RESUMEN
Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.
Asunto(s)
Distrofia Miotónica , Animales , Quinasas Ciclina-Dependientes , Modelos Animales de Enfermedad , Humanos , Ratones , Distrofia Miotónica/tratamiento farmacológico , Distrofia Miotónica/genética , ARN , Empalme del ARN/genética , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.
Asunto(s)
Antimaláricos/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Resistencia a Medicamentos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antimaláricos/química , Antimaláricos/metabolismo , Sitios de Unión , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Femenino , Hepatocitos/citología , Hepatocitos/metabolismo , Hepatocitos/parasitología , Humanos , Imidazoles/química , Estadios del Ciclo de Vida/efectos de los fármacos , Metabolómica , Ratones , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteómica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
CRISPR/Cas9-based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40-300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN/genética , Silenciador del Gen/fisiología , Células Hep G2 , Humanos , Mutación INDEL/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.
Asunto(s)
Antituberculosos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Sulfonamidas/farmacología , Animales , Diseño de Fármacos , Descubrimiento de Drogas , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Mutación , Conformación Proteica , Conejos , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinética , Tuberculosis/tratamiento farmacológicoRESUMEN
Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.