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Identification, Characterization and Evaluation of the Novel Class of Warbicin Oncostatic Glucose Transport Inhibitors
Boek - Dissertatie
Catabolic breakdown of glucose through glycolysis is one the most ancient and conserved ways to fuel and maintain bioenergetic homeostasis. Both yeast and cancer cells ferment glucose to ethanol or lactate, respectively, to support vigorous cell division. Cancerous growth poses an important threat to human health as currently millions of people yearly succumb to its consequences. Fast proliferating cancer cells often rely on strong glucose uptake to support aerobic glycolysis, named the Warburg effect, which is positively correlated with its aggressiveness, metastatic potential and increased resistance to chemotherapeutics and radiation. Therefore, finding novel therapeutic targets or agents to combat the disease are still needed. As glycolytic cancers have an increased dependence on glucose uptake, inhibiting glucose transport has the potential to effectively kill off or reduce the overall fitness of these cells, re-sensitizing them to additional therapies. In this research, we worked with a specific Saccharomyces cerevisiae mutant that displays overactive flux through upper glycolysis when grown on glucose. We used this mutant as an exaggerated and translational model for the Warburg effect. As such, the tps1∆ strain lost its ability to feedback inhibit the hexokinases due to loss of trehalose-6-phosphate production. Consequently, growth on glucose results in extreme sugar phosphate buildup, Ras protein overactivation and eventually the induction of apoptosis. By screening a compound library using the tps1∆ mutant at growth inhibitory concentrations of glucose, we isolated one compound, which we named Warbicin (WBC) -A that was able to rescue the growth of this mutant. Mode-of-action studies revealed that WBC-A functions as a glucose transport inhibitor, thereby normalizing glycolytic deregulation of this mutant. To determine the structure-activity relationship and isolate compounds with enhanced efficacy, a library of about 200 structural analogs was constructed and screened using the tps1∆ mutant. This led to the identification of WBC-2C, an analog that inhibits glucose transport around 6 times stronger than WBC-A in S. cerevisiae. WBC-A activity was found conserved in human cells as growth and glucose transport of A549 lung adenocarcinoma was dose-dependently inhibited. The same analog library was screened for growth inhibition of A549 cells, leading to the isolation of three analogs WBC-15C, WBC-4C and especially WBC-11C, that inhibited glucose transport more strongly. Besides on A549 cells, these WBC compounds inhibited the growth of MCF10A breast epithelial cells and the U266 and KMS-PE-12 multiple myeloma cell lines, showcasing its broad-spectrum activity range. Moreover, when evaluating these compounds for 2-deoxyglucose uptake (2DG) in A549 cells for their modes-of-inhibition, different mechanisms were revealed: WBC-A and WBC-4C inhibited 2DG uptake noncompetitively, whereas WBC-15C inhibited competitively and WBC-11C uncompetitively. Mice experiments will determine whether besides the proven in vitro efficacy, the compounds hold promise to treat cancer progression, in vivo. Finally, using S. cerevisiae as a superior genetic model, transport activity inhibition by WBC compounds was studied in great detail. As such, WBC-A was found to inhibit all Hxt carriers in S. cerevisiae that are involved in glucose uptake. Moreover, glucose transport inhibition was dependent on the presence of functional hexokinase activity. WBC-A inhibited transport less strongly when the respective hexokinases were deleted. In addition, we showed that WBC-A likely reduces transport rate, not only by targeting the carrier, but also by decreasing the ability of hexokinase to directly phosphorylate incoming glucose. Given the structural similarities of WBC compounds with ATP, we propose that this novel family of synthetic inhibitors mimic ATP binding to the carrier. In light of the transport-associated phosphorylation (TAP) hypothesis in yeast, WBC compounds might decrease hexokinase activity in a spatio-temporal way, by displacing available ATP bound to the carrier. We argue that TAP-related processes might support strong glycolytic flux in cancer cells and that normal cells would not rely heavily on this process, thereby creating a novel specific therapeutic target. As such, WBC compounds might differentiate themselves from already pre-existing glucose transport inhibitors by targeting the coordinated uptake and phosphorylation of hexose sugars. In this work, the identification, characterization and evaluation of a novel class of glucose transport inhibitors is described. The mode-of-action was studied in detail and was found to be conserved from yeast to human. Likely by mimicking ATP binding, flux through glucose uptake and phosphorylation is lowered. WBC compounds provide a novel tool in the molecular toolbox to study facilitative hexose transport functioning in yeast and human cells. Finally, these compounds hold great therapeutic promise for the treatment of glycolytic-dependent cancer cells, alone or in combination with already existing cancer treatments.