Hexakisphosphate (IP6) potentiates HIV-1 maturation inhibitor binding at the CA-SP1 junction: insights from molecular...

Bevirimat (BVM) and related compounds act as HIV maturation inhibitors (MIs), impeding protease-mediated cleavage of the Gag polyprotein by binding to and stabilizing the capsid (CA) and spacer peptide 1 (SP1) domains (CA-SP1). The CA-SP1 six-helix bundle (6HB) within Gag hexamers is a critical interaction site for MIs and the anchoring of IP6, the cellular polyanion inositol hexakisphosphate. IP6 binds to the immature Gag lattice and neutralizes lysine-rich rings, stabilizing the 6HB at the CA-SP1 junction; it also promotes the assembly of the mature capsid lattice during later stages. However, how IP6 influences the activity of MIs on CA-SP1 remains incompletely understood. Our previous structural modification of beesioside I yielded a potent anti-HIV derivative, (20S,24S)-15β,16β-diacetoxy-18,24;20,24-diepoxy-9,19-cyclolanostane-3β,25-diol 3-O-3',3'-dimethylsuccinate (DSC), which demonstrated efficacy analogous to BVM but with reduced toxicity. Computational analysis suggested that DSC works as a potential MI to inhibit HIV by targeting CA_CTD-SP1. This study applied multiple ligands simultaneous docking (MLSD), multiple ligands molecular dynamics simulations, and a suite of biophysical techniques including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, as well as scanning electron microscopy (SEM), to explore the binding interaction of the HIV MIs DSC/BVM to CA_CTD-SP1 in the context of IP6. The results indicated that IP6 markedly enhances inhibitor binding and capsid stability: in the presence of IP6, both BVM and DSC form much more stable complexes with the CA_CTD-SP1 hexamer. Notably, DSC-bound CA_CTD-SP1 hexamers were even more rigid and stable than BVM-bound ones, indicating that DSC further reinforces the 6HB and more effectively prevents its unfolding. These results reveal a previously unrecognized synergy between IP6 and a CA_CTD-SP1-targeting inhibitor, highlighting a novel cooperative mechanism to block HIV-1 maturation and providing insights to guide the design of improved MIs.