Potency assay (off target):
UNC0638 was inactive against other H3K9 (SUV39H1 and SUV39H2), H3K27 (EZH2), H3K4 (SETD7, MLL and SMYD3), H3K79 (DOT1L) and H4K20 (SETD8) methyltransferases, as well as PRDM1, PRDM10 and PRDM12. In addition, UNC0638 was inactive against protein arginine methyltransferases PRMT1 and PRMT3, and HTATIP, a histone acetyltransferase. UNC0638 had weak but measurable activity against JMJD2E (IC50 = 4,500 ± 1,100 nM (n = 3)), a Jumonji protein demethylase and DNA methyltransferase DNMT1 (IC50 = 107,000 ±
6,000 nM (n = 2)). Nevertheless, the selectivity of UNC0638 for EHMT2 and EHMT1 over JMJD2E was >200-fold, and selectivity for EHMT2 and EHMT1 over DNMT1 was >5,000-fold.
Probe Selectivity in
Vitro:
Selectivity of UNC0638 was evaluated across non-epigenetic targets, including GPCRs, ion channels, transporters and kinases. UNC0638 was clean (<30% inhibition at 1 μM) against 26 out of 29 targets in the Ricerca Selectivity Panel. At 1 μM concentration, UNC0638 showed 64%, 90% and 69% inhibition of muscarinic M2, adrenergic α1A and adrenergic α1B receptors, respectively. Because UNC0638 hit three GPCRs in the Ricerca Selectivity Panel, we further assessed its selectivity against GPCRs by testing UNC0638 in the NIMH's Psychoactive Drug Screen Program Selectivity Panel, which consists of a total of 45 targets, including 36 GPCRs. UNC0638 had <50% inhibition at 1 μM against 39 targets in the panel, and >50% inhibition at 1 μM against six targets in the panel. Ki in the radioligand binding assay for each of the six interacting GPCRs was subsequently determined. The Ki measurements showed UNC0638 was at least 100-fold selective for EHMT2 over these six GPCRs. In M1, M2 and M4 functional assays, UNC0638 had no agonist activity, low antagonist potency against M1 and M4 (IC50 > 10,000 nM (n = 2)), and modest antagonist potency against M2 (IC50 = 480 ± 10 nM (n = 2)). Furthermore, UNC0638 was tested against a panel of 24 kinases and showed <10% inhibition at 1 μM against these kinases.
