This work was supported by a grant from the UK Engineering and Physical Sciences Research Council (Studentship award: EP/S023518/1).

1. Fragment library construction
<ol>
	<li>Design a library of electrophilic fragments and either synthesize or purchase them. Electrophilic fragment libraries typically consist of mildly reactive electrophiles (such as acrylamides, chloroacetamides, and vinyl sulfonamides) appended to a diverse array of chemical scaffolds. Electrophilic fragment libraries are widely available from commercial suppliers, and more detail about their design and construction is reviewed elsewhere5,11.</li>
	<li>Dissolve each compound in DMSO to a concentration of 50 mM, then dispense a different compound into each well of columns 3 to 22 of a 384-well reservoir plate, termed the Fragment Library Plate.
	<ol>
		<li>Dispense DMSO into each well of columns 1, 2, 23, and 24 for the positive and negative controls. Plate layout can be varied depending on the number of compounds being screened (Figure 2). The volume added to the library plate will depend on the number of screens to be performed, considering approximately 1.8 &#956;L from each well is required per qIT screen. Prepare this Fragment Library Plate in advance and store it at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67178/67178fig02.jpg" /> 
Figure 2: Plate layout. An overview of qIT assay 384-well plate layout. <a href="https://www.jove.com/files/ftp_upload/67178/67178fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Assay setup (Figure 3)
<ol>
	<li>Prepare the following solutions: recombinant protein solution (target protein in any buffer), qIT assay buffer (150 mM NaCl, 25 mM Hepes (pH 8)), qIT assay quench buffer (150 mM NaCl, 25 mM Hepes (pH 7.5)), and compound stock solutions (electrophilic fragment (50 mM) in DMSO). 
	NOTE: A pH of 8 is used in the reaction buffer to accelerate the reaction between the target cysteine residue and fragments, allowing the rate of reaction to be measured even for extremely mildly reactive fragments. A slightly lower pH of 7.5 is used in the quench buffer as the thioether bond formed between the maleimide of CPM and the cysteine thiol is prone to hydrolysis at more basic pHs (for example, previous work found cysteine-maleimide conjugates are far more stable at pH 7.3 than pH 812).</li>
	<li>Remove the DMSO fragment stock solutions from storage and leave them at room temperature for a minimum of 2 h to allow compound stocks to thaw.</li>
	<li>Degas qIT assay buffer by bubbling with argon gas for approximately 30 min.</li>
	<li>Thoroughly exchange the target protein solution into degassed qIT assay buffer. Crucially, reducing agents such as DTT and TCEP must be thoroughly removed to concentrations &#60; 100 nM as they will interfere with thiol quantification. This step can be performed via a repetitive series of buffer exchanges, as described below.
	<ol>
		<li>Dilute protein solution in excess of degassed qIT assay buffer in a spin filter unit, concentrate using centrifugation (4000 x g, 4 &#176;C, 15 min) and repeat. Typically, 4 rounds of 1 in 15 dilutions are required to sufficiently remove DTT (1 mM) from 1 mL of recombinant protein stock solution, although this step can be adjusted depending on the reducing agent concentration in the original recombinant protein solution. 
		NOTE: Alternatively, dialyzing the target protein solution against a large excess of degassed qIT assay buffer can achieve the same outcome.</li>
	</ol>
	</li>
	<li>Dilute the target protein solution (produced in step 2.1) to a protein concentration of 15 &#956;M in degassed qIT assay buffer. Approximately 9 mL of target protein solution is required to fill a 384-well plate with sufficient dead volume.</li>
	<li>Prepare a 1.5% (v/v) suspension of TCEP-agarose in degassed qIT assay buffer. Approximately 9 mL of TCEP-agarose suspension is required per screening plate.</li>
	<li>Dispense 20 &#956;L of TCEP-agarose solution into every well of two 384-well plates.</li>
	<li>Prepare a 15 &#956;M glutathione stock in degassed qIT assay buffer. This should be prepared immediately before plating as glutathione rapidly oxidizes in solution without reducing agent.</li>
	<li>Dispense 20 &#956;L of qIT assay buffer into columns 1 and 2 of both 384-well plates.</li>
	<li>Dispense 20 &#956;L of 15 &#956;M protein or glutathione solution (in accordance with the intended plate) into columns 3-24 of the 384-well plate. These are termed the Protein Reaction Plate and the Glutathione Reaction Plate. Leave to reduce at RT for 1 h.</li>
	<li>Dispense 58.2 &#956;L of degassed qIT assay buffer into each well of a 384-well plate, then transfer 1.8 &#956;L from each corresponding well of the Fragment Library Plate to create a Compound Dilution Plate, where each compound is diluted to a concentration of 1.5 mM in qIT assay buffer plus 3% DMSO. 
	NOTE: This step is made easier with a 384-pipetting station, which allows compound stocks to be transferred for every well in one operation.</li>
