{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T14:00:16Z","timestamp":1785765616217,"version":"3.56.0"},"posted":{"date-parts":[[2026,7,30]]},"group-title":"Biophysics","reference-count":41,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T00:00:00Z","timestamp":1785369600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Science Foundation","award":["MCB-2141824"],"award-info":[{"award-number":["MCB-2141824"]}]},{"DOI":"10.13039\/100001021","name":"Damon Runyon Cancer Research Foundation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100001021","id-type":"DOI","asserted-by":"crossref"}]},{"award":["K99 CA286801"],"award-info":[{"award-number":["K99 CA286801"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P30 CA008748"],"award-info":[{"award-number":["P30 CA008748"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["R35 GM152017"],"award-info":[{"award-number":["R35 GM152017"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Marie Sk\u0142odowska-Curie Actions, Horizon Europe","award":["Marie Sk\u0142odowska-Curie postdoctoral fellowship"],"award-info":[{"award-number":["Marie Sk\u0142odowska-Curie postdoctoral fellowship"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,30]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The binding affinity of a ligand to a protein is influenced by the protonation and tautomeric states of both partners. However, this relationship remains under-investigated due to the limited availability of computational tools capable of considering all charge and tautomer states in a scalable manner to study clinically relevant systems. Here, we use Multi-Conformation Continuum Electrostatics (MCCE) to calculate the protonation and tautomer distributions of nine kinase domains bound to 18 FDA-approved inhibitors while considering their Boltzmann-ensemble. Our simulations show that protein net charge and proton distribution remain largely stable even upon binding charged inhibitors. Our results find that individual inhibitor charges are dynamic, frequently increasing, or decreasing upon binding a specific protein target. Kinase-inhibitor binding significantly shifts the relative probabilities of low-energy states (\u0394\n                  <jats:italic>G &lt;<\/jats:italic>\n                  2.5 kcal\/mol), though it does not recruit higher-energy conformers into the bound population. Our consideration of all possible charge states and tautomers enable us to identify when tautomer have significant significant free binding energy differentials (3\u20136kcal\/mol). In turn, we find that minority species can become the dominant component in the bound state, emphasizing the necessity of considering ensemble-wide protonation and tautomer states to accurately predict protein-ligand binding energetics.\n                <\/jats:p>","DOI":"10.64898\/2026.07.27.741060","type":"posted-content","created":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:05:15Z","timestamp":1785434715000},"source":"Crossref","is-referenced-by-count":0,"title":["Kinase inhibitors can change protonation or tautomeric state upon binding"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9558-1204","authenticated-orcid":false,"given":"Gehan A.","family":"Ranepura","sequence":"first","affiliation":[{"name":"Ph.D. Program in Physics, The Graduate Center, City University of New York, New York, NY 10016, USA"},{"name":"Department of Physics, City College of New York, New York, NY 10031, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-7613-4095","authenticated-orcid":false,"given":"Saqibul I.","family":"Chowdhury","sequence":"additional","affiliation":[{"name":"Department of Physics, City College of New York, New York, NY 10031, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8464-8813","authenticated-orcid":false,"given":"Elizabeth A.","family":"Rosenzweig","sequence":"additional","affiliation":[{"name":"Department of Physics, City College of New York, New York, NY 10031, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7616-3701","authenticated-orcid":false,"given":"Ari\u00ebn S.","family":"Rustenburg","sequence":"additional","affiliation":[{"name":"Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA"},{"name":"Graduate Program in Physiology, Biophysics, and Systems Biology, Weill Cornell Medical College, New York, New York 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2668-7405","authenticated-orcid":false,"given":"Raquel","family":"L\u00f3pez-R\u00edos de Castro","sequence":"additional","affiliation":[{"name":"Department of Physics, Freie Universit\u00e4t Berlin, Arnimallee 14, Berlin, 14195, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3106-3018","authenticated-orcid":false,"given":"Junjun","family":"Mao","sequence":"additional","affiliation":[{"name":"Benjamin Levich Institute for Physico-Chemical Hydrodynamics, City College of New York, New York, NY 10031"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0542-119X","authenticated-orcid":false,"given":"John