Proteasome inhibition measurements: clinical application.

BACKGROUND: PS-341, a selective inhibitor of the proteasome, currently is under evaluation as an anticancer agent in multiple phase I clinical trials. In animal-model studies, PS-341 was rapidly removed from the vascular compartment and distributed widely, quickly approaching the limits of detection. An accurate pharmacodynamic assay has been developed as an alternative or complement to pharmacokinetic measurements. METHODS: Fluorogenic kinetic assays for both the chymotryptic and tryptic activities of the proteasome have been optimized for both whole blood and blood cells. Using the ratio of these activities and the catalytic mechanism of the proteasome, we developed a novel method of calculating percentage of inhibition, using two structurally unrelated inhibitors (PS-341 and lactacystin). RESULTS: This ratio method was demonstrated to be sensitive (detection limit of 13% inhibition with 10 microgram of cell lysate), specific to the proteasome (PS-341 provides >98% inhibition), accurate (112% analyte recovery), and precise (0% +/- 5% inhibition at 0 nmol/L PS-341 and 74.5% +/- 1.7% inhibition at 200 nmol/L PS-341). Using these assays, we found that both erythrocytes and leukocytes contain proteasome at 3 micromol/L. Pharmacodynamic results for PS-341 obtained from the whole-blood ratio method were comparable to those using leukocytes determined by another method. CONCLUSIONS: The described assay provides a reliable method for studying the pharmacodynamics of proteasome inhibitors and is now in use in concurrent phase I clinical trials with PS-341.

Proteasome inhibition: A novel mechanism to combat asthma.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a critical transcription factor required for the regulation of many genes involved in inflammatory responses to noxious stimuli. On activation, NF-kappaB induces the transcription of numerous proinflammatory cytokines, enzymes, and cellular adhesion molecules. Blockade of the proteasome with selective inhibitors attenuates the effects of NF-kappaB, leading to suppression of the inflammatory response. OBJECTIVE: We sought to determine whether proteasome inhibitors would be active in a model of asthma. METHODS: The mouse delayed-type hypersensitivity model was used to screen a panel of compounds for in vivo activity. The proteasome inhibitor, PS-519, was shown to be the most active in this model and was selected for further development. Allergen-induced pulmonary eosinophilia in Brown Norway rats was used subsequently to determine anti-inflammatory activity in an animal model. RESULTS: Direct administration of PS-519 into the lungs significantly reduced leukocyte numbers, particularly the selective increase in eosinophils. Because steroids are the mainstay anti-inflammatory therapy in asthma, and data is available to suggest their possible interaction to suppress the activation of NF-kappaB, rats were also treated by inhalation with combinations of a steroid and the proteasome inhibitor. In both the delayed-type hypersensitivity and the animal eosinophil model, low doses of proteasome inhibitors were shown to be effective when given with low doses of steroids. CONCLUSION: Taken together, the present data suggest that proteasome inhibition may represent a novel strategy for the treatment of inflammatory lung diseases such as asthma.

A new model of cancer cachexia: contribution of the ubiquitin-proteasome pathway.

A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-proteasome pathway was investigated. Cloning of the colon-26 tumor produced a cell line, termed R-1, which induced cytokine (noninterleukin-1beta, interleukin-6 and tumor necrosis factor-alpha)-independent cachexia. Implantation of R-1 cells in mice elicited significant (20-30%) weight loss and decreased blood glucose by 70%, and adipose tissue levels declined by 95% and muscle weights decreased by 20-25%. Food intake was unaffected. The decrease in muscle weight reflected a decline in insoluble, but not soluble, muscle protein that was associated with a significant increase in net protein degradation. The rate of ubiquitin conjugation of proteins was significantly elevated in muscles of cachectic mice. Furthermore, the proteasome inhibitor lactacystin blocked the increase in protein breakdown but had no significant effect on proteolysis. Several markers of the ubiquitin-proteasome pathway, E2(14k) mRNA and E2(14k) protein and ubiquitin-protein conjugates, were not elevated. Future investigations with this new model should gain further insights into the mechanisms of cachexia and provide a background to evaluate novel and more efficacious therapies.

Kinetic studies of the branched chain amino acid preferring peptidase activity of the 20S proteasome: development of a continuous assay and inhibition by tripeptide aldehydes and clasto-lactacystin beta-lactone.

We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu-Ala bond by the 20S proteasome with a kc/Km value of 13 000 M-1 s-1. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (lambda ex = 340 nm, lambda em = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a series of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)Phe, and Trp we observe biphasic or partial inhibition of the BrAAP activity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate Ki values of 120 nM and 12 nM, respectively. The inhibitors that exhibit simple inhibition kinetics for BrAAP activity are also approximately equipotent for inhibition of the chymotrypsin-like (ChT-L) and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities, dissociation constants varying by less than 25-fold, whereas the inhibitors that exhibit biphasic inhibition kinetics for BrAAP activity are >300-fold more potent for inhibiting ChT-L activity than for PGPH activity. Inactivation of the BrAAP activity of the proteasome by clasto-lactacystin beta-lactone is also biphasic. beta-Lactone inactivates approximately 60% of the BrAAP activity rapidly, with kinetics indistinguishable from its inactivation of the chymotrypsin-like activity. The remaining 40% of the BrAAP activity is inactivated by beta-lactone at a 50-fold slower rate, with kinetics indistinguishable from its inactivation of the PGPH activity. These results suggest a mechanism in which hydrolysis of Abz-Gly-Pro-Ala-Leu-Ala-Nba (i.e., BrAAP activity) occurs at two different active sites in the 20S proteasome, and that these two active sites are the same ones that catalyze the previously described ChT-L and PGPH activities.

Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells.

The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.