In vitro and computational studies of the β-lactamase inhibition and β-lactam potentiating properties of plant secondary metabolites

β-lactam resistance in bacteria is primarily mediated through the production of β-lactamases. Among the several strategies explored to mitigate the issue of β-lactam resistance, the use of plant secondary metabolites in combination with existing β-lactams seem promising. The present study aims to identify possible β-lactam potentiating plant secondary metabolites following <i>in vitro</i> and <i>in silico</i> approaches. Among 180 extracts from selected 30 medicinal plants, acetone extract of <i>Ficus religiosa</i> (FRAE) bark recorded the least IC₅₀ value of 3.9 mg/ml. Under <i>in vitro</i> conditions, FRAE potentiated the activity of ampicillin, which was evidenced by the significant reduction in IC₅₀ values of ampicillin against multidrug resistant bacteria. Metabolic profiling following HR-LCMS analysis revealed the presence of diverse metabolites viz. flavonoids, alkaloids, terpenoids, etc. in FRAE. Further, ensemble docking of the FRAE metabolites against four Class A β-lactamase (SHV1, TEM1, KPC2 and CTX-M-27) showed quercetin, taxifolin, myricetin, luteolin, and miquelianin as potential inhibitors with the least average binding energy. In molecular dynamic simulation studies, myricetin formed the most stable complex with SHV1 and KPC-2 while miquelianin with TEM1 and CTX-M-27. Further, all five metabolites interacted with amino acid residue Glu166 in Ω loop of β-lactamase, interfering with the deacylation step, thereby disrupting the enzyme activity. The pharmacokinetics and ADMET profile indicate their drug-likeness and non-toxic nature, making them ideal β-lactam potentiators. This study highlights the ability of metabolites present in FRAE to act as β-lactamase inhibitors. Communicated by Ramaswamy H. Sarma

细菌β-内酰胺耐药主要由β-内酰胺酶介导。在目前已探索的多种缓解β-内酰胺耐药问题的策略中，植物次生代谢物与现有β-内酰胺类药物联用的方案颇具应用前景。本研究旨在通过体外（in vitro）与虚拟（in silico）实验方法，鉴定具备β-内酰胺增敏活性的植物次生代谢物。从筛选出的30种药用植物中获取的180份提取物里，菩提树（Ficus religiosa，FRAE）树皮的丙酮提取物的半最大抑制浓度（IC₅₀）最低，为3.9 mg/ml。体外实验条件下，FRAE可增强氨苄青霉素的抗菌活性：针对多重耐药细菌时，氨苄青霉素的IC₅₀值显著降低，证实了这一增效作用。经高效液相色谱-质谱联用（HR-LCMS）进行代谢组分析后发现，FRAE中含有多种代谢物，即黄酮类、生物碱类、萜类等化合物。进一步针对4种A类β-内酰胺酶（SHV1、TEM1、KPC2与CTX-M-27）对FRAE中的代谢物开展集成对接分析，结果显示槲皮素、紫杉叶素、杨梅素、木犀草素及米奎林（miquelianin）为潜在抑制剂，其平均结合能最低。分子动力学模拟实验表明，杨梅素可与SHV1和KPC-2形成最稳定的复合物，而米奎林则可与TEM1和CTX-M-27形成稳定复合物。此外，上述5种代谢物均能与β-内酰胺酶Ω环中的Glu166氨基酸残基相互作用，干扰脱酰基步骤，进而破坏酶的催化活性。药代动力学及吸收、分布、代谢、排泄与毒性（ADMET）特性分析结果显示，这些代谢物具备成药性且无毒性，是理想的β-内酰胺增敏剂。本研究证实了FRAE中所含代谢物可作为β-内酰胺酶抑制剂。本文由Ramaswamy H. Sarma通讯供稿。