The Locus Derepression (LDR) assay detects the derepression of a GFP reporter that is stably integrated in the mouse mammary carcinoma line, C127. In the vector, GFP transcription is controlled by a CMV promoter, which normally is strong and constitutively active. However, this cell line (referred to as LDR cells) was selected for lack of constitutive expression of the GFP reporter [12
] presumably due to epigenetic silencing of the integration locus and/or methylation of the CMV promoter. GFP production can be induced by incubating the cells with HDAC inhibitors such as TSA or butyrate [13
] or with DNMT inhibitors such as 5-aza-deoxycytidine [12
] but not by general activators of transcriptional pathways such as serum, insulin or steroids (data not shown). Derepression of this reporter locus was measured by enumerating GFP-positive cells using a laser scanning microplate cytometer, the Acumen Explorer [13
To find new small molecules that modulate epigenetic control of transcription, the LDR cells were screened as outlined in , against 69,137 compounds using qHTS, a method that assays compounds at multiple concentrations [14
]. Following curve fit and classification of the titration-response data, 411 compounds (0.6% of the library) were found as activators that induced GFP expression (). Fourteen actives showed complete titration curves, of which 11 displayed full efficacy relative to control (response >80 %; Class 1.1 in the qHTS classification) (Inglese et al., 2006
) and three showed partial efficacy (response <80 %, Class 1.2). An additional 51 compounds produced incomplete titration-response curves containing only the lower asymptote; of these, 35 displayed full efficacy (Class 2.1) and 16 showed partial efficacy (Class 2.2). The remaining 346 active compounds showed activity only at the highest concentration tested, or were associated with poorly fit curves (Class 3). Nine actives had half-maximal activity concentrations (EC50
) of ≤ 1 uM and 161 compounds had an EC50
between 1 and 10 uM ().
Potency and curve class of qHTS actives
To determine structure-activity relationships among the actives, compounds associated with Class 1 and 2.1 curves were clustered and maximal common substructures (MCS) were extracted from clusters containing three or more actives. Each MCS was then used to search the entire library to recover all analogs, including inactives. In addition, the core structure for each Class 1.1 compound was searched against the collection to find all related structures. The combined approaches yielded six series () for further investigation.
Activities and potencies of selected classes of LDR actives
For follow-up studies, 13 qHTS actives, representing five of the six series and two singletons, a combinatorial library containing the sixth series, and 35 commercially available analogs were chosen. These compounds were first counter screened against the parental C127 cells (which do not contain the GFP transgene) to identify fluorescent compounds. Each compound was titrated in 24 two-fold dilutions in duplicate beginning at 46 uM while the combinatorial library was screened at the three highest qHTS concentrations (9, 2, and 0.4 uM). The compound containing the 7-aminochromen-2-one core (series 4) and the pteridin-7-one-containing compounds from the combinatorial library (series 6) showed activity in the control cells ( and data not shown), indicating these were fluorescent molecules that bound or permeated the cells. These series were therefore classified as false positives and eliminated from further consideration. All the other tested compounds showed no activity in the parental cells ( and data not shown).
Activities of selected compounds on LDR and parental cells
As a further counter screen to identify potential fluorescent false positives, compound-treated LDR cells were assayed for nuclear translocation of the GFP reporter. In LDR cells, GFP is fused to a glucocorticoid estrogen receptor chimeric protein (GFP-GER) that is retained predominantly in the cytosol by the glucocorticoid receptor portion. However, upon estradiol binding to the ligand binding domain of the estrogen receptor portion, GFP-GER undergoes nuclear translocation [19
]. The cytosol-to-nuclear translocation of GFP-GER provided an easy means to confirm that the fluorescence induced by active compounds in the LDR cells arose from the expression of the GFP-GER reporter. LDR cells were treated with compounds or vehicle, incubated overnight at 37 °C and the following day imaged by fluorescent microscopy, before and after estradiol stimulation. Cells treated with 200nM TSA to induce GFP-GER showed cytosolic fluorescence that became nuclear after estradiol addition (). In contrast, LDR cells treated with the fluorescent but inactive compound 4
showed cytosolic fluorescence in either the presence or absence of estradiol stimulation. Treatment of LDR cells with actives from series 1, 2 or 3 resulted in a predominantly cytosolic signal that became clearly nuclear in the presence of estradiol (), supporting the hypothesized induction of GFP-GER expression by these compounds.
Nuclear localization of GFP-GER in compound-treated LDR cells upon addition of estradiol, an ER ligand
Compounds that were negative in the fluorescence assays were next tested on the LDR cells. One of the two singletons showed activity upon retest of the library samples. This compound, 2-butyl-N-(3,4-dimethoxyphenyl) cyclopropanecarboxamide, displayed a poorly-fit Class 3 curve of 4 nM EC50 in the qHTS that upon retest, displayed a Class 2.1 curve with a 14 uM EC50 (). Of the three compounds retested from the 1,3,5-triazine series identified in the qHTS (series 5), one compound showed no activity, and two did not show consistent activity at different times or with freshly prepared independent samples. In addition, five analogs showed no activity (). As the activity of series 5 could not be consistently reproduced, these compounds were not investigated further.
