We thank Thomas Lundb&#228;ck and members of Thomas Helleday&#39;s laboratory for advice and support. Part of this work was facilitated by the Protein Science Facility at Karolinska Institutet/SciLifeLab (http://ki.se/psf), and we acknowledge the National Cancer Institute (NCI), Division of Cancer Treatment and Diagnosis (DCTD), and Developmental Therapeutics Program (DTP) (http://dtp.cancer.gov) for providing a compound. Funding was provided by grants awarded to S.G.R. from the Swedish Research Council (2018-02114), the Swedish Cancer Society (19-0056-JIA, 20-0879-PJ), the Swedish Childhood Cancer Fund (PR2019-0014), and Karolinska Institutet.

A schematic overview of the methods below is depicted in Figure 1 and a detailed list of materials and reagents is available in the Table of Materials.
1. Preparation of assay buffers.
<ol>
	<li>Preparation of stock buffers. 
	NOTE: As the assay is sensitive to the detection of phosphates, which can be commonplace, rinse glassware three times with ultrapure or double-distilled water to avoid contamination. All buffers can be stored at room temperature (RT).
	<ol>
		<li>Prepare 1 L of SAMHD1 reaction buffer (RB) stock solution (25 mM Tris-Acetate pH 8, 40 mM NaCl, 1 mM MgCl2) by dissolving 4.5 g Tris Acetate, 2.3 g NaCl, and 0.2 g MgCl2, in approximately 800 mL of water before adjusting to pH 8 and final volume.</li>
		<li>Prepare 5 mL of 0.1 M TCEP stock solution by diluting 1 mL of 0.5 M TCEP into 4 mL of water.</li>
		<li>Prepare 50 mL of 11% Tween-20 stock solution by diluting 5 mL of 100% Tween-20 into 44.5 mL of water. 
		NOTE:&#160;Tween-20 is light sensitive.</li>
		<li>Prepare 50 mL of 0.5 M EDTA stop solution by dissolving 9.3 g EDTA in approximately 40 mL of water before adjusting to pH 8 and final volume.</li>
		<li>Prepare Malachite Green (MG) stock solution (3.2 mM malachite green in H2SO4) by slowly adding 60 mL concentrated sulfuric acid to 300 mL water in a brown glass bottle. Cool the solution to RT and dissolve 0.44 g malachite green. 
		CAUTION: The reaction of sulfuric acid with water is exothermic and so the bottle may heat up causing a build-up of pressure; ensure this pressure is released frequently. 
		NOTE: The resulting orange solution is light sensitive (hence brown bottle) and stable for at least 1 year at RT. Precipitate may form over time, ensure only the supernatant is used.</li>
		<li>Prepare 50 mL of 7% ammonium molybdate stock solution by dissolving 3.75 g ammonium molybdate in 50 mL of water. 
		&#8203;NOTE: Precipitate may form over time, ensure only the supernatant is used.</li>
	</ol>
	</li>
	<li>Preparation of complete assay buffers 
	NOTE: This should be done on the day of the experiment
	<ol>
		<li>Prepare complete SAMHD1 RB (25 mM Tris-Acetate pH 8, 40 mM NaCl,&#160;1 mM MgCl2, 0.3 mM TCEP, 0.005% Tween-20). Use previously prepared 11% Tween-20 and 0.1 M TCEP stocks to add these components at a final concentration of 0.005% for Tween-20 and 0.3 mM for TCEP to the SAMHD1 RB stock.</li>
		<li>Prepare EDTA stop solution (25 mM Tris-Acetate pH 8, 40 mM NaCl,&#160;1 mM MgCl2, 0.3 mM TCEP, 0.005% Tween-20, 7.9 mM EDTA). To complete SAMHD1 RB, use 0.5 M EDTA stock solution to add EDTA to a final concentration of 7.9 mM.</li>
		<li>Prepare MG working solution (2.5 mM malachite green, 1.4% ammonium molybdate, 0.18% Tween-20) by mixing 10 parts of MG stock solution with 2.5 parts of 7% ammonium molybdate and 0.2 parts of 11% Tween-20.</li>
	</ol>
	</li>
</ol>
2. SAMHD1 inhibition assay and determination of compound IC50
NOTE: Final assay conditions are shown in Table 1.
<ol>
	<li>Preparation of compounds in assay plate 
	NOTE: Small molecular weight compounds are typically dissolved in 100% DMSO and nucleotide analogues in water. Stock concentration ranges from 10 to 100 mM and is influenced by the potency and solubility of the compounds, together with the DMSO tolerance of the assay. Check that the final DMSO concentration in the reaction does not exceed 1% to ensure enzyme activities are not affected by this solvent. It is good practice to test the tolerance of the assay to the solvent prior to the experiment.
	<ol>
		<li>Prepare serially diluted test compounds at 100x final concentration in the relevant solvent (e.g., 100% DMSO for small molecules or water for nucleotide analogues) in a clear round-bottomed polypropylene 96-well plate using either a multichannel pipette or automated liquid handling equipment. 
		NOTE: Depending upon compound stability, dilution plates can be prepared in advance, sealed, and stored at -20 &#176;C. Allow plates to equilibrate to RT before continuing the protocol.</li>
		<li>Using complete SAMHD1 RB, dilute compounds to 25x final concentration (to maintain the final solvent concentration below 1%) and transfer 5 &#181;L to the appropriate wells of a clear 384-well flat-bottomed assay plate. Repeat the procedure with solvent-only control samples.</li>
	</ol>
	</li>
	<li>Preparation of reaction components 
	NOTE: This should be done on the day of the assay. Recombinant human SAMHD1 and E. coli pyrophosphatase (PPase) aliquots are stored long term at -80 &#176;C diluted at 9.1 mg/mL and 23.0 mg/mL, respectively, in storage buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP). Once thawed, aliquots are stored short-term at -20 &#176;C.
