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Background and Purpose: Necroptosis is a form of programmed, caspaseindependent, cell death, mediated by receptor-interacting protein kinases, RIPK1 and RIPK3, and the mixed lineage kinase domain-like (MLKL). Necroptosis contributes to the pathophysiology of various inflammatory, infectious, and degenerative diseases. Thus, identification of low MW inhibitors for necroptosis has broad therapeutic relevance. Here, we identified that the pan-Raf inhibitor TAK-632 was also an inhibitor of necroptosis. We have further generated a more selective, highly potent analogue of TAK-632 by targeting RIPK1 and RIPK3. Experimental Approach: Cell viability was measured by MTT, propidium staining, or CellTiter-Glo luminescent assays. Effects of TAK-632 on necroptosis signalling pathways were investigated by western blotting, immunoprecipitation, and in vitro kinase assays. Downstream targets of TAK-632 were identified by a drug affinity responsive target stability assay and a pull-down assay with biotinylated TAK-632. A mouse model of TNF-α-induced systemic inflammatory response syndrome (SIRS) was further used to explore the role of TAK-632 in protecting against necroptosisassociated inflammation in vivo.Key Results: TAK-632 protected against necroptosis in human and mouse cells but did not protect cells from apoptosis. TAK-632 directly bound with RIPK1 and RIPK3 to inhibit kinase activities of both enzymes. In vivo, TAK-632 alleviated TNF-induced SIRS. Furthermore, we performed a structure-activity relationship analysis of TAK-632 analogues and generated SZM594, a highly potent inhibitor of RIPK1/3. Abbreviations: DARTS, drug affinity responsive target stability assay; Nec-1, necrostatin-1; SAR, structure-activity relationship; SIRS, systemic inflammatory response syndrome Xiaofei Chen, Chunlin Zhuang and Yibin Ren are contributed equally to this work.

Cell membrane chromatography (CMC) has been widely used for characterizing the interaction between drugs and membrane receptors to screen target components from herbal medicines. However, the column life, stability, and the efficiency cannot meet the needs of high-throughput screening purpose. In this study, a P-glycoprotein immobilized cell membrane stationary phase (P-gp/CMSP) was prepared with a simple and mild twostep aldehyde modification, realizing the covalent bonding between cell membrane and stationary phase. The column life and stability were significantly enhanced compared with the unmodified columns. The P-gp/CMC column was equipped into a comprehensive 2D P-gp/CMC/Capcell-C18/TOFMS system, which actualizes the automated and high-throughput analytical process and rapid identification of complex chemical samples with no data loss. Five compounds with significant retention were screened out and unambiguously identified by the comprehensive 2D analytical system. Baicalin was confirmed as a P-gp inhibitor with ATP depletion inhibition ratio of 83.4%. Moreover, the reversal index of baicalin on DOX significantly increased to 11.13 when its concentration reached 25 μM, revealing that baicalin could effectively reverse the MDR cell model induced by DOX. The integrated system is a practical drug discovery platform and could be applied to other transmembrane protein models.

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.