CHEMBL5734971

Inhibition of Human SHMT: Inhibition of human SHMT by compounds in this disclosure is evaluated, for example, in their racemic forms in an in vitro assay. For example, Compound 61 (also referred to as KDG-30) was tested and demonstrated inhibition in vitro for both SHMT1 and SHMT2 with IC50 values at 3.2 nM and 0.78 nM respectively, shown in FIGS. 2A and 2B.Of note, certain compounds in this disclosure are selective for SHMT. These compounds show activity against SHMT2 in vitro but do not inhibit human MTHFD2 in enzymatic assays.Enzymatic Assays:For the SHMT1 and SHMT2 in vitro enzymatic assays, the rate of 5,10-methylene tetrahydrofolate formation catalyzed by SHMT1/2 was indirectly evaluated by coupling with excess MTHFD2, which converts NAD+ to NADH allowing for reaction monitoring by absorption at 340 nm. The reaction was started by addition of serine (1 mM final) to either human SHMT1 or human SHMT2 (2 mcg/mL), and human MTHFD2 (25 mcg/mL) in a buffer of 50 mM potassium phosphate (pH 7.4), 0.3 mM tetrahydrofolate, 7.5 mM dithiothreitol, 1.25 mM NAD+, and 4% DMSO. Inhibition of initial reaction velocity was determined by adding various inhibitors at different concentrations and monitored as described. IC50 may be calculated based on this assay.