{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T16:23:45Z","timestamp":1793291025273,"version":"4.5.0"},"reference-count":50,"publisher":"Elsevier BV","issue":"8","license":[{"start":{"date-parts":[[2025,8,1]],"date-time":"2025-08-01T00:00:00Z","timestamp":1754006400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/www.elsevier.com\/tdm\/userlicense\/1.0\/"},{"start":{"date-parts":[[2025,8,1]],"date-time":"2025-08-01T00:00:00Z","timestamp":1754006400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/www.elsevier.com\/legal\/tdmrep-license"},{"start":{"date-parts":[[2025,7,17]],"date-time":"2025-07-17T00:00:00Z","timestamp":1752710400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003977","name":"Israel Science Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100003977","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100006245","name":"Ministry of Science and Technology, Israel","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100006245","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100024250","name":"Israel Innovation Authority","doi-asserted-by":"publisher","award":["0002310"],"award-info":[{"award-number":["0002310"]}],"id":[{"id":"10.13039\/501100024250","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["cell.com","elsevier.com","sciencedirect.com"],"crossmark-restriction":true},"short-container-title":["Cell Reports Methods"],"published-print":{"date-parts":[[2025,8]]},"DOI":"10.1016\/j.crmeth.2025.101139","type":"journal-article","created":{"date-parts":[[2025,8,7]],"date-time":"2025-08-07T14:32:30Z","timestamp":1754577150000},"page":"101139","update-policy":"https:\/\/doi.org\/10.1016\/elsevier_cm_policy","source":"Crossref","is-referenced-by-count":3,"title":["Optimized pipeline and designer cells for synthetic-biology-based high-throughput screening of viral protease inhibitors"],"prefix":"10.1016","volume":"5","author":[{"given":"Shlomi","family":"Edri","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shayma","family":"El-Atawneh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tehila","family":"Ernst","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maayan","family":"Elnekave","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chaja","family":"Katzman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tali","family":"Lanton","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ido","family":"Aldar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Omri","family":"Wolk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noa","family":"Stern","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amiram","family":"Goldblum","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lior","family":"Nissim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"78","reference":[{"key":"10.1016\/j.crmeth.2025.101139_bib1","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1038\/s41564-018-0296-2","article-title":"Tracking virus outbreaks in the twenty-first century","volume":"4","author":"Grubaugh","year":"2019","journal-title":"Nat. Microbiol."},{"key":"10.1016\/j.crmeth.2025.101139_bib2","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.1021\/acsptsci.0c00108","article-title":"Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-Throughput Screening","volume":"3","author":"Zhu","year":"2020","journal-title":"ACS Pharmacol. Transl. Sci."},{"key":"10.1016\/j.crmeth.2025.101139_bib3","doi-asserted-by":"crossref","DOI":"10.1016\/j.cplett.2020.137489","article-title":"Identification of potential binders of the main protease 3CL(pro) of the COVID-19 via structure-based ligand design and molecular modeling","volume":"750","author":"Macchiagodena","year":"2020","journal-title":"Chem. Phys. Lett."},{"key":"10.1016\/j.crmeth.2025.101139_bib4","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1038\/s42003-020-01577-x","article-title":"Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents","volume":"4","author":"Mody","year":"2021","journal-title":"Commun. Biol."},{"key":"10.1016\/j.crmeth.2025.101139_bib5","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1146\/annurev.biophys.27.1.249","article-title":"Inhibitors of HIV-1 protease: a major success of structure-assisted drug design","volume":"27","author":"Wlodawer","year":"1998","journal-title":"Annu. Rev. Biophys. Biomol. Struct."