	<li>Following 1 h incubation of protein and glutathione with TCEP-agarose, transfer 20 &#956;L of compound solution from each well of the Compound Dilution Plate into both the protein and glutathione reaction plates to begin the assay. This step is also made easier with a 384-pipetting station. The assay steps are summarized in Figure 3. 
	NOTE: The fragment concentration can be varied as required, and qIT assays can be repeated for the hits at lower concentrations, if desired, to confirm the protein affinity is not an artifact of the high fragment concentration. The relatively high fragment concentration is used to elevate the kpro&#160;and kGSH&#160;to ensure that accurate rates are determinable for mildly reactive compounds (which can display t1/2(GSH) &#62; 24 h under the assay conditions). Moreover, although a fragment concentration of 500 &#181;M is high, high &#181;M to low mM concentrations have been used in fragment screening, for example13.</li>
	<li>Start a timer - the assay has begun, and quenches should be performed at regular time points in accordance with the section below.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67178/67178fig03v2.jpg" /> 
Figure 3: qIT assay summary. A summary illustrating the key steps of qIT assay reaction well setup. (created using BioRender). <a href="https://www.jove.com/files/ftp_upload/67178/67178fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. CPM quenches
NOTE: CPM quenches are performed to quantify the amount of unreacted thiol remaining in the protein and glutathione reaction plates. These should be performed at discrete time points (for example, the following time points are typically used: 7, 15, 30, 60, 120, 240, 360, 1220, and 1440 min) to allow reaction kinetics to be monitored.
<ol>
	<li>Prepare CPM stock solutions (500 &#956;M) in DMSO, dispense them into 111.2 &#181;L aliquots in microcentrifuge tubes, and store them at -20 &#176;C. These CPM stocks can be prepared in advance, and 1 aliquot will be required per quench time point.</li>
	<li>Prepare CPM quench solution (1.4 &#956;M) by thawing a frozen CPM stock solution (500 &#956;M) and adding it to 40 mL qIT assay quench buffer.</li>
	<li>Dispense 27 &#181;L of CPM quench solution into each well of two 384-well plates-two are required for quenching both the protein and glutathione reaction plates. These are termed CPM-Quench Plates, prepare them a maximum of 30 min before use.</li>
	<li>Centrifuge the protein reaction plate at 200 x g for 3 min to ensure TCEP-agarose is pelleted.</li>
	<li>At pre-determined time points, transfer a 3 &#181;L of sample from every well of the Protein Reaction Plate to a pre-filled-CPM Quench Plate. This step is also simplified using a 384-pipetting station.</li>
	<li>Mix the CPM Quench Plate by aspirating and dispensing 3 x 25 &#181;L. It is important to remove the sample from the top of the well to avoid aspirating the TCEP-agarose at the bottom, which will interfere with CPM quenching.</li>
	<li>Repeat steps 3.4-3.6 with the Glutathione Reaction Plate.</li>
	<li>Incubate the CPM Quench Plates (prepared in 3.4-3.6) at room temperature for 1 h.</li>
	<li>Measure the fluorescence intensity of the CPM quench plate (excitation at 384 nm and emission at 470 nm).</li>
</ol>
4. Data analysis and hit selection
<ol>
	<li>For each quench, calculate Z&#39; as a measure of assay quality. Any CPM quenches with Z&#39; &#60; 0.5 should be disregarded. 
	Z&#39; = 1 - ((3&#963;positive control + 3&#963;negative control) / (&#181;positive control - &#181;negative control)) 
	Where &#963; = fluorescence intensity standard deviation and &#181; = fluorescence intensity mean</li>
	<li>Calculate mean fluorescence values for the negative and positive control wells (columns 1 and 2, and 23 and 24, respectively).</li>
	<li>Normalize each reaction well to the average fluorescence values of the positive and negative controls: 
	%&#160;modified = 100 x (compound well fluorescence - negative control fluorescence) / (positive control fluorescence - negative control fluorescence)</li>
	<li>Separately plot % modified versus time for each compound reacting with glutathione and with target protein.</li>
	<li>Fit normalized % modified versus time data to a one-phase exponential decay model for both the reaction with protein and with glutathione using the equation: 
	% Modified = ae - kt + c 
	Where a = the range of % modified values, k = rate of reaction, t = time, c = the % modified value at infinite time. Curve fitting and parameter determination can be performed using commercial software (such as Graphpad).</li>
	<li>Use the rate at which fragments react with protein (kpro) and glutathione (kGSH) to calculate the Rate Enhancement Factor (REF) value for each fragment: 
	REF = kpro / kGSH</li>
	<li>Use the REF values to select hit fragments. This can be performed by setting a REF threshold (e.g., REF &#62; 3), an arbitrary hit rate (e.g., top 2%), or by selecting fragments with REF 3 standard deviations greater than the geometric mean.</li>
</ol>

