Fifteen novel non-peptide HIV-1 protease inhibitors were identified by flexible 3D database pharmacophore searching of the NCI DIS 3D database. The pharmacophore query used in the search was derived directly from the X-ray determined structures of protease/inhibitor complexes. These 15 inhibitors, belonging to nine different chemical classes, are promising leads for further development. The two best inhibitors found, NSC 32180, a "dimer" of 4-hydroxycoumarin, and NSC 117027, a "tetramer" of 2-hydroxy quinone, had ID50 values of 0.32 and 0.75 microM for HIV-1 protease inhibition, respectively, and two other inhibitors had ID50 values close to 1 microM. Among the potent inhibitors, NSC 158393 not only demonstrated activity against HIV-1 protease (ID50 1.7 microM) but also exhibited promising antiviral activity in HIV-1-infected CEM-SS cells (EC50 = 11.5 microM). Validation of the pharmacophore used in the search was accomplished by conformational analysis. The binding modes of the most potent inhibitor found in our studies, NSC 32180, were predicted employing docking and molecular dynamics techniques.