Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs

Gao, Jinxu; Mfuh, Adelphe; Amako, Yuka; Woo, Christina M.

doi:10.1021/jacs.7b11639

Cited by 89 publications

(92 citation statements)

References 51 publications

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“…Targeted database searching yielded binding site maps of photo-celecoxib (2) to COX-2 in vitro and in the global proteome in Jurkat and K562 cells. 11 In the course of these studies, we found that CuAAC with a standard biotin azide often yielded higher overall anti-biotin signal than CBA 5, which may have been caused by incomplete CuAAC with the small molecule-labeled proteome. Inspired by chelation-assisted catalysis, 13 we thus designed CBPA 6 to improve the throughput of binding site hotspot mapping ( Figure 1D, Scheme S1).…”

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confidence: 87%

Discovery of a celecoxib binding site on PTGES with a cleavable chelation-assisted biotin probe

Miyamoto¹,

Flaxman²,

Wu³

et al. 2019

Preprint

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The coxibs are a subset of non-steroidal anti-inflammatory drugs (NSAIDs) that primarily target cyclooxygenase-2 (COX-2) to inhibit prostaglandin signaling and reduce inflammation. However, mechanisms to inhibit other members of the prostaglandin signaling pathway may improve selectivity and reduce off-target toxicity. Here, we report a novel binding site for celecoxib on prostaglandin E synthase (PTGES), an enzyme downstream of COX-2 in the prostaglandin signaling pathway, using a cleavable chelation-assisted biotin probe 6. Evaluation of the multifunctional probe 6 revealed significantly improved tagging efficiencies attributable to the embedded picolyl functional group. Application of the probe 6 within the small molecule interactome mapping by photo-affinity labeling (SIM-PAL) platform using photo-celecoxib as a reporter for celecoxib identified PTGES and other membrane proteins in the top eight enriched proteins from A549 cells. Carbonic anhydrase 12, a known protein target of celecoxib, was also enriched. Four binding sites to photo-celecoxib were additionally mapped by the probe 6, including a binding site with PTGES. The binding interaction with PTGES was validated by competitive displacement with celecoxib and known PTGES inhibitor licofelone. The binding site of photo-celecoxib on PTGES enabled the development of a structural model of the interaction and will inform the design of new selective inhibitors of the prostaglandin signaling pathway. Main textInflammation is a major immune response to injury or infection that leads to short-term symptoms of swelling and pain and, when dysregulated, long-term diseases including arthritis, autoimmune disorders, and neurodegeneration. Induction of the acute inflammatory response is primarily mediated by prostaglandin signaling. 1 The prostaglandins are produced by the cyclooxygenases COX-1 and COX-2, which transform arachidonic acid into prostaglandin H2 (PGH2) for further tailoring by prostaglandin synthases ( Figure 1A). The specific inhibition of prostaglandin E2 (PGE2) production and signaling has been an anti-inflammatory target since the development of the first non-steroidal anti-inflammatory drug (NSAID), aspirin, and the subsequent introduction of selective COX-2 inhibitors known as the "coxibs". 2 While therapeutic inhibition of COX-1 and COX-2 is associated with gastrointestinal toxicity, the selective COX-2 inhibitor rofecoxib was withdrawn due to cardiovascular toxicity. 3 This cardiovascular toxicity may arise from the simultaneous suppression of PGE2 and the cardiovascular protectant PGI2 by selective COX-2 inhibitors. 4 As a result, identification of PGE2-selective inhibitors by targeting the inducible prostaglandin E synthase (PTGES) has been the focus of several efforts. 5-8 Here, we report the discovery of a direct interaction between PTGES and celecoxib using binding site hotspot mapping with an isotopically-coded cleavable chelation-assisted biotin probe (CBPA, 6).Chemical proteomics methods have enabled the unbiased identification of protein...