	<ol>
		<li>Prepare enzyme (SAMHD1/PPase) master mix by diluting recombinant human SAMHD1 protein and recombinant PPase in complete SAMHD1 RB to 4x desired final concentration, thus 1.4 &#181;M SAMHD1 and 50 U/mL PPase.</li>
		<li>Prepare activator/substrate dGTP by diluting dGTP stock (typically 10 or 100 mM in water) in complete SAMHD1 RB to 2x final concentration, thus 50 &#181;M dGTP.</li>
	</ol>
	</li>
	<li>Perform the assay 
	NOTE: All assay components should be equilibrated to RT. Liquid additions can be performed with either a multichannel pipette or a bulk reagent liquid dispenser.
	<ol>
		<li>To 384-well assay plate containing compound dilutions and solvent only controls, dispense 5 &#181;L of SAMHD1/PPase master mix. To no enzyme control wells, dispense 5 &#181;L of complete SAMHD1 RB. Pre-incubate enzyme and compounds for 10 min at RT.</li>
		<li>To all wells, dispense 10 &#181;L of 2x dGTP solution to start the reaction.</li>
		<li>Incubate the reaction for 20 min at RT.</li>
		<li>Stop the reaction by dispensing 20 &#181;L EDTA stop solution to all wells. 
		NOTE: The experiment can be paused here if desired.</li>
		<li>Add 10 &#181;L MG working solution to all wells. 
		CAUTION: MG working solution contains sulfuric acid.</li>
		<li>Ensure mixing of well contents using an orbital microwell plate shaker and centrifugation at 1,000 x g for 1 min.</li>
		<li>Incubate the plate for 20 min at RT.</li>
		<li>Read the absorption at 630 nm wavelength in a microwell plate reader.</li>
	</ol>
	</li>
	<li>Data visualization and analysis
	<ol>
		<li>Calculate the average and standard deviation of the positive and negative control wells (positive, complete reaction with solvent; negative, dGTP alone with solvent). Calculate Z-factor37&#160;as an indicator of assay quality.</li>
		<li>Normalize each absorbance value to the mean values of the positive and negative controls, setting the positive control as 100% SAMHD1 activity and the negative control as 0% SAMHD1 activity.</li>
		<li>Plot SAMHD1 activity (%) as a function of compound concentration and fit a four-parameter variable slope dose-response curve, allowing determination of compound IC50.</li>
	</ol>
	</li>
</ol>
3. SAMHD1 activator and substrate screen
NOTE: Final assay conditions are shown in Table 2. Recombinant SAMHD1 and PPase aliquots are stored long term at -80 &#176;C diluted at 9.1 mg/mL and 23.0 mg/mL, respectively, in storage buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP) at -80 &#176;C. Once thawed, aliquots are stored short term at -20 &#176;C.
<ol>
	<li>Preparation of nucleotide analogues in assay plate
	<ol>
		<li>Dilute nucleotide analogue stocks (typically 10 or 100 mM in water) to 4x final concentration in complete SAMHD1 RB, in our case 800 &#181;M nucleotide analogue, and transfer 5 &#181;L to the appropriate wells of a 384-well assay plate.</li>
	</ol>
	</li>
	<li>Preparation of reaction components 
	NOTE: This should be done on the day of the assay
	<ol>
		<li>Prepare enzyme (SAMHD1/PPase) master mix by diluting recombinant human SAMHD1 protein and recombinant E. coli PPase in complete SAMHD1 RB to 2x desired final concentration, thus 0.7 &#181;M SAMHD1 and 25 U/mL PPase.</li>
		<li>Prepare PPase alone solution by diluting recombinant E. coli PPase in complete SAMHD1 RB to 2x desired final concentration, thus 25 U/mL PPase.</li>
		<li>Prepare activators GTP (AS1) and dGTP&#945;S (AS1 and AS2) diluting stock (typically 10 or 100 mM in water) in complete SAMHD1 RB to 4x final concentration, thus 50 &#181;M GTP or dGTP&#945;S.</li>
	</ol>
	</li>
	<li>Perform the assay 
	NOTE: All assay components should be equilibrated to RT. Liquid additions can be performed with either a multichannel pipette or a bulk reagent liquid dispenser.
	<ol>
		<li>To 384-well assay plate containing nucleotide analogues, dispense 5 &#181;L of the activator (either GTP or dGTP&#945;S) or complete SAMHD1 RB to the appropriate wells.</li>
		<li>Start the reaction by dispensing 10 &#181;L of SAMHD1/PPase master mix, PPase alone, or complete SAMHD1 RB to the appropriate wells.</li>
		<li>Incubate the reaction for 20 min at RT.</li>
		<li>Stop the reaction by dispensing 20 &#181;L EDTA stop solution to all the wells. 
		NOTE: The experiment can be paused here if desired.</li>
		<li>Add 10 &#181;L MG working solution to all the wells. 
		CAUTION: MG working solution contains sulfuric acid.</li>
		<li>Ensure mixing of well contents using an orbital microwell plate shaker and centrifugation at 1,000 x g for 1 min.</li>
		<li>Incubate the plate for 20 min at RT.</li>
		<li>Read the absorption at 630 nm wavelength in a microwell plate reader.</li>
	</ol>
	</li>
	<li>Data visualization and analysis
	<ol>
		<li>Calculate the average absorbance values for the PPase only reaction wells (negative control or background signal). 
		NOTE:&#160;As a positive control of a SAMHD1 allosteric activator and substrate, dGTP can be included in the plate. In this case, you may use this condition to calculate Z-factor as an indicator of assay quality.</li>
		<li>Subtract the background value from the corresponding wells in the SAMHD1/PPase reactions.</li>
		<li>Plot corrected absorbance values for each nucleotide analogue with buffer, GTP, and dGTP&#945;S conditions.</li>
	</ol>
	</li>
</ol>

