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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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><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.remotexs.ntu.edu.sg/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 ...

covalent-fragment-screening-using-quantitative-irreversible-tethering

Nanyang Technological University

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JoVE Journal

Biochemistry

452 Views.  Imperial College London, Molecular Sciences Research Hub. We werken samen tussen de departementen chemie en biowetenschappen en ontwikkelen nieuwe assisterende reactieve covalente liganden voor een reeks doelwitten tegen kanker. Het is te hopen dat deze moleculen in de toekomst zullen worden gebruikt, hetzij als therapieën, hetzij mogelijk als chemische sondes. Er bestaan dus verschillende technologieën voor het screenen van covalente liganden.Deze kunnen grofweg worden opgesplitst in proteomische technieke...

Video: Screening van covalente fragmenten met behulp van de kwantitatieve onomkeerbare tethering-assay

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 ...

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

Cancer Research

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 ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

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 ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into lead-like compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen.
Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification.
XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turn-around for the main protease.

This paper describes the complete XChem process for crystal-based fragment screening, starting from applying for access and all subsequent steps to data dissemination.

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify ...

Chemistry

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

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