HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibit Subnanomolar Binding to Drug-Resistant Variants

Abstract

The acquisition of drug-resistance mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wildtype protease for the best binders, from K i of 30-50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6-13 fold affinity relative to wild type. Testing a subset of secondround compounds against the panel of resistant variants revealed three classes of inhibitorsrobust binders (maximum affinity loss of 14-16 fold), moderate binders (35-80 fold), and susceptible binders (greater than 100 fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance.

Evaluating the Substrate-Envelope Hypothesis: Structural Analysis of Novel HIV-1 Protease Inhibitors Designed To Be Robust against Drug Resistance

“…Thus, the Inv1 inhibitors which were designed using the substrate-bound protease structure (1KJG [micromolar affinity]) yielded compounds with micromolar affinities, and retrospective calculations were unable to distinguish high-from low-affinity inhibitors (2). In contrast, when the crystal structure of protease bound to the picomolar affinity inhibitor DRV (1T3R) was used for designing PIs, high-affinity inhibitors were obtained and the correlation between calculations and experiments was improved.…”

“…To evaluate the substrate-envelope hypothesis, new protease inhibitors were designed based on the (R)-(hydroxyethylamino) sulfonamide isostere (Table 1), the scaffold present in the clinical inhibitors APV and DRV (1,2,4,20). This scaffold fits predominately within the substrate envelope and has three sites to which various substituent groups can be easily attached to manipulate functional characteristics.…”

“…Based on retrospective analysis of the Inv1 library, a second library (the Inv2 library) was designed by reoptimizing the computational algorithm, using successful substituent R groups from the first round and the crystal structure of a DRV-protease complex (1T3R) instead of the crystal structure of a substrate-protease complex (1KJG). In the second round of Inv, 36 inhibitors were synthesized with binding affinities to wild-type protease ranging from 4.2 nM to 14 pM (3 orders of magnitude better than the binding affinities seen with the first-round inhibitors) (2). Compounds in the third library (SAR) were designed based on the same consensus scaffold, (R)-(hydroxyethylamino)sulfonamide isostere, without using any explicit substrate-envelope constraint.…”

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

“…Structure-based design strategies can utilize this model as an added constraint to develop inhibitors that fit within the substrate envelope. In fact, previous work in our laboratory provides proof-of-concept for the successful incorporation of the substrate envelope in the design of unique HIV protease inhibitors, which maintain high affinities against a panel of multidrug resistant variants of HIV-1 protease (42,(48)(49)(50)(51)(52)(53). As a general paradigm, design efforts incorporating the substrate envelope would facilitate a more rationale evaluation of drug candidates and lead to the development of more robust inhibitors that are less susceptible to resistance.…”

Intracellular Antiviral Activity of Low-Dose Ritonavir in Boosted Protease Inhibitor Regimens

“…The calculation of [I]/K i was performed with two different K i values for RTV: the highest K i reported in the literature (55 pM) (17), as the K i values for all PIs were evaluated in the same work, and a mean K i calculated from different works reported in the literature (37.5 pM) (18)(19)(20). Moreover, once the observed [I]/K i values for RTV and for the concomitant PI in each sample were obtained, the ratio between these two values was calculated (RTV 1/concomitant PI 1).…”