Furthermore to address the concern that the observed positiv
Furthermore, to address the concern that the observed positive in vitro activity results might be stymied by the propensity of such assays to display false positives due to the inherent noxiousness of the tested compounds, all analogs were evaluated against Vero cells in an MTT assay. Some of the most PSA–active compounds in this study (9 and 10, Table 1) did not inhibit the growth of Vero cells and furnished CC50 values greater than 100 μM. This effect seems to emanate from the higher steric constraints afforded by the bulky cysteinyl substituents for these compounds . Amongst the known inhibitors of aminopeptidases, while bestatin did not display harmful effects against Vero cells (CC50 > 100 μM), the toxicity–profile of tosedostat (CC50 = 13 μM) raises concerns about the selectivity it furnishes in achieving its therapeutic objectives.
While several factors, such as changes in pH, apoptosis, undesirable inhibition of other enzymes, etc., could be responsible for toxicity against Vero cells, our results also eliminate the possibility of protein synthesis inhibition being the cause for this effect for compounds 11, 12, and 13.
Antitumor Cell Proliferation Studies. Encouraged by the reactivity and toxicity profiles of the tested analogs, we proceeded to study the effects of these compounds in tumor cell lines. As mentioned earlier, studies by Wheatly and coworkers demonstrated that inducing nutrient stress via depriving cells of essential cantharidin sale could be utilized to target various malignant phenotypes . Human promyelocytic leukemia (HL60), and human acute lymphoblastic leukemia (MOLT4) cells were identified as most susceptible to this effect. The cells were maintained in RPMI 1640 growth media supplemented with 10% FBS, 1% Penicillin/Streptomycin and 1% Glutamax–1. For both cell lines, the measurement of cell viability was carried out using a modified method of Mosmann based on 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT) . Compounds were diluted in culture medium, added to the wells in triplicate, and incubated for a further 72 h at 37 °C in a 5% CO2/95% air humidified atmosphere, followed by removal of media and addition of freshly prepared MTT (at 1 mg/mL in serum–free, phenol red–free RPMI 1640 media). The MTT was removed after 3 h and formazan crystals were solubilized with isopropanol. Plates were read on a Molecular Devices SpectraMax i3 spectrophotometer at 570 nm for formazan and 690 nm for background subtraction.
The observed EC50 values revealed significant antitumor activity against these cell lines for several analogs (Table 1). The parent puromycin molecule (1) seemed to inhibit the proliferation of both HL60 and MOLT4 cells. This result is attributable to the protein synthesis inhibitory action of puromycin. Excellent activity of compound 9 against PSA translated into single–digit micromolar efficacy against the ALL cell line (MOLT4). Another addition of an aromatic ring in the structural motif led to further decrease in potency with S–triphenylmethyl analog (10) found to be inactive in both the cell lines. Among other structural congeners, the –isomer analogs were found to be inactive. Conceivably, the lack of potency for non–nucleoside compound 17 against PSA and APN, translated into attenuated activity against these malignant cell lines. These SAR trends will be indispensable in refining the structural features required for better potency in assays targeting AML (HL60) and ALL (MOLT4) and other hematologically compromised cell lines.
Conclusions Inhibitors of aminopeptidases based on the structural template of puromycin were prepared and studied for their potency against two broad–specificity aminopeptidases – PSA and APN. Clear trends from the SAR data confirmed a noteworthy correlation of the steric bulk with the corresponding anti–aminopeptidase potency. The steric requirement allows the inhibitory efficacy to reach maxima but continuing the increase in steric bulk after crossing that threshold, leads to a comparative attenuation of potency. Compounds 8 and 9 display exceptional anti–aminopeptidase potency against the tested aminopeptidases. Compounds 9 and 10 did not inhibit protein synthesis and also did not show toxicity against Vero cells. The apparent better potency of compound 8 against the cancer cell lines is attributable to its inherent toxicity (30% protein synthesis inhibition @ 10 μM; Vero cell CC50 = 0.88 ± 0.067 μM). These data clearly support compound 9 as the compound of interest for further studies.