Departamento de Física, Universidade Estadual Paulista (UNESP), São José do Rio Preto, São Paulo CEP 15054-000, Brazil.; Computational Biology and Biomolecular Dynamics Laboratory, Center for Protein Studies, Faculty of Biology, University of Havana, Havana, Cuba.
Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín (UNSAM) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, Buenos Aires, Argentina.
Biochim Biophys Acta Gen Subj. 2018 Dec;1862(12):2911-2923. doi: 10.1016/j.bbagen.2018.09.015. Epub 2018 Sep 22.
Falcipain 2 (FP-2) is the hemoglobin-degrading cysteine protease of Plasmodium falciparum most extensively targeted to develop novel antimalarials. However, no commercial antimalarial drugs based on FP-2 inhibition are available yet due to the low selectivity of most FP-2 inhibitors against the human cysteine proteases.
A structure-based virtual screening (SVBS) using Maybridge HitFinder™ compound database was conducted to identify potential FP-2 inhibitors. In vitro enzymatic and cell-growth inhibition assays were performed for the top-scoring compounds. Docking, molecular dynamics (MD) simulations and free energy calculations were employed to study the interaction of the best hits with FP-2 and other related enzymes.
Two hits based on 4-(9H-fluoren-9-yl) piperazin-1-yl) methanone scaffold, HTS07940 and HTS08262, were identified as inhibitors of FP-2 (half-maximal inhibitory concentration (IC) = 64 μM and 14.7 μM, respectively) without a detectable inhibition against the human off-target cathepsin K (hCatK). HTS07940 and HTS08262 inhibited the growth of the multidrug-resistant P. falciparum strain FCR3 in culture (half-maximal inhibitory concentrations (IC50) = 2.91 μM and 34 μM, respectively) and exhibited only moderate cytotoxicity against HeLa cells (Half-maximal cytotoxic concentration (CC50) = 133 μM and 350 μM, respectively). Free energy calculations reproduced the experimental affinities of the hits for FP-2 and explained the selectivity with respect to hCatK.
To the best of our knowledge, HTS07940 stands among the most selective FP-2 inhibitors identified by SBVS reported so far, displaying moderate antiplasmodial activity and low cytotoxicity against human cells. Hence, this compound constitutes a promising lead for the design of more potent and selective FP-2 inhibitors.
裂殖体蛋白酶 2（FP-2）是恶性疟原虫中降解血红蛋白的半胱氨酸蛋白酶，是开发新型抗疟药物的主要靶点。然而，由于大多数 FP-2 抑制剂对人半胱氨酸蛋白酶的选择性较低，因此尚无基于 FP-2 抑制作用的商业抗疟药物。
使用 Maybridge HitFinder™化合物数据库进行基于结构的虚拟筛选（SVBS），以鉴定潜在的 FP-2 抑制剂。对得分最高的化合物进行体外酶和细胞生长抑制测定。采用对接、分子动力学（MD）模拟和自由能计算研究最佳命中物与 FP-2 和其他相关酶的相互作用。
鉴定出两种基于 4-（9H-芴-9-基）哌嗪-1-基甲酮骨架的化合物 HTS07940 和 HTS08262 为 FP-2 的抑制剂（半最大抑制浓度（IC）分别为 64 μM 和 14.7 μM），对人靶标组织蛋白酶 K（hCatK）没有可检测的抑制作用。HTS07940 和 HTS08262 抑制了培养中的多药耐药恶性疟原虫株 FCR3 的生长（半最大抑制浓度（IC50）分别为 2.91 μM 和 34 μM），对 HeLa 细胞仅有中度细胞毒性（半最大细胞毒性浓度（CC50）分别为 133 μM 和 350 μM）。自由能计算再现了命中物对 FP-2 的实验亲和力，并解释了相对于 hCatK 的选择性。
据我们所知，HTS07940 是迄今为止通过 SBVS 鉴定的选择性最高的 FP-2 抑制剂之一，对 FP-2 具有中等的抗疟活性和对人细胞的低细胞毒性。因此，该化合物是设计更有效和选择性更高的 FP-2 抑制剂的有前途的先导化合物。
