In vitro and in silico antimicrobial evaluation of N- methyl-2-phenylmaleimides

Abstract

Due to COVID-19, many other pandemics have been overlooked in the past few years. Antimicrobial resistance (AMR) is a pandemic that have killed more people than Human Immunodeficiency Virus (HIV) and malaria in 2019 and ranked only behind COVID-19 and tuberculosis. In addition, it is estimated that the mortality rate due to AMR will increase to 10 million deaths by the year 2050. Despite the increase in AMR, it is now evident that there is a drastic decline in the development of novel antibiotics which further exacerbates the problem. While antibiotic stewardship as well as infection prevention is important to combat AMR, only novel antibiotics can treat resistant bacterial strains. Due to the high cost and attrition rates with regards to the development of new drugs, drug repurposing is increasingly being used to identify novel antibiotics. Maleimides (1H-pyrrole-2,5-dione) are a fusion of maleic acid and imides and have been found to exhibit antibacterial properties, however their antibacterial mechanism of action is unknown. Agirbas and co-workers (2007:2324) synthesised 2,3,5-substituted perhydropyrrolo[3,4-d]isoxazole-4,6-diones derivatives of maleimide and proved they had antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. These derivatives are similar to a series of N-Methyl-2-phenyl-maleimide (1H-pyrrole-2,5-dione) (NMP) derivatives that were previously synthesised by our research group and tested for activity against monoamine oxidase B (MAO-B). The aim of this study was to firstly use computer aided drug design (CADD) to create and validate a pharmacophore model of the maleimide derivatives synthesised by Agirbas and co-workers (2007:2324). The pharmacophore model was validated using the enrichment value (EF), hit rate (HR) and the area under the receiver operating characteristic (AUC-ROC) curve as metrics. The validated pharmacophore model was used to estimate the probability of the NMP derivatives to also have antibacterial activity, whereafter the in vitro activity of the NMP derivatives was determined against Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. Lastly, PharmMapper was used to conduct target fishing in an effort to identify potential antibacterial targets. A common feature pharmacophore model was created (rank score: 120.5; max. fit value: 4), which was able to accurately identify active analogues out of the decoy set (EF20%: 4.3, HR20%: 86.4%, AUC-ROC: 0.9 ± 0.03). Three hydrogen bond acceptors and a ring aromatic region was identified as important for in vitro antibacterial activity. The NMP compounds only had antibacterial activity against S. aureus. The most active compound, i.e., 3, had a minimum

inhibitory concentration (MIC) of 4 μg/ml, whilst the MIC of the other compounds ranged from 8 μg/ml to 16 μg/ml. Compound 5 was found to be bactericidal, whilst all other compounds were bacteriostatic. A statistically significant correlation was observed between the log P and the MIC of each compound, indicating that more lipophilic compounds have greater antibacterial activity. Using PharmMapper three possible antibacterial targets were identified, i.e., the malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT), the signal peptidase I (SPase) and topoisomerase VI. Further investigation will be needed to confirm the PharmMapper findings.