Quantitative-Irreversible Tethering: Screening Electrophilic Ligands Efficiently

<ol>
	<li>Boike, L., Henning, N. J., Nomura, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+covalent+drug+discovery.">Advances in covalent drug discovery.</a> Nat Rev Drug Discov. 21, 881-898 (2022).</li><li>Gehringer, M., Laufer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+and+re-emerging+warheads+for+targeted+covalent+inhibitors:+Application+in+medicinal+chemistry+and+chemical+biology.">Emerging and re-emerging warheads for targeted covalent inhibitors: Application in medicinal chemistry and chemical biology.</a> J Med Chem. 62, 5673-5724 (2019).</li><li>Strelow, J. 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+perspective+on+the+kinetics+of+covalent+and+irreversible+inhibition.">A perspective on the kinetics of covalent and irreversible inhibition.</a> SLAS Discov. 22 (1), 3-20 (2016).</li><li>Burger, J. A., Buggy, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruton+tyrosine+kinase+inhibitor+Ibrutinib+(PCI-32765).">Bruton tyrosine kinase inhibitor Ibrutinib (PCI-32765).</a> Leukemia Lymphoma. 54 (11), 2385-2391 (2013).</li><li>Hocking, B., Armstrong, A., Mann, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+fragment+libraries+in+drug+discovery+-+Design,+synthesis+and+screening+methods.">Covalent fragment libraries in drug discovery - Design, synthesis and screening methods.</a> Prog Med Chem. 62, 105-146 (2023).</li><li>Lucas, S. C. C., 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=Covalent+hits+and+where+to+find+them.">Covalent hits and where to find them.</a> SLAS Discov. 29 (3), 100142 (2024).</li><li>Craven, 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=High-throughput+kinetic+analysis+for+target-directed+covalent+ligand+discovery.">High-throughput kinetic analysis for target-directed covalent ligand discovery.</a> Angewandte Chemie. 130, 5355-5359 (2018).</li><li>Kathman, S. G., Xu, Z., Statsyuk, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Fragment-based+method+to+discover+irreversible+covalent+inhibitors+of+cysteine+proteases.">A Fragment-based method to discover irreversible covalent inhibitors of cysteine proteases.</a> J Med Chem. 57 (11), 4969-4974 (2014).</li><li>Hallenbeck, K. K., 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=A+liquid+chromatography/mass+spectrometry+method+for+screening+disulfide+tethering+fragments.">A liquid chromatography/mass spectrometry method for screening disulfide tethering fragments.</a> SLAS Discov. 23 (2), 183-192 (2019).</li><li>Craven, 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=Multiparamter+kinetic+analysis+for+covalent+fragment+optimization+by+using+quantitative+irreversible+tethering+(qIT).">Multiparamter kinetic analysis for covalent fragment optimization by using quantitative irreversible tethering (qIT).</a> Chem Bio Chem. 21, 3417-3422 (2020).</li><li>Keeley, A., 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=Covalent+fragment+libraries+in+drug+discovery.">Covalent fragment libraries in drug discovery.</a> Drug Discov Today. 25 (6), 983-996 (2020).</li><li>Gober, I. N., Sharan, R., Villain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+stability+of+thiol-maleimide+bioconjugates+via+the+formation+of+a+thiazine+structure.">Improving the stability of thiol-maleimide bioconjugates via the formation of a thiazine structure.</a> J Pept Sci. 29 (10), e3495 (2023).</li><li>Olp, M. D., 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=Covalent-fragment+screening+of+BRD4+identifies+a+ligandable+site+orthogonal+to+the+acetyl-lysine+binding+sites.">Covalent-fragment screening of BRD4 identifies a ligandable site orthogonal to the acetyl-lysine binding sites.</a> ACS Chem Biol. 15 (4), 1036-1049 (2020).</li><li>Qin, B., 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=Acrylamide+fragment+inhibitors+that+induce+unprecedented+conformational+distorions+in+Entereovirus+71+3C+and+SARS-CoV-2+main+protease.">Acrylamide fragment inhibitors that induce unprecedented conformational distorions in Entereovirus 71 3C and SARS-CoV-2 main protease.</a> Acta Pharmaceutica Sinica B. 12 (10), 3924-3999 (2022).</li><li>Jamshidiha, 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=Identification+of+the+first+structurally+validated+covalent+ligands+of+the+small+GTPase+RAP27A.">Identification of the first structurally validated covalent ligands of the small GTPase RAP27A.</a> RSC Med Chem. 13 (2), 150-155 (2022).</li><li>Flanagan, 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=Chemical+and+computational+methods+for+the+characterization+of+covalent+reactive+groups+for+the+prospective+design+of+irreversible+inhibitors.">Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors.</a> J Med Chem. 57, 10072-10079 (2014).</li></ol>

