Development and applications of chimera platforms for tyrosine phosphorylation

Development and applications of chimera platforms for tyrosine phosphorylation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Development and applications of chimera platforms for tyrosine phosphorylation View ORCID Profile Rajaiah Pergu , View ORCID Profile Veronika M. Shoba , View ORCID Profile Santosh K. Chaudhary , Dhanushka N. P. Munkanatta Godage , View ORCID Profile Arghya Deb , View ORCID Profile Santanu Singha , View ORCID Profile Uttam Dhawa , View ORCID Profile Viktoriya Anokhina , View ORCID Profile Sameek Singh , Sachini U. Siriwardena , View ORCID Profile Amit Choudhary doi: https://doi.org/10.1101/2023.03.05.531183 Rajaiah Pergu 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rajaiah Pergu Veronika M. Shoba 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Veronika M. Shoba Santosh K. Chaudhary 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Santosh K. Chaudhary Dhanushka N. P. Munkanatta Godage 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Arghya Deb 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arghya Deb Santanu Singha 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Santanu Singha Uttam Dhawa 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Uttam Dhawa Viktoriya Anokhina 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Viktoriya Anokhina Sameek Singh 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sameek Singh Sachini U. Siriwardena 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amit Choudhary 1 Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard , Cambridge, MA 02142, USA 2 Department of Medicine, Harvard Medical School , Boston, MA 02115, USA 3 Divisions of Renal Medicine and Engineering, Brigham and Women’s Hospital , Boston, MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Amit Choudhary For correspondence: achoudhary{at}bwh.harvard.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Chimeric small molecules that induce post-translational modification (PTM) on a target protein by bringing it in proximity to a PTM-inducing enzyme are furnishing novel modalities to perturb protein function. Despite recent advances, such molecules are unavailable for a critical PTM, tyrosine phosphorylation. Furthermore, the contemporary design paradigm of chimeric molecules, formed by joining a non-inhibitory binder of the PTM-inducing enzyme with the binder of the target protein, prohibits the recruitment of most PTM-inducing enzymes as their non-inhibitory binders are unavailable. Here, we report two platforms to generate phosphorylation-inducing chimeric small molecules (PHICS) for tyrosine phosphorylation. We generate PHICS from both non-inhibitory binders (scantily available, platform 1) and kinase inhibitors (abundantly available, platform 2) using cysteine-based group transfer chemistry. PHICS triggered phosphorylation on tyrosine residues in diverse sequence contexts and target proteins (e.g., membrane-associated, cytosolic) and displayed multiple bioactivities, including initiation of a growth receptor signaling cascade and death of drug-resistant cancer cells. These studies provide an approach to induce biologically relevant PTM and lay the foundation for pharmacologic PTM editing (i.e., induction or removal) on target proteins using abundantly available inhibitors of PTM-inducing or erasing enzymes. Download figure Open in new tab INTRODUCTION Protein phosphorylation is the most frequent and well-studied post-translational modification (PTMs) 1 , 2 , 3 , and its dysregulation is associated with various pathologies. 4 , 5 In particular, phosphorylated tyrosine (pTyr) is vital 6 , 7 in many diverse cellular functions and signaling, including highly dynamic processes often controlled using small molecules. The human genome codes 90 tyrosine kinases, 6 107 tyrosine phosphatases, 8 and over hundreds of pTyr-recognition domains (e.g., SH2 domain) 9 - 11 and the immune system can produce antibodies that selectively recognize pTyr over phosphorylated serine or threonine. These attributes have led to suggestions to consider pTyr a functionally separate PTM class. 12 While molecules that inhibit tyrosine phosphorylation exist and constitute one of the largest classes of drugs 13 - 15 , there are no small molecules that can induce tyrosine phosphorylation on a given target of interest, even though these compounds will be of immense utility in both basic research and biomedicine. 16 Recently, we reported on an approach to induce phosphorylation using heterobifunctional small molecules termed phosphorylation-inducing chimeric small molecules (PHICS) that recruit endogenous Ser/Thr kinases to a target protein of interest. 17 - 19 These PHICS were formed by joining small-molecule ligands of targeted protein and a kinase via a linker. Our previously reported PHICS brought Ser/Thr kinases (AMPK or PKC) in proximity to a target protein, inducing the phosphorylation of the latter even for non-substrates (i.e., neo-substrates) of the kinase. However, these PHICS did not recruit endogenous tyrosine kinase or induce a downstream signaling event. Furthermore, these PHICS require non-inhibitory kinase binders that are unavailable for most kinases, limiting recruitment of only two of ≈538 human kinases. 20 Beyond phosphorylation, heterobifunctional molecules that can induce or remove other PTMs, including acetylation and glycosylation, by recruiting PTM inducing or erasing enzymes are needed. 