Background:  Drug resistance at the molecular level 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. In studying this we previously made 2 observations:  HIV protease recognizes its diverse substrate sequences through a conserved shape, which we defined as the substrate envelope. This shape is discernable only from substrate-protease crystal structures. Furthermore, the 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 (PI) 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.

Results:  We have successfully designed, synthesized, assayed, and analyzed the crystal structures of novel HIV-1 protease inhibitors that bind the enzyme with nanomolar to picomolar affinity. Our initial libraries utilize a scaffold similar to aprenavir/darunavir (APV/DRV). The most successful inhibitor in terms of resistant profile has a flat (sub-nanomolar) binding profile to a variety of representative variants that include among them resistant mutations:  I84V, V82A, G48V, D30N, and I50V. The crystal structure of this inhibitor shows that it does indeed fit within the substrate envelope.

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