RESUMO
Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia. Prior to the discovery of TDI-11055, existing inhibitors of ENL YEATS showed in vitro potency, but had not shown efficacy in in vivo animal models. During the course of the medicinal chemistry campaign described here, we identified ENL YEATS inhibitor TDI-11055 that has an improved pharmacokinetic profile and is appropriate for in vivo evaluation of the ENL YEATS inhibition mechanism in AML.
RESUMO
Free energy perturbation is a computational technique that can be used to predict how small changes to an inhibitor structure will affect the binding free energy to its target. In this paper, we describe the utility of free energy perturbation with FEP+ in the hit-to-lead stage of a drug discovery project targeting soluble adenyl cyclase. The project was structurally enabled by X-ray crystallography throughout. We employed free energy perturbation to first scaffold hop to a preferable chemotype and then optimize the binding affinity to sub-nanomolar levels while retaining druglike properties. The results illustrate that effective use of free energy perturbation can enable a drug discovery campaign to progress rapidly from hit to lead, facilitating proof-of-concept studies that enable target validation.
Assuntos
Adenilil Ciclases , Descoberta de Drogas , Termodinâmica , EntropiaRESUMO
Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.
Assuntos
Adenilil Ciclases , Motilidade dos Espermatozoides , Animais , Masculino , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Oócitos/metabolismo , Transdução de Sinais/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Anticoncepcionais Masculinos/química , Anticoncepcionais Masculinos/farmacologiaRESUMO
The chromatin reader eleven-nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9-mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line- and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach. SIGNIFICANCE: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.
Assuntos
Leucemia Mieloide Aguda , Lisina , Humanos , Leucemia Mieloide Aguda/genética , Histonas/metabolismo , Cromatina , Proteína de Leucina Linfoide-Mieloide/metabolismoRESUMO
Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received U.S. Food and Drug Administration approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain penetrance, limiting their use in patients with central nervous system malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity, and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound, we discovered TDI-11904 (compound 21), a novel, highly potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.
RESUMO
Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.
RESUMO
Cancer cells reprogram their metabolism to support growth and to mitigate cellular stressors. The serine synthesis pathway has been identified as a metabolic pathway frequently altered in cancers and there has been considerable interest in developing pharmacological agents to target this pathway. Here, we report a series of indole amides that inhibit human 3-phosphoglycerate dehydrogenase (PHGDH), the enzyme that catalyzes the first committed step of the serine synthesis pathway. Using X-ray crystallography, we show that the indole amides bind the NAD+ pocket of PHGDH. Through structure-based optimization we were able to develop compounds with low nanomolar affinities for PHGDH in an enzymatic IC50 assay. In cellular assays, the most potent compounds inhibited de novo serine synthesis with low micromolar to sub-micromolar activities and these compounds successfully abrogated the proliferation of cancer cells in serine free media. The indole amide series reported here represent an important improvement over previously published PHGDH inhibitors as they are markedly more potent and their mechanism of action is better defined.
Assuntos
Amidas/química , Inibidores Enzimáticos/química , Indóis/química , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Serina/biossíntese , Amidas/metabolismo , Amidas/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Dinâmica Molecular , Fosfoglicerato Desidrogenase/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of E. coli DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of E. coli and Pseudomonas aeruginosa DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.
Assuntos
Antibacterianos/farmacologia , DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Fluoroquinolonas/farmacologia , Testes de Sensibilidade Microbiana , Domínios Proteicos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologiaRESUMO
We have been focused on identifying a structurally different next generation inhibitor to MK-5172 (our Ns3/4a protease inhibitor currently under regulatory review), which would achieve superior pangenotypic activity with acceptable safety and pharmacokinetic profile. These efforts have led to the discovery of a novel class of HCV NS3/4a protease inhibitors containing a unique spirocyclic-proline structural motif. The design strategy involved a molecular-modeling based approach, and the optimization efforts on the series to obtain pan-genotypic coverage with good exposures on oral dosing. One of the key elements in this effort was the spirocyclization of the P2 quinoline group, which rigidified and constrained the binding conformation to provide a novel core. A second focus of the team was also to improve the activity against genotype 3a and the key mutant variants of genotype 1b. The rational application of structural chemistry with molecular modeling guided the design and optimization of the structure-activity relationships have resulted in the identification of the clinical candidate MK-8831 with excellent pan-genotypic activity and safety profile.
