Discovery of a chemical probe for PRDM9

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.

Fig. 1. PRDM9 inhibition by MRK-740.

Chemical structures of (a) MRK-740 and (b) MRK-740-NC are presented. Inhibitory effect of (circle) MRK-740 and (triangle) MRK-740-NC on the methyltransferase activity of (c) PRDM9 and (d) PRDM7 were evaluated. Experiments were performed in triplicate using biotinylated H3 (1–25) peptide as a substrate as described in Methods. Data points are the average ± standard deviation from a minimum of three repeats (n = 3). Source data are provided as a Source Data file.

Fig. 2. Orthogonal confirmation of MRK-740 binding to PRDM9.

Binding affinity of MRK-740 was determined using SPR in the presence of 350 µM SAM (5xK_{m SAM}, 70 ± 10 µM). a A representative sensorgram (solid green) is shown with the kinetic fit (black dots). From kinetic fitting, a K_d value of 87 ± 5 nM, k_on of 1.2 ± 0.1 × 10⁺⁶ M⁻¹ S⁻¹ and k_off of 0.1 ± 0.01 s⁻¹ were obtained from quadruplicate experiments (n = 4). b The steady state response (black circles) obtained from A is shown with the steady state 1:1 binding model fitting (red dashed line). Biotinylated PRDM9 (amino acids 195–415) was immobilized on the flow cell of a SA sensor chip in 1x HBS-EP buffer, yielding 5700 RU. Using the buffer with 0.5% DMSO, 350 µM SAM and single cycle kinetic with 60 s contact time and a dissociation time of 120 s at a flow rate of 75 µL/min. MRK-740 was tested at 1 µM as the highest concentration with dilution factor of 0.33 for five tested concentrations. c Binding and selectivity of MRK-740 against PRDM family members were tested by DSF as described in Methods. Only binding to PRDM9 and PRDM7 with ∆T_m of higher than 2 °C was observed. Mechanism of action of MRK-740 was also evaluated by determining the IC₅₀ values (d) in the presence of fixed peptide (20 μM) and varying SAM concentrations as well as (e) fixed concentration of SAM (350 µM) and varying concentrations of peptide. The data indicate that MRK-740 is a SAM-dependent peptide-competitive inhibitor. All (c–e) experiments were performed in triplicate (n = 3) and data points are presented as average ± standard deviation. Source data are provided as a Source Data file.

Fig. 3. Co-crystal structure of MRK-740 bound to PRDM9.

a Surface representation of PRDM9 PR domain (white) bound by SAM (blue) and MRK-740. b Intermolecular interactions between MRK-740 and its binding pocket. Hydrophobic interactions (light blue), pi–pi interactions (yellow), CH–pi interactions (pink) and polar interactions (green) are represented by dashes. Structural alignment with the mouse holoenzyme (PDB accession code 4C1Q) showing steric clashes between MRK-740 and (c) the substrate lysine residue (green) and (d) the post-SET substrate recognition helix when in an active, substrate-bound conformation (orange). The corresponding helix in the inhibited, human structure is shown in (teal).

Fig. 4. MRK-740 binding is unique among methyltransferase inhibitors.

a Atoms tallied at the interface (distance < 4 Å) between SAM or SAH and small molecule inhibitors. Rossmann-type methyltransferases targeting nitrogen or oxygen are indicated in blue or orange, respectively. The tallied atoms (red) at the interface between SAM or SAH (teal) and small molecule inhibitors (pink) are shown for (b) PRDM9 with MRK-470, (c) SUV420H1 with 9ZY, (d) SETD7 with (R)-PFI-2, (e) SMYD2 with AZ506, (f) HNMT with Quinacrine, (g) PRMT5 with EPZ015666, and (h) COMT with a Mg²⁺ cation and 43J. The post-SET helices are indicated in purple. PDB accession codes: 6NM4, 5WBV, 4JLG, 5KJN, 1JQE, 4 × 61 and 4XUE, respectively. Data are from Supplementary Table 2.

Fig. 5. MRK-740 engages PRMD9 in a cell-based thermal stability assay.

a Effect of MRK-740 (10 µM) or MRK-740-NC control compound (10 µM) on the thermal stability of NanoLuc-tagged PRDM9 (residues 135–285) in cells. Shown is the mean ± SEM of three biological replicates. b T_m-shift (∆T_m) of NanoLuc-tagged PRDM9 in response to MRK-740 (10 µM) or MRK-740-NC control compound (10 µM). Shown is the mean ± SEM of three biological replicates. Boxes indicate the mean values. P-values calculated using an unpaired Welch’s two-tailed t-test, *p-value = 0.017. Source data are provided as a Source Data file.

Fig. 6. MRK-740 inhibits PRDM9 activity in cells.

a MRK-740 but not MRK740-NC decreases PRDM9-dependent K4 trimethylation of exogenous histone H3. HEK293T cells were co-transfected with H3-GFP and PRDM9-FLAG and treated with compounds at indicated concentrations for 20 h. Mut denotes PRDM9 catalytic mutant (Y357S). First and last lanes are controls at 0 µM of compound. b The graph represents non-linear fit of H3K4me3 fluorescence intensities normalized to intensities of GFP (MRK-740 n = 10, 4 separate experiments, MRK-740-NC n = 4, 2 separate experiments). c MRK-740 and MRK-740-NC do not affect HEK293T cell growth up to 10 µM. Cells were treated with compounds for 1 day. Cell number was measured using IncuCyte™ ZOOM live cell imaging device. The results are mean ± sd, n = 3. Source data are provided as a Source Data file.

Fig. 7. Concentration-dependent inhibition of PRDM9-dependent H3K4 trimethylation using MRK-740.

ChIP qPCR analyses of H3K4me3 methylation levels of known PRDM9-bound loci and control loci. Cells transfected with empty vector control, or a vector overexpressing wild-type PRDM9 were treated with DMSO, increasing concentrations of MRK-740 or MRK-740-NC. Concentrations used (µM) are indicated below each plot. (a–d) represent reported loci of PRDM9 methylation; (e, f) are transcriptional start sites (TSSs) which are not known loci of PRDM9 methylation. Data are normalized to total H3 and are presented as the mean ± upper and lower limits from two replicates. Representative plots of two independent experiments are shown in this figure. The coordinates and the genomic features associated with the assayed loci are indicated above each plot. Left Tailed Student's t-tests were performed by comparing DMSO (0 µM) treated cells to compound treated cells that have been transfected with the same plasmids. *p-value < 0.05 and **p-value < 0.01. Note that here we show a representative of two independent experiments (mean ± s.d of technical replicates) for our ChIP-qPCR experiments. Data obtained using this technique are inherently noisy due to stochastic differences in gene activity/histone positioning. Data are thus typically presented as representative rather than averaged. Source data are provided as a Source Data file.