Mitochondrial ATP-dependent proteases are vital for maintaining cellular homeostasis and the response to environmental stress. These enzymes are ATP-powered proteolytic machines that selectively degrade abnormal proteins and regulate metabolic processes. In many disease states and aging, the increased generation of reactive oxygen species within mitochondria results in protein oxidation and aggregation. As a counter-measure, the mitochondrial Lon protease for example, degrades oxidized aconitase thereby preventing its irreversible accumulation and aggregation. The oxidative damage of proteins, nucleic acids and lipids is directly linked to aging, heart disease and neuromuscular disorders;whereas protein aggregation is common to many neurodegenerative disorders. In addition, recent work suggests that Lon-mediated proteolysis may be important in tumorigenesis and the adaptation of intratumoral cells to hypoxia. Unfortunately, there are no specific high affinity inhibitors or activators of mitochondrial ATP-dependent proteases. The goals of this proposal are to develop assays for identifying and validating compounds that selectively activate or inhibit the Lon protease.
Aim 1 is to develop and optimize a primary screening assay for measuring Lonmediated degradation of reporter peptide substrate using time-resolved fluorometry. The feasibility and reproducibility of these assays will be demonstrated using commercially available chemical libraries.
Aim 2 is to develop and optimize secondary- and counter- screening assays, which will provide information about the mechanism of compound activity, and distinguish compounds that are Lon-specific from those that target other mitochondrial or non-mitochondrial proteases. Small molecule activators of mitochondrial ATP-dependent proteolysis have potential application in the treatment of neurodegenerative and/or myocardial dysfunctions linked to mitochondrial protein aggregation. Our preliminary data suggest that inhibitors of mitochondrial ATP-dependent proteolysis may function as anti-cancer agents, with potential clinical application either alone or in combination with other chemotherapeutic strategies. Taken together, Lon and other mitochondrial ATP-dependent proteases may be new and viable drug targets.
Significance. Mitochondrial ATP-dependent proteases are potentially new targets for drug development and clinical application. Our preliminary data show that anti-cancer agents such as CDDO and Velcade block Lonmediated proteolysis (see Figs. 3-8, 10-11). However, a fundamental question remains unanswered: Does inhibition of Lon contribute to the chemotherapeutic effects of CDDO and Velcade, or, might this inhibition lead to uncharacterized and undesirable side effects of these compounds? The identification of specific and potent Lon inhibitors will help us to answer these key questions. If we discover that Lon activity promotes tumor growth and that Lon-specific inhibition is anti-tumorigenic, then strategies for testing Lon inhibitors alone or in combination with other agents will be developed for clinical application. In addition, knowledge of the chemical structure(s), mechanism(s) of action and physiological effects of inhibitors and activators of mitochondrial Lon, will also permit the rational design of compounds that specifically target fungal and bacterial Lon, which may result in the development of new antibiotic agents.
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| Lu, Bin; Lee, Jae; Nie, Xiaobo et al. (2013) Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease. Mol Cell 49:121-32 |
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| Fishovitz, Jennifer; Li, Min; Frase, Hilary et al. (2011) Active-site-directed chemical tools for profiling mitochondrial Lon protease. ACS Chem Biol 6:781-8 |
