RESUMO
KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Histonas/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Transdução de Sinais , Linhagem Celular TumoralRESUMO
The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (ß, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, ß, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and ß Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19 , Lactamas/química , SARS-CoV-2 , Inibidores de Protease Viral/química , COVID-19/virologia , Proteases 3C de Coronavírus , Cisteína Endopeptidases/metabolismo , Humanos , Leucina , Nitrilas , Pandemias , Prolina , SARS-CoV-2/efeitos dos fármacos , Proteínas Virais/metabolismoRESUMO
Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein, we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious, and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound 23.
Assuntos
Inibidores de Proteínas Quinases/química , Receptor trkA/antagonistas & inibidores , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Microssomos Hepáticos/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Estrutura Terciária de Proteína , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacocinética , Ratos , Receptor trkA/metabolismo , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure-activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16-19 h.
Assuntos
Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indóis/farmacologia , Succinimidas/farmacologia , Animais , Linhagem Celular , Cristalografia por Raios X , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indóis/química , Indóis/farmacocinética , Macaca fascicularis , Masculino , Camundongos , Simulação de Acoplamento Molecular , Ratos , Relação Estrutura-Atividade , Succinimidas/química , Succinimidas/farmacocinéticaRESUMO
The design of potent Pin1 inhibitors has been challenging because its active site specifically recognizes a phospho-protein epitope. The de novo design of phosphate-based Pin1 inhibitors focusing on the phosphate recognition pocket and the successful replacement of the phosphate group with a carboxylate have been previously reported. The potency of the carboxylate series is now further improved through structure-based optimization of ligand-protein interactions in the proline binding site which exploits the H-bond interactions necessary for Pin1 catalytic function. Further optimization using a focused library approach led to the discovery of low nanomolar non-phosphate small molecular Pin1 inhibitors. Structural modifications designed to improve cell permeability resulted in Pin1 inhibitors with low micromolar anti-proliferative activities against cancer cells.
Assuntos
Benzimidazóis/farmacologia , Ácidos Carboxílicos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase/antagonistas & inibidores , Fosfatos/química , Benzimidazóis/síntese química , Benzimidazóis/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Domínio Catalítico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Relação Estrutura-AtividadeRESUMO
The M2 isoform of pyruvate kinase is an emerging target for antitumor therapy. In this letter, we describe the discovery of 2-((1H-benzo[d]imidazol-1-yl)methyl)-4H-pyrido[1,2-a]pyrimidin-4-ones as potent and selective PKM2 activators which were found to have a novel binding mode. The original lead identified from high throughput screening was optimized into an efficient series via computer-aided structure-based drug design. Both a representative compound from this series and an activator described in the literature were used as molecular tools to probe the biological effects of PKM2 activation on cancer cells. Our results suggested that PKM2 activation alone is not sufficient to alter cancer cell metabolism.
Assuntos
Benzimidazóis/química , Proteínas de Transporte/agonistas , Proteínas de Membrana/agonistas , Pirimidinonas/química , Hormônios Tireóideos/agonistas , Sítios de Ligação , Proteínas de Transporte/metabolismo , Linhagem Celular , Desenho Assistido por Computador , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Pirimidinonas/síntese química , Pirimidinonas/metabolismo , Relação Estrutura-Atividade , Hormônios Tireóideos/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
High throughput cell-based screening led to the identification of 3-aryloxy lactams as potent androgen receptor (AR) antagonists. Refinement of these leads to improve the ADME profile and remove residual agonism led to the discovery of 12, a potent full antagonist with greater oral bioavailability. Improvements in the ADME profile were realized by designing more ligand-efficient molecules with reduced molecular weights and lower lipophilicities.
