RESUMO
We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinaseâ A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.
Assuntos
Benzotiazóis/farmacologia , Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Benzotiazóis/síntese química , Benzotiazóis/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Quinases Associadas a rho/metabolismoRESUMO
The deuteration of proteins and selective labeling of side chain methyl groups has greatly enhanced the molecular weight range of proteins and protein complexes which can be studied using solution NMR spectroscopy. Protocols for the selective labeling of all six methyl group containing amino acids individually are available, however to date, only a maximum of five amino acids have been labeled simultaneously. Here, we describe a new methodology for the simultaneous, selective labeling of all six methyl containing amino acids using the 115 kDa homohexameric enzyme CoaD from E. coli as a model system. The utility of the labeling protocol is demonstrated by efficiently and unambiguously assigning all methyl groups in the enzymatic active site using a single 4D (13)C-resolved HMQC-NOESY-HMQC experiment, in conjunction with a crystal structure. Furthermore, the six fold labeled protein was employed to characterize the interaction between the substrate analogue (R)-pantetheine and CoaD by chemical shift perturbations, demonstrating the benefit of the increased probe density.
Assuntos
Aminoácidos/química , Ressonância Magnética Nuclear Biomolecular , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Nucleotidiltransferases/química , Coloração e RotulagemRESUMO
Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.
Assuntos
Antineoplásicos/química , Descoberta de Drogas/métodos , Mutação/genética , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Antineoplásicos/farmacologia , Desenho de Fármacos , Descoberta de Drogas/tendências , Humanos , Simulação de Acoplamento Molecular/métodos , Simulação de Acoplamento Molecular/tendências , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
In the preceding manuscript [ Moreau et al. 2018 , 10.1021/acs.jmedchem.7b01691 ] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/metabolismo , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Sítios de Ligação , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mutação , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ligação Proteica , Pirimidinonas/síntese química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Triazóis/síntese química , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/metabolismo , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Sítios de Ligação , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Pirimidinonas/síntese química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Triazóis/síntese química , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Inibidores do Citocromo P-450 CYP2C9/química , Inibidores do Citocromo P-450 CYP2C9/farmacologia , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Meia-Vida , Humanos , Camundongos Endogâmicos C57BL , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/patologia , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Quinases Associadas a rho/químicaRESUMO
Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.
Assuntos
Hipotensão/induzido quimicamente , Indazóis/síntese química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirazóis/síntese química , Piridinas/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Cães , Indazóis/efeitos adversos , Indazóis/farmacologia , Camundongos , Modelos Moleculares , Conformação Proteica , Ramos Subendocárdicos/efeitos dos fármacos , Ramos Subendocárdicos/fisiologia , Pirazóis/efeitos adversos , Pirazóis/farmacologia , Piridinas/efeitos adversos , Piridinas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
ATR, a protein kinase in the PIKK family, plays a critical role in the cell DNA-damage response and is an attractive anticancer drug target. Several potent and selective inhibitors of ATR have been reported showing significant antitumor efficacy, with most advanced ones entering clinical trials. However, due to the absence of an experimental ATR structure, the determinants contributing to ATR inhibitors' potency and specificity are not well understood. Here we present the mutations in the ATP-binding site of PI3Kα to progressively transform the pocket to mimic that of ATR. The generated PI3Kα mutants exhibit significantly improved affinity for selective ATR inhibitors in multiple chemical classes. Furthermore, we obtained the X-ray structures of the PI3Kα mutants in complex with the ATR inhibitors. The crystal structures together with the analysis on the inhibitor affinity profile elucidate the roles of individual amino acid residues in the binding of ATR inhibitors, offering key insights for the binding mechanism and revealing the structure features important for the specificity of ATR inhibitors. The ability to obtain structural and binding data for these PI3Kα mutants, together with their ATR-like inhibitor binding profiles, makes these chimeric PI3Kα proteins valuable model systems for structure-based inhibitor design.
Assuntos
Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Sítios de Ligação , Classe I de Fosfatidilinositol 3-Quinases , Cristalografia por Raios X , Modelos Moleculares , Proteínas Mutantes/química , Fosfatidilinositol 3-Quinases/química , Ligação Proteica , Conformação ProteicaRESUMO
RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.
Assuntos
2,2'-Dipiridil/análogos & derivados , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Quinases raf/antagonistas & inibidores , Proteínas ras/genética , 2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Benzamidas/química , Cristalografia por Raios X , Cães , Desenho de Fármacos , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Concentração Inibidora 50 , Camundongos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (K(i) = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses approximately 2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting.
Assuntos
Indazóis/farmacologia , Indóis/farmacologia , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Indazóis/química , Indazóis/uso terapêutico , Indóis/química , Indóis/uso terapêutico , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Piridinas/química , Piridinas/farmacologia , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.
Assuntos
Farmacorresistência Viral , Inibidores da Protease de HIV/síntese química , HIV-1/efeitos dos fármacos , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , HIV-1/genética , HIV-1/isolamento & purificação , Ligação de Hidrogênio , Técnicas In Vitro , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Mutação , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of symmetry-based HIV protease inhibitors was designed and synthesized. Modification of the core regiochemistry and stereochemistry significantly affected the potency, metabolic stability, and oral bioavailability of the inhibitors, as did the variation of a pendent arylmethyl P3 group. Optimization led to the selection of two compounds, 10c (A-790742) and 9d (A-792611), for advancement to preclinical studies. Both compounds displayed low nanomolar potency against wild type HIV in the presence of human serum, low rates of metabolism in human liver microsomes, and high oral bioavailability in animal models. The compounds were examined in a preclinical model for the hyperbilirubinemia observed with some HIV PIs, and both exhibited less bilirubin elevation than comparator compounds. X-ray crystallographic analyses of the new cores were used to examine differences in their binding modes. The antiviral activity of the compounds against protease inhibitor resistant strains of HIV was also determined.
Assuntos
Carbamatos/síntese química , Dipeptídeos/síntese química , Inibidores da Protease de HIV/síntese química , Putrescina/análogos & derivados , Piridinas/síntese química , Animais , Sítios de Ligação , Disponibilidade Biológica , Células CACO-2 , Carbamatos/metabolismo , Carbamatos/farmacologia , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Dipeptídeos/efeitos adversos , Dipeptídeos/farmacologia , Cães , Farmacorresistência Viral , Protease de HIV/genética , Inibidores da Protease de HIV/efeitos adversos , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Hiperbilirrubinemia/induzido quimicamente , Hiperlipidemias/induzido quimicamente , Hiperlipidemias/metabolismo , Técnicas In Vitro , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Mutação , Putrescina/síntese química , Putrescina/metabolismo , Putrescina/farmacologia , Piridinas/efeitos adversos , Piridinas/farmacologia , Ratos , Ratos Gunn , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Thr-211 is one of three different amino acid residues in the kinase domain of protein kinase B/Akt as compared to protein kinase A (PKA), a closely related analog in the same AGC family. In an attempt to improve the potency and selectivity of our indazole-pyridine series of Akt inhibitors over PKA, efforts have focused on the incorporation of a chemical functionality to interact with the hydroxy group of Thr-211. Several substituents including an oxygen anion, amino, and nitro groups have been introduced at the C-6 position of the indazole scaffold, leading to a significant drop in Akt potency. Incorporation of a nitrogen atom into the phenyl ring at the same position (i.e., 9f) maintained the Akt activity and, in some cases, improved the selectivity over PKA. The structure-activity relationships of the new pyridine-pyrazolopyridine series of Akt inhibitors and their structural features when bound to PKA are also discussed.