RESUMEN
RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.
Asunto(s)
Poliovirus/enzimología , ARN Polimerasa Dependiente del ARN/química , Proteínas Virales/química , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Simulación de Dinámica Molecular , Mutación , Mutación Missense , Nucleótidos/química , Unión Proteica , Estructura Secundaria de Proteína , ARN Viral/química , ARN Viral/fisiología , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genéticaRESUMEN
The ability of an RNA virus to exist as a population of genetically distinct variants permits the virus to overcome events during infections that would otherwise limit virus multiplication or drive the population to extinction. Viral genetic diversity is created by the ribonucleotide misincorporation frequency of the viral RNA-dependent RNA polymerase (RdRp). We have identified a poliovirus (PV) RdRp derivative (H273R) possessing a mutator phenotype. GMP misincorporation efficiency for H273R RdRp in vitro was increased by 2-3-fold that manifested in a 2-3-fold increase in the diversity of the H273R PV population in cells. Circular sequencing analysis indicated that some mutations were RdRp-independent. Consistent with the population genetics theory, H273R PV was driven to extinction more easily than WT in cell culture. Furthermore, we observed a substantial reduction in H273R PV virulence, measured as the ability to cause paralysis in the cPVR mouse model. Reduced virulence correlated with the inability of H273R PV to sustain replication in tissues/organs in which WT persists. Despite the attenuated phenotype, H273R PV was capable of replicating in mice to levels sufficient to induce a protective immune response, even when the infecting dose used was insufficient to elicit any visual signs of infection. We conclude that optimal RdRp fidelity is a virulence determinant that can be targeted for viral attenuation or antiviral therapies, and we suggest that the RdRp may not be the only source of mutations in a RNA virus genome.
Asunto(s)
Aptitud Genética , Poliovirus/genética , Poliovirus/patogenicidad , ARN Polimerasa Dependiente del ARN/genética , Animales , Secuencia de Bases , Genoma Viral/genética , Células HeLa , Humanos , Inmunidad , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Poliomielitis/inmunología , Poliomielitis/virología , Poliovirus/enzimología , Poliovirus/ultraestructura , Virulencia , Ensamble de Virus , Replicación ViralRESUMEN
We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.
Asunto(s)
Benzamidas/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Descubrimiento de Drogas , Lisina/análogos & derivados , Sondas Moleculares/farmacología , Piperidinas/farmacología , Benzamidas/química , Benzamidas/metabolismo , Unión Competitiva/efectos de los fármacos , Cristalografía por Rayos X , Proteínas de Unión al ADN/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Lisina/antagonistas & inhibidores , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Estructura Molecular , Piperidinas/química , Piperidinas/metabolismo , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismo , Relación Estructura-Actividad , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Lysine methylation is a key epigenetic mark, the dysregulation of which is linked to many diseases. Small-molecule antagonism of methyl-lysine (Kme) binding proteins that recognize such epigenetic marks can improve our understanding of these regulatory mechanisms and potentially validate Kme binding proteins as drug-discovery targets. We previously reported the discovery of 1 (UNC1215), the first potent and selective small-molecule chemical probe of a methyl-lysine reader protein, L3MBTL3, which antagonizes the mono- and dimethyl-lysine reading function of L3MBTL3. The design, synthesis, and structure-activity relationship studies that led to the discovery of 1 are described herein. These efforts established the requirements for potent L3MBTL3 binding and enabled the design of novel antagonists, such as compound 2 (UNC1679), that maintain in vitro and cellular potency with improved selectivity against other MBT-containing proteins. The antagonists described were also found to effectively interact with unlabeled endogenous L3MBTL3 in cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Lisina/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Diseño de Fármacos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.
RESUMEN
Proteins which bind methylated lysines ("readers" of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small-molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first cocrystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design.