Department of Chemistry and Biochemistry, Montclair State University, Montclair, NJ, United States of America.
PLoS One. 2018 May 22;13(5):e0197173. doi: 10.1371/journal.pone.0197173. eCollection 2018.
Filariasis is a tropical disease caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. Known inhibitors of dihydrofolate reductase (DHFR) have been previously shown to kill Brugia malayi nematodes and to inhibit Brugia malayi DHFR (BmDHFR) at nanomolar concentrations. These data suggest that BmDHFR is a potential target for the treatment of filariasis. Here, protocols for cloning, expression and purification of Wuchereria bancrofti DHFR (WbDHFR) were developed. The Uniprot entry J9F199-1 predicts a 172 amino acid protein for WbDHFR but alignment of this sequence to the previously described BmDHFR shows that this WbDHFR sequence lacks a crucial, conserved 13 amino acid loop. The presence of the loop in WbDHFR is supported by a noncanonical splicing event and the loop sequence was therefore included in the gene design. Subsequently, the KM for dihydrofolate (3.7 ± 2 μM), kcat (7.4 ± 0.6 s-1), and pH dependence of activity were determined. IC50 values of methotrexate, trimethoprim, pyrimethamine, raltitrexed, aminopterin, (-)-epicatechin gallate, (-)-epicatechin, and vitexin were measured for WbDHFR and BmDHFR. Methotrexate and structurally related aminopterin were found to be effective inhibitors of WbDHFR, with an KI of 1.2 ± 0.2 nM and 2.1 ± 0.5 nM, respectively, suggesting that repurposing of known antifolate compound may be an effective strategy to treating filariasis. Most compounds showed similar inhibition profiles toward both enzymes, suggesting that the two enzymes have important similarities in their active site environments and can be targeted with the same compound, once a successful inhibitor is identified.
丝虫病是一种由寄生线虫班氏吴策线虫和马来布鲁线虫引起的热带疾病。先前已表明，二氢叶酸还原酶 (DHFR) 的已知抑制剂以纳摩尔浓度杀死马来布鲁线虫并抑制马来布鲁线虫 DHFR (BmDHFR)。这些数据表明 BmDHFR 是治疗丝虫病的潜在靶标。在这里，开发了班氏吴策线虫 DHFR (WbDHFR) 的克隆、表达和纯化方案。Uniprot 条目 J9F199-1 预测 WbDHFR 是一种 172 个氨基酸的蛋白质，但将该序列与先前描述的 BmDHFR 进行比对表明，该 WbDHFR 序列缺少一个关键的、保守的 13 个氨基酸环。WbDHFR 中环的存在得到了非规范剪接事件的支持，因此将环序列包含在基因设计中。随后，测定了二氢叶酸 (3.7±2μM)、kcat (7.4±0.6 s-1) 和活性的 pH 依赖性的 KM 值。测定了 WbDHFR 和 BmDHFR 的甲氨蝶呤、甲氧苄啶、乙胺嘧啶、雷替曲塞、氨蝶呤、(-)-表儿茶素没食子酸酯、(-)-表儿茶素和牡荆素的 IC50 值。发现甲氨蝶呤和结构上相关的氨蝶呤是 WbDHFR 的有效抑制剂，KI 值分别为 1.2±0.2 nM 和 2.1±0.5 nM，这表明重新利用已知的抗叶酸化合物可能是治疗丝虫病的有效策略。大多数化合物对两种酶表现出相似的抑制谱，这表明两种酶在其活性位点环境中具有重要的相似性，并且一旦确定了有效的抑制剂，就可以用相同的化合物靶向它们。
