Isatin and rosmarinic acid derivatives as antibacterial agents : molecular design, synthesis, structure-activity relationship and target validation

Abstract

The increasing antimicrobial resistance has become a global concern as a threat to human health and a burden to the healthcare system. To deal with this problem, a logical solution is to increase antibiotic candidates by developing new and novel inhibitory compounds against new drug targets. Bacterial peptidoglycan glycosyltransferases (GTs) have long been regarded as a promising target for the development of new antibiotics. GT functions as the polymerase of the lipid II precursor to elongate the glycan strands in the synthesis of bacterial cell walls. Similar to the transpeptidases (TPs), GT is located outside the cell membrane, enabling easy access to inhibitors. In addition, the encoded genes of GTs are highly conserved among Gram-positive and Gramnegative bacteria. Furthermore, mammalian cells have no cell walls and GTs have no counterpart among human cells, which make it possible to kill pathogenic microorganisms without harming the human cells. However, the only known natural GT inhibitor, moenomycin, is only used as a food additive for livestock because of its poor pharmacokinetic properties and bioavailability. The lacking of antibiotics targeting at GT is attributed to limited chemotypes as GT inhibitors. With advances in computer-aided drug design (CADD), hits could be screened out from compound libraries to reduce the time and cost used in the drug discovery process. Recently, through CADD virtual high-throughput screening, isatin derivatives were identified as GT inhibitors with potent antibacterial profiles and PBP 1b inhibitory effect. We have further modified the isatin core taking advantage of the molecular scaffold which allows 3-site modifications to maximize the antibacterial potency. Our results showed that substitution at the C-5 position of the isatin core with electron-donating groups, such as methyl and methoxy, resulted in compound IS 2 with the strongest antibacterial effect. Molecular docking suggests a strong hydrophobic interaction between the substituted isatin core of compound IS 2 and the PRO226 residue of the active site of GT. Moreover, replacement of the 4-n-butyl-phenyl group with alkyl chains of 10 or 11 carbons retains the antibacterial activity. Although replacing aminoguanidine moiety in the isatin core with thiosemicarbazide will reduce the cytotoxicity to human cells, the antibacterial effect is also impaired. Based on molecular docking, rosmarinic acid (RA) was predicted to bind to the active site of GT, while a benzimidazole moiety is predicted to bind to the undecaprenyl-pyrophosphate-interacting residues of GT, indicating that it is likely to obtain a GT inhibitor by linking benzimidazole-like structures with rosmarinic acid. Structure-activity relationship (SAR) studies revealed that rosmarinic acid derivative RA 15 with 6-trifluoromethoxy-benzothiazole moiety probably provides the right amphipathic property to interact with its target. However, in view of the results of protein inhibition tests, RA 15 may bind to targets other than PBP 1b non-specifically. To validate the target of RA 15, resistance induction experiments, SYTOX Green assays, and membrane integrity experiments were carried out. The results indicated that RA 15 may kill bacterial pathogens by damaging the cell membranes.