{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:22:14Z","timestamp":1785410534217,"version":"3.56.0"},"posted":{"date-parts":[[2025,9,30]]},"group-title":"Pharmacology and Toxicology","reference-count":39,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T00:00:00Z","timestamp":1759190400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH","award":["R01GM145793"],"award-info":[{"award-number":["R01GM145793"]}]},{"name":"NIH","award":["DP1CA174428"],"award-info":[{"award-number":["DP1CA174428"]}]},{"name":"NIH","award":["P30CA006973"],"award-info":[{"award-number":["P30CA006973"]}]},{"name":"NIH","award":["S10OD034217"],"award-info":[{"award-number":["S10OD034217"]}]},{"award":["N\/A"],"award-info":[{"award-number":["N\/A"]}],"id":[{"id":"https:\/\/ror.org\/00ybhm522","id-type":"ROR","asserted-by":"publisher"}]},{"name":"IBBS, Johns Hopkins School of Medicine"},{"name":"Rapafusyn Pharmaceuticals"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,30]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>The 26S proteasome is an essential regulator of protein homeostasis and a clinically validated therapeutic target in multiple myeloma (MM). Rapaprotin, a novel macrocycle identified from a rapamycin-inspired rapafucin library, disrupts 26S proteasome function by inducing disassembly of the 19S regulatory particle in the 26S proteasome, leading to apoptosis in MM cells. Its bioactivation requires prolyl endopeptidase (PREP)\u2013mediated cleavage to generate Rapaprotin-L, a negatively charged, linear metabolite with potent proteasome-disassembly activity. Using the PRISM cancer cell line profiling platform, we identified high P-glycoprotein (P-gp\/ABCB1) expression as a major determinant of Rapaprotin resistance in solid tumor cell lines. Efflux assays confirmed Rapaprotin-L, but not its parent Rapaprotin, as a high-affinity P-gp substrate. Co-treatment with the third-generation P-gp inhibitor tariquidar restored the intracellular accumulation of Rapaprotin-L, reinstating proteasome inhibition and consequent apoptosis of Rapaprotin-resistant colorectal cancer cell lines. Strong synergy between Rapaprotin and tariquidar was observed in a 3D spheroid model. These results establish P-gp as a key mediator of resistance to Rapaprotin and identify a rare example of a negatively charged Rapaprotin-L as a P-gp substrate. Together, these findings expand the potential therapeutic scope of Rapaprotin beyond hematologic malignancies to a broader range of solid tumors.<\/jats:p>","DOI":"10.1101\/2025.09.28.679086","type":"posted-content","created":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T20:55:19Z","timestamp":1759265719000},"source":"Crossref","is-referenced-by-count":0,"title":["Overcoming rapaprotin resistance through inhibition of P-glycoprotein"],"prefix":"10.64898","author":[{"given":"Qian","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Asbell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A. V. Subba","family":"Rao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kalyan Kumar","family":"Pasunooti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew G.","family":"Rees","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer A.","family":"Roth","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun O.","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025100321350653000_2025.09.28.679086v1.1","doi-asserted-by":"crossref","first-page":"e09777","DOI":"10.1016\/j.heliyon.2022.e09777","article-title":"P-glycoprotein: new insights into structure, physiological function, regulation and alterations in disease","volume":"8","year":"2022","journal-title":"Heliyon"},{"key":"2025100321350653000_2025.09.28.679086v1.2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1168750"},{"key":"2025100321350653000_2025.09.28.679086v1.3","doi-asserted-by":"publisher","DOI":"10.4137\/DTI.S12519"},{"key":"2025100321350653000_2025.09.28.679086v1.4","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-062917-011931"},{"key":"2025100321350653000_2025.09.28.679086v1.5","doi-asserted-by":"crossref","first-page":"1273","DOI":"10.3389\/fonc.2020.01273","article-title":"Drug