Assuntos
Descoberta de Drogas , Lactamas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/química , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Humanos , Lactamas/síntese química , Lactamas/química , Masculino , Modelos Moleculares , Estrutura Molecular , Neoplasias da Próstata/cirurgia , Receptores Androgênicos/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Analogues substituted with various amines at the 6-position of the pyrazine ring on (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyrazin-2-ylmethanone were discovered as potent and selective inhibitors of PDK1 with potential as anticancer agents. An early lead with 2-pyridine-3-ylethylamine as the pyrazine substituent showed moderate potency and selectivity. Structure-based drug design led to improved potency and selectivity against PI3Kα through a combination of cyclizing the ethylene spacer into a saturated, five-membered ring and substituting on the 4-position of the aryl ring with a fluorine. ADME properties were improved by lowering the lipophilicity with heteroatom replacements in the saturated, five-membered ring. The optimized analogues have a PDK1 Ki of 1 nM and >100-fold selectivity against PI3K/AKT-pathway kinases. The cellular potency of these analogues was assessed by the inhibition of AKT phosphorylation (T308) and by their antiproliferation activity against a number of tumor cell lines.
Assuntos
Antineoplásicos/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/síntese química , Pirróis/síntese química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Etilaminas/síntese química , Etilaminas/química , Etilaminas/farmacologia , Humanos , Modelos Moleculares , Fosforilação , Conformação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/química , Piridinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
An aryloxy tetramethylcyclobutane was identified as a novel template for androgen receptor (AR) antagonists via cell-based high-throughput screening. Follow-up to the initial "hit" established 5 as a viable lead. Further optimization to achieve full AR antagonism led to the discovery of 26 and 30, both of which demonstrated excellent in vivo tumor growth inhibition upon oral administration in a castration-resistant prostate cancer (CRPC) animal model.
Assuntos
Antagonistas de Androgênios/síntese química , Antineoplásicos/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Ciclobutanos/síntese química , Pirazóis/síntese química , Administração Oral , Antagonistas de Androgênios/farmacocinética , Antagonistas de Androgênios/farmacologia , Androgênios/síntese química , Androgênios/farmacocinética , Androgênios/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Ciclobutanos/farmacocinética , Ciclobutanos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Pirazóis/farmacocinética , Pirazóis/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Phosphoinositide-dependent kinase-1 (PDK1) is a critical enzyme in the PI3K/AKT pathway and to the activation of AGC family protein kinases, including S6K, SGK, and PKC. Dysregulation of this pathway plays a key role in cancer cell growth, survival and tumor angiogenesis. As such, inhibitors of PDK1 offer the promise of a new therapeutic modality for cancer treatment. Fragment based drug screening has recently become a viable entry point for hit identification. In this work, NMR spectroscopy fragment screening of PDK1 afforded novel chemotypes as orthogonal starting points from HTS screening hits. Compounds identified as hits by NMR spectroscopy were tested in a biochemical assay, and fragments with activity in both assays were clustered. The Pfizer compound file was mined via substructure and 2D similarity search, and the chemotypes were prioritized by ligand efficiency (LE), SAR mining, chemical attractiveness, and chemical enablement of promising vectors. From this effort, an isoquinolone fragment hit, 5 (IC(50) 870 µM, LE = 0.39), was identified as a novel, ligand efficient inhibitor of PDK1 and a suitable scaffold for further optimization. Initially in the absence of crystallographic data, a fragment growing approach efficiently explored four vectors of the isoquinolone scaffold via parallel synthesis to afford a compound with crystallographic data, 16 (IC(50) 41.4 µM, LE = 0.33). Subsequent lead optimization efforts provided 24 (IC(50) 1.8 µM, LE = 0.42), with greater than fivefold selectivity against other key pathway kinases.
Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Humanos , Ligação de Hidrogênio , Ligantes , Imageamento por Ressonância Magnética , Fragmentos de Peptídeos/química , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
Intra-molecular hydrogen bonding was introduced to the quinazoline motif to form a pseudo ring (intra-molecular H-bond scaffold, iMHBS) to mimic our previous published core structures, pyrido[2.3-D]pyrimidin-7-one and pteridinone, as PI3K/mTOR dual inhibitors. This design results in potent PI3K/mTOR dual inhibitors and the purposed intra-molecular hydrogen bonding structure is well supported by co-crystal structure in PI3Kγ enzyme. In addition, a novel synthetic route was developed for these analogs.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Quinazolinas/química , Serina-Treonina Quinases TOR/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Químicos , Modelos Moleculares , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/síntese química , Quinazolinas/farmacologia , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismoRESUMO
Pteridinones were designed based on a non-selective kinase template. Because of the uniqueness of the PI3K and mTOR binding pockets, a methyl group was introduced to C-4 position of the peteridinone core to give compounds with excellent selectivity for PI3K and mTOR. This series of compounds were further optimized to improve their potency against PI3Kα and mTOR. Finally, orally active compounds with improved solubility and robust in vivo efficacy in tumor growth inhibition were identified as well.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Pteridinas/química , Pteridinas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pteridinas/administração & dosagem , Pteridinas/farmacologia , Solubilidade , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/metabolismoRESUMO
Pin1 is a member of the cis-trans peptidyl-prolyl isomerase family with potential anti-cancer therapeutic value. Here we report structure-based de novo design and optimization of novel Pin1 inhibitors. Without a viable lead from internal screenings, we designed a series of novel Pin1 inhibitors by interrogating and exploring a protein crystal structure of Pin1. The ligand efficiency of the initial concept molecule was optimized with integrated SBDD and parallel chemistry approaches, resulting in a more attractive lead series.
