Protein immobilization confers the advantages of biological systems to a more chemical setting and has applications in catalysis, sensors, and materials development. While numerous immobilization techniques exist, it is optimal to develop a well-defined and chemically stable methodology to allow for full protein function. This paper describes the utilization of unnatural amino acid technologies to introduce bioorthogonal handles in a site-specific fashion for protein immobilization. To develop this approach a range of solid-supports, organic linkers, and protein immobilization sites have been investigated using a GFP reporter system. Overall, a sepharose resin derivatized with propargyl alcohol has afforded the highest yields of immobilized protein. Moreover, an unnatural amino acid residue protein context has been demonstrated, signifying a necessity to consider the protein site of immobilization. Finally, a resin-conferred stabilization was demonstrated in several organic solvents.
The NSD family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting significant challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in development of the first-in-class irreversible small molecule inhibitors of the NSD1 SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead, compound BT5, demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of H3K36 dimethylation and downregulation of target genes, and impairs colony formation in NUP98-NSD1 patient sample. This study will facilitate development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.
The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.
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