| dc.description.abstract | Branched-chain amino acid transaminase 1 (BCAT1) is a protein known to be upregulated in severe cancers such as brain-, pancreatic-, and breast cancer, as well as in the neurodegenerative Alzheimer’s disease. Via structural-, activity-, and cell-based analyses, this master´s project aims to identify and characterize 22 potential BCAT1 inhibitors.
A coupled BCAT1/leucine dehydrogenase enzymatic assay was performed to characterize the inhibitory traits of the compounds to BCAT1, resulting in six compounds with IC50 values below 57 µM. Compound 12 was the most effective BCAT1 inhibitor with an IC50 value of 15 ± 2 µM, the compound was additionally BCAT1 specific since it did not inhibit the other isoform despite increasing concentration (IC50 > 125 µM). The MTT cell proliferation assay was performed to analyze the cytotoxicity of the 12 initial compounds to a pancreatic cancer cell line and human control cells. The analysis revealed interesting results for four of the compounds (3, 4, 7, and 10) as the cancer cells were more sensitive than the control cells. Compound 12 did not affect either of the cell lines in the tested concentration range (0-50 µM), while compounds 4 and 10 were nearly 20- and 10-fold more toxic to the cancer cells, respectively. Comparing the in vitro and in vivo results, compounds 3 and 4 were found to be both BCAT1 and BCAT2 inhibitors, as well as being toxic to the cancer model. Compound 3 was particularly interesting as the LD50 value for the cancer model was 3- and 90-fold higher than the IC50 values for BCAT1 and BCAT2, respectively. Compounds 7 and 10 were not effective BCAT1 inhibitors with IC50 values over 100 µM. The X-ray crystallography experiments posed a variety of difficulties, such as demanding crystallization conditions and poor BCAT1-crystal quality and resolution. Homology modeling was performed with three solved complexes from the BCAT2 isoform as templates, demonstrating that π-π interactions and hydrogen bonds increased the affinity. The modeled BCAT/ligand complexes showed that the compounds formed different contacts with the two proteins, despite near-identical active sites. However, these models are approximate, and high-resolution crystal structures are needed for a detailed analysis of the interactions between BCAT1 and the inhibitors.
In summary, compound 12 stood out as the most effective and selective BCAT1 inhibitor, while compounds 3 and 4 worked as both BCAT1 and BCAT2 inhibitors and were more toxic to the cancer model in the MTT analyses. The results presented in this thesis form a great basis for the development of BCAT1 inhibitors with affinities in the nM range. | en_US |
