It’s been a busy couple of weeks in and out of the lab for me which is why it’s been some time since my last post. I previously identified small molecule compounds of interest using a 19F NMR spectroscopy assay. To quantify and validate these small molecule compounds, I used a surface plasmon resonance (SPR) assay to determine binding affinities of compounds to the protein domain of interest. In the SPR assay the protein (USP5) is attached via a linker to a chip. When compounds bind to or disengage from the protein, there is a change in the level of organic matter at the surface of the chip, which results in a change in the angle at which an incident ray of light is reflected off a gold layer lining on the sensor chip surface. This allows for the measurement of binding constants. You can see experimental details on Zenodo.
All 12 compounds that were active in the 19F NMR assay were included in the SPR assay. Four of the compounds (DAT180, DAT194, DAT198, DAT201) showed weak binding with affinities in the micromolar range (Figure 1).
Figure 1. Replicate binding curves of DAT180 and USP5 Zf-UBD
It’s encouraging to see that the small molecules from the initial screen are binding weakly to USP5 Zf-UBD; however, there was a 3-10 fold variation between my first and second experimental repeats, so I definitely need to re-run this assay a few more times to have confidence in the binding affinities, as well as test these compounds with a secondary assay such as isothermal titration calorimetry. More on this later!