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 能在浓度为1.6-50 μM时剂量依赖性地增加40 kDa蛋白探针的标记。GAPDH-IN-1 (50 μM, 4 h) 与GAPDH的Asp35共价结合，而不是半胱氨酸残基，这会促进Cys152随后与iodoacetamide-based (IA) 探针的反应。在HEK293细胞中，GAPDH-IN-1 (25-50 μM, 24 h) 对探针和GAPDH反应的增强低于在细胞裂解物中的增强。在HEK293细胞裂解液中，GAPDH-IN-1 (0-100 μM, 24 h) 能浓度依赖性地失活GAPDH，IC 50为39.31 μM。它也能抑制HEK293细胞的活力，IC 50为50.64 μM。添加过量NAD + 可以减少GAPDH-IN-1-GAPDH加合物，这可通过银染色凝胶中内源性GAPDH的条带移动证明。