Structure elucidation of the inhibitor-bound plasmodial lactate transporter, PfFNT, by cryo-EM

在前一个项目中，我们成功地合成了针对疟疾寄生虫恶性疟原虫重要的乳酸转运蛋白PfFNT的新型类药物抑制剂。我们通过筛选抗疟疾化合物文库发现了第一批PfFNT抑制剂；然而，当用亚致死剂量处理时，这些药物在培养的寄生虫中产生了耐药性。这种耐药性是基于PfFNT基因的一个突变导致了Gly107Ser交换。丝氨酸使抑制物亲和力降低2个数量级，可能是由于结合口袋中的空间较小所致。我们产生了五个新的化合物集，目的是绕过耐药性突变，并最终通过最新一代化合物实现了这一目标。最初生成的化合物线被设计成用空间较小的部分来取代苯，例如脂肪链或5元杂环，或者通过引入短连接基来连接苯。大多数新化合物对PfFNT野生型蛋白非常有效，但未能改善与抗药性PfFNT G107S突变体的结合。在这一点上，我们想到的想法不仅是避免丝氨酸侧链，而且通过形成一个额外的氢键来利用它。为此，我们回到了6元环，现在以氮原子作为氢键受体。事实上，这些化合物第一次以与野生型相似的功效抑制耐药转运蛋白。重要的是，用亚致死剂量处理的培养寄生虫无法逃脱，也没有形成新的抗药性。这些新化合物被一项欧洲专利和一项最近发布的美国专利所涵盖。通过这次更新申请，我们希望解决与抑制剂结合的PfFNT的高分辨率结构，并分析两个亲脂收缩位点与PfFNT运输路径的功能作用；其中一个结合我们的抑制剂。我们已经与伦敦克里克研究所的彼得·罗森塔尔建立了联系，他将提供低温EM服务。我们已经制定了制备高质量PfFNT蛋白质样本的方案，如负染电子显微镜所示，并获得了PfFNT的第一张冷冻EM图像。因此，这项建议被构建为三个主要目标：1.我们将在无细胞的系统中生产PfFNT，如果需要，还将在酵母系统中生产PfFNT，确定适合溶解的洗涤剂，通过光谱、功能重组、负染电子显微镜评估蛋白质质量，然后发送样本进行低温EM2。我们也可以选择将PfFNT重组成纳米盘，以获得更自然的脂质环境。我们将对PfFNT在收缩部位的广泛突变进行生物物理学表征。我们期望在两个层面上获得洞察力：1.通过了解PfFNT与PfFNT的结合模式，进一步开发抗疟疾药物的应用水平；2.FNT运输机制的基本水平，通过看到在下部收缩中通过抑制剂相互作用的侧链运动。

In the previous project, we succeeded in generating novel drug-like inhibitors against the vital lactate transporter, PfFNT, of malaria parasites, Plasmodium falciparum. We discovered first PfFNT-inhibitors by screening of an antimalarial compound library; yet these gave rise to resistance in cultured parasites when treated with sublethal doses. The resistance is based on a single mutation in the PfFNT gene resulting in a Gly107Ser exchange. The serine decreases inhibitor affinity by 2 orders of magnitude probably due to less space in the binding pocket. We generated five new compound sets with the aim to circumvent the resistance mutation, and eventually achieved this goal with the latest generation of compounds. The initially generated compound lines were designed to replace a benzene by less spacious moieties, such aliphatic chains or 5-membered heterocycles, or by introducing a short linker to attach the benzene. Most of the new compounds were perfectly effective at the PfFNT wildtype protein but failed to improve binding to the resistant PfFNT G107S mutant. At this point we came up with the idea not only to avoid the serine sidechain but to make use of it via formation of an additional hydrogen bond. For this, we went back to 6-membered rings, now carrying nitrogen atoms as hydrogen bond acceptors. Indeed, for the first time, these compounds inhibit the resistant transporter with similar efficacy as the wildtype. Importantly, cultured parasites treated with sublethal doses were unable to escape and no new resistance was formed. The new compounds are covered by a European and a just recently issued US patent.With this renewal application, we want to solve the high-resolution structure of inhibitor-bound PfFNT, and to analyze the functional role of two lipophilic constriction sites with the PfFNT transport path; to one of which bind our inhibitors. We have established contact with Peter Rosenthal, Crick Institute, London, who will provide access to cryo-EM as a service. We have already created protocols for the preparation of high-quality PfFNT protein samples as shown by negative stain electron microscopy, and obtained first cryo-EM images of PfFNT.Hence, this proposal is structured into three main objectives:1. We will produce PfFNT in a cell-free and, if required, a yeast-based system, identify suitable detergents for solubilisation, assess the protein quality by spectroscopy, functional reconstitution, negative-stain electron microscopy before sending samples for cryo-EM.2. We will alternatively reconstitute PfFNT into nanodiscs for a more natural lipid environment.3. We will biophysically characterize PfFNT with widening mutations in the constriction sites.We expect insight on two levels: i. applied level for further antimalarial drug development by understanding the binding mode to PfFNT, ii. fundamental level of the FNT transport mechanism by seeing the sidechain movement through inhibitor interaction in the lower constriction.