Background
Our client asked to characterize the interaction of the oncology clinical candidate MRTX1719 with two forms of Protein Arginine Methyl Transferase 5 (PRMT5:MEP50), bound to either the cofactor SAM or the metabolite MTA (PRMT5•SAM and PRMT5•MTA). The goal was to determine the affinity and kinetic properties of MRTX1719 for each PRMT5 form and compare this with its known specificity for killing MTAP-deleted cells versus wild-type cells.

Challenge
When characterizing the affinity of MRTX1719 for PRMT5•SAM and PRMT5•MTA, we found that the dissociation rate (koff) of the complex was too slow to be accurately determined by SPR. In single-cycle kinetics experiments with a prolonged dissociation period, MRTX1719 showed minimal dissociation, preventing the measurement of sufficient dissociation to determine koff. Compounds with such slow dissociation rates are rare, and since SPR is the primary method for measuring small molecule dissociation, such cases present unique challenges.

Solution
We used an adapted SPR chaser assay to indirectly determine the dissociation rate constant of MRTX1719, due to the inability to directly observe its dissociation. This method, enables the measurement of small molecule dissociation with a half-life greater than 24 hours. We introduced three chaser compounds with suitable kinetic properties that compete with MRTX1719 for binding to PRMT5. By assessing the binding of the chaser molecules at different dissociation points, we determined the remaining fraction of MRTX1719 bound to the protein, based on the amount of chaser binding. The KD and half-life were determined by fitting MRTX1719 occupancy data to an exponential decay function.

Achievements
Using the chaser assay, we successfully determined the dissociation rate constant (koff) and affinity (KD) values of MRTX1719 for both forms of PRMT5, which was not achievable with a direct binding assay. MRTX1719 showed a long dissociation half-life when bound to PRMT5•MTA and a shorter half-life when bound to PRMT5•SAM. These represent some of the longest complex half-lives reported in SPR experiments at the time of publication.

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