Glucose-6-phosphate dehydrogenase (G6PDH) is the rate-limiting
enzyme in the pentose phosphate pathway (PPP) and plays a crucial role in the maintenance of redox homeostasis by producing
nicotinamide adenine dinucleotide phosphate (
NADPH), the major intracellular
reductant. G6PDH has been shown to be a
biomarker and potential therapeutic target for
renal cell carcinoma (RCC). Here, we report a previously unknown biochemical mechanism through which
caffeine, a well-known natural small molecule, regulates G6PDH activity to disrupt cellular redox homeostasis and suppress RCC development and progression. We found that
caffeine can inhibit G6PDH enzymatic activity. Mechanistically,
caffeine directly binds to G6PDH with high affinity (K D = 0.1923 μM) and competes with the
coenzyme NADP+ for G6PDH binding, as demonstrated by the decreased binding affinities of G6PDH for its
coenzyme and substrate. Molecular docking studies revealed that
caffeine binds to G6PDH at the structural NADP+ binding site, and chemical cross-linking analysis demonstrated that
caffeine inhibits the formation of dimeric G6PDH. G6PDH inhibition abrogated the inhibitory effects of
caffeine on RCC cell growth. Moreover, inhibition of G6PDH activity by
caffeine led to a reduction in the intracellular levels of
NADPH and
reactive oxygen species (ROS), and altered the expression of redox-related
proteins in RCC cells. Accordingly,
caffeine could inhibit
tumor growth through inhibition of G6PDH activity in vivo. Taken together, these results demonstrated that
caffeine can target G6PDH to disrupt redox homeostasis and inhibit RCC
tumor growth, and has potential as a therapeutic agent for the treatment of RCC.