RESUMO
Target-based approaches have traditionally been used in the search for new anti-infective molecules. Target selection process, a critical step in Drug Discovery, identifies targets that are essential to establish or maintain the infection, tractable to be susceptible for inhibition, selective towards their human ortholog and amenable for large scale purification and high throughput screening. The work presented herein validates the Plasmodium falciparum mRNA 5' triphosphatase (PfPRT1), the first enzymatic step to cap parasite nuclear mRNAs, as a candidate target for the development of new antimalarial compounds. mRNA capping is essential to maintain the integrity and stability of the messengers, allowing their translation. PfPRT1 has been identified as a member of the tunnel, metal dependent mRNA 5' triphosphatase family which differs structurally and mechanistically from human metal independent mRNA 5' triphosphatase. In the present study the essentiality of PfPRT1 was confirmed and molecular biology tools and methods for target purification, enzymatic assessment and target engagement were developed, with the goal of running a future high throughput screening to discover PfPRT1 inhibitors.
Assuntos
Antimaláricos , Descoberta de Drogas , Plasmodium falciparum , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/enzimologia , Descoberta de Drogas/métodos , Humanos , Ensaios de Triagem em Larga Escala/métodos , RNA Mensageiro/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Inibidores Enzimáticos/farmacologia , Hidrolases Anidrido ÁcidoRESUMO
The inhibition of kallikrein 5 (KLK5) has been identified as a potential strategy for treatment of the genetic skin disorder Netherton syndrome, in which loss-of-function mutations in the SPINK5 gene lead to down-regulation of the endogenous inhibitor LEKTI-1 and profound skin-barrier defects with severe allergic manifestations. To aid in the development of a medicine for this target, an X-ray crystallographic system was developed to facilitate fragment-guided chemistry and knowledge-based drug-discovery approaches. Here, the development of a surrogate crystallographic system in place of KLK5, which proved to be challenging to crystallize, is described. The biochemical robustness of the crystallographic surrogate and the suitability of the system for the study of small nonpeptidic fragments and lead-like molecules are demonstrated.
Assuntos
Benzamidinas/química , Calicreínas/química , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Benzamidinas/farmacologia , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Descoberta de Drogas , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Calicreínas/antagonistas & inibidores , Calicreínas/genética , Calicreínas/metabolismo , Cinética , Modelos Moleculares , Mutação , Síndrome de Netherton/tratamento farmacológico , Síndrome de Netherton/enzimologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Eletricidade Estática , Especificidade por SubstratoRESUMO
RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Pirazóis/síntese química , Pirazóis/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Disponibilidade Biológica , Linhagem Celular , Doença Crônica , Desenho de Fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Haplorrinos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Esclerose Múltipla/tratamento farmacológico , Pirazóis/farmacocinética , Ratos , Retinose Pigmentar/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.
Assuntos
Carcinoma Ductal Pancreático/imunologia , Tolerância Imunológica/imunologia , Macrófagos/imunologia , Neoplasias Pancreáticas/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , Tolerância Imunológica/genética , Células L , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Transcrição STAT1/metabolismo , Células Th1/citologia , Células Th17/citologiaRESUMO
A novel series of P2-P4 macrocyclic HCV NS3/4A protease inhibitors with α-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around α-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action.
Assuntos
Compostos de Boro/química , Ácidos Borônicos/química , Compostos Macrocíclicos/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Compostos de Boro/síntese química , Compostos de Boro/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Hepacivirus/efeitos dos fármacos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
We have designed and synthesized a novel series of alpha-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described.