Plasmodium Whole-genome Data And Molecular Docking Method To Predict The Antimalarial Target

There has been a long history of struggle against malaria. However, malaria has stillbeen one of the most widely distributed and epidemic parasitic diseases in the world.The emergency of malarial parasite resistance has rendered many traditional drugssuch as quinine, chloroquine and mefloquine ineffective. Qinghaosu (QHS), extractedfrom sweet wormwood Artemisia annua L, has been named as the 'biggest hope foreradicating malaria' by WHO. As artemisinin has limitations in terms of short halflife, low oral bioavailability and high recurrence, general interest of chemist andpharmaceutist is concerntrated on design and synthesis to develop new QHS-typedrugs with improved efficacy and low toxicity. Development process of novelQHS-type antimalarial drugs has stagnated in recent years. The basic reason is unclearantimalarial target of this compound. Identification of targets is the basis for rationaldrug design. Molecular docking and bioinformatics related methods are useful toolsfor drug target discovery, which have many successful cases in the application.Therefore, our theoretical investigations have focused on searching for antimalarialtargets of QHS. On the one hand, this will help in designing new QHS analogues; onthe other hand, it will also promote innovative chemotherapy of malaria.Chapter 1 reviewed all the viewpoints in this field and discussed the antimalarialaction mode of QHS. The results indicated multi-target mechanism of action in manyways. But SERCA-type calcium-dependent ATPase (pfATP6) is generally known as amolecular target for QHS. There were no equally reliable evidences that indentifycysteine proteinase (Falcipain-2, FP2), translationally controlled tumor protein (TCTP)and His-rich protein 2 (HRP2) as antimalarial targets.In Chapter 2, we performed homology modeling, molecular docking and quantitative structure-activity relationship (QSAR) to elucidate whether FP2,TCTP and HRP2 were antimalarial targets for QHS derivatives. Before we started molecular docking study, homology modeling had been applied to predict three-dimenision structures of pfATP6 and TCTP. Quality evaluation has proved the rationality of models.Compared with five computational methods including semi-empirical, ab initio and density functional theory, the advanced density functional theory both in speed and accuracy was employed to calculate the geometry optimization of ligands prior to molecular docking in the thesis. Docking between QHS and its known target pfATP6 was carried out to test the reliability of docking method. This laid the foundation of methodology for the following docking and QSAR study. Binding mode of QHS, cysteine proteinase specific inhibitor E64 and deoxyartemisinin (DQHS) with FP2, respectively, was compared by means of Autodock package. The docking results showed that binding mode of QHS was similar to that of E64 and completely different from that of DQHS. The preliminary study indicated that FP2 was the possible antimalarial target for QHS. Further study was conducted to investigate the complex between four series of 34 typical QHS derivates with FP2. The docking results showed all the lignds bind to FP2 in similar conformation. Furthermore, the binding free energy exhibited a good linear correlation with antimalarial activity. On the basis of previous study, QSAR model was built between binding free energy and many descriptors. According toQSAR model, introducing substitution to increase the degree of branching may enhance antimalarial activity. Moreover, the more formation of hydrogen bonds with FP2, the better the antimalarial activity. The negative charge of peroxide bridge oxygen atoms will benefit for improving antimalarial activity. On the contrary, increasing molecular weight will decrease antimalarial activity. The obtained model is not only significant, but also has good ability of external prediction. Combined with the results of molecular docking and QSAR, we confirmed FP2 was other molecular target for QHS, in addition to pfATP6. Theoretical study on the mechanism of QHS reaction with FP2 suggested that secondary radical was the possible active species. No regularity of binding model was found when docking QHS and its 34 derivates with TCTP. The distance between endoperoxide atoms and key residue Cys14 was too far to form chemical bonds. From this point of view, we assumed that TCTP was not the target for QHS derivates. The verification of results needs to be further validated. All the QHS derivates bind to HRP2 in the same pocket. Because of limited knowledge of active sites of HRP2, the definite conclusion can not be reached. The research still needs to be improved. The theoretical inhibition constants between artemisinin and HRP2 obtained from docking studies are very close to the experimental data. This proved the credibility and reasonability of docking method.Since the complete genome sequence of Plasmodium falciparum has been published, Chapter 3 enlarged the artemisinin target research into the entire genomics. In order to obtain valuable antimalarial target of QHS, we exploited available bioinformatics resources from the following two aspects: homologous proteins and signaling pathways. Combination of homologous proteins searching, bioinformatics analysis with homology modeling, two highly homologous proteins for FP2, theoretical antimalarial targets of QHS, were found. There are FP2B and FP3. These proteins are not only similar in the primary sequence, but also highly conserved in the structure domain and the three-dimension structure. Comparing the binding patterns between three homologous cysteine proteases with QHS, we found that QHS can bind to the active pockets of different receptors. And the binding energies are at same level. At whole genome level, it is hard for QHS to selectively inhibit the three homologous cysteine proteases of malarial parasite in vivo. This suggested that we should consider the inhibition of whole cysteine proteases family during screening and designing QHS-type artimalarials. In particular, Signal pathway is the physical basis of organisms responding to stimulation from environment. Based on signal pathway database, exploratory experiments have been conducted to search the possible antimalarial targets and related signal pathways of QHS. 8 critical proteases in signal pathway were collected to dock with QHS. We assumed that pfPNP, pfPDF, and pfRpiA maybe the possible antimalarial targets. Signal pathways that might be interfered by QHS are purine metabolism, pyrimidine metabolism, Glyoxylate and dicarboxylate metabolism, methionine metabolism and pentose phosphate pathway.