GAPDH-IN-1 (Compound F8) is a GAPDH inhibitor, with an IC50 value of 39.31 μM for GAPDH enzyme activity. It forms a covalent adduct with the aspartic acid in the active site, displacing the enzyme's cofactor NAD+, and simultaneously enhances the reactivity of cysteine-reactive probes with the catalytic cysteine.

GAPDH-IN-1 exhibits dose-dependent effects, enhancing 40 kDa protein probe labeling within the range of 1.6-50 μM. At a concentration of 50 μM for 4 hours, GAPDH-IN-1 forms a covalent bond with Asp35 in GAPDH, rather than with cysteine residues, which subsequently stimulates a reaction between Cys152 and an iodoacetamide-based (IA) probe. When applied at concentrations of 25-50 μM for 24 hours, the enhancement of probe and GAPDH reaction by GAPDH-IN-1 in HEK293 cells is less significant compared to enhancements observed in cell lysates. Furthermore, GAPDH-IN-1 inactivates GAPDH in HEK293 cell lysates in a concentration-dependent manner, exhibiting an IC50 of 39.31 μM over 24 hours in the range of 0-100 μM. It also inhibits the viability of HEK293 cells, with an IC50 of 50.64 μM in the same concentration range. The GAPDH-IN-1-GAPDH adduct can be reduced by the addition of excess NAD+, as evidenced by the shift in endogenous GAPDH bands observed in silver-stained gels.