The identification of active compounds in <i>Ganoderma lucidum var. antler</i> extract inhibiting dengue virus serine protease and its computational studies

The number of global dengue incidences is alarmingly high in recent years. The global distribution of four dengue serotypes has also added economic burden in the dengue-endemic countries. To discover the potent dengue virus inhibitors in the antler form of <i>Ganoderma lucidum</i> (Lingzhi or Reishi), the water extraction of normal <i>G. lucidum</i> and its antler form were conducted and the chemical compounds were identified by LC-MS. Six distinct chemical compounds identified in high abundance were hesperetin, thymidine, lucidenic acid, 11-aminoundecanoic acid, 5-carboxyvanillic acid and ganocin B. The water extracts of <i>G. lucidum</i> in its antler form inhibited the DENV2 NS2B-NS3 protease activity at 84.6 ± 0.7%, higher than the normal <i>G. lucidum</i>. Then, molecular docking was performed on the homology model built from an in-house sequence. Docking simulation results showed that hesperetin and ganocin B were the best leads to bind at the catalytic triad of DENV2 NS2B-NS3pro via hydrogen bonding, van der Waals and pi-pi interactions. Extensive overlapping of HOMO-LUMO orbitals at the ringed regions of hesperetin helped to facilitate the entry of ligand to the catalytic triad cleft. LC-MS, molecular docking and density functional theory analyses confirmed that hesperetin was the strongest inhibitor against NS2B-NS3 protease. Communicated by Ramaswamy H. Sarma近年来，全球登革热发病例数居高不下，形势严峻。四种登革热血清型的全球流行也给登革热流行国家带来了额外的经济负担。为从鹿角形赤芝（<i>Ganoderma lucidum</i>，俗称灵芝（Lingzhi）或Reishi）中发掘强效登革病毒抑制剂，研究人员对普通赤芝及其鹿角形赤芝进行了水提取，并通过液相色谱-质谱联用（LC-MS）鉴定了其中的化学成分。最终在提取物中鉴定出六种高丰度的独特化学成分，分别为橙皮素（hesperetin）、胸苷（thymidine）、灵芝酸（lucidenic acid）、11-氨基十一烷酸（11-aminoundecanoic acid）、5-羧基香草酸（5-carboxyvanillic acid）以及ganocin B。鹿角形赤芝的水提取物对登革病毒2型（DENV2）NS2B-NS3蛋白酶的抑制活性可达84.6 ± 0.7%，优于普通赤芝提取物。随后，研究人员基于内部序列构建的同源模型开展了分子对接实验。对接模拟结果显示，橙皮素与ganocin B是最佳的候选配体，可通过氢键、范德华力以及π-π相互作用结合于DENV2 NS2B-NS3pro的催化三联体位点。橙皮素环状区域的最高占据分子轨道-最低未占据分子轨道（HOMO-LUMO）轨道存在大量重叠，可促进配体进入催化三联体裂隙。液相色谱-质谱联用、分子对接以及密度泛函理论（DFT）分析均证实，橙皮素是针对NS2B-NS3蛋白酶的最强抑制剂。本文由Ramaswamy H. Sarma通讯。