A Rationally Designed CDK5 Inhibitor Selectively Erases an EndMT-Competent State in Primary Lung Endothelium

Endothelial-to-mesenchymal transition (EndMT) plays a vital role in driving vascular remodeling in pulmonary arterial hypertension (PAH), yet the molecular regulators that commit endothelial cells to this fate, and whether they represent important pharmacological targets, remain poorly defined. Here, we integrate structure-based drug discovery, multidimensional functional biology, and high-resolution single-cell RNA sequencing (scRNA-seq) to establish cyclin-dependent kinase 5 (CDK5) as a nodal regulator of endothelial fate conversion and to demonstrate its pharmacological reversibility. CDK5 was significantly upregulated in the Su5416/hypoxia rat model of PAH together with the EndMT-associated markers FOXC2, TRPV2, and α-SMA, implicating CDK5 in disease-driven endothelial remodeling. Guided by the crystal structure of CDK5/p25, we designed and synthesized TK22, a novel reversible ATP-competitive CDK5 inhibitor (IC50 ~ 181.3 nM) that outperformed commercial CDK5 inhibitors in suppressing TGF-β1-induced EndMT in primary GFP+ murine lung endothelial cells (mLECs). TK22 reversed EndMT across morphological, proteomic, and functional readouts, normalizing mesenchymal marker expression (SM22, α-SMA, Calponin), restoring angiogenic tube formation, and abolishing the smooth muscle-like contractility acquired by EndMT cells in collagen gel assays. To resolve the cellular mechanism at single-cell resolution, we performed scRNA-seq on GFP-sorted mLECs under control, TGF-β1, and TGF-β1 + TK22 conditions. Unsupervised clustering identified transcriptionally distinct endothelial subpopulations. Cross-validated trajectory inference using PHATE and Monocle3 revealed a directional, lineage-committed fate transition originating in a highly proliferative Mki67+ endothelial subpopulation and leading to a TGF-β1-expanded EndMT state marked by Tnnt2, Ptgs2, and Serpine1. Kernel density estimation confirmed that TGF-β1 drives the emergence of a stable EndMT cell-state attractor that is selectively and completely abolished by TK22, without disrupting the remaining endothelial subpopulations. This population-level precision, disrupting a pathological fate branch point while preserving normal endothelial heterogeneity, is invisible to bulk approaches and positions scRNA-seq trajectory analysis as an essential tool for evaluating endothelial-targeted therapeutics. Together, these findings establish CDK5 as a gatekeeper of the proliferative-to-mesenchymal fate decision in lung endothelial cells and nominate TK22 as a mechanistically precise agent for therapeutic intervention in PAH and other EndMT-driven vascular diseases.