19 , 21 However, the necessity of non-inhibitory ligands for these enzymes has been a major roadblock in the development of such heterobifunctional molecules. Finally, the previously reported PHICS did not explore the covalent binders for the target protein or kinase which will have relatively simple two-body equilibrium vs. the complex three-body equilibrium displayed by non-covalent binders. 22 - 26 Herein, we report tyrosine PHICS that recruit Abelson Kinase (ABL) or Bruton’s Tyrosine Kinase (BTK) to induce the phosphorylation of neo-substrates in cells. We report five non-inhibitory molecular scaffolds to recruit ABL to a target protein, and PHICS generated from these scaffolds display hallmarks of contemporarly heterobifunctional molecules, including hook effect, turnover, and ternary complex formation in cells. Importantly, we showed that ABL PHICS was able to initiate a signaling pathway by inducing phosphorylation of epidermal growth factor receptor (EGFR). Furthermore, we report a new design paradigm of a heterobifunctional molecule consisting of a BTK inhibitor connected to the target binder through a linker that covalently labels BTK with the target-protein binder while releasing the inhibitor from BTK and enabling phosphorylation of the target by BTK. We demonstrated this group transfer chemistry with two commonly employed covalent inhibitor scaffolds (i.e., aliphatic amine and aryl amine). Using BTK or ABL PHICS, we phosphorylated tyrosine residues in a diverse sequence context, including those surrounded by neutral, acidic, or basic residues. Finally, we generated ABL-BTK PHICS using a covalent inhibitor of BTK (i.e., ibrutinib) to induce its ABL-mediated phosphorylation; these PHICS effectively kill ibrutinib-resistant cancer cell lines and inhibit downstream survival signaling of BTK, highlighting the therapeutic potential of this class of molecules. RESULTS AND DISCUSSION Identification of ABL binders for generation of BRD4 PHICS For the tyrosine kinase recruited by PHICS, we first chose ABL, as it can phosphorylate wide-ranging target sequences and has several non-inhibitory, selective, and allosteric binders that also have co-crystal structures to aid the rational design of PHICS. 27 - 30 We selected four scaffolds that competitively bind to the myristoyl pocket of ABL: dihydropyrazole (scaffold 1, Figure S1A), thiazole (scaffold 2, Figure S1B), pyrazole (scaffold 3, Figure S1C), 30 and hydantoin (scaffold 4, Figure S2). 29 Using molecular docking studies, we identified the solvent-exposed 3-amino group as the linker attachment site for the dihydropyrazole, pyrazole, and thiazole scaffolds (Figure S1A‒C, PDB ID: 6NPV and 6NPE). 30 For the hydantoin scaffold (PDB ID: 3PYY), 29 linkers were grown from the ortho, meta , or para positions of the solvent-exposed aryl ring (Figure S2). Previous reports for synthesizing hydantoin scaffold 29 involving the Vilsmer-Haack reaction provided poor yield 31 and were sensitive to substitutions on the aryl ring (Figure S3A) preventing exploration of linker growth from - ortho , - meta , or - para positions from aryl ring. We developed a new synthetic route with fewer steps to rapidly access hydantoin scaffold precursors by involving Cu-mediated high-yielding C─N bond coupling 32 between pyrazole and appropriately substituted phenylboronic acids (Figure S3B). By applying this synthetic strategy, we were able to rapidly generate a library of hydantoin-based PHICS with linkers through the - ortho , - meta , and - para positions of the aryl ring. To assess the efficacy of these scaffolds, we synthesized four different PHICS, each containing one of the four binders of ABL, a linker comprised of four polyethylene glycol units, and the small molecule ( S )-JQ1 (Figures S3C and S4), which binds our proof-of-concept target bromodomain-containing protein 4 (BRD4). 33 With these compounds in hand, we measured PHICS-induced phosphorylation using recombinant ABL and BRD4 proteins. Dihydropyrazole induced the most BRD4 phosphorylation followed by PHICS based on thiazole, while the pyrazole scaffold (an oxidized form of the dihydropyrazole) induced significantly less phosphorylation (Figure S5). Hydantoin-derived PHICS induced less phosphorylation than the dihydropyrazole-derived PHICS with the meta-substituted analog showing the highest phosphorylation levels compared to the ortho- and para-substituted analogs (Figures S5 and S6). These studies identified the dihydropyrazole scaffold as the most efficient for inducing BRD4 phosphorylation. To more deeply characterize the ability of the dihydropyrazole scaffold to induce BRD4 phosphorylation, PHICS 1 ( Figure 1A ) was selected for further studies together with three controls: 2 generated by replacing ( S )-JQ1 in 1 with inactive enantiomer ( R )-JQ1, 3 formed by replacing the dihydropyrazole in 1 with pyrazole scaffold, and 4 a dihydropyrazole scaffold with a short linker. In biochemical settings, we confirmed that tyrosine phosphorylation of BRD4 occurred only in the presence of all components of the ternary complex, namely 1 , BRD4, and ABL ( Figure 1B ). We then assessed the formation of the ternary complex between 1 , BRD4-GST, and ABL-His was assessed using Amplified Luminescent Proximity Homogenous assay (AlphaScreen), 34 wherein 1 , but not 2 , showed a “hook-effect” which is a hallmark of 3-body