Academic Journal
Analysis of ligand recognition by choline O-acetyltransferase reveals thiol-reactive assay interference and weak ligand affinity in solution.
| Τίτλος: | Analysis of ligand recognition by choline O-acetyltransferase reveals thiol-reactive assay interference and weak ligand affinity in solution. |
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| Συγγραφείς: | Forsgren N; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden., Jonsson F; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden., Carlsson M; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden., Sander RA; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden., Larsson A; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden., Lindén P; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden., Linusson A; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden; Department of Chemistry, Umeå University, Umeå, Sweden., Engdahl CS; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden., Wiktelius D; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden., Ekström F; CBRN Defence and Security, Swedish Defence Research Agency, Umeå, Sweden. Electronic address: fredrik.ekstrom@foi.se. |
| Πηγή: | The Journal of biological chemistry [J Biol Chem] 2026 Jun; Vol. 302 (6), pp. 113115. Date of Electronic Publication: 2026 May 07. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 2985121R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1083-351X (Electronic) Linking ISSN: 00219258 NLM ISO Abbreviation: J Biol Chem Subsets: MEDLINE |
| Imprint Name(s): | Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Original Publication: Baltimore, MD : American Society for Biochemistry and Molecular Biology |
| Ιατρικοί όροι (MeSH): | Sulfhydryl Compounds*/metabolism , Sulfhydryl Compounds*/chemistry , Choline O-Acetyltransferase*/metabolism , Choline O-Acetyltransferase*/chemistry, Ligands ; Crystallography, X-Ray ; Models, Molecular ; Solutions |
| Περίληψη: | Choline O-acetyltransferase (ChAT) catalyzes the biosynthesis of acetylcholine and is a cysteine-rich enzyme that has been investigated using a range of biochemical, biophysical and structural approaches. Existing ChAT ligands rely on electrophilic or unstable scaffolds that limit their suitability for biological systems. Prior work established that arylvinylpyridiniums (AVPs) are substrate mimics that undergo ChAT-catalyzed hydrothiolation with CoA to form covalent AVP-CoA adducts. Here, we applied a structure-guided strategy to design nonreactive ligands intended to mimic key features of the AVP-CoA binding pose while avoiding covalent reactivity. Nineteen analogs were synthesized and evaluated across complementary biochemical, structural, and biophysical assays. X-ray crystallography confirmed that the new ligands bind within the ChAT tunnel similar to AVP-CoAs. Importantly, the high cysteine content of ChAT, especially within a reactive CXCXXC motif, rendered the enzyme susceptible to modification by the widely used 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) reagent used for measuring ChAT activity, leading to confounding results in thiol-dependent activity assays. Enzyme-free counter-screens demonstrated that all apparent inhibitory activity arose from interference with assay readout rather than true enzymatic inhibition. Surface plasmon resonance measurements established that none of the designed ligands display detectable reversible affinity for ChAT, despite their crystallographically validated poses, and no selectivity over the related enzyme carnitine O-acetyltransferase (CrAT) was observed. These findings demonstrate that confirmed binding with X-ray crystallography is insufficient to establish functional interaction with ChAT and highlight the susceptibility of this enzyme to thiol-reactive assay artefacts. More broadly, this work underscores the necessity of integrating orthogonal biophysical validation when studying ligand binding to cysteine-rich enzymes. (Copyright © 2026 The Authors. Published by Elsevier Inc. All rights reserved.) |
| Competing Interests: | Conflict of interest Daniel Wiktelius is an employee of AstraZeneca. The other authors declare that they have no conflicts of interest with the contents of this article. |
| Contributed Indexing: | Keywords: CoA mimicry; X-ray crystallography; biochemical assay interference; carnitine O-acetyltransferase (CrAT); choline O-acetyltransferase (ChAT); cysteine reactivity; structural biology; surface plasmon resonance (SPR) |
| Substance Nomenclature: | 0 (Ligands) 0 (Sulfhydryl Compounds) EC 2.3.1.6 (Choline O-Acetyltransferase) 0 (Solutions) |
| Entry Date(s): | Date Created: 20260508 Date Completed: 20260701 Latest Revision: 20260726 |
| Update Code: | 20260726 |
| PubMed Central ID: | PMC13255071 |
| DOI: | 10.1016/j.jbc.2026.113115 |
| PMID: | 42103233 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1083-351X |
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| DOI: | 10.1016/j.jbc.2026.113115 |