RESUMO
Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.
Assuntos
Amidas/química , Antivirais/síntese química , Inibidores Enzimáticos/síntese química , Hepacivirus/efeitos dos fármacos , Tiofenos/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/síntese química , Amidas/farmacologia , Antivirais/química , Antivirais/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologia , Proteínas não Estruturais Virais/metabolismoRESUMO
We have designed and synthesized a novel series of 2,8-diaryl-quinoxalines as Janus kinase 2 inhibitors. Many of the inhibitors show low nanomolar activity against JAK2 and potently suppress proliferation of SET-2 cells in vitro. In addition, compounds from this series have favorable rat pharmacokinetic properties suitable for in vivo efficacy evaluation.
Assuntos
Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinoxalinas/química , Quinoxalinas/farmacologia , Administração Oral , Animais , Linhagem Celular , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacocinética , Quinoxalinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Janus kinase (JAK) inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms, and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type I binding mode can lead to an increase in JAK activation loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type II inhibition acts in the opposite manner, leading to a loss of activation loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation loop may or may not be elicited.
Assuntos
Janus Quinases/antagonistas & inibidores , Janus Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/química , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Estrutura Terciária de Proteína , Fator de Transcrição STAT5/metabolismoRESUMO
We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a rationale for this unprecedented selectivity based on the X-ray crystal structure of an analogue of 1 bound to the ATP-binding site of Jak3. While equally potent compared to the Pfizer pan Jak inhibitor CP-690,550 (2) in an enzymatic Jak3 assay, compound 1 was found to be 20-fold less potent in cellular assays measuring cytokine-triggered signaling through cytokine receptors containing the common γ chain (γC). Contrary to compound 1, compound 2 inhibited Jak1 in addition to Jak3. Permeability and cellular concentrations of compounds 1 and 2 were similar. As Jak3 always cooperates with Jak1 for signaling, we speculate that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine signal transduction required for strong immunosuppression.
Assuntos
Indóis/síntese química , Janus Quinase 3/antagonistas & inibidores , Maleimidas/síntese química , Linhagem Celular , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Humanos , Imunossupressores/síntese química , Imunossupressores/química , Imunossupressores/farmacologia , Indóis/química , Indóis/farmacologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 3/química , Maleimidas/química , Maleimidas/farmacologia , Modelos Moleculares , Estrutura Molecular , Fosforilação , Piperidinas , Pirimidinas/farmacologia , Pirróis/farmacologia , Fator de Transcrição STAT5/metabolismo , Relação Estrutura-AtividadeRESUMO
The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2(V617F) mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2(V617F) and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2(V617F)-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2(V617F) cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats.
Assuntos
Proliferação de Células/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Policitemia/prevenção & controle , Quinoxalinas/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Eritropoese/efeitos dos fármacos , Humanos , Janus Quinase 2/química , Janus Quinase 2/genética , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Modelos Moleculares , Estrutura Molecular , Mutação , Fosforilação/efeitos dos fármacos , Policitemia/metabolismo , Policitemia/patologia , Estrutura Terciária de Proteína , Quinoxalinas/química , Ratos , Fator de Transcrição STAT5/metabolismo , Esplenomegalia/metabolismo , Esplenomegalia/patologia , Esplenomegalia/prevenção & controleRESUMO
p53 tumor suppressor activity is negatively regulated through binding to the oncogenic proteins Hdm2 and HdmX. The p53 residues Leu(26), Trp(23), and Phe(19) are crucial to mediate these interactions. Inhibiting p53 binding to both Hdm2 and HdmX should be a promising clinical approach to reactivate p53 in the cancer setting, but previous studies have suggested that the discovery of dual Hdm2/HdmX inhibitors will be difficult. We have determined the crystal structures at 1.3 A of the N-terminal domain of HdmX bound to two p53 peptidomimetics without and with a 6-chlorine substituent on the indole (which binds in the same subpocket as Trp(23) of p53). The latter compound is the most potent peptide-based antagonist of the p53-Hdm2 interaction yet to be described. The x-ray structures revealed surprising conformational changes of the binding cleft of HdmX, including an "open conformation" of Tyr(99) and unexpected "cross-talk" between the Trp and Leu pockets. Notably, the 6-chloro p53 peptidomimetic bound with high affinity to both HdmX and Hdm2 (K(d) values of 36 and 7 nm, respectively). Our results suggest that the development of potent dual inhibitors for HdmX and Hdm2 should be feasible. They also reveal possible conformational states of HdmX, which should lead to a better prediction of its interactions with potential biological partners.
Assuntos
Materiais Biomiméticos/química , Complexos Multiproteicos/química , Proteínas Nucleares/química , Peptídeos/química , Proteínas Proto-Oncogênicas/química , Proteína Supressora de Tumor p53/química , Sítios de Ligação/fisiologia , Materiais Biomiméticos/metabolismo , Proteínas de Ciclo Celular , Cristalografia por Raios X , Humanos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV) polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb- and a palm-binding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Replicon/genética , Ácidos Carboxílicos , Quimioterapia Combinada , Variação Genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hepacivirus/genética , Mutação de Sentido Incorreto , Seleção Genética , Tiofenos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação ViralRESUMO
To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.
Assuntos
Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Técnicas Biossensoriais/métodos , Antígeno Prostático Específico/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/normas , Humanos , Cinética , Ligantes , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/imunologia , Ligação Proteica , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos , Ressonância de Plasmônio de Superfície/normas , Fatores de TempoRESUMO
Carbohydrate-protein interactions play a key role in many biological processes. Cramoll is a lectin purified from Cratylia mollis seeds that is taxonomically related to concanavalin A (Con A). Although Cramoll and Con A have the same monosaccharide specificity, they have different glycoprotein binding profiles. We report the primary structure of Cramoll, determined by Edman degradation and mass spectrometry and its 1.77 A crystallographic structure and compare it with the three-dimensional structure of Con A in an attempt to understand how differential binding can be achieved by similar or nearly identical structures. We report here that Cramoll consists of 236 residues, with 82% identity with Con A, and that its topological architecture is essentially identical to Con A, because the Calpha positional differences are below 3.5 A. Cramoll and Con A have identical binding sites for MealphaMan, Mn2+, and Ca2+. However, we observed six substitutions in a groove adjacent to the extended binding site and two in the extended binding site that may explain the differences in binding of oligosaccharides and glycoproteins between Cramoll and Con A.
Assuntos
Fabaceae/química , Lectinas/química , Sementes/química , Sequência de Aminoácidos , Sítios de Ligação , Carboidratos/química , Concanavalina A/química , Cristalografia por Raios X , Ligação de Hidrogênio , Metais/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Introdução: O Lúpus Eritematoso Sistêmico (LES) é uma doença de origem auto-imune, de etiologia desconhecida, podendo afetar um ou mais órgãos. Apresenta incidência relativamente comum. Objetivo: O trabalho aborda os aspectos epidemiologicos de pacientes lúpicos,demonstrando a variedade das manifestações clínicas e o comportamento dessa patologia em nossa região. Métodos: Nos 104 pacientes internados no Hospital Ofir Loyola (HOL) no período de 1986 a 2000, foram analisados os seguintes dados; idade, sexo, raça, critérios diagnósticados do LES, manifestações clínicas, motivo das internações, tempo de doença e causas de óbito. Resultado: Foi observado a maior incidência em mulheres na idade fértil e nos miscigenados, a frequência do comprometimento osteoarticular, a importância do envolvimento renal e a elevada mortalidade na segunda década de vida. Conclusão: A ocorrência dos variados aspectos pesquisados, é semelhante as encontradas na literatura