This protocol is useful for fragmented-base lead discovery campaigns as an additional tool for selecting promising fragments as heat candidates. Nano DSO for fragment screening uses a limited amount of unlabeled protein sample and can be routinely used for almost all protein targets. This method can be used to screen any collections of ligands.
or to verify heats from the other methods. Take out a fragment plate from the minus 20 or minus 80 degrees Celsius freezer and let it thaw at room temperature with gentle shaking on a bench top shaker. Centrifuge the plate at 500 times G for 30 seconds to collect any drops sticking to the side of the wells.
Take an MRC 2-well crystallization plate and pipette 15 microliters of the protein stock solution into each sub well using a multi-channel pipette. To check fragment and protein plate definitions on the Mosquito, switch on the nano dispenser and make sure that there are no obstacles around the moving components. Open the graphical user interface, click on the Setup tab and under Deck Configuration check whether the type of plate in which the compounds are supplied and the one in which the protein is transferred are already present in the list of available plates.
If not, click Options and then Plates and create a new plate definition by filling in the correct values for the property type. Under the Setup tab, specify the plate positions under Deck configuration. Using the dropdown menu, choose Grainier U bottom plate for position one and MRC 2-well plate for position two and save the protocol.
Place the fragment plate at position one and the protein plate at position two of the deck. In the protocol tab, click on File and choose the protocol saved in the previous step for dispensing the fragments. Define the volume of the fragments to be dispensed as 0.3 microliters.
For a typical plate where columns one and 12, do not contain fragments, define the Start location to column two, and the End location to column 11. In some plates, if columns two or 11 are empty, use columns three and 10 as start and end values. Click on Run to start the program.
After dispensing, which takes about two minutes is completed, remove the protein and the fragment plates from the robot and seal them with an adhesive sealing film. Briefly centrifuge the protein plate at 500 times G for 30 seconds to collect any drop sticking to the sides the wells before proceeding to the next step. Visually inspect the wells of the protein plate for precipitation that might've occurred due to the addition of the fragments.
If precipitation is observed in many wells, reduce the concentration of the fragments and repeat the experiment. Switch on the Prometheus instrument. On the touch screen, press Open Drawer to access the capillary loading module of the instrument.
Remove the magnetic strip from the loading module and clean the mirror with ethanol to remove any dust particles. To transfer a solution from the protein fragments plate to the capillaries, place the protein plate and the capillaries next to the instrument for easy access to the capillary loading module. Take one capillary holding it at one end and touch the solution in the protein plate with the other end of the capillary to transfer the sample.
Always wear gloves and make sure not to touch the capillary in the middle as impurities from the gloves would affect the measurement. Place the capillary in the designated position of the holder, making sure it is properly aligned and centered, repeat these steps to fill all positions in the loading module. At the end, place the magnetic strip on top of the capillaries to hold them in place and press Close Drawer to start the Nano-DSF experiment.
Open the Prometheus application PR.ThermControl and create a new project by clicking on Start New Session. First, do a discovery scan to detect the fluorescence of the samples. Alter the fluorescent signal of the samples by adjusting the excitation power of the laser.
For a thermal denaturation, click on the Melting Scan tab, set the start temperature to 20 degrees Celsius, the end temperature to 95 degrees Celsius and the temperature slope to one degree Celsius per minute. Then click on Start Measurement. Frequency distribution of the shift and melting temperature for the outer, kinetic, or highly expressed in cancer one protein, regulatory tetratricopeptide repeat domain of the monopolar spindle kinase 1, and SARS-CoV-2 3c-like protease are shown here.
Negative values indicate a reduction in the melting temperature in the presence of a fragment, while a positive value indicates an increase in melting temperature. For Nsp5, all fragments have a de-stabilizing effect, whereas for Hec1 and Mps1, both stabilizing and de-stabilizing hits are observed. This is a cheap and quick method to screen fragment libraries.
Other methods such as NMR or X-ray Crystallography can show the exact binding of fragments and they're also available through INX discovery. The results with coronavirus protease helped propose new compounds that can be developed into drugs against COVID-19.
