f 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 (K i of 12.7 pM with PR WT ) and GRL008 (K i 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 [EC 50 ] of >1 M) while GRL008 was highly active (EC 50 of 0.04 M) 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.

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 [EC 50

No abstract