Withanolide-Type Steroids from Physalis nicandroides Inhibit HIV Transcription

Abstract

The aim of the present study is to report the isolation, structural elucidation, and antiviral evaluation of four new withanolide-type steroids, named nicansteroidins A-D (1-4), together with nine related known compounds (5-13) isolated from the aerial parts of Physalis nicandroides. Their structures were established based on an extensive spectroscopic analysis, including 1D and 2D NMR techniques. Outstandingly, nicansteroidins A and B possess an unusual side chain with an exocyclic double bond on the δ-lactone system, whereas nicansteroidins C and D have an uncommon cycloperoxide functionality in ring A as distinct structural motifs. Their biological evaluation as inhibitors of human immunodeficiency virus type 1 replication revealed that two compounds from this series, 7 and 13, displayed strong inhibition of HIV-1 replication with IC₅₀ values lower than 2 μM. Moreover, cellular mechanism experiments showed that the main target of these compounds in the HIV replication cycle is viral transcription. This study is the first report of withanolide-type steroids as HIV inhibitors and provides insight into their potential as candidates for further preclinical studies.

Scheme 1. Structures of Withasteroids 1–13 Isolated from Physalis nicandroides

Figure 1

Selected HMBC (¹H–¹³C) long-range correlations (→) and NOE effects (↔) for compounds 1 (left) and 3 (right).

Figure 2

(A) In vitro evaluation of the anti-HIV activity and cytotoxicity of isolated compounds. MT-2 cells were infected with an X4 recombinant HIV (NL4.3-Ren, 100.000 RLUs or 20 ng p24/well) or with a VSV pseudotyped HIV (VSV-HIV, 100.000 RLUs or 20 ng p24/well) in the presence of different concentrations of test compounds or the fusion inhibitor enfuvirtide for 48 h. The same concentration of vehicle (DMSO or water) was used as a nontreated control (100%). The cell culture was then lysed, and relative luminescence units (RLUs) were measured in a luminometer. Cell viability was evaluated in mock-infected cells in parallel using the CellTiter Glo reagent (Promega). (B) Effect of isolated compounds on HIV transcription. MT-2 cells were transfected with a pNL4.3-Luc plasmid (1 ng/10⁶ cells) and treated or nontreated with serial dilutions of the test compounds for 48 h. The same concentration of vehicle (DMSO) was used as a nontreated control (100%). The cell culture was then lysed, and RLUs were measured in a luminometer. Results were analyzed using GraphPad software.