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.

Public Health Relevance

Our goal is to develop high throughput screening assays for identifying small molecules that specifically and potently inhibit or activate the mitochondrial ATP-dependent Lon protease. These molecular tools will allow us to determine the role that Lon plays in mitochondrial dysfunction and apoptosis, which are associated with numerous neuromuscular disorders, cardiomyopathies, cancers and aging. Inhibitors of Lon have potential application as anti- cancer agents used alone or in combination with other chemotherapies. This possibility is supported by preliminary data demonstrating that compounds used to treat leukemia and solid tumors also inhibit Lon-mediated proteolysis. Activators of Lon can be potentially exploited in the treatment of neurodegenerative disorders linked to mitochondrial protein aggregation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Instrumentation and Systems Development Study Section (ISD)
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Dunsmore, Sarah
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University of Medicine & Dentistry of NJ
Schools of Medicine
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Strauss, Kevin A; Jinks, Robert N; Puffenberger, Erik G et al. (2015) CODAS syndrome is associated with mutations of LONP1, encoding mitochondrial AAA+ Lon protease. Am J Hum Genet 96:121-35
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
Bernstein, Steven H; Venkatesh, Sundararajan; Li, Min et al. (2012) The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood 119:3321-9
Venkatesh, Sundararajan; Lee, Jae; Singh, Kamalendra et al. (2012) Multitasking in the mitochondrion by the ATP-dependent Lon protease. Biochim Biophys Acta 1823:56-66
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