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D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+NUDT5+inhibitors+block+hormone+signaling+in+breast+cancer+cells.">Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells.</a> Nature Communications. 9 (1), 250 (2018).</li><li>Zhang, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+chemical+probe+against+NUDT15.">Development of a chemical probe against NUDT15.</a> Nature Chemical Biology. 16 (10), 1120-1128 (2020).</li><li>Michel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+druggability+assessment+of+the+NUDIX+hydrolase+protein+family+as+a+workflow+for+target+prioritization.">In silico druggability assessment of the NUDIX hydrolase protein family as a workflow for target prioritization.</a> Frontiers in Chemistry. 8, 443 (2020).</li><li>Zhang, J., Chung, T., Oldenburg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> Journal of Biomolecular Screening. 4 (2), 67 (1999).</li><li>Baykov, A. A., Evtushenko, O. A., Avaeva, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+malachite+green+procedure+for+orthophosphate+determination+and+its+use+in+alkaline+phosphatase-based+enzyme+immunoassay.">A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay.</a> Analytical Biochemistry. 171 (2), 266-270 (1988).</li><li>Markossian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+guidance+manual.">Assay guidance manual.</a> Eli Lilly &amp; Company and the National Center for Advancing Translational Sciences. , (2004).</li><li>Holdgate, G. A., Meek, T. D., Grimley, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+enzymology+in+drug+discovery:+a+fresh+perspective.">Mechanistic enzymology in drug discovery: a fresh perspective.</a> Nature Reviews Drug Discovery. 17 (2), 115-132 (2018).</li><li>Tsesmetzis, N., Paulin, C. B. J., Rudd, S. G., Herold, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleobase+and+nucleoside+analogues:+resistance+and+re-sensitisation+at+the+level+of+pharmacokinetics,+pharmacodynamics+and+metabolism.">Nucleobase and nucleoside analogues: resistance and re-sensitisation at the level of pharmacokinetics, pharmacodynamics and metabolism.</a> Cancers. 10 (7), 240 (2018).</li></ol>

The authors have nothing to disclose.

The enzyme-coupled activity assay detailed here is a high-throughput-amenable colorimetric assay allowing the indirect measurement of dNTP hydrolysis by SAMHD1. This method exploits the ability of inorganic PPase from E. coli, which when included in excess in the reaction mixture, converts each inorganic triphosphate generated by SAMHD1 into three individual free phosphates that can be quantified using the simple and economical malachite green reagent. We provide this assay in a 384 microwell plate format, which is ideal for screening of compound libraries, and demonstrate the applicability and versatility of this technique in the identification and characterization of SAMHD1 inhibitors, activators, and substrates.
As with all in vitro biochemical screening assays, there are a number of critical steps and important considerations, and many of these are discussed in-depth in the freely available Assay Guidance Manual39. Integrity of the purified recombinant enzymes, both SAMHD1 and the coupled enzyme inorganic PPase, is extremely important, and should be confirmed prior to establishing the assay. And accordingly, each new purification of these enzymes should be subject to some level of batch testing, as batch-to-batch variabilities could introduce inconsistencies in results. Ideally, use of an orthogonal direct assay, such as HPLC, which allows detection of both the substrate and reaction product, should be used to verify the dNTP triphosphohydrolase activity of the purified recombinant SAMHD1 being used.
Regarding limitations of this assay, the principle one is that it measures the dNTPase activity of SAMHD1 in an indirect manner, exploiting the activity of inorganic PPase, which has a number of implications. It is important to confirm that PPase possesses little to no activity toward nucleotides used in the assay, and likewise, that inhibitory small molecules identified possess no activity toward PPase. Thus, with regard to screening, a counter-screen against PPase can be an important consideration. The presence of PPase in the reaction also makes it critical to use an orthogonal assay to confirm findings. With regard to direct activity assays, a number of them have been reported to date, including TLC9,20,23 and HPLC9,21, which accurately detect substrate exhaustion and product formation. Additionally, the b4NPP assay21, which is also high-throughput, could be used to test potential inhibitors; however, it is not ideal to test substrates or allosteric activators. Biophysical assays, such as differential scanning fluorimetry (DSF), which we have previously reported with SAMHD118, can also be particularly powerful in identifying and characterizing ligands. Another limitation of the assay, specifically as shown in the setup here for identifying substrates and activators, is the use of the non-hydrolysable dGTP analog dGTP&#945;S as an AS1 and AS2 activator. While this allows activation of SAMHD1 with no observable activity in the assay, dGTP&#945;S is a competitive inhibitor of SAMHD1, and thus the use of high concentrations will inactivate the enzyme. As our understanding of SAMHD1 progresses, future studies could utilize molecules that exclusively occupy each site of SAMHD1, thus negating this potential issue.
As we have shown here, this method is versatile and can be used to address a number of biochemical questions. We have described two variations of this assay, one for the identification of allosteric regulators and substrates of SAMHD1, and another for the characterization of inhibitors, but further adaptations can be made. With regard to potential inhibitors, this assay, being microwell plate-based, makes it well suited for downstream mechanism of action studies39,40. Similarly, for further characterization of substrates and allosteric regulators, this technique can be used to determine kinetic parameters of catalysis, as we performed for the active metabolite of cytarabine and clofarabine7. However, one drawback is that the enzyme-coupled assay reported here is an endpoint assay, and so, although well-suited for screening, a continuous assay would be better suited for some mechanistic studies. Arnold et al. reported a continuous enzyme-coupled assay that utilizes the biosensor MDCC-PBP6, which relies on the use of the periplasmic phosphate binding protein (PBP) labeled with coumarin maleimide (MDCC) fluorophore that can bind to a free phosphate group. MDCC-PBP is very sensitive and enables the quantification of very low phosphate concentrations, with the sensor&#39;s time of response being in the millisecond to second timescale.
SAMHD1 plays a number of important functions in human health and disease2 many of which could be linked to its central role in the maintenance of intracellular dNTP levels1. Thus, identification of a high-quality chemical probe toward the dNTPase activity of SAMHD1 would be a powerful tool in defining these links, and the enzyme-coupled assay reported here could be readily employed to identify such probes. Furthermore, as nucleoside-based drugs, many of which are modulated by SAMHD1, are a diverse and important group of therapeutic41; chemical probes could be further developed into drugs to target SAMHD1 in the clinical setting, with the aim of enhancing the efficacy of these therapies. It is also critical to understand the full extent of the interaction of these nucleoside-based compounds with SAMHD1, a question which can be addressed utilizing this enzyme-coupled assay too. Taken together, the enzyme-coupled SAMHD1 activity assay as reported here, is a low-cost, versatile, high-throughput assay that can be used to further our understanding of this important enzyme.

