Computational Biology Lab, National Center for Bioinformatics, Quaid-i-Azam University, Islamabad, Pakistan.
University of Maryland Computer-Aided Drug Design Center, Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, United States of America.
PLoS One. 2021 Jan 15;16(1):e0244967. doi: 10.1371/journal.pone.0244967. eCollection 2021.
β-lactam antibiotics are the most widely used antimicrobial agents since the discovery of benzylpenicillin in the 1920s. Unfortunately, these life-saving antibiotics are vulnerable to inactivation by continuously evolving β-lactamase enzymes that are primary resistance determinants in multi-drug resistant pathogens. The current study exploits the strategy of combination therapeutics and aims at identifying novel β-lactamase inhibitors that can inactivate the β-lactamase enzyme of the pathogen while allowing the β-lactam antibiotic to act against its penicillin-binding protein target. Inhibitor discovery applied the Site-Identification by Ligand Competitive Saturation (SILCS) technology to map the functional group requirements of the β-lactamase CMY-10 and generate pharmacophore models of active site. SILCS-MC, Ligand-grid Free Energy (LGFE) analysis and Machine-learning based random-forest (RF) scoring methods were then used to screen and filter a library of 700,000 compounds. From the computational screens 74 compounds were subjected to experimental validation in which β-lactamase activity assay, in vitro susceptibility testing, and Scanning Electron Microscope (SEM) analysis were conducted to explore their antibacterial potential. Eleven compounds were identified as enhancers while 7 compounds were recognized as inhibitors of CMY-10. Of these, compound 11 showed promising activity in β-lactamase activity assay, in vitro susceptibility testing against ATCC strains (E. coli, E. cloacae, E. agglomerans, E. alvei) and MDR clinical isolates (E. cloacae, E. alvei and E. agglomerans), with synergistic assay indicating its potential as a β-lactam enhancer and β-lactamase inhibitor. Structural similarity search against the active compound 11 yielded 28 more compounds. The majority of these compounds also exhibited β-lactamase inhibition potential and antibacterial activity. The non-β-lactam-based β-lactamase inhibitors identified in the current study have the potential to be used in combination therapy with lactam-based antibiotics against MDR clinical isolates that have been found resistant against last-line antibiotics.
β-内酰胺类抗生素自 20 世纪 20 年代发现苄青霉素以来，一直是应用最广泛的抗菌药物。不幸的是，这些救命抗生素容易被不断进化的β-内酰胺酶灭活，而β-内酰胺酶是多药耐药病原体中主要的耐药决定因素。本研究利用联合治疗策略，旨在鉴定新型β-内酰胺酶抑制剂，该抑制剂既能使病原体的β-内酰胺酶失活，又能使β-内酰胺类抗生素作用于其青霉素结合蛋白靶标。抑制剂的发现应用配体竞争饱和的位点鉴定（SILCS）技术来绘制β-内酰胺酶 CMY-10 的功能基团要求，并生成活性位点的药效团模型。然后，使用 SILCS-MC、配体网格自由能（LGFE）分析和基于机器学习的随机森林（RF）评分方法对 70 万种化合物库进行筛选和过滤。通过计算筛选，有 74 种化合物进行了实验验证，其中包括β-内酰胺酶活性测定、体外药敏试验和扫描电子显微镜（SEM）分析，以探索其抗菌潜力。鉴定出 11 种化合物为增强剂，7 种化合物为 CMY-10 的抑制剂。其中，化合物 11 在β-内酰胺酶活性测定、针对 ATCC 菌株（大肠杆菌、阴沟肠杆菌、成团肠杆菌、蜂房哈夫尼菌）和 MDR 临床分离株（阴沟肠杆菌、蜂房哈夫尼菌和成团肠杆菌）的体外药敏试验中表现出良好的活性，协同试验表明其具有作为β-内酰胺增强剂和β-内酰胺酶抑制剂的潜力。对活性化合物 11 进行结构相似性搜索，得到了 28 种更多的化合物。这些化合物大多数也表现出β-内酰胺酶抑制潜力和抗菌活性。本研究中鉴定的非β-内酰胺类β-内酰胺酶抑制剂有可能与基于β-内酰胺的抗生素联合用于治疗对最后一线抗生素耐药的 MDR 临床分离株。
