Inherited mitochondrial respiratory chain disorders are progressive, life-threatening conditions for which there are limited supportive treatment options and no approved drugs. Because of this unmet medical need, as well as the implication of mitochondrial dysfunction as a contributor to more common age-related and neurodegenerative disorders, mitochondrial diseases represent an important therapeutic target. Thirteen children and one adult with genetically-confirmed mitochondrial disease (polymerase γ deficiency, n=4; Leigh syndrome, n=4; MELAS, n=3; mtDNA deletion syndrome, n=2; Friedreich ataxia, n=1) at risk for progressing to end-of-life care within 90 days were treated with EPI-743, a novel para-benzoquinone therapeutic, in a subject controlled, open-label study. Serial measures of safety and efficacy were obtained that included biochemical, neurological, quality-of-life, and brain redox assessments using technetium-99m-hexamethylpropyleneamine oxime (HMPAO) single photon emission computed tomography (SPECT) radionuclide imaging. Twelve patients treated with EPI-743 have survived; one polymerase γ deficiency patient died after developing pneumonia and one patient with Surf-1 deficiency died after completion of the protocol. Of the 12 survivors, 11 demonstrated clinical improvement, with 3 showing partial relapse, and 10 of the survivors also had an improvement in quality-of-life scores at the end of the 13-week emergency treatment protocol. HMPAO SPECT scans correlated with clinical response; increased regional and whole brain HMPAO uptake was noted in the clinical responders and the one subject who did not respond clinically had decreased regional and whole brain HMPAO uptake. EPI-743 has modified disease progression in >90% of patients in this open-label study as assessed by clinical, quality-of-life, and non-invasive brain imaging parameters. Data obtained herein suggest that EPI-743 may represent a new drug for the treatment of inherited mitochondrial respiratory chain disorders. Prospective controlled trials will be undertaken to substantiate these initial promising observations. Furthermore, HMPAO SPECT imaging may be a valuable tool for the detection of central nervous system redox defects and for monitoring response to treatments directed at modulating abnormal redox.

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Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive ⑀PKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed⑀RACK. An amino acid difference between the ⑀RACK sequence in ⑀PKC and its homologous sequence in ⑀RACK constitutes a change from a polar non-charged amino acid (asparagine) in ⑀RACK to a polar charged amino acid (aspartate) in ⑀PKC. Here we show that mutating the aspartate to asparagine in ⑀PKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone-or PMAinduced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to ⑀RACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the ⑀RACK site and RACK-binding site within ⑀PKC is critical and rate limiting in the process of PKC translocation.The protein kinase C (PKC) 1 family of phospholipid (PL) -dependent serine/threonine kinases undergoes a conformational change and translocation, or movement, from the cytosolic to the cell particulate fraction upon activation (1, 2). Conformational changes in PKC from an inactive to an active state results in exposure of domains required for PKC anchoring to the particulate fraction and in increased sensitivity of the enzyme to proteases (1-3, 41). Therefore, the inactive state exists in a closed conformation, with the proteolytic sites protected, whereas the active state is in an open conformation with exposed proteolytic sites. Structural alterations from the closed to open states involve disruption of intramolecular interactions within the enzyme.An intramolecular interaction in inactive PKC between the catalytic site and a site in the regulatory domain that resembles a substrate phosphorylation site but lacks a serine or threonine phosphoacceptor (pseudosubstrate site) has been previously identified (3, 6). Deletion of the pseudosubstrate ( -substrate) site generated a constitutively active enzyme (6) and mutations of the basic residues in the -substrate site reduced the affinity of the catalytic site to the -substrate site generating a constitutively active enzyme, preferentially localized to the cell particulate fraction (6). Furthermore, conversion of the alanine in the -substrate site to a glutamic acid, mimicking a phosphorylated amino acid, resulted in loss of binding of the -substrate site to the catalytic site, creating a constitutively active enzyme (6). Finally, a peptide corresponding to the -substrate site is a competitive inhibitor of PKC catalytic activity (6).We previously demonstrated that translocation of PKC is associated with binding of each activated PKC isozyme to...

Protein kinase C (PKC) is a family of kinases that are critical in many cellular events. These enzymes are activated by lipid-derived second messengers, are dependent on binding to negatively charged phospholipids and some members also require calcium to attain full activation. The interaction with lipids and calcium activators is mediated by binding to the regulatory domains C1 and C2. In addition, many protein-protein interactions between PKC and other proteins have been described. These include interactions with adaptor proteins, substrates and cytoskeletal elements. Regulation of the interactions between PKC, small molecules and other proteins is essential for signal transduction to occur. Finally, a number of auto-inhibitory intramolecular protein-protein interactions have also been identified in PKC. This chapter focuses on mapping the sites for many of these inter and intramolecular interactions and how this information may be used to generate selective inhibitors and activators of PKC signaling.

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