Shao Jinfeng, Chu Jonathan, Mohammad Kashif, Desai Sanjay A
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
Pathogens. 2025 May 23;14(6):520. doi: 10.3390/pathogens14060520.
To facilitate intracellular growth and replication, the virulent human malaria parasite remodels its host erythrocyte by exporting many proteins into the host cell cytosol. Along with a few other exported proteins, the parasite CLAG3 protein is then inserted in the host erythrocyte membrane, exposing a small variant loop to host plasma and contributing to essential nutrient acquisition via the plasmodial surface anion channel (PSAC). To explore trafficking mechanisms and develop therapies that block host cell remodeling, we have now used a split NanoLuc reporter and performed a high-throughput screen for inhibitors of parasite CLAG3 trafficking and insertion at the host membrane. We screened ~52,000 small molecules and uncovered 65 chemically diverse hits. Hits that inhibit the NanoLuc reporter without blocking protein export were filtered out by a secondary screen whose signal does not depend on protein export. Because chemicals that interfere with parasite maturation were found to compromise CLAG3 export indirectly, a third screen using a NanoLuc reporter-tagged intracellular protein was used to evaluate nonspecific toxicity. Although our relatively small chemical screen did not identify bona fide inhibitors of CLAG3 host membrane insertion, these studies establish a framework for larger screens to identify novel export inhibitors. Such novel inhibitors will provide important insights into how remodel their host cells and may seed the development of therapies that block the export and membrane insertion of proteins needed for intracellular parasite survival.
为促进细胞内生长和复制，恶性疟原虫通过向宿主细胞质中输出多种蛋白质来重塑其宿主红细胞。然后，寄生虫CLAG3蛋白与其他一些输出蛋白一起插入宿主红细胞膜，将一个小的可变环暴露于宿主血浆中，并通过疟原虫表面阴离子通道（PSAC）促进必需营养物质的获取。为了探索运输机制并开发阻断宿主细胞重塑的疗法，我们现在使用了一种分裂型纳米荧光素酶报告基因，并对寄生虫CLAG3在宿主膜上的运输和插入抑制剂进行了高通量筛选。我们筛选了约52000个小分子，发现了65个化学结构不同的活性化合物。通过二次筛选滤除了那些不阻断蛋白质输出但抑制纳米荧光素酶报告基因的活性化合物，该二次筛选的信号不依赖于蛋白质输出。由于发现干扰寄生虫成熟的化学物质会间接损害CLAG3的输出，因此使用标记有纳米荧光素酶报告基因胞内蛋白的第三次筛选来评估非特异性毒性。尽管我们相对较小规模的化学筛选未鉴定出CLAG3插入宿主膜的真正抑制剂，但这些研究为更大规模的筛选建立了框架，以鉴定新型输出抑制剂。此类新型抑制剂将为寄生虫如何重塑其宿主细胞提供重要见解，并可能推动开发阻断细胞内寄生虫生存所需蛋白质输出和膜插入的疗法发展。