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a central regulator of nucleotide homeostasis in mammalian cells1 with many roles in human health and disease2. This enzyme is capable of hydrolyzing deoxynucleoside triphosphates (dNTPs) into their cognate deoxynucleoside and inorganic triphosphate molecules3,4, with this activity being allosterically regulated by (d)NTP abundance (reviewed in reference5). Each SAMHD1 monomer contains two allosteric sites (AS1 and AS2) and one catalytic site, and the formation of the active enzyme requires the ordered assembly of a homotetramer upon (d)NTP binding. Dimerization of SAMHD1 monomers is first triggered through the binding of a guanine triphosphate (GTP or dGTP) to AS1, and subsequent tetramerization is achieved when an additional dNTP molecule binds to AS2, enabling substrate access to the catalytic site and subsequent hydrolysis.
SAMHD1 substrates include the four canonical dNTPs3,4 together with some base and sugar modified nucleotides, including the triphosphate metabolites of several nucleoside-based drugs used in the treatment of viral infections and cancer, several of which can also serve as allosteric activators6,7,8,9,10,11. In consequence SAMHD1 modulates the efficacy of many of these compounds in disease models7,8,9,10,11,12,13,14,15, and furthermore, in the case of the deoxycytidine analogue cytarabine (ara-C), which has remained standard-of-care therapy for acute myeloid leukemia (AML) for decades, actually dictates treatment efficacy in this disease7,8,16. SAMHD1 is thus a potential biomarker and therapeutic target to improve the efficacy of nucleoside-based therapies17, and accordingly, we and others have sought to identify strategies to inactivate SAMHD1 in cells. We proposed the use of viral protein X (Vpx) as a biological inhibitor to target SAMHD1 for degradation inside cancer cells7, however, this approach has a number of limitations (discussed in reference12), and we also recently reported an indirect approach to suppress SAMHD1 activity via inhibition of ribonucleotide reductase which we demonstrated in various models of AML18. A number of studies have sought to identify small molecules capable of directly inhibiting SAMHD1, and to date, several such molecules have been reported, however, only documenting inhibition in vitro6,9,19,20,21,22. In consequence, a lack of small molecules that potently inhibit SAMHD1 activity in cells coupled with the complex mechanisms of SAMHD1 catalysis of nucleoside-based therapeutics, underscores the need for further investigation. Thus robust and ideally high-throughput amenable methods for probing small molecule interaction with SAMHD1 are ideal in order to identify substrates, allosteric regulators and inhibitors, of this clinically relevant enzyme.
Several methodologies are available that directly measure the dNTPase activity of SAMHD1, such as thin-layer chromatography (TLC)9,20,23&#160;and high-performance liquid chromatography (HPLC)9,21, but these are not readily amenable to high-throughput setups. One exception is the assay reported by Mauney et al., which exploits the ability of SAMHD1 to hydrolyze bis (4-nitrophenyl) phosphate (b4NPP) to p-nitrophenol and p-nitrophenyl phosphate when Mn2+ is used as the activating cation, resulting in a colorimetric change that can be readily measured in a microwell plate21. This assay has been successfully used for the identification and characterization of SAMHD1 inhibitors, but it should be noted that hydrolysis does occur in the absence of (d)NTP activators and in the presence of a likely non-physiological activating cation, both being important caveats to consider. This also renders this assay less applicable to the study and identification of allosteric regulators of SAMHD1.
In this context, an enzyme-coupled approach combined with the malachite green reagent, as detailed in this report, can be a versatile method to indirectly measure the dNTPase activity of SAMHD1 and, furthermore, interrogate the impact of various small molecules upon it. The malachite green assay is a robust and reliable colorimetric technique for the detection of free inorganic phosphate (Pi), based on the formation of a molybdophosphoric acid complex that leads to a colorimetric change measured at 620 nm24. As SAMHD1 hydrolysis releases the triphosphate group from nucleotide substrates, it is thus necessary to couple this reaction with a (pyro)phosphatase activity, which will generate free inorganic phosphate, prior to the addition of the malachite green reagent. The malachite green assay is sensitive and cost effective and has been widely used for the identification and characterization of inhibitors and substrates for enzymes that release inorganic phosphate groups either in their reactions or in the presence of a coupling enzyme. It has been widely applied in characterization of the ATPase activities of helicases25,26,27,&#160;or the study of CD73 enzymatic activity, which mediates the degradation of AMP to adenosine and inorganic phosphate28. Additionally, when coupled, it has been employed in the discovery of antibiotic drugs targeting the UDP-2,3-diacylglucosamine pyrophosphatase LpxH, an essential enzyme in most Gram-negative pathogens29. With regards to cancer research, the enzyme-coupled approach has been extensively deployed against the NUDIX hydrolases, a family of nucleotide metabolizing enzymes, both in the characterization of substrates30,31,32 and in the identification and development of drugs and chemical probes33,34,35,36.
With regards to the dNTPase SAMHD1, this approach has been utilized in several reports. Using exopolyphosphatase Ppx1 from Saccharomyces cerevisiae as the coupling enzyme, this assay was used to test several nucleotide analogues as either substrates, activators, or inhibitors of SAMHD1, and resulted in the identification of the triphosphate metabolite of the anti-leukemic drug clofarabine as an activator and substrate6. Additionally, with inorganic pyrophosphatase from Escherichia coli as the coupling enzyme, it has been employed in the screening of a library of clinically approved compounds against SAMHD1 to identify inhibitors20. In our research, we utilized this approach to show that ara-CTP, the active metabolite of ara-C, is a SAMHD1 substrate but not allosteric activator7 and subsequently used this assay to show that several small molecules that could sensitize AML models to ara-C in a SAMHD1-dependent manner, actually did not directly inhibit SAMHD118. In this report, we will detail this versatile method and demonstrate its applicability, in a high-throughput amenable setup, for the identification of inhibitors, activators, and substrates of SAMHD1.

<table><tbody><tr><td>2&#039;-deoxyadenosine-5&#039;-triphosphate (dATP)</td><td>Jena bioscience</td><td>NU-1001</td><td>Compound tested in SAMHD1 activator/substrate assay</td></tr><tr><td>2&#039;-deoxycytidine-5&#039;-triphosphate (dCTP)</td><td>Jena bioscience</td><td>NU-1002</td><td>Compound tested in SAMHD1 activator/substrate assay</td></tr><tr><td>2&#039;-Deoxyguanosine-5&#039;-(&amp;alpha;-thio)-triphosphate (dGTP&amp;alpha;S)</td><td>Jena bioscience</td><td>NU-424</td><td>Non-hydrolyzable dGTP analogue for SAMHD1 activator/substrate assay</td></tr><tr><td>2&#039;-deoxyguanosine-5&#039;-triphosphate (dGTP)</td><td>GE Healthcare</td><td>27-1870-04</td><td>SAMHD1 allosteric activator and substrate used in inhibition assay and activator/substrate assay</td></tr><tr><td>2&#039;-Deoxythymidine-5&#039;-[(&amp;alpha;,&amp;beta;)-imido]triphosphate (dTMPNPP)</td><td>Jena bioscience</td><td>NU-907-1</td><td>Compound tested in SAMHD1 inhibition assay</td></tr><tr><td>2&#039;-deoxythymidine-5&#039;-triphosphate (dTTP)</td><td>Jena bioscience</td><td>NU-1004</td><td>Compound tested in SAMHD1 activator/substrate assay</td></tr><tr><td>2&#039;Deoxyguanosine mohohydrate (dGuo)</td><td>Sigma-Aldrich</td><td>D0901</td><td>Compound tested in SAMHD1 inhibition assay</td></tr><tr><td>384 well clear flat-bottom&amp;nbsp; microplate</td><td>Thermo Fisher Scientific</td><td>262160</td><td>Assay plate</td></tr><tr><td>96 well clear U-bottom polypropylene&amp;nbsp; microplate</td><td>Thermo Fisher Scientific</td><td>267245</td><td>Compound dilution plate</td></tr><tr><td>Ammonium heptamolybdate tetrahydrate</td><td>Sigma-Aldrich</td><td>A1343</td><td>Reagent required for malachite green working reagent</td></tr><tr><td>ara-Cytidine-5&#039;-triphosphate (ara-CTP)</td><td>Jena bioscience</td><td>NU-1170</td><td>Compound tested in SAMHD1 activator/substrate assay</td></tr><tr><td>Clofarabine-5&#039;-triphosphate (Cl-F-ara-ATP)</td><td>Jena bioscience</td><td>NU-874</td><td>Compound tested in SAMHD1 activator/substrate assay</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>VWR</td><td>23486.297</td><td>Solvent</td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td>EDTA stop solution component</td></tr><tr><td>Gemcitabine-5&#039;-triphosphate (dF-dCTP)</td><td>Jena bioscience</td><td>NU-1607</td><td>Compound tested in SAMHD1 activator/substrate assay</td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software</td><td>Prism 8</td><td>Data analysis and visualisation</td></tr><tr><td>Guanosine 5&amp;prime;-triphosphate (GTP) sodium salt hydrate</td><td>Sigma-Aldrich</td><td>G8877</td><td>Allosteric activator for SAMHD1 activator/substrate assay</td></tr><tr><td>His-tagged E. coli inorganic pyrophosphatase&amp;nbsp; (PPase)</td><td>Generated in house using Protein Science Facility, Karolinska Institutet</td><td>-</td><td>Recombinant PPase protein, hydrolises inorganic triphosphate and pyrophosphate to orthophosphate so it can form complex with malachite green</td></tr><tr><td>His-tagged human SAMHD1</td><td>Generated in house using Protein Science Facility, Karolinska Institutet</td><td>-</td><td>Recombinant SAMHD1 protein, hydrolizes dNTPs into its corresponding nucleoside and inorganic triphosphate</td></tr><tr><td>Hydroxyurea</td><td>Sigma-Aldrich</td><td>H8627</td><td>Compound tested in SAMHD1 inhibition assay</td></tr><tr><td>Lomofungin</td><td>National Cancer Institute (NCI)/Division of Cancer Treatment and Diagnosis (DCTD)/Developmental Therapeutics Program (DTP)</td><td>NSC106995</td><td>Compound tested in SAMHD1 inhibition assay</td></tr><tr><td>Magnesium Chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M2670</td><td>SAMHD1 reaction buffer component</td></tr><tr><td>Malachite green Carbinol hydrochloride</td><td>Sigma-Aldrich</td><td>213020</td><td>Malachite green stock component</td></tr><tr><td>Microplate Reader</td><td>Hidex</td><td>Hidex Sense Microplate reader</td><td>Data acquisition, absorption read at 630 nm wavelength</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>31434</td><td>SAMHD1 reaction buffer component</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>567530</td><td>SAMHD1 reaction buffer component</td></tr><tr><td>Sodium phosphate (Na&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>342483</td><td>Required for phosphate standard curve</td></tr><tr><td>Sulphuric acid 95-97%</td><td>Sigma-Aldrich</td><td>84720</td><td>Malachite green stock component</td></tr><tr><td>Tris-Acetate salt</td><td>Sigma-Aldrich</td><td>T1258</td><td>SAMHD1 reaction buffer component</td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Sigma-Aldrich</td><td>C4706</td><td>SAMHD1 reaction buffer component</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P1379</td><td>SAMHD1 reaction buffer and malachite green working reagent component</td></tr></tbody></table>

