Study Of The Resistant Mechanism Of Naphthoquine Phosphate

Malaria is a global public health priority which remains one of the leading causes of morbidity and mortality in tropical and subtropical regions. In four human malaria parasites, Plasmodium falciparum (P.falciparum) causes the most severe symptoms and it is still endemic in Hainan and Yunnan provinces of China. Since the resistance to chloroquine of Plasmodium falciparum appeared, drug-resistance has been spreading in the parasites epidemics and the level of resistance has continuously increased. In some epidemics, drug-resistance, even multi-drug resistance and cross resistance, has seriously hampered the control of malaria and the choice of antimalarials. The US military selected mefloquine as the trump card to antimalarias'market from 250 000 compounds in clinical studies since 1963 to 1974, but only six months later the mefloquine resistance strain of P. falciparum had emerged in Thailand. Therefore, researchers carried out extensive studies about the antimalarials resistance mechanisms. But so far, only anti-folate antimalaria resistance way of P. falciparum has been definite which was considered to dihydrofolate reductase (Dihyrofolateredctase, DHFR) gene mutations and other antimalarials such as Chloroquine phosphate and other quinoline antimalarial resistance mechanisms are not yet clear.Naphthoquine phosphate (NQ) synthesized in our lab is a new anti-malarial drug, which is a 4-amino-quinoline antimalarial as chloroquine phosphate (CQ). Experimental results show that naphthoquine phosphate has a high efficacy, but there is a probability of resistance to NQ with its long half-life (255h). So there is a need to understand the resistance mechanism of NQ to avoid the resistance emergence in clinical use.Up to date, there are no reportes about NQ resistant mechanism, but the resistance mechanism of CQ has conducted extensive researches. It's generally agreed that P. falciparum multidrug resistance gene 1(Plasmodium falciparum multidrug resistance genel, pfmdrl), cg2 and P. falciparum chloroquine resistance transporter protein (Plasmodium falciparum chloroquine resistance transporter, pfcrt) gene are related with the chloroquine resistance and the research focus among of them is the crt gene. It is considered that mutations of crt is the key fact in the resistanc emergence of CQ. Therefore, our study of NQ resistance mechanism will start from the crt gene by gene targeting technology.The way to study gene function most direct and effective is to carry out allele replacement, Currently, the allele replacement experiments of malaria parasites in vivo mostly executed by gene transfection. PPyrFlu (Figure 2A) is an useful vector in malaria parasites gene transfection. In our study, CRT targeting recombinant plasmid was constructed by the crt gene and pPyrFlu vector and by gene transfection we studied the relationship between crt gene and naphthoquine phosphate resistance mechanism with the single crossover of recombinant plasmid (Figure 2B).Methods:(1) Selection the naphthoquine-resistant rodent malaria parasite (Plasmodium berghei Keyberg 173 naphthoquine-resistant strain, NQR) under continuous NQ drug pressure in vivo.50% and 90% effective doses (ED50 and ED90, respectively) were measured by'4-day inhibition assays'in which the parasites were exposed to consecutive administrations for four days. The stability was tested by 10 serial passages in drug-free medium and cryo-preserved over a period of 12 months. And the cross-resistance of the NQR to other antimalarials, such as ARCO(?), artemisinin (QHS), chloroquine (CQ), lumefantrine (LM) and pyrimethamine (PM)-were also determined following the 4-day treatment regimen. (2) Referringe to the crt gene of Plasmodium berghei ANKA strain, PCR primers were designed and crt genes of K173 strain, NQR strain, CQR strain were got by RT-PCR for the first time. Then analyze the sequences compared with other Plasmodiums'CRT. And identify the differences between drug-sensitive strain and drug-resistant strain. (3) Construction of recombinant strains and identification the antagonistic phenotype. First, building the crt targetting plasmid. Cloning the crt of K173 strain or the resistant strain into pPyrFlu which was a plasmid for Plasmodium berghei transfection. Then, double digestion and PCR were used to identify the integrated plasmid. Second, optimize the transfection conditions. The recombinant plasmid was linearized and parasites were cultured in vitro. Then, a set transfer systems (100-400ul) were chosed to transfected. Pyrimethamine injection intraperitoneal and fluorescence microscope were used to screen the right transformants. Finally, the susceptibilities of recombinant K173-NQR/CQR strain, NQR-K173 strain and CQR-K173 strain to naphthoquine and chloroquine were determined.Results:(1) Through exposing a susceptible strain to sub-curative doses of the drug in 100 sequential passages over a period of 700 days, we got NQR strain showed ED50 and ED90 to NQ were 41.25mg/kg and 152.23mg/kg, yielding I50 and I90 were 105.8 and 200.3, respectivelye. And the resistance phenotype was stable after 10 serial passages in drug-free medium and cryo-preserved over a period of 12 months with I90 over 200-fold. Through the cross-resistance tests, we found the activity of chloroquine against NQR was decreased by 14.5-fold, a high-level cross-resistance. (2) Through RT-PCR, we got the crt gene of P.b.K173,1278bp long, for the first time which was a transporter protein with 10 transmembrane structures and encoded 426 animo acides. Compared with K173 strain, there were 41 mutations and three-base deletion of crt in NQR. The crt sequence of CQR was same as the sequence of NQR. (3) The crt gene integrated into pPyrFlu was proven by the double digestion and PCR identifications. Optimum conditions for the Plasmodium berghei culture in vitro contained 1640 with 20% fetal bovine serum,1% hematocrit,37℃and 18-22h in candle jar. Gradient centrifugation and magnetic cells separation (MACs) were used to enrich the mature schizonts. There were no great differences between the two ways but the operation time, gradient density centrifugation need 30min and MACs required more than 2h.100-400ul transfer systems were determained under the conditions of 1.1kv,25uF and infinite resistance and we could screen the right recombinations just in 300ul transfer system. And we identified the parasites with green fluorescence by fluorescence microscopy after pyrimethamine injection intraperitoneal for twe times. We also got the 1.75kb fragment expected by PCR which indicated the combinant plasmid had been integrated into the genome of P.berghei and expressed completely. (4) The ED90 of recombinant K173-NQR/CQR strain, NQR-K173 strain and CQR-K173 strain to naphthoquine were 36.06±2.79mg/kg,2.76±0.79mg/kg and 4.66±1.32mg/kg, respectively, yielding I90 of 60.1,4.6 and 2.2 and the ED90 to chloroquine were 93.70±8.83mg/kg, 6.92±1.07mg/kg and 19.08±3.39mg/kg, respectively, yielding I90 of 44.2,11.5 and 9.0.Conclutions:(1) With the protocol of ST, we could select the stable and high-level resistant strain of naphthoquine. However, the high-level cross-resistance of CQ against NQR suggested the clinical application of NQ in epidemics of chloroquine-resistant P.falciparum should be cautious, and the drug susceptibility should be monitored closely. (2) We obtained the crt gene of P.berghei K173 for the first time and the mutations of crt in drug-resistance strain pointed the relationship between crt and naphthoquine-resistance and the resistant of NQ might share similar mechanism with CQR partly. (3) We had established the gene targeting technology of Plasmodium berghei. (4)The distinct changes of resistant phenotypes of recombinant K173-NQR/CQR strain, NQR-K173 strain and CQR-K173 strain represented the key point of crt in the emergence of naphthoquine resistance. (5) Maybe other factors also impact the malaria resistance to naphthoquine besides crt which need to be studied further more.