We have characterized the structural and molecular interactions of CC-chemokine receptor 5 (CCR5) with three CCR5 inhibitors active against R5 human immunodeficiency virus type 1 (HIV-1) including the potent in vitro and in vivo CCR5 inhibitor aplaviroc (AVC). The data obtained with saturation binding assays and structural analyses delineated the key interactions responsible for the binding of CCR5 inhibitors with CCR5 and illustrated that their binding site is located in a predominantly lipophilic pocket in the interface of extracellular loops and within the upper transmembrane (TM) domain of CCR5. Mutations in the CCR5 binding sites of AVC decreased gp120 binding to CCR5 and the susceptibility to HIV-1 infection, although mutations in TM4 and TM5 that also decreased gp120 binding and HIV-1 infectivity had less effects on the binding of CC-chemokines, suggesting that CCR5 inhibition targeting appropriate regions might render the inhibition highly HIV-1-specific while preserving the CC chemokine-CCR5 interactions. The present data delineating residue by residue interactions of CCR5 with CCR5 inhibitors should not only help design more potent and more HIV-1-specific CCR5 inhibitors, but also give new insights into the dynamics of CC-chemokine-CCR5 interactions and the mechanisms of CCR5 involvement in the process of cellular entry of HIV-1.Highly active antiretroviral therapy has brought about a major impact on the acquired immunodeficiency syndrome (AIDS) epidemics in industrially advanced nations (1, 2), however, eradication of HIV-1 2 appears to be currently impossible mainly because of the viral reservoirs remaining in blood and infected tissues (3). Successful antiviral drugs, in theory, exert their virus-specific effects by interacting with viral components such as viral genes or their transcripts without disturbing cellular metabolisms or functions (2). However, at present, no antiretroviral drugs or agents have been demonstrated to be completely specific for HIV-1 and devoid of toxicity or side effects in the therapy of AIDS (4). Limitations of antiviral therapy of AIDS are exacerbated by complicated regimens, emergence of drug-resistant HIV-1 variants (1), and a number of inherent adverse effects (5).Thus, identification of new antiretroviral drugs that have unique mechanisms of action and produce no or least minimal side effects remains an important therapeutic objective (2, 4). CCR5 is a member of the G protein-coupled, seven-transmembrane segment receptors, which comprise the largest superfamily of proteins in the body (6). In 1996, it was revealed that CCR5 serves as one of the two essential coreceptors for HIV-1 entry to human CD4 ϩ cells, thereby serving as an attractive target for possible intervention of HIV-1 infection (7-10). Aplaviroc (AVC; AK602/ONO4128/873140; Fig. 1), a novel spirodiketopiperazine derivative, represents a CCR5 inhibitor that specifically binds to human CCR5 with a high affinity, greatly blocks HIV-1-gp120/ CCR5 binding, and exerts potent activity against a wide spectrum of ...

We attempted to select HIV-1 variants resistant to darunavir (DRV), which potently inhibits the enzymatic activity and dimerization of protease and has a high genetic barrier to HIV-1 development of resistance to DRV. We conducted selection using a mixture of 8 highly multi-protease inhibitor (PI)-resistant, DRV-susceptible clinical HIV-1 variants (HIV-1MIX) containing 9 to 14 PI resistance-associated amino acid substitutions in protease. HIV-1MIX became highly resistant to DRV, with a 50% effective concentration (EC50) ∼333-fold greater than that against HIV-1NL4-3. HIV-1MIX at passage 51 (HIV-1MIXP51 ) replicated well in the presence of 5 μM DRV and contained 14 mutations. HIV-1MIXP51 was highly resistant to amprenavir, indinavir, nelfinavir, ritonavir, lopinavir, and atazanavir and moderately resistant to saquinavir and tipranavir. HIV-1MIXP51 had a resemblance with HIV-1C of the HIV-1MIX population, and selection using HIV-1C was also performed; however, its DRV resistance acquisition was substantially delayed. The H219Q and I223V substitutions in Gag, lacking in HIV-1CP51 , likely contributed to conferring a replication advantage on HIV-1MIXP51 by reducing intravirion cyclophilin A content. HIV-1MIXP51 apparently acquired the substitutions from another HIV-1 strain(s) of HIV-1MIX through possible homologous recombination. The present data suggest that the use of multiple drug-resistant HIV-1 isolates is of utility in selecting drug-resistant variants and that DRV would not easily permit HIV-1 to develop significant resistance; however, HIV-1 can develop high levels of DRV resistance when a variety of PI-resistant HIV-1 strains are generated, as seen in patients experiencing sequential PI failure, and ensuing homologous recombination takes place. HIV-1MIXP51 should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents.

The structure-based design, synthesis and biological evaluation of a series of nonpeptidic macrocyclic HIV protease inhibitors are described. The inhibitors are designed to effectively fill in the hydrophobic pocket in the S1′ S2′ subsites and retain all major hydrogen bonding with the protein backbone similar to darunavir (1) or inhibitor 2. The ring size, the effect of methyl substitution and unsaturation within the macrocyclic ring structure were assessed. In general, cyclic inhibitors were significantly more potent than their acyclic homologues, saturated rings were less active than their unsaturated analogs and a preference for 10 and 13-membered macrocylic rings was revealed. The addition of methyl substituents resulted in the reduction of potency. Both inhibitors 14b and 14c exhibited marked enzyme inhibitory and antiviral activity and they exerted potent activity against multi-drug resistant HIV-1 variants. Protein-ligand X-ray structures of inhibitors 2 and 14c provided critical molecular insights into the ligand-binding site interactions.

dWe 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 [EC 50 ], 0.0005 to 0.0007 M) with minimal cytotoxicity (50% cytotoxic concentration [CC 50 ], 44.6 M). GRL-0519 blocked the infectivity and replication of HIV-1 NL4-3 variants selected by up to a 5 M concentration of ritonavir, lopinavir, or atazanavir (EC 50 , 0.0028 to 0.0033 M). GRL-0519 was also potent against multi-PIresistant 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-2 ROD . 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-PIresistant 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.

Reliable and robust prediction of binding affinity for drug molecules continues to be a daunting challenge. We have simulated the binding interactions and free energy of binding of nine protease inhibitors (PIs) with wild-type and various mutant proteases by performing GBSA simulations, in which each PI’s partial charge was determined by quantum mechanics (QM) and the partial charge accounts for the polarization induced by the protease environment. We employed a hybrid solvation model that retains selected explicit water molecules in the protein with surface generalized Born (SGB) implicit solvent. We examined the correlation of the free energy with antiviral potency of PIs with regard to amino acid substitutions in protease. The GBSA free energy thus simulated showed strong correlations (r > 0.75) with antiviral IC50 values of PIs when amino acid substitutions were present in the protease active site. We also simulated the binding free energy of PIs with P2-bis-tetrahydrofuranylurethane (bis-THF) or related cores, utilizing a bis-THF-containing protease crystal structure as a template. The free energy showed a strong correlation (r = 0.93) with experimentally determined anti-HIV-1 potency. The present data suggest that the presence of selected explicit water in protein, and protein polarization-induced quantum charges for the inhibitor, compared to lack of explicit water and a static force field-based charge model, can serve as an improved lead optimization tool, and warrants further exploration.