Proteasomal Degradation of XPB as a Novel Mechanism for Treating Inflammation

National Heart, Lung, And Blood Institute

Investigators

Abstract

In collaboration with the NCATS Functional Genomics Lab, we have created and validated a high-throughput luminescent reporter assay suitable for identifying small molecule degraders of XPB and genome-wide RNAi screening. Using this state-of-the-art high-throughput assay we will: 1) Identify novel, small-molecule XPB degraders with potent anti-inflammatory effects that can be accelerated into clinical trials; and 2) Characterize molecular partners necessary for drug-induced XPB degradation in order to construct a more generalizable pharmaceutical paradigm. This innovative assay platform will enable discovery of clinically relevant XPB degraders that modulate lung vascular inflammation in PAH patients, and the NIH Clinical Center is ideally suited for spearheading trials testing these first-in-class anti-inflammatory drugs. Project Milestones: - Screen the NCATS Small Molecule Library (up to 150,000 compounds) for drugs that induce XPB-HiBiT degradation. Candidate drugs will be selected through bio/chemi-informatic analyses, validated at 11 concentrations, and undergo counter screens to exclude cytotoxic effects and/or assay interference. Top candidates will be resynthesized and their activity confirmed. - Perform a second, high-throughput screen of candidate XPB degraders for potent anti-inflammatory effects across heterogeneous signaling pathways. Novel drugs identified in both screens will be further examined for their effects on inflammatory gene transcription and cytokine production in cellular models of PAH-associated endothelial cell dysfunction. - Perform a genome-wide RNAi screen to define the molecular mechanism underlying drug-induced XPB degradation. Target deconvolution of candidate drugs that induce proteasome-dependent XPB degradation and the NCATS pharmacologically annotated chemical toolbox will be used to substantiate gene silencing effects. Candidate XPB degradation genes will be confirmed in primary human pulmonary vascular endothelium. - Apply state-of-the-art medicinal chemistry techniques to optimize pharmacokinetics, potency, and efficacy of lead anti-inflammatory drugs for translation into the clinic.