A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19

Immunogen design for HIV-1 and influenza

Vaccines provide the most cost effective defense against pathogens. Although vaccines have been designed for a number of viral diseases, a vaccine against HIV-1 still remains elusive. In contrast while there are excellent influenza vaccines, these need to be changed every few years because of antigenic drift and shift The recent discovery of a large number of broadly neutralizing antibodies (bNAbs) and structural characterization of the conserved epitopes targeted by them presents an opportunity for structure based HIV-1 and influenza A vaccine design. We discuss strategies to design immunogens either targeting a particular antigenic region or focusing on native structure stabilization. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. (C) 2014 Elsevier B.V. All rights reserved

Glycosylation of the core of the HIV-1 envelope subunit protein gp120 is not required for native trimer formation or viral infectivity

The gp120 subunit of the HIV-1 envelope (Env) protein is heavily glycosylated at similar to 25 glycosylation sites, of which similar to 7-8 are located in the V1/V2 and V3 variable loops and the others in the remaining core gp120 region. Glycans partially shield Env from recognition by the host immune system and also are believed to be indispensable for proper folding of gp120 and for viral infectivity. Previous attempts to alter glycosylation sites in Env typically involved mutating the glycosylated asparagine residues to structurally similar glutamines or alanines. Here, we confirmed that such mutations at multiple glycosylation sites greatly diminish viral infectivity and result in significantly reduced binding to both neutralizing and non-neutralizing antibodies. Therefore, using an alternative approach, we combined evolutionary information with structure-guided design and yeast surface display to produce properly cleaved HIV-1 Env variants that lack all 15 core gp120 glycans, yet retain conformational integrity and multiple-cycle viral infectivity and bind to several broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies and a germline-reverted version of the bNAb VRC01. Our observations demonstrate that core gp120 glycans are not essential for folding, and hence their likely primary role is enabling immune evasion. We also show that our glycan removal approach is not strain restricted. Glycan-deficient Env derivatives can be used as priming immunogens because they should engage and activate a more divergent set of germlines than fully glycosylated Env. In conclusion, these results clarify the role of core gp120 glycosylation and illustrate a general method for designing glycan-free folded protein derivatives

Design of an Escherichia coli expressed HIV-1 gp120 fragment Immunogen that binds to b12 and induces broad and potent neutralizing antibodies

b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC50 cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals

Host genes that affect progression of AIDS/HIV in India and novel gene therapeutic approaches against HIV

A multitude of host and viral factors play critical role in susceptibility to HIV-1 infection and its subsequent progression to AIDS. Various host factors involved in HIV-1 infection include the chemokine receptors CCR5, CX3CR1, their ligands, RANTES, SDF-1 and cytokines like IL-10, IL-4, among others. The CCR5Δ32 allele is the most important genetic factor known to confer resistance to HIV-1 infection. However, other mutations in CCR5, CX3CR1 and SDF-1 have also been identified in Indian population. Polymorphisms in DC-SIGN, MHC class-I and II molecules are also known to affect HIV-1 progression. These polymorphisms can be utilized as genetic markers for evaluating disease progression and developing effective therapeutics. The review also describes the development of anti-viral therapy, involving the use of catalytic nucleic acids like DNA-enzymes and ribozymes and the expression of ribozymes and si-RNA using lentiviral vectors for stem cell based anti-HIV therapy

Bacterially expressed HIV-1 gp120 outer-domain fragment immunogens with improved stability and affinity for CD4-binding site neutralizing antibodies

