Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 μM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).

Multidrug resistance, the phenomenon by which cells treated with a drug become resistant to the cytotoxic effect of a variety of other structurally and functionally unrelated drugs, is often associated with the expression of P-glycoprotein, an efflux membrane pump coded by the MDR1 (ABCB1) gene. Transcription from MDR1 can start at 2 promoters: a well-characterized downstream promoter and an as yet uncharacterized upstream promoter (USP). We have previously determined that the USP is activated in some drug-resistant cell lines, in primary breast tumors and in metastatic epithelial cells isolated from the lymph nodes of breast cancer patients. In this study, we report the cloning and characterization of the MDR1 USP and studied its association with chemotherapy response in breast cancer patients. Deletion analysis indicated that a nearby endogenous retroviral long terminal repeat is not responsible for promoter activation, and that the region within the first 400 nucleotides upstream from the transcription start point contained all the elements necessary for promoter activity in drug-resistant cells. We identified an element recognized by the transcription factor NF-IL6 (activated upon interleukin-6 exposure) which is necessary for promoter activity in drug-resistant cells and plays a role in the activation of the promoter in response to interleukin-6 in breast cancer MCF-7 cells. Although transcripts from this promoter are associated with translating polyribosomes, their low abundance makes the amount of synthesized P-glycoprotein insufficient to affect the response to first-line chemotherapy in patients with advanced breast cancer. ' 2007 Wiley-Liss, Inc.

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.

Overexpression of P-glycoprotein, encoded by the MDR1 (multidrug resistance 1) gene, is often responsible for multidrug resistance in acute myeloid leukaemia. We have shown previously that MDR1 (P-glycoprotein) mRNA levels in K562 leukaemic cells exposed to cytotoxic drugs are up-regulated but P-glycoprotein expression is translationally blocked. In the present study we show that cytotoxic drugs down-regulate the Akt signalling pathway, leading to hypophosphorylation of the translational repressor 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] and decreased eIF4E availability. The 5'-end of MDR1 mRNA adopts a highly-structured fold. Fusion of this structured 5'-region upstream of a reporter gene impeded its efficient translation, specifically under cytotoxic stress, by reducing its competitive ability for the translational machinery. The effect of cytotoxic stress could be mimicked in vivo by blocking the phosphorylation of 4E-BP by mTOR (mammalian target of rapamycin) using rapamycin or eIF4E siRNA (small interfering RNA), and relieved by overexpression of either eIF4E or constitutively-active Akt. Upon drug exposure MDR1 mRNA was up-regulated, apparently stochastically, in a small proportion of cells. Only in these cells could MDR1 mRNA compete successfully for the reduced amounts of eIF4E and translate P-glycoprotein. Consequent drug efflux and restoration of eIF4E availability results in a feed-forward relief from stress-induced translational repression and to the acquisition of drug resistance.