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  • br Table br Antiproliferative activity of

    2020-08-10


    Table 1
    Antiproliferative activity of compounds 9–22 against human cancer cell lines.
    Compounds Cell viability
    Prostate Colorectal Liver
    a SD: standard deviation, all experiments were independently performed at least three times.
    more potent than the 3X FLAG standard (2) against Hep3B cell lines. A com-parative analysis of the cytotoxic profile of the fused construct (11) with compounds 9, 10 and 12 confirmed the favorable activity trend encountered with the N-ethylation of the amide NH. Compounds 9 (bearing unsubstituted amide NH), 10 (N-CH3, amide) and 12 (N-C3H7, amide) exhibit relatively diminished activity profiles against the cell lines tested. A decline in the activity profile was seen on translocating the resorcinol fragment from position 3 to 4, 6 and 8 (compare 11 with 14, 19 and 22). Keeping the N-ethyl substitution pattern intact and shifting the site of resorcinol fusion from position 3 to 6 (19) retained the cytotoxic effects against the PC-3 (GI50 = 0.46 µM), HCT-116 (GI50 = 1.87 µM) and Hep3B (GI50 = 0.69 µM) cell lines, but the effi-cacy was less pronounced than that of 3X FLAG 11. The 8-quinolinyl substituted compound with an N-ethyl substituent (22) also demon-strated moderate inhibition of the growth of tumor cell lines. The in-vitro cytotoxicity assay clearly indicates that the sites of attachment of the quinoline scaffold and the resorcinol fragment are critical for in-duction of the antiproliferative effects. Overall, the fusion of the structural motifs at position 3 of the quinoline moiety and N-ethyl
    Table 2
    HSP90 inhibitory activity of compounds 9–22.
    Compounds IC50 ± SD(nMa)
    a SD: standard deviation, all experiments were in-dependently performed at least three times.  Bioorganic Chemistry 91 (2019) 103119
    substitution pattern (amide NH) played a key role in inducing the an-tiproliferative effects.
    To validate the underlying mechanism of the antiproliferative ac-tivity of constructs 9–22, an in-vitro assay was performed and the HSP90 inhibitory potential was evaluated. The data in Table 2 made it evident that only the cytotoxic compounds (11, 19, 22) are able to modulate the function of the chaperone protein. The compounds unable to induce inhibitory effects against the cell lines tested were also found to be devoid of any activity against HSP90 (IC50 > 10000 nM) with the exception compound 12. Thus the assay results establish a strong de-pendence of cell growth inhibitory effects of the conjugates on their capacity to inhibit the chaperone protein. Compound 11, which is en-dowed with substantial antiproliferative effects, exhibits significant HSP90 inhibitory activity with an IC50 value of 149 nM and the results with either of the standards employed were comparable. Compound 22 (quinoline-resorcinol: site of linkage – C8) also inhibited the chaperone activity with IC50 = 172 nM. In addition to the activity profile of 11, the promising cellular and chaperone function inhibitory profile of compound 19 is an important finding (Tables 1 and 2). Compound 19 with IC50 = 124 nM was found to be the most potent HSP90 inhibitor. Correlating the results of antiproliferative activity (Table 1) with those from the HSP90 inhibitory assay (Table 2) revealed a similar preference of structural features for HSP90 inhibition as that for cytotoxicity. The N-ethyl substitution with positions 3 or 6 as the critical point of at-tachment of the quinoline scaffold and the resorcinol ring were the favored structural attributes. In light of these assay results, the most potent antiproliferative agent (11) with substantial HSP90 inhibitory potential was further investigated to ascertain its HSP90-mediated suppression of prostate cancer cell growth.
    2.2.3. Effect of test compounds on HSP90-regulated client proteins
    To further establish whether the most potent fused construct (11) possesses the signatory features of known HSP90 inhibitors in terms of its capacity to modulate the expression of cellular markers, a western blot analysis was performed. The ability of compound 11 to trigger the downregulation of representative HSP90 client protein expression and induction of HSP70 protein levels was determined in a PC-3 cell line. Western immunoblotting data revealed that 11 induces HSP70 ex-pression and depletes the protein expression levels of client proteins, such as EGFR, AKT, ERK, FAK and Rb indicating selective HSP90 in-hibition (Fig. 3A). The underlying mechanism of arrest of PC-3 cells by 11 on the G2/M phase was explored and the expressions of cell cycle related proteins were examined. Induction of p-MPM2 along with cyclin B1 and downregulation of Cdc2 and its phosphorylation of Tyr15 was observed after a 24 h treatment with compound 11 and BIIB021 (2) (Fig. 3B). These results suggested that the HSP90 inhibitor (11) induces mitotic arrest in prostate cancer cells. Moreover, compound 11 triggers cell apoptosis at 48 h leading to PARP and caspase activations as evi-dent from the levels of cleaved Caspase 3 and PARP (Fig. 3C). The activation of Caspase 3 and PARP is considered to be a hallmark of apoptosis and the results of the western blot analysis clearly indicate the apoptosis inducing ability of 11. These observations coupled with the results presented in Tables 1 and 2 confirm that mediation of the cell growth inhibitory effects of 11 is a consequence of Hsp90 cha-perone inhibition and also confirms the mitotic arrest in prostate cancer cells.