Cayman scientists perform structure-based mutational analysis to study the preferential binding mechanisms of inhibitors to an enzyme involved in pancreatic cancer cell metabolism.

Melissa C. Holt, Zahra Assar, Reza Beheshti Zavareh, Lin Lin, Justin Anglin, Oksana Mashadova, Daniel Haldar, Edouard Mullarky, Daniel M. Kremer, Lewis C. Cantley, Alec C. Kimmelman, Adam J. Stein, Luke L. Lairson, and Costas A. Lyssiotis

Highlights

• Inhibition of GOT1—an essential enzyme in the metabolic pathway of pancreatic cancer cells—prevents the growth of pancreatic ductal adenocarcinoma cells and tumors but is well tolerated by normal pancreatic cells and fibroblasts.
• In silico docking analysis of inhibitor–GOT1 interactions revealed that GOT1 inhibitors compete directly for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1.
• X-ray crystallography and thermal shift assays of GOT1 enzymes mutated to prevent PLP binding exposed several residues necessary for GOT1 catalytic activity.
• Insight into this mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.

Abstract

Pancreatic cancer cells are characterized by deregulated metabolic programs that facilitate growth and resistance to oxidative stress. Among these programs, pancreatic cancers preferentially utilize a metabolic pathway through the enzyme aspartate aminotransferase 1 [also known as glutamate oxaloacetate transaminase 1 (GOT1)] to support cellular redox homeostasis. As such, small molecule inhibitors that target GOT1 could serve as starting points for the development of new therapies for pancreatic cancer. We ran a high-throughput screen for inhibitors of GOT1 and identified a small molecule, iGOT1-01, with in vitro GOT1 inhibitor activity. Application in pancreatic cancer cells revealed metabolic and growth inhibitory activity reflecting a promiscuous inhibitory profile. We then performed an in silico docking analysis to study inhibitor–GOT1 interactions with iGOT1-01 analogs that possess improved solubility and potency properties. These results suggested that the GOT1 inhibitor competed for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1. To analyze how the GOT1 inhibitor bound to GOT1, a series of GOT1 mutant enzymes that abolished PLP binding were generated. Application of the mutants in X-ray crystallography and thermal shift assays again suggested but were unable to formally conclude that the GOT1 inhibitor bound to the PLP site. Mutational studies revealed the relationship between PLP binding and the thermal stability of GOT1 while highlighting the essential nature of several residues for GOT1 catalytic activity. Insight into the mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.

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