Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

Joyce Sayegh,Jian Cao,Mike Ran Zou,Alfonso Morales,Lauren P Blair,Michael Norcia,Denton Hoyer,Alan J Tackett,Jane S Merkel,Qin Yan

doi:10.1074/jbc.m112.419861

Abstract

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.

Highlights

JARID1B is an H3K4 histone demethylase and an attractive target for cancer therapy
The current study is the first report of a high throughput screen for inhibitors of the Jumonji AT-rich interactive domain 1 (JARID1) family of demethylases
We showed that the apparent Km for bio-H3K4me3 is 15 nM. This is much lower than the reported Km for the JARID1B catalytic core [23], suggesting that other domains of JARID1B contribute to folding of the protein or substrate recognition and can be targeted for inhibition

Summary

Background

JARID1B is an H3K4 histone demethylase and an attractive target for cancer therapy. Results: High throughput screen identified novel compounds that can inhibit JARID1B demethylase activity. 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. Small molecule inhibitor screens of other JmjC domain-containing demethylases employed methods including detection of the reaction byproduct formaldehyde [17, 18], mass spectrometry [19], AlphaScreen [20], and LANCE Ultra and AlphaLISA assays [21] In these studies, ␣-KG analogues were reported to inhibit the JmjC demethylases [22]. Did not inhibit the H3K27me demethylases ubiquitously transcribed tetratricopeptide repeat protein, X-linked (UTX) or JmjC domain-containing protein 3 (JMJD3), indicating that PBIT is specific for the JARID1 enzymes This small molecule is able to modulate H3K4me levels in cells and attenuate proliferation of UACC-812 breast cancer cells. These studies reveal the identification of novel inhibitors of JARID1B in vitro with therapeutic implications for breast cancer

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION