{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:52:55Z","timestamp":1782280375262,"version":"3.54.5"},"posted":{"date-parts":[[2025,10,31]]},"group-title":"Biochemistry","reference-count":43,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T00:00:00Z","timestamp":1761868800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"crossref","award":["R01AI148844"],"award-info":[{"award-number":["R01AI148844"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"crossref","award":["R01AI078958"],"award-info":[{"award-number":["R01AI078958"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NIH","award":["T32GM138826"],"award-info":[{"award-number":["T32GM138826"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,31]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>The inhibition of a specific protein-protein interaction is often difficult to achieve in targeted drug design. We report the development and optimization of a general-purpose, readily implemented enzyme-linked immunosorbent assay (ELISA) for high-throughput screening to identify small- molecule inhibitors of protein interactions. This ELISA does not involve the use of any capture antibodies, probes, or compounds coated on the plate and represents a general strategy to identify inhibitors of a given protein-protein interaction. We demonstrate its utility in blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook by targeting the native form of the FlgE protein, which differs from the strategies used in previous assays. The flagellar hook protein FlgE self-catalyzes the formation of a lysinoalanine (Lal) inter-subunit crosslink that is essential for the motility, and thus, infectivity of spirochetes. Prevention of Lal crosslinking through inhibition with small molecules thus represents an avenue for therapeutic development against spirochete-related diseases, such as Lyme and syphilis. Screening a library of \u223c700 compounds with the ELISA confirmed that hexachlorophene, currently the only known inhibitor of Lal crosslinking in FlgE, effectively inhibits the crosslinking reaction. In addition, the assay identified two new potential inhibitors, honokiol and zafirlukast, and several activators which belong to well-known classes of antibiotics.<\/jats:p>","DOI":"10.1101\/2025.10.31.685777","type":"posted-content","created":{"date-parts":[[2025,11,1]],"date-time":"2025-11-01T02:55:11Z","timestamp":1761965711000},"source":"Crossref","is-referenced-by-count":0,"title":["An ELISA for discovering protein-protein interaction inhibitors: blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook as a test case"],"prefix":"10.64898","author":[{"given":"Maithili","family":"Deshpande","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"Lynch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kurni","family":"Kurniyati","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunhao","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian R.","family":"Crane","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025110308251087000_2025.10.31.685777v1.1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201100563"},{"key":"2025110308251087000_2025.10.31.685777v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2014.11.021"},{"key":"2025110308251087000_2025.10.31.685777v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(20)30251-8"},{"key":"2025110308251087000_2025.10.31.685777v1.4","doi-asserted-by":"publisher","DOI":"10.7298\/3j8d-8357"},{"key":"2025110308251087000_2025.10.31.685777v1.5","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.4c00749"},{"key":"2025110308251087000_2025.10.31.685777v1.6","doi-asserted-by":"publisher","DOI":"10.1055\/s-0029-1188114"},{"key":"2025110308251087000_2025.10.31.685777v1.7","first-page":"528","article-title":"Vorl\u00e4ufiger Bericht \u00fcber das Vorkommen f\u00fcr Spirochaeten in syphilitischen Krankheitsprodukten und bei Papillomen","volume":"22","year":"1905","journal-title":"Arb Gesundh AMT Berlin"},{"key":"2025110308251087000_2025.10.31.685777v1.8","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.19.1.29-49.2006"},{"key":"2025110308251087000_2025.10.31.685777v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/nrdp.2016.90"},{"key":"2025110308251087000_2025.10.31.685777v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2009.03.012"},{"key":"2025110308251087000_2025.10.31.685777v1.11","doi-asserted-by":"publisher","DOI":"10.1111\/j.1600-0757.2005.00108.x"},{"key":"2025110308251087000_2025.10.31.685777v1.12","unstructured":"Castellani A . On the presence of spirochaetes in some cases of parangi (yaws, framboesia tropica) preliminary note. J Ceylon Branch Brit Med Assoc. 1905;2:54."},{"key":"2025110308251087000_2025.10.31.685777v1.13","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.15-0147"},{"key":"2025110308251087000_2025.10.31.685777v1.14","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01095-09"},{"key":"2025110308251087000_2025.10.31.685777v1.15","doi-asserted-by":"publisher","DOI":"10.1186\/s13071-019-3495-7"},{"key":"2025110308251087000_2025.10.31.685777v1.16","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-micro-092611-150145"},{"key":"2025110308251087000_2025.10.31.685777v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/j.semcdb.2015.10.015"},{"key":"2025110308251087000_2025.10.31.685777v1.18","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.03728-24"},{"key":"2025110308251087000_2025.10.31.685777v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/nmicrobiol.2016.134"},{"key":"2025110308251087000_2025.10.31.685777v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-019-0341-3"},{"key":"2025110308251087000_2025.10.31.685777v1.21","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0098338"},{"key":"2025110308251087000_2025.10.31.685777v1.22","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01421-07"},{"key":"2025110308251087000_2025.10.31.685777v1.23","doi-asserted-by":"publisher","DOI":"10.1159\/000077867"},{"key":"2025110308251087000_2025.10.31.685777v1.24","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.01228-12"},{"key":"2025110308251087000_2025.10.31.685777v1.25","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.13403"},{"key":"2025110308251087000_2025.10.31.685777v1.26","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2010.07078.x"},{"key":"2025110308251087000_2025.10.31.685777v1.27","doi-asserted-by":"publisher","DOI":"10.1111\/cmi.12617"},{"key":"2025110308251087000_2025.10.31.685777v1.28","unstructured":"Olsen DAM Anna Dey, and Dr Cathy. Advantages of ELISA for Accurate and Accelerated Drug Discovery [Podcast]. https:\/\/www.moleculardevices.com\/lab-notes\/microplate-readers\/advantages-of-elisa-for-accurate-and-accelerated-drug-discovery"},{"key":"2025110308251087000_2025.10.31.685777v1.29","doi-asserted-by":"publisher","DOI":"10.1201\/9781351071840"},{"key":"2025110308251087000_2025.10.31.685777v1.30","doi-asserted-by":"publisher","DOI":"10.1021\/acssensors.0c02415"},{"key":"2025110308251087000_2025.10.31.685777v1.31","doi-asserted-by":"publisher","DOI":"10.1039\/D2AN00921H"},{"key":"2025110308251087000_2025.10.31.685777v1.32","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2022.116605"},{"key":"2025110308251087000_2025.10.31.685777v1.33","doi-asserted-by":"publisher","DOI":"10.1177\/03000605251315913"},{"key":"2025110308251087000_2025.10.31.685777v1.34","doi-asserted-by":"publisher","DOI":"10.1093\/pnasnexus\/pgad349"},{"key":"2025110308251087000_2025.10.31.685777v1.35","doi-asserted-by":"publisher","DOI":"10.1021\/tx960145c"},{"key":"2025110308251087000_2025.10.31.685777v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/0041-008X(73)90307-4"},{"key":"2025110308251087000_2025.10.31.685777v1.37","doi-asserted-by":"publisher","DOI":"10.2174\/156652412803833508"},{"key":"2025110308251087000_2025.10.31.685777v1.38","doi-asserted-by":"publisher","DOI":"10.3390\/cancers12010048"},{"key":"2025110308251087000_2025.10.31.685777v1.39","doi-asserted-by":"publisher","DOI":"10.2165\/00003495-199855010-00008"},{"key":"2025110308251087000_2025.10.31.685777v1.40","doi-asserted-by":"publisher","DOI":"10.2165\/00003088-200241020-00003"},{"key":"2025110308251087000_2025.10.31.685777v1.41","unstructured":"Karunarathna I , Aluthge P , Perera N , et al. Zafirlukast: A Comprehensive Review of Indications, Mechanisms, Adverse Effects, and Role in Asthma Management. Published online 2025."},{"key":"2025110308251087000_2025.10.31.685777v1.42","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.12.6.792-795.1980"},{"key":"2025110308251087000_2025.10.31.685777v1.43","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6968.1986.tb01527.x"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.10.31.685777","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:01:57Z","timestamp":1782277317000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.10.31.685777"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,31]]},"references-count":43,"URL":"https:\/\/doi.org\/10.1101\/2025.10.31.685777","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1016\/j.slasd.2025.100292","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,10,31]]},"subtype":"preprint"}}