RESUMO
The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.
Assuntos
Desenho de Fármacos , Agonismo Inverso de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Piridinas/administração & dosagem , Piridinas/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Piridinas/farmacocinética , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.
Assuntos
Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/enzimologia , Quinazolinas/farmacologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Atrofia Muscular Espinal/genética , Regiões Promotoras Genéticas , Quinazolinas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/deficiência , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Despite its diverse applications, such as identification of the protein binding partners of small molecules and investigation of intracellular drug-target engagement, photoaffinity labelling (PAL) is intrinsically challenging, primarily due to the difficulty in discovering functionally active photoaffinity probes. Here we describe the creation of a chemoproteomic library to discover a novel photoaffinity probe for DcpS, an mRNA decapping enzyme that is a putative target for Spinal Muscular Atrophy. This library approach expedites the discovery of photoaffinity probes and expands the chemical biology toolbox to include RNA cap-binding proteins.
Assuntos
Endorribonucleases/metabolismo , Sondas Moleculares/química , Marcadores de Fotoafinidade/química , Sítios de Ligação , Endorribonucleases/química , Biblioteca Gênica , Humanos , Modelos Moleculares , Sondas Moleculares/metabolismo , Quinazolinas/químicaRESUMO
This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.
Assuntos
Endorribonucleases/metabolismo , Inibidores Enzimáticos/farmacologia , Sondas Moleculares/química , Ácidos Sulfínicos/química , Tirosina/metabolismo , Domínio Catalítico , Células Cultivadas , Técnicas de Química Sintética , Cristalografia por Raios X , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Sondas Moleculares/síntese química , Terapia de Alvo Molecular/métodos , Relação Estrutura-Atividade , Tirosina/químicaRESUMO
The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/química , Inibidores Enzimáticos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Ácidos Hidroxâmicos/química , Amidoidrolases/metabolismo , Antibacterianos/síntese química , Antibacterianos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/química , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Relação Estrutura-Atividade , Sulfonas/químicaRESUMO
The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/síntese química , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Ácidos Hidroxâmicos/síntese química , Piridonas/síntese química , Ácidos Sulfônicos/síntese química , Animais , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Cristalografia por Raios X , Humanos , Ácidos Hidroxâmicos/farmacocinética , Ácidos Hidroxâmicos/farmacologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Piridonas/farmacocinética , Piridonas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Ácidos Sulfônicos/farmacocinética , Ácidos Sulfônicos/farmacologiaRESUMO
In this paper, we present the synthesis and SAR as well as selectivity, pharmacokinetic, and infection model data for representative analogues of a novel series of potent antibacterial LpxC inhibitors represented by hydroxamic acid.