Potency and curve class of qHTS and follow-up actives
Compounds containing 8-hydroxy quinoline, quinoline-8-thiol and 1,3,5-thiadiazinane-2-thione cores (series 1-3), confirmed activity on LDR cells (, ). For the 8-hydroxy quinolines (series 1), 28 unique compounds with substitutions at the R1, R2, R4, and R5 positions were tested (). Three of four qHTS actives reproduced activity with similar or lower potencies. Of the 24 analogs containing the 8-hydroxy quinoline core, seven showed Class 1.1 curves with EC50 between 3 and 16 uM, five displayed Class 2 curves of 38 uM EC50 or greater, and twelve were inactive (). Analog 1c () was initially scored as inactive on LDR cells as detected by a laser-scanning imager, yet by fluorescence microscopy was active, indicating this molecule to be a weak positive (data not shown). No discernible SAR from series 1 could be ascertained.
Structures of selected compounds from the qHTS and follow up testing
For series 2, which contained a quinoline-8-thiol core, both qHTS actives (2a and 2b, ) confirmed as independent samples having potencies of 7 and 9 uM ( and ). The testing of three analogs indicated that a 1-(pyrrolidin-1-yl)ethanone substitution at R1 did not alter potency () while phenyl- or benzyl-acetamide substitutions were inactive (data not shown).
Series 3 comprised two actives containing the1,3,5-thiadiazinane-2-thione core. The 3,5-dimethyl form (3a, ), identified from the qHTS, and a 3,5 diethyl analog (3b, ) showed potencies of about 7 uM. A second analog (3c, ), with phenyl substitutions at R1 and R2, was inactive (, ).
In summary, the follow-up investigation showed that of the initial six series, three were confirmed active by testing the original and/or independent samples of library compounds, as well as their analogs. Of the remaining three series, one showed inconclusive activity, and two were false positive due to fluorescence. Eight of thirteen (62%) library compounds identified by the qHTS confirmed activity upon retest including one fluorescent compound, while three were inactive, and two were inconclusive due to inconsistent activity (). Thirty-five analogs from four series were tested and 14 (40%) were active with their potencies ranging from 3 uM to above 46 uM, the highest tested concentration.
Since known HDAC inhibitors such as butyrate and TSA induce GFP-GER in LDR cells [12
], we examined whether the LDR actives inhibit HDAC activity in vitro
. While 5 uM TSA completely inhibited HDAC enzymatic activity in HeLa nuclear extracts, none of the tested actives blocked activity when assayed at 48 uM (). In addition, these compounds did not block HDAC activity in whole cell extracts of LDR cells (data not shown). NSC3852 (5-nitroso-8-quinolinol) is a reported HDAC inhibitor [20
] and shares the 8-hydroxy quinoline core of series 1. While this molecule induced GFP-GER with a 2.8 uM EC50
(), it did not inhibit HDAC activity in HeLa extracts at 48 uM (4A). Though NSC3852 could inhibit HDAC activity by 80 % at 190 uM, it also inhibited an unrelated protease enzyme assay by 40 % (data not shown), indicating some nonspecific activity at this concentration. These results suggest that series 1-3 are not general HDAC inhibitors but rather may target different epigenetic enzymes or specific HDACs of low abundance in HeLa or LDR extracts.
Effect of LDR compounds on HDAC activity, gene expression and H358 viability
We next ascertained whether the LDR actives could reactivate the expression of endogenous genes silenced by promoter methylation. Human non-small cell lung cancer (NSCLC) H358 cells harbor methylated CpG islands at the CDH13 and p16 promoters, with the latter being densely methylated and fully silenced by this modification [21
]. H358 cells were incubated with compounds for 3 days and CDH13 and p16 transcript levels 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 (). Nicotinamide, an inhibitor of sirtuins [23
], and series 2 and 3 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.
As known epigenetic drugs inhibit cancer cell growth in vitro
and in some cases in vivo
], we evaluated LDR actives for potential anti-tumor activity. H358 cells were treated with compounds for four days and viability was assessed by MTS reduction. TSA terminated cells with an IC50
of 40 nM while 5-azadeoxycytidine showed incomplete killing at 10 uM, the highest tested concentration. Series 1 compounds killed cells with IC50
values ranging from 0.1 to 2.4 uM while series 2 and 3 showed little or no effect (, ).
Summary of biological activities of selected LDR compounds
To test whether the LDR actives were selective against tumor cells, we tested two NSCLC and their matched normal bronchial epithelial cells, both derived from patient samples (see Materials and Methods for cell line development details). 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 (). One active and one inactive compound were tested from series 2 and 3, all of which showed little to no activity (). For one matched set, 2a decreased viability in both tumor and normal lines by about 50% at 20 uM. Of the three series 1 compounds tested, 1a and 1b were potent and selective for both tumor lines (, ). 1a was 10-to 30-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.
Effect of LDR compounds on matched patient-derived lung tumor and normal cells