Identification of 2-(4-N,N-Dimethylaminophenyl)-5-methyl-1-phenethyl-1H-benzimidazole targeting HIV-1 CA capsid protein and inhibiting HIV-1 replication in cellulo

Abstract

The capsid (CA) subunit of the HIV-1 Gag polyprotein is involved in several steps of the viral cycle, from the assembly of new viral particles to the protection of the viral genome until it enters into the nucleus of newly infected cells. As such, it represents an interesting therapeutic target to tackle HIV infection. In this study, we screened hundreds of compounds with a low cost of synthesis for their ability to interfere with Gag assembly in vitro. Representatives of the most promising families of compounds were then tested for their ability to inhibit HIV-1 replication in cellulo. From these molecules, a hit compound from the benzimidazole family with high metabolic stability and low toxicity, 2-(4-N,N-dimethylaminophenyl)-5-methyl-1-phenethyl-1H-benzimidazole (696), appeared to block HIV-1 replication with an IC50 of 3 µM. Quantitative PCR experiments demonstrated that 696 does not block HIV-1 infection before the end of reverse transcription, and molecular docking confirmed that 696 is likely to bind at the interface between two monomers of CA and interfere with capsid oligomerization. Altogether, 696 represents a promising lead molecule for the development of a new series of HIV-1 inhibitors.

Keywords: Benzimidazole; CA; Capsid; HIV-1; Inhibitor; p24.

Fig. 1

Inhibition of HIV-1 CA assembly in vitro at 50 µM. Results are expressed as the percentage of assembly at 30 min of measure, compared to the maximum assembly-level reached with untreated CA(negative control DMSO 0.5%). Data for PF74 was acquired in the same condition from Xu J. P. 2018 et al. [31]

Fig. 2

Chemical structures of the best compounds and toxicology summary [, –33] compared to reference anti-CA drug PF-74. Detailed synthesis and characterization of 696 and 314 are available in supporting information. EC₅₀ (concentration of a drug that gives half-maximal response) corresponds to the cytotoxicity of the compounds. LD₅₀ correspond to the oral acute toxicity in rat or mice, and AMES test predicts mutagenicity

Fig. 3

a Dose–response inhibition of HIV-1 replication in U87-CD4-CCR5 cells after 11 days. Each curve displays the mean and SD of 4 independent wells of a representative experiment (n = 3). Viral replication was determined by p24 production in the culture supernatant and expressed as % of the DMSO-treated control. b Toxicity of compound 696 on U87-CD4-CCR5 cells measured by MTT assay after 11 days displayed as percentage live cells (mean and SD of 4 wells) relative to control

Fig. 4

Measure of the affinity for HIV-1 CA of 696 by MST. It is showed one representative experiment out of three

Fig. 5

Inhibition by 696 of early steps in the viral replication cycle by the analysis of reverse transcription by qPCR detecting viral DNA. Each bar displays the mean and SD of 4 wells of qPCR for the HIV-1 pol gene of a representative two independent experiments (see Materials and methods). Stars demonstrate the significance of the difference compared to DMSO-treated samples (unpaired two tails Mann–Whitney test) with *: p < 0.05 and N.S.: non-significant (p > 0.05). Results are expressed as the copy number of pol viral DNA per 1000 copies of β-actin

Fig. 6

Molecular docking of compound 696 on HIV-1 CA monomer (top) or dimer (bottom). Compound 696 and its surfaces are displayed in cyan and the chain of the dimer used for docking is displayed in dark orange. HIV-1 CA monomers and dimers are extracted from PDB entry 4XFZ [19]

Fig. 7

Molecular docking of compound 696 in a CA dimeric interface (top) compared to the published structure of PF74 in a complex with a CA hexamer (bottom, PDB entry 4XFZ [19]). The coloring of the amino-acid chains is identical to Fig. 6