Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

We work between the departments of chemistry and life sciences, developing novel assisting reactive covalent ligands for a range of anti-cancer drug targets. It's hoped that these molecules will be used in the future, either as therapeutics or possibly as chemical probes. So several technologies exist for screening covalent ligands.

These can be broadly split into proteomic techniques whereby compounds are incubated with cultured cells and mass spectrometry used to determine which proteins the molecules label and target-based methods where libraries of electrophiles are incubated with individual proteins. A significant experimental challenge relates to the reactivity of covalent ligands. Overly reactive molecules will often appear as hits during screening assays, but are unsuitable as they'll non-selectively label many different proteins in the cell.

Compound of activity is, therefore, crucial to be considered at every step of covalent drug discovery. So the Mann and Armstrong groups at Imperial have developed a new method of screening electrophiles against a protein target called the quantitative irreversible tethering or the qIT assay. Our protocol provides a much-needed, new method of screening electrophiles against a protein target, which is scalable, does not require mass spectrometry, and controls for compound intrinsic reactivity.

To begin, prepare all the required stock solutions and leave them at room temperature for two hours before the experiment. Degas quantitative, irreversible tethering, or qIT assay buffer with argon gas for approximately 30 minutes. For a performing buffer exchange, dilute the protein solution in excess of degassed qIT assay buffer in a spin filter unit, and spin at 4, 000 G for 15 minutes at four degrees Celsius to concentrate the protein.

Then dilute the target protein solution to a concentration of 15 micro molars in degassed qIT assay buffer to prepare nine milliliters of the solution. Dispense TCEP-agraose solution, qIT buffer, and glutathione stock into the appropriate wells of the 384-well plates. Transfer 1.8 microliters from each corresponding well of the fragment library plate to create a compound dilution plate diluting each compound to 1.5 millimolar in qIT assay buffer, plus 3%DMSO.

Use a 384 pipetting station to transfer 20 microliters of compound solution from each well of the compound dilution plate into both the reaction plates. To begin, set up the quantitative irreversible tethering or qIT assay with the desired protein and glutathione. Dispense 500 Micromolar CPM stock into 111.2 microliter aliquots in microcentrifuge tubes and store them at 20 degrees Celsius.

After thawing the CPM stock, add it to 40 milliliters of qIT assay quench buffer to prepare a 1.4 micromolar solution Dispense 27 microliters of CPM Quench solution into each well of two, fresh 384-well plates. Centrifuge the protein reaction plate at 200 G for three minutes to pellet TCEP-agarose. At predetermined time points, use a 384 pipetting station to transfer three microliters of sample from every well of the protein reaction plate to the CPM quench plate.

Remove the sample from the top of the well avoiding aspirating TCEP-agarose at the bottom. Incubate the CPM quench plates at room temperature for one hour, and measure the fluorescence intensity with an excitation at 384 nanometers and emission at 470 nanometers. To begin, perform quantitative-irreversible tethering or the qIT assay with the desired protein and quench the samples in CPM plate.

For each quench, calculate Z prime as a measure of assay quality. Then calculate the mean fluorescence values for the negative control wells in columns one and two and the positive control wells in columns 23 and 24. Normalize each reaction well to the average fluorescence values of the positive and negative controls, and separately plot the percent modified versus time for each compound reacting with glutathione and with the target protein.

Using GraphPad, fit normalized percent modified versus time data to a one phase exponential decay model for both the reactions using the equation, AE to the power of KT C.Use the rate at which fragments react with protein and glutathione to calculate the rate enhancement factor or REF value for each fragment. Finally, to select Hit fragments, set an REF threshold, an arbitrary Hit rate, or select fragments with REF three standard deviations greater than the geometric mean. Compound one reacted with the cysteine residue on the target protein at a six-fold greater rate than with glutathione, meeting the Hit criterion of REF greater than three.

Compound two did not show an accelerated reaction rate with the target cysteine residue compared to glutathione, indicating a lack of productive non-covalent interactions and was, therefore, not selected as a hit.