{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T12:49:04Z","timestamp":1776084544838,"version":"3.50.1"},"posted":{"date-parts":[[2026,4,9]]},"group-title":"Neuroscience","reference-count":44,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T00:00:00Z","timestamp":1775692800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01 NS131663"],"award-info":[{"award-number":["R01 NS131663"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  PrP lowering is a validated therapeutic hypothesis in prion disease. To identify small molecules that reduce PrP levels, we performed phenotypic screening in cultured cells. To prioritize PrP specificity in our primary screen, we generated mouse N2a cells stably expressing GFP and used high content imaging analysis to select compounds that lowered PrP without affecting GFP signal or cell viability. Screening a curated library of 3,492 compounds with annotated mechanisms of action identified two small molecules, EYH (PubChem CID: 71678945) and LCZ (PubChem CID: 24970350), that selectively and dose-dependently lowered PrP. Proteomics on whole cell lysates identified PrP as the #1 or #2 most potently downregulated out of 8,722 proteins detected. Both compounds minimally affected\n                  <jats:italic>Prnp<\/jats:italic>\n                  mRNA, reduced expression of exogenously transfected PrP, and remained potent in non-dividing primary cells, consistent with a post-translational mechanism. Co-treatment with the proteasome inhibitor MG132 yielded accumulation of unglycosylated PrP, demonstrating proteasome clearance of PrP. However, both compounds showed limited or no activity in human cell lines, and failed to reduce PrP in vivo after 14 days of treatment. These findings highlight the challenges associated with mechanism-agnostic phenotypic screening for PrP-lowering compounds and support prioritizing compounds with known mechanisms of action.\n                <\/jats:p>","DOI":"10.64898\/2026.04.07.716919","type":"posted-content","created":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T06:00:19Z","timestamp":1775800819000},"source":"Crossref","is-referenced-by-count":0,"title":["Phenotypic screening for small molecules that lower PrP in cultured cells"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0004-4022","authenticated-orcid":false,"given":"Jeannine A","family":"Frei","sequence":"first","affiliation":[{"name":"Program in Brain Health, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrew G","family":"Reidenbach","sequence":"additional","affiliation":[{"name":"Program in Brain Health, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leo MH","family":"Xu","sequence":"additional","affiliation":[{"name":"Program in Brain Health, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raja Mohan","family":"Gopalakrishnan","sequence":"additional","affiliation":[{"name":"Program in Brain Health, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dominick","family":"Casalana","sequence":"additional","affiliation":[{"name":"Facilitated Access to Screening Technologies (FAST) Lab, Novartis Institutes for Biomedical Research, Cambridge, MA, 02139, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel A","family":"Sprague","sequence":"additional","affiliation":[{"name":"Program in Brain Health, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9748-3592","authenticated-orcid":false,"given":"Mark-Anthony","family":"Bray","sequence":"additional","affiliation":[{"name":"Facilitated Access to Screening Technologies (FAST) Lab, Novartis Institutes for Biomedical Research, Cambridge, MA, 02139, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amy Q.","family":"Wang","sequence":"additional","affiliation":[{"name":"Early Translation Branch, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville, MD, 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1929-477X","authenticated-orcid":false,"given":"Vanessa","family":"Laversenne","sequence":"additional","affiliation":[{"name":"Program in Brain Health, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brian","family":"Erickson","sequence":"additional","affiliation":[{"name":"IQ Proteomics, Framingham, MA, 01702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Craig","family":"Braun","sequence":"additional","affiliation":[{"name":"IQ Proteomics, Framingham, MA, 01702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samarjit","family":"Patnaik","sequence":"additional","affiliation":[{"name":"Early Translation Branch, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville, MD, 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mckenzie","family":"Hall","sequence":"additional","affiliation":[{"name":"Early Translation Branch, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville, MD, 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Douglas","family":"Auld","sequence":"additional","affiliation":[{"name":"Facilitated Access to Screening Technologies (FAST) Lab, Novartis Institutes for Biomedical Research, Cambridge, MA, 02139, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2206-1608","authenticated-orcid":false,"given":"Eric Vallabh","family":"Minikel","sequence":"additional","affiliation":[{"name":"Program in Brain Health, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA"},{"name":"McCance Center for Brain Health and Department of Neurology, Massachusetts General Hospital, Boston, MA, 02114, USA"},{"name":"Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA"},{"name":"Prion Alliance, Cambridge, MA, 02139, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3824-2702","authenticated-orcid":false,"given":"Sonia M","family":"Vallabh","sequence":"additional","affiliation":[{"name":"Program in Brain Health, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA"},{"name":"McCance Center for Brain Health and Department of Neurology, Massachusetts General Hospital, Boston, MA, 02114, USA"},{"name":"Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA"},{"name":"Prion Alliance, Cambridge, MA, 02139, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041305000810000_2026.04.07.716919v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/S1474-4422(19)30403-X"},{"key":"2026041305000810000_2026.04.07.716919v1.2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.23.13573"},{"key":"2026041305000810000_2026.04.07.716919v1.3","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.69.12.7586-7592.1995"},{"issue":"1","key":"2026041305000810000_2026.04.07.716919v1.4","first-page":"1","article-title":"High