A.A., and D.J.M. are co-inventors on a patent covering the qIT assay: PCT/GB2017/052456.

The qIT assay provides an effective and convenient platform for screening cysteine-reactive electrophilic fragments against a target protein. However, there are critical considerations that may hamper assay performance if not adequately accounted for. Most notably, target proteins must only include a single surface-exposed cysteine to be suitable for qIT screening, as other cysteine residues will react with CPM and interfere with fluorescence measurements. In previous cases where target proteins contain multiple surface-exposed cysteines (such as for the SARS-CoV-2 main protease, the small GTPase RAP27A, and Cyclin-dependent kinase 2), screening constructs with interfering residues removed have previously been used successfully. However, the effectiveness of this strategy will vary depending on the protein10,14,15.
Additionally, as the qIT assay measures the decrease in thiol available to react with CPM over time, any factor that reduces the proportion of available thiols, such as thiol oxidation or protein aggregation, may hamper assay performance. Buffer degassing and the use of an immobilized reducing agent (TCEP-agarose) ensure thiol oxidation is slow, and any remaining oxidation should be mitigated by normalizing fluorescence measurements to that of the positive control wells. However, problems can arise when an uneven distribution of TCEP-agarose between wells causes non-uniform thiol oxidation. Consistent TCEP-agarose plating is therefore essential for assay performance, which can be challenging as TCEP-agarose particles sink in pipetting reservoirs.
Protein aggregation can also be a problem, as the assay conditions (24 h incubation at room temperature) are relatively harsh. Troubleshooting strategies such as fusing target proteins to solubilizing tags, altering reaction buffers, or performing the assay at lower temperatures can reduce aggregation and may solve protein stability issues. Certain fragments may also induce protein aggregation, which again reduces the proportion of thiol available to react with CPM and creates false positive hits. Orthogonal hit validation using intact-protein mass spectrometry is an effective method of detecting these cases, as false positive hits will create a small or non-existent population of labeled species when incubated with a target protein.
When these considerations are controlled for, the qIT assay has some clear advantages over other covalent fragment screening methods. Hit selection using REF ensures that hits are only selected if they form productive non-covalent interactions with the target protein. Fragment electrophilicity varies over large ranges, both between different warheads and within warhead classes; for example, previous work has found several hundred-fold variations in the rate of glutathione reactivity between acrylamides10. This wide range of electrophilicity can make it challenging to distinguish fragments with high kpro values due to a templating effect from those which are simply highly reactive, which can lead to highly reactive fragments being selected as false positive hits. For example, 2 reacted with the target cysteine residue more rapidly than 1 and, without considering intrinsic reactivity, may be selected as a hit. However, 2 does not react with the target cysteine residue at a greater rate than with glutathione, indicating the greater kpro of 2 relative to 1 is due to greater fragment intrinsic reactivity, and not a templating effect.
The simple fluorescence readout used by the qIT assay to quantify the remaining thiol is also highly advantageous. Other methods of covalent fragment screening use intact-protein mass spectrometry to determine the rate at which fragments react with a target protein and LC-MS to determine the rate at which glutathione reacts with fragments - both of which require specialized equipment and lack throughput8,16. The convenient fluorescence readout makes the qIT assay highly scalable and requires only commonly available laboratory equipment.
Combined, these innovations create a robust and scalable method of covalent fragment screening, which negates the need for mass-spectrometry equipment.