Assuntos
Inibidores Enzimáticos/química , Peptidilprolil Isomerase/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Técnicas de Química Combinatória , Simulação por Computador , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Relação Estrutura-AtividadeRESUMO
The penultimate catalytic step of the purine de novo synthesis pathway is the conversion of aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formyl-AICAR that requires the cofactor N(10)-formyl-tetrahydrofolate as the formyl donor. This reaction is catalyzed by the AICAR transformylase domain of the bifunctional enzyme AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC). Identification of the location of the AICAR transformylase active site was previously elucidated from the crystal structure of the avian ATIC with bound substrate AICAR; however, due to the absence of any bound folate, the folate binding region of the active site could not be identified. Here, we have determined the homodimeric crystal structure of avian ATIC in complex with the ATIC-specific multisubstrate adduct inhibitor beta-DADF to 2.5 A resolution. Beta-DADF encompasses both the AICAR and folate moieties into a single covalently linked entity, thereby allowing for the characterization of the folate binding pocket of the AICAR transformylase active site. Beta-DADF is intimately bound at the dimer interface of the transformylase domains with the majority of AICAR moiety interactions occurring within one subunit, whereas the primary interactions to the folate occur with the opposing subunit. The crystal structure suggests that a buried Lys(267) is transiently protonated during formyl transfer allowing for the stabilization of the oxyanion transition state and subsequent protonation of N10 on the tetrahydrofolate leaving group. Furthermore, the beta-DADF-bound structure provides a more optimal three-dimensional scaffold to improve the design of specific antineoplastic agents.
Assuntos
Ácido Fólico/química , Ácido Glutâmico/química , Hidroximetil e Formil Transferases/química , Ribonucleotídeos/química , Animais , Sítios de Ligação , Aves , Carbono/química , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Escherichia coli/metabolismo , Ácido Glutâmico/análogos & derivados , Cinética , Lisina/química , Modelos Químicos , Modelos Moleculares , Fosforribosilaminoimidazolcarboxamida Formiltransferase , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Purinas/química , Tetra-Hidrofolatos/química , Água/químicaRESUMO
Glycinamide ribonucleotide transformylase (GAR Tfase) is a key folate-dependent enzyme in the de novo purine biosynthesis pathway and, as such, has been the target for antitumor drug design. Here, we describe the crystal structures of the human GAR Tfase (purN) component of the human trifunctional protein (purD-purM-purN) at various pH values and in complex with its substrate. Human GAR Tfase exhibits pH-dependent enzyme activity with its maximum around pH 7.5-8. Comparison of unliganded human GAR Tfase structures at pH 4.2 and pH 8.5 reveals conformational differences in the substrate binding loop, which at pH 4.2 occupies the binding cleft and prohibits substrate binding, while at pH 8.5 is permissive for substrate binding. The crystal structure of GAR Tfase with its natural substrate, beta-glycinamide ribonucleotide (beta-GAR), at pH 8.5 confirms this conformational isomerism. Surprisingly, several important structural differences are found between human GAR Tfase and previously reported E. coli GAR Tfase structures, which have been used as the primary template for drug design studies. While the E. coli structure gave valuable insights into the active site and formyl transfer mechanism, differences in structure and inhibition between the bacterial and mammalian enzymes suggest that the human GAR Tfase structure is now the appropriate template for the design of anti-cancer agents.