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

Abstract

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.

C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir

“…Structure solutions were obtained using the molecular replacement (MR) method as described previously (36,37). Briefly, MR was performed using MOLREP (38) through the CCP4 (39,40) interface with WT PR taken from PDB ID 4HLA as a search model.…”

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

“…The compound GRL-008 is a derivative of DRV and contains the benzene carboxamide substitution instead of aniline at P2’ [65]. The crystal structure in complex with protease exhibits a new water-mediated connection between the NH of the carboxamide and the backbone carbonyl oxygen of Gly48 in the flexible flap [65].…”

“…The crystal structure in complex with protease exhibits a new water-mediated connection between the NH of the carboxamide and the backbone carbonyl oxygen of Gly48 in the flexible flap [65]. The structure also forms weaker hydrogen bonds to the backbone of conserved residues Asp29, Asp30/30’.…”

A Modified P1 Moiety Enhances In Vitro Antiviral Activity against Various Multidrug-Resistant HIV-1 Variants and In Vitro Central Nervous System Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413

“…Compounds with apparent permeability coefficients of Ͼ20 ϫ 10 Ϫ6 cm/s are thought to have reasonably efficient penetration across the BBB, those with values of 10 ϫ 10 Ϫ6 to 20 ϫ 10 Ϫ6 cm/s are thought to have a moderate degree of penetration, and those with values of Ͻ10 ϫ 10 Ϫ6 cm/s are thought to not effectively penetrate the BBB (34).…”

“…Expression, purification, and refolding of the wild-type PR (PR WT ) protein were performed as described previously (34). Briefly, inclusion bodies isolated from Escherichia coli containing PR WT were extracted with 3 M guanidine HCl (GndHCl) and centrifuged, and the supernatant was loaded on a Sephadex-200 column that was preequilibrated with 4 M GndHCl.…”