Novel transketolase inhibitor oroxylin A suppresses the non‐oxidative pentose phosphate pathway and hepatocellular carcinoma tumour growth in mice and patient‐derived organoids

Abstract

Background Transketolase (TKT), a key rate‐limiting enzyme in the non‐oxidative branch of the pentose phosphate pathway (PPP), provides more than 85% of the ribose required for de novo nucleotide biosynthesis and promotes the development of hepatocellular carcinoma (HCC). Pharmacologic inhibition of TKT could impede HCC development and enhance treatment efficacy. However, no safe and effective TKT inhibitor has been approved. Methods An online two‐dimensional TKT protein immobilised biochromatographic system was established for high‐throughput screening of TKT ligands. Oroxylin A was found to specifically bind TKT. Drug affinity responsive target stability, cellular thermal shift assay, surface plasmon resonance, molecular docking, competitive displacement assay, and site mutation were performed to identify the binding of oroxylin A with TKT. Antitumour effects of oroxylin A were evaluated in vitro, in human xenograft mice, diethylnitrosamine (DEN)‐induced HCC mice, and patient‐derived organoids (PDOs). Metabolomic analysis was applied to detect the enzyme activity. Transcriptome profiling was conducted to illustrate the anti‐HCC mechanism of oroxylin A. TKT knocking‐down HCC cell lines and PDOs were established to evaluate the role of TKT in oroxylin A‐induced HCC suppression. Results By targeting TKT, oroxylin A stabilised the protein to proteases and temperature extremes, decreased its activity and expression, resulted in accumulation of non‐oxidative PPP substrates, and activated p53 signalling. In addition, oroxylin A suppressed cell proliferation, induced apoptosis and cell‐cycle arrest, and inhibited the growth of human xenograft tumours and DEN‐induced HCC in mice. Crucially, TKT depletion exerted identical effects to oroxylin A, and the promising inhibitor also exhibited excellent therapeutic efficacy against clinically relevant HCC PDOs. Conclusions These results uncover a unique role for oroxylin A in TKT inhibition, which directly targets TKT and suppresses the non‐oxidative PPP. Our findings will facilitate the development of small‐molecule inhibitors of TKT and novel therapeutics for HCC.

Skullcapflavone II, a novel NQO1 inhibitor, alleviates aristolochic acid I-induced liver and kidney injury in mice

“…The actual inhibitory effect of an agent to the target in cells can not be simulated completely by pure protein affinity experiment since the microenvironment is much more complicated in cells. In addition, oroxylin A, as a multi target inhibitor, has many binding targets, such as L-plastin (LPL) [ 33 ], transketolase (TKT) [ 34 ], angiotensin converting enzyme II (ACE2) [ 35 ], cyclooxygenase 2 (Cox-2), inducible nitric oxide synthase (iNOS), glycogen synthase kinase-3β (GSK-3β) [ 36 ], etc. These targets might competitively bind oroxylin A with NQO1, which may impair the binding of oroxylin A to NQO1.…”

Bioengineered Organoids Offer New Possibilities for Liver Cancer Studies: A Review of Key Milestones and Challenges

“…Oroyxlin A (transketolase inhibitor) is a novel anti-cancer drug studied using liver organoids. Its impact on inhibiting transketolase, a rate-limiting enzyme in de novo nucleotide synthesis, suppressed growth and cell death [128]. Lim and coworkers employed liver organoids to observe and deduce rational drug combinations for proteosome inhibitors, thus overcoming the limited furtherance in combinational therapy, along with discovering enhanced efficacy and clinical outcomes [129].…”

Research progress in the metabolic reprogramming of hepatocellular carcinoma (Review)

“…Similarly, transketolase (TKT), a key enzyme in the non-oxidative branch of the PPP, promotes HCC progression through significantly increasing the level of glucose flux and NADPH, which maintaining redox homeostasis of HCC cells ( 62 ). Oroxylin A, a small molecule inhibitor of TKT, directly targets TKT and leads to accumulation of glycolytic intermediates in the non-oxidative PPP, which inhibits HCC proliferation by inducing apoptosis and cell cycle arrest, providing a novel approach for the treatment of HCC ( 63 ). Therefore, in addition to the rate-limiting enzymes G6PD and TKT, miR-122 and PGLS could also be used as novel potential therapeutic targets to inhibit HCC progression by regulating HCC cell metabolism.…”