Development of Novel Antimalarials

我们已经在大肠杆菌中表达了恶性疟原虫的二氢叶酸还原酶-胸苷酸合成酶（TS）复合物的二氢叶酸还原酶（DHFR）部分，通过用改变基因密码子使用的合成寡核苷酸构建基因。合成基因在大肠杆菌细胞中的诱导表达产生了占细菌总蛋白约30%的产物。产物在细胞中以包涵体形式沉淀。经盐酸胍变性、复性后，酶活力恢复。制备在位置108处具有Ser或Thr的重组DHF。动力学分析表明，DHFRSer 108对NADPH和二氢叶酸的亲和力低于DHFRThr 108。利用在大肠杆菌中表达的重组恶性疟原虫DHFRs，结合抗叶酸剂抗性株DHFR基因的氨基酸改变，筛选了120种抗叶酸剂。屏幕的有效性，与 ...更多信息 培养的寄生虫的生长抑制通过随后检测几种取代的吡咯并[2，3-D]嘧啶的抗疟疾活性来验证。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。测试化合物对于具有环胍和乙胺嘧啶抗性的pfDHFR分别给出范围为12-550 nM和8-1800 nM的50%抑制浓度。与敏感酶相比，耐药酶对吡咯并衍生物的敏感性降低倍数分别为0.8-7.5和3.6-29 ;对乙胺嘧啶和环胍的敏感性降低倍数分别为308和400。同样，对培养的恶性疟原虫的生长抑制，对于环胍抗性（FCR 3）和乙胺嘧啶抗性（K1）菌株，分别给出0.15-49 nM和5.4-1500 nM的50%抑制浓度。与敏感菌株（3D 7）相比，在测试化合物的情况下，抗性寄生虫的敏感性降低倍数为0.9-2和15-50;对于环胍和乙胺嘧啶的那些为690和18640。此外，代号为T-49172的吡咯衍生物以50%有效剂量浓度抑制伯氏疟原虫在小鼠体内的生长，与环胍的抑制作用相当。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