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Increasing hydrophobicity of residues in an anti-HIV-1 Env peptide synergistically improves potency.

ABSTRACT: T-20/Fuzeon/Enfuvirtide (ENF), a peptide inhibitor of HIV-1 infection, targets the grooves created by heptad repeat 2 (HR2) of Env's coiled-coil, but mutants resistant to ENF emerge. In this study, ENF-resistant mutants--V38A, N43D, N43D/S138A, Q40H/L45M--were combined with modified inhibitory peptides to identify what we believe to be novel ways to improve peptide efficacy. V38A did not substantially reduce infectivity, but was relatively resistant to inhibitory peptides. N43D was more resistant to inhibitory peptides than wild-type, but infectivity was reduced. The additional mutation S138A (N43D/S138A) increased infectivity and further reduced peptide inhibitory potency. It is concluded that S138A increased binding of HR2/ENF into grooves and that S138A compensated for electrostatic repulsion between N43D and HR2. The six-helix bundle structure indicated that E148A should increase hydrophobic interactions between the coiled-coil and peptide. Importantly, the modifications S138A and E148A in the same peptide retained potency against ENF-escape mutants. The double mutant's increase in potency was greater than the increases from the sum of S138A and E148A individually, showing that these two altered residues synergistically contributed to peptide binding. Isothermal titration calorimetry established that hydrophobic substitutions at positions S138 and E148 improved potency of inhibitory peptides against escape mutants by increasing enthalpic release of energy upon peptide binding.

SUBMITTER: Leung MY

PROVIDER: S-EPMC3077694 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Increasing hydrophobicity of residues in an anti-HIV-1 Env peptide synergistically improves potency.

Leung Michael Y K MY Cohen Fredric S FS

Biophysical journal 20110401 8

T-20/Fuzeon/Enfuvirtide (ENF), a peptide inhibitor of HIV-1 infection, targets the grooves created by heptad repeat 2 (HR2) of Env's coiled-coil, but mutants resistant to ENF emerge. In this study, ENF-resistant mutants--V38A, N43D, N43D/S138A, Q40H/L45M--were combined with modified inhibitory peptides to identify what we believe to be novel ways to improve peptide efficacy. V38A did not substantially reduce infectivity, but was relatively resistant to inhibitory peptides. N43D was more resistan ...[more]

PMID: 21504732

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Project description: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.

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Dataset Information

Increasing hydrophobicity of residues in an anti-HIV-1 Env peptide synergistically improves potency.

Publications

Increasing hydrophobicity of residues in an anti-HIV-1 Env peptide synergistically improves potency.

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