Cysteine-reactive targeted covalent ligands (molecules that exert a biological effect by reacting with a select cysteine residue on a specific protein) have become increasingly popular as chemical tools and therapeutics over recent decades1. These typically consist of a mildly reactive electrophile (such as an acrylamide, chloroacetamide, or vinyl sulfone) appended to a ligand moiety with a high affinity for a protein target2. Ideally, ligand-protein interaction proceeds via a two-step mechanism involving initial non-covalent ligand binding, followed by irreversible covalent bond formation between the target cysteine and electrophile3.
Several early targeted covalent ligands, including Bruton&#39;s Tyrosine Kinase inhibitor Ibrutinib, were developed by furnishing a previously identified reversible ligand with an electrophilic handle4. However, this approach is not always successful, and other attempts have found electrophile installation to require significant optimization5. Instead, electrophile-first approaches, particularly using electrophilic fragments, have become more popular in recent years. These typically involve incubating a library of electrophiles with a protein and then selecting compounds with an enhanced rate of reaction towards a cysteine, which is assumed to be induced by productive non-covalent interactions between the ligand and binding pocket6. Notably, these methods were successfully applied during the development of the KRAS (G12C) inhibitor Sotorasib1.
Electrophile-first screening poses several unique challenges when compared to traditional non-covalent ligand screening, principally due to the high variability in reactivity within and between warhead classes7. Most cysteine-reactive electrophiles will, over time, react with all solvent-exposed cysteine residues, regardless of any selective interaction for the target protein. Ligand occupancy will, therefore, increase over time for almost all electrophiles, and without controlling for reactivity, hit selection will often be biased towards more reactive fragments that engage a target more rapidly8.
Moreover, electrophile-first screening methods often use mass spectrometry to detect protein-ligand adduct formation, which is limited in throughput9. Regular reaction sampling is required over an extended time period (hours to days) to properly characterize reaction kinetics for a library of compounds with varied electrophilicity10. A screen of 300 fragments sampled at 10-time points will, therefore, require 3000 mass spectra to be performed, exceeding the throughput of most protein mass spectrometry systems. Higher-throughput methods of monitoring protein-ligand adduct formation are, therefore, desirable.
The approach outlined here, the quantitate irreversible tethering (qIT) assay, is an electrophile-first screening method that controls intrinsic reactivity and utilizes a simple fluorescence readout7. Protein, or glutathione (an unstructured, thiol-containing tripeptide used as a control for intrinsic reactivity) are incubated with a library of electrophilic fragments, and regular samples taken for quenching into a solution of CPM (7-Diethylamino-3-(4&#39;-Maleimidylphenyl)-4-Methylcoumarin, a fluorogenic thiol-quantification agent). Adduct formation between the cysteine and electrophilic fragment reduces the amount of thiol available for reaction with CPM, reducing the resulting fluorescence on quenching. CPM fluorescence thus acts as a proxy for unreacted thiol, allowing the kinetics of thiol reaction with each electrophilic fragment to be determined. Electrophilic fragments with significantly enhanced protein reactivity over glutathione are selected as hits and prioritized for further development (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67178/67178fig01.jpg" /> 
Figure 1: Schematic of qIT. A schematic overview of the qIT assay. (created using BioRender). <a href="https://www.jove.com/files/ftp_upload/67178/67178fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Hit selection using REF values controls for intrinsic reactivity and ensures that fragments are only selected as hits if they form productive non-covalent interactions with a protein target. The simple fluorescence readout also means the qIT assay is highly scalable and does not require mass spectrometry-based methods of reaction monitoring.
Here, a comprehensive procedure for the qIT assay is outlined, with example results included to illustrate assay performance and hit selection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Argon Gas</td>
			<td>BOC</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well, low-flange, flat-bottom assay plates</td>
			<td>Corning</td>
			<td>3575</td>
			<td></td>
		</tr>
		<tr>
			<td>CPM</td>
			<td>Invitrogen</td>
			<td>D346</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilized TCEP-agarose</td>
			<td>Pierce</td>
			<td>77712</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro plate reader</td>
			<td>CLARIOstar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Plate Seals</td>
			<td>BioRad</td>
			<td>MSA5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced L-glutathione</td>
			<td>Sigma-Aldrich</td>
			<td>G4251</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>RES6007H-A7</td>
			<td></td>
		</tr>
	</tbody>
</table>

