Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

Nutrient ingestion increased mTOR signaling, but not hVps34 activity in human skeletal muscle after sprint exercise.

Nutrient provision after sprint exercise enhances mammalian target of rapamycin (mTOR) signaling. One suggested that nutrient sensor is the class III phosphatidylinositol 3-kinase, vacuolar protein sorting 34 (Vps34), not previously studied in human skeletal muscle. It is hypothesized that oral ingestion of essential amino acids (EAA) and carbohydrates (Carb) increases Vps34 activity and mTOR signaling in human skeletal (hVps34) muscle after sprint exercise. Nine subjects were performed 3 × 30-sec all-out sprints with or without ingestion of EAA + Carb or placebo drinks in a randomized order with a month interval. Muscle biopsies were performed at rest and 140 min after last sprint and analyzed for p-mTOR, p-p70S6k, p-eEF2 and for hVps34 activity and hVps34 protein content. Venous blood samples were collected and analyzed for amino acids, glucose, lactate, and insulin. During the sprint exercise session, EAA, glucose, and insulin in blood increased significantly more in EAA + Carb than in placebo. P-mTOR and p-p70S6k were significantly increased above rest in EAA + Carb (P = 0.03, P = 0.007) 140 min after last sprint, but not in placebo. Activity and protein expression of hVps34 were not significantly changed from rest in EAA + Carb 140 min after the last sprint. However, hVps34 activity and protein expression tended to increase in placebo (both P = 0.08). In conclusion, on the contrary to the hypothesis, no increase in activation of hVps34 was found following sprint exercise in EAA + Carb condition. In spite of this, the results support an activation of mTOR during this condition. However, this does not exclude the permissive role of hVps34 in mediating the amino acid-induced activation of mTOR and muscle protein synthesis.