Design, synthesis, and biological evaluation of AV6 derivatives as novel dual reactivators of latent HIV-1

Abstract

The "shock and kill" strategy might be a promising therapeutic approach for HIV/AIDS due to the existence of latent viral reservoirs. A major challenge of the "shock and kill" strategy arises from the general lack of clinically effective latency-reversing agents (LRAs). The 2-methylquinoline derivative, antiviral 6 (AV6) has been reported to induce latent HIV-1 expression and act synergistically with a HDAC inhibitor VA to reverse HIV latency. We report herein the design and identification of AV6 analogues which possess the zinc-binding group of HDAC inhibitors and have dual acting mechanism for the reactivation of HIV-1 from latency. Evaluation of compounds for the reactivation of HIV-1 latency identified two excellent active compounds 12c and 12d. Further bioassays revealed that these two compounds reactivated latent HIV-1 through dual mechanism, the inhibition of HDACs and NFAT-required for early HIV-1 gene expression. Additionally, it was found that 12c and 12d could reactivate HIV-1 transcription by releasing P-TEFb from the inactive complex 7SK snRNP. At last, molecular docking identified their orientation and binding interactions at the active site of HDAC2. This experimental data suggests that 12c and 12d can be served as effective HIV-1 LRAs which can be taken up for further studies.

Fig. 1. Several HIV reactivators. HDAC inhibitors: short-chain aliphatic acids (butyric acid and valproic acid), hydroxamic acids (vorinostat and givinostat). AV6: its target protein is not available now.

Fig. 2. Design of multi-targeted HIV activator based AV6 and HDAC inhibitors.

Scheme 1. Synthesis of 2-methyl-N-arylquinolin-4-amine 7a–7w. Reagents and conditions: (a) ethyl 3-oxobutanoate, EtOH, reflux, 4 h; (b) diphenyl ether, 1 h; (c) POCl₃, 120 °C, 2 h; (d) BBr₃, DCM, r.t., 2 h; (e) CH₃CH₂Br or CH₃CHBrCH₃, NaH, DMF, r.t., overnight; (f) substituted aromatic amines, butyl alcohol, conc. HCl, 120 °C, 5 h.

Scheme 2. Synthesis of 3-((4-((3,4-dichlorophenyl)amino)-2-methylquinolin-6-yl)oxy)-alkanoic acid/hydroxyl acid. Reagents and conditions:(a) BBr₃, DCM, r.t., 2 h; (b) appropriate alkyl halide, NaH, DMF, r.t., overnight; (c) ethyl or methyl bromoalkanoate, NaH, DMF, r.t., overnight; (d) EtOH, 40% NaOH aqueous solution, 50 °C, 1 h; (e) NH₂OH, NaOH, CH₃OH, 0 °C to r.t.

Fig. 3. HIV latency can be reversed by 12c-12d in different latency cell models. (A) J-Lat A2 cells, (B) 2D10 cells, (C) the HeLa-based NH1 cells. (D) The NH2 cells expressing Tat. All tested cells were treated with 0.1% DMSO or the indicated concentrations of the drugs (12c, 12d, SAHA and AV6) for 24 h. The expression of GFP for tested J-Lat A2 and 2D10 cells was measured by FACS. Whole cell extracts for tested NH1 and NH2 cells were prepared and examined for the luciferase activities. The error bars in all panels represent mean ± SD based on at least three independent experiments. The P value was defined as **p < 0.01, and ***p < 0.001 vs. control.

Fig. 4. 12c retains the reactivation mechanism of AV6. (A) J-Lat A2 cells were treated with AV6, SAHA, 12c alone, or in combination for 24 h, and then analyzed by flow cytometry to determine the percentages of GFP(+) cells. (B) J-Lat A2 cells were treated with SAHA, AV6 or 12c for 24 h, in the presence or absence of the calcineurin inhibitor FK506 (20 μM). The percentages of GFP(+) cells were evaluated by FACS and plotted. Results shown are the means ± SD from three independent experiments. The P value was defined as **p < 0.01, and ***p < 0.001 vs. control.

Fig. 5. Western blotting analysis of the acetylation levels of histones H3 and H4 in A549 cells (A), HeLa cells (B) and J-Lat A2 cells (C) after 12c and 12d treatment respectively for 24 h.

Fig. 6. The HeLa-based F1C2 cells stably expressing the Flag-tagged CDK9 were treated with 12c, 12d, or DMSO for 24 h. Nuclear extracts (NE) and anti-Flag immunoprecipitates (IP) derived from NE were analyzed by western blotting for the indicated proteins.

Fig. 7. SAHA (A) and 12c (B) docked into the active site of HDAC2 (PDB entry 4LXZ). SAHA and 12c are shown in stick with yellow and brown carbon, respectively. The catalytic zinc in magenta CPK representation. The HDAC2 binding site is presented with a coloured ribbon. Neighbouring amino acids were displayed in lines within a distance of 5 Å approximately to ligand. 2D binding models of SAHA (C) and 12c (D) with HDAC2. Hydrogen bonds are indicated with solid arrows, colour lines around ligands (SAHA and 12c) stand for the binding pocket and the residues in colours nearby established the pocket. The green colour denotes the hydrophobic nature of amino acids, the red colour denotes the acid amino acids, the purple denotes the alkalinity of amino acids, the cyan denotes the polar amino acids, and the grey points of ligand atoms denotes the solvent accessibility.