},{"key":"10.1016\/j.crmeth.2025.101139_bib6","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1038\/s41586-019-0917-9","article-title":"Ultra-large library docking for discovering new chemotypes","volume":"566","author":"Lyu","year":"2019","journal-title":"Nature"},{"key":"10.1016\/j.crmeth.2025.101139_bib7","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.2214168120","article-title":"Turning high-throughput structural biology into predictive inhibitor design","volume":"120","author":"Saar","year":"2023","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"10.1016\/j.crmeth.2025.101139_bib8","doi-asserted-by":"crossref","first-page":"1795","DOI":"10.1016\/j.chembiol.2021.05.018","article-title":"An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 M(pro) inhibitor","volume":"28","author":"Zaidman","year":"2021","journal-title":"Cell Chem. Biol."},{"key":"10.1016\/j.crmeth.2025.101139_bib9","doi-asserted-by":"crossref","DOI":"10.1126\/science.abo7201","article-title":"Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors","volume":"382","author":"Boby","year":"2023","journal-title":"Science"},{"key":"10.1016\/j.crmeth.2025.101139_bib10","doi-asserted-by":"crossref","first-page":"6499","DOI":"10.1021\/acs.jmedchem.2c00117","article-title":"Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19","volume":"65","author":"Unoh","year":"2022","journal-title":"J. Med. Chem."},{"key":"10.1016\/j.crmeth.2025.101139_bib11","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1016\/j.tibtech.2010.05.002","article-title":"Lighting up multiprotein complexes: lessons from GPCR oligomerization","volume":"28","author":"Ciruela","year":"2010","journal-title":"Trends Biotechnol."},{"key":"10.1016\/j.crmeth.2025.101139_bib12","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1016\/j.tibtech.2006.10.008","article-title":"Recent progress in developing FRET-based intracellular sensors for the detection of small molecule nutrients and ligands","volume":"24","author":"Medintz","year":"2006","journal-title":"Trends Biotechnol."},{"key":"10.1016\/j.crmeth.2025.101139_bib13","doi-asserted-by":"crossref","first-page":"1245","DOI":"10.1021\/acscentsci.1c00519","article-title":"Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay","volume":"7","author":"Ma","year":"2021","journal-title":"ACS Cent. Sci."},{"key":"10.1016\/j.crmeth.2025.101139_bib14","doi-asserted-by":"crossref","first-page":"e01265-20","DOI":"10.1128\/JVI.01265-20","article-title":"Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay","volume":"94","author":"Froggatt","year":"2020","journal-title":"J. Virol."},{"key":"10.1016\/j.crmeth.2025.101139_bib15","doi-asserted-by":"crossref","first-page":"1636","DOI":"10.1016\/j.apsb.2021.10.026","article-title":"Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays","volume":"12","author":"Ma","year":"2022","journal-title":"Acta Pharm. Sin. B"},{"key":"10.1016\/j.crmeth.2025.101139_bib16","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.slasd.2023.01.001","article-title":"High throughput screening for drugs that inhibit 3C-like protease in SARS-CoV-2","volume":"28","author":"Smith","year":"2023","journal-title":"SLAS Discov."},{"key":"10.1016\/j.crmeth.2025.101139_bib17","doi-asserted-by":"crossref","DOI":"10.1128\/spectrum.03372-23","article-title":"Potent small molecule inhibitors against the 3C protease of foot-and-mouth disease virus","volume":"12","author":"Kim","year":"2024","journal-title":"Microbiol. Spectr."},{"key":"10.1016\/j.crmeth.2025.101139_bib18","doi-asserted-by":"crossref","first-page":"8179","DOI":"10.1021\/acs.jmedchem.0c00413","article-title":"A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays","volume":"63","author":"Kuhl","year":"2020","journal-title":"J. Med. Chem."},{"key":"10.1016\/j.crmeth.2025.101139_bib19","doi-asserted-by":"crossref","first-page":"e02754-20","DOI":"10.1128\/mBio.02754-20","article-title":"Engineering a Reliable and Convenient SARS-CoV-2 Replicon System for Analysis of Viral RNA Synthesis and Screening of Antiviral Inhibitors","volume":"12","author":"Luo","year":"2021","journal-title":"mBio"},{"key":"10.1016\/j.crmeth.2025.101139_bib20","doi-asserted-by":"crossref","first-page":"1075","DOI":"10.1038\/s41564-024-01618-9","article-title":"Preclinical evaluation of the SARS-CoV-2 M(pro) inhibitor RAY1216 shows improved pharmacokinetics compared with nirmatrelvir","volume":"9","author":"Chen","year":"2024","journal-title":"Nat. Microbiol."},{"key":"10.1016\/j.crmeth.2025.101139_bib21","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/j.tibtech.2014.12.002","article-title":"Manifesting synthetic biology","volume":"33","author":"Andrianantoandro","year":"2015","journal-title":"Trends Biotechnol."},{"key":"10.1016\/j.crmeth.2025.101139_bib22","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1016\/j.tibtech.2015.05.001","article-title":"Synthetic biology in cell-based cancer immunotherapy","volume":"33","author":"Chakravarti","year":"2015","journal-title":"Trends Biotechnol."