RESUMO
Understanding protein conformational variability remains a challenge in drug discovery. The issue arises in protein kinases, whose multiple conformational states can affect the binding of small-molecule inhibitors. To overcome this challenge, we propose a comprehensive computational framework based on Markov state models (MSMs). Our framework integrates the information from explicit-solvent molecular dynamics simulations to accurately rank-order the accessible conformational variants of a target protein. We tested the methodology using Abl kinase with a reference and blind-test set. Only half of the Abl conformational variants discovered by our approach are present in the disclosed X-ray structures. The approach successfully identified a protein conformational state not previously observed in public structures but evident in a retrospective analysis of Lilly in-house structures: the X-ray structure of Abl with WHI-P154. Using a MSM-derived model, the free energy landscape and kinetic profile of Abl was analyzed in detail highlighting opportunities for targeting the unique metastable states.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas c-abl/química , Trifosfato de Adenosina/química , Sítio Alostérico , Cinética , Cadeias de Markov , Ácido Mirístico/química , Conformação Proteica , TermodinâmicaRESUMO
To date, IL-17A antibodies remain the only therapeutic approach to correct the abnormal activation of the IL-17A/IL-17R signaling complex. Why is it that despite the remarkable success of IL-17 antibodies, there is no small molecule antagonist of IL-17A in the clinic? Here we offer a unique approach to address this question. In order to understand the interaction of IL-17A with its receptor, we combined peptide discovery using phage display with HDX, crystallography, and functional assays to map and characterize hot regions that contribute to most of the energetics of the IL-17A/IL-17R interaction. These functional maps are proposed to serve as a guide to aid in the development of small molecules that bind to IL-17A and block its interaction with IL-17RA.
Assuntos
Colífagos/metabolismo , Interleucina-17/metabolismo , Peptídeos/metabolismo , Receptores de Interleucina-17/metabolismo , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Células HT29 , Humanos , Interleucina-17/química , Modelos Moleculares , Receptores de Interleucina-17/química , Ressonância de Plasmônio de SuperfícieRESUMO
Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4ß-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.
Assuntos
Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Agonistas de Aminoácidos Excitatórios/síntese química , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Animais , Compostos Bicíclicos com Pontes/farmacocinética , Cristalografia por Raios X , AMP Cíclico/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacocinética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenciclidina/antagonistas & inibidores , Fenciclidina/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.
Assuntos
Compostos Bicíclicos com Pontes/química , Receptores de Glutamato Metabotrópico/agonistas , Triazóis/química , Regulação Alostérica , Animais , Ligação Competitiva , Compostos Bicíclicos com Pontes/farmacocinética , Compostos Bicíclicos com Pontes/farmacologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Cães , Agonismo Parcial de Drogas , Humanos , Masculino , Camundongos , Modelos Moleculares , Atividade Motora/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Estereoisomerismo , Triazóis/farmacocinética , Triazóis/farmacologiaRESUMO
Microsomal prostaglandin E synthase 1 (mPGES-1) is an α-helical homotrimeric integral membrane inducible enzyme that catalyzes the formation of prostaglandin E2 (PGE2) from prostaglandin H2 (PGH2). Inhibition of mPGES-1 has been proposed as a therapeutic strategy for the treatment of pain, inflammation, and some cancers. Interest in mPGES-1 inhibition can, in part, be attributed to the potential circumvention of cardiovascular risks associated with anti-inflammatory cyclooxygenase 2 inhibitors (coxibs) by targeting the prostaglandin pathway downstream of PGH2 synthesis and avoiding suppression of antithrombotic prostacyclin production. We determined the crystal structure of mPGES-1 bound to four potent inhibitors in order to understand their structure-activity relationships and provide a framework for the rational design of improved molecules. In addition, we developed a light-scattering-based thermal stability assay to identify molecules for crystallographic studies.
Assuntos
Analgésicos/química , Anti-Inflamatórios/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Imidazóis/química , Oxirredutases Intramoleculares/química , Sequência de Aminoácidos , Analgésicos/metabolismo , Analgésicos/uso terapêutico , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Humanos , Oxirredutases Intramoleculares/metabolismo , Microssomos/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Prostaglandina-E Sintases , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.
Assuntos
Domínio Catalítico/genética , Doença/genética , Mutação , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Células Sf9 , SpodopteraRESUMO
The sirtuin SIRT1 is a NAD(+)-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241-516) bound to NAD(+) and the 27 analogue compound 35. 35 binds deep in the catalytic cleft, displacing the NAD(+) nicotinamide and forcing the cofactor into an extended conformation. The extended NAD(+) conformation sterically prevents substrate binding. The SIRT1/NAD(+)/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.
Assuntos
Carbazóis/farmacologia , Inibidores de Histona Desacetilases/farmacologia , NAD/metabolismo , Sirtuína 1/metabolismo , Carbazóis/química , Domínio Catalítico , Cristalografia por Raios X , Inibidores de Histona Desacetilases/química , Humanos , Modelos Moleculares , Conformação Proteica , Sirtuína 1/química , Ressonância de Plasmônio de SuperfícieRESUMO
Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteína-Arginina N-Metiltransferases/química , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.
Assuntos
Trifosfato de Adenosina/farmacologia , Neoplasias/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridazinas/farmacologia , Triazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Catálise/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Cinética , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridazinas/química , Triazóis/química , Carga Tumoral/efeitos dos fármacosRESUMO
Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.