Inactivation of HIV-1 and SARS-CoV-2 viruses by targeting metastable virus spike proteins

Aakansha Nangarlia

doi:10.17918/00001316

Despite the development of COVID-19 vaccines and HIV's highly active antiretroviral therapy (HAART), new strains of drug-resistant coronaviruses (CoV) and human immunodeficiency viruses (HIV) are emerging at an alarming rate. This calls for an urgent need to develop potent antiviral agents capable of inhibiting infection and disease progression by targeting the viruses prior to host cell interaction. SARS-CoV-2, responsible for COVID, and HIV-1, responsible for HIV, 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 the host cell receptor, CD4, results in a series of Env spike conformational changes that leads to viral - host cell membrane fusion and infection. Furthermore, the glycan residues that decorate the virion's Env spikes play a crucial role in host cell recognition and shielding the virus from neutralization. The structural and functional similarities between the SARS-CoV-2 and HIV-1 Env spike led to the premise for my research work. We demonstrated that lectin-based Env targeting inactivator (EI), cyanovirin -N (CVN), causes an irreversible inactivation of SARS-CoV-2 pseudoviruses by engaging with the two conserved glycan residues on the S1 subunit of the SARS-CoV-2 Env Spike. Correspondingly, the development of bifunctional classes of EIs, namely cyclic peptide triazole thiols (cPTTs) and small molecule dual-lytic inactivators (smDLIs), were found to cause irreversible inactivation of HIV-1 pseudoviruses potentially via two different dual-engagement mechanisms. For the first part of the research, we identified potent EIs that can inactivate SARS-CoV-2 virions and hinder COVID-19 disease progression. Surprisingly, we discovered that the lectin CVN causes an irreversible inactivation of SARS-CoV-2 pseudoviruses. Additionally, 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, one near the receptor binding domain (RBD) and one near the S1/S2 furin cleavage site, 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 ("stable") and open ("unstable/receptor-binding") conformational states of the Env spike and to also be within the CVN's binding range while the Env spike is in an open conformation. On the other hand, the distance within the second glycan cluster near the S1/S2 furin cleavage site did not significantly change between the closed and open state of the Env spike but fell within the CVN binding range. The overall findings from this study led to the conclusion that CVN-based irreversible inactivation requires lectin 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 ("unstable") state. In the next part of this research, we investigated the development of next-generation classes of bifunctional EIs for HIV treatment. PT-thiols (PTTs) have previously been found to target the HIV-1 virus at the entry step via dual inhibition of the viral Env to the receptor, CD4, and co-receptors, CCR5 or CXCR4, on the target cells. Interestingly, only the PTTs, in contrast to their non-thiolated derivatives, can cause combined Env spike shedding and CD4 independent virolytic activity. Mechanistic studies revealed that this adverse effect is due to the interaction of the active pharmacophore of the PTTs with the CD4 Phe43 binding pocket and the thiol group with one of the disulfide bonds in the Env spike subunit, gp120. However, whether the dual interaction occurs within the same protomer or between neighboring protomers of an Env spike trimer is yet to be understood. Moreover, despite the potent antiviral activity of PTs and PTTs, they remained nonresistant to proteolytic degradation. Thus, as part of this work, we focused on synthesizing the next generation of protease resistant PTTs called cyclic peptide triazole thiols (cPTTs). The results from the functional assays confirmed that, like PTTs, cPTT retained the ability to bind to the gp120 subunit of the HIV-1 Env spike and irreversibly inactivate HIV-1 pseudoviruses. Furthermore, the mechanism of action is dependent on the dual engagement of the active pharmacophore and thiol to the CD4 Phe43 binding pocket in the gp120 subunit and a disulfide bond within the HIV-1 Env Spike, respectively. To determine if the dual engagement is within or cross protomer of the trimeric Env spike, we conducted surface density direct binding analysis on surface plasmon resonance (SPR). The results from both SPR direct binding and structural analysis concluded that cPTTs cause irreversible inactivation of HIV-1 pseudoviruses via dual engagement of the pharmacophore and thiol components within the same protomer of the Env spike. Additionally, a drastic decrease in off-rate was observed for thiol-containing compounds only, suggesting a tight-irreversible interaction. Simultaneously, we successfully synthesized another class of bifunctional EIs, small molecule dual lytic inactivators (smDLIs), composed of two subunits connected by a linker, Lx, capable of simultaneous binding to two sites on the HIV-1 Env spike. One component of the conjugates 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 gp41 Membrane Proximal External Region (MPER), which was previously found to bind to the gp41 subunit of the HIV-1 Env spike. Our findings showed that smDLI, BNM-III-170-Lx-Trp3, can cause irreversible inactivation of HIV-1 virions. Through mechanistic studies, we confirmed that the virolysis observed by the BNM-III-170-Lx-Trp3 smDLIs was dependent on the covalent linkage of the BNM-III-170 and Trp3 domains. Interestingly, a significant magnitude of virolysis was observed for Env negative pseudoviruses, suggesting that the irreversible inactivation effect is partially due to Env and membrane interaction. Computational modeling studies supported the feasibility of a strong membrane binding activity by BNM-III-170. To investigate the extent of membrane disruption by BNM-III-170-Lx-Trp3 smDLIs, cell-based cytotoxicity assays were conducted in which the HIV-1 Env expressing HEK 293T cells were found to exhibit a dose-dependent and specific cytotoxic effect with BNM-III-170-Lx-Trp3 smDLIs. Lastly, molecular dynamic and modeling studies showed that the BNM-III-170-Lx-Trp3 binds to the gp120 and gp41 subunits in tandem with the open-state Env trimers and induce relative motion of the gp120 subunits consistent with models of Env inactivation. Thus, our findings show that despite the membrane effect seen with pseudoviruses, BNM-III-170-Lx-Trp3 smDLIs cause specific irreversible inactivation of HIV-1 infected cells via dual engagement with the open ("activated") state of the Env spike. Overall, this study establishes the feasibility for the development of EIs that can lead to inactivation of enveloped virions like SARS-CoV-2 and HIV-1 viruses. The common mechanism by which the EIs cause the irreversible inactivation is via targeting the Env spike in an open ("activated") state and hijacking the metastability of the Env spike's protein. In conclusion, this work lays the groundwork for advancing Env targeting virus inhibitors as potential therapeutic leads for eradicating Env-expressing viruses responsible for infectious diseases like COVID-19 and HIV-1.

Inactivation of HIV-1 and SARS-CoV-2 viruses by targeting metastable virus spike proteins

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Inactivation of HIV-1 and SARS-CoV-2 viruses by targeting metastable virus spike proteins

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