In silico design of knowledge-based Plasmodium falciparum epitope ensemble vaccines

Malaria is a global health burden, and a major cause of mortality and morbidity in Africa. Here we designed a putative malaria epitope ensemble vaccine by selecting an optimal set of pathogen epitopes. From the IEDB database, 584 experimentally-verified CD8+ epitopes and 483 experimentally-verified CD4+ epitopes were collected; 89% of which were found in 8 proteins. Using the PVS server, highly conserved epitopes were identified from variability analysis of multiple alignments of Plasmodium falciparum protein sequences. The allele-dependent binding of epitopes was then assessed using IEDB analysis tools, from which the population protection coverage of single and combined epitopes was estimated. Ten conserved epitopes from four well-studied antigens were found to have a coverage of 97.9% of the world population: 7 CD8+ T cell epitopes (LLMDCSGSI, FLIFFDLFLV, LLACAGLAYK, TPYAGEPAPF, LLACAGLAY, SLKKNSRSL, and NEVVVKEEY) and 3 CD4+ T cell epitopes (MRKLAILSVSSFLFV, KSKYKLATSVLAGLL and GLAYKFVVPGAATPYE). The addition of four heteroclitic peptides − single point mutated epitopes − increased HLA binding affinity and raised the predicted world population coverage above 99%

Revealing druggable cryptic pockets in the Nsp1 of SARS-CoV-2 and other β-coronaviruses by simulations and crystallography

Non-structural protein 1 (Nsp1) is a main pathogenicity factor of α- and β-coronaviruses. Nsp1 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suppresses the host gene expression by sterically blocking 40S host ribosomal subunits and promoting host mRNA degradation. This mechanism leads to the downregulation of the translation-mediated innate immune response in host cells, ultimately mediating the observed immune evasion capabilities of SARS-CoV-2. Here, by combining extensive molecular dynamics simulations, fragment screening and crystallography, we reveal druggable pockets in Nsp1. Structural and computational solvent mapping analyses indicate the partial crypticity of these newly discovered and druggable binding sites. The results of fragment-based screening via X-ray crystallography confirm the druggability of the major pocket of Nsp1. Finally, we show how the targeting of this pocket could disrupt the Nsp1-mRNA complex and open a novel avenue to design new inhibitors for other Nsp1s present in homologous β-coronaviruses

Selection-based design of in silico dengue epitope ensemble vaccines

Dengue virus affects approximately 130 countries. 25% of infections result in febrile, self‐limiting illness; heterotypic infection results in potentially fatal Dengue Haemorrhagic Fever or Dengue Shock Syndrome. Only one vaccine is currently available. Its efficacy is very variable. Thus, to target Dengue, we used an innovative immunoinformatic protocol to design a putative epitope ensemble vaccine by selecting an optimal set of highly‐conserved epitopes with experimentally‐verified immunogenicity. From 1597 CD4+ and MHC II epitopes, 6 MHC Class I epitopes (RAVHADMGYW, GPWHLGKLEM, GLYGNGVVTK, NMIIMDEAHF, KTWAYHGSY, WAYHGSYEV) and 9 MHC Class II epitopes (LAKAIFKLTYQNKVV, GKIVGLYGNGVVTTS, AAIFMTATPPGSVEA, AAIFMTATPPGTADA, GKTVWFVPSIKAGND, KFWNTTIAVSMANIF, RAIWYMWLGARYLEF, VGTYGLNTFTNMEVQ, WTLMYFHRRDLRLAA) were selected; this candidate vaccine achieved a world population coverage of 92.49%

Toward the Discovery of Inhibitors Targeting Dengue and Zika virus Methyltransferase Using Fragment-based Screening

BACKGROUND: The Zika and dengue viruses, members of the family Flaviviridae, present threats to global health, yet no effective therapy is currently available against these human pathogens. The Dengvaxia vaccine has recently become available to prevent dengue, but its application is limited to certain age groups and depends on the recipients’ pre-vaccination infection status. The highly conserved non-structural protein 5 (NS5), encompassing the RNA-dependent RNA polymerase and the N7 and 2’-O methyltransferase (MTase) domains, offers a potential antiviral target. AIM: This study aimed to identify and develop compounds targeting the MTase domain of NS5 through fragment-based drug discovery. METHODOLOGY: To identify fragment hits as starting points, a fragment library was screened against dengue virus serotype 3 (DENV3) MTase using X-ray crystallography. This project presents the results of the fragment-based screening, including the optimisation of crystallisation conditions to yield the high-quality crystals required for screening, diffraction data collection, structure determination and refinement. The crystallographic fragment screening was followed by orthogonal biophysical assays, such as microscale thermophoresis and thermal shift assays, to identify novel ligand binding pockets on the DENV3 MTase. Finally, the structureactivity relationship by catalogue methodology was employed in an effort to improve the affinity of the identified hits. RESULTS AND CONCLUSION: Almost 600 crystals of DENV3 MTase with an average resolution of 2.5 Å were produced for fragment-based screening. The primary crystallographic screening identified 20 fragment hits that showed clear electron densities. These fragments bind to 10 binding sites in total, eight of which are unpublished and two of which were previously discovered. A structure-activity relationship by catalogue study with 66 analogues improved the affinity of six of the fragment hits. Further investigation and optimisation of these analogues will significantly expand our options for developing small molecules with favourable druglike properties targeting the DENV3 MTase domain

Two Ligand-Binding Sites on SARS-CoV-2 Non-Structural Protein 1 Revealed by Fragment-Based X-ray Screening

The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 by suppressing its replication or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against the N-terminal domain of SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.99 Å). Binding affinities of hits against nsp1 and potential stabilisation were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal shift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medically relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1