Abstract: Objective To screen the HIV-1 entry inhibitors targeting HIV-1 gp120 from the IBS natural product database by
virtual screening based on the binding mode of the neutralizing antibody VRC01 with HIV-1 gp120 and investigate the
anti-viral activities of the inhibitors and their action mechanisms. Methods The binding interaction of the candidate molecules
binding gp120 and changes of the binding free energy were analyzed by MM-PBSA calculation. The anti-HIV-1 activities of the
tested compounds were detected by HIV-1 pseudotyped virus, laboratory-adapted HIV-1 and a cell-cell fusion assay. The
cytotoxicity of the studied molecules was examined by XTT colorimetric assay. The mechanisms of the anti-viral activities of
the candidate molecules were analyzed using enzyme-linked immunosorbent assay. Results A total of 19 molecules with
distinct reduction of the binding free energy after binding with gp120 were screened from 40000 molecules. Among them,
NC-2 showed anti-HIV-1 activities against HIV-1 pseudotyped virus and laboratory-adapted HIV-1, and was capable of
blocking HIV-1 envelope-mediated cell-cell fusion. The IC50 of NC-2 for inhibiting HIV-1IIIB and pseudotyped HIV-1JRFL
infection were 1.95±0.44 μmol/L and 10.58±0.13 μmol/L, respectively. The results of ELISA suggested that NC-2 could inhibit
the binding of HIV-1 gp120 to CD4 without blocking the formation of gp41 six-helix bundle in vitro. Conclusion This
computer-based virtual screening method can be used to screen HIV-1 entry inhibitors targeting gp120. Using this virtual
screening approach combined with anti-viral activity screening, we obtained a potent HIV-1 entry inhibitor NC-2 with novel
structure.