Date of Award

2017

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

John J. Lemasters

Second Advisor

Robert M. Gemmill

Third Advisor

Eduardo M. Maldonado

Fourth Advisor

Campbell McInnes

Fifth Advisor

Patrick M. Woster

Abstract

Enhancement of aerobic glycolysis and suppression of mitochondrial metabolism characterize the proliferation-favoring Warburg phenotype of cancer cells, and suppression of mitochondrial metabolism contributes to a low cytosolic ATP/ADP ratio favoring enhanced aerobic glycolysis. High free tubulin in cancer cells closes voltage dependent anion channels (VDAC) to decrease mitochondrial membrane potential (ΔΨ), an effect antagonized by erastin, the canonical promotor of ferroptosis, a type of non-apoptotic, iron-dependent oxidative cell death. Here, we identified six compounds (X1-X6) that also block tubulin-dependent mitochondrial depolarization. The three most potent lead compounds were structurally very similar, each containing a phenylpyrimidine carboxamide scaffold. We performed a retrospective analysis of our drug screen data and found three additional ChemBridge small molecules containing the same backbone that were also screened individually, but their activity did not meet the lead selection threshold. Using this information, we generated a preliminary pharmacophore model. In addition, lead compounds and erastin did not promote microtubule stabilization, so changes in ΔΨ were independent of tubulin dynamics. The most potent lead compound also decreased lactate formation in cancer cells. We hypothesized that VDAC opening after erastin and X1-X6 increases mitochondrial metabolism and reactive oxygen species (ROS) formation, leading to ROS-dependent mitochondrial dysfunction, bioenergetic failure and cell death. Accordingly, we characterized erastin and the two most potent structurally unrelated lead compounds, X1 and X4, on ROS formation, mitochondrial function, activation of c-jun N-terminal kinase (JNK) and cell viability. Erastin, X1 and X4 increased ΔΨ, followed closely by an increase of mitochondrial ROS generation and JNK activation. Subsequently, mitochondria began to depolarize indicating onset of mitochondrial dysfunction. N-acetylcysteine (glutathione precursor and ROS scavenger) and MitoQ (mitochondrially targeted antioxidant) blocked increased ROS formation after X1 and prevented mitochondrial dysfunction. Our two most potent structurally unrelated lead compounds, X1 and X4, selectively promoted cell killing in HepG2 and Huh7 human hepatocarcinoma cells compared to primary rat hepatocytes. X1 and X4-dependent cell death was blocked by NAC. These results suggest that ferroptosis induced by erastin and our erastin-like lead compounds was caused by VDAC opening, leading to increased ΔΨ, mitochondrial ROS generation and oxidative stress-induced cell death.

Rights

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