Sardarian Alireza, Douglas Kenneth T, Read Martin, Sims Paul F, Hyde John E, Chitnumsub Penchit, Sirawaraporn Rachada, Sirawaraporn Worachart
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, UK M13 9PL.
Org Biomol Chem. 2003 Mar 21;1(6):960-4. doi: 10.1039/b211636g.
Pyrimethamine acts against malarial parasites by selectively inhibiting their dihydrofolate reductase-thymidylate synthase. Resistance to pyrimethamine in Plasmodium falciparum is due to point mutations in the DHFR domain, initially at residue 108 (S108N), with additional mutations imparting much greater resistance. Our previous work, the development of a simple rational drug design strategy to overcome such resistance, used suitable meta-substituents in the pyrimethamine framework to avoid the unfavorable steric clash with mutant side chains at position 108. Interestingly, the meta-chloro analog of pyrimethamine not only overcame the resistance due to S108N, but also that contributed by the more remote mutation, C59R. The present work improves on this by means of other meta-substituents. Against wild type DHFR, double mutant types A16V + S108T and C59R + S108T, and the highly pyrimethamine/cycloguanil-resistant quadruple-mutant form N51I + C59R + S108N + I164L, pyrimethamine itself gave Ki values of 1.5, 2.4, 72.3 and 859 nM, respectively. The meta-substituted analogs, especially the meta-bromo analog, were much more powerful inhibitors of these DHFRs, including the quadruple-mutant form (meta-bromo analog, Ki 5.1 nM). For comparison, the dihydropyrazine antifolate, WR99210, gave Ki values of 0.9, 3.2, 0.8 and 0.9 nM, respectively. Ki values were also measured against recombinant human DHFR, as were their activities against the growth of Plasmodium falciparum cultures bearing the double mutations (FCB and K1 strains) and quadruple mutation (V1/S) and the wild type (3D7). The meta-analogs were highly active against all of these, with the meta-bromo again being the strongest, having an IC50 of 37 nM against V1/S, compared to > 5000 nM for pyrimethamine itself and 1.1 nM for WR99210.
乙胺嘧啶通过选择性抑制疟原虫的二氢叶酸还原酶-胸苷酸合成酶来对抗疟原虫。恶性疟原虫对乙胺嘧啶的耐药性是由于二氢叶酸还原酶（DHFR）结构域中的点突变，最初是第108位残基（S108N）发生突变，其他突变会导致更强的耐药性。我们之前的工作是开发一种简单合理的药物设计策略来克服这种耐药性，在乙胺嘧啶骨架中使用合适的间位取代基以避免与第108位突变侧链产生不利的空间冲突。有趣的是，乙胺嘧啶的间氯类似物不仅克服了由S108N导致的耐药性，还克服了更远处突变C59R导致的耐药性。目前的工作通过其他间位取代基对此进行了改进。对于野生型DHFR、双突变类型A16V + S108T和C59R + S108T，以及对乙胺嘧啶/环氯胍高度耐药的四重突变形式N51I + C59R + S108N + I164L，乙胺嘧啶自身的抑制常数（Ki）值分别为1.5、2.4、72.3和859 nM。间位取代类似物，尤其是间溴类似物，是这些DHFR的更强抑制剂，包括四重突变形式（间溴类似物，Ki为5.1 nM）。作为比较，二氢吡嗪抗叶酸剂WR99210的Ki值分别为0.9、3.2、0.8和0.9 nM。还测定了它们对重组人DHFR的Ki值，以及它们对携带双突变（FCB和K1菌株）、四重突变（V1/S）和野生型（3D7）的恶性疟原虫培养物生长的活性。间位类似物对所有这些都具有高活性，间溴类似物再次表现最强，对V1/S的半数抑制浓度（IC50）为37 nM，相比之下乙胺嘧啶自身大于5000 nM，WR99210为1.1 nM。