equilibrium 22 , 23 ( Figure 1C ). Here, as the concentration of 1 increases, there is an increase in the population of the ternary complex. However, after a certain concentration, the binary complexes between 1 and ABL and 1 and BRD4 dominate the equilibrium over the ternary complex, resulting in a hook-shape dose curve. Download figure Open in new tab Figure 1. (A) Structures of ABL-BRD4 PHICS 1 and control compounds 2-4. (B) PHICS-induced phosphorylation of BRD4 by ABL in vitro . (C) PHICS-induced ternary-complex formation between BRD4 and ABL observed by Alpha Screen assay. (D) PHICS-induced phosphorylation of BRD4 by ABL in HEK293T cells; co-immunoprecipitation (Co-IP) of ABL with BRD4 in the presence of PHICS. (E) ADPGlo assay for BRD4 phosphorylation by ABL in the presence of 1 and its inactive isomer 2 . To demonstrate BRD4 phosphorylation in cells using PHICS, we used a construct of BRD4 lacking an intrinsic nuclear localization signal 35 and incubated HEK293T cells transfected with both HA-BRD4 and ABL-FLAG plasmids with bifunctional molecules and controls. Here, HA-based immunoprecipitation (IP) showed significantly higher co-immunoprecipitation of ABL-FLAG with 1 compared to the control compounds (i.e., 2, 3 , or 4 ), suggesting ternary-complex formation is specific to 1 ( Figure 1D ). Probing the immunoprecipitated HA-BRD4 with a pan phospho-tyrosine antibody showed significantly higher phosphorylation levels in cells treated with 1 than in the control compounds, confirming that the observed phosphorylation is arising from PHICS-mediated ternary complex formation between ABL and BRD4. To confirm that the reversible binding of PHICS to both BRD4 and ABL allows it to exhibit catalytic turnover, we used an ADP-Glo assay 36 to determine the amount of ADP generated per molecule of ABL using active molecule 1 and control 2 ( Figure 1E ). Here, the amount of ADP generated in the reaction with 1 was 2261 ± 411 nM higher than the amount of ADP generated in the reaction with 2 at a limiting concentration of ABL kinase (30 nM), confirming that PHICS exhibits turnover, like our previously reported Ser/Thr PHICS. 17 PHICS can activate EGFR signaling EGFR is a tyrosine kinase receptor that responds to the epidermal growth factor (EGF) binding to initiate a signaling pathway that regulates the growth, proliferation, survival, and differentiation of cells. 37 , 38 Upon EGF binding, EGFR oligomerizes and autophosphorylates tyrosine residues, which acts as docking site of proteins with phosphotyrosine-binding SH2 or PTB domains leading to a series of molecular events and cellular response. 39 We were interested in determining if ABL PHICS can trigger signaling by phosphorylating relevant EGFR tyrosines. Some kinases prefer specific residues surrounding the phosphosite while others do not. Since signaling relevant EGFR tyrosines are present in diverse sequence environments (i.e. surrounded by acidic, basic, or neutral residues), we were interested in determining if ABL PHICS can phosphorylate these diverse tyrosines and trigger signaling. We constructed a catalytically inactive intracellular domain of the EGFR variant fused to FKBP F36V ( iEGFR-FKBP F36V -FLAG ), and synthesized PHICS 5 using the FKBP F36V binder AP1867 ( Figure 2A , S7). 40 HEK293T cells transfected with iEGFR-FKBP F36V -FLAG and ABL-HA were treated with 5 or DMSO. Immunoblotting of cell lysates and probing with site-specific phospho-tyrosine antibodies suggested that ABL PHICS could phosphorylate tyrosines present in diverse sequence environments ( Figure 2B ) in line with previous reports that ABL lacks sequence motifs. 28 , 41 Similarly, we observed phosphorylation when using catalytically inactive HER2, another receptor tyrosine kinase (Figure S8), pointing to the generality of the designed PHICS. Download figure Open in new tab Figure 2. (A) Structure of ABL-FKBP F36V bifunctional molecule 5 used in PHICS-induced phosphorylation of EGFR-FKBP F36V construct. (B) Diverse sequence environments of Tyr phosphorylated by ABL in cells in the presence of 5 detected using antibodies specific for phospho-EGFR. (C) Induction of luciferase-based reporter gene with serum response element by PHICS 5 , but not by DMSO or ABL binder 4 . To evaluate the effect of PHICS 5 on downstream EGFR signaling, we used a previously described luciferase-based reporter gene assay with serum response element (SRE). 42 Here, HEK293T cells were co-transfected with iEGFR-FKBP F36V -FLAG and pGL4.33[luc2P/SRE/Hygro] vectors, treated with PHICS 5 , or controls (DMSO or 4 ); the activation of EGFR signaling was quantified using Promega’s ONE-Glo™ Luciferase Assay System. PHICS 5 induced a two-fold higher luciferase activity than DMSO or ABL binder 4 , indicating activation of EGFR by 5 ( Figure 2C ). These studies confirm that cytoplasmic ABL can be recruited to membrane-localized targets to induce functionally relevant tyrosine phosphorylations flanked by various amino-acid sequences and further confirms the broad range of phosphorylating motifs available to ABL kinase. 43 , 44 PHICS can be developed from covalent binder of the target protein While covalency at the kinase end allows phosphorylation of multiple molecules of the target protein per molecule of PHICS owing to turnover, the use of covalent binder of the target protein will block such turnover by PHICS and may dramatically reduce the efficacy of PHICS to induce phosphorylation. We were interested in determining if efficacious PHICS can be generated using a covalent binder of the target protein. For this purpose, several bifunctional molecules were designed by connecting hydantoin or dihydropyrazole binders of ABL to chloroalkane linkers that covalently bind HaloTag ( Figure 3A and S9). 