Characterization of the Biochemical Activity of Ktx-1001, a Selective Small Molecule NSD2 Inhibitor, in Surface Plasmon Resonance (SPR)

Background: NSD2 (Nuclear Receptor Binding SET Domain Protein 2), also known as MMSET (Multiple Myeloma SET Domain-Containing Protein) is a Histone Lysine-Methyl Transferase (HMT) that specifically produces mono- and di-methylated histone 3 at Lysine 36 and regulates gene transcription. NSD2 is implicated in multiple malignancies due to either overexpression resulting from translocation of chromosomes 4 and 14 (t(4;14)) as in multiple myeloma, or from the presence of gain of function mutations in acute lymphocytic leukemia and multiple solid tumors, including prostate cancer. These genomic aberrations that impact NSD2 are associated with poor prognosis, making NSD2 an attractive target for drug development. Development of NSD2 small molecule inhibitors is challenging, primarily due to: 1) conservation of the SAM binding pocket, which poses selectivity hurdles for the HMT family members and 2) competition with high intracellular SAM concentrations, for binding pocket inhibitors. A potent small-molecule NSD2 inhibitor, KTX-1001, is currently an experimental drug in clinical development (NCT05651932).

Study Design and Methods: Evaluation of the nature of binding of KTX-1001 to its target was conducted using surface plasmon resonance (SPR) to quantify on-rate, off-rate, and equilibrium dissociation constant utilizing the NSD2 SET domain as the immobilized target. In this assay format, no saturable or specific binding could be observed, despite the potent activity in the enzymatic assay using either full length NSD2 or the SET domain sequence, and radiolabeled SAM. To interrogate the discordance between potent enzymatic activity and the lack of detectable binding in the aforementioned assay, a series of experiments were designed in which KTX-1001 was tethered to the surface of the chip, with target (NSD2 SET domain) applied in the solution phase, with and without nucleosomes and with and without cofactor, in a “reverse” type SPR format.

Results: The mean binding affinity (K_D) was determined for the tethered KTX-1001 molecule to SET, SET/nucleosomes, SET/SAM, and a combination of SET/SAM/nucleosomes. A mean K_D of ~70 nM for SET, versus an IC₅₀ of 2.31 nM for untethered KTX-1001 in the biochemical assay, is indicative of the tethered KTX-1001 successfully binding to the SET domain in the “reverse” SPR format. Repetition of the experiment in the presence of nucleosomes resulted in a K_D of ~15 nM, a nearly 5-fold tighter binding to SET. SET preincubated with SAM bound tethered KTX-1001 with a K_D of ~18 nM, demonstrative of non-interference of SAM with binding. Measurement of SET, SAM, and nucleosomes provided a K_D of ~6 nM for the tethered KTX-1001 for an 11-fold tighter binding compared to SET alone, approaching the IC₅₀ of 2.3 nM measured in the biochemical assay.

Conclusion: The experiments demonstrated that the tethered molecule bound to the NSD2 SET domain in a specific and saturable manner, with an affinity comparable to the IC50 determined in the enzymatic assay, and identified KTX-1001 as a unique binder of NSD2 in the presence of cofactor and substrate. These data also identify that SPR in “reverse” mode is a useful tool to characterize small molecule binding events not amenable to standard SPR format. A Phase 1 study evaluating KTX-1001 in relapsed/refractory multiple myeloma is ongoing (NCT05651932).