Screening and characterization of BCAT inhibitors ¨C potential novel anti-cancer agents

Introduction and background

The enzymes BCAT (cytosolic isoform BCAT1 and mitochondrial isoform BCAT2) are catalyzing the conversion of branched chain amino acids to their corresponding keto acids for further degradation in human cells.

Maple Syrup Urine Disease (MSUD) is an inborn metabolic disease caused by the inability to degrade the branched chain amino acids leucine, isoleucine and valine, resulting in accumulation of toxic keto acid intermediates. By blocking the mitochondrial BCAT2 enzyme we hope to reduce the level of toxic keto acids in MSUD patients, reducing the risk of developing severe neurological damage during episodes of increased levels of protein degradation.

Interestingly, the second, cytosolic isoform BCAT1, has recently been implied to support growth of ERa-negative breast cancer (1), as well as promote resistance to bevacizumab in glioblastoma (aggressive brain cancer) (2). Further, BCAT1 inhibition is associated with increased sensitivity to CDDP treatment in prostate cancer (3) , and BCAT1 is also upregulated in gastric cancer (4)

Given these roles of BCAT in various pathological disorders, it is of interest to identify and develop inhibitors for both BCAT isoforms. We have already a small in-house panel of inhibitors and aim to expand this set of compounds and develop even more potent molecules.

Aim of project

The aim of this project is to characterize present in-house BCAT1/2 inhibitors and search for new compounds with even higher potency than our current set of compounds. This will be achieved using both enzymatic assays, cell based tests with exposure to cancer cells as well as structural studies and modeling of enzyme-inhibitor complexes.

Project plan and methods

The project will consist of the following tasks and methods

1. Express recombinant human BCAT isoforms using E. coli as host and purify the enzymes using chromatographic principles.
2. Determine inhibitory effects (IC₅₀-values) and protein affinities (K_D-values) for potential BCAT inhibitors. IC₅₀-values will be determined using an enzyme assay, and K_D-values will be determined using various biophysical methods (MST, SPR, DSF or similar).
3. Test inhibitors in cell-based assays to determine toxicity and cancer cell specificity.
4. Test in vivo targeting of BCAT using a cell assay based on tracing of radioisotopes.   

Organisation / collaboration

The project is a collaboration between group leaders Bj?rn Dalhus and Lars Eide at the Department of Medical Biochemistry, University of Oslo and Oslo University Hospital (OUS). Our laboratory is located at Rikshospitalet, where we have access to relevant technologies to complete the project. Dalhus is an experienced structural biologist, with interest in drug design, and Eide is an expert in metabolism and mitochondrial biology. Hans-Petter Hersleth is internal supervisor at IBV.