A Computer-Aided Approach for the Discovery of D-Peptides as Inhibitors of SARS-CoV-2 Main Protease

The SARS-CoV-2 main protease, also known as 3-chymotrypsin-like protease (3CLpro), is a cysteine protease responsible for the cleavage of viral polyproteins pp1a and pp1ab, at least, at eleven conserved sites, which leads to the formation of mature nonstructural proteins essential for the replication of the virus. Due to its essential role, numerous studies have been conducted so far, which have confirmed 3CLpro as an attractive drug target to combat Covid-19 and have reported a vast number of inhibitors and their co-crystal structures. Despite all the ongoing efforts, D-peptides, which possess key advantages over L-peptides as therapeutic agents, have not been explored as potential drug candidates against 3CLpro. The current work fills this gap by reporting an in silico approach for the discovery of D-peptides capable of inhibiting 3CLpro that involves structure-based virtual screening (SBVS) of an in-house library of D-tripeptides and D-tetrapeptides into the protease active site and subsequent rescoring steps, including Molecular Mechanics Generalized-Born Surface Area (MM-GBSA) free energy calculations and molecular dynamics (MD) simulations. In vitro enzymatic assays conducted for the four top-scoring D-tetrapeptides at 20 μM showed that all of them caused 55-85% inhibition of 3CLpro activity, thus highlighting the suitability of the devised approach. Overall, our results present a promising computational strategy to identify D-peptides capable of inhibiting 3CLpro, with broader application in problems involving protein inhibition

A comparative pan-genomic analysis of 53 C. pseudotuberculosis strains based on functional domains

Corynebacterium pseudotuberculosis is a pathogenic bacterium with great veterinary and economic importance. It is classified into two biovars: ovis, nitrate-negative, that causes lymphadenitis in small ruminants and equi, nitrate-positive, causing ulcerative lymphangitis in equines. With the explosive growth of available genomes of several strains, pan-genome analysis has opened new opportunities for understanding the dynamics and evolution of C. pseudotuberculosis. However, few pan-genomic studies have compared biovars equi and ovis. Such studies have considered a reduced number of strains and compared entire genomes. Here we conducted an original pan-genome analysis based on protein sequences and their functional domains. We considered 53 C. pseudotuberculosis strains from both biovars isolated from different hosts and countries. We have analysed conserved domains, common domains more frequently found in each biovar and biovar-specific (unique) domains. Our results demonstrated that biovar equi is more variable; there is a significant difference in the number of proteins per strains, probably indicating the occurrence of more gene loss/gain events. Moreover, strains of biovar equi presented a higher number of biovar-specific domains, 77 against only eight in biovar ovis, most of them are associated with virulence mechanisms. With this domain analysis, we have identified functional differences among strains of biovars ovis and equi that could be related to niche-adaptation and probably help to better understanding mechanisms of virulence and pathogenesis. The distribution patterns of functional domains identified in this work might have impacts on bacterial physiology and lifestyle, encouraging the development of new diagnoses, vaccines, and treatments for C. pseudotuberculosis diseases.Communicated by Ramaswamy H. Sarma

A Review of Omics Studies on Arboviruses: Alphavirus, Orthobunyavirus and Phlebovirus

Since the intricate and complex steps in pathogenesis and host-viral interactions of arthropod-borne viruses or arboviruses are not completely understood, the multi-omics approaches, which encompass proteomics, transcriptomics, genomics and metabolomics network analysis, are of great importance. We have reviewed the omics studies on mosquito-borne viruses of the Togaviridae, Peribuyaviridae and Phenuiviridae families, specifically for Chikungunya, Mayaro, Oropouche and Rift Valley Fever viruses. Omics studies can potentially provide a new perspective on the pathophysiology of arboviruses, contributing to a better comprehension of these diseases and their effects and, hence, provide novel insights for the development of new antiviral drugs or therapies

Discovery of All- d -Peptide Inhibitors of SARS-CoV-2 3C-like Protease

During the replication process of SARS-CoV-2, the main protease of the virus [3-chymotrypsin-like protease (3CLpro)] plays a pivotal role and is essential for the life cycle of the pathogen. Numerous studies have been conducted so far, which have confirmed 3CLpro as an attractive drug target to combat COVID-19. We describe a novel and efficient next-generation sequencing (NGS) supported phage display selection strategy for the identification of a set of SARS-CoV-2 3CLpro targeting peptide ligands that inhibit the 3CL protease, in a competitive or noncompetitive mode, in the low μM range. From the most efficient l-peptides obtained from the phage display, we designed all-d-peptides based on the retro-inverso (ri) principle. They had IC50 values also in the low μM range and in combination, even in the sub-micromolar range. Additionally, the combination with Rutinprivir decreases 10-fold the IC50 value of the competitive inhibitor. The inhibition modes of these d-ri peptides were the same as their respective l-peptide versions. Our results demonstrate that retro-inverso obtained all-d-peptides interact with high affinity and inhibit the SARS-CoV-2 3CL protease, thus reinforcing their potential for further development toward therapeutic agents. The here described d-ri peptides address limitations associated with current l-peptide inhibitors and are promising lead compounds. Further optimization regarding pharmacokinetic properties will allow the development of even more potent d-peptides to be used for the prevention and treatment of COVID-19

Design of D-Amino Acids SARS-CoV-2 Main Protease Inhibitors Using the Cationic Peptide from Rattlesnake Venom as a Scaffold

The C30 endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and, hence, is a promising drug target. Molecules isolated from animals, insects, plants, or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus, has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial, and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low µM range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro

Purification, crystallization and preliminary X-ray diffraction analysis of crotamine, a myotoxic polypeptide from the Brazilian snake Crotalus durissus terrificus

Crotamine, a highly basic myotoxic polypeptide (molecular mass 4881 Da) isolated from the venom of the Brazilian rattlesnake Crotalus durissus terrificus, causes skeletal muscle contraction and spasms, affects the functioning of voltage-sensitive sodium channels by inducing sodium influx and possesses antitumour activity, suggesting potential pharmaceutical applications. Crotamine was purified from C. durissus terrificus venom; the crystals diffracted to 1.9 Å resolution and belonged to the orthorhombic space group I2(1)2(1)2(1) or I222, with unit-cell parameters a = 67.75, b = 74.4, c = 81.01 Å. The self-rotation function indicated that the asymmetric unit contained three molecules. However, structure determination by molecular replacement using NMR-determined coordinates was unsuccessful and a search for potential derivatives has been initiated