},{"key":"10.1016\/j.crmeth.2025.101139_bib23","doi-asserted-by":"crossref","first-page":"6786","DOI":"10.1038\/s41467-021-27072-3","article-title":"Design of modular autoproteolytic gene switches responsive to anti-coronavirus drug candidates","volume":"12","author":"Franko","year":"2021","journal-title":"Nat. Commun."},{"key":"10.1016\/j.crmeth.2025.101139_bib24","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.tibtech.2024.07.014","article-title":"Programming mammalian cell behaviors by physical cues","volume":"43","author":"Huang","year":"2025","journal-title":"Trends Biotechnol."},{"key":"10.1016\/j.crmeth.2025.101139_bib25","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1038\/s41551-018-0215-0","article-title":"Synthetic gene circuits for the detection, elimination and prevention of disease","volume":"2","author":"Sedlmayer","year":"2018","journal-title":"Nat. Biomed. Eng."},{"key":"10.1016\/j.crmeth.2025.101139_bib26","doi-asserted-by":"crossref","first-page":"8133","DOI":"10.1073\/pnas.1604391113","article-title":"Cellular heterogeneity mediates inherent sensitivity-specificity tradeoff in cancer targeting by synthetic circuits","volume":"113","author":"Morel","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"10.1016\/j.crmeth.2025.101139_bib27","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1038\/s41580-018-0024-z","article-title":"Designing cell function: assembly of synthetic gene circuits for cell biology applications","volume":"19","author":"Xie","year":"2018","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"10.1016\/j.crmeth.2025.101139_bib28","doi-asserted-by":"crossref","first-page":"1359","DOI":"10.1038\/s41598-020-80451-6","article-title":"Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity","volume":"11","author":"Neefjes","year":"2021","journal-title":"Sci. Rep."},{"key":"10.1016\/j.crmeth.2025.101139_bib29","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1007\/s11307-017-1152-0","article-title":"Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay","volume":"20","author":"Kalinina","year":"2018","journal-title":"Mol. Imaging Biol."},{"key":"10.1016\/j.crmeth.2025.101139_bib30","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1089\/adt.2008.165","article-title":"A dual-fluorescence high-throughput cell line system for probing multidrug resistance","volume":"7","author":"Brimacombe","year":"2009","journal-title":"Assay Drug Dev. Technol."},{"key":"10.1016\/j.crmeth.2025.101139_bib31","doi-asserted-by":"crossref","DOI":"10.3390\/ijms20184331","article-title":"Molecular Docking: Shifting Paradigms in Drug Discovery","volume":"20","author":"Pinzi","year":"2019","journal-title":"Int. J. Mol. Sci."},{"key":"10.1016\/j.crmeth.2025.101139_bib32","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.tibtech.2012.11.006","article-title":"From gene switches to mammalian designer cells: present and future prospects","volume":"31","author":"Auslander","year":"2013","journal-title":"Trends Biotechnol."},{"key":"10.1016\/j.crmeth.2025.101139_bib33","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0027228","article-title":"Profiling of substrate specificities of 3C-like proteases from group 1, 2a, 2b, and 3 coronaviruses","volume":"6","author":"Chuck","year":"2011","journal-title":"PLoS One"},{"key":"10.1016\/j.crmeth.2025.101139_bib34","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1038\/s41564-023-01356-4","article-title":"Therapeutics for COVID-19","volume":"8","author":"Toussi","year":"2023","journal-title":"Nat. Microbiol."},{"key":"10.1016\/j.crmeth.2025.101139_bib35","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0013197","article-title":"Profiling of substrate specificity of SARS-CoV 3CL","volume":"5","author":"Chuck","year":"2010","journal-title":"PLoS One"},{"key":"10.1016\/j.crmeth.2025.101139_bib36","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1038\/s41586-020-2223-y","article-title":"Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors","volume":"582","author":"Jin","year":"2020","journal-title":"Nature"},{"key":"10.1016\/j.crmeth.2025.101139_bib37","doi-asserted-by":"crossref","DOI":"10.1126\/scitranslmed.abq7360","article-title":"SARS-CoV-2 3CL(pro) mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376","volume":"15","author":"Heilmann","year":"2023","journal-title":"Sci. Transl. Med."},{"key":"10.1016\/j.crmeth.2025.101139_bib38","unstructured":"Enamine HTS Collection; accessed Dec 15, 2021; https:\/\/enamine.net\/(2021). https:\/\/enamine.net\/."},{"key":"10.1016\/j.crmeth.2025.101139_bib39","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1021\/acs.jcim.2c00920","article-title":"Predicting the Likelihood of Molecules to Act as Modulators of Protein-Protein Interactions","volume":"63","author":"Wolk","year":"2023","journal-title":"J. Chem. Inf. Model."