Protein minimization is an attractive approach for designing vaccines against rapidly evolving pathogens such as human immunodeficiency virus, type 1 (HIV-1), because it can help in focusing the immune response toward conserved conformational epitopes present on complex targets. The outer domain (OD) of HIV-1 gp120 contains epitopes for a large number of neutralizing antibodies and therefore is a primary target for structure-based vaccine design. We have previously designed a bacterially expressed outer-domain immunogen (ODEC) that bound CD4-binding site (CD4bs) ligands with 3-12 m affinity and elicited a modest neutralizing antibody response in rabbits. In this study, we have optimized ODEC using consensus sequence design, cyclic permutation, and structure-guided mutations to generate a number of variants with improved yields, biophysical properties, stabilities, and affinities (K-D of 10-50 nm) for various CD4bs targeting broadly neutralizing antibodies, including the germline-reverted version of the broadly neutralizing antibody VRC01. In contrast to ODEC, the optimized immunogens elicited high anti-gp120 titers in rabbits as early as 6 weeks post-immunization, before any gp120 boost was given. Following two gp120 boosts, sera collected at week 22 showed cross-clade neutralization of tier 1 HIV-1 viruses. Using a number of different prime/boost combinations, we have identified a cyclically permuted OD fragment as the best priming immunogen, and a trimeric, cyclically permuted gp120 as the most suitable boosting molecule among the tested immunogens. This study also provides insights into some of the biophysical correlates of improved immunogenicity

ASHD alters the expression of apoptotic proteins in Reh and K562 cells.

<p><b>A–C</b>. Cell lysates were prepared from Reh cells after incubating with ASHD (100 µM) for 24, 48 and 72 h. DMSO treated cell lysate was used as control. Approximately, 40 µg of protein per sample was resolved on SDS-PAGE and transferred to a PVDF membrane. The membrane was probed for the expression of BAD, BCL2 (<b>A</b>), PARP (<b>B</b>) and caspase 3, caspase 9 and caspase 8 (<b>C</b>) with specific primary antibodies and appropriate secondary antibodies. <b>D–E</b>. Cell lysates were prepared from K562 cells after incubating with ASHD (30 µM) for 48 h and used for western blotting. The proteins studied are BAD, BAX, PARP (<b>D</b>), and caspase 8, caspase 9 and cytochrome C (<b>E</b>). The α-tubulin was used as an internal loading control in all the panels. <b>F–G</b>. Effect of pancaspase inhibitor (z-VAD-FMK) on Reh cells treated with ASHD. Approximately 0.75×10⁵ cells/ml were cultured and incubated with 30 µM ASHD, with or without 50 µM z-VAD-FMK. DMSO treated cells were used as vehicle control. <b>F</b>. MTT assay showing effect of ASHD on cell proliferation following treatment with z-VAD-FMK at 24 and 48 h. <b>G</b>. Trypan blue assay showing cell viability. For other details refer Fig. 1 legend. Error bars in panels, <b>F</b> and <b>G</b> are based on three independent batches of experiments.</p

Detection of apoptosis induced by ASHD using flow cytometry and confocal microscopy.

<p>Reh (<b>A</b>) and K562 (<b>B</b>) cells were cultured with ASHD (50 and 250 μM) for 72 h and processed for Annexin V- FITC/PI double-staining. The cells were then quantitatively or qualitatively monitored. In panels <b>A</b> and <b>B</b>, lower left quadrant shows cells which are negative for both Annexin V-FITC and PI, lower right shows Annexin V positive cells which are in the early stage of apoptosis, upper left shows only PI positive cells which are dead, and upper right shows both Annexin V and PI positive, which are in the stage of late apoptosis or necrosis. The values mentioned in the quadrants show the percentage of cells positive for both the Annexin V and PI (Top) or Annexin V alone (Bottom). In both panels <b>A</b> and <b>B</b>, cells treated with DMSO (a), ASHD, 50 μM (b), and ASHD 250 μM (c) are shown. In both panels, bar diagram showing comparison of early and late apoptotic cells at different doses of ASHD treatment are presented (d). (<b>C</b>) and (<b>D</b>) shows confocal microscopy visualization of Reh or K562 cells, following treatment with ASHD. Cells incubated with DMSO alone (<b>a</b>), or ASHD 50 μM (<b>b, c</b>) and 250 μM (<b>d, e</b>) respectively are used for the study.</p

IC₅₀ value of ASHD calculated at different time points.

<p>IC₅₀ value of ASHD calculated at different time points.</p