Substrate envelope-designed potent HIV-1 protease inhibitors to avoid drug resistance

Abstract

The rapid evolution of HIV under selective drug pressure has led to multidrug resistant (MDR) strains that evade standard therapies. We designed highly potent HIV-1 protease inhibitors (PIs) using the substrate envelope model, which confines inhibitors within the consensus volume of natural substrates, providing inhibitors less susceptible to resistance because a mutation affecting such inhibitors will simultaneously affect viral substrate processing. The designed PIs share a common chemical scaffold but utilize various moieties that optimally fill the substrate envelope, as confirmed by crystal structures. The designed PIs retain robust binding to MDR protease variants and display exceptional antiviral potencies against different clades of HIV as well as a panel of 12 drug-resistant viral strains. The substrate envelope model proves to be a powerful strategy to develop potent and robust inhibitors that avoid drug resistance.

Figure 1

Structure of the designed protease inhibitors. (a) The chemical scaffold, and P1′ and P2′ groups of the 10 inhibitors. (b) Superimposed x-ray crystal structures of all 10 inhibitors in complex with WT HIV-1 protease. See Table S1 for crystallographic statistics. (c) Fit of inhibitors within the substrate envelope. The substrate envelope is in blue space filling representation, and the superimposed inhibitors are displayed as sticks. Protrusions beyond the substrate envelope are in red.

Figure 2

Synthesis of protease inhibitors. Reagents and Conditions: (a) R¹NH₂, EtOH, 80 °C, 3 h; (b) aq. Na₂CO₃, CH₂Cl₂, 0 °C to rt, overnight; (c) CH₂Cl₂, Et₃N, 0 °C to rt, overnight; (d) NaBH₄, MeOH, 0 °C, 15 min; (e) TFA, CH₂Cl₂, 1 h; (f) Py, p-NO₂-PhOCOCl, 0 °C to rt, 24 h; (g) DIEA, CH₃CN, 0 °C to rt, 24 h; (h) SnCl₂.2H₂O, EtOAc, 70 °C, 3 h. See Supplemental experimental procedures for more details.

Figure 3

Binding affinities of FDA-approved drugs and the 10 designed protease inhibitors to WT and drug resistant variants of HIV-1 protease; the Ki values for the FDA-approved drugs are from Nalam et al.(Nalam, et al., 2010) Inhibitory activity was determined by a FRET-based enzymatic assay; Ki values are the average of at least three independent measurements. See also Table S2.

Figure 4

Comparison of van der Waals contacts with the protease for P1′ groups in PIs. (a) Superposition of DRV, PI 10a and PI 11a structures bound to WT HIV-1 protease. (b) Isobutyl P1′ group in DRV (c) Isopentyl P1′ group in PI 10a (d) Isohexyl P1′ group in PI 11a. The longer P1′ groups make enhanced van der Waals contacts with the protease.

Figure 5

Resistance profile of DRV and the 10 PIs designed. Antiviral potencies (EC₅₀ values) were obtained for wild-type HIV from clades A, B and C, and 12 different patient-derived drug-resistant variants of the virus. The PhenoSense™ HIV assays were performed by Monogram Biosciences. See also Table S3 and Figure S1.