RESUMO
Hepatitis C virus (HCV) replication is dependent on the formation of specialized membrane structures; however, the host factor requirements for the formation of these HCV complexes remain unclear. Herein, we demonstrate that inhibition of stearoyl-CoA desaturase 1 (SCD-1) halts the biosynthesis of unsaturated fatty acids, such as oleic acid, and negatively modulates HCV replication. Unsaturated fatty acids play key roles in membrane curvature and fluidity. Mechanistically, we demonstrate that SCD-1 inhibition disrupts the integrity of membranous HCV replication complexes and renders HCV RNA susceptible to nuclease-mediated degradation. Our work establishes a novel function for unsaturated fatty acids in HCV replication.
Assuntos
Hepacivirus/metabolismo , Membranas/metabolismo , Membranas/virologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Linhagem Celular Tumoral , Ácidos Graxos Insaturados/metabolismo , Hepacivirus/efeitos dos fármacos , Humanos , Membranas/efeitos dos fármacos , Estearoil-CoA Dessaturase/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotypeâ 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phaseâ 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection.
Assuntos
Antivirais/química , Benzofuranos/química , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Imidazóis/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/farmacocinética , Benzofuranos/síntese química , Benzofuranos/farmacocinética , Cães , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Meia-Vida , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Imidazóis/síntese química , Imidazóis/farmacocinética , Indóis/química , Mutação , Pan troglodytes , Ligação Proteica , Ratos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a ß-lactam potentiation screen to identify small molecules that augment the activity of ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the ß-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive ß-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Peptidoglicano Glicosiltransferase/metabolismo , Pirazóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Esteróis/farmacologia , Simulação por Computador , Farmacorresistência Bacteriana , Inibidores Enzimáticos/farmacologia , Humanos , Microscopia de Fluorescência , Modelos Moleculares , Pirazóis/química , Staphylococcus aureus/enzimologia , Esteróis/químicaRESUMO
P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Agonistas do Receptor Purinérgico P2/farmacologia , Antagonistas do Receptor Purinérgico P2/farmacologia , Receptores Purinérgicos P2/metabolismo , Animais , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Ligantes , Camundongos , Pan troglodytes , Ligação Proteica , Receptores Purinérgicos P2/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Our series of competitive antagonists against the G-protein coupled receptor P2Y(14) were found to be highly shifted in the presence of serum (>99% protein bound). A binding assay using 2% human serum albumin (HSA) was developed to guide further SAR studies and led to the identification of the zwitterion 2, which is substantially less shifted (18-fold) than our previous lead compound 1 (323-fold). However, as the bioavailability of 2 was low, a library of ester pro-drugs was prepared (7a-7j) and assessed in vitro. The most interesting candidates were then profiled in vivo and led to the identification of the pro-drug 7j, which possesses a substantially improved pharmacokinetic profile.
Assuntos
Pró-Fármacos/química , Antagonistas do Receptor Purinérgico P2/química , Receptores Purinérgicos P2/química , Disponibilidade Biológica , Humanos , Microssomos Hepáticos/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/farmacocinética , Ligação Proteica , Antagonistas do Receptor Purinérgico P2/síntese química , Antagonistas do Receptor Purinérgico P2/farmacocinética , Receptores Purinérgicos P2/metabolismo , Relação Estrutura-AtividadeRESUMO
A weak, UDP-competitive antagonist of the pyrimidinergic receptor P2RY(14) with a naphthoic acid core was identified through high-throughput screening. Optimization provided compounds with improved potency but poor pharmacokinetics. Acylglucuronidation was determined to be the major route of metabolism. Increasing the electron-withdrawing nature of the substituents markedly reduced glucuronidation and improved the pharmacokinetic profile. Additional optimization led to the identification of compound 38 which is an 8 nM UDP-competitive antagonist of P2Y(14) with a good pharmacokinetic profile.
