Background:  Drug resistance is a subtle change in the balance of recognition events between the relative affinity of the enzyme to bind inhibitors and its ability to bind and cleave substrates. We previously made 2 observations:  HIV protease recognizes its diverse substrate sequences through a conserved shape, which we defined as the substrate envelope; and0 most active-site drug-resistant mutations within HIV protease occur where the inhibitors protrude beyond the “substrate envelope” and contact the protease. Those protease residues are prime positions for drug resistance to occur, as they are more important for inhibitor binding than for substrate binding. These observations led us hypothesize that HIV-1 protease inhibitors that fit within the substrate envelope would be less susceptible to drug resistant mutations.

Methods:  Computational inhibitor design was utilized to predict inhibitors that would fit within the confines of the substrate envelope. Synthetic chemistry was utilized to actually make the inhibitors. The resulting inhibitors were then tested for inhibitory activity in enzymatic and calorimetric studies against a panel of wild-type and resistant HIV-1 protease variants. Their crystal structures were also determined in complex with HIV-1 protease. Finally, the best inhibitors were tested by Monogram Biosciences, Inc using their PhenoSense^TM assay.

Results:  We have successfully designed, synthesized, assayed, and analyzed the crystal structures of novel HIV-1 protease inhibitors the best of which bind with single-digit picomolar affinity. The most successful inhibitors in terms of resistant profile have a flat binding profile to a variety of representative variants that include among them resistant mutations:  I84V, V82A, G48V, D30N, and I50V. The crystal structure of these inhibitors shows that they do indeed fit within the substrate envelope. The PhenoSense^TM assay demonstrated that 10 of these inhibitors were consistently more potent than Darunavir against a diverse panel of wild-type and resistant viruses.

Conclusions:  This design effort of highly potent HIV-1 PI validates the substrate envelope hypothesis that it is possible to avoid susceptibility to drug resistant variants by designing inhibitors to fit within the substrate envelope. These results outline a new paradigm for developing new robust inhibitors that are less susceptible to resistant variants against quickly evolving therapeutic targets.