A conserved hydrogen-bonding network of P2 bis-tetrahydrofuran-containing HIV-1 protease inhibitors (PIs) with a protease active-site amino acid backbone aids in their activity against PI-resistant HIV.

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 µM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 µM) and tipranavir (TPV; EC50, 0.364 µM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRV(R)P20), with a 2.6-fold increase in its EC50 (0.097 µM) compared to its corresponding EC50 (0.038 µM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30' of HIVA02 protease (PRA02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30' due to an absence of polar groups. The P2' thiazolyl moiety of RTV showed two conformations in the crystal structure of the PRA02-RTV complex, one of which showed loss of contacts in the S2' binding pocket of PRA02, supporting RTV's compromised antiviral activity (EC50, >1 µM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30' most likely contributes to its persistently greater antiviral activity against HIVWT, HIVA02, and HIVDRV(R)P20.

P2' benzene carboxylic acid moiety is associated with decrease in cellular uptake: evaluation of novel nonpeptidic HIV-1 protease inhibitors containing P2 bis-tetrahydrofuran moiety.

GRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2' moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PR(WT)). Both GRL007 (Ki of 12.7 pM with PR(WT)) and GRL008 (Ki of 8.9 pM) inhibited PR(WT) with high potency in vitro. X-ray crystallographic analysis of PR(WT) in complex with GRL007 or GRL008 showed that the bis-THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PR(WT). The P2' moiety of both compounds showed one direct contact with the backbone of Asp30' and a bridging polar contact with Gly48' through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (50% effective concentration [EC50] of >1 µM) while GRL008 was highly active (EC50 of 0.04 µM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme-inhibitory activity and protease binding profile, exhibited a lack of antiviral activity in cell-based assays, most likely due to its compromised cellular uptake associated with its P2' benzene carboxylic acid moiety. The anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2' moiety may improve its antiretroviral features.