JH-RE-06

JH-RE-06 disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced-toxicity in cultured human and mouse cell lines.^[1]

Clonogenic survival assay—300 cells are plated in triplicate in 6-well plates for 24 hours. Cisplatin is added to relevant wells for 24 hours. All plates are incubated at 37℃ for 24 hours. Media are changed the next day and in fresh media JH-RE-06 (at 1.5 μM concentration) is added to untreated or cisplatin-treated cells for another 24 hours. Media are changed at the end of these combination treatments, and cells are allowed to recover for 7 days. To stain the resulting colonies, media are aspirated and the fixative (50% methanol and 10% glacial acetic acid) is added for 10 minutes, followed by the addition of 0.02% Coomassie brilliant blue R-250 stain in methanol: acetic acid: water in a ratio of 46.5:7:46.5 (v/v/v). Colonies that stained blue and contained at least 40 cells are counted. Relative cell survival or colony formation is calculated by dividing colony counts from treated samples by the DMSO or untreated controls.

JH-RE-06 improves tumor cell response to cisplatin in vivo. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft human melanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.^[1]