a high-throughput enzyme-coupled activity assay to probe small molecule interaction with the dntpase samhd1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

The protocol outlined in Figure 1 describes the basic workflow for utilizing the enzyme-coupled malachite green assay to probe the interaction of small molecules with the dNTPase SAMHD1 and can be adapted in a number of ways to interrogate different biochemical questions. In the representative results discussed in the below paragraphs, we illustrate examples of using this assay to determine the inhibitory properties of small molecules toward SAMHD1 and to test whether different nucleotide analogues are substrates and/or activators of this enzyme.
The results shown in Figure 2 illustrate several core principles of this assay. The malachite green reagent allows the colorimetric detection of inorganic phosphate through the formation of a phosphomolybdate malachite green complex, and accordingly, this approach can be applied to the study of enzymatic reactions whose product is phosphate. To demonstrate the sensitivity of this method to detect free inorganic phosphate, Figure 2A shows the absorbance values obtained with increasing concentrations of Na3PO4 following a 20 min incubation with the malachite green reagent. While the signal reaches saturation at 0.25 mM Na3PO4, the linear detection range of phosphate is visible from 0.004 to 0.03 mM (Figure 2A, right panel), in agreement with other studies that reported a phosphate linear range up to 10-20 &#956;M using the malachite green assay38.
SAMHD1 is a dNTPase that releases inorganic triphosphate when hydrolyzing a dNTP molecule, and thus in order to generate free inorganic phosphate for detection by malachite green, a coupling enzyme is required. Inorganic pyrophosphatase (PPase) from E. coli has been shown useful for this purpose, both with regard to SAMHD17,20, but also other nucleotide metabolizing enzymes30,33,35. Additionally, SAMHD1 is an active dNTPase when as a homotetramer, and this requires allosteric activation by (d)NTPs, specifically a guanine triphosphate (GTP or dGTP) at AS1 and any dNTP at&#160;AS2. Subsequently, the catalytic site becomes accessible for substrate binding and the enzymatic reaction takes place. As dGTP fulfils the requirements for binding to AS1 and AS2, and is a substrate, use of this nucleotide in the inhibition assay greatly simplifies the workflow. Figure 2B illustrates the requirement of the different assay components to achieve measurable SAMHD1 activity indicated by an increase in absorbance at 630 nm. Neither SAMHD1 nor PPase alone are capable of generating inorganic phosphate in the presence of dGTP, consistent with the documented activities of these enzymes. However, in the condition in which all the assay components are present (SAMHD1, PPase, and the dGTP activator/substrate) we observe an increase in signal. The Z factor37&#160;of the example shown here (taking no enzymes + dGTP as a negative control and SAMHD1/PPase + dGTP as a positive control) was 0.74, indicating a robust assay.
One of the potential applications of the enzyme-coupled SAMHD1 activity assay is the identification of inhibitors through high-throughput screening (HTS). Thus, in this report, we validate the detection of SAMHD1 inhibition in this assay using a diverse set of compounds already described in the literature. Seamon et al. evaluated the dose-dependent inhibition of canonical nucleosides toward SAMHD1 using a similar assay as shown here, and found that deoxyguanosine (dGuo) was the only canonical nucleoside able to significantly inhibit SAMHD1, with an IC50 value of 488 &#181;M20. A HTS of FDA-approved drugs performed with the direct b4NPP assay revealed several hits that inhibited SAMHD1 activity at micromolar concentrations, from which lomofungin was the molecule that most potently inhibited SAMHD1 dNTPase activity in vitro, exhibiting an IC50 of 20.1 &#181;M when determined in the presence of dGTP as a substrate21. Additionally, the four &#945;,&#946;-imido-dNTP analogs have also been identified as competitive inhibitors of SAMHD1 using the MDCC-PBP sensor and SAMHD1 coupled to Ppx activity, which showed that the inhibitory constants of the dNMPNPP analogs were in the low micromolar / high nanomolar range6,22. Thus, to demonstrate that the enzyme-coupled SAMHD1 activity assay can be used to identify SAMHD1 inhibitors, dGuo, lomofungin and 2&#39;-deoxythymidine-5&#39;-[(&#945;,&#946;)-imido]triphosphate (dTMPNPP), were used to validate the technique. Figure 3A illustrates the dose-response curves obtained for these compounds, showing that increasing concentrations effectively inhibit SAMHD1 activity. The mean IC50 values obtained for these molecules from three independent experiments (&#177; standard deviation) were as follows: dGuo = 361.9 &#177; 72.8 &#181;M, lomofungin 6.78 &#177; 3.9 &#181;M, and dTMPNPP = 2.10 &#177; 0.9 &#181;M. As an example of a negative result, the impact of hydroxyurea (HU) on SAMHD1 activity was also determined. HU is an inhibitor of ribonucleotide reductase, and, although it limits SAMHD1 ara-CTPase activity in various AML models, the effects of HU on SAMHD1 were shown to be indirect and rely on perturbing the allosteric regulation of SAMHD118. The dose response curve of HU is shown in Figure 3B, and no changes in SAMHD1 activity were observed with increasing HU doses, demonstrating that HU does not inhibit SAMHD1 activity in vitro.