45 To confirm the cell permeability of these compounds and their ability to efficiently label HaloTag, we used a competition assay wherein HEK293T cells stably expressing HaloTag were treated with compounds 7, 9, 11 , or 12 and controls ( 10 and 4 ), and the lysates were subsequently labeled with tetramethylrhodamine (TMR)-HaloTag, a fluorescent probe. 46 We found that the lysates from cells pretreated with HaloTag-PHICS were not labeled by TMR-HaloTag, whereas control-treated lysates ( 10 and 4 ) showed efficient TMR-labelling (Figure S10), indicating that HaloTag-PHICS are cell-permeable and efficiently label the HaloTag protein. To assess the relative ability of these covalent PHICS to induce tyrosine phosphorylation, we transiently expressed the ABL-HA construct in HEK293T cells stably expressing the HaloTag protein and treated these cells with various covalent PHICS (compounds 7, 9, 11 , and 12 ) and controls (compounds 6, 8, 10 , and 4 ). We observed significant tyrosine phosphorylation on HaloTag in the presence of active PHICS compared to control-treated samples ( Figure 3B ). These results demonstrate that irreversible binding is tolerated by both the kinase and the target to generate a functional PHICS, opening avenues for the use of covalent inhibitors, a burgeoning class of chemical matter. 47 Download figure Open in new tab Figure 3. (A) Structures of hydantoin and dihydropyrazole-derived HaloTag bifunctional and control compounds. (B) PHICS-induced phosphorylation of HaloTag by ABL in cells. PHICS can be developed using kinase inhibitors All the reported PHICS were developed using non-inhibitory allosteric binders, which are rare, though kinase inhibitors are plentiful and form an important class of therapeutic. 48 A PHICS can be designed 49 - 51 from a kinase inhibitor in which a nucleophile (e.g., cysteine) near the inhibitor-binding pocket can initiate a group transfer chemistry 52 - 59 resulting in appending the target-protein binder to the kinase while releasing the inhibitor from the kinase’s active site ( Figure 4A ). The kinase binder in such cysteine-triggered PHICS can be derived from abundantly available acrylamide-based inhibitors, where the amide nitrogen is often an aryl-amine 60 ( 13 , Figure 4B ) or less frequently aliphatic amine 61 ( 14 , Figure 4B ). 47 Using these BTK inhibitor scaffolds, we synthesized BRD4 PHICS 17 (aryl amine scaffold, Figure 4B ) and 19 (aliphatic amine scaffold, Figure 4B ), wherein the inhibitor is connected to the BRD4 binder JQ1 via a cleavable methacrylamide linker (Figure S11). We also synthesized inactive controls ( 18 and 20 , Figure 4B ) for BRD4 using ( R )-JQ1 and tested whether 17 or 19 can rewire BTK’s specificity and induce the phosphorylation of BRD4. Indeed, we observed much higher BRD4 phosphorylation in the presence of 17 ( Figure 4C ) or 19 ( Figure 4D ) than with inactive controls ( 18 or 20 ) in cells. Using the same assessments of ternary-complex formation as previously, we observed significantly higher levels of co-immunoprecipitated BTK-FLAG and higher levels of BRD4 phosphorylation in samples treated with 17 or 19 compared to samples treated with inactive controls, 18 or 20 ( Figures 4C-D ). Download figure Open in new tab Figure 4. (A) Schematic representation of BTK tagging via inhibitor-directed addition-elimination, ternary complex formation, and phosphorylation of BRD4. (B) Structures of known BTK inhibitors 13 and 14 , corresponding alkyne-containing analogs 15 and 16 , and BTK-BRD4 PHICS 17 and 19 with their inactive analogs 18 and 20. (C-D) PHICS-induced phosphorylation of BRD4 by BTK in cells and co-IP of BTK-FLAG with HA-BRD4 upon treatment with 17 - 18 ( C ) or 19 - 20 ( D ). Next, we performed target engagement and labeling studies designed to support the mechanism outlined in Figure 4A . Using compounds that have an alkyne handle in place of JQ1 ( 15 and 16 , Figure 4B ), we confirmed that the scaffolds of 17 or 19 ( Figure 4B ) covalently engaged BTK in cells. Briefly, HEK293T cells transiently expressing BTK were treated with alkyne-containing compounds 15 and 16 for 4 h. After washing with PBS, Cu-catalyzed click reaction was performed on the lysates using sulfo-Cy5.5 azide; BTK labeling was confirmed via in-gel fluorescence ( Figure 5A ). To demonstrate that the BTK inhibitor scaffold is released from the ATP-binding pocket, we used a reporter assay based on Bioluminescence Resonance Energy Transfer (BRET) 62 between a nanoluciferase (nanoLuc) and a fluorophore probe that binds to the ATP pocket; a higher inhibitor occupancy in this ATP pocket will prevent the binding of the tracer, and lower the BRET signal 63 ( Figure 5B ). As expected, 13 and 14 dramatically lowered the BRET signal indicating blockage of the ATP pocket, while their derivatives with cleavable methacrylamide linkers (alkynes 15 and 16 , BRD4-BTK PHICS 17 and 19 ) did not affect the binding of tracer ( Figure 5C-D ). Agreeing with these findings, we observed higher autophosphorylation of BTK with PHICS compounds 17 and 19 compared to the parent inhibitors 13 and 14 (Figure S12). These