Microtubule affinity regulating kinase 4 (MARK4) is a Ser/Thr kinase belonging to AMPK-like family, has recently become an important drug target against cancer and neurodegenerative disorders. In this study, we have evaluated different natural dietary polyphenolics including rutin, quercetin, ferulic acid, hesperidin, gallic acid and vanillin as MARK4 inhibitors. All compounds are primarily binds to the active site cavity of MARK4. In silico observations were further complemented by the fluorescence-binding studies and isothermal titration calorimetry (ITC) measurements. We found that rutin and vanillin bind to MARK4 with a reasonably high affinity. ATPase and tau-phosphorylation assay further suggesting that rutin and vanillin inhibit the enzyme activity of MARK4 to a great extent. Cell proliferation, ROS quantification and Annexin-V staining studies are clearly providing sufficient evidences for the apoptotic potential of rutin and vanillin. In conclusion, rutin and vanillin may be considered as potential inhibitors for MARK4 and further exploited to design novel therapeutic molecules against MARK4 associated diseases.

Microtubule affinity regulating kinase (MARK4) is a potential drug target for different types of cancer as it controls the early step of cell division. In this study, we have screened a series of natural compounds and finally identified rosmarinic acid (RA) as a potential inhibitor of MARK4. Molecular docking and 500 ns all-atom simulation studies suggested that RA binds to the active site pocket of MARK4, forming enough number of non-covalent interactions with critical residues and MARK4-RA complex is stable throughout the simulation trajectory. RA shows an excellent binding affinity to the MARK4 with a binding constant (K) of 107 M−1. Furthermore, RA significantly inhibits MARK4 activity (IC50 = 6.204 µM). The evaluation of enthalpy change (∆H) and entropy change (∆S) suggested that the MARK4-RA complex formation is driven by hydrogen bonding and thus complexation process is seemingly specific. The consequence of MARK4 inhibition by RA was further evaluated by cell-based tau-phosphorylation studies, which suggested that RA inhibited the phosphorylation of tau. The treatment of cancer cells with RA significantly controls cell growth and subsequently induces apoptosis. Our study provides a rationale for the therapeutic evaluation of RA and RA-based inhibitors in MARK4 associated cancers and other diseases.

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Design and development of potential pyruvate dehydrogenase kinase 3 (PDK3) inhibitors have gained attention because of their possible therapeutic uses in lung cancer therapy. In the present study, the binding affinity of naturally occurring alkaloids, hordenine, vincamine, tryptamine, cinchonine, and colcemid was measured with PDK3. The molecular docking and fluorescence binding studies suggested that all these compounds show a considerable binding affinity for PDK3. Among them, the affinity of hordenine to the PDK3 was excellent (K = 106 M−1) which was further complemented by isothermal titration calorimetric measurements. Hordenine binds in the active site pocket of PDK3 and forms a significant number of non-covalent interactions with functionally important residues. All-atom molecular dynamics (MD) simulation study suggested that the PDK3-hordenine complex is stabilized throughout the trajectory of 100ns and leads to fewer conformational changes. The enzyme inhibition studies showed that hordenine inhibits the activity of PDK3 with an IC50 value of 5.4 µM. Furthermore, hordenine showed a cytotoxic effect on human lung cancer cells (A549 and H1299) with an admirable IC50 value. However, it did not inhibit the growth of HEK293 cells up to 200 µM, indicating its non-toxicity to non-cancerous cell lines. In summary, our findings provide the basis for the therapeutic implication of hordenine and its derivatives in lung cancer and PDK3-related diseases after required in vivo validation.

Microtubule affinity regulating kinase 4 (MARK4) becomes a unique anti-cancer drug target as its overexpression is responsible for different types of cancers. In quest of novel, effective MARK4 inhibitors, some acridone derivatives were synthesized, characterized and evaluated for inhibitory activity against human MARK4. Among all the synthesized compounds, three (7b, 7d and 7f) were found to have better binding affinity and enzyme inhibition activity in µM range as shown by fluorescence binding, ITC and kinase assays. Here we used functional assays of selected potential lead molecules with commercially available panel of 26 kinases of same family. A distinctive kinase selectivity profile was observed for each compound. The selective compounds were identified with submicromolar cellular activity against MARK4. Furthermore, in vitro antitumor evaluation against cancerous cells (MCF-7 and HepG2) revealed that compounds 7b, 7d and 7f inhibit cell proliferation and predominantly induce apoptosis in MCF-7 cells, with IC50 values of 5.2 ± 1.2 μM, 6.3 ± 1.2 μM, and 5.8 ± 1.4 μM respectively. In addition, these compounds significantly upsurge the oxidative stress in cancerous cells. Our observations support our approach for the synthesis of effective inhibitors against MARK4 that can be taken forward for the development of novel anticancer molecules targeting MARK4.