D.","family":"Chodera","sequence":"additional","affiliation":[{"name":"Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1914-4955","authenticated-orcid":false,"given":"Sukrit","family":"Singh","sequence":"additional","affiliation":[{"name":"Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1120-5776","authenticated-orcid":false,"given":"M. R.","family":"Gunner","sequence":"additional","affiliation":[{"name":"Ph.D. Program in Physics, The Graduate Center, City University of New York, New York, NY 10016, USA"},{"name":"Department of Physics, City College of New York, New York, NY 10031, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026080306151124000_2026.07.27.741060v1.1","first-page":"249","article-title":"INHIBITORS OF HIV-1 PROTEASE: A Major Success of Structure-Assisted Drug Design1","volume":"27","year":"1998","journal-title":"Annual Review of Biophysics"},{"key":"2026080306151124000_2026.07.27.741060v1.2","doi-asserted-by":"crossref","first-page":"103257","DOI":"10.1016\/j.sbi.2026.103257","article-title":"More protein-ligand data are needed for AlphaFold-like models to enable drug discovery","volume":"98","year":"2026","journal-title":"Current Opinion in Structural Biology"},{"key":"2026080306151124000_2026.07.27.741060v1.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.abo7201"},{"key":"2026080306151124000_2026.07.27.741060v1.4","doi-asserted-by":"crossref","first-page":"2882","DOI":"10.1021\/acs.jpcb.4c07794","article-title":"Prospective Evaluation of Structure-Based Simulations Reveal Their Ability to Predict the Impact of Kinase Mutations on Inhibitor Binding","volume":"129","year":"2025","journal-title":"The Journal of Physical Chemistry B"},{"key":"2026080306151124000_2026.07.27.741060v1.5","first-page":"18378","article-title":"Best Practices for Alchemical Free Energy Calculations [Article v1.0]","volume":"2","year":"2020","journal-title":"Living Journal of Computational Molecular Science"},{"key":"2026080306151124000_2026.07.27.741060v1.6","doi-asserted-by":"crossref","first-page":"2871","DOI":"10.1021\/acs.jctc.3c01173","article-title":"Free Energy Density of a Fluid and Its Role in Solvation and Binding","volume":"20","year":"2024","journal-title":"Journal of Chemical Theory and Computation"},{"key":"2026080306151124000_2026.07.27.741060v1.7","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1516652112"},{"key":"2026080306151124000_2026.07.27.741060v1.8","doi-asserted-by":"crossref","unstructured":"Czodrowski, P . Blind, one-eyed, or eagle-eyed? pKa calculations during blind predictions with staphylococcal nuclease. Proteins: Structure, Function, and Bioinformatics 2011, n\/a\u2013n\/a, Type: Journal Article.","DOI":"10.1002\/prot.23110"},{"key":"2026080306151124000_2026.07.27.741060v1.9","doi-asserted-by":"crossref","first-page":"5453","DOI":"10.1016\/j.bmc.2012.03.009","article-title":"Who cares for the protons?","volume":"20","year":"2012","journal-title":"Bioorg Med Chem"},{"key":"2026080306151124000_2026.07.27.741060v1.10","doi-asserted-by":"publisher","DOI":"10.1017\/S0033583513000024"},{"key":"2026080306151124000_2026.07.27.741060v1.11","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1608118113"},{"key":"2026080306151124000_2026.07.27.741060v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2005.03.051"},{"key":"2026080306151124000_2026.07.27.741060v1.13","doi-asserted-by":"crossref","first-page":"5554","DOI":"10.1021\/jm500401x","article-title":"Chasing protons: how isothermal titration calorimetry, mutagenesis, and pKa calculations trace the locus of charge in ligand binding to a tRNA-binding enzyme","volume":"57","year":"2014","journal-title":"J Med Chem"},{"key":"2026080306151124000_2026.07.27.741060v1.14","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1007\/s10822-016-9920-5","article-title":"The importance of protonation and tautomerization in relative binding affinity prediction: a comparison of AMBER TI and Schr\u00f6dinger FEP","volume":"30","year":"2016","journal-title":"Journal of computer-aided molecular design"},{"key":"2026080306151124000_2026.07.27.741060v1.15","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jcim.0c00900"},{"key":"2026080306151124000_2026.07.27.741060v1.16","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1007\/s10555-019-09791-8","article-title":"Intracellular pH dynamics and charge-changing somatic mutations in cancer","volume":"38","year":"2019","journal-title":"Cancer Metastasis Reviews"},{"key":"2026080306151124000_2026.07.27.741060v1.17","doi-asserted-by":"crossref","first-page":"eadt3018","DOI":"10.1126\/scisignal.adt3018","article-title":"Ionizable networks mediate pH-dependent allostery in the SH2 domain\u2013containing signaling proteins SHP2 and SRC","volume":"18","year":"2025","journal-title":"Science Signaling"},{"key":"2026080306151124000_2026.07.27.741060v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-020-0082-8"},{"key":"2026080306151124000_2026.07.27.741060v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.phrs.2026.108107"},{"key":"2026080306151124000_2026.07.27.741060v1.20","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0811223106"},{"key":"2026080306151124000_2026.07.27.741060v1.21","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/1472-6807-11-6","article-title":"Graphical