(320a) Roles of Variable Linker Length in Dual Acting Virucidal Entry Inhibitors on HIV-1 Potency Via on-the-Fly Free Energy Molecular Simulations

Type-1 Human Immunodeficiency Virus (HIV-1), the viral agent responsible for AIDS, leads to the death of over two million people each year and represents a global health epidemic. Despite the success of highly active antiretroviral therapies (HAART) in extending lifespans of infected individuals, strategies still are needed for prevention of transmission to uninfected individuals as well as a cure for those already infected. We recently developed a novel microbicidal recombinant fusion protein comprising the lectin cyanovirin-N (CVN) to the amphipathic peptide membrane-proximal external region (MPER) of gp41, attached by a flexible polypeptide linker of controllable length (Contarino et al. 2013 Antimicrob. Agents Ch. 57:4743-4750). This class of molecules, termed “dual-acting virucidal entry inhibitors” (DAVEI’s), induce poration of the virus evidently by prematurely triggering the viral fusion machinery of the envelope spike complexes to which they bind. Recent work on optimizing DAVEI’s has focused on understanding where on virus the DAVEI MPER binds, and we recently showed that the binding site is likely to involve endogenous MPER segments (Parajuli et al. 2016; Biochemistry 55:6100-6114). In this work, we sought to use molecular modeling and simulation to determine the distribution of end-to-end distances sampled by DAVEI linkers to determine a functional length of DAVEI. Our approach consists of first building a model of DAVEI using the CVN PDB entry (Bewley et al. 1998; Nat. Struct. Mol. Biol. 5:571-578) and growing a linker and MPER segment onto this structure. On-the-fly parameterization (OTFP) calculations were then performed on this structure to generate a free energy profile on the end-to-end distance of the linker. It has been observed experimentally that DAVEI with a linker length of 0 (i.e., only the HIS₆ tag) is ineffective against the virus, showing little lytic activity, while DAVEI with a repeat linker length of 2 (i.e., 2 GLY₄SER units plus the HIS₆ tag) does show lytic activity with an EC₅₀ of 182.6 ± 28.5 nM (Parajuli et al. 2016; Biochemistry 55:6100-6114). Preliminary results show that a linker length of zero repeat units most often samples end-to-end distances between 12 and 18 Ã…. DAVEI with two repeat units in the linker has a global minimum at ≈ 20 Ã… and a local minimum at ≈ 35 Ã…. It is possible that 35 Ã… represents an active conformation of DAVEI. In a trimeric crystal structure of gp120 and gp41 in complex with the PGT122 antibody (PDB 5I8H), distances between sugar groups and the beginning of the MPER sequence were found to be 47-51 Ã…, 80-83 Ã…, ≈ 100 Ã… and ≈ 110 Ã… (Kong et al. 2016; Science 352: 828-833). Given that the distance from the N-terminus of the linker to the glycan binding site on the CVN component of DAVEI is 23 Ã…, it is possible then that DAVEI could achieve dual engagement of the gp120 glycans and gp41 MPER region given these distances when the linker is extended to 35 Å. Continuing work will involve refining these free-energy profiles and computing them for other linker lengths and types with the aim of reducing the overall molecular weight of DAVEI.