Dynamic protein-protein interactions drive most cellular processes. Determining the precise timing of these interactions is crucial for elucidating molecular function. Herein, we developed a trifunctional small-molecule probe to study protein-protein interactions in a time-resolved manner. We take advantage of a destabilized dihydrofolate reductase domain (dDHFR) that can be fused to a protein of interest. Addition of the ligand trimethoprim (TMP) can stabilize the dDHFR protein fusion, preventing proteasomal degradation, and can thereby induce protein accumulation in cells. We synthesized analogs of TMP that maintained the trimethoprim moiety, thus stabilizing the fusion construct, and appended a terminal alkyne which is amenable to further functionalization (<i>e.g</i>. Click chemistry). Furthermore, our probe design includes different thiol reactive groups which can covalently react with a cysteine residue placed on the dDHFR by site-directed mutagenesis. We synthesized a panel of probe compounds with varying carbon linkers and thiol reactive groups and evaluated the stabilization and reactivity of dDHFR-yellow fluorescence (YFP) protein in human cell lines. Furthermore, we tested locations of engineered cysteine residues on the dDHFR protein to determine the most effective small molecule structure to stabilize and covalently engage the dDHFR-YFP fusion protein. Validation experiments have shown that this technology can rescue and capture a dDHFR-KRas fusion, confirming that the technique can be utilized in biologically relevant systems. With this molecule initiating protein accumulation, the Click handle can be derivatized with biotin for streptavidin pulldowns enabling mass spectrometric identification of interacting partners in a time-resolved manner. We also plan to utilize this tool to elucidate the timing of host-pathogen protein-protein interactions.

Synthesis and application of a trifunctional small-molecule probe to study time-resolved protein-protein interactions

ACS Spring Meeting 2021

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