analysis of pHdependent properties of proteins predicted using PROPKA","volume":"11","year":"2011","journal-title":"BMC structural biology"},{"key":"2026080306151124000_2026.07.27.741060v1.22","doi-asserted-by":"crossref","first-page":"6134","DOI":"10.1021\/acs.jctc.2c00517","article-title":"Best Practices in Constant pH MD Simulations: Accuracy and Sampling","volume":"18","year":"2022","journal-title":"Journal of Chemical Theory and Computation"},{"key":"2026080306151124000_2026.07.27.741060v1.23","doi-asserted-by":"crossref","first-page":"6148","DOI":"10.1021\/acs.jctc.2c00516","article-title":"Scalable Constant pH Molecular Dynamics in GROMACS","volume":"18","year":"2022","journal-title":"Journal of Chemical Theory and Computation"},{"key":"2026080306151124000_2026.07.27.741060v1.24","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2016.05.052"},{"key":"2026080306151124000_2026.07.27.741060v1.25","doi-asserted-by":"crossref","first-page":"3306","DOI":"10.1002\/prot.23124","article-title":"MCCE analysis of the pKas of introduced buried acids and bases in staphylococcal nuclease","volume":"79","year":"2011","journal-title":"Proteins: Structure, Function, and Bioinformatics"},{"key":"2026080306151124000_2026.07.27.741060v1.26","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21222"},{"key":"2026080306151124000_2026.07.27.741060v1.27","doi-asserted-by":"crossref","first-page":"2638","DOI":"10.1002\/prot.22779","article-title":"The measured and calculated affinity of methyland methoxy-substituted benzoquinones for the QA site of bacterial reaction centers","volume":"78","year":"2010","journal-title":"Proteins: Structure, Function, and Bioinformatics"},{"key":"2026080306151124000_2026.07.27.741060v1.28","doi-asserted-by":"crossref","first-page":"1025","DOI":"10.1016\/j.bpj.2017.07.025","article-title":"Two Cl Ions and a Glu Compete for a Helix Cage in the CLC Proton\/ClAntiporter","volume":"113","year":"2017","journal-title":"Biophys J"},{"key":"2026080306151124000_2026.07.27.741060v1.29","doi-asserted-by":"publisher","DOI":"10.1007\/s10822-020-00280-7"},{"key":"2026080306151124000_2026.07.27.741060v1.30","doi-asserted-by":"crossref","first-page":"10974","DOI":"10.1021\/acs.jpcb.3c06273","article-title":"Computing the Relative Affinity of Chlorophylls a and b to Light-Harvesting Complex II","volume":"127","year":"2023","journal-title":"The Journal of Physical Chemistry B"},{"key":"2026080306151124000_2026.07.27.741060v1.31","doi-asserted-by":"crossref","first-page":"149496","DOI":"10.1016\/j.bbabio.2024.149496","article-title":"Assignment of chlorophyll d in the ChlD1 site of the electron transfer chain of far-red light acclimated photosystem II supported by MCCE binding calculations","volume":"1865","year":"2024","journal-title":"Biochimica et Biophysica Acta (BBA) Bioenergetics"},{"key":"2026080306151124000_2026.07.27.741060v1.32","doi-asserted-by":"publisher","DOI":"10.1021\/bi034482d"},{"key":"2026080306151124000_2026.07.27.741060v1.33","doi-asserted-by":"publisher","DOI":"10.1007\/s10822-007-9133-z"},{"key":"2026080306151124000_2026.07.27.741060v1.34","doi-asserted-by":"publisher","DOI":"10.1007\/s10822-010-9349-1"},{"key":"2026080306151124000_2026.07.27.741060v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(97)78851-9"},{"key":"2026080306151124000_2026.07.27.741060v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/0301-4622(94)00062-X"},{"key":"2026080306151124000_2026.07.27.741060v1.37","doi-asserted-by":"publisher","DOI":"10.1021\/ja00105a030"},{"key":"2026080306151124000_2026.07.27.741060v1.38","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1002\/prot.21123","article-title":"Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins: Structure","volume":"65","year":"2006","journal-title":"Function, and Bioinformatics"},{"key":"2026080306151124000_2026.07.27.741060v1.39","doi-asserted-by":"crossref","first-page":"2476","DOI":"10.1021\/acs.jpcb.2c00139","article-title":"Characterizing protein protonation microstates using Monte Carlo sampling","volume":"126","year":"2022","journal-title":"The Journal of Physical Chemistry B"},{"key":"2026080306151124000_2026.07.27.741060v1.40","doi-asserted-by":"publisher","DOI":"10.1126\/science.7761829"},{"key":"2026080306151124000_2026.07.27.741060v1.41","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1814279116"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.07.27.741060","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T13:15:23Z","timestamp":1785762923000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.07.27.741060"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,30]]},"references-count":41,"URL":"https:\/\/doi.org\/10.64898\/2026.07.27.741060","relation":{},"subject":[],"published":{"date-parts":[[2026,7,30]]},"subtype":"preprint"}}