Inactivation of HIV-1 and SARS-CoV-2 viruses by Targeting Metastable Virus Spike Proteins
Wednesday, June 1, 2022
12:00 PM-2:00 PM
BIOMED PhD Thesis Defense
Title:
Inactivation of HIV-1 and SARS-CoV-2 viruses by Targeting Metastable Virus Spike Proteins
Speaker:
Aakansha Nangarlia, PhD Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University
Advisors:
Irwin Chaiken, PhD
Professor of Biochemistry and Molecular Biology
College of Medicine
Drexel University
Ken Barbee, PhD
Professor, Senior Associate Dean, and Associate Dean for Research
School of Biomedical Engineering, Science and Health Systems
Drexel University
Abstract:
New strains of drug-resistant coronaviruses (CoV) and human immunodeficiency virus (HIV) are emerging at an alarming rate, despite development of COVID-19 vaccines and HIV’s highly active antiretroviral therapy. This calls for a critical need to develop potent antiviral agents capable of inhibiting initial infection and disease progression. SARS-CoV-2, responsible for COVID, and HIV-1, for AIDS, are both enveloped virions that rely on their surface envelope (Env) spike protein for viral infection. The interaction of the SARS-CoV-2 Env spike with the host cell receptor, ACE2, and HIV-1 Env spike with CD4 results in a series of conformational changes that leads to membrane fusion and infection. The glycan residues that decorate the virion’s Env spikes play a crucial role in shielding virus neutralization and host cell recognition. Structural and functional similarities between the SARS-CoV-2 and HIV-1 Env spike led to the premise for my work.
In Aim 1, we identified potent Env targeting inactivators (EI)s that can inactivate SARS-CoV-2 and hinder disease progression. We discovered that the lectin CVN causes an irreversible inactivation of SARS-CoV-2 pseudoviruses. Mechanistic studies using single-site N-linked glycan mutations and CVN washout experiments identified two glycan clusters in the S1 subunit of the SARS-CoV-2 Env spike to be vital for CVN-based inactivation. Structural analysis to evaluate the feasibility of CVN-lectin engagement pinpointed the glycan cluster near the RBD to have the maximum change in distance between the closed and open Env conformational states and to be within the CVN’s bivalent binding range in an open conformation. The distance within the second glycan cluster near the S1/S2 furin cleavage site did not significantly change between the Env spike states but fell within CVN’s binding range. The findings from this study led to the conclusion that CVN-based irreversible inactivation requires engagement with two glycan clusters on the S1 subunit and to a theory that the cooperativity effect seen between the glycan clusters leads to CVN based stabilization of the Env spike in an open state.
In Aim 2, we investigated the development of bifunctional EIs for HIV treatment. Peptide triazoles (PTs) target HIV-1 at its entry step via dual inhibition of the viral Env to both CD4 and co-receptors, CCR5 or CXCR4, on the target cells. Additionally, only PT- thiols (PTTs), in contract to PTs, cause combined Env spike shedding and CD4 independent virolysis. Mechanistic studies revealed that this adverse effect by PTTs is due to dual engagement of PTT’s active pharmacophore and thiol with the CD4 Phe43 binding pocket and one of the disulfide bonds within the HIV-1 Env, respectively. However, whether the dual interaction occurs within the same or neighboring protomers of the HIV-1’s trimeric Env was yet to be fully understood. Moreover, despite the potent antiviral activity of PTs and PTTs, they remain nonresistant to protease. Thus, we focused on synthesizing the next generation of protease-resistant PTTs called cyclic-PTTs (cPTTs). The results from the functional assays confirmed that cPTTs retain the ability to bind to the gp120 subunit of HIV-1’s Env spike and irreversibly inactivate the pseudoviruses. Furthermore, the mechanism of action, like PTTs, was found to be dependent on the dual engagement of the cPTT’s active pharmacophore and thiol with the Env spike. Additional structural correlation concluded that cPTTs cause irreversible inactivation of HIV-1 pseudoviruses via dual engagement within the same protomer.
In Aim 3, we successfully synthesized another class of bifunctional EIs, smDLIs, composed of two domain components connected by a linker, Lx. One component is derived from BNM-III-170, a small-molecule CD4 mimic that binds to gp120, and the second component is derived from the N-terminus of the HIV-1’s gp41 Membrane Proximal External Region (MPER). Our findings showed that smDLIs, BNM-III-170-Lx-Trp3, cause an irreversible inactivation of HIV-1 virions via dual engagement. Furthermore, mechanistic studies confirmed that the virolytic effect observed is dependent on the covalent linkage of BNM-III-170 and Trp3 domains. Significant virolysis was also observed for Env-negative pseudoviruses, suggesting that the irreversible inactivation effect is partially due to Env and membrane interaction. Cell-based cytotoxicity assays to investigate the extent of membrane disruption by BNM-III-170-Lx-Trp3 showed specific cytotoxic effect with BNM-III-170-Lx-Trp3 and HIV-1 Env expressing cells. Lastly, computational modeling studies supported BNM-III-170 related strong membrane interaction and the feasibility of BNM-III-170-Lx-Trp3 to bind to gp120 and gp41 subunits in tandem with the open-state Env trimers. Thus, our findings show that despite the membrane effect seen with pseudoviruses, BNM-III-170-Lx-Trp3 causes specific irreversible inactivation of HIV-1 infected cells via dual engagement with the open-state of the Env spike.
In conclusion, this study shows the feasibility of EIs to cause inactivation of enveloped virions like SARS-CoV-2 and HIV-1 via targeting the Env spike in an open (“activated”) state and hijacking the metastability of the Env spike’s protein. Overall, this work lays the groundwork for advancing EIs as potential therapeutic leads for eradicating infectious diseases like COVID-19 and HIV-1.
Contact Information
Natalia Broz
njb33@drexel.edu