Testing the Substrate-Envelope Hypothesis with Designed Pairs of Compounds

Acquired resistance to therapeutic agents is a significant barrier to the development of clinically effective treatments for diseases in which evolution occurs on clinical time scales, frequently arising from target mutations. We previously reported a general strategy to design effective inhibitors for rapidly mutating enzyme targets, which we demonstrated for HIV-1 protease inhibition [Altman et al. J. Am. Chem. Soc. 2008, 130, 6099–6113]. Specifically, we developed a computational inverse design procedure with the added constraint that designed inhibitors bind entirely inside the substrate envelope, a consensus volume occupied by natural substrates. The rationale for the substrate-envelope constraint is that it prevents designed inhibitors from making interactions beyond those required by substrates and thus limits the availability of mutations tolerated by substrates but not by designed inhibitors. The strategy resulted in subnanomolar inhibitors that bind robustly across a clinically derived panel of drug-resistant variants. To further test the substrate-envelope hypothesis, here we have designed, synthesized, and assayed derivatives of our original compounds that are larger and extend outside the substrate envelope. Our designs resulted in pairs of compounds that are very similar to one another, but one respects and one violates the substrate envelope. The envelope-respecting inhibitor demonstrates robust binding across a panel of drug-resistant protease variants, whereas the envelope-violating one binds tightly to wild type but loses affinity to at least one variant. This study provides strong support for the substrate-envelope hypothesis as a design strategy for inhibitors that reduce susceptibility to resistance mutations.National Science Foundation (U.S.) (NSF grant 0821391)National Institute of General Medical Sciences (U.S.) (NIH (GM066524))National Institute of General Medical Sciences (U.S.) (GM065418)National Institute of General Medical Sciences (U.S.) (the NIH (GM082209)National Institute of General Medical Sciences (U.S.) (AI41404)National Institute of General Medical Sciences (U.S.) (AI43198

New approaches to HIV protease inhibitor drug design II: testing the substrate envelope hypothesis to avoid drug resistance and discover robust inhibitors

PURPOSE OF REVIEW: Drug resistance results when the balance between the binding of inhibitors and the turnover of substrates is perturbed in favor of the substrates. Resistance is quite widespread to the HIV-1 protease inhibitors permitting the protease to process its 10 different substrates. This processing of the substrates permits the virus HIV-1 to mature and become infectious. The design of HIV-1 protease inhibitors that closely fit within the substrate-binding region is proposed to be a strategy to avoid drug resistance. RECENT FINDINGS: Cocrystal structures of HIV-1 protease with its substrates define an overlapping substrate-binding region or substrate envelope. Novel HIV-1 protease inhibitors that were designed to fit within this substrate envelope were found to retain high binding affinity and have a flat binding profile against a panel of drug-resistant HIV-1 proteases. SUMMARY: The avoidance of drug resistance needs to be considered in the initial design of inhibitors to quickly evolving targets such as HIV-1 protease. Using a detailed knowledge of substrate binding appears to be a promising strategy for achieving this goal to obtain robust HIV-1 protease inhibitors

Hydrophobic core flexibility modulates enzyme activity in HIV-1 protease

Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity

Crystal Structure of Lysine Sulfonamide Inhibitor Reveals the Displacement of the Conserved Flap Water Molecule in Human Immunodeficiency Virus Type 1 Protease▿

Human immunodeficiency virus type 1 (HIV-1) protease has been continuously evolving and developing resistance to all of the protease inhibitors. This requires the development of new inhibitors that bind to the protease in a novel fashion. Most of the inhibitors that are on the market are peptidomimetics, where a conserved water molecule mediates hydrogen bonding interactions between the inhibitors and the flaps of the protease. Recently a new class of inhibitors, lysine sulfonamides, was developed to combat the resistant variants of HIV protease. Here we report the crystal structure of a lysine sulfonamide. This inhibitor binds to the active site of HIV-1 protease in a novel manner, displacing the conserved water and making extensive hydrogen bonds with every region of the active site

Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity

Discovery of HIV-1 protease inhibitors with picomolar affinities incorporating N-aryl-oxazolidinone-5-carboxamides as novel P2 ligands

Here, we describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors incorporating N-phenyloxazolidinone-5-carboxamides into the (hydroxyethylamino)sulfonamide scaffold as P2 ligands. Series of inhibitors with variations at the P2 phenyloxazolidinone and the P2\u27 phenylsulfonamide moieties were synthesized. Compounds with the (S)-enantiomer of substituted phenyloxazolidinones at P2 show highly potent inhibitory activities against HIV-1 protease. The inhibitors possessing 3-acetyl, 4-acetyl, and 3-trifluoromethyl groups at the phenyl ring of the oxazolidinone fragment are the most potent in each series, with K(i) values in the low picomolar (pM) range. The electron-donating groups 4-methoxy and 1,3-dioxolane are preferred at P2\u27 phenyl ring, as compounds with other substitutions show lower binding affinities. Attempts to replace the isobutyl group at P1\u27 with small cyclic moieties caused significant loss of affinities in the resulting compounds. Crystal structure analysis of the two most potent inhibitors in complex with the HIV-1 protease provided valuable information on the interactions between the inhibitor and the protease enzyme. In both inhibitor - enzyme complexes, the carbonyl group of the oxazolidinone ring makes hydrogenbond interactions with relatively conserved Asp29 residue of the protease. Potent inhibitors from each series incorporating various phenyloxazolidinone based P2 ligands were selected and their activities against a panel of multidrug-resistant (MDR) protease variants were determined. Interestingly, the most potent protease inhibitor starts out with extremely tight affinity for the wild-type enzyme (K(i) = 0.8 pM), and even against the MDR variants it retains picomolar to low nanomolar K(i), which is highly comparable with the best FDA-approved protease inhibitors

Design, synthesis, and biological and structural evaluations of novel HIV-1 protease inhibitors to combat drug resistance

A series of new HIV-1 protease inhibitors (PIs) were designed using a general strategy that combines computational structure-based design with substrate-envelope constraints. The PIs incorporate various alcohol-derived P2 carbamates with acyclic and cyclic heteroatomic functionalities into the (R)-hydroxyethylamine isostere. Most of the new PIs show potent binding affinities against wild-type HIV-1 protease and three multidrug resistant (MDR) variants. In particular, inhibitors containing the 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone, and oxazolidinone moieties at P2 are the most potent with K(i) values in the picomolar range. Several new PIs exhibit nanomolar antiviral potencies against patient-derived wild-type viruses from HIV-1 clades A, B, and C and two MDR variants. Crystal structure analyses of four potent inhibitors revealed that carbonyl groups of the new P2 moieties promote extensive hydrogen bond interactions with the invariant Asp29 residue of the protease. These structure-activity relationship findings can be utilized to design new PIs with enhanced enzyme inhibitory and antiviral potencies

Structure-based design, synthesis, and structure-activity relationship studies of HIV-1 protease inhibitors incorporating phenyloxazolidinones

A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different phenyloxazolidinone P2 ligands were designed and synthesized. Variation of phenyl substitutions at the P2 and P2\u27 moieties significantly affected the binding affinity and antiviral potency of the inhibitors. In general, compounds with 2- and 4-substituted phenyloxazolidinones at P2 exhibited lower binding affinities than 3-substituted analogues. Crystal structure analyses of ligand-enzyme complexes revealed different binding modes for 2- and 3-substituted P2 moieties in the protease S2 binding pocket, which may explain their different binding affinities. Several compounds with 3-substituted P2 moieties demonstrated picomolar binding affinity and low nanomolar antiviral potency against patient-derived viruses from HIV-1 clades A, B, and C, and most retained potency against drug-resistant viruses. Further optimization of these compounds using structure-based design may lead to the development of novel protease inhibitors with improved activity against drug-resistant strains of HIV-1