prion and PrPSc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene","volume":"1","year":"1994","journal-title":"Mol Med Camb Mass"},{"key":"2026041305000810000_2026.04.07.716919v1.5","doi-asserted-by":"publisher","DOI":"10.1172\/jci.insight.131175"},{"key":"2026041305000810000_2026.04.07.716919v1.6","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa616"},{"key":"2026041305000810000_2026.04.07.716919v1.7","doi-asserted-by":"publisher","DOI":"10.1101\/2024.12.05.627039"},{"key":"2026041305000810000_2026.04.07.716919v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-024-03466-w"},{"key":"2026041305000810000_2026.04.07.716919v1.9","doi-asserted-by":"publisher","DOI":"10.1101\/2025.03.05.636713"},{"key":"2026041305000810000_2026.04.07.716919v1.10","unstructured":"Ionis Pharmaceuticals, Inc. A Phase 1\/2a Study to Evaluate the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of Intrathecally Administered ION717 in Patients With Prion Disease [Clinical trial registration] [Internet]. clinicaltrials.gov; 2024 Dec [cited 2025 Jul 2]. Report No.: NCT06153966. Available from: https:\/\/clinicaltrials.gov\/study\/NCT06153966"},{"key":"2026041305000810000_2026.04.07.716919v1.11","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1010013"},{"key":"2026041305000810000_2026.04.07.716919v1.12","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.234393"},{"key":"2026041305000810000_2026.04.07.716919v1.13","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1303510110"},{"key":"2026041305000810000_2026.04.07.716919v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmc.2014.01.001"},{"key":"2026041305000810000_2026.04.07.716919v1.15","unstructured":"Information NC for B, Medicine USNL of, Pike 8600 Rockville, Bethesda, MD20894, USA. AID 488894 - Broad Institute MLPCN Screen of the Prion (PrP 5\u2019 Untranslated region) Inhibitor Probe Project - PubChem [Internet]. 2015 [cited 2015 Nov 6]. Available from: https:\/\/pubchem.ncbi.nlm.nih.gov\/bioassay\/488894"},{"key":"2026041305000810000_2026.04.07.716919v1.16","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jnatprod.5b00947"},{"key":"2026041305000810000_2026.04.07.716919v1.17","unstructured":"AID 743200 - TRFRET-based cell-based high throughput confirmation assay to identify inhibitors of cell surface Prion Protein (PRPC) - PubChem [Internet]. [cited 2026 Mar 26]. Available from: https:\/\/pubchem.ncbi.nlm.nih.gov\/bioassay\/743200"},{"key":"2026041305000810000_2026.04.07.716919v1.18","unstructured":"Minikel E. A look at PrP knockdown screen data. CureFFI.org. 2015."},{"key":"2026041305000810000_2026.04.07.716919v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2020.07.004"},{"key":"2026041305000810000_2026.04.07.716919v1.20","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw1099"},{"key":"2026041305000810000_2026.04.07.716919v1.21","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-21-1295"},{"key":"2026041305000810000_2026.04.07.716919v1.22","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmcl.2018.08.027"},{"key":"2026041305000810000_2026.04.07.716919v1.23","doi-asserted-by":"publisher","DOI":"10.3390\/biom11111695"},{"key":"2026041305000810000_2026.04.07.716919v1.24","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejphar.2013.06.014"},{"key":"2026041305000810000_2026.04.07.716919v1.25","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.63.1.175-181.1989"},{"issue":"3","key":"2026041305000810000_2026.04.07.716919v1.26","first-page":"3","article-title":"Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells","volume":"110","year":"1990","journal-title":"J Cell Biol"},{"key":"2026041305000810000_2026.04.07.716919v1.27","doi-asserted-by":"publisher","DOI":"10.1101\/2024.11.12.623215"},{"key":"2026041305000810000_2026.04.07.716919v1.28","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2006.10.032"},{"key":"2026041305000810000_2026.04.07.716919v1.29","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M116.773283"},{"key":"2026041305000810000_2026.04.07.716919v1.30","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20172045"},{"key":"2026041305000810000_2026.04.07.716919v1.31","doi-asserted-by":"publisher","DOI":"10.1124\/jpet.113.212407"},{"key":"2026041305000810000_2026.04.07.716919v1.32","doi-asserted-by":"publisher","DOI":"10.1089\/nat.2023.0027"},{"key":"2026041305000810000_2026.04.07.716919v1.33","doi-asserted-by":"publisher","DOI":"10.1002\/alz.084521"},{"key":"2026041305000810000_2026.04.07.716919v1.34","doi-asserted-by":"publisher","DOI":"10.1126\/science.adm8386"},{"key":"2026041305000810000_2026.04.07.716919v1.35","doi-asserted-by":"publisher","DOI":"10.1126\/science.ado7082"},{"key":"2026041305000810000_2026.04.07.716919v1.36","doi-asserted-by":"publisher","DOI":"10.1126\/science.adp3043"},{"key":"2026041305000810000_2026.04.07.716919v1.37","doi-asserted-by":"publisher","DOI":"10.1093\/biostatistics\/kxx069"},{"key":"2026041305000810000_2026.04.07.716919v1.38","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2006-7-10-r100"},{"key":"2026041305000810000_2026.04.07.716919v1.39","doi-asserted-by":"publisher","DOI":"10.1177\/1087057110370895"},{"key":"2026041305000810000_2026.04.07.716919v1.40","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btw438"},{"key":"2026041305000810000_2026.04.07.716919v1.41","doi-asserted-by":"publisher","DOI":"10.1101\/060012"},{"key":"2026041305000810000_2026.04.07.716919v1.42","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty441"},{"key":"2026041305000810000_2026.04.07.716919v1.43","doi-asserted-by":"publisher","DOI":"10.1172\/jci.insight.156532"},{"key":"2026041305000810000_2026.04.07.716919v1.44","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA120.014905"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.04.07.716919","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T12:00:20Z","timestamp":1776081620000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.04.07.716919"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,9]]},"references-count":44,"URL":"https:\/\/doi.org\/10.64898\/2026.04.07.716919","relation":{},"subject":[],"published":{"date-parts":[[2026,4,9]]},"subtype":"preprint"}}