},{"key":"10.1016\/j.crmeth.2025.101139_bib40","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1038\/s42003-022-03090-9","article-title":"Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay","volume":"5","author":"Narayanan","year":"2022","journal-title":"Commun. Biol."},{"key":"10.1016\/j.crmeth.2025.101139_bib41","doi-asserted-by":"crossref","first-page":"2013","DOI":"10.1038\/s41467-019-09974-5","article-title":"A modular degron library for synthetic circuits in mammalian cells","volume":"10","author":"Chassin","year":"2019","journal-title":"Nat. Commun."},{"key":"10.1016\/j.crmeth.2025.101139_bib42","doi-asserted-by":"crossref","first-page":"950","DOI":"10.1016\/j.cels.2022.11.005","article-title":"The sound of silence: Transgene silencing in mammalian cell engineering","volume":"13","author":"Cabrera","year":"2022","journal-title":"Cell Syst."},{"key":"10.1016\/j.crmeth.2025.101139_bib43","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1038\/gt.2017.20","article-title":"Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells","volume":"24","author":"Hoffmann","year":"2017","journal-title":"Gene Ther."},{"key":"10.1016\/j.crmeth.2025.101139_bib47","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The Protein Data Bank","volume":"28","author":"Berman","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"10.1016\/j.crmeth.2025.101139_bib48","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1007\/s10822-013-9644-8","article-title":"Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments","volume":"27","author":"Sastry","year":"2013","journal-title":"J. Comput. Aided Mol. Des."},{"key":"10.1016\/j.crmeth.2025.101139_bib44","doi-asserted-by":"crossref","first-page":"698","DOI":"10.1016\/j.molcel.2014.04.022","article-title":"Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR\/Cas toolkit in human cells","volume":"54","author":"Nissim","year":"2014","journal-title":"Mol. Cell"},{"key":"10.1016\/j.crmeth.2025.101139_bib45","unstructured":"Schr\u00f6dinger Release 2018-2; LigPrep. (2018-2). https:\/\/ir.schrodinger.com\/press-releases\/news-details\/2018\/Announcing-Schrdinger-Software-Release-2018-2-06-06-2018\/default.aspx."},{"key":"10.1016\/j.crmeth.2025.101139_bib46","doi-asserted-by":"crossref","first-page":"681","DOI":"10.1007\/s10822-007-9133-z","article-title":"Epik: a software program for pK( a ) prediction and protonation state generation for drug-like molecules","volume":"21","author":"Shelley","year":"2007","journal-title":"J. Comput. Aided Mol. Des."},{"key":"10.1016\/j.crmeth.2025.101139_bib49","doi-asserted-by":"crossref","first-page":"6177","DOI":"10.1021\/jm051256o","article-title":"Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes","volume":"49","author":"Friesner","year":"2006","journal-title":"J. Med. Chem."},{"key":"10.1016\/j.crmeth.2025.101139_bib50","unstructured":"Schr\u00f6dinger Release 2020-2; Maestro. (2020-2). https:\/\/www.exxactcorp.com\/blog\/News\/schrodinger-software-release-2020-2."}],"container-title":["Cell Reports Methods"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.elsevier.com\/content\/article\/PII:S2667237525001754?httpAccept=text\/xml","content-type":"text\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/api.elsevier.com\/content\/article\/PII:S2667237525001754?httpAccept=text\/plain","content-type":"text\/plain","content-version":"vor","intended-application":"text-mining"}],"deposited":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T22:49:02Z","timestamp":1755557342000},"score":1,"resource":{"primary":{"URL":"https:\/\/linkinghub.elsevier.com\/retrieve\/pii\/S2667237525001754"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8]]},"references-count":50,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2025,8]]}},"alternative-id":["S2667237525001754"],"URL":"https:\/\/doi.org\/10.1016\/j.crmeth.2025.101139","relation":{},"ISSN":["2667-2375"],"issn-type":[{"value":"2667-2375","type":"print"}],"subject":[],"published":{"date-parts":[[2025,8]]},"assertion":[{"value":"Elsevier","name":"publisher","label":"This article is maintained by"},{"value":"Optimized pipeline and designer cells for synthetic-biology-based high-throughput screening of viral protease inhibitors","name":"articletitle","label":"Article Title"},{"value":"Cell Reports Methods","name":"journaltitle","label":"Journal Title"},{"value":"https:\/\/doi.org\/10.1016\/j.crmeth.2025.101139","name":"articlelink","label":"CrossRef DOI link to publisher maintained version"},{"value":"article","name":"content_type","label":"Content Type"},{"value":"\u00a9 2025 The Author(s). Published by Elsevier Inc.","name":"copyright","label":"Copyright"}],"article-number":"101139"}}