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 μM for HIV-1 protease inhibition, respectively, and two other inhibitors had ID50 values close to 1 μM. Among the potent inhibitors, NSC 158393 not only demonstrated activity against HIV-1 protease (ID50 1.7 μM) but also exhibited promising antiviral activity in HIV-1-infected CEM-SS cells (EC50 = 11.5 μM). 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.

The p75 NTR functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with the nonsteroidal anti-inflammatory drug, indomethacin, induced p75 NTR expression in the T24 cancer cell line leading to p75 NTR -mediated decreased survival. Utilizing the indole moiety of indomethacin as a pharmacophore, we identified in rank-order with least efficacy, ketorolac, etodolac, indomethacin, 5-methylindole-3-acetic acid, indole-3-carbinol, and 3,3′-diindolylmethane (DIM) exhibiting greatest activity for induction of p75 NTR levels and inhibition of cell survival. Prostate (PC-3, DU-145) and bladder (T24) cancer cells were more sensitive to DIM induction of p75 NTR -associated loss of survival than breast (MCF7) and fibroblast (3T3) cells. Transfection of the PC-3 prostate cell line with a dominant-negative form of p75 NTR before DIM treatment significantly rescued cell survival demonstrating a cause and effect relationship between DIM induction of p75 NTR levels and inhibition of survival. Furthermore, siRNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75 NTR by DIM in the PC-3 prostate cell line. DIM treatment induced phosphorylation of p38 MAPK as early as within 1 minute. Collectively, we identify DIM as an indole capable of inducing p75 NTR -dependent apoptosis via the p38 MAPK pathway in prostate cancer cells.

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A33 A comparison of the indoles etodolac, indomethacin, 5-methylindole-3-acetic acid, indole-3-carbinol, 3,3’diindolylmethane (DIM) and the related ketorolac molecule showed superior efficacy of DIM, a dietary indole, to induce expression of the p75NTR tumor suppressor and inhibit survival of the PC-3 prostate cancer cell line. Each indole that induced p75NTR expression exhibited the same rank-order for inhibition of prostate cell survival. Comparison of the activity of DIM, the most potent of the indoles, between several cell lines, showed that DIM induced p75NTR at low concentrations in prostate cells (PC-3 , DU-145), followed by bladder (T24), with breast (MCF-7) and fibroblast cells (3T3) being the least responsive to DIM. DIM induction of p75NTR levels in the various cell lines occurred in the same rank-order as inhibition of cell survival. Since the consumption of cruciferous vegetables has been reported to reduce the risk of prostate cancer through the release of indole-3-carbinol (I3C) which is subsequently metabolized in the acidic environment of the stomach to DIM, the superior activity of DIM to induce expression of the p75NTR tumor suppressor protein and inhibit cell survival suggests a mechanism of action for the anticancer activity of DIM in the prevention of prostate cancer. In order to demonstrate a cause and effect between DIM and p75NTR inhibition of survival, we used a dominant negative of p75NTR to rescue DIM induced loss of cell survival. In order to investigate the mechanism of action by which p75NTR transduces a biochemical signaling cascade leading to cell death of prostate cancer cells we utilized knockdown with p38 MAPK siRNA to rescue DIM induction of p75NTR thereby implicating p38 MAPK in DIM induction of cell death. Moreover, DIM treatment induced phosphorylation of p38 MAPK within one minute, suggesting the target of DIM is proximal to p38 MAPK. Hence, these results suggest that the anti-cancer activity of cruciferous vegetables may be mediated in part by DIM and that the mechanism of action occurs, in part, by activation of the p38 MAPK pathway leading to expression of the p75NTR death receptor with subsequent apoptosis of prostate cancer cells. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A33.