Voacanga globosa Spirobisindole Alkaloids Exert Antiviral Activity in HIV Latently Infected Cell Lines by Targeting the NF-kB Cascade: In Vitro and In Silico Investigations

Abstract

Since the efficiency in the transcription of the HIV genome contributes to the success of viral replication and infectivity, we investigated the downregulating effects of the spirobisindole alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) from the endemic Philippine medicinal plant, Voacanga globosa, during HIV gene transcription. Alkaloids 1-3 were explored for their inhibitory activity on TNF-α-induced viral replication in two latently HIV-infected cell lines, OM10.1 and J-Lat. The induction of HIV replication from OM10.1 and J-Lat cells elicited by TNF-α was blocked by globospiramine (1) within noncytotoxic concentrations. Furthermore, globospiramine (1) was found to target the NF-ĸB activation cascade in a dose-dependent manner when the transcriptional step at which inhibitory activity is exerted was examined in TNF-α-induced 293 human cells using transient reporter (luciferase) gene expression systems (HIV LTR-luc, ĸB-luc, and mutant ĸB-luc). Interrogation through molecular docking against the NF-ĸB p50/p65 heterodimer and target sites of the subunits comprising the IKK complex revealed high binding affinities of globospiramine (1) against the S281 pocket of the p65 subunit (BE = -9.2 kcal/mol) and the IKKα activation loop (BE = -9.1 kcal/mol). These findings suggest globospiramine (1) as a molecular inspiration to discover new alkaloid-based anti-HIV derivatives.

Keywords: HIV latency; NF-ĸB; Voacanga globosa; anti-HIV; globospiramine; spirobisindole alkaloids.

Figure 1

Spirobisindole alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) from Voacanga globosa.

Figure 2

Effect of compounds in TNF-α-induced HIV latently infected cell lines (A) OM10.1 and (B) J-Lat. Cells were pretreated with varying concentrations of compounds 1, 2, and 3 for 4 h. Two sets were setup: one set of each cell lines was stimulated with 1-ng/mL TNF-α for HIV production and incubated for 48 h at 37 °C; the other set was not stimulated. The cells were dislodged, and the contents were spun at low speed; cell viability was determined using WST, while the supernate was tested for p24 antigen by ELISA to test for viral production. As expected, there was no significant HIV production (data not shown) in non-stimulated cells. For the other set, IC₅₀ and CC₅₀ were determined. Values shown are representative of the mean value ± SD of 3 independent experiments (n = 3). Asterisk (*) is at p < 0.05 for a representative experiment. Cytotoxic Concentration at 50% (CC₅₀), Inhibitory Concentration at 50% (IC₅₀), non-inhibitory (ni), and enzyme linked immunosorbent assay (ELISA).

Figure 3

Effect of spirobisindole alkaloids 1–3 and roscovitine on the HIV-1 LTR-mediated transcription and on the ĸB promoter. Two hundred and ninety-three cells in a 24-well plate at 8 × 10⁴ cells per mL were transfected with the reporter plasmids: pCD12-Luc for HIV-1 LTR, pGK3-4ĸB-Luc for the wild-type ĸB promoter, and pGL3-ĸBmut-Luc for mutated ĸB sites using the Fugene 6^® Transfection Reagent. Cells were treated with 10-fold dilutions of the compounds 1–3 and roscovitine for 4 h, then treated and non-treated cells were stimulated with 5-ng/mL TNF-α for 24 h. Luminescence from treated cells of each concentration was compared against the untreated cells. Relative luciferase activity is shown as % fold activity. Values shown are representative of the mean value ± SD of 3 independent experiments (n = 3).

Figure 4

Globospiramine (1) docked against the p65 subunit (red) in complex with the p50 subunit (violet) of NF-κB (PDB ID: 1VKX). κB DNA was added for visualization.

Figure 5

Globospiramine (1) docked against the activation loop of the IKKα kinase domain (PDB ID: 5EBZ).