{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"Research Square"}],"indexed":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T19:06:42Z","timestamp":1784833602690,"version":"3.55.0"},"posted":{"date-parts":[[2026,7,23]]},"group-title":"In Review","reference-count":0,"publisher":"Springer Science and Business Media LLC","license":[{"start":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T00:00:00Z","timestamp":1784764800000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,2]]},"abstract":"<title>Abstract<\/title>\n                <p>Endothelial-to-mesenchymal transition (EndMT) drives vascular remodeling in pulmonary arterial hypertension (PAH), yet the regulators that commit endothelial cells to this fate and whether they are pharmacologically addressable remain poorly defined. We report TK22, a small molecule that selectively erases the EndMT-competent endothelial state and reverses experimental pulmonary hypertension. TK22 was developed by structure-based design as an ATP-competitive CDK5 inhibitor (IC\u2085\u2080 181 nM). Unbiased profiling across 394 kinases, however, revealed that TK22 inhibits the interphase cell-cycle kinases CDK2\/3\/4\/6 node as well as a pro-mesenchymal node of AMPK-related kinases (NUAK1 and SIK1\/2\/3) together with PDGFR\u03b2 and MLK3, placing its activity at the intersection of proliferative and EndMT control rather than at a single node. Owing to this dual-node action, TK22 produced a strikingly clean cellular phenotype. In primary murine lung endothelial cells, TK22 reversed TGF-\u03b21-induced EndMT across morphological, proteomic, and functional readouts, normalizing \u03b1-SMA, SM22, and Calponin, restoring angiogenic tube formation, abolishing acquired smooth-muscle-like contractility, and returning TGF-\u03b21-driven hyperproliferation to baseline without suppressing normal endothelial growth. Single-cell RNA sequencing resolved the basis of this precision: TGF-\u03b21 redirected a cycling Mki67\u207a endothelial subpopulation into an EndMT-committed state (Tnnt2, Ptgs2, Serpine1) and induced an associated metabolic-stress program (Egln3, Pfkfb3, Pdk1, Bnip3). Cross-validated PHATE and Monocle3 trajectory inference defined a directional proliferative-to-mesenchymal fate transition, and kernel density estimation revealed a stable EndMT cell-state attractor. TK22 selectively eliminated the EndMT-competent and metabolic-stress subpopulations while leaving the remaining endothelial landscape intact. This population-level precision of TK22 is consistent with its dual engagement of the anti-cell-cycle and anti-mesenchymal kinase nodes. In the Su5416\/hypoxia rat PH model, TK22 normalized right ventricular systolic pressure, Fulton index, and cardiac function. Together, these findings reframe pharmacologic EndMT control: inhibition at the proliferative-to-mesenchymal fate branch point, through coordinated suppression of cell-cycle and mesenchymal kinases, achieves high selectivity for erasing a pathological endothelial fate state. These findings propose TK22 as a mechanistically precise agent for EndMT-driven vascular disease and establish single-cell trajectory analysis as an essential readout of cellular target engagement and cell-state precision.<\/p>","DOI":"10.21203\/rs.3.rs-9306408\/v2","type":"posted-content","created":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T18:27:06Z","timestamp":1784831226000},"source":"Crossref","is-referenced-by-count":0,"title":["Polypharmacologic Disruption of the Proliferative-to-Mesenchymal Fate Branch Point Reverses EndMT and Pulmonary Hypertension"],"prefix":"10.21203","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5950-4076","authenticated-orcid":false,"given":"Ruslan","family":"Rafikov","sequence":"first","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dinesh","family":"Bharti","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tetiana","family":"Kolodiazhna","sequence":"additional","affiliation":[{"name":"University of Houston"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sedat","family":"Kacar","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mathews","family":"Varghese","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takanori","family":"Sano","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sruthi","family":"Radhakrishnan","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joel","family":"James","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"AJ","family":"Hinkle","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maki","family":"Niihori","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olga","family":"Rafikova","sequence":"additional","affiliation":[{"name":"Indiana University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Statsyuk","sequence":"additional","affiliation":[{"name":"University of Houston"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","container-title":[],"original-title":[],"link":[{"URL":"https:\/\/www.researchsquare.com\/article\/rs-9306408\/v2","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.researchsquare.com\/article\/rs-9306408\/v2.html","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T18:27:16Z","timestamp":1784831236000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.researchsquare.com\/article\/rs-9306408\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,23]]},"references-count":0,"URL":"https:\/\/doi.org\/10.21203\/rs.3.rs-9306408\/v2","relation":{"is-version-of":[{"id-type":"doi","id":"10.21203\/rs.3.rs-9306408\/v1","asserted-by":"subject"}],"has-version":[{"id-type":"doi","id":"10.21203\/rs.3.rs-9306408\/v1","asserted-by":"object"}]},"subject":[],"published":{"date-parts":[[2026,7,23]]},"subtype":"preprint"}}