Another use of the enzyme coupled SAMHD1 activity assay is to interrogate whether nucleotides and their analogs are substrates and/or allosteric activators of this enzyme, which is illustrated in Figure 4. In this experiment, canonical nucleotides, as well as the active metabolites of several anti-cancer nucleoside analogs, such as cytarabine (ara-CTP), clofarabine (Cl-F-ara-ATP), and gemcitabine (dF-dCTP), were tested as SAMHD1 substrates and activators. Due to the complex allosteric regulation of SAMHD1, the reaction is performed in the presence of GTP as an AS1 activator or the non-hydrolysable dGTP analog 2&#39;-deoxyguanosine-5&#39;-(&#945;-thio)-triphosphate (dGTP&#945;S), which can occupy AS1 and AS2. SAMHD1 activity in the presence of the tested nucleotide analog and GTP indicates that the nucleotide is able to bind to the secondary allosteric site and catalytic site (i.e., AS2 activator and substrate), while SAMHD1 activity with the nucleotide analog and dGTP&#945;S indicates the nucleotide can only occupy the catalytic site (i.e., only a substrate). If the nucleotide is able to bind to both the AS1 and AS2 allosteric sites and to the catalytic site, SAMHD1 will be active in the presence of the nucleotide alone, as shown in the case of dGTP. The results show that all canonical dNTPs are able to bind to the AS2 site and to the catalytic site. In the case of nucleotide analogs, clofarabine triphosphate is an AS2 activator and a substrate, whereas cytarabine triphosphate is only able to occupy the catalytic site. On the other hand, no activity was observed with gemcitabine triphosphate, suggesting that under the conditions tested gemcitabine triphosphate is not able to act as allosteric effector nor substrate. Although this result is consistent with previous predictions9, later crystallization and kinetic studies10 revealed that gemcitabine triphosphate is able to bind the SAMHD1 catalytic pocket, and that it is indeed a substrate of the enzyme. However, in the latter study10, the authors show that the hydrolysis rate is considerably lower compared to other reported substrates, such as cytarabine triphosphate, thus explaining why we were not able to observe this with this screening setup.
Altogether, these representative results validate the use of the enzyme coupled SAMHD1 activity assay as a robust technique for the identification and characterization of SAMHD1 inhibitors, allosteric regulators, and substrates. However, similar to all experimental approaches, this method has its caveats, and so orthogonal assays (e.g., using a different assay technology) should be used to further validate findings.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62503/62503fig01.jpg" /> 
Figure 1: Schematic overview of the protocol described in this article. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62503/62503fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62503/62503fig02.jpg" /> 
Figure 2: Enzyme-coupled SAMHD1 activity assay. (A) Na3PO4 standard curve in the malachite green assay. Na3PO4 serial dilution (2-fold) was prepared from 1 mM to 0.004 mM in triplicate and incubated with malachite green reagent for 20 min. Raw absorbance values over the full range of tested concentrations are shown in the left panel and the linear range in the right panel. Representative of two independent experiments shown. (B) Validation of enzyme-coupled activity assay. SAMHD1 (0.35 &#181;M) and/or PPase (12.5 U/mL) in the presence or absence of activator/substrate dGTP (25 &#181;M) were incubated for 20 min in the enzyme-coupled activity assay. Quadruplets from a representative of two independent experiments shown with raw absorbance values plotted, bars, and error bars indicate mean and SD. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62503/62503fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62503/62503fig03.jpg" /> 
Figure 3:&#160;Evaluating compounds for SAMHD1 inhibition in the enzyme-coupled activity assay. Dose response of lomofungin (0.78-100 &#181;M), 2&#39;-deoxythymidine-5&#39;-[(&#945;,&#946;)-imido]triphosphate (dTMPNPP, 0.01-100 &#181;M) and deoxyguanosine (dGuo, 10-1,500 &#181;M) (A) or hydroxyurea (HU) (0.78-100 &#181;M) (B) in the enzyme-coupled SAMHD1 activity assay with dGTP (25 &#181;M) as activator/substrate. Percentage activity relative to reaction controls from individual replicates plotted (DMSO + SAMHD1/PPase + dGTP = 100% activity, DMSO + dGTP = 0% activity) with a representative of three experiments shown. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62503/62503fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62503/62503fig04.jpg" /> 
