RESUMO
Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. Here, we report the discovery of small-molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, 3 compounds were selected for evaluation in 2 experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2.
Assuntos
Descoberta de Drogas , Proteína 1 Semelhante a Receptor de Interleucina-1/efeitos dos fármacos , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Proteômica , Receptores de Citocinas/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Biomarcadores , Linhagem Celular Tumoral , Biologia Computacional , Avaliação Pré-Clínica de Medicamentos , Doença Enxerto-Hospedeiro , Ensaios de Triagem em Larga Escala , Interleucina-33/metabolismo , Leucemia/tratamento farmacológico , Camundongos , Modelos Animais , Transplante de Células-TroncoRESUMO
We pursued a structure-guided approach toward the development of improved dihydroorotate dehydrogenase (DHODH) inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two potential residues, T63 and Y356, suitable for novel H-bonding interactions, were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of potent quinoline-based analogues 41 (DHODH IC50 = 9.71 ± 1.4 nM) and 43 (DHODH IC50 = 26.2 ± 1.8 nM). A cocrystal structure between 43 and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to the 1,7-naphthyridine 46 (DHODH IC50 = 28.3 ± 3.3 nM) that forms a novel H-bond with Y356. Importantly, compound 41 possesses significant oral bioavailability ( F = 56%) and an elimination t1/2 = 2.78 h (PO dosing). In conclusion, the data supports further preclinical studies of our lead compounds toward selection of a candidate for early-stage clinical development.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Quinolinas/química , Administração Oral , Animais , Compostos de Bifenilo/química , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/síntese química , Feminino , Células HCT116 , Meia-Vida , Humanos , Ligação de Hidrogênio , Camundongos Endogâmicos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Piridinas/química , Pirimidinas/química , Solubilidade , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Förster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed "Dual-Readout F(2) assay" with F(2) for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F(2) assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.
Assuntos
Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala/métodos , Apoptose/efeitos dos fármacos , Bioensaio , Técnicas de Laboratório Clínico , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Humanos , Microscopia , Miniaturização , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/análise , Fatores de TempoRESUMO
Lafora disease (progressive myoclonus epilepsy of Lafora type) is an autosomal recessive neurodegenerative disorder resulting from defects in the EPM2A gene. EPM2A encodes a 331-amino acid protein containing a carboxyl-terminal phosphatase catalytic domain. We demonstrate that the EPM2A gene product also contains an amino-terminal carbohydrate binding domain (CBD) and that the CBD is critical for association with glycogen both in vitro and in vivo. The CBD domain localizes the phosphatase to specific subcellular compartments that correspond to the expression pattern of glycogen processing enzyme, glycogen synthase. Mutations in the CBD result in mis-localization of the phosphatase and thereby suggest that the CBD targets laforin to intracellular glycogen particles where it is likely to function. Thus naturally occurring mutations within the CBD of laforin likely result in progressive myoclonus epilepsy due to mis-localization of phosphatase expression.