Technical Notes

2 TECHNICAL NOTE ©2020 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. In this technical note, well-known interactions between carbonic anhydrase II (CAII) and a set of small molecule inhibitors are analyzed to demonstrate the ability of the Monolith Pico instrument to characterize interactions in both a labeled and label-free way. First, the interaction of CAII with Furosemide is analyzed under native conditions using the LabelFree detector. Next, the interaction of labeled CAII with three small molecule inhibitors — Furosemide, Acetazolamide and Benzenesulfonamide — are measured using the RED detector. This allows for the precise quantification of the lower nM K d of Acetazolamide. The analysis shows Monolith Pico instrument's ability to provide the flexibility needed to cover most projects in the pipeline. analyzed with the MO.Affinity Analysis 3 so ware. Figure 1 shows the resulting dose-response curve that was fitted to a one-site binding model to extract the K d of 344 ± 40 nM, which is in good agreement with literature values 1,2 . In a complementary approach, CAII was fluorescently labeled using the RED-NHS 2nd Generation dye (Cat# MO-LO11), and its affinity for three small molecule inhibitors was measured. To do this, a 16-step 1:1 serial dilution of compounds with 10 µL volume in each sample was prepared, starting with a highest concentration of 100 µM of Furosemide and Benzenesulfonamide and 5 µM of Acetazolamide. Next, 10 µL of 20 nM labeled CAII was added to all dilutions. MST measurements were performed using the RED Figure 1: Label-free interaction analysis of CAII and Furosemide showed K d value in agreement with the literature. Furosemide was titrated against non-labeled CAII, inducing significant changes in the initial fluorescence signal. This initial fluorescence change was used for analysis and a K d value of 344 ± 40 nM was obtained (n = 3 independent measurements, error bars represent the standard deviation). 0. 1 1 1 0 1 00 1 0 3 10 4 1 0 5 1 0 6 Ligand Concentration [nM] 4000 6000 8000 10000 12000 Raw Fluorescence [counts] Results For the label-free affinity quantification of CAII against Furosemide, a 100 µM solution of the ligand Furosemide was prepared. This solution was used for a 16-step 1:1 serial dilution with 10 µL volume in each sample. Next, 10 µL of 400 nM unlabeled CAII was added to all vials of the serial dilution. Samples were mixed by pipetting up and down, were loaded into capillaries and were then transferred into the Monolith Pico instrument. MST measurement was carried out using the LabelFree detector at a LED power of 60% and medium MST power. A ligand-induced fluorescence change was observed, verified by the SD-Test (data not shown) and the data was

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