N-Substituted Pyrrole Derivative 12m Inhibits HIV-1 Entry by Targeting Gp41 of HIV-1 Envelope Glycoprotein

Abstract

The combination of three or more antiviral agents that act on different targets is known as highly active antiretroviral therapy (HAART), which is widely used to control HIV infection. However, because drug resistance and adverse effects occur after long-term administration, an increasing number of HIV/AIDS patients do not tolerate HAART. It is necessary to continue developing novel anti-HIV drugs, particularly HIV entry/fusion inhibitors. Our group previously identified a small-molecule compound, NB-64, with weak anti-HIV activity. Here, we found that N-substituted pyrrole derivative 12m (NSPD-12m), which was derived from NB-64, had strong anti-HIV-1 activity, and NSPD-12m-treated cells showed good viability. The mechanism of action of NSPD-12m might be targeting the gp41 transmembrane subunit of the HIV envelope glycoprotein, thus inhibiting HIV entry. Site-directed mutagenesis confirmed that a positively charged lysine residue (K574) located in the gp41 pocket region is pivotal for the binding of NSPD-12m to gp41. These findings suggest that NSPD-12m can serve as a lead compound to develop novel virus entry inhibitors.

Keywords: HIV-1 entry inhibitor; cell–cell fusion; gp41 envelope; six-helix bundle; small molecular compound.

Figure 1

Chemical structures of NB-64 and NSPD-12m.

Figure 2

The inhibitory activity of NSPD-12m on infection by HIV-1_SF162 (A), HIV-1_JR-FL (B), HIV-1_HXB2 (C), and VSV-G (D) pseudotyped virus. The samples were tested in triplicate, and the data were presented in mean ± SD.

Figure 3

The function of NSPD-12m as a viral entry inhibitor confirmed by HIV-1-mediated cell-cell fusion (A), cell–cell HIV-1 X4 transmission (B), cell–cell HIV-1 R5 transmission (C), and cell–cell HIV-1 IIIB transmission (D) assay. Each sample on was tested in triplicate and data were presented in means ± SD.

Figure 4

NSPD-12m inhibited the interaction between the NHR and CHR peptides to form the gp41 six-helix bundle formation. (A) The schematic view of the gp41 functional regions. (B) 6-HB formation was measured by a sandwich ELISA with mAb NC-1, NB-64 is a positive control. (C) 6-HB formation was measured by N-PAGE. Lane 1 is the peptide C34 and lane 2 is the mixture of peptide N36 and C34. The peptide N36 was incubated with NSPD-12m at graded concentrations (400, 200, 100, 50 and 25 mM for lanes 3 to 7, respectively) at 37°C for 30 min before addition of the peptide C34. Lane 8 is a positive control by ADS-J1 at 1 mM.

Figure 5

NSPD-12m interfered with the α-helical conformation formed by NHR and CHR peptides as detected by CD spectroscopy and SPR analysis. (A) N36 was incubated with ADS-J1 or PBS before the addition of C34 by CD. (B) N36 was incubated with NSPD-12m before or after the addition of C34 by CD. Dose-dependent binding of N36 with NSPD-12 m (C) or T20 (D) by SPR assay.

Figure 6

Site-directed mutagenesis analysis. (A) NSPD-12m and its docking conformation inside the hydrophobic pocket of HIV-1 gp41. (B) Inhibitory activity of NSPD-12m on infection of the mutant HIV-1 W571A, K574A, Q577A, and R579A pseudoviruses. (C) Inhibitory activity of ADS-J1 on infection of the mutant HIV-1 K574A pseudoviruses. The samples were tested in triplicate, and the data are presented in mean ± SD.