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The pKa of a targetable cysteine residue in a disease-related protein is an important physiochemical parameter in covalent drug discovery, as it influences the fraction of nucleophilic thiolate amenable to chemical protein modification. Traditional structure-based in silico tools are limited in their predictive accuracy of cysteine pKa\u2019s relative to other titratable residues. Additionally, there are limited comprehensive benchmark assessments for cysteine pKa predictive tools. This raises the need for extensive assessment and evaluation of methods for cysteine pKa prediction. Here, we report the performance of several computational pKa methods, including single-structure and ensemble-based approaches, on a diverse test set of experimental cysteine pKa\u2019s retrieved from the PKAD database. The dataset consisted of 16 wildtype and 10 mutant proteins with experimentally measured cysteine pKa values. Our results highlight that these methods are varied in their overall predictive accuracies. Among the test set of wildtype proteins evaluated, the best method (MOE) yielded a mean absolute error of 2.3 pK units, highlighting the need for improvement of existing pKa methods for accurate cysteine pKa estimation. Given the limited accuracy of these methods, further development is needed before these approaches can be routinely employed to drive design decisions in early drug discovery efforts.<\/jats:p>","DOI":"10.1021\/acs.jcim.3c00004","type":"journal-article","created":{"date-parts":[[2023,3,30]],"date-time":"2023-03-30T15:08:37Z","timestamp":1680188917000},"page":"2170-2180","source":"Crossref","is-referenced-by-count":30,"title":["Benchmarking\n                    <i>In Silico<\/i>\n                    Tools for\nCysteine p\n                    <i>K<\/i>\n                    a Prediction"],"prefix":"10.1021","volume":"63","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9127-8539","authenticated-orcid":true,"given":"Ernest","family":"Awoonor-Williams","sequence":"first","affiliation":[{"name":"Novartis Institutes for BioMedical Research , 181 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrei A.","family":"Golosov","sequence":"additional","affiliation":[{"name":"Novartis Institutes for BioMedical Research , 181 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2344-9586","authenticated-orcid":true,"given":"Viktor","family":"Hornak","sequence":"additional","affiliation":[{"name":"Novartis Institutes for BioMedical Research , 181 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2023,3,30]]},"reference":[{"key":"2026081804443892900_cit1","doi-asserted-by":"publisher","first-page":"91","DOI":"10.3109\/10409238509085133","article-title":"PH-Dependent Processes in Proteins","volume":"18","author":"Matthew","year":"1985","journal-title":"CRC Crit. 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Ed."},{"key":"2026081804443892900_cit10","doi-asserted-by":"publisher","first-page":"61","DOI":"10.1016\/j.cbpa.2015.11.004","article-title":"Covalent\nTargeting of Acquired Cysteines\nin Cancer","volume":"30","author":"Visscher","year":"2016","journal-title":"Curr. Opin. Chem. Biol."},{"key":"2026081804443892900_cit11","doi-asserted-by":"publisher","first-page":"1268","DOI":"10.1016\/j.bbrc.2016.08.109","article-title":"Cysteinome: The First Comprehensive Database for Proteins with Targetable\nCysteine and Their Covalent Inhibitors","volume":"478","author":"Wu","year":"2016","journal-title":"Biochem.\nBiophys. Res. Commun."},{"key":"2026081804443892900_cit12","doi-asserted-by":"publisher","first-page":"1664","DOI":"10.1016\/j.bbapap.2017.05.009","article-title":"Modeling Covalent-Modifier Drugs","volume":"1865","author":"Awoonor-Williams","year":"2017","journal-title":"Biochim. Biophys. 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Toxicol."