br Effects of test compounds on cell cycle distribution
2.2.4. Effects of test compounds on cell cycle distribution
The effect of 11 on the cell cycle progression was evaluated by flow cytometry to validate the underlying mechanism of cell growth re-pression in PC-3 cells. The results of the experiment indicate that compound 11 and BIIB021 (2) cause cell arrest (G2M phase) at 24 h followed by a concentration-dependent apoptotic subG1 accumulation at 48 h (Fig. 4A and B). In addition, it was observed that 11 is more
Fig. 3. The effects of 11 on client proteins of HSP90, cell cycle regulated and apoptotic protein levels. PC3 cells were treated with DMSO (Control; C) or 0.03–3 μM of test compounds (11 or BIIB021) for 24 h. Cells were harvested and sub-jected to western blot analysis for the detection of various HSP90 client proteins (A) and cell cycle related proteins (B). (C) PC3 cells were incubated with DMSO (Control; C) or 0.03–3 μM of test compounds (11 or BIIB021) for 48 h to detect the apoptotic proteins. Actin was used as the internal control; B: BIIB021. (G256 represents 11).
potent than BIIB021 (2) as at 0.3 μM, it causes the initiation of the increase in G2M and subG1 phase population (Fig. 4C and D). These results indicate that the hybrid compound 11 causes dose-dependent G2M arrest and apoptosis in prostate cancer cells.
2.2.5. Molecular modeling studies
A molecular docking study was performed to elucidate the inter-actions between compound 11 and amino BODIPY 493/503 residues of the cha-perone protein HSP90. Initially, the validation of the docking protocol was performed by redocking the co-crystallized ligand (Fig. 1S. in Supporting Information – SI). This showed that the redocked ligand has a docking pose similar to that of the co-crystallized ligand and indicates that the docking protocol used has a favorable prediction acuity. Compound 11 was then docked into the HSP90 binding site and it was found that it is anchored within the binding site and can be separated into three distinct segments with respect to its HSP90 docking pose (Fig. 5). At Site 1 (S1), the 2,4‐dihydroxy-5-isopropylbenzoyl moiety is held in place through hydrophobic interactions with residues Met98, Leu107 and Phe138. In addition, the hydroxyl groups form hydrogen bonds with Asn51 and Asp93, and the carbonyl forms a hydrogen bond with Thr184. At Site 2 (S2), the quinoline moiety of compound 11 occupies a hydrophobic pocket with Ala55 and Tyr61. At Site 3 (S3), the two pharmacophores are connected via an N-ethyl amide bond. This ethanamine moiety has hydrophobic interactions with Ala55, Ile96, Met98 and Tyr61. In contrast, compounds 9 and 10, with an un-substituted amide linkage (–NH-CO–) and an N-methyl amide linkage respectively, do not inhibit HSP90 as effectively as 11, suggesting that the N-ethyl substitution is a prerequisite for activity. Together, these interactions shed light on the potency of compound 11 against HSP90.
2.2.6. In vivo antitumor efficacy in human xenografts
Compound 11 was evaluated for its in vivo efficacy versus tumor xenografts in nude mice bearing the human PC-3 cancer cell line. PC3-tumor-bearing nude mice were treated with compound 11 (50, 100 and 200 mg/kg/d, by oral gavage qd) and tumor was excised when the tumor size reached 1200 mm3. It was found that treatment of PC3 xe-nograft-bearing nude mice with compound 11 resulted in dose depen-dent decrease of the tumor progression. Construct 11 led to the sup-pression of tumor growth by a tumor growth inhibition factor (% TGI = 50.1%) without loss of their body weight (Fig. 6). Thus the in vivo animal model experiments demonstrated that compound 11 pos-sess moderate in vivo potential against prostate cancer.
A novel class of inhibitors that potently inhibits HSP90 is reported. By fusing key structural motifs known to induce cell growth inhibitory effects by modulation of the HSP90 chaperone protein, a series of fused quinoline-resorcinol compounds were designed and synthesized. A correlation of the tumor growth inhibition exerted through HSP90 in-hibition was established from the results of in vitro cytotoxicity studies, HSP90 inhibitory assays and western blot analysis. The site of linkage of the resorcinol ring to the quinoline scaffold via an amide bond and N-alkylation of amide bond was found to be extremely critical for both the cytotoxicity as well as the modulation of chaperone function. These attempts led us to speculate about the structure activity relationships with ethylation at the amide NH and position 3 (the quinoline ring) as the site of attachment being the structural prerequisites for activity. The main finding of the study is the identification of 11 as an HSP90 in-hibitor endowed with apoptosis inducing ability and substantial in vitro