This method allows us to characterize which chemotherapy drugs are hydrolyzed by SAMHD1 and can be used as a screening assay to identify small molecule inhibitors of this enzyme. The main advantage of this technique is that it is simple, inexpensive, and yields vast amount of information about how different small molecules interact with this enzyme. Demonstrating the procedure will be Miriam, a postdoc from the group.
To begin, prepare complete SAMHD1 reaction buffer by adding Tween-20 and TCEP to the final concentrations of 0.005%and 0.3 millimolar respectively. Prepare EDTA stop solution by adding EDTA to a final concentration of 7.9 millimolar in complete SAMHD1 reaction buffer. Prepare malachite green, or MG working solution by mixing 10 parts of MG stock solution with 2.5 parts of 7%ammonium molybdate and 0.2 parts of 11%Tween-20.
Using a multichannel pipette, serially dilute test compounds in the relevant solvent at 100 times the final concentration in a clear round bottom polypropylene 96-well plate. further dilute these compounds to 25 times the final concentration with complete SAMHD1 reaction buffer to maintain the final solvent concentration below 1%Transfer five microliters of diluted component to the appropriate wells of a clear 384-well flat bottomed assay plate and repeat the procedure with solvent only control samples. For the enzyme master mix preparation, dilute recombinant human SAMHD1 protein and recombinant pyrophosphatase in complete SAMHD1 reaction buffer to four times the desired final concentration.
Prepare activator or substrate dGTP by diluting the solution in complete SAMHD1 reaction buffer to attain 50 micromolar concentration. To the wells containing compound dilutions and solvent only control, dispense five microliters of SAMHD1 pyrophosphatase master mix, and to the no enzyme control wells, add five microliters of complete SAMHD1 reaction buffer. Then dispense 10 microliters of dGDP solution into all wells to start the reaction and incubate for 20 minutes at room temperature.
To stop the reaction, add 20 microliters EDTA stop solution. After adding 10 microliters of MG working solution to all the wells, mix the content using an orbital microwell plate shaker and centrifuge the plate at 1000 x g for one minute. Incubate the plate for 20 minutes at room temperature and read the absorption at 630 nanometers in a microwell plate reader.
Calculate the standard deviation of the positive control wells and then calculate the average. Similarly for the negative control wells, calculate the standard deviation and then calculate the average. Calculate the Z factor is an indicator of the assay quality.
Normalize each absorbance value to the mean values of positive and negative controls. Setting the positive control as 100%SAMHD1 activity and the negative control as 0%SAMHD1 activity. Plot SAMHD1 activity is a function of compound concentration and fit a four parameter variable slope dose response curve, allowing determination of the compound's half maximal inhibitory concentration.
Dilute nucleotide analog stocks and complete SAMHD1 reaction buffer to four times the final concentration and transfer five microliters to the appropriate wells of a 384-well assay plate. To prepare enzyme SAMHD1 or pyrophosphatase master mix, dilute recombinant human SAMHD1 protein and Escherichia coli pyrophosphatase in complete SAMHD1 reaction buffer to twice the desired final concentration. Prepare only pyrophosphatase solution by diluting recombinant Escherichia coli pyrophosphatase to twice the desired final concentration in complete SAMHD1 reaction buffer.
Similarly, prepare activators GTP and dGTP alpha S by diluting the stock in complete SAMHD1 reaction buffer to four times the final concentration. Dispense five microliters of the activator, either GDP or dGTP alpha S or complete SAMHD1 reaction buffer to the appropriate wells of a 384-well assay plate containing the nucleotide analogs. Start the reaction by dispensing 10 microliters of SAMHD1 pyrophosphatase master mix, pyrophosphatase alone or complete SAMHD1 reaction buffer to the appropriate wells and incubate for 20 minutes at room temperature.
After stopping the reaction using EDTA stop solution, add 10 microliters of MG working solution to all the wells. Once the contents are mixed, centrifuge the plate at 1000 x g for one minute and incubate for 20 minutes at room temperature. Then measure the absorbance at 630 nanometers.
Calculate the average absorbance values for the pyrophosphatase only reaction wells, which will be the background value. Then subtract the background value from the corresponding wells in the SAMHD1 or pyrophosphatase reactions. Finally, plot the corrected absorbance values for each nucleotide analog with buffer GTP and dGTP alpha S conditions.
In the present study, the absorbent increased with increasing sodium phosphate concentration following incubation with malachite green reagent and reached saturation at 0.25 millimolar concentration. The linear detection range of phosphate is visible from 0.004 to 0.03 millimolar. The requirement of assay components to achieve measurable SAMHD1 activity is illustrated.
Neither SAMHD1 nor pyrophosphatase alone could generate an organic phosphate in the presence of dGTP. However, when all the assay components were present, an increase in signal was observed. The dose response curves obtained for SAMHD1 inhibitor compounds showed that increasing concentrations effectively inhibit SAMHD1 activity.
Hydroxyurea does not inhibit SAMHD1 activity in vitro, which was indicated by unaffected SAMHD1 activity with increasing hydroxyurea doses. dGTP binding to both allosteric sites activate SAMHD1, allowing subsequent hydrolysis of this nucleotide, whereas the other three canonical dNTPs first require GTP activation of the first allosteric site before they can bind the second allosteric site and then be hydrolyzed at the catalytic site. For nucleotide analogs, clofarabine triphosphate is an allosteric site two activator and a substrate as activity is observed in the presence of GTP.
Whereas cytarabine triphosphate is only able to occupy the catalytic site as dGTP alpha S is required to observe activity. No activity was observed with gemcitabine triphosphate. Following the identification of small molecules inhibiting SAMHD1 in this assay by physical approaches, such as thermal shift assays, can be used to interrogate target engagement.
The method here shown helped us understand which of the cancer drugs can be hydrolyzed by SAMHD1 and establish this enzyme as a potential therapeutical target to overcome drug resistance.