Assuntos
Ácidos Carboxílicos/síntese química , Naftalenos/síntese química , Antagonistas do Receptor Purinérgico P2/síntese química , Receptores Purinérgicos P2 , Difosfato de Uridina , Animais , Ligação Competitiva , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacocinética , Ácidos Carboxílicos/farmacologia , Camundongos , Estrutura Molecular , Naftalenos/química , Naftalenos/farmacocinética , Naftalenos/farmacologia , Pan troglodytes , Ligação Proteica/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2/química , Antagonistas do Receptor Purinérgico P2/farmacocinética , Antagonistas do Receptor Purinérgico P2/farmacologia , Receptores Purinérgicos P2Y , Relação Estrutura-AtividadeRESUMO
A weak antagonist of the pyrimidinergic receptor P2Y(14) containing a dihydropyridopyrimidine core was identified through high-throughput screening. Subsequent optimization led to potent, non-UTP competitive antagonists and represent the first reported non-nucleotide antagonists of this receptor. Compound 18q was identified as a 10 nM P2Y(14) antagonist with good oral bioavailability and provided sufficient exposure in mice to be used as a tool for future in vivo studies.
Assuntos
Antagonistas do Receptor Purinérgico P2/síntese química , Pirimidinas/síntese química , Receptores Purinérgicos P2/química , Administração Oral , Animais , Disponibilidade Biológica , Camundongos , Estrutura Molecular , Pan troglodytes , Antagonistas do Receptor Purinérgico P2/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Receptores Purinérgicos P2Y , Relação Estrutura-AtividadeRESUMO
We describe herein a novel series of 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and selective inhibitors against the CXCR2 chemokine receptor and IL-8-mediated chemotaxis of a CXCR2-expressing cell line. Furthermore, these alkyl-hydrazine series inhibitors such as 5b demonstrated acceptable metabolic stability when incubated in human and rat microsomes.
Assuntos
Anti-Inflamatórios/síntese química , Hidrazinas/síntese química , Receptores de Interleucina-8B/antagonistas & inibidores , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Células CHO , Quimiotaxia/efeitos dos fármacos , Cricetinae , Cricetulus , Desenho de Fármacos , Humanos , Hidrazinas/química , Hidrazinas/farmacologia , Interleucina-8/metabolismo , Microssomos Hepáticos/metabolismo , Ratos , Receptores de Interleucina-8B/metabolismo , Relação Estrutura-AtividadeRESUMO
The discovery of the potent and selective prostaglandin D2 (PGD2) receptor (DP) antagonist [(3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (13) is presented. Initial lead antagonists 6 and 7 were found to be potent and selective DP antagonists (DP Ki = 2.0 nM for each); however, they both suffered from poor pharmacokinetic profiles, short half-lives and high clearance rates in rats. Rat bile duct cannulation studies revealed that high concentrations of parent drug were present in the biliary fluid (Cmax = 1100 microM for 6 and 3900 microM for 7). This pharmacokinetic liability was circumvented by replacing the 7-methylsulfone substituent present in 6 and 7 with a fluorine atom resulting in antagonists with diminished propensity for biliary excretion and with superior pharmacokinetic profiles. Further optimization led to the discovery of the potent and selective DP antagonist 13.
Assuntos
Indóis/síntese química , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Obstrução das Vias Respiratórias/tratamento farmacológico , Animais , Bile/metabolismo , Ligação Competitiva , Cães , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Indóis/farmacocinética , Indóis/farmacologia , Macaca fascicularis , Masculino , Camundongos , Microssomos/metabolismo , Descongestionantes Nasais/síntese química , Descongestionantes Nasais/farmacocinética , Descongestionantes Nasais/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Ovinos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A novel indole series of PGD2 receptor (DP receptor) antagonists is presented. Optimization of this series led to the identification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with Ki values of 2.6 and 1.8 nM, respectively. These two antagonists are also potent in a DP functional assay where they inhibit the PGD2 induced cAMP production in platelet rich plasma with IC50 values of 7.9 and 8.6 nM, respectively. The structure-activity relationships of this indole series of DP receptor antagonists will also be discussed.