The crucial functions of HIV-1 nucleocapsid-p7 protein (NC-p7) at different stages of HIV replication are dependent on its nucleic acid binding properties. In this study, a search has been made to identify antagonists of the interaction between NC-p7 and d(TG)(4). A chemical library of similar to2000 small molecules (the NCI Diversity Set) was screened, of the 26 active inhibitors that were identified, five contained a xanthenyl ring structure. Further analysis of 63 structurally related compounds led to the identification of 2,3,4,5-tetrachloro-6-(4',5',6'-trihydroxy-3'-oxo-3H-xanthen-9'-yl)benzo ic acid, which binds to NC-p7 stoichiometrically. This compound exerted a significant anti-HIV activity in vitro with an IC50 of 16.6 +/- 4.3 muM (means +/- SD). Synthetic variants lacking the two hydroxyls at positions 4' and 5' in the xanthenyl ring system failed to bind NC-p7 and showed significantly less protection against HIV infection. Molecular modeling predicts that these hydroxyl groups would bind to the amide nitrogen of Gly(35) with other contacts at the carbonyl oxygens of Gly(40) and Lys(33). (C) 2002 Elsevier Science (USA). All rights reserved.