Study On The Antimalarial Mechanism Of Artemisinin And Artemisinin-Based Combination Therapy

Malaria is a deadly infectious disease caused by protozoan parasites of the genus Plasmodium,which is widely prevalent in more than 90 countries around the world,especially in tropical and subtropical regions.Nearly half of the world's population is at risk of contracting malaria.The World Health Organization(WHO)ranks malaria together with AIDS and tuberculosis as the world's three major public health problems.The latest world malaria report shows that in 2018,about 228 million people worldwide were infected with malaria and caused about 405,000 deaths,of which children under 5 accounted for 67%of the deaths.The prevention and control of malaria mainly focuses on three aspects:vector control,drug control and vaccination.However,with the widespread resistance of malaria parasites to pyrethroids,Dichloro-Diphenyl-Trichloroethane(DDT)and other insecticides and the lack of clinically approved vaccines,drug therapy has become the most important means for the treatment of malaria.The early antimalarial drugs were mainly quinolines based on quinine,such as chloroquine.However,in the 1950s,due to the abuse of quinoline and other antimalarial drugs,the malaria parasite developed a wide range of drug resistance,which made the world face the threat of malaria once again.Therefore,the emergence of more efficient and safe antimalarial drugs has become particularly urgent.Artemisinin(ART)was first discovered and isolated from Artemisia annua by researcher Tu Youyou of the Institute of traditional Chinese Medicine in our institute in 1971.Artemisinin was used in clinical treatment of malaria in the 1980s and has been recommended by the World Health Organization as a first-line drug for the treatment of malaria since 2001.The discovery of artemisinin and the introduction of artemisinin-based combination therapy(ACT)have effectively controlled the global epidemic of malaria,alleviated the severe situation of widespread resistance to chloroquine and other drugs,and saved millions of lives.Tu Youyou also won the 2015 Nobel Prize in Physiology or Medicine for her discovery of artemisinin.Artemisinin is a sesquiterpene lactone compound containing 1,2,4-trioxane and internal peroxide bridge.It has a very good killing effect on many kinds of malaria parasites,especially on Plasmodium falciparum,which causes the most death.Although ART has been used in clinic for nearly 40 years,the exact target and antimalarial mechanism are still not very clear,which greatly limits the clinical application of artemisinin and the development of new indications.Therefore,it is of great significance to clarify the antimalarial mechanism of artemisinin as soon as possible.ART is currently the only antimalarial drug that has not yet developed extensive drug resistance,and there are no other antimalarial drugs to replace it.However,drug resistance to ART and ACT has been reported in the Greater Mekong Subregion at the beginning of this century,which is characterized by reduced clearance rate of Plasmodium,prolonged clearance time and recurrence after treatment.Once there is widespread resistance to artemisinin,human beings will face the situation of no cure.The study on the antimalarial mechanism of artemisinin is an important basis for unraveling its drug resistance.Therefore,to study and clarify the antimalarial mechanism of ART is an urgent problem to be solved,and it is also the biggest challenge currently facing.At the same time,clarifying the antimalarial mechanism of ART plays an important role in revealing the mechanism of ACT,which is conducive to optimize the clinical application of artemisinin and ACT,expanding new indications and developing new ACT paired drugs.On the basis of the previous research in the laboratory,this paper mainly focuses on the study on the antimalarial mechanism of artemisinin and ACT.It was mainly studied by activity-based protein profiling technology(ABPP),cellular thermal shift assay(CETSA),exogenous expression and purification of protein and cell co-localization imaging.1.A novel photo-crosslinking probe AP2 based on artemisinin was synthesized to explore whether artemisinin has a non-covalent binding target protein in Plasmodium.Its activity was detected by detecting its IC50 against Plasmodium falciparum strain 3D7 and chloroquine-resistant Plasmodium strain 7G8.The results showed that ART maintained its high efficiency and specific antimalarial activity after modification with diacridine group with photoaffinity.The results of in situ fluorescence labeling showed that after 365nm UV irradiation,the photoaffinity group was activated and cross-linked with the adjacent proteins,which could bind to more proteins than the non-UV irradiation group.It is suggested that artemisinin drugs not only alkylate the target protein by carbon free radical activated by heme,then combine with the target protein in the way of covalent binding to produce antimalarial effect,there may also be target proteins that bind in a non-covalent binding manner.2.CETSA was used to study the effect of artesunate(ATS),a derivative of artemisinin,on the thermal stability of total protein of malaria parasite in order to identify the target protein.Firstly,the melting curve experiment(Melt curve),was used to monitor the change of soluble protein content of Plasmodium under the heating condition of 37??73?,and the melting temperature(Tm,50%protein denaturation temperature)was determined to be 52?.Then isothermal dose response(ITDR),was used to monitor the content of soluble proteins at different drug concentrations at non-denaturing temperature 37? and denaturing temperature 52 ?.145 Plasmodium proteins with significantly increased or decreased thermal stability after binding to ATS were identified,indicating that ATS has a variety of target proteins in the antimalarial process and produces antimalarial effect by interfering with a variety of physiological processes.The bioinformatics analysis of the target protein shows that artemisinin drugs may produce efficient and specific antimalarial effect by affecting the hemoglobin uptake and energy metabolism of Plasmodium through the digestive vesicle(DV).3.Using the active probe AP1-AQP,based on artesunate-amodiaquine(ATS-AQ)in ACT,which is commonly used in clinic,and purifying Plasmodium protein by exogenous expression,in situ fluorescence labeling and cell co-localization imaging were used to study the interaction between ACT drugs.The results showed that among the ATS-AQ drug pairs,ATS played a leading role in the antimalarial process,and had stronger binding efficiency and binding ability to Plasmodium protein.The compatible drug AQ can not only enhance the antimalarial effect by prolonging the half-life of drugs in blood,but also enhance the binding ability of ATS to proteins in Plasmodium,so as to improve the antimalarial ability of ATS and play a synergistic effect.The results also show that heme can not only activate artemisinin,but also enhance the binding ability of AQ to Plasmodium protein,indicating that heme may have an important synergistic effect in ACT therapy.The above results are of great significance for the further study of the antimalarial mechanism of ACT,the optimization of clinical drug use and the development of new ACT paired drugs.To sum up,artemisinin drugs have not only covalent binding target proteins,but also some non-covalent binding target proteins in Plasmodium parasites.and it mainly affects the hemoglobin uptake,organic metabolism and energy metabolism of the parasite in the digestive vesicles of the parasite to produce antimalarial effect.In ACT therapy,artemisinin plays an important role in antimalaria.compatible drugs can not only enhance the antimalarial effect by prolonging the existence time of antimalarial drugs in blood,but also enhance the binding ability of artemisinin to protein in malaria parasite,so as to improve antimalarial ability and play a synergistic effect.