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confidence: 87%

Discovery of a celecoxib binding site on PTGES with a cleavable chelation-assisted biotin probe

Miyamoto¹,

Flaxman²,

Wu³

et al. 2019

Preprint

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“…Comparison between samples can be achieved using quantification methods (e.g., label‐free, SILAC, tandem mass tag [TMT]). These approaches have been widely used to characterize the protein interactomes of metabolites, fragment libraries, bioactive small molecules, and drugs (Das, ; Flaxman & Woo, ; Gao, Mfuh, Amako, & Woo, ; Gertsik et al., ; MacKinnon, Garrison, Hegde, & Taunton, ; Parker et al., ). Cleavage of the enrichment handle by chemical or enzymatic means then allows for recovery of the small molecule–conjugated peptide for analysis of the binding site itself (Speers & Cravatt, ; Szychowski et al., ).…”

Section: Introductionmentioning

confidence: 99%

“…The major steps of the procedure are: (1) generation of small molecule–conjugated proteins either in live cells or cell lysate; (2) attachment of an acid‐cleavable, isotopically coded biotin picolyl azide via CuAAC and enrichment with streptavidin‐agarose beads, followed by collection of trypsin and acid cleavage fractions for protein and conjugation site identification, respectively; and (3) LC‐MS/MS analysis to identify the interacting proteins and conjugated peptides. The example data shown was generated by implementing the SIM‐PAL procedure following treatment of A549 cells with photo‐celecoxib (Gao et al., ; Miyamoto et al., ).…”

Section: Introductionmentioning

confidence: 99%

“…To ameliorate the complexity challenge in binding site assignment, we developed a method termed small molecule interactome mapping by PAL (SIM-PAL), summarized in Figure 2A. The SIM-PAL workflow maps both the interacting proteins and the underlying binding site, thus providing direct validation of and structural insight into small moleculeprotein interactions (Flaxman, Chang, Wu, Nakamoto, & Woo, 2019;Flaxman & Woo, 2018;Gao et al, 2018). Key to this method is the use of a multifunctional, acid-cleavable,…”

Section: Introductionmentioning

confidence: 99%

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Small Molecule Interactome Mapping by Photo‐Affinity Labeling (SIM‐PAL) to Identify Binding Sites of Small Molecules on a Proteome‐Wide Scale

Flaxman

Miyamoto

Woo

2019

CP Chemical Biology

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Identification and characterization of small molecule–protein interactions is critical to understanding the mechanism of action of bioactive small molecules. Photo‐affinity labeling (PAL) enables the capture of noncovalent interactions for enrichment and unbiased analysis by mass spectrometry (MS). Quantitative proteomics of the enriched proteome reveals potential interactions, and MS characterization of binding sites provides validation and structural insight into the interactions. Here, we describe the identification of the protein targets and binding sites of a small molecule using small molecule interactome mapping by PAL (SIM‐PAL). Cells are exposed to a diazirine‐alkyne‐functionalized small molecule, and binding interactions are covalently captured upon UV irradiation. An isotopically coded, acid‐cleavable biotin azide handle is attached to the conjugated proteins using copper‐catalyzed azide‐alkyne cycloaddition. Biotin‐labeled proteins are enriched for on‐bead digestion and quantitative proteomics. Acid cleavage of the handle releases the bead‐bound conjugated peptides for MS analysis and isotope‐directed assignment of the binding site. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a small molecule–conjugated protein sample following treatment of live cells Alternate Protocol: Generation of a small molecule–conjugated protein sample following treatment of cell lysate Basic Protocol 2: Copper‐catalyzed azide‐alkyne cycloaddition functionalization and enrichment of labeled peptides Support Protocol 1: Synthesis of acid‐cleavable, isotopically coded biotin picolyl azide handle Support Protocol 2: Monitoring enrichment by immunoblotting Basic Protocol 3: Mass spectrometry analysis to identify interacting proteins and conjugation sites

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“…The biotin portion of the AZO linker is cleaved off upon reduction of the azobenzene group using sodium dithionite resulting in the formation of a residual aminophenol moiety on the cleaved peptide. To date, the applications of the DADPS linker included: identification of glycosylation sites 23 , profiling the deubiquitinase family of proteins 24 , and mapping the sites of small-molecule protein interactions [25][26][27] . The biotin portion of the DADPS linker is cleaved using formic acid leaving a hydroxyl moiety on the peptide adduct.…”

Section: Introductionmentioning

confidence: 99%

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

Rabalski

Bogdan

Baranczak

2019

Preprint

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Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely-used chemically-cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, a mass tolerant database search strategy using MSFragger software was performed. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.

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Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs

Cited by 89 publications

References 51 publications

Discovery of a celecoxib binding site on PTGES with a cleavable chelation-assisted biotin probe

Discovery of a celecoxib binding site on PTGES with a cleavable chelation-assisted biotin probe

Small Molecule Interactome Mapping by Photo‐Affinity Labeling (SIM‐PAL) to Identify Binding Sites of Small Molecules on a Proteome‐Wide Scale

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

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