g-52 and amprenavir

Extreme drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with the clinical inhibitor amprenavir (1) and two potent antiviral investigational inhibitors GRL-02031 (2) and GRL-0519 (3). Clinical inhibitors are >1000-fold less active on PR20 than on wild-type enzyme, which is consistent with dissociation constants (KL) from isothermal titration calorimetry of 40 nM for 3, 178 nM for amprenavir, and 960 nM for 2. High resolution crystal structures of PR20-inhibitor complexes revealed altered interactions compared with the corresponding wild-type PR complexes in agreement with relative inhibition. Amprenavir lacks interactions due to PR20 mutations in the S2/S2' subsites relative to PR. Inhibitors 2 and 3 lose interactions with Arg8' in PR20 relative to the wild-type enzyme because Arg8' shifts to interact with mutated L10F side chain. Overall, inhibitor 3 compares favorably with darunavir in affinity for PR20 and shows promise for further development.

Topics: Binding Sites; Calorimetry, Differential Scanning; Carbamates; Crystallization; Darunavir; Drug Resistance, Multiple; Drug Resistance, Viral; Escherichia coli; Furans; Genes, Synthetic; HIV Protease; HIV Protease Inhibitors; HIV-1; Humans; Models, Molecular; Mutation; Pyrrolidinones; Structure-Activity Relationship; Sulfonamides

We report that GRL-0519, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing tris-tetrahydrofuranylurethane (tris-THF) and a sulfonamide isostere, is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0005 to 0.0007 μM) with minimal cytotoxicity (50% cytotoxic concentration [CC50], 44.6 μM). GRL-0519 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to a 5 μM concentration of ritonavir, lopinavir, or atazanavir (EC50, 0.0028 to 0.0033 μM). GRL-0519 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, highly darunavir (DRV)-resistant variants, and HIV-2ROD. The development of resistance against GRL-0519 was substantially delayed compared to other PIs, including amprenavir (APV) and DRV. The effects of nonspecific binding of human serum proteins on GRL-0519's antiviral activity were insignificant. Our analysis of the crystal structures of GRL-0519 (3OK9) and DRV (2IEN) with protease suggested that the tris-THF moiety, compared to the bis-THF moiety present in DRV, has greater water-mediated polar interactions with key active-site residues of protease and that the tris-THF moiety and paramethoxy group effectively fill the S2 and S2' binding pockets, respectively, of the protease. The present data demonstrate that GRL-0519 has highly favorable features as a potential therapeutic agent for treating patients infected with wild-type and/or multi-PI-resistant variants and that the tris-THF moiety is critical for strong binding of GRL-0519 to the HIV protease substrate binding site and appears to be responsible for its favorable antiretroviral characteristics.

Topics: Amino Acid Sequence; Atazanavir Sulfate; Binding Sites; Carbamates; Cell Line, Tumor; Darunavir; Drug Resistance, Viral; Furans; HIV Protease; HIV Protease Inhibitors; HIV-1; Humans; Inhibitory Concentration 50; Lopinavir; Molecular Docking Simulation; Molecular Sequence Data; Oligopeptides; Protein Binding; Pyridines; Ritonavir; Structure-Activity Relationship; Sulfonamides; T-Lymphocytes; Virus Replication

Topics: Carbamates; Crystallography, X-Ray; Darunavir; Dimerization; Drug Design; Drug Resistance, Multiple, Viral; Furans; Heterocyclic Compounds, 3-Ring; HIV Infections; HIV Protease; HIV Protease Inhibitors; HIV-1; Humans; Ligands; Proteins; Structure-Activity Relationship; Sulfonamides