RESUMO
Toll-like receptor (TLR) 7 and TLR8 are transmembrane receptors that recognize single-stranded RNA. Activation of these receptors results in immune cell stimulation and inflammatory cytokine production, which is normally a protective host response. However, aberrant activation of TLR7/8 is potentially pathogenic and linked to progression of certain autoimmune diseases such as lupus. Thus, we hypothesize that an inhibitor that blocks TLR7/8 would be an effective therapeutic treatment. Prior efforts to develop inhibitors of TLR7/8 have been largely unsuccessful as a result of the challenge of producing a small-molecule inhibitor for these difficult targets. Here, we report the characterization of M5049 and compound 2, molecules which were discovered in a medicinal chemistry campaign to produce dual TLR7/8 inhibitors with drug-like properties. Both compounds showed potent and selective activity in a range of cellular assays for inhibition of TLR7/8 and block synthetic ligands and natural endogenous RNA ligands such as microRNA and Alu RNA. M5049 was found to be potent in vivo as TLR7/8 inhibition efficaciously treated disease in several murine lupus models and, interestingly, was efficacious in a disease context in which TLR7/8 activity has not previously been considered a primary disease driver. Furthermore, M5049 had greater potency in disease models than expected based on its in vitro potency and pharmacokinetic/pharmacodynamic properties. Because of its preferential accumulation in tissues, and ability to block multiple TLR7/8 RNA ligands, M5049 may be efficacious in treating autoimmunity and has the potential to provide benefit to a variety of patients with varying disease pathogenesis. SIGNIFICANCE STATEMENT: This study reports discovery of a novel toll-like receptor (TLR) 7 and TLR8 inhibitor (M5049); characterizes its binding mode, potency/selectivity, and pharmacokinetic and pharmacodynamic properties; and demonstrates its potential for treating autoimmune diseases in two mouse lupus models. TLR7/8 inhibition is unique in that it may block both innate and adaptive autoimmunity; thus, this study suggests that M5049 has the potential to benefit patients with autoimmune diseases.
Assuntos
Autoimunidade/efeitos dos fármacos , Descoberta de Drogas , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/antagonistas & inibidores , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Proteica , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/química , Receptor 8 Toll-Like/metabolismoRESUMO
Fragment-based screening by SPR enabled the discovery of chemical diverse fragment hits with millimolar binding affinities to the peptidyl-prolyl isomerase Cyclophilin D (CypD). The CypD protein crystal structures of 6 fragment hits provided the basis for subsequent medicinal chemistry optimization by fragment merging and linking yielding three different chemical series with either urea, oxalyl or amide linkers connecting millimolar fragments in the S1' and S2 pockets. We successfully improved the in vitro CypD potencies in the biochemical FP and PPIase assays and in the biophysical SPR binding assay from millimolar towards the low micromolar and submicromolar range by >1000-fold for some fragment derivatives. The initial SAR together with the protein crystal structures of our novel CypD inhibitors provide a suitable basis for further hit-to-lead optimization.
Assuntos
Ciclofilinas/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Lactamas/farmacologia , Cristalografia por Raios X , Ciclofilinas/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Lactamas/síntese química , Lactamas/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
For oligonucleotide-based therapeutics, a thorough understanding of the thermodynamic properties of duplex formation is critical to developing stable and potent drugs. For unmodified small interfering RNA (siRNA), DNA antisense oligonucleotide (AON) and locked nucleic acid (LNA), DNA/LNA modified oligonucleotides, nearest neighbor (NN) methods can be effectively used to quickly and accurately predict duplex thermodynamic properties such as melting point. Unfortunately, for chemically modified olignonucleotides, there has been no accurate prediction method available. Here we describe the potential of estimating melting temperature (T(m)) for nonstandard oligonucleotides by using the correlation of the experimental T(m) with the calculated duplex binding energy (BE) for oligonucleotides of a given length. This method has been automated into a standardized molecular dynamics (MD) protocol through Pipeline Pilot (PP) using the CHARMm component in Discovery Studio (DS). Results will be presented showing the correlation of the predicted data with experiment for both standard and chemically modified siRNA and AON.
Assuntos
Química Farmacêutica/métodos , DNA/análise , Terapia Genética/métodos , Simulação de Dinâmica Molecular , Oligonucleotídeos Antissenso/análise , Oligonucleotídeos/análise , Preparações Farmacêuticas/análise , RNA Interferente Pequeno/análise , Automação Laboratorial , DNA/química , DNA/metabolismo , Estabilidade de Medicamentos , Humanos , Terapia de Alvo Molecular/métodos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Preparações Farmacêuticas/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Espectrofotometria , Termodinâmica , Temperatura de TransiçãoRESUMO
Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Descoberta de Drogas , Doenças do Sistema Imunitário/tratamento farmacológico , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Administração Oral , Tirosina Quinase da Agamaglobulinemia/metabolismo , Relação Dose-Resposta a Droga , Humanos , Doenças do Sistema Imunitário/metabolismo , Estrutura Molecular , Piperidinas/administração & dosagem , Piperidinas/química , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
This study explores employee burnout within and across the four administrative regions of the Natural Resource Conservation Service (NRCS) based on the hypothesis that burnout levels will vary systematically with workload levels. Approximately 1100 District Conservationists (DCs), the front-line operating officers of the NRCS, were surveyed. Results indicate significant variation in workload and burnout levels but no significant association between these two variables within and across regions. Further analysis revealed that a number of important variables help to explain burnout across regions including job design, functional unit cooperation, and adequate staffing. Within regions, the variables that explain burnout are under the categories of leadership, job design, human resource systems, and agency policy. The authors make use of the research findings to develop a set of organization development (OD) recommendations to help the agency deal with burnout within and across regions.
Assuntos
Esgotamento Profissional/psicologia , Órgãos Governamentais/organização & administração , Conservação dos Recursos Naturais , Comportamento Cooperativo , Humanos , Liderança , Gestão de Recursos Humanos/métodos , Estados Unidos , Carga de Trabalho/psicologiaRESUMO
In this chapter we present an application of in silico quantitative structure-activity relationship (QSAR) models to establish a new ligand-based computational approach for generating virtual libraries. The Free-Wilson methodology was applied to extract rules from two data sets containing compounds which were screened against either kinase or PDE gene family panels. The rules were used to make predictions for all compounds enumerated from their respective virtual libraries. We also demonstrate the construction of R-group selectivity profiles by deriving activity contributions against each protein target using the QSAR models. Such selectivity profiles were used together with protein structural information from X-ray data to provide a better understanding of the subtle selectivity relationships between kinase and PDE family members.
Assuntos
Técnicas de Química Combinatória/métodos , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Humanos , Modelos Lineares , Modelos Moleculares , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Análise de Regressão , Reprodutibilidade dos Testes , Especificidade por Substrato , Interface Usuário-ComputadorRESUMO
The design, synthesis and utility of fluorescence probes that bind to the DFG-out conformation of p38alpha kinase are described. Probes that demonstrate good affinity for p38alpha, have been identified and one of the probes, PF-04438255, has been successfully used in an high throughput screening (HTS) assay to identify two novel non-classical p38alpha inhibitors. In addition, a cascade activity assay was utilized to validate the selective binding of these non-classical kinase inhibitors to the unactive form of the enzyme.
Assuntos
Corantes Fluorescentes/síntese química , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/síntese química , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Ensaios de Triagem em Larga Escala , Cinética , Proteína Quinase 14 Ativada por Mitógeno/química , Naftalenos/química , Naftalenos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.