Dataset Information

Therapeutic potential of allosteric HECT E3 ligase inhibition

ABSTRACT: Supplemental proteomics data Abstract: Inhibition of ubiquitin E3-ligases is therapeutically attractive; however, the absence of an active-site pocket impedes computational approaches to inhibitor identification. In a large unbiased physical screen, we discovered inhibitors that bind a cryptic cavity distant from the protruding SMURF1 catalytic cysteine. Structural and biochemical analyses, and engineered escape mutants, revealed that the inhibitors restrict an essential catalytic motion by extending an α-helix over a conserved glycine-hinge. SMURF1 is overexpressed in patients with pulmonary arterial hypertension (PAH), a disease caused by mutation of bone morphogenetic protein receptor-2 (BMPR2). We demonstrated that SMURF1 inhibition prevents direct BMPR2 ubiquitylation, normalizes BMP-signaling, restores pulmonary vascular cell homeostasis and reverses pathology in established, experimental PAH. We leveraged this deep mechanistic understanding to undertake a machine learning-based screen that identified inhibitors of the prototypic HECT E6AP, and confirm glycine-hinge-dependent allosteric activity. Inhibition of HECT E3s and other glycine-hinge proteins opens a new druggable space.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Pulmonary Artery Endothelial Cell

DISEASE(S): Primary Pulmonary Hypertension

SUBMITTER: Tomas Rejtar

LAB HEAD: Tomas Rejtar

PROVIDER: PXD051134 | Pride | 2025-05-26

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	PXP0629_73A_55C_30cm_TMT11_1uL_fr10_CLU2_AH_20181121_1493.raw	Raw
	PXP0629_73A_55C_30cm_TMT11_1uL_fr11_CLU2_AH_20181121_1480.raw	Raw
	PXP0629_73A_55C_30cm_TMT11_1uL_fr12_CLU2_AH_20181121_1494.raw	Raw
	PXP0629_73A_55C_30cm_TMT11_1uL_fr13_CLU2_AH_20181121_1481.raw	Raw
	PXP0629_73A_55C_30cm_TMT11_1uL_fr14_CLU2_AH_20181121_1495.raw	Raw

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Publications

Therapeutic potential of allosteric HECT E3 ligase inhibition.

Rothman Alexander M K AMK Florentin Amir A Zink Florence F Quigley Catherine C Bonneau Olivier O Hemmig Rene R Hachey Amanda A Rejtar Tomas T Thaker Maulik M Jain Rishi R Huang Shih-Min SM Sutton Daniel D Roger Jan J Zhang Ji-Hu JH Weiler Sven S Cotesta Simona S Ottl Johannes J Srivastava Salil S Kordonsky Alina A Avishid Reut R Yariv Elon E Rathi Ritu R Khvalevsky Oshrit O Troxler Thomas T Binmahfooz Sarah K SK Kleifeld Oded O Morrell Nicholas W NW Humbert Marc M Thomas Matthew J MJ Jarai Gabor G Beckwith Rohan E J REJ Cobb Jennifer S JS Smith Nichola N Ostermann Nils N Tallarico John J Shaw Duncan D Guth-Gundel Sabine S Prag Gali G Rowlands David J DJ

Cell 20250402 10

Targeting ubiquitin E3 ligases is therapeutically attractive; however, the absence of an active-site pocket impedes computational approaches for identifying inhibitors. In a large, unbiased biochemical screen, we discover inhibitors that bind a cryptic cavity distant from the catalytic cysteine of the homologous to E6-associated protein C terminus domain (HECT) E3 ligase, SMAD ubiquitin regulatory factor 1 (SMURF1). Structural and biochemical analyses and engineered escape mutants revealed that ...[more]

PMID: 40179885

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Project description:Pulmonary arterial hypertension (PAH) is a progressive disorder leading to occlusive vascular remodeling. Current PAH therapies improve quality of life but do not reverse structural abnormalities in the pulmonary vasculature. Here, we used a high-throughput drug screen combined with in silico analyses of existing transcriptomic datasets to identify a promising lead compound to reverse PAH. Induced pluripotent stem cell-derived endothelial cells (iPSC-EC) generated from six patients with PAH were exposed to 4,500 compounds and assayed for improved cell survival after serum withdrawal using a chemiluminescent caspase assay. Subsequent validation of caspase activity and improved angiogenesis combined with in silico analyses using the Gene Expression Omnibus (GEO ) and Library of Integrated Network-Based Cellular Signatures (LINCS) databases revealed that the lead compound AG1296 was positively associated with an anti-PAH gene signature. AG1296 increased abundance of bone morphogenetic protein receptors (BMPR2 and Ia), downstream signaling and gene expression, and suppressed PAH smooth muscle cell proliferation. AG1296 induced regression of pulmonary arterial (PA) neointimal lesions in lung organ culture and PA occlusive changes in the Sugen/hypoxia rat model and reduced right ventricular systolic pressure. Moreover, AG1296 improved vascular function and BMPR2 signaling and showed better correlation with the anti-PAH gene signature than other tyrosine kinase inhibitors (TKIs) such as imatinib. Specifically, AG1296 upregulated the transcription factors and small mothers against decapentaplegic (SMAD)1/5 co-activators, cAMP-response element binding protein (CREB)3 and CREB5: CREB3 induced inhibitor of DNA binding 1 (ID1) and downstream genes that improved vascular function. Thus, drug discovery for PAH can be accelerated by combining a phenotypic screen using patient-specific iPSC-derived vascular cells with in silico analyses of publicly available datasets.