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

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.

Fig 1

Structures of GRL007, GRL008, and DRV. Structures of GRL007, GRL008, and DRV are shown with the P2 (bis-THF) and P2′ moieties labeled. GRL007, GRL008, and DRV contain benzene carboxylic acid, benzene carboxamide, and aniline as P2′ moieties, respectively. All three PIs have a phenyl group at the P1 and an isopropyl group at the P1′ positions with a transition state-mimetic hydroxyl group in between.

Fig 2

Polar contacts between PR^WT and inhibitors. Polar contacts of GRL007, GRL008, and DRV with PR^WT are shown in panels a, b, and c, respectively. The carbon atoms of PIs and PR^WT are shown as white and green sticks, respectively. Oxygen and nitrogen atoms are shown as red and blue sticks, respectively. Polar contacts in each panel are shown as yellow dashed lines. Conserved crystallographic waters are shown as red spheres. The P2 bis-THF moiety shows three conserved polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30. The P2′ moiety of each inhibitor shows at least one conserved polar contact, each with the backbone of Asp30′. In addition, GRL007 shows two direct polar contacts and DRV shows one direct polar contact with the δ-oxygen atom from the side chain of Asp30′. While two stable water molecules are seen bridging between the aniline moiety of DRV and Gly48′ of PR^WT (panel c), only one stable water is seen in the case of GRL007 (panel a) and GRL008 (panel b). This loss of a water molecule is compensated by the benzene carboxylic acid and benzene carboxamide groups. All polar contacts were calculated as the distances between the heavy atoms.

Fig 3

Distribution of hydrophobic contacts for DRV, GRL007, and GRL008. The amino acid residues from PR^WT that are involved in hydrophobic interactions with DRV, GRL007, or GRL008 are shown here in green, red, and blue, respectively. All three PIs show a conserved trend of contacts in the S1 binding pocket but variable contacts in the S1′ binding pocket due to altered binding orientations of the P1′ isopropyl group. The bis-THF moiety of each PI shows contacts with at least one residue in the S2 binding pocket. GRL007 and GRL008 show more contacts in the S1′ and S2′ pockets than DRV. Residues that show conserved contacts with two or all three PIs are boxed in black.

Fig 4

RP-HPLC/MS chromatograms of PIs. Overlays of chromatograms (GRL007, GRL008, and DRV) obtained from RP-HPLC/ESI-MS analysis of MT-2 and MT-4 cell extracts are shown, as indicated. Peak elution time (x axis) was plotted against the peak abundance on the y axis. Each chromatogram shows two prominent peaks (GRL008 and DRV) and a small peak (GRL007). An enlarged view of the small peak (GRL007) along with background is shown for clarity in insets. The smaller peak for GRL007 in either cell line indicates its poor cell penetration property compared to that of GRL008 or DRV.

Fig 5

Percent areas under the curve determined for GRL007 and GRL008. Values for the percent area under the curve (AUC) of GRL007 and GRL008 determined using MT-2 and MT-4 cell extracts are shown, as indicated. Among the three inhibitors, GRL007 shows the lowest AUC, suggesting the poor cellular uptake of the inhibitor. DRV shows better uptake than GRL008 in MT-2 cell extracts, while the AUC for GRL008 in MT-4 cell extracts is relatively greater than that of DRV.