zaprinast

The mitochondrial pyruvate carrier (MPC) is a protein complex composed of two subunits, MPC1 and MPC2. This carrier is at the interface between glycolysis and mitochondrial metabolism and plays an essential role in hepatic glucose production.

Here we describe an in vitro screen for small molecule inhibitors of the MPC using a strain of Lactococcus lactis that has been engineered to co-express the two subunits of the human MPC and is able to import exogenous 14C-pyruvate. We then tested the top candidates for potential antidiabetic effects through the repression of gluconeogenesis.

By screening the Prestwick compound library of 1'200 drugs approved by the Food and Drug Administration for inhibitors of pyruvate uptake, twelve hit molecules were identified. In a secondary screen, the most potent inhibitors were found to inhibit pyruvate-driven oxygen consumption in mouse C2C12 muscle cells. Assessment of gluconeogenesis showed that Zaprinast, as well as the established MPC inhibitor UK5099, inhibited in vitro and in vivo hepatic glucose production. However, when tested acutely in mice without the administration of gluconeogenic substrates, MPC inhibitors raised blood glucose levels, pointing to liver-independent effects. Furthermore, chronic treatment with Zaprinast failed to correct hyperglycemia in both lean and obese diabetic mouse models.

New MPC inhibitors have been identified, showing inhibitory effects on hepatic glucose production.

For potential antidiabetic applications, MPC inhibitors should target the liver without undesired inhibition of mitochondrial pyruvate metabolism in the skeletal muscles or pancreatic beta-cells in order to avoid dual effects on glycemia.

Retinitis pigmentosa (RP) is one of the most common inherited diseases of the retina (retinopathies) that is estimated to affect 1 in 4,000 people in the United States and worldwide. This collection of genetic diseases leads to progressive photoreceptor cell degeneration and ultimately vision loss, with more than 60 genes implicated in molecular pathways towards photoreceptor cell death. Currently, there are no treatments available and, due to its vast diverse genetic background, a putative gene‐based therapy is challenging. Consequently, mutation‐independent strategies to protect photoreceptors against continued damage and progressive vision loss are appealing approaches. One of them is the use of survival, neurotrophic and neuroprotective factors that target common signaling pathways of cell death in the different types of RP. The aim of this study is to optimize methods to evaluate the efficacy of compounds to protect photoreceptor cells against death.We used PSVue® to optimize the cell death read‐out in cultured retinal explants and in retinas of a mouse model for a human inherited autosomal recessive RP, the rd10 mouse. Retinal degeneration in the rd10 mice is caused by a spontaneous mutation in the gene for the β‐subunit of phosphodiesterase. PSVue® is a robust probe for detecting phosphatidyl serine on the surface of cells undergoing death. We used retinal tissue explanted from wild type mouse eyes, and cultured it ex vivo. Induction of photoreceptor cell death was achieved with Zaprinast, an inhibitor of phosphodiesterase, to mimic the rd10 photoreceptor cell death. Low probe concentrations and short incubation times resulted in a reproducible detection of photoreceptor death in the retina by confocal super resolution microscopy. The PSVue® detection method yielded lower fluorescence background than the TUNEL detection method. Pigment epithelium‐derived factor (PEDF), a photoreceptor survival protein, was used as control to prevent photoreceptor cell death induced by Zaprinast. PEDF supplementation prior to Zaprinast treatment decreased cell death detection, as expected. We used the mouse model rd10 to evaluate photoreceptor death in vivo. PSVue® eye‐drops were administered to the left eye of rd10 mice of P17‐P25 postnatal ages, and fluorescence was detected by fundoscopy using MICRON III a day later. HBBS eye‐drops were administered to the right eye as a control reference. The fluorescence of the probe detected in the back of mouse eyes increased between ages P17 and P23 relative to that of HBBS‐treated eyes. The results revealed a peak of photoreceptor cell degeneration that occurred at P25 days of age in living rd10 mouse models.We have optimized methods to determine photoreceptor cell death in the ex vivo and in vivo experimental systems described here using a fluorescent probe. The methodologies are simple, fast, and complementary, and together they will prove to be suitable for screening factors and drugs against RP.