Figure 4:&#160;Evaluating nucleotide analogs as SAMHD1 allosteric activators and substrates in the enzyme-coupled activity assay. Canonical nucleotides and selected triphosphate metabolites of anticancer drugs cytarabine (ara-CTP), clofarabine (Cl-F-ara-ATP), and gemcitabine (dF-dCTP), were tested at 200 &#181;M in the enzyme-coupled SAMHD1 activity assay in the presence or absence of GTP or non-hydrolysable dGTP analog dGTP&#945;S (12.5 &#181;M). Normalized absorbance values from individual experimental replicates plotted, mean and SD are indicated. Representative of two independent experiments shown, adapted from our previous study7. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62503/62503fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Reagent</td>
			<td>Volume dispensed (&#181;L)</td>
			<td>Final reaction volume (&#181;L)</td>
			<td>Concentration dispensed</td>
			<td>Fold dilution in reaction</td>
			<td>Final concentration in reaction</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Inhibitor</td>
			<td>5</td>
			<td rowspan="3">20</td>
			<td>0.4 mM</td>
			<td>4</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>2</td>
			<td>SAMHD1+PPase mix</td>
			<td>5</td>
			<td>1.4 &#181;M SAMHD1, 50 U/mL PPase</td>
			<td>4</td>
			<td>0.35 &#181;M SAMHD1, 12.5 U/mL Ppase</td>
		</tr>
		<tr>
			<td>3</td>
			<td>dGTP</td>
			<td>10</td>
			<td>50&#160; &#181;M</td>
			<td>2</td>
			<td>25 &#181;M</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="6">Incubation for 20 minutes</td>
		</tr>
		<tr>
			<td>5</td>
			<td>EDTA solution</td>
			<td>20</td>
			<td>40</td>
			<td>7.9 mM</td>
			<td>2</td>
			<td>3.95 mM</td>
		</tr>
		<tr>
			<td>6</td>
			<td>MG reagent</td>
			<td>10</td>
			<td>50</td>
			<td>2.5 mM Malachite green, 64.4 mM Ammonium Molybdate, 0.18% Tween-20</td>
			<td>5</td>
			<td>0.5 mM Malachite green, 12.9 mM Ammonium Molybdate, 0.036% Tween-20</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="6">Incubation for 20 minutes</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="6">Read @ 630 nm</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of the final conditions in the enzyme-coupled assay for inhibitors screening.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Reagent</td>
			<td>Volume dispensed (&#181;L)</td>
			<td>Final reaction volume (&#181;L)</td>
			<td>Concentration dispensed</td>
			<td>Fold dilution in reaction</td>
			<td>Final concentration in reaction</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Allosteric regulator</td>
			<td>5</td>
			<td rowspan="3">20</td>
			<td>800 &#181;M</td>
			<td>4</td>
			<td>200 &#181;M</td>
		</tr>
		<tr>
			<td>2</td>
			<td>GTP or dGTP&#945;S</td>
			<td>5</td>
			<td>50 &#181;M</td>
			<td>4</td>
			<td>12.5 &#181;M</td>
		</tr>
		<tr>
			<td>3</td>
			<td>SAMHD1 and/or PPase</td>
			<td>10</td>
			<td>0.7 &#181;M SAMHD1, 25 U/mL PPase</td>
			<td>2</td>
			<td>0.35 &#181;M SAMHD1, 12.5 U/mL PPase</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="6">Incubation for 20 minutes</td>
		</tr>
		<tr>
			<td>5</td>
			<td>EDTA solution</td>
			<td>20</td>
			<td>40</td>
			<td>7.9 mM</td>
			<td>2</td>
			<td>3.95 mM</td>
		</tr>
		<tr>
			<td>6</td>
			<td>MG reagent</td>
			<td>10</td>
			<td>50</td>
			<td>2.5 mM Malachite green, 64.4 mM Ammonium Molybdate, 0.18% Tween-20</td>
			<td>5</td>
			<td>0.5 mM Malachite green, 12.9 mM Ammonium Molybdate, 0.036% Tween-20</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="6">Incubation for 20 minutes</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="6">Read @ 630 nm</td>
		</tr>
	</tbody>
</table>
Table 2: Summary of the final conditions in the enzyme-coupled assay for allosteric regulators screening

Watch this Scientific Journal Video about A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1 at JoVE.com

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

2.2K Views.  Karolinska Institutet. SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Video: A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

Chemistry

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...

Immunology and Infection

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

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2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

NMR-Based Activity Assays for Determining Compound Inhibition, IC₅₀ Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Measuring In Vitro ATPase Activity for Enzymatic Characterization