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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2&#39;-deoxyadenosine-5&#39;-triphosphate (dATP)</td>
			<td>Jena bioscience</td>
			<td>NU-1001</td>
			<td>Compound tested in SAMHD1 activator/substrate assay</td>
		</tr>
		<tr>
			<td>2&#39;-deoxycytidine-5&#39;-triphosphate (dCTP)</td>
			<td>Jena bioscience</td>
			<td>NU-1002</td>
			<td>Compound tested in SAMHD1 activator/substrate assay</td>
		</tr>
		<tr>
			<td>2&#39;-Deoxyguanosine-5&#39;-(&#945;-thio)-triphosphate (dGTP&#945;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&#39;-deoxyguanosine-5&#39;-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&#39;-Deoxythymidine-5&#39;-[(&#945;,&#946;)-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&#39;-deoxythymidine-5&#39;-triphosphate (dTTP)</td>
			<td>Jena bioscience</td>
			<td>NU-1004</td>
			<td>Compound tested in SAMHD1 activator/substrate assay</td>
		</tr>
		<tr>
			<td>2&#39;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&#160; microplate</td>
			<td>Thermo Fisher Scientific</td>
			<td>262160</td>
			<td>Assay plate</td>
		</tr>
		<tr>
			<td>96 well clear U-bottom polypropylene&#160; 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&#39;-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&#39;-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&#39;-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&#8242;-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&#160; (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 (MgCl2)</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 (Na3PO4)</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://www.jove.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://www.jove.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://www.jove.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://www.jove.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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2.1K 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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

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 ...

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.

A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...