Repositioning for P-Glycoprotein Mediated Co-Expression Networks in Colorectal Cancer","volume":"10","year":"2020","journal-title":"Front Oncol"},{"key":"2025100321350653000_2025.09.28.679086v1.6","doi-asserted-by":"publisher","DOI":"10.1023\/A:1026556119020"},{"key":"2025100321350653000_2025.09.28.679086v1.7","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.1003-21.2021"},{"key":"2025100321350653000_2025.09.28.679086v1.8","doi-asserted-by":"publisher","DOI":"10.1053\/j.seminoncol.2005.02.005"},{"key":"2025100321350653000_2025.09.28.679086v1.9","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1038\/s43018-019-0018-6","article-title":"Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling","volume":"1","year":"2020","journal-title":"Nat Cancer"},{"key":"2025100321350653000_2025.09.28.679086v1.10","doi-asserted-by":"crossref","first-page":"1273","DOI":"10.1007\/s00280-015-2845-1","article-title":"Pharmacokinetic and pharmacodynamic study of tariquidar (XR9576), a P-glycoprotein inhibitor, in combination with doxorubicin, vinorelbine, or docetaxel in children and adolescents with refractory solid tumors","volume":"76","year":"2015","journal-title":"Cancer Chemother Pharmacol"},{"key":"2025100321350653000_2025.09.28.679086v1.11","first-page":"321","article-title":"Where is it and How Does it Get There - Intracellular Localization and Traffic of P-glycoprotein","volume":"3","year":"2013","journal-title":"Front Oncol"},{"key":"2025100321350653000_2025.09.28.679086v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/s41557-018-0187-4"},{"key":"2025100321350653000_2025.09.28.679086v1.13","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.beha.2012.04.007","article-title":"Proteasome inhibitors in mantle cell lymphoma","volume":"25","year":"2012","journal-title":"Best Pract Res Clin Haematol"},{"key":"2025100321350653000_2025.09.28.679086v1.14","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/j.htct.2018.07.003","article-title":"New proteasome inhibitors in the treatment of multiple myeloma","volume":"41","year":"2019","journal-title":"Hematol Transfus Cell Ther"},{"key":"2025100321350653000_2025.09.28.679086v1.15","doi-asserted-by":"crossref","unstructured":"Kim, C.H. , Lee, S. , Choi, J.Y. , Lyu, M.J. , Jung, H.M. , Goo, Y.T. , Kang, M.J. , Choi, Y.W ., 2023. Functionalized Lipid Nanocarriers for Simultaneous Delivery of Docetaxel and Tariquidar to Chemoresistant Cancer Cells. Pharmaceuticals (Basel) 16.","DOI":"10.3390\/ph16030349"},{"key":"2025100321350653000_2025.09.28.679086v1.16","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2009-07-229492"},{"key":"2025100321350653000_2025.09.28.679086v1.17","doi-asserted-by":"crossref","first-page":"1230","DOI":"10.1002\/j.1875-9114.1998.tb03142.x","article-title":"Pharmacokinetics of dexamethasone and valspodar, a P-glycoprotein (mdr1) modulator: implications for coadministration","volume":"18","year":"1998","journal-title":"Pharmacotherapy"},{"key":"2025100321350653000_2025.09.28.679086v1.18","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1186\/2050-7771-1-13","article-title":"The resistance mechanisms of proteasome inhibitor bortezomib","volume":"1","year":"2013","journal-title":"Biomark Res"},{"key":"2025100321350653000_2025.09.28.679086v1.19","doi-asserted-by":"publisher","DOI":"10.1111\/febs.12613"},{"key":"2025100321350653000_2025.09.28.679086v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/emboj.2008.73"},{"key":"2025100321350653000_2025.09.28.679086v1.21","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-19325-x"},{"key":"2025100321350653000_2025.09.28.679086v1.22","doi-asserted-by":"crossref","unstructured":"Nguyen, T.T. , Duong, V.A. , Maeng, H.J ., 2021. Pharmaceutical Formulations with P-Glycoprotein Inhibitory Effect as Promising Approaches for Enhancing Oral Drug Absorption and Bioavailability. Pharmaceutics 13.","DOI":"10.3390\/pharmaceutics13071103"},{"key":"2025100321350653000_2025.09.28.679086v1.23","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1292\/jvms.16-0315","article-title":"CAN a P-gp modulator assist in the control of