Peptide Laboratory, Department of Chemistry, Faculty of Science, Universiti Malaya, Kuala Lumpur 50603, Malaysia.
Drug Design & Development Research Group, Department of Chemistry, Faculty of Science, Universiti Malaya, Kuala Lumpur 50603, Malaysia.
Molecules. 2022 May 18;27(10):3233. doi: 10.3390/molecules27103233.
Data from the World Health Organisation show that the global incidence of dengue infection has risen drastically, with an estimated 400 million cases of dengue infection occurring annually. Despite this worrying trend, there is still no therapeutic treatment available. Herein, we investigated short peptide fragments with a varying total number of amino acid residues (peptide fragments) from previously reported dengue virus type 2 (DENV2) peptide-based inhibitors, DN58wt (GDSYIIIGVEPGQLKENWFKKGSSIGQMF), DN58opt (TWWCFYFCRRHHPFWFFYRHN), DS36wt (LITVNPIVTEKDSPVNIEAE), and DS36opt (RHWEQFYFRRRERKFWLFFW), aided by in silico approaches: peptide-protein molecular docking and 100 ns of molecular dynamics (MD) simulation via molecular mechanics using Poisson-Boltzmann surface area (MMPBSA) and molecular mechanics generalised Born surface area (MMGBSA) methods. A library of 11,699 peptide fragments was generated, subjected to in silico calculation, and the candidates with the excellent binding affinity and shown to be stable in the DI-DIII binding pocket of DENV2 envelope (E) protein were determined. Selected peptides were synthesised using conventional Fmoc solid-phase peptide chemistry, purified by RP-HPLC, and characterised using LCMS. In vitro studies followed, to test for the peptides' toxicity and efficacy in inhibiting the DENV2 growth cycle. Our studies identified the electrostatic interaction (from free energy calculation) to be the driving stabilising force for the E protein-peptide interactions. Five key E protein residues were also identified that had the most interactions with the peptides: (polar) LYS36, ASN37, and ARG350, and (nonpolar) LEU351 and VAL354; these residues might play crucial roles in the effective binding interactions. One of the peptide fragments, DN58opt_8-13 (PFWFFYRH), showed the best inhibitory activity, at about 63% DENV2 plague reduction, compared with no treatment. This correlates well with the in silico studies in which the peptide possessed the lowest binding energy (-9.0 kcal/mol) and was maintained steadily within the binding pocket of DENV2 E protein during the MD simulations. This study demonstrates the use of computational studies to expand research on lead optimisation of antiviral peptides, thus explaining the inhibitory potential of the designed peptides.
世界卫生组织的数据显示，全球登革热感染发病率急剧上升，估计每年有 4 亿例登革热感染病例。尽管存在这种令人担忧的趋势，但目前仍没有有效的治疗方法。在此，我们研究了具有不同氨基酸残基数的短肽片段（肽片段），这些片段源自先前报道的登革热病毒 2 型（DENV2）基于肽的抑制剂 DN58wt（GDSYIIIGVEPGQLKENWFKKGSSIGQMF）、DN58opt（TWWCFYFCRRHHPFWFFYRHN）、DS36wt（LITVNPIVTEKDSPVNIEAE）和 DS36opt（RHWEQFYFRRRERKFWLFFW），并借助于计算机辅助方法：肽-蛋白分子对接和 100 ns 的分子动力学（MD）模拟，通过使用泊松-玻尔兹曼表面积（MMPBSA）和分子力学广义 Born 表面积（MMGBSA）方法的分子力学。生成了一个包含 11699 个肽片段的文库，进行了计算机计算，然后确定了与 DENV2 包膜（E）蛋白的 DI-DIII 结合口袋具有优异结合亲和力并表现出稳定的候选肽片段。选择的肽使用常规 Fmoc 固相肽化学合成，通过 RP-HPLC 纯化，并通过 LCMS 进行表征。随后进行了体外研究，以测试肽的毒性和抑制 DENV2 生长周期的功效。我们的研究确定静电相互作用（来自自由能计算）是 E 蛋白-肽相互作用的主要稳定力。还鉴定了五个与肽片段相互作用最多的关键 E 蛋白残基：（极性）LYS36、ASN37 和 ARG350，以及（非极性）LEU351 和 VAL354；这些残基可能在有效的结合相互作用中发挥关键作用。肽片段中的一种，DN58opt_8-13（PFWFFYRH）表现出最佳的抑制活性，与未处理相比，DENV2 斑减少约 63%。这与计算机研究结果相符，该研究表明，该肽具有最低的结合能（-9.0 kcal/mol），并且在 MD 模拟过程中能够稳定地保留在 DENV2 E 蛋白的结合口袋内。该研究表明，可利用计算机研究来扩展抗病毒肽的先导优化研究，从而解释设计肽的抑制潜力。
