Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy

Abstract

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and reroutes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.(F) IF co-staining of distal tubule in MKD patient kidney organoid for MUC1-wt (red), MUC1-fs (green), E-cadherin (blue), and Na + /K + -ATPase (yellow). MUC1-fs localized intracellularly (middle) compared to apical MUC1-wt (left). (G) IF co-staining in P cells for MUC1-fs (green), MUC1-wt (red), and Hoechst (gray). MUC1-fs localized intracellularly (middle) compared to MUC1-wt on the plasma membrane (left). See also Figures S1, S2, and S3 and Table S1.

A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury during the COVID-19 pandemic

“…These data were corroborated by the expression of MUC1 mRNA in human lung reported by the HPA, GTEx and FANTOM5 databases ( Figure 1B) and by the Human Lung Atlas project (Muus, 2020) We screened 3,713 compounds of the Repurposing Library for their ability to reduce MUC1 protein levels (Figure 2A). The screen employed high-content immunofluorescence (IF) imaging of an immortalized kidney tubular epithelial cell line (P cells) that express endogenous MUC1 on the plasma membrane, to simultaneously assess MUC1 protein abundance and cell number as an index of cell toxicity (Dvela-Levitt et al, 2019). The bromodomain inhibitor JQ1 served as positive control, as preliminary experiments demonstrated complete transcriptional suppression of MUC1 by JQ1.…”

“…Prompted by the connection between elevated MUC1 and ALI, we investigated the possibility of identifying MUC1-reducing drugs for rapid repurposing. We had originally screened the Broad Repurposing Library (comprised of 3,713 compounds at different stages of pre-clinical and clinical development (Corsello et al, 2017)) to identify compounds capable of reducing a mutant MUC1 neo-protein (MUC1-fs) causing autosomal dominant tubulo-interstitial kidney disease-mucin1 (ADTKD-MUC1 or MUC1 kidney disease, MKD) (Dvela-Levitt et al, 2019). In this context wildtype MUC1 (MUC1-wt) served as a control, as we sought compounds that specifically reduced the mutant, but not the wildtype form of MUC1.…”

“…As the number of COVID-19 cases increased globally, we turned our attention to identifying MUC1-reducing compounds and mined this dataset to identify approved drugs that reduce expression of MUC1wt. We searched for MUC1-reducing compounds based on the following criteria: 1) a drug that reduces MUC1-wt protein in a dose-dependent manner; 2) a drug with a favorable toxicity profile; 3) a drug that reduces MUC1-wt by non-transcriptional mechanisms (Dvela-Levitt et al, 2019), unlike transcriptional suppressors such as vitamin D agonists that have proven ineffective in the clinic (Castro et al, 2014); and 4) a drug that is US Food and Drug Administration (FDA)approved. Based on these criteria, our screen identified R406, the active metabolite of Fostamatinib (R788, an oral prodrug rapidly converted to R406), as a repurposing candidate for the treatment of ALI.…”

The cellular prion protein is a stress protein secreted by renal tubular cells and a urinary marker of kidney injury

“…Remarkably, mutations in the UMOD gene encoding Uromodulin, which is the most abundant protein secreted in normal urine and has multiple roles in kidney physiology, as well as mutations in the MUC1 gene which encodes the transmembrane epithelial mucin 1 protein supporting roles in epithelial barrier protection, lead to mutant proteins retention and aggregation in the ER, likely due to protein misfolding. The activation of the UPR in those inherited diseases leads to progressive tubular damage, triggering inflammation and interstitial fibrosis 32,33 . The identification and validation of ER stress markers is extremely important in this area, as it will provide a useful tool for diagnostic purpose and monitoring disease activity.…”

A translational kidney organoid system bolsters human relevance of clinical development candidate

“…New opportunities for human preclinical target evaluation have arisen through the development of iPSC-derived organoids including kidney organoids grown in vitro (Dvela-Levitt et al, 2019;Morizane et al, 2015;Taguchi and Nishinakamura, 2017;Takasato et al, 2016b) or transplanted in vivo (Sharmin et al, 2016;Subramanian et al, 2019;van den Berg et al, 2018). However, the use of organoids in drug discovery has been limited by problems with scalability, and by the fact that organoids grown in vitro could not be used for pharmacokinetic (PK) and pharmacodynamic (PD) studies.…”