Abstract 14275: Allosteric Inhibition Restricts Motion of the SMURF1 Glycine Hinge and Reverses Experimental Pulmonary Arterial Hypertension

Abstract

Background: Reduced expression of bone morphogenetic protein receptor 2 (BMPR2) predisposes to pulmonary vascular remodelling and the development of pulmonary arterial hypertension (PAH). The HECT E3 ligase SMURF1 is a key negative regulator of this pathway that is overexpressed in the pulmonary vasculature of patients with PAH. However, the absence of an active-site pocket renders E3s undruggable. Hypothesis: We hypothesized that a large, unbiased screen would be needed to identify SMURF1 inhibitors and that a SMURF1 inhibition would augment BMP signaling and treat experimental PAH. Aims: We aimed to: identify SMURF1 inhibitors through a large, unbiased screen; determine mechanism of action; examine the effects on BMP signalling, cell phenotype and experimental models of PAH. Methods: We constructed a time-resolved fluorescence resonance energy transfer-based assay reporting SMURF1 self-ubiquitylation and undertook a 1.1 million compound screen. Primary hits were rationalised based on biochemical selectivity and cell-based assays designed to prioritize and optimize molecules for specific inhibition of SMURF1. Crystal structure of SMURF1 and SMURF2 were determined in the bound and unbound state at 2.05-2.75 Å. Target interaction was examined in a split-CAT based system. The effect on signaling and phenotype was examined in primary pulmonary artery endothelial and smooth muscle cells, and in rats with PAH induced by monocrotaline and sugen hypoxia. Results: Screening identified lead compounds that selectively inhibited SMURF1. Structures reveal that inhibitor binding induces an α-helix elongation over an invariant glycine-containing hinge, limiting a motion mandatory for catalytic activity. We affirmed this structure-based model with mutants engineered to resist inhibition and demonstrated that inhibition prevents direct BMPR2 ubiquitylation, normalized BMP signaling and restored homeostasis in pulmonary vascular cells from patients with PAH. Finally, we demonstrated that SMURF1 inhibition reverses established, experimental PAH. Conclusions: Our structural understanding enables design of HECT and other glycine-hinge protein inhibitors opening a new druggable space.