covalent fragment screening using the quantitative irreversible tethering assay

Compounds that form covalent bonds with specific target proteins offer a variety of advantages&#160;as chemical probes and therapeutic agents. Most commonly, mildly reactive, electrophilic small molecules are employed to form covalent bonds with select cysteine side chains in specific proteins. Electrophile-first approaches of ligand discovery, whereby a library of electrophilic small molecules are screened against a protein target, have become popular as they avoid the need for time-consuming downstream installation of an electrophilic warhead. Such screening is complicated, however, as electrophilic ligands can exhibit a wide range of different rates of spontaneous reaction with cysteines. Quantitative-irreversible tethering (qIT) offers a fluorescence-based method for hit identification and development that normalizes data for these differences in intrinsic compound reactivity. Rates of reaction of individual compounds with a target protein are determined and compared to compound reactivity with the unstructured tripeptide glutathione (this being a proxy for spontaneous compound reaction), enabling the identification of compounds that preferentially react with the protein of interest. This methodology has been successfully applied to identify selective covalent fragments against several drug targets, including SARS-CoV-2 main protease, cyclin-dependent kinase 2, and RAP27A. Here, we demonstrate the application of qIT to a target protein to generate a quantitative and robust data set, allowing prioritization of hit ligands for future development.

The qIT assay is used here to identify covalent fragment hits for an unnamed, single cysteine protein. MgCl2 (10 mM) was added to the reaction buffers as the target protein is known to bind to Mg2+ ions. The screening was performed in triplicate, and the rate at which each fragment reacted with both the target protein (kpro) and glutathione (kGSH) determined. Z&#39; values were greater than 0.5 for all quench plates and averaged 0.75 for protein quenches and 0.79 for glutathione quenches, indicating excellent assay performance. REF values were then calculated for each fragment, and hits selected where REF &#62; 3.
The representative results (Figure 4), provide an example of hit selection using the qIT assay. Compound 1 reacted at a 6.0-fold greater rate with the cysteine residue on the target protein than with glutathione and, using a criterion of REF &#62; 3, was selected as a hit (Figure&#160;4B). This rate acceleration indicates the presence of a templating effect, whereby non-covalent interactions position 1 in the correct orientation for the cysteine on the target protein to react with the chloroacetamide warhead. In contrast, 2 is an example of a non-hit fragment as the reaction between 2 and the target cysteine residue is not accelerated relative to the reaction between 2 and glutathione. The lack of rate acceleration indicates there are not productive non-covalent interactions positioning 2 in the correct orientation to react with the target cysteine residue.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67178/67178fig04v2.jpg" /> 
Figure 4: Representative results. The results show protein and glutathione reaction monitoring by qIT for (A)&#160;A high intrinsic reactivity, a possible false-positive hit fragment, and (B)a hit fragment. <a href="https://www.jove.com/files/ftp_upload/67178/67178fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

The quantitative irreversible tethering (qIT) assay is a fluorescence-based method of identifying covalent fragments that selectively engage with a target protein. Here, a detailed protocol is outlined to describe the qIT assay, with example results included.

Compounds that form covalent bonds with specific target proteins offer a variety of advantages&#160;as chemical probes and therapeutic agents. Most ...

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Biochemistry

373 Views.  Imperial College London, Molecular Sciences Research Hub. The quantitative irreversible tethering (qIT) assay is a fluorescence-based method of identifying covalent fragments that selectively engage with a target protein. Here, a detailed protocol is outlined to describe the qIT assay, with example results included.

Video: Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

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 ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties 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 ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

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Chapters in this video

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Real-time Tracking of DNA Fragment Separation by Smartphone

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

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

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin