Background:  Dimerization of HIV-1 protease subunits is an essential process for its proteolytic activity, which plays a critical role in HIV-1 replication. We discovered a group of non-peptidyl small molecule protease dimerization inhibitors (PDI).

Methods:  An intermolecular fluorescence resonance energy transfer (FRET) -based HIV-1-expression assay that employs cyan and yellow fluorescent protein-tagged HIV-1 protease monomer subunits was generated to evaluate the disruption of HIV-1 protease dimerization. Using this FRET-based HIV-1-expression assay, we characterized effects of amino acid residue substitutions on protease dimerization and determined interactions of mutant protease and various PDI.

Results:  Using the FRET-based-HIV-1-expression assay, we identified a group of PDI, including darunavir (DRV) and tipranavir (TPV), while other conventional protease inhibitors (PI) failed to block dimerization. Such PDI dose-responsively blocked protease dimerization at concentrations of as low as 0.01 µM and blocked HIV-1 replication with IC₅₀ values of 0.0002 to 0.48 µM. When a single mutation was introduced into the N- and C- termini of protease, I3A, L5A, T96A, L97A, and F99A disrupted protease dimerization. R8Q/A, T26A, D29N/A, and R87K substitutions outside the N- and C- termini also disrupted protease dimerization. However, D25A, A28S, and D30N in the active site did not disrupt dimerization. A single substitution—such as P1A, Q2A, T4A, D25N, D30N, and N98A—allowed protease to undergo dimerization, which DRV effectively inhibited at 1 µM, suggesting that these amino acids are not significantly involved in the binding of DRV to the protease monomer subunit. A single mutation—such as V32I, L33F, I54M, and I84V, which are known to be associated with HIV-1’s DRV resistance—also allowed protease to undergo dimerization, and DRV effectively blocked dimerization. Protease with an A28S mutation or 4 mutations (V32I, L33F, I54M, I84V) underwent dimerization, which DRV and TPV failed to block, strongly suggesting that such mutations altered the conformation of the monomer subunit binding site of DRV and TPV.

Conclusions:  The data suggest that dual inhibition mechanisms of PDI against HIV-1 protease are substantially different from conventional PI. The data should not only help design and examine agents that potentially inhibit HIV-1 protease dimerization, but also should give new insights into the process and dynamics of HIV-1 protease dimerization per se.