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JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC50 of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.

// Molly Gale 1 , Joyce Sayegh 1, 3 , Jian Cao 1 , Michael Norcia 2 , Peter Gareiss 2 , Denton Hoyer 2 , Jane S. Merkel 2 , Qin Yan 1 1 Department of Pathology, Yale School of Medicine, New Haven, CT, USA 2 Yale Center for Molecular Discovery, Yale University, West Haven, CT, USA 3 Current address: Department of Biology and Chemistry, Azusa Pacific University, Azusa, CA, USA Correspondence to: Qin Yan, email: qin.yan@yale.edu Keywords: histone demethylase inhibitor, KDM5A, anti-cancer drug, drug resistance, epigenetics Received: February 23, 2016 Accepted: May 05, 2016 Published: May 21, 2016 ABSTRACT Lysine demethylase 5A (KDM5A/RBP2/JARID1A) is a histone lysine demethylase that is overexpressed in several human cancers including lung, gastric, breast and liver cancers. It plays key roles in important cancer processes including tumorigenesis, metastasis, and drug tolerance, making it a potential cancer therapeutic target. Chemical tools to analyze KDM5A demethylase activity are extremely limited as available inhibitors are not specific for KDM5A. Here, we characterized KDM5A using a homogeneous luminescence-based assay and conducted a screen of about 9,000 small molecules for inhibitors. From this screen, we identified several 3-thio-1,2,4-triazole compounds that inhibited KDM5A with low μM in vitro IC 50 values. Importantly, these compounds showed great specificity and did not inhibit its close homologue KDM5B (PLU1/JARID1B) or the related H3K27 demethylases KDM6A (UTX) and KDM6B (JMJD3). One compound, named YUKA1, was able to increase H3K4me3 levels in human cells and selectively inhibit the proliferation of cancer cells whose growth depends on KDM5A. As KDM5A was shown to mediate drug tolerance, we investigated the ability of YUKA1 to prevent drug tolerance in EGFR-mutant lung cancer cells treated with gefitinib and HER2+ breast cancer cells treated with trastuzumab. Remarkably, this compound hindered the emergence of drug-tolerant cells, highlighting the critical role of KDM5A demethylase activity in drug resistance. The small molecules presented here are excellent tool compounds for further study of KDM5A’s demethylase activity and its contributions to cancer.

A fermentation broth of an unidentified fungus (N983-46) was found to produce DNA gyrase inhibitors, CJ-12, 371 (1) and CJ-12, 372 (2). Following isolation by solvent extraction and silica gel and ODS (reverse phase) chromatographies, the structures were determined to be novel spiro-ketal compounds with S-configuration at position C-1. CJ-12, 371 and CJ-12, 372 inhibit both DNA supercoiling and relaxation mediated by Escherichia coli DNA gyrase. The interaction of these compounds with DNA gyrase appears to be novel in that the compounds inhibit supercoiling and relaxation without blocking religation; thus, no cleavage intermediate of double strand DNA is observed. Both compounds have antibacterial activity against several species of pathogenic Grampositive bacteria, with MICs between 25 and 100μg/ml. These results suggest that the antibacterial potency of CJ-12, 371 and CJ-12, 372 is attributed to the inhibition of DNA gyrase. However, the compounds did not inhibit DNA gyrase selectively, as they also inhibited eukaryotic topoisomerase II-mediated relaxation. Semi-synthetic modifications to the dihydroxy motif in CJ-12, 371 altered both gyrase- and topoisomerase Il-inhibitory activities, but did not enhance selectivity.

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.

N,N-dimethylglycylamido (DMG) derivatives of 6-demethyl-6-deoxytetracycline and doxycycline bind 5-fold more effectively than tetracycline to the tetracycline high-affinity binding site on the Escherichia coli 70S ribosome, which correlates with a 10-fold increase in potency for inhibition of E. coli cell-free translation. The potencies of DMG-doxycycline and DMG-6-demethyl-6-deoxytetracycline were unaffected by the ribosomal tetracycline resistance factors Tet(M) and Tet(O) in cell-free translation assays and whole-cell bioassays with a conditional Tet(M)-producing E. coli strain.

A fermentation broth of an unidentified fungus (N983-46) was found to produce DNA gyrase inhibitors, CJ-12,371 (1) and CJ-12,372 (2). Following isolation by solvent extraction and silica gel and ODS (reverse phase) chromatographies, the structures were determined to be novel spiro-ketal compounds with S-configuration at position C-1. CJ-12,371 and CJ-12,372 inhibit both DNA supercoiling and relaxation mediated by Escherichia coli DNA gyrase. The interaction of these compounds with DNA gyrase appears to be novel in that the compounds inhibit supercoiling and relaxation without blocking religation; thus, no cleavage intermediate of double strand DNA is observed. Both compounds have antibacterial activity against several species of pathogenic Gram-positive bacteria, with MICs between 25 and 100 micrograms/ml. These results suggest that the antibacterial potency of CJ-12,371 and CJ-12,372 is attributed to the inhibition of DNA gyrase. However, the compounds did not inhibit DNA gyrase selectively, as they also inhibited eukaryotic topoisomerase II-mediated relaxation. Semi-synthetic modifications to the dihydroxy motif in CJ-12,371 altered both gyrase- and topoisomerase II-inhibitory activities, but did not enhance selectivity.