methotrexate concentrations in the rat brain? -inhibitory effects of rhodamine 123, a specific substrate for P-gp, on methotrexate excretion from the rat brain and its optimal route of administration","volume":"79","year":"2017","journal-title":"J Vet Med Sci"},{"key":"2025100321350653000_2025.09.28.679086v1.24","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1200\/JCO.1987.5.4.641","article-title":"Verapamil and adriamycin in the treatment of drug-resistant ovarian cancer patients","volume":"5","year":"1987","journal-title":"J Clin Oncol"},{"key":"2025100321350653000_2025.09.28.679086v1.25","doi-asserted-by":"crossref","unstructured":"Peng, H. , Guo, Z. , Wang, W.L. , Yin, D. , Zou, S. , Asbell, T. , Ullman, B.R. , Thakar, M. , Zhang, F. , Hong, S.Y. , Rao, A.V.S. , Wang, K. , Zhang, S. , Wu, Z. , Li, X. , Margolis, S.S. , Matsui, W.H. , Gocke, C.B. , Mao, Y. , Liu, J.O ., 2025. Rapaprotin is Activated by an Endopeptidase to Disassemble 26S Proteasome. bioRxiv.","DOI":"10.1101\/2025.02.21.639179"},{"key":"2025100321350653000_2025.09.28.679086v1.26","doi-asserted-by":"crossref","unstructured":"Pilotto Heming, C. , de Souza Barbosa, I. , Lyra Miranda, R. , Nogueira Ugarte, O ., Santorio de Sao Jose, V ., Moura Neto, V ., Aran, V ., 2025. P-Glycoprotein Drives Glioblastoma Survival and Chemotherapy Resistance: Potential as a Promising Liquid Biopsy Biomarker. Am J Pathol.","DOI":"10.1016\/j.ajpath.2024.12.004"},{"key":"2025100321350653000_2025.09.28.679086v1.27","doi-asserted-by":"crossref","first-page":"e11171","DOI":"10.1016\/j.heliyon.2022.e11171","article-title":"P-glycoprotein and cancer: what do we currently know?","volume":"8","year":"2022","journal-title":"Heliyon"},{"key":"2025100321350653000_2025.09.28.679086v1.28","doi-asserted-by":"publisher","DOI":"10.1002\/cncr.21227"},{"key":"2025100321350653000_2025.09.28.679086v1.29","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1002\/1878-0261.13318","article-title":"ERBB and P-glycoprotein inhibitors break resistance in relapsed neuroblastoma models through P-glycoprotein","volume":"17","year":"2023","journal-title":"Mol Oncol"},{"key":"2025100321350653000_2025.09.28.679086v1.30","doi-asserted-by":"crossref","first-page":"6077","DOI":"10.1016\/S0021-9258(18)53221-X","article-title":"Human P-glycoprotein transports cyclosporin A and FK506","volume":"268","year":"1993","journal-title":"J Biol Chem"},{"key":"2025100321350653000_2025.09.28.679086v1.31","doi-asserted-by":"crossref","unstructured":"Sanchez, P.R. , Head, S.A. , Qian, S. , Qiu, H. , Roy, A. , Jin, Z. , Zheng, W. , Liu, J.O ., 2023. Modulation of the Endomembrane System by the Anticancer Natural Product Superstolide\/ZJ-101. Int J Mol Sci 24.","DOI":"10.3390\/ijms24119575"},{"key":"2025100321350653000_2025.09.28.679086v1.32","doi-asserted-by":"publisher","DOI":"10.1158\/1541-7786.MCR-19-1026"},{"key":"2025100321350653000_2025.09.28.679086v1.33","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0045078"},{"key":"2025100321350653000_2025.09.28.679086v1.34","doi-asserted-by":"publisher","DOI":"10.2183\/pjab.85.12"},{"key":"2025100321350653000_2025.09.28.679086v1.35","doi-asserted-by":"publisher","DOI":"10.1124\/pr.117.015370"},{"key":"2025100321350653000_2025.09.28.679086v1.36","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-3083.1996.d01-52.x"},{"key":"2025100321350653000_2025.09.28.679086v1.37","doi-asserted-by":"publisher","DOI":"10.1016\/j.semcancer.2019.12.013"},{"key":"2025100321350653000_2025.09.28.679086v1.38","doi-asserted-by":"publisher","DOI":"10.1023\/A:1018993312773"},{"key":"2025100321350653000_2025.09.28.679086v1.39","doi-asserted-by":"publisher","DOI":"10.1211\/jpp.59.9.0011."}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.09.28.679086","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:42:06Z","timestamp":1768470126000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.09.28.679086"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,30]]},"references-count":39,"URL":"https:\/\/doi.org\/10.1101\/2025.09.28.679086","relation":{},"subject":[],"published":{"date-parts":[[2025,9,30]]},"subtype":"preprint"}}