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Browsing by Author "Smith, Jeffrey S."

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    Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane
    Kenwood, Brandon M.; Weaver, Janelle L.; Bajwa, Amandeep; Poon, Ivan K.; Byrne, Frances L.; Murrow, Beverley A.; Calderone, Joseph A.; Huang, Liping; Divakaruni, Ajit S.; Tomsig, Jose L.; Okabe, Kohki; Lo, Ryan H.; Coleman, G. Cameron; Columbus, Linda; Yan, Zhen; Saucerman, Jeffrey J.; Smith, Jeffrey S.; Holmes, Jeffrey W.; Lynch, Kevin R.; Ravichandran, Kodi S.; Uchiyama, Seiichi; Santos, Webster L.; Rogers, George W.; Okusa, Mark D.; Bayliss, Douglas A.; Hoehn, Kyle L. (Elsevier, 2013)
    Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.
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    Saccharomyces cerevisiae as a platform for assessing sphingolipid lipid kinase inhibitors
    Kharel, Yugesh; Agah, Sayeh; Huang, Tao; Mendelson, Anna J.; Eletu, Oluwafunmilayo T.; Barkey-Bircannl, Peter; Gesualdil, James; Smith, Jeffrey S.; Santos, Webster L.; Lynch, Kevin R. (PLOS, 2018-04-19)
    Successful medicinal chemistry campaigns to discover and optimize sphingosine kinase inhibitors require a robust assay for screening chemical libraries and for determining rank order potencies. Existing assays for these enzymes are laborious, expensive and/or low throughput. The toxicity of excessive levels of phosphorylated sphingoid bases for the budding yeast, Saccharomyces cerevisiae, affords an assay wherein inhibitors added to the culture media rescue growth in a dose-dependent fashion. Herein, we describe our adaptation of a simple, inexpensive, and high throughput assay for assessing inhibitors of sphingosine kinase types 1 and 2 as well as ceramide kinase and for testing enzymatic activity of sphingosine kinase type 2 mutants. The assay was validated using recombinant enzymes and generally agrees with the rank order of potencies of existing inhibitors.