Comparative binding and activity analysis of known serine hydroxymethyltransferase inhibitors in biochemical assays.

Serine hydroxymethyltransferase (SHMT) is a crucial enzyme in folate metabolism, transferring a methylene group from serine to tetrahydrofolate (THF). It plays a key role in one-carbon metabolism, particularly in purine and thymidine biosynthesis, which has been linked to various cancers. Recently, a small number of small-molecule inhibitors distinct from traditional antifolates have been identified, such as SHIN1, SHIN2, AGF347 and W478. However, comprehensive cell-free biochemical data for binding and activity analysis of these inhibitors are lacking. By using thoroughly characterized recombinant SHMT1/2, this study investigates direct inhibitor binding by protein thermal shift assay and aims to expand the use of a known biochemical coupled enzyme approach, which has not yet been applied to determine the inhibitory activity of chemical test compounds, to evaluate the activity of these inhibitors against SHMT1 and SHMT2. Our work provides a comprehensive analysis of nine different SHMT1/2 inhibitors, thereby presenting a new methodological strategy that fills a gap in the existing literature on SHMT inhibition as well as opening up new avenues for the development and investigation of SHMT1/2 inhibitors.