Biochemical Characterization and Structure-Based Mutational Analysis Provide Insight into the Binding and Mechanism of Action of Novel Aspartate Aminotransferase Inhibitors
[7];
[8]
- Cayman Chemical Company, Ann Arbor, MI (United States)
- California Inst. for Biomedical Research, La Jolla, CA (United States)
- Univ. of Michigan Medical School, Ann Arbor, MI (United States)
- Weill Cornell Medical College, New York, NY (United States)
- Harvard Medical School, Boston, MA (United States)
- NYU Langone Medical Center, New York, NY (United States)
- California Inst. for Biomedical Research, La Jolla, CA (United States); The Scripps Research Inst., La Jolla, CA (United States)
- Univ. of Michigan, Ann Arbor, MI (United States)
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 analogues that possess improved solubility and potency properties. Here, 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.
- Research Organization:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Organization:
- Cancer Center Support; AACR-Bayer Innovation and Discovery Grant; National Inst. of Health
- Grant/Contract Number:
- P30 CA046592; 16-80-44LYSS; DK097153; R35-CA197588
- OSTI ID:
- 1499730
- Journal Information:
- Biochemistry, Vol. 57, Issue 47; ISSN 0006-2960
- Publisher:
- American Chemical Society (ACS)Copyright Statement
- Country of Publication:
- United States
- Language:
- ENGLISH
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