RESUMO
Salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 are serine/threonine kinases and form a subfamily of the protein kinase AMP-activated protein kinase (AMPK) family. Inhibition of SIKs in stimulated innate immune cells and mouse models has been associated with a dual mechanism of action consisting of a reduction of pro-inflammatory cytokines and an increase of immunoregulatory cytokine production, suggesting a therapeutic potential for inflammatory diseases. Following a high-throughput screening campaign, subsequent hit to lead optimization through synthesis, structure-activity relationship, kinome selectivity, and pharmacokinetic investigations led to the discovery of clinical candidate GLPG3312 (compound 28), a potent and selective pan-SIK inhibitor (IC50: 2.0 nM for SIK1, 0.7 nM for SIK2, and 0.6 nM for SIK3). Characterization of the first human SIK3 crystal structure provided an understanding of the binding mode and kinome selectivity of the chemical series. GLPG3312 demonstrated both anti-inflammatory and immunoregulatory activities in vitro in human primary myeloid cells and in vivo in mouse models.
Assuntos
Proteínas Quinases Ativadas por AMP , Proteínas Serina-Treonina Quinases , Camundongos , Animais , Humanos , Expressão Gênica , CitocinasRESUMO
Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).
Assuntos
Códon sem Sentido , Doenças Genéticas Inatas , Guanidinas , Quinazolinas , Linhagem Celular , Códon sem Sentido/efeitos dos fármacos , Códon sem Sentido/genética , Códon de Terminação/efeitos dos fármacos , Códon de Terminação/genética , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Gentamicinas/farmacologia , Guanidinas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Quinazolinas/farmacologiaRESUMO
Protein synthesis is a complex multistep process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single-molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labeling of the ribosomes are much more complicated. Here, we use a noncanonical translation initiation based on internal ribosome entry sites (IRES), and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of cricket paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones, suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility of studying both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.
Assuntos
Dicistroviridae/genética , Dicistroviridae/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , Animais , Gryllidae/virologia , Humanos , Técnicas In Vitro , Cinética , Microscopia de Fluorescência , Modelos Biológicos , Elongação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , RNA Viral/genética , Coelhos , Ribossomos/metabolismo , Imagem Individual de MoléculaRESUMO
Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity.
