Proteasomal Degradation of XPB as a Novel Mechanism for Treating Inflammation

National Heart, Lung, And Blood Institute

Investigators

Abstract

Spironolactone (SPL) increases survival in left heart failure by improving endothelial function and reducing inflammation. Unlike other mineralocorticoid receptor antagonists, SPL induces protein degradation of xeroderma pigmentosum type B (XPB), a subunit of a key transcription factor, TFIIH. While SPL-induced depletion of XPB does not impact normal gene expression, our group demonstrated that SPL suppresses expression of inflammatory genes as a direct result of inducing XPB degradation. Therefore, SPL is a prototype for a new class of anti-inflammatory drugs that degrade specific protein targets. Nonetheless, the concentrations of SPL necessary for XPB degradation in vitro (3-20 uM) are challenging to achieve clinically due to dose-dependent adverse side effects. Compounds that selectively promote XPB degradation at therapeutically relevant concentrations with a more favorable safety profile would be an innovative strategy for treating vascular inflammation in PAH. Through collaboration with the National Center for Advancing Translational Sciences (NCATS), we have successfully miniaturized our XPB-HiBiT assay to a 1536-well format and the assay has been optimized and validated for high-throughput screening. To date, we have screened three annotated, small molecule libraries that include FDA-approved chemical entities and other investigational agents, totaling over 15,000 compounds. Initial screening has unveiled a relatively exclusive number of compounds that may induce XPB degradation and has provided proof-of-concept data that support our approach. In addition, we have created and validated a novel assay system that is suitable for high-throughput RNAi screening. Targeted screening of a ubiquitin/proteasome RNAi library has identified new candidate genes required for spironolactone-induced XPB degradation and verified previously known molecular partners. Based on the assay reproducibility demonstrated here, we plan to proceed with genome-wide RNAi screening. In a complimentary approach to siRNA screening, we plan to identify spironolactone binding partners that are necessary for proteasome dependent XPB degradation. Lastly, we recently initiated a collaboration with the NHLBI Chemical Synthesis Center to develop structural analogues of spironolactone that can tested for their ability to induce XPB degradation and suppress inflammatory gene transcription. Novel analogues with greater potency compared to spironolactone have been identified. Comparisons across these analogues will inform structure-function relationships necessary for inducing XPB degradation. We look forward to filing a provisional patent application in the coming months.