Summary /

Inhibition of HDAC activity, derepression of methylated gene expression, and killing of lung cancer lines. ML065 was not observed to inhibit HDAC activity in HeLa cell extracts at 48 uM (Figure 6A).  This suggested that this compound is not a general HDAC inhibitors, but rather may target different epigenetic enzymes or specific HDACs of low abundance in HeLa or LDR extracts.

Reactivation of the expression of endogenous genes silenced by promoter methylation. Human non-small cell lung cancer H358 cells harbor methylated CpG islands at the CDH13 and p16 promoters, with the latter being densely methylated and fully silenced by this modification (Phelps 1996; Virmani 2002). H358 cells were incubated with compounds for 3 days, and CDH13 and p16 transcript levels were measured by real time quantitative RT PCR. The HDAC inhibitors, depsipeptide and TSA, and the DNMT inhibitor, 5-azadeoxycytidine, reversed CDH13 silencing by 10 to 1000 fold, while only 5-azadeoxycytidine reactivated p16 expression (Figure 6B and 6C). Nicotinamide, an inhibitor of sirtuins (Bitterman 2002), and Series 2 (2a = ML065) and 3 (3a = ML064) compounds did not derepress either gene. Like depsipeptide and TSA, Series 1 compounds induced CDH13 but not p16 gene expression. Of this series, NSC3852 was the most potent, inducing expression 85 fold over basal levels while the others induced expression by 4 to 12 fold. These results indicate the LDR actives do not behave as DNMT inhibitors to derepress transcription of both p16 and CDH13 genes.

Summary /

Selective against tumor cells: two non-small cell lung cancer lines and their matched normal bronchial epithelial cells, both derived from patient samples. After four days of treatment, TSA killed both NSCLC lines and one normal line with 0.1-0.4 uM IC50, but did not reduce the viability of normal line 2 at concentrations up to 0.4 uM (Figure 6D). One active and one inactive compound were tested from Series 2 and 3, all of which showed little to no activity (Table 4). For one matched set, 2a (ML065) decreased viability in both tumor and normal lines by about 50% at 20 uM. Of the three series 1 compounds tested, 1a (ML066) and 1b were potent and selective for both tumor lines (Figure 7, Table 4). ML066 was 10- to 20-fold selective for the tumor lines with an IC50 between 0.2-0.3 uM, while 1b was 7- to 12-fold selective with an IC50 of about 1 uM. 1c displayed 3- to 10-fold selectivity for the normal lines with a potency of 1-2 uM.