Virtual Screening Of PNKP Enzyme Inhibitors And Research On Enzyme Activity Detection Methods

DNA is the carrier of genetic information.DNA is often threatened by various endogenous and exogenous damages.These damages can lead to DNA strand breaks and form the formation of 3’-phosphate/5’-hydroxyl damage gaps at the ends of DNA,which is one of the main inducing factors for normal cells to transform into tumor cells.The human body has formed an efficient DNA repair system in the process of evolution,which can keep the amount of DNA damage in normal cells at a low level,this response is an early barrier to prevent cell canceration.Thus,DNA damage repair plays an vital function in the treatment of tumors.On the other hand,chemotherapy and radiotherapy have become the mainstream methods for the treatment of cancer,and most of these therapeutics through damaging DNA.However,cancer cells can evasion from apoptosis through the initiation of the DNA repair pathways,finally leading to therapeutic resistance.Therefore,pharmacological inhibition of DNA-repair processes would be an attractive means of enhancing the cytotoxic effects in the treatment of cancers.Among those DNA repair enzymes,PNKP(polynucleotide kinase/phosphatase)is a critical human DNA repair enzyme that repairs DNA strand breaks by catalyzing the restoration of 5’-phosphate and 3’-hydroxyl termini that are required for subsequent processing by DNA ligases and polymerases.PNKP is the only protein that repairs the3′-hydroxyl group and 5′-phosphate group.In addition,PNKP is the only DNA repair enzyme with 5’-kinase activity,this unique feature makes the kinase domain of PNKP a potential drug target for the development of specific PNKP kinase inhibitors.However,there are neither crystal structures of human PNKP nor the kinase inhibitors reported so far.Therefore,in this study,computer-aided drug design and biochemical experiments were used to develop new PNKP kinase inhibitors.The highlights of this study are as follows:(1)First,the homology modeling method was used to construct the structure of human PNKP protein,molecular dynamics simulation was conducted to optimize the homology PNKP model,and then generate multiple PNKP conformations to construct a multi-conformation virtual screening model.Second,a sequential virtual screening method was constructed,which integrated semi-flexible docking,flexible docking,and MM-GBSA,to screen out new PNKP inhibitors.Subsequently,the molecule with the highest score was subjected to molecular dynamics simulation and binding free energy calculation to reveal the binding mechanism between the PNKP kinase domain and the inhibitor,and find out the characteristic residues which have key contributions to the specific binding of the ligand.Finally,the virtual screened compounds were tested for activity at the cell level,and the results showed that several compounds have strong tumor inhibitory effects.Compared with the reported PNKP inhibitor A12B4C3,some compounds have stronger therapeutic potential for pancreatic cancer.(2)In order to sensitively and accurately detect the enzymatically activity of these screened compounds,we established a DNA molecular beacon fluorescence amplification sensing strategy.The results show that this detection method can amplify the action process of PNKP,thereby realizing real-time monitoring,which is conducive to the quantitative detection of PNKP inhibitor activity.This method has a simple operation and sensitive detection,and shows great potential in the large-scale screening of PNKP inhibitors.