Many environmental, pharmaceutical, and industrial compounds negatively affect human health by deleterious effects on mitochondrial function. Mitochondrial toxicity is defined as a decrease in the function and /or number of mitochondria, leading to decreased respiration and energy production. Severe loss of mitochondrial function can result in damage to vital organs due to cellular injury, apoptosis, or necrosis. Currently there is no database of chemically related compounds that cause mitochondrial dysfunction, and there are no reliable methods for predicting mitochondrial toxicity. To develop a database of mitochondrial toxicants, a novel respirometric assay of 1760 diverse compounds was conducted using primary cultures of rabbit renal proximal tubule cells (RPTC) and the Seahorse Biosciences Extracellular Flux (XF) analyzer. The results of this screen identified 22 compounds that diminish uncoupled mitochondrial respiration, a stress test for mitochondrial function. From these 22 compounds, five chemical clusters were identified based on molecular similarity between three or more compounds. One of these clusters has been aligned to identify a preliminary group of chemical structures related to mitochondrial toxicity, defined as a toxicophore. We hypothesize that discrete toxicophores defined by distinct chemical entities can identify previously unknown and future mitochondrial toxicants. We will examine this hypothesis in three Specific Aims.
Specific Aim 1 is to elucidate the toxicophores for the five identified clusters of mitochondrial toxicants using cheminformatic analysis.
Specific Aim 2 is to identify mitochondrial toxicants related to the elucidated toxicophores through an in silico screen of a diverse library of 50,000 small molecules. The resulting compounds will be examined for their effects on mitochondrial respiration using RPTC and a 96-well XF instrument. Confirmed mitochondrial toxicants will be included into the cheminformatic models and used to refine the toxicophores.
Specific Aim 3 is to analyze the mechanism of mitochondrial damage for the identified toxicophores by examining the effects of representative compounds on uncoupling oxidative phosphorylation, disruption of the electron transport chain, and/or altering the mitochondrial permeability transition pore (MPT). The long-term goal of this research is to design toxicophores that can reliably predict mitochondrial toxicity for both chemical discovery and risk assessment. The training that I will acquire by completing the outlined proposal will enhance my research expertise, and will prepare me to apply innovative concepts to the field of toxicology.
We hypothesize that discrete toxicophores defined by distinct chemical entities can identify previously unknown and future mitochondrial toxicants. A high-throughput respirometric screen of a diverse chemical library in primary cultures of rabbit renal proximal tubule cells identified five clusters of molecularly similar mitochondrial toxicants. The identified clusters will be used to define toxicophores that can reliably predict mitochondrial toxicity for chemical design, drug discovery, and human health risk assessment.
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