Biochemical and functional characterization of the p.A165T missense variant of mitochondrial amidoxime-reducing component 1.

Recent genome-wide association studies have identified a missense variant p.A165T in mitochondrial amidoxime-reducing component 1 (mARC1) that is strongly associated with protection from all-cause cirrhosis and improved prognosis in nonalcoholic steatohepatitis (NASH). The precise mechanism of this protective effect is unknown. Substitution of alanine 165 with threonine is predicted to affect mARC1 protein stability and to have deleterious effects on its function. To investigate the mechanism, we have generated a knock-in mutant mARC1 A165T and a catalytically dead mutant C273A (as a control) in human hepatoma HepG2 cells, enabling characterization of protein subcellular distribution, stability, and biochemical functions of the mARC1 mutant protein expressed from its endogenous locus. Compared to wild-type (WT) mARC1, we found that the A165T mutant exhibits significant mislocalization outside of its traditional location anchored in the mitochondrial outer membrane and reduces protein stability, resulting in lower basal levels. We evaluated the involvement of the ubiquitin proteasome system in mARC1 A165T degradation and observed increased ubiquitination and faster degradation of the A165T variant. In addition, we have shown that HepG2 cells carrying the MTARC1 p.A165T variant exhibit lower N-reductive activity on exogenously-added amidoxime substrates in vitro. The data from these biochemical and functional assays suggest a mechanism by which the MTARC1 p.A165T variant abrogates enzyme function which may contribute to its protective effect in liver disease.

SZN-413, a FZD4 Agonist, as a Potential Novel Therapeutic for the Treatment of Diabetic Retinopathy.

Purpose: There remains a high unmet need for therapies with new mechanisms of action to achieve reperfusion of ischemic retina in diabetic retinopathy. We examined whether a novel frizzled class receptor 4 (FZD4) agonist could promote regeneration of functional blood vessels in animal models of retinopathy. Methods: We developed a novel Norrin mimetic (SZN-413-p) targeting FZD4 and low-density lipoprotein receptor-related protein 5 (LRP5) and examined its effect on retinal and brain endothelial cells in vitro. SZN-413-p was subsequently humanized, resulting in the therapeutic candidate SZN-413, and was examined in animal models of retinopathy. In an oxygen-induced retinopathy mouse model, avascular and neovascularization areas were measured. Furthermore, in a vascular endothelial growth factor (VEGF)-induced retinal vascular leakage rabbit model, the impact on vascular leakage by SZN-413 was examined by measuring fluorescein leakage. Results: SZN-413-p induced Wnt/ß-catenin signaling and upregulated blood-brain barrier/blood-retina barrier gene expressions in endothelial cells. In the oxygen-induced retinopathy mouse model, SZN-413-p and SZN-413 significantly reduced the neovascularization area size (P < 0.001) to a level comparable to, or better than the positive control aflibercept. Both agonists also showed a reduction in avascular area size compared to vehicle (P < 0.001) and aflibercept groups (P < 0.05 and P < 0.01 for SZN-413-p and SZN-413, respectively). In the VEGF-induced retinal vascular leakage rabbit model, SZN-413 reduced retinal vascular leakage by ∼80%, compared to the vehicle-treated group (P < 0.01). Conclusions: Reduction of neovascular tufts and avascular areas and of VEGF-driven retinal vascular leakage suggests that SZN-413 can simultaneously address retinal non-perfusion and vascular leakage. Translational Relevance: FZD4 signaling modulation by SZN-413 is a novel mechanism of action that can offer a new therapeutic strategy for diabetic retinopathy.