studies demonstrate that PHICS can be generated from cysteine-targeting covalent inhibitors, which are available for many kinases 47 , 64 with diverse cellular and tissue localization, sequence preferences, and successful clinical outcomes. Download figure Open in new tab Figure 5. (A) Demonstration of covalent labeling of BTK in cells by 13 and 14 at 1 µM via in-gel fluorescence. (B) Schematic representation of nanoBRET assay for evaluation of BTK binding pocket occupancy. (C-D) NanoBRET assay for binding pocket occupancy for known BTK inhibitors 13 ( C ) and 14 ( D ), and their derivatives designed to label BTK via group transfer chemistry. PHICS induce death of drug-resistant cancer cells After validating two classes of Tyr-PHICS (ABL- and BTK-derived), we explored the functional capabilities of bifunctional molecules consisting of binders of both kinases since these kinases are often overexpressed in cancer cells, and we have reported that Ser/Thr PHICS can induce inhibitory phosphorylation on BTK. 18 We designed compound 21 , connecting the reversible binder of ABL with the covalent binder of BTK via a PEG3 linker ( Figure 6A and S13) and used this PHICS along with a mixture of the separate binders ( 22 and 4 ) for the treatment of HEK293T cells co-transfected with ABL-HA and BTK-FLAG plasmids. Western blotting with a pan anti-phospho-tyrosine antibody revealed increased levels of BTK phosphorylation in the presence of 21 when compared to the control (mixture of binders 22 and 4 ) ( Figure 6B ). Surprisingly, the level of ABL phosphorylation remained unchanged. This phosphorylation outcome motivated us to evaluate BTK-ABL bifunctional 21 in cancer cell lines that depend on ABL or BTK, particularly those that were resistant to the known BTK-targeting drug, ibrutinib 65 . When tested in BTK-dependent ibrutinib-resistant Mino and Raji cell lines, 21 reduced their viability with an EC 50 of 1.7 µM and 4.9 µM, respectively ( Figure 6C ). 65 In contrast, 21 did not impact the viability of BCR-ABL dependent K562 cells (Figure S14), in agreement with our expectation based on the unaffected phosphorylation of ABL in HEK293T-based studies ( Figure 6B ). We noted that neither the mixture of individual binders ( 22 and 4 ) nor ibrutinib induced the same effect of cell viability in BTK-dependent and ibrutinib-resistant cancer cells as compound 21 . These results with ibrutinib are especially interesting, as this BTK inhibitor is an approved therapeutic agent for several B-cell cancers. However, the emergence of resistance has resulted in a need for next-generation therapeutics; 66 the promising activity of BTK-ABL PHICS in ibrutinib-resistant cell lines opens such an alternative therapeutic strategy. Download figure Open in new tab Figure 6. (A) Structures of ABL-BTK bifunctional molecule 21 and BTK binder 22. (B) PHICS-induced phosphorylation of BTK by ABL in HEK293T cells. (C) Effect of PHICS 21 on the viability of Mino and Raji cells. CONCLUSION To address the lack of technologies for the facile editing of PTMs (i.e., addition or removal) on a given protein of interest, we report a new class of bifunctional molecules that induce functionally relevant tyrosine phosphorylation by recruiting ABL or BTK. PHICS triggered activation of EGFR, which belongs to the receptor tyrosine kinase superfamily that includes insulin receptor. A PHICS that similarly activates insulin receptor signaling may furnish an orally available, small molecule substitute for the insulin that requires injection and may offer an alternative therapeutic modality to patients with insulin resistance. Furthermore, PHICS induced death of drug-resistant cancer cells, offering a potentially novel and alternative therapeutic modality to kinase inhibitors against which resistance has developed. The CRISPR-Cas system 67 , 68 has furnished technologies for facile editing of the DNA or RNA, but methods for facile editing of post-translational modifications (i.e., addition or removal) on a given protein of interest is still challenging. We report an approach to induce phosphorylation on the target protein by generating PHICS that utilize kinase inhibitors and cysteine-triggered group transfer chemistries. Since inhibitors for several PTM-inducing or removing enzymes are available, and group transfer chemistries can be implemented using other nucleophilic residues (e.g., lysine, tyrosine, methionine); 69 - 71 these studies lay the foundation for pharmacologic editing of PTMs on proteins. Overall, these studies further highlight the power of bifunctional molecules to endow neo-functions to proteins in cells with value in basic research and medicine. AUTHOR CONTRIBUTIONS These authors contributed equally to this work. COMPETING FINANCIAL INTERESTS Broad Institute has filed patent applications for the work described herein, some of which were licensed to Photys Therapeutics. A.C. is the scientific founder and is on the scientific advisory board of Photys Therapeutics. ACKNOWLEDGMENTS We thank Dr. Mary O’Reilly (Pattern, Broad Institute) for contributive discussions and help with figure design. This work was supported by the Merkin Institute of Transformative Technologies in Healthcare, Mark Foundation, Burroughs Wellcome Fund (Career Award at the Scientific Interface), DARPA (N66001-17-2-4055 and HR0011-21-2-0010), and NIH (R21AI154099). V.M.S. was supported by Damon Runyon Postdoctoral Fellowship. REFERENCES (1). ↵ Cohen , P. The origins of protein phosphorylation . Nat Cell Biol 2002 , 4 ( 5 ), E127 – 130 . OpenUrl CrossRef PubMed Web of Science (2). ↵ Gschwind , A. ; Fischer , O. M. ; Ullrich , A. The discovery of receptor tyrosine kinases: targets for cancer therapy . Nat Rev Cancer 2004 , 4 ( 5 ), 361 – 370 . OpenUrl CrossRef PubMed Web of Science (3). ↵ Fuhs , S. R. ; Hunter , T. pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification . Curr Opin Cell Biol 2017 , 45 , 8 – 16 . OpenUrl (4). ↵ Lahiry , P. ; Torkamani , A. ; Schork , N. J. ; Hegele , R. A. Kinase mutations in human disease: interpreting genotype-phenotype relationships . Nat Rev Genet 2010 , 11 ( 1 ), 60 – 74 . OpenUrl CrossRef PubMed (5). ↵ Zarrin , A. A. ; Bao , K. ; Lupardus , P. ; Vucic , D. Kinase inhibition in autoimmunity and inflammation . Nat Rev Drug Discov 2021 , 20 ( 1 ), 39 – 63 . OpenUrl CrossRef (6). ↵ Hunter , T. Tyrosine phosphorylation: thirty years and counting . Curr Opin Cell Biol 2009 , 21 ( 2 ), 140 – 146 . OpenUrl CrossRef PubMed Web of Science (7). ↵ Hunter , T. The genesis of tyrosine phosphorylation . Cold Spring Harbor perspectives in biology 2014 , 6 ( 5 ), a020644 . OpenUrl Abstract / FREE Full Text (8). ↵ Mustelin , T. ; Vang , T. ; Bottini , N. Protein tyrosine phosphatases and the immune response . Nat Rev Immunol 2005 , 5 ( 1 ), 43 – 57 . OpenUrl CrossRef PubMed Web of Science (9). ↵ Yaffe , M. B. Phosphotyrosine-binding domains in signal transduction . Nat Rev Mol Cell Biol 2002 , 3 ( 3 ), 177 – 186 . OpenUrl CrossRef PubMed Web of Science (10). Kaneko , T. ; Joshi , R. ; Feller , S. M. ; Li , S. S. Phosphotyrosine recognition domains: the typical, the atypical and the versatile . Cell Commun Signal 2012 , 10 ( 1 ), 32 . OpenUrl CrossRef PubMed (11). ↵ Liu , B. A. ; Nash , P. D. Evolution of SH2 domains and phosphotyrosine signalling networks . Philos Trans R Soc Lond B Biol Sci 2012 , 367 ( 1602 ), 2556 – 2573 . OpenUrl CrossRef PubMed (12). ↵ Sharma , K. ; D’Souza , R. C. ; Tyanova , S. ; Schaab , C. ; Wiśniewski , J. R. ; Cox , J. ; Mann , M. Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling . Cell reports 2014 , 8 ( 5 ), 1583 – 1594 . OpenUrl (13). ↵ Levitzki , A. ; Mishani , E. Tyrphostins and other tyrosine kinase inhibitors . Annu Rev Biochem 2006 , 75 , 93 – 109 . OpenUrl CrossRef PubMed Web of Science (14). Levitzki , A. Tyrosine kinase inhibitors: views of selectivity, sensitivity, and clinical performance . Annu Rev Pharmacol Toxicol 2013 , 53 , 161 – 185 . OpenUrl CrossRef PubMed (15). ↵ Ferguson , F. M. ; Gray , N. S. Kinase inhibitors: the road ahead . Nature Reviews Drug Discovery 2018 , 17 ( 5 ), 353 – 377 . OpenUrl CrossRef PubMed (16). ↵ Pottier , C. ; Fresnais , M. ; Gilon , M. ; Jérusalem , G. ; Longuespée , R. ; Sounni , N. E. Tyrosine Kinase Inhibitors in Cancer: Breakthrough and Challenges of Targeted Therapy . Cancers (Basel) 2020 , 12 ( 3 ). (17). ↵ Siriwardena , S. U. ; Munkanatta Godage , D. N. P. ; Shoba , V. M. ; Lai , S. ; Shi , M. ; Wu , P. ; Chaudhary , S. K. ; Schreiber , S. L. ; Choudhary , A. Phosphorylation-Inducing Chimeric Small Molecules . J Am Chem Soc 2020 , 142 ( 33 ), 14052 – 14057 . OpenUrl CrossRef (18). ↵ Shoba , V. M. ; Munkanatta Godage , D. N. P.; Chaudhary , S. K. ; Deb , A. ; Siriwardena , S. U. ; Choudhary , A. Synthetic reprogramming of kinases expands cellular activities of proteins . Angew Chem Int Ed Engl 2022 , 61 ( 29 ), e202202770 . OpenUrl (19). ↵ Lai , S. ; Modell , A. E. ; Choudhary , A. Proximity-Inducing Bifunctional Molecules Beyond PROTACs . In Protein Homeostasis in Drug Discovery , 2022 ; pp 419 – 446 . (20). ↵ Zhang , H. ; Cao , X. ; Tang , M. ; Zhong , G. ; Si , Y. ; Li , H. ; Zhu , F. ; Liao , Q. ; Li , L. ; Zhao , J. ; et al. A subcellular map of the human kinome . Elife 2021 , 10 . (21). ↵ Hua , L. ; Zhang , Q. ; Zhu , X. ; Wang , R. ; You , Q. ; Wang , L. Beyond proteolysis-targeting chimeric molecules: Designing heterobifunctional molecules based on functional effectors . J Med Chem 2022 , 65 ( 12 ), 8091 – 8112 . OpenUrl (22). ↵ Douglass , E. F. , Jr . .; Miller , C. J. ; Sparer , G. ; Shapiro , H. ; Spiegel , D. A. A comprehensive mathematical model for three-body binding equilibria . J Am Chem Soc 2013 , 135 ( 16 ), 6092 – 6099 . OpenUrl CrossRef PubMed (23). ↵ Lu , C. ; Wang , Z. X. Quantitative analysis of ligand induced heterodimerization of two distinct receptors . Anal Chem 2017 , 89 ( 13 ), 6926 – 6930 . OpenUrl (24). Roy , M. J. ; Winkler , S. ; Hughes , S. J. ; Whitworth , C. ; Galant , M. ; Farnaby , W. ; Rumpel , K. ; Ciulli , A. SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate . ACS Chem Biol 2019 , 14 ( 3 ), 361 – 368 . OpenUrl CrossRef (25). Han , B. A suite of mathematical solutions to describe ternary complex formation and their application to targeted protein degradation by heterobifunctional ligands . J Biol Chem 2020 , 295 ( 45 ), 15280 – 15291 . OpenUrl Abstract / FREE Full Text (26). ↵ McCann , H. M. ; Lake , B. P. M. ; Hoffman , K. S. ; Davola , M. E. ; Mossman , K. L. ; Rullo , A. F. Covalent immune proximity-induction strategy using SuFEx-engineered bifunctional viral peptides . ACS Chem Biol 2022 , 17 ( 5 ), 1269 – 1281 . OpenUrl (27). ↵ Rychlewski , L. ; Kschischo , M. ; Dong , L. ; Schutkowski , M. ; Reimer , U. Target specificity analysis of the Abl kinase using peptide microarray data . J Mol Biol 2004 , 336 ( 2 ), 307 – 311 . OpenUrl CrossRef PubMed (28). ↵ Colicelli , J. ABL tyrosine kinases: evolution of function, regulation, and specificity . Sci Signal 2010 , 3 ( 139 ), re6 . OpenUrl Abstract / FREE Full Text (29). ↵ Yang , J. ; Campobasso , N. ; Biju , M. P. ; Fisher , K. ; Pan , X. Q. ; Cottom , J. ; Galbraith , S. ; Ho , T. ; Zhang , H. ; Hong , X. ; et al. Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site . Chem Biol 2011 , 18 ( 2 ), 177 – 186 . OpenUrl CrossRef PubMed (30). ↵ Simpson , G. L. ; Bertrand , S. M. ; Borthwick , J. A. ; Campobasso , N. ; Chabanet , J. ; Chen , S. ; Coggins , J. ; Cottom , J. ; Christensen , S. B. ; Dawson , H. C. ; et al. Identification and optimization of novel small c-Abl kinase activators using fragment and HTS methodologies . J Med Chem 2019 , 62 ( 4 ), 2154 – 2171 . OpenUrl (31). ↵ Baraldi , P. G. ; Tabrizi , M. A. ; Preti , D. ; Bovero , A. ; Fruttarolo , F. ; Romagnoli , R. ; Zaid , N. A. ; Moorman , A. R. ; Varani , K. ; Borea , P. A. New 2-arylpyrazolo[4,3-c]quinoline derivatives as potent and selective human A3 adenosine receptor antagonists . J Med Chem 2005 , 48 ( 15 ), 5001 – 5008 . OpenUrl PubMed (32). ↵ Vantourout , J. C. ; Miras , H. N. ; Isidro-Llobet , A. ; Sproules , S. ; Watson , A. J. Spectroscopic studies of the Chan-Lam amination: A mechanism-inspired solution to boronic ester reactivity . J Am Chem Soc 2017 , 139 ( 13 ), 4769 – 4779 . OpenUrl (33). ↵ Filippakopoulos , P. ; Qi , J. ; Picaud , S. ; Shen , Y. ; Smith , W. B. ; Fedorov , O. ; Morse , E. M. ; Keates , T. ; Hickman , T. T. ; Felletar , I. ; et al. Selective inhibition of BET bromodomains . Nature 2010 , 468 ( 7327 ), 1067 – 1073 . OpenUrl CrossRef PubMed Web of Science (34). ↵ Bielefeld-Sevigny , M. AlphaLISA immunoassay platform-the “no-wash” high-throughput alternative to ELISA . Assay Drug Dev Technol 2009 , 7 ( 1 ), 90 – 92 . OpenUrl CrossRef PubMed Web of Science (35). ↵ Fukazawa , H. ; Masumi , A. The conserved 12-amino acid stretch in the inter-bromodomain region of BET family proteins functions as a nuclear localization signal . Biol Pharm Bull 2012 , 35 ( 11 ), 2064 – 2068 . OpenUrl CrossRef PubMed (36). ↵ Zegzouti , H. ; Zdanovskaia , M. ; Hsiao , K. ; Goueli , S. A. ADP-Glo: A bioluminescent and homogeneous ADP monitoring assay for kinases . Assay Drug Dev Technol 2009 , 7 ( 6 ), 560 – 572 . OpenUrl CrossRef PubMed Web of Science (37). ↵ Sabbah , D. A. ; Hajjo , R. ; Sweidan , K. Review on Epidermal Growth Factor Receptor (EGFR) structure, signaling pathways, interactions, and recent updates of EGFR inhibitors . Curr Top Med Chem 2020 , 20 ( 10 ), 815 – 834 . OpenUrl CrossRef (38). ↵ Avraham , R. ; Yarden , Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops . Nat Rev Mol Cell Biol 2011 , 12 ( 2 ), 104 – 117 . OpenUrl CrossRef PubMed Web of Science (39). ↵ Kolch , W. ; Pitt , A. Functional proteomics to dissect tyrosine kinase signalling pathways in cancer . Nat Rev Cancer 2010 , 10 ( 9 ), 618 – 629 . OpenUrl CrossRef PubMed Web of Science (40). ↵ Clackson , T. ; Yang , W. ; Rozamus , L. W. ; Hatada , M. ; Amara , J. F. ; Rollins , C. T. ; Stevenson , L. F. ; Magari , S. R. ; Wood , S. A. ; Courage , N. L. ; et al. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity . Proc Natl Acad Sci U S A 1998 , 95 ( 18 ), 10437 – 10442 . OpenUrl Abstract / FREE Full Text (41). ↵ Hornbeck , P. V. ; Zhang , B. ; Murray , B. ; Kornhauser , J. M. ; Latham , V. ; Skrzypek , E. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations . Nucleic Acids Res 2015 , 43 ( Database issue ), D512 – 520 . OpenUrl CrossRef PubMed (42). ↵ Cheng , Z. ; Garvin , D. ; Paguio , A. ; Stecha , P. ; Wood , K. ; Fan , F. Luciferase reporter assay system for deciphering GPCR pathways . Curr Chem Genomics 2010 , 4 , 84 – 91 . OpenUrl CrossRef PubMed (43). ↵ Ubersax , J. A. ; Ferrell , J. E. , Jr . . Mechanisms of specificity in protein phosphorylation . Nat Rev Mol Cell Biol 2007 , 8 ( 7 ), 530 – 541 . OpenUrl CrossRef PubMed Web of Science (44). ↵ Till , J. H. ; Chan , P. M. ; Miller , W. T. Engineering the substrate specificity of the Abl tyrosine kinase . J Biol Chem 1999 , 274 ( 8 ), 4995 – 5003 . OpenUrl Abstract / FREE Full Text (45). ↵ Los , G. V. ; Encell , L. P. ; McDougall , M. G. ; Hartzell , D. D. ; Karassina , N. ; Zimprich , C. ; Wood , M. G. ; Learish , R. ; Ohana , R. F. ; Urh , M. ; et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis . ACS Chem Biol 2008 , 3 ( 6 ), 373 – 382 . OpenUrl CrossRef PubMed Web of Science (46). ↵ England , C. G. ; Luo , H. ; Cai , W. HaloTag technology: a versatile platform for biomedical applications . Bioconjug Chem 2015 , 26 ( 6 ), 975 – 986 . OpenUrl CrossRef PubMed (47). ↵ Abdeldayem , A. ; Raouf , Y. S. ; Constantinescu , S. N. ; Moriggl , R. ; Gunning , P. T. Advances in covalent kinase inhibitors . Chem Soc Rev 2020 , 49 ( 9 ), 2617 – 2687 . OpenUrl CrossRef (48). ↵ Cohen , P. ; Cross , D. ; Jänne , P. A. Kinase drug discovery 20 years after imatinib . Nat Rev Drug Discov 2022 . (49). ↵ Choudhary , A. L. , Sophia; Shoba , Veronika ; Kokkonda , Praveen ; Munkanatta Godage , Dhanushka ; Siriwardena , Sachini . Multi-functional chimeric molecules . WO2021142351A1 . July 15 , 2021 . (50). Choudhary , A. S. , Veronika; Deb , Argya ; Nguyen , Tuan ; Lai , Sophia ; Munkanatta Godage , Dhanushka ; Tiwari , Praveen ; Modell , Ashely ; Siriwardene , Sachini . Bifunctional molecules for selective modification of target substrates . WO2022225728A9 . October 27 , 2022 . (51). ↵ Choudhary , A. Bifunctional chimeric molecules for labeling of kinases with target binding moieties and methods use thereof . US provisional Patent application 63/173, 351 . April 19 , 2021 . (52). ↵ Matsuo , K. ; Nishikawa , Y. ; Masuda , M. ; Hamachi , I. Live-cell protein sulfonylation based on proximity-driven N-sulfonyl pyridone chemistry . Angew Chem Int Ed Engl 2018 , 57 ( 3 ), 659 – 662 . OpenUrl (53). Tamura , T. ; Ueda , T. ; Goto , T. ; Tsukidate , T. ; Shapira , Y. ; Nishikawa , Y. ; Fujisawa , A. ; Hamachi , I. Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide . Nature Communications 2018 , 9 ( 1 ), 1870 . OpenUrl (54). Ward , R. A. ; Anderton , M. J. ; Ashton , S. ; Bethel , P. A. ; Box , M. ; Butterworth , S. ; Colclough , N. ; Chorley , C. G. ; Chuaqui , C. ; Cross , D. A. ; et al. Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR) . J Med Chem 2013 , 56 ( 17 ), 7025 – 7048 . OpenUrl CrossRef PubMed (55). Zhuang , J. ; Zhao , B. ; Meng , X. ; Schiffman , J. D. ; Perry , S. L. ; Vachet , R. W. ; Thayumanavan , S. A programmable chemical switch based on triggerable Michael acceptors . Chem Sci 2020 , 11 ( 8 ), 2103 – 2111 . OpenUrl (56). Reddi , R. N. ; Resnick , E. ; Rogel , A. ; Rao , B. V. ; Gabizon , R. ; Goldenberg , K. ; Gurwicz , N. ; Zaidman , D. ; Plotnikov , A. ; Barr , H. ; et al. Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry . J Am Chem Soc 2021 , 143 ( 13 ), 4979 – 4992 . OpenUrl (57). Reddi , R. N. ; Rogel , A. ; Resnick , E. ; Gabizon , R. ; Prasad , P. K. ; Gurwicz , N. ; Barr , H. ; Shulman , Z. ; London , N. Site-specific labeling of endogenous proteins Using CoLDR chemistry . J Am Chem Soc 2021 , 143 ( 48 ), 20095 – 20108 . OpenUrl (58). Matos , M. J. ; Oliveira , B. L. ; Martínez-Sáez , N. ; Guerreiro , A. ; Cal , P. ; Bertoldo , J. ; Maneiro , M. ; Perkins , E. ; Howard , J. ; Deery , M. J. ; et al. Chemo- and regioselective lysine modification on native proteins . J Am Chem Soc 2018 , 140 ( 11 ), 4004 – 4017 . OpenUrl (59). ↵ Tamura , T. ; Hamachi , I. Chemistry for covalent modification of endogenous/native proteins: from test tubes to complex biological systems . J Am Chem Soc 2019 , 141 ( 7 ), 2782 – 2799 . OpenUrl CrossRef (60). ↵ Kim , K. H. ; Maderna , A. ; Schnute , M. E. ; Hegen , M. ; Mohan , S. ; Miyashiro , J. ; Lin , L. ; Li , E. ; Keegan , S. ; Lussier , J. ; et al. Imidazo[1,5-a]quinoxalines as irreversible BTK inhibitors for the treatment of rheumatoid arthritis . Bioorg Med Chem Lett 2011 , 21 ( 21 ), 6258 – 6263 . OpenUrl CrossRef PubMed Web of Science (61). ↵ Honigberg , L. A. ; Smith , A. M. ; Sirisawad , M. ; Verner , E. ; Loury , D. ; Chang , B. ; Li , S. ; Pan , Z. ; Thamm , D. H. ; Miller , R. A. ; et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy . Proc Natl Acad Sci U S A 2010 , 107 ( 29 ), 13075 – 13080 . OpenUrl Abstract / FREE Full Text (62). ↵ Pfleger , K. D. ; Eidne , K. A. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET) . Nat Methods 2006 , 3 ( 3 ), 165 – 174 . OpenUrl CrossRef PubMed Web of Science (63). ↵ Vasta , J. D. ; Corona , C. R. ; Wilkinson , J. ; Zimprich , C. A. ; Hartnett , J. R. ; Ingold , M. R. ; Zimmerman , K. ; Machleidt , T. ; Kirkland , T. A. ; Huwiler , K. G. ; et al. Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement . Cell Chem Biol 2018 , 25 ( 2 ), 206 – 214 e211 . OpenUrl (64). ↵ Hillebrand , L. ; Gehringer , M. Never Gonna Give You Up – Current Developments in Covalent Protein Kinase Inhibitors . Chimia 2022 , 76 ( 5 ). (65). ↵ Yu , H. ; Wang , X. ; Li , J. ; Ye , Y. ; Wang , D. ; Fang , W. ; Mi , L. ; Ding , N. ; Wang , X. ; Song , Y. ; et al. Addition of BTK inhibitor orelabrutinib to rituximab improved anti-tumor effects in B cell lymphoma . Mol Ther Oncolytics 2021 , 21 , 158 – 170 . OpenUrl (66). ↵ George , B. ; Chowdhury , S. M. ; Hart , A. ; Sircar , A. ; Singh , S. K. ; Nath , U. K. ; Mamgain , M. ; Singhal , N. K. ; Sehgal , L. ; Jain , N. Ibrutinib Resistance Mechanisms and Treatment Strategies for B-Cell lymphomas . Cancers (Basel) 2020 , 12 ( 5 ). (67). ↵ Modell , A. E. ; Lim , D. ; Nguyen , T. M. ; Sreekanth , V. ; Choudhary , A. CRISPR-based therapeutics: current challenges and future applications . Trends Pharmacol Sci 2022 , 43 ( 2 ), 151 – 161 . OpenUrl (68). ↵ Shivram , H. ; Cress , B. F. ; Knott , G. J. ; Doudna , J. A. Controlling and enhancing CRISPR systems . Nat Chem Biol 2021 , 17 ( 1 ), 10 – 19 . OpenUrl (69). ↵ Tamura , T. ; Ueda , T. ; Goto , T. ; Tsukidate , T. ; Shapira , Y. ; Nishikawa , Y. ; Fujisawa , A. ; Hamachi , I. Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide . Nat Commun 2018 , 9 ( 1 ), 1870 . OpenUrl (70). Hahm , H. S. ; Toroitich , E. K. ; Borne , A. L. ; Brulet , J. W. ; Libby , A. H. ; Yuan , K. ; Ware , T. B. ; McCloud , R. L. ; Ciancone , A. M. ; Hsu , K. L. Global targeting of functional tyrosines using sulfur-triazole exchange chemistry . Nat Chem Biol 2020 , 16 ( 2 ), 150 – 159 . OpenUrl (71). ↵ Lin , S. ; Yang , X. ; Jia , S. ; Weeks , A. M. ; Hornsby , M. ; Lee , P. S. ; Nichiporuk , R. V. ; Iavarone , A. T. ; Wells , J. A. ; Toste , F. D. ; et al. Redox-based reagents for chemoselective methionine bioconjugation . Science 2017 , 355 ( 6325 ), 597 – 602 . OpenUrl Abstract / FREE Full Text Back to top Previous Next Posted March 05, 2023. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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