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.