},{"key":"2026081804443892900_cit18","first-page":"203","article-title":"Measuring and Predicting Warhead and Residue Reactivity","volume-title":"Annual Reports in Medicinal Chemistry","author":"Awoonor-Williams","year":"2021"},{"key":"2026081804443892900_cit19","doi-asserted-by":"publisher","DOI":"10.1002\/wcms.1636","article-title":"Mechanistic\nAspects of Thiol Additions\nto Michael Acceptors: Insights from Computations","volume":"13","author":"Roseli","year":"2023","journal-title":"WIREs Comput. Mol. Sci."},{"key":"2026081804443892900_cit20","doi-asserted-by":"publisher","first-page":"8898","DOI":"10.1039\/D2OB01369J","article-title":"A Mechanistic\nStudy of Thiol Addition to N -Phenylacrylamide","volume":"20","author":"Watt","year":"2022","journal-title":"Org. Biomol. 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Insights"},{"key":"2026081804443892900_cit22","doi-asserted-by":"publisher","first-page":"368","DOI":"10.1093\/nar\/gki464","article-title":"H++: A Server for Estimating\nPKas and Adding Missing\nHydrogens to Macromolecules","volume":"33","author":"Gordon","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"2026081804443892900_cit23","doi-asserted-by":"publisher","first-page":"537","DOI":"10.1093\/nar\/gki464","article-title":"H++ 3.0: Automating PK Prediction\nand the Preparation of Biomolecular Structures for Atomistic Molecular\nModeling and Simulations","volume":"40","author":"Anandakrishnan","year":"2012","journal-title":"Nucleic Acids Res."},{"key":"2026081804443892900_cit24","doi-asserted-by":"publisher","first-page":"2231","DOI":"10.1002\/jcc.21222","article-title":"MCCE2: Improving Protein PKa Calculations with Extensive\nSide Chain Rotamer Sampling","volume":"30","author":"Song","year":"2009","journal-title":"J. Comput. 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Theory\nComput."},{"key":"2026081804443892900_cit39","doi-asserted-by":"publisher","first-page":"1277","DOI":"10.1002\/prot.25608","article-title":"DelPhiPKa: Including Salt in the\nCalculations and Enabling Polar Residues to Titrate","volume":"86","author":"Pahari","year":"2018","journal-title":"Proteins: Struct., Funct., Bioinf."},{"key":"2026081804443892900_cit40","doi-asserted-by":"publisher","first-page":"3689","DOI":"10.1021\/acs.jctc.0c00258","article-title":"Predicting Reactive Cysteines with Implicit-Solvent-Based\nContinuous Constant PH Molecular Dynamics in Amber","volume":"16","author":"Harris","year":"2020","journal-title":"J. Chem. 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Chem."},{"key":"2026081804443892900_cit51","doi-asserted-by":"publisher","first-page":"12860","DOI":"10.1021\/bi801718d","volume-title":"Cysteine p K Values for the Bacterial Peroxiredoxin\nAhpC Cysteine pKa Values for the Bacterial Peroxiredoxin AhpC \u2020,\u2021","author":"Nelson","year":"2008"},{"key":"2026081804443892900_cit52","doi-asserted-by":"publisher","first-page":"4713","DOI":"10.1016\/S0021-9258(18)42891-8","article-title":"Characterization\nof Recombinant Rat Cathepsin B and Nonglycosylated Mutants Expressed\nin Yeast. New Insights into the PH Dependence of Cathepsin B-Catalyzed\nHydrolyses","volume":"267","author":"Hasnain","year":"1992","journal-title":"J. Biol. Chem."},{"key":"2026081804443892900_cit53","doi-asserted-by":"publisher","first-page":"7430","DOI":"10.1021\/bi800282d","article-title":"Cysteine PKa Depression\nby a Protonated Glutamic Acid in Human DJ-1","volume":"47","author":"Witt","year":"2008","journal-title":"Biochemistry"},{"key":"2026081804443892900_cit54","doi-asserted-by":"publisher","first-page":"11698","DOI":"10.1021\/bi011208f","article-title":"An Unusually\nLow p K a for Cys282 in the Active Site of Human Muscle Creatine Kinase\n\u2020","volume":"40","author":"Wang","year":"2001","journal-title":"Biochemistry"},{"key":"2026081804443892900_cit55","doi-asserted-by":"publisher","first-page":"25596","DOI":"10.1074\/jbc.M112.369116","article-title":"A Low PK Cysteine at the Active Site of Mouse Methionine\nSulfoxide Reductase A","volume":"287","author":"Lim","year":"2012","journal-title":"J. Biol. Chem."},{"key":"2026081804443892900_cit56","doi-asserted-by":"crossref","unstructured":"Guengerich, F. 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