Regenerative Biology Research Laboratory, Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey.
Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahçeşehir University, Istanbul, Turkey.
Biochem J. 2021 Sep 30;478(18):3445-3466. doi: 10.1042/BCJ20210481.
OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to characterize and develop first-in-class inhibitors of P. falciparum, P. vivax, and P. yoelii OTU-like proteins. These Plasmodium OTU-like proteins have highly conserved residues in the catalytic and inhibition pockets similar to viral OTU proteins. Plasmodium OTU proteins demonstrated Ubiquitin and ISG15 deconjugation activities as evident by intracellular ubiquitinated protein content analyzed by western blot and flow cytometry. We screened a library of small molecules to determine plasmodium OTU inhibitors with potent anti-malarial activity. Enrichment and correlation studies identified structurally similar molecules. We have identified two small molecules that inhibit P. falciparum, P. vivax, and P. yoelii OTU proteins (IC50 values as low as 30 nM) with potent anti-malarial activity (IC50 of 4.1-6.5 µM). We also established enzyme kinetics, druglikeness, ADME, and QSAR model. MD simulations allowed us to resolve how inhibitors interacted with plasmodium OTU proteins. These findings suggest that targeting malarial OTU-like proteases is a plausible strategy to develop new anti-malarial therapies.
OTU 蛋白酶拮抗宿主细胞中的细胞防御，并参与发病机制。有趣的是，疟原虫、间日疟原虫和约氏疟原虫具有未被描述的高度保守的病毒 OTU 样蛋白。然而，它们的结构、功能或抑制剂尚未被报道。为此，我们进行了结构建模、小分子筛选、去缀合测定，以表征和开发疟原虫、间日疟原虫和约氏疟原虫 OTU 样蛋白的首创类抑制剂。这些疟原虫 OTU 样蛋白在催化和抑制口袋中具有高度保守的残基，类似于病毒 OTU 蛋白。疟原虫 OTU 蛋白表现出泛素和 ISG15 的去缀合活性，这可以通过 Western blot 和流式细胞术分析细胞内泛素化蛋白含量来证明。我们筛选了小分子文库，以确定具有强大抗疟活性的疟原虫 OTU 抑制剂。富集和相关性研究确定了结构相似的分子。我们已经鉴定出两种抑制疟原虫、间日疟原虫和约氏疟原虫 OTU 蛋白的小分子（IC50 值低至 30 nM），具有强大的抗疟活性（IC50 为 4.1-6.5 μM）。我们还建立了酶动力学、药物性质、ADME 和 QSAR 模型。MD 模拟使我们能够确定抑制剂与疟原虫 OTU 蛋白的相互作用方式。这些发现表明，靶向疟原虫 OTU 样蛋白酶是开发新抗疟疗法的一种可行策略。
