HIV-1 infection begins with the binding of envelope glycoprotein surface subunit 120 to CD4 and a co-receptor CXCR4 or CCR5,triggering a series of conformational changes in the Env transmembrane subunit gp41:i) fusion peptide inserts into the host cell membrane; ii) three N-heptad repeats associate to form an NHR-trimer,a prehairpin fusion intermediate (FIM); and iii) three C-heptad repeats interact bind to the NHR-trimer to form a table six-helix bundle (6-HB),which brings the viral envelope and target cell membrane into close proximity for fusion.Peptides derived from the CHR region,such as C34 and T20 (Enfuvirtide),the first FDA-approved peptidic HIV fusion inhibitor,inhibit HIV-1 fusion by binding to the viral gp41 NHR-trimer to block the formation of fusion-active core of gp41.However,the clinical application of peptidic drugs is limited due to the lack of oral availability and high cost of production.Therefore,it is essential to develop orally available and inexpensive small molecule HIV-1 fusion/entry inhibitors targeting the gp41 FIM.Using a molecular modeling-based virtual screening method in combination with ELISA,we identified the first small molecule HIV fusion/entry inhibitor targeting gp41 FIM,ADS-J1,which inhibits HIV-l-mediated membrane fusion and HIV-1 replication with IC50 of~10 碌M.We then screened a chemical library consisting of 33,040 drug-like compounds using a florescent-linked immunosorbent assay-based high-throughput screening assay and identified two N-substituted pyrroles,NB-2 (MW=231) and NB-64 (MW=222),which have potent inhibitory activity against 6-HB formation and HIV-1 replication (IC50=~1,LM).