Discovery of a chemical probe for PRDM9

Abdellah Allali-Hassani,John M Sanders,Solomon D Kattar,Peter J Brown,David Dilworth,Philipp Kaldis,Dalia Baršytė-Lovejoy ,Matthieu Schapira,Magdalena M Szewczyk,Ernesto Guccione,Genna M Luciani,Nathan Palmer,Qin Wu,Evelyne Lima‐Fernandes ,Mohammad S Eram,Levi L Blazer ,S Zakiah A Talib,L Halabelian ,Jabez Bok,Danton Ivanochko,David Jonathan Bennett ,Fengling Li,Benjamin Nicholson,Shawna Organ ,Ronan O’hagan ,P Loppnau ,Jessica Ho ,C.h Arrowsmith ,Masoud Vedadi

doi:10.1038/s41467-019-13652-x

Abstract

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.

Highlights

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36
In order to identify small molecule inhibitors of PRDM9, we used a radioactivity-based methyltransferase assay to screen a library of 7500 compounds, including some of the most diverse compounds in the MSD compound collection, compounds similar to known methyltransferase inhibitors, and candidate compounds from a virtual screening campaign
An extensive follow up SAR campaign led to the identification of MRK-740 as a potent PRDM9 inhibitor with IC50 value of 80 ± 16 nM (Fig. 1a, c, Supplementary Data 2 and 3, and Supplementary Methods)

Summary

Introduction

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. PRDM9 is normally expressed solely in germ cells entering meiotic prophase in female fetal gonads and in postnatal testis[8] It was first identified as a lysine methyltransferase with activity toward histone 3 lysine 4 (H3K4)[8]. In PRDM9-deficient testis, lower levels of H3K4 trimethylation and altered meiotic gene transcription led to impairment of double-stranded breaks and pairing between homologous chromosomes. These data identified an essential function for PRDM9 in spermatocytes and progression of meiotic prophase[8].

Methods

Results

Conclusion