RESUMO
Deubiquitinylating enzymes (DUBs) regulate the deubiquitinylation process of post-translationally modified proteins and thus control protein signaling in various cellular processes. The DUB Cezanne-1 catalyzes the cleavage of the iso-peptide bond of Lys11-linked polyubiquitin chains with high selectivity. Crystal structures of Cezanne-1 in different states provide important insight regarding the complex formation and global changes during the catalytic cycle but are lacking details of dynamics and control of activation. Activity-based probes are used to isolate intermediate states upon forming covalent bonds with the DUB active site. Those, however, may lead to structures that are non-native. Conformational changes of Cezanne-1, during its process of activation and proteolytic activity, are investigated using all-atom molecular dynamics (MD) simulations of the ubiquitin-free, diubiquitin-bound, and monoubiquitin-bound Cezanne-1 DUB for a total of â¼18 µs. Our results show that ubiquitin-free Cezanne-1 dynamically shuttles between catalytically competent and incompetent states which suggests that its activation is independent of substrate binding. The catalytically competent substrate-free Cezanne-1 promotes distal ubiquitin substrate access to the catalytic center. The subsequent binding of the proximal ubiquitin shifts the equilibrium toward the catalytically competent state of the dyad, thereby promoting proteolysis of the iso-peptide bond. After cleavage of the scissile bond, sequential dissociation of first the proximal ubiquitin induces the inactivation of Cezanne-1. The subsequent release of the distal ubiquitin fully reconstitutes the inactive substrate-free state of Cezanne-1. The process of activation and catalytic turnover of DUB Cezanne-1 is a multistage cycle with several critical dynamic transitions that cannot be characterized based on protein structures alone. Activity-based probes of cysteine proteases lead to non-native protein-protein contacts, which need to be resolved in order to be able to issue statements about physiological states and substrate binding.
Assuntos
Endopeptidases , Ubiquitina , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Poliubiquitina/metabolismo , Peptídeos/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.
Assuntos
COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Dimetil Sulfóxido/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , SARS-CoV-2/enzimologia , Simulação por Computador , Proteases 3C de Coronavírus/genética , Humanos , Técnicas Analíticas Microfluídicas , Peptídeos/metabolismo , Estabilidade ProteicaRESUMO
G protein-coupled receptors (GPCRs) are involved in a wide variety of physiologicalprocesses. Therefore, approximately 40% of currently prescribed drugs have targeted this receptorfamily. Discovery of ß-arrestin mediated signaling and also separability of G protein and b-arrestinsignaling pathways have switched the research focus in the GPCR field towards development ofbiased ligands, which provide engagement of the receptor with a certain effector, thus enrichinga specific signaling pathway. In this review, we summarize possible factors that impact signalingprofiles of GPCRs such as oligomerization, drug treatment, disease conditions, genetic background,etc. along with relevant molecules that can be used to modulate signaling properties of GPCRssuch as allosteric or bitopic ligands, ions, aptamers and pepducins. Moreover, we also discuss theimportance of inclusion of pharmacogenomics and molecular dynamics simulations to achieve aholistic understanding of the relation between genetic background and structure and function ofGPCRs and GPCR-related proteins. Consequently, specific downstream signaling pathways can beenriched while those that bring unwanted side effects can be prevented on a patient-specific basis.This will improve studies that centered on development of safer and personalized therapeutics,thus alleviating the burden on economy and public health.
Assuntos
Desenvolvimento de Medicamentos , Medicina de Precisão , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica , Animais , Sítios de Ligação , Desenvolvimento de Medicamentos/métodos , Humanos , Ligantes , Modelos Moleculares , Terapia de Alvo Molecular , Polimorfismo de Nucleotídeo Único , Medicina de Precisão/métodos , Ligação Proteica , Multimerização Proteica , Receptores Acoplados a Proteínas G/genética , Relação Estrutura-AtividadeRESUMO
Coronavirus disease-2019 (COVID-19) was firstly reported in Wuhan, China, towards the end of 2019, and emerged as a pandemic. The spread and lethality rates of the COVID-19 have ignited studies that focus on the development of therapeutics for either treatment or prophylaxis purposes. In parallel, drug repurposing studies have also come into prominence. Herein, we aimed at having a holistic understanding of conformational and dynamical changes induced by an experimentally characterized inhibitor on main protease (Mpro) which would enable the discovery of novel inhibitors. To this end, we performed molecular dynamics simulations using crystal structures of apo and α-ketoamide 13b-bound Mpro homodimer. Analysis of trajectories pertaining to apo Mpro revealed a new target site, which is located at the homodimer interface, next to the catalytic dyad. Thereafter, we performed ensemble-based virtual screening by exploiting the ZINC and DrugBank databases and identified three candidate molecules, namely eluxadoline, diosmin, and ZINC02948810 that could invoke local and global conformational rearrangements which were also elicited by α-ketoamide 13b on the catalytic dyad of Mpro. Furthermore, ZINC23881687 stably interacted with catalytically important residues Glu166 and Ser1 and the target site throughout the simulation. However, it gave positive binding energy, presumably, due to displaying higher flexibility that might dominate the entropic term, which is not included in the MM-PBSA method. Finally, ZINC20425029, whose mode of action was different, modulated dynamical properties of catalytically important residue, Ala285. As such, this study presents valuable findings that might be used in the development of novel therapeutics against Mpro.Communicated by Ramaswamy H. Sarma.
Assuntos
Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus , Reposicionamento de Medicamentos/métodos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2RESUMO
Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.
Assuntos
Quinases raf , Proteínas ras , Humanos , Células HEK293 , Ligantes , Nucleotídeos/metabolismo , Fosforilação , Proteínas ras/metabolismo , Proteínas Mutantes , Quinases raf/metabolismoRESUMO
RAS mutants have been extensively studied as they are associated with development of cancer; however, H-RASG12P mutant has remained untouched since it does not lead to transformation in the cell. To the best of our knowledge, this is the first study where structural/dynamical properties of H-RASG12P have been investigated -in comparison to H-RASWT, H-RASG12D, RAF-RBD-bound and GAP-bound H-RASWT- using molecular dynamics simulations (total of 9 µs). We observed remarkable differences in dynamics of Y32. Specifically, it is located far from the nucleotide binding pocket in the catalytically-active GAP-bound H-RASWT, whereas it makes close interaction with the nucleotide in signaling-active systems (H-RASG12D, KRAS4BG12D, RAF-RBD-bound H-RASWT) and H-RASWT. The accessibility of Y32 in wild type protein is achieved upon GAP binding. Interestingly; however, it is intrinsically accessible in H-RASG12P. Considering the fact that incomplete opening of Y32 is associated with cancer, we propose that Y32 can be targeted by means of small therapeutics that can displace it from the nucleotide binding site, thus introducing intrinsic GTPase activity to RAS mutants, which cannot bind to GAP. Therefore, mimicking properties of H-RASG12P in RAS-centered drug discovery studies has the potential of improving success rates since it acts as a molecular switch per se.