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

在之前的项目中，我们成功地开发了针对疟疾寄生虫恶性疟原虫的重要乳酸转运蛋白PfFNT的新型药物样抑制剂。我们通过筛选抗疟化合物库发现了第一个PfFNT抑制剂;然而，当用亚致死剂量治疗时，这些抑制剂在培养的寄生虫中引起了耐药性。耐药性是基于PfFNT基因中的单一突变导致Gly 107 Ser交换。丝氨酸使抑制剂亲和力降低2个数量级，可能是由于结合口袋中的空间较小。我们产生了五个新的化合物组，目的是规避耐药突变，并最终用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为通过较小空间的部分（例如脂肪族链或5元杂环）或通过引入短连接体来连接苯来替换苯。大多数新化合物对PfFNT野生型蛋白完全有效，但未能改善与抗性PfFNT G107 S突变体的结合。在这一点上，我们提出了这样的想法，不仅要避免丝氨酸侧链，而且要通过形成额外的氢键来利用它。为此，我们回到六元环，现在携带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，也没有形成新的耐药性。这些新化合物已获得欧洲和美国的专利。通过此次更新申请，我们希望解析与通道结合的PfFNT的高分辨率结构，并分析两个亲脂性收缩位点在PfFNT转运路径中的功能作用;其中一个位点与我们的抑制剂结合。我们已经与伦敦克里克研究所的Peter Rosenthal建立了联系，他将提供低温EM服务。我们已经创建了用于制备高质量PfFNT蛋白样品的方案，如负染电子显微镜所示，并获得了PfFNT的第一个cryo-EM图像。我们将在无细胞系统中生产PfFNT，如果需要，还将在基于酵母的系统中生产PfFNT，确定用于溶解的合适洗涤剂，通过光谱学、功能重建、负染电子显微镜评估蛋白质质量，然后将样品送去进行冷冻EM。我们将替代性地将PfFNT重组成纳米盘以获得更天然的脂质环境。我们将从生物病理学的角度来研究在缩窄部位有变宽突变的PfFNT。通过了解PfFNT的结合模式，进一步开发抗疟药物的应用水平，ii. FNT运输机制的基本水平，通过观察侧链运动，通过抑制剂的相互作用，在较低的收缩。

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.