ChemMedChem. 2013 Mar;8(3):426-32.

Identification of a small-molecule inhibitor of HIV-1 assembly that targets the phosphatidylinositol (4,5)-bisphosphate binding site of the HIV-1 matrix protein.

Zentner I, Sierra LJ, Fraser AK, Maciunas L, Mankowski MK, Vinnik A, Fedichev P, Ptak RG, Martín-García J, Cocklin S.

Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102, USA.



The development of drug resistance remains a critical problem for current HIV-1 antiviral therapies, creating a need for new inhibitors of HIV-1 replication. We previously reported on a novel anti-HIV-1 compound, N(2)-(phenoxyacetyl)-N-[4-(1-piperidinylcarbonyl)benzyl]glycinamide (14), that binds to the highly conserved phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) binding pocket of the HIV-1 matrix (MA) protein. In this study, we re-evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV-1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2-(4-{[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]methyl})-1-piperazinyl)-N-(4-methylphenyl)acetamide (7), 3-(2-ethoxyphenyl)-5-[[4-(4-nitrophenyl)piperazin-1-yl]methyl]-1,2,4-oxadiazole (17), and N-[4-ethoxy-3-(1-piperidinylsulfonyl)phenyl]-2-(imidazo[2,1-b][1,3]thiazol-6-yl)acetamide (18), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV-1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P(2) for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P(2) binding site of MA decreased the antiviral effect of compound 7. Additionally, compound 7 displays a broadly neutralizing anti-HIV activity, with IC(50) values of 7.5-15.6 μM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti-HIV-1 therapeutics.

PMID: 23361947



HIV-1 matrix (MA) is a highly conserved and multifunctional protein that is primarily responsible for recruiting HIV-1 Gag to the plasma membrane (PM) during the assembly of the virus. The recruitment of HIV-1 Gag to the PM is centered around the HIV-1 MA interaction with phosphatidylinositol 4,5-biphosphate (PI[4,5]P2). We previously performed a virtual screen in combination with a biophysical technique (surface plasmon resonance) to discover potential inhibitors of this HIV-1 MA/ PI[4,5]P2 interface. We identified a compound that directly bound to HIV-1 MA and displayed broad-range antiviral activity. In hopes to identify a more potent inhibitor, the initial hits from our virtual screen were re-evaluated using antiviral potency as our main filter. Modifying our initial screening methodology lead to the identification of three new scaffolds, wherein compound 7 showed the highest potency.

Similar to our initial HIV-1 MA inhibitor, compound 7 exhibited a broad-range therapeutic spectrum having a median IC50 value of 11µM across the major clades (A,B and C). Further investigation revealed that the antiviral effect of compound 7 occurred during the late stage of the viral life cycle, primarily through inhibiting the HIV-1 MA/ PI[4,5]P2 interface. Mutagenesis of key amino acids within the PI[4,5]P2 binding pocket of HIV-1 MA negated the antiviral activity of compound 7 giving credence to our target site and allowed for a refined binding model (Figure 1).

Taken together, using an antiviral potency assay as the primary filter for potential hits from the virtual screen, allowed the discovery of a more effective inhibitor. Compound 7 can be used as a biological tool to more closely define the function of HIV-1 MA in the viral life cycle, as well as be the starting point for lead drug design of a novel HIV-1 therapeutic.

Isaac Zentner-2Figure 1: Refined predicted binding mode of compound 7 in the PI(4,5)P2 binding site of HIV-1 MA. Binding site residues (L21, R22, W36, R76, T81, and K98) are represented as dark sticks, whereas compound 7 is shown in green sticks. Residues highlighted in blue are thought to be critical for compound binding. Yellow dashed lines represent hydrogen bonds.

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