Development of Novel Antimalarials

我们用人工合成的寡核苷酸构建了一个改变了二氢叶酸还原酶(DHFR)密码子使用的基因，在大肠杆菌中表达了恶性疟原虫二氢叶酸还原酶(DHFR)-胸苷合成酶(TS)复合体的一部分。合成基因在大肠杆菌细胞中的诱导表达产生了一种约占细菌总蛋白30%的产物。产物在细胞内以包涵体形式沉淀。用盐酸胍变性和复性后，其酶活性得以恢复。制备第108位含丝氨酸或苏氨酸的重组DHFRs。动力学研究表明，DHFRSer108对NADPH和二氢叶酸的亲和力低于DHFRThr108。利用在大肠杆菌中表达的重组恶性疟原虫DHFRs，结合抗叶酸抗性株DHFR基因的氨基酸变化，我们筛选了120种可能的抗疟疾药物。与…相比，屏幕的有效性通过随后对几种取代的吡咯并[2，3-d]嘧啶类化合物的抗疟疾活性的检测，证实了培养的寄生虫对生长的更多抑制。对耐药酶有效的化合物相应地抑制了培养的寄生虫的生长。受试化合物对环鸟胺和乙胺嘧啶抗性的pfDHFRs的半数抑制浓度分别为12-550 nM和8-1800 nM。与敏感酶相比，抗性酶对吡咯衍生物的敏感度分别降低0.8~7.5倍和3.6~29倍，对乙胺嘧啶和环鸟苷的敏感度分别降低308倍和400倍。同样，培养的恶性疟原虫对环鸟嘌呤抗性株(FCR3)和乙胺嘧啶抗性株(K1)的半数抑制浓度分别为0.15-49 nM和5.4-1500 nM。与敏感品系(3D7)相比，在受试化合物中，抗性寄生虫的敏感性分别下降了0.9%~2倍和15%~50%，对环鸟苷和乙胺嘧啶的敏感性分别下降了690%和18640倍。此外，代号为T-49172的吡咯衍生物对伯氏疟原虫在小鼠体内的生长有抑制作用，其有效剂量浓度为50%，与环鸟苷相当。较少

We have expressed the dihydrofolate reductase (DHFR) part of the DHFR-thymidylate synthetase (TS) complex of P.falciparum in E.coli, by constructing a gene with synthetic oligonucleotides that changed the gene's codon usages. The induced expression in an E.coli cell of the synthetic gene yielded a product that constituted about 30% of the total bacterial protein. The product was precipitated in an inclusion body in a cell. Its enzymatic activity was restored after denaturation and renaturation procedures with guanidine-HCl. Recombinant DHFRs with Ser or Thr at position 108 were prepared. Kinetic characterization showed that the DHFRSer108 has less of an affinity for NADPH and dihydrofolate than the DHFRThr108.By using the recombinant Plasmodium falciparum DHFRs expressed in Escherichia coli, with amino acid alterations found in DHFR genes of the antifolate resistant strains, we screened 120 kinds of antifolate for possible antimalarials. The validity of the screen, as compared with the … More growth inhibition of the cultured parasite was verified by the subsequent examination of several substituted pyrrolo [2,3-d] pyrimidines for their antimalarial activity. The chemical compounds that were effective against the drug resistant enzymes correspondingly inhibited the growth of the cultured parasites. The test compounds gave 50% inhibitory concentration of the ranged 12-550 nM and 8-1800 nM respectively for the pfDHFRs with cycloguanil and pyrimethamine resistance. As compared to the sensitive enzyme, the fold decrease in sensitivity of the resistant enzymes to the pyrrolo-derivatives were 0.8-7.5 and 3.6-29 ; those for pyrimethamine and cycloguanil were 308 and 400. Similary, the growth inhibition of the cultured P.falciparum gave 50% inhibition concentration of 0.15-49 nM and 5.4-1500 nM respectively for the cycloguanil resistant (FCR3) , and pyrimethamine resistant (K1) strains. As contrasted with the sensitive strain (3D7) , the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds ; those for cycloguanil and pyrimethamine were 690 and 18640. Moreover, the pyrroloderivative code named T-49172 inhibited the growth of P.berghei in mice with 50% effective dose concentration that was comparable to that inhibited by cycloguanil. Less