Potent HIV-1 PI Resilient to Resistance Developed Using the Substrate Envelope Hypothesis
M Nalam1, A Ali1, G Reddy1, M Altman2, S Chellappan3, S Anjum1, T Rana4, M Gilson3, B Tidor2, and Celia Schiffer*1
1Univ of Massachusetts Med Sch, Worcester, US; 2Massachusetts Inst of Tech, Cambridge, US; 3Ctr for Advanced Res in Biotech, Univ of Maryland, Rockville, US; and 4Burham Inst for Med Res, La Jolla, CA, US
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 PhenoSenseTM 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
PhenoSenseTM 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.