Small-molecule drugs arise as a prospective area to treat different types of cancer. A promising target is the NSD protein family. These proteins have been related to cancers like myeloid leukemia, multiple myeloma, prostate, lung, and breast cancer. However, their treatment is limited to chemotherapy, radiotherapy, and surgical operation that could affect the patient's life quality. In 2020, Huang and collaborators developed a novel kind of inhibitor for NSD1 protein, BT5. This inhibitor covalently binds to the SET domain of the NSD family proteins. However, there is a high affinity for NSD1 than their counterparts. These proteins share a similar structure, but their dynamics could explain the affinity difference. In this project, we compare the NSD family protein dynamics by measuring NMR relaxation experiments. We identify a higher percentage binding for NSD1 and NSD3 to BT5 than NSD2. We also determine the perturbed chemical shifts under the presence of BT5 in NSD1, where the most affected regions are the SET and post-SET domain (auto-inhibitory loop) and the beginning region of the AWS domain. By comparing different NMR relaxation measurements, we identify that the three proteins share high dynamics in the auto-inhibitory loop region, especially in NSD1, and in the AWS domain for NSD1 and NSD3. These motions corresponds to the obtained results by adding BT5 in NSD1, which could indicated a relationship between the AWS dynamics and the auto-inhibitory loop, and the protein affinity.

Mutations in epigenetic regulators such as the SET domain-containing methyltransferase NSD2 are of high interest among the research community nowadays. The involvement of this mutations in multiple diseases put them in the spotlight. Interestingly, the change of the glutamic acid residue in the position 1099 of the NSD2 SET domain for a lysine residue has been recurrently found in multiple myeloma patients. This mutation produces a hyperactive enzyme that hypermethylate the natural enzymatic substrate: the lysine in the position 36 of the basic tail of the histone 3 in the nucleosomal context. Apparently, this hyperactivation may be related to the disruption of a critical salt-bridge that stabilize an autoregulatory loop of the NSD2 catalytic site. However, despite the extensive research that have been done around this phenomenon, the molecular mechanism behind this hyperactivation still remains unknow. For this reason, in this study we addressed this matter from a structural point of view by evaluating the structure and dynamics of the protein in solution by high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy and biophysical techniques. We found increased local segmental motions in us to ms timescale that induced protein flexibility that may correlate with gain-of-function of E1099K miss-sense mutation. Further functional studies with the native substrates in vitro and in vivo are needed to understand this observation.