br STAR Methods br Author Contributions br
Acknowledgments This work was supported by the Intramural Research Program of the National Heart Lung and Blood Institute in the National Institutes of Health. E.D.A. was supported by NIH grant HL73167. The LCR-HCR rat model system was funded by Office of Research Infrastructure Programs grant P40OD021331 (to L.G.K. and S.L.B.) from the NIH. This research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), which is funded by the Ministry of Health & Welfare of the Republic of Korea (grant number HI14C1176). We acknowledge the expert care of the rat colony provided by Molly Kalahar and Lori Heckenkamp. L.G.K. ([email protected]) or S.L.B. ([email protected]) may be contacted for information on the LCR and HCR rats; these rat models are maintained as an international resource with support from the Department of Anesthesiology at the University of Michigan in Ann Arbor, Michigan. We thank Benoit Viollet for AMPKα2-KO mice, Dalton Saunders for his help with drug studies, and Zu-Xi Yu for assistance with dpn mg microscopy. We also thank Adam Weidenhammer and Yiying Tsai for their technical assistance.
Introduction DNA-dependent protein kinase (DNA-PK) is a multicomponent serine/threonine protein kinase and considered a member of the phosphatidylinositol (PI) 3-kinase related kinase (PIKK family). This enzyme plays a critical role in the repair of mammalian DNA double strand breaks (DSBs) through the non-homologous end joining pathway of DNA repair , . It was proved that human cell lines defective in DNA-PK function are hypersensitive to agents that elicit DNA DSBs , . Selective DNA-PK inhibitors of DNA DSBs properties have potential application as radio and chemo potentiators in the treatment of cancer , , , , . Importantly, tumor cell lines defective in DSB repair as a consequence of compromised DNA-PK activity are highly sensitive to topoisomerase II inhibitors and ionizing radiation (IR) whereas over-expression of DNA-PKcs confers resistance to these agents by enhancing DSB repair . Taken together, these data provide compelling evidence that DNA-PK is an attractive therapeutic target for the modulation of DNA DSB repair in cancer therapy, and preliminary results with inhibitors have proved encouraging . In the drug discovery process, certain ligand-based approaches were introduced as advanced tools for design of new active hits , . Of these methods, 2D QSAR and pharmacophore modeling processes which in turn were able to predict the activity of proposed structures for saving the time and money . The validation procedures of the 2D and 3D models are essential for the whole protocol application and were indicated by different parameters like linearity of the correlation, root-mean-square error (RMSE), and the correlation factor, R2). Here, we report the design, synthesis, characterization, and modeling studies for novel small molecule DNA-PK inhibitors based upon structure-activity relationship analysis of different reference ligands , , , , . The protocol was designed through the insertion of different linker and terminal moieties to the reference L-27 structure that have potent DNA-PK inhibitory activity . The molecular docking of compound L-27 in the DNA-PK homology model revealed the importance of this side chain which was responsible for its activity. The interaction analysis showed stable bidirectional hydrophobic and hydrogen bonding interactions to Lysine, Arginine, and Serine residues. Different steps were followed subjecting the compound library resulted for pharmacophore analysis and activity prediction by 2D QSAR model, synthesized, and finally tested for both DNA-PK and cytotoxic screening, Fig. 1.