The Dual-Action Virolytic Entry Inhibitors, or "DAVEI's," are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15-residue fragment from the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEI's trigger rupture of HIV-1 virions, and the interaction site between DAVEI MPER and HIV-1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free-energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER . We found that a W666A mutation had a large positive ΔΔG for all three protomers, while W672A had a large positive ΔΔG for only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER . Our findings are consistent with our previous experimental study of W-to-A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV-1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.

A strategy has been established for the synthesis of a family of bifunctional HIV-1 inhibitor covalent conjugates with the potential to bind simultaneously to both the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein trimeric complex (Env). One component of the conjugates is derived from BNM-III-170, a small-molecule CD4 mimic that binds to gp120. The second component, comprised of the peptide DKWASLWNW (“Trp3”), was derived from the N-terminus of the HIV-1 gp41 Membrane Proximal External Region (MPER) and found previously to bind to the gp41 subunit of Env. The resulting bifunctional conjugates were shown to inhibit virus cell infection with low micromolar potency and to induce lysis of the HIV-1 virion. Crucially, virolysis was found to be dependent on the covalent linkage of the BNM-III-170 and Trp3 domains, as coadministration of a mixture of the un-cross-linked components proved to be nonlytic. However, a significant magnitude of lytic activity was observed in Env-negative and other control pseudoviruses, suggesting parallel mechanisms of action of the conjugates involving Env interaction and direct membrane disruption. Computational modeling suggested strong membrane-binding activity of BNM-III-170, which may underly the nonspecific virolytic effects of the conjugates. To investigate the scope of the membrane effect, cell-based cytotoxicity and membrane permeability assays were performed employing flow cytometry. Here, we observed a dose-dependent and specific cytotoxic effect on HIV-1 Env-expressing cells by the small-molecule bifunctional inhibitor. Most importantly, Env-negative cells were not susceptible to the cytotoxic effect upon exposure to this construct at concentrations where cell-killing effects were observed for Env-positive cells. Computational structural modeling supports a mechanism in which the bifunctional inhibitors bind to the gp120 and gp41 subunits in tandem in open-state Env trimers and induce relative motion of the gp120 subunits consistent with models of Env inactivation. This observation supports the idea that the cell-killing effect of the small-molecule bifunctional inhibitor is due to specific Env conformational triggering. This work lays important groundwork to advance a small-molecule bifunctional inhibitor approach for eliminating Env-expressing infected cells and the eradication of HIV-1.

The Dual-Acting Virolytic Entry Inhibitors, or DAVEI's, are a class of recombinant chimera fusion proteins consisting of a lectin, a flexible polypeptide linker, and a fragment of the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEIs trigger virolysis of HIV-1 virions through interactions with the trimeric envelope glycoprotein complex (Env), though the details of these interactions are not fully determined as yet. The purpose of this work was to use structural modeling to rationalize a dependence of DAVEI potency on the molecular length of the linker connecting the two components. We used temperature accelerated molecular dynamics and on-the-fly parameterization to compute free energy versus end-to-end distance for two different linker lengths, DAVEI L0 (His 6) and DAVEI L2 ([Gly 4 Ser] 2 His 6). Additionally, an envelope model was created based on a cryo-electron microscopy-derived structure of a cleaved, soluble Env construct, with high-mannose glycans added which served as putative docking locations for the lectin, along with MPER added that served as a putative docking location for the MPER region of DAVEI (MPER DAVEI). Using MD simulation, distances between the lectin C-terminus and Env gp41 MPER were measured. We determined that none of the glycans were close enough to gp41 MPER to allow DAVEI L0 to function, while one, N448, will allow DAVEI L2 to function. These findings are consistent with the previously determined dependence of lytic function on DAVEI linker lengths. This supports the hypothesis that DAVEI's engage Env at both glycans and the Env MPER in causing membrane poration and lysis.

The design, synthesis and validation of a family of small molecule “Dual-Action Virucidal EntryInhibitors” (DAVEIs) has been achieved that result in irreversible lytic inactivation of HIV-1 virions. These constructs contained two functional components that endow the capacity to bindsimultaneously to both the gp120 and gp41 subunits of the HIV-1 Envelope glycoprotein (Env). One component is derived from BNM-III-170, a small molecule CD4 mimic warhead that binds togp120. The second component, a Trp3 peptide, is a 9-amino acid segment based on the gp41 Membrane Proximal External Region (MPER) that has been proposed to bind to the gp41 MPERdomain of the Env. The resulting smDAVEIs both inhibit infection with low micromolar potency and induce lysis of the HIV-1 virion. The lytic activity was selective for functional HIV-1 virions. Crucially, virolysis was found to be dependent on covalent tethering of the BNM-III-170 and Trp3 domains with various spacers, as coadministration of the un-crosslinked components proved not to be lytic. Computational modeling supports a mechanism in which DAVEIs bind to open-state Env trimers and induce relative motion of gp120 subunits that further opens the trimers. Overall, this work represents a promising new step toward the use of small-molecule DAVEIs for eradication of HIV.

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