Bravo Patricia, Bizzarri Lorenzo, Steinbrunn Dominik, Lohse Jonas, Hirsch Anna K H, Mäser Pascal, Rottmann Matthias, Hahne Hannes
Swiss Tropical and Public Health Institute, Kreuzstrasse 2, 4123 Allschwil, Switzerland.
Universität Basel, Petersplatz 1, 4003 Basel, Switzerland.
ACS Infect Dis. 2024 Dec 13;10(12):4073-4086. doi: 10.1021/acsinfecdis.4c00418. Epub 2024 Dec 4.
The limited understanding of the mechanism of action (MoA) of several antimalarials and the rise of drug resistance toward existing malaria therapies emphasizes the need for new strategies to uncover the molecular target of compounds in . Integral solvent-induced protein precipitation (iSPP) is a quantitative mass spectrometry-based (LC-MS/MS) proteomics technique. The iSPP leverages the change in solvent-induced denaturation of the drug-bound protein relative to its unbound state, allowing identification of the direct drug-protein target without the need to modify the drug. Here, we demonstrate proof-of-concept of iSPP in (Pf) lysate. At first, we profiled the solvent-induced denaturation behavior of the Pf proteome, generating denaturation curves and determining the melting concentration () of 2712 proteins. We then assessed the extent of stabilization of three antimalarial target proteins in multiple organic solvent gradients, allowing for a rational selection of an optimal solvent gradient. Subsequently, we validated iSPP by successfully showing target-engagement of several standard antimalarials. The iSPP assay allows the testing of multiple conditions within reasonable LC-MS/MS measurement time. Furthermore, it requires a minimal amount of protein input, reducing culturing time and simplifying protein extraction. We envision that iSPP will be useful as a complementary tool for MoA studies for next-generation antimalarials.
对几种抗疟药物作用机制（MoA）的了解有限，以及对现有疟疾疗法耐药性的增加，凸显了采用新策略来揭示化合物分子靶点的必要性。整体溶剂诱导蛋白沉淀（iSPP）是一种基于定量质谱（LC-MS/MS）的蛋白质组学技术。iSPP利用药物结合蛋白相对于其未结合状态时溶剂诱导变性的变化，无需对药物进行修饰即可鉴定直接的药物-蛋白质靶点。在此，我们展示了iSPP在恶性疟原虫（Pf）裂解物中的概念验证。首先，我们分析了Pf蛋白质组的溶剂诱导变性行为，生成变性曲线并确定了2712种蛋白质的解链浓度（）。然后，我们评估了三种抗疟靶点蛋白在多种有机溶剂梯度中的稳定程度，从而合理选择最佳溶剂梯度。随后，我们通过成功展示几种标准抗疟药物的靶点结合情况，验证了iSPP。iSPP分析能够在合理的LC-MS/MS测量时间内测试多种条件。此外，它所需的蛋白质输入量极少，减少了培养时间并简化了蛋白质提取过程。我们设想iSPP将作为一种补充工具，用于下一代抗疟药物的作用机制研究。
