H-(benzotriazol-1-yl)pyrans 10, benzo[b]pyrans 16, or xanthenes 23. Novel anion precursors 10, 16, and 23 undergo smooth lithiations at the positions alpha to the benzotriazol-1-yl function, i.e., at the para-position of the O-heterocycle. Subsequent trapping with different alkyl halides gives intermediates 14, 21, and 25, which are converted by mineral acid into para-functionalized pyrylium 11-13, benzo[b]pyrylium 17-20, and the xanthylium 24 salts in good to excellent yields.