Discovery and characterization of natural product luteolin as an effective inhibitor of human pyridoxal kinase.

Pyridoxal kinase (PDXK) is an essential enzyme in the synthesis of pyridoxal 5-phosphate (PLP), the active form of vitamin B6, which plays a pivotal role in maintaining the enzyme activity necessary for cell metabolism. Thus, PDXK has garnered attention as a potential target for metabolism regulation and tumor therapy. Despite this interest, existing PDXK inhibitors have faced limitations, including weak suppressive activity, unclear mechanisms of action, and associated toxic side effects. In this study, we present the discovery of a novel PDXK inhibitor, luteolin, through a high-throughput screening approach based on enzyme activity. Luteolin, a natural product, exhibits micromolar-level affinity for PDXK and effectively inhibits the enzyme's activity in vitro. Our crystal structures reveal that luteolin occupies the ATP binding pocket through hydrophobic interactions and a weak hydrogen bonding pattern, displaying reversible characteristics as confirmed by biochemical assays. Moreover, luteolin disrupts vitamin B6 metabolism by targeting PDXK, thereby inhibiting the proliferation of leukemia cells. This research introduces a novel screening method for identifying high-affinity and potent PDXK inhibitors and sheds light on clarification of the structural mechanism of PDXK-luteolin for subsequent structure optimization of inhibitors.

Natural product P57 induces hypothermia through targeting pyridoxal kinase.

Induction of hypothermia during hibernation/torpor enables certain mammals to survive under extreme environmental conditions. However, pharmacological induction of hypothermia in most mammals remains a huge challenge. Here we show that a natural product P57 promptly induces hypothermia and decreases energy expenditure in mice. Mechanistically, P57 inhibits the kinase activity of pyridoxal kinase (PDXK), a key metabolic enzyme of vitamin B6 catalyzing phosphorylation of pyridoxal (PL), resulting in the accumulation of PL in hypothalamus to cause hypothermia. The hypothermia induced by P57 is significantly blunted in the mice with knockout of PDXK in the preoptic area (POA) of hypothalamus. We further found that P57 and PL have consistent effects on gene expression regulation in hypothalamus, and they may activate medial preoptic area (MPA) neurons in POA to induce hypothermia. Taken together, our findings demonstrate that P57 has a potential application in therapeutic hypothermia through regulation of vitamin B6 metabolism and PDXK serves as a previously unknown target of P57 in thermoregulation. In addition, P57 may serve as a chemical probe for exploring the neuron circuitry related to hypothermia state in mice.

Ethiadin Induces Apoptosis and Suppresses Growth of MCF-7 Breast Cancer Cells by Regulating the Phosphorylation of Glycogen Synthase Kinase 3 Beta (GSK3beta).

Glycogen synthase kinase 3 beta (GSK3ß) has emerged as a therapeutic target for breast cancer. As inhibitors of GSK-3ß, 1,2,4-thiadiazole-3,5-dione (TDZD) family members have been reported as potential candidates for cancer treatment. In this study, the anticancer effects of ethiadin (ETD-174), one of the chemical synthesis compounds of TDZD, were investigated in MCF-7 human breast cancer cells. MCF-7 cells incubated with different doses of ETD-174 for different time periods. CCK-8 assays were carried out to test the effect of ETD-174 on the proliferation of MCF-7 cells. The occurrence of apoptosis was detected by Hoechst 33258 staining and flow cytometry. ETD-174 on cell migration and colony formation were examined by wound healing experiments and soft agar assays. Relative protein expressions were conducted with immunoblot assay. ETD-174 demonstrated a higher degree of cytotoxicity in MCF-7 cells. Topical morphological changes of apoptotic body formation after ETD-174 treatment were observed. Meanwhile, apoptosis was elicited by ETD-174. Also, ETD-174 could inhibit the migration and clonality of MCF-7 cells. After the treatment with ETD-174, the level of phosphorylation of GSK3ßSer9 in MCF-7 cells increased significantly, and the enzymatic activity of GSK3ß decreased. ETD-174 is likely to have an effective suppressor role in breast cancer, suggesting that pharmacological inhibition of GSK3ß as a novel treatment modality for breast cancer should warrant further investigation.

Ciliary localization of folliculin mediated via a kinesin-2-binding motif is required for its functions in mTOR regulation and tumor suppression.

Folliculin (FLCN) is a tumor suppressor protein involved in many cellular processes, including cell signaling, apoptosis, and autophagy. In ciliated cells, FLCN localizes to primary cilia and controls mTORC1 signaling in response to flow stress. Here, we show that the ciliary localization of FLCN requires its interaction with kinesin-2, the motor protein for anterograde intraflagellar transport. FLCN binds to kinesin-2 through a loop region in the middle of the protein. Single point mutations within this region of FLCN disrupt its kinesin-2 binding and ciliary entry. The mutants lose the ability to suppress the abnormal mTORC1/2 signaling activities and anchorage-independent growth of FLCN-deficient tumor cells. These observations suggest that ciliary localization of FLCN is essential for its function as a tumor suppressor.