Biochemical evaluation of novel antibacterial compounds

Introduction

Antimicrobial resistance has emerged as a defining public health crisis of the twenty-first century, with projections suggesting drug-resistant infections could cause ten million deaths annually by 2050 if current trends persist [1] . The World Health Organization has designated antimicrobial resistance among the top ten global health threats, emphasizing the urgent need for novel therapeutic approaches [2] . Staphylococcus aureus exemplifies this challenge, with methicillin-resistant strains now accounting for over forty percent of healthcare-associated infections in many regions [3] . The benzothiazole scaffold has attracted considerable attention in medicinal chemistry due to its presence in numerous biologically active compounds and favorable pharmacokinetic properties [4] . Aminobenzothiazole derivatives demonstrate particular promise as antibacterial agents, with several compounds showing activity against resistant organisms through mechanisms distinct from existing drug classes [5] . Structure-activity relationship analyses have identified key pharmacophoric features influencing both potency and selectivity [6] . Successful antibacterial development requires compounds demonstrating not only potent antimicrobial activity but also acceptable safety profiles in mammalian systems. The selectivity index, calculated as the ratio of mammalian cytotoxicity to bacterial minimum inhibitory concentration, provides a useful metric for evaluating therapeutic potential during early discovery phases [7] . Compounds with selectivity indices exceeding one hundred are generally considered to possess adequate margins for further development [8] . Understanding mechanisms of antibacterial action facilitates optimization efforts and helps predict resistance emergence patterns. Bacterial DNA gyrase represents a validated target inhibited by fluoroquinolones, while membrane-active compounds offer alternative approaches less susceptible to resistance through target modification [9] . Compounds acting through multiple mechanisms may demonstrate reduced resistance development frequencies compared to singletarget agents [10] . This research aimed to characterize the biochemical properties and antibacterial activities of eight novel aminobenzothiazole derivatives synthesized in our laboratory. Primary objectives included determination of minimum inhibitory concentrations against clinically relevant pathogens, assessment of mammalian cytotoxicity, and preliminary mechanistic investigation of the most promising compound [11] .

Reagents and chemicals

Test compounds ABX-101 through ABX-108 were synthesized in-house following established protocols with purity exceeding 98% as confirmed by high-performance liquid chromatography analysis. Reference antibiotics including vancomycin, ciprofloxacin, and gentamicin were obtained from Sigma-Aldrich (St. Louis, MO, USA) as analytical standards. Mueller-Hinton broth and agar were purchased from BD Difco (Franklin Lakes, NJ, USA). Dimethyl sulfoxide used for compound dissolution was molecular biology grade from Merck (Darmstadt, Germany). Cell culture media including Dulbecco's Modified Eagle Medium, fetal bovine serum, and penicillinstreptomycin were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The MTT reagent for cytotoxicity assays was purchased from Invitrogen [12] . Bacterial strains were obtained from the American Type Culture Collection including S. aureus ATCC 29213, methicillin-resistant S. aureus ATCC 43300, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. HepG2 hepatocellular carcinoma cells and HEK293 human embryonic kidney cells for cytotoxicity testing were maintained in the institutional cell bank. All reagents were stored according to manufacturer specifications until use.

Material and Methods

Material

This research was conducted at the Westbrook Institute of Medical Research Antimicrobial Discovery Laboratory, Brussels, Belgium, between January and August 2023. The compound library consisted of eight aminobenzothiazole derivatives varying in substitution patterns on the benzothiazole core and pendant aromatic rings. Stock solutions were prepared at 10 milligrams per milliliter in dimethyl sulfoxide and stored at negative twenty degrees Celsius. Working dilutions were prepared fresh in appropriate media immediately before each experiment with final dimethyl sulfoxide concentrations not exceeding one percent [13] . Bacterial cultures were maintained on appropriate agar media and subcultured weekly. Overnight broth cultures in Mueller-Hinton medium served as inocula for susceptibility testing. Mammalian cell lines were maintained in complete medium at 37 degrees Celsius in humidified atmosphere containing five percent carbon dioxide. Cells were passaged at seventy to eighty percent confluence using trypsin-EDTA and used within twenty passages of original stocks [14] .

Methods

Minimum inhibitory concentrations were determined by broth microdilution following Clinical and Laboratory Standards Institute guidelines. Two-fold serial dilutions of test compounds ranging from 64 to 0.03 micrograms per milliliter were prepared in 96-well microplates. Bacterial suspensions adjusted to 0.5 McFarland standard were diluted to yield final inocula of approximately five times ten to the fifth colony-forming units per milliliter. Plates were incubated at 35 degrees Celsius for 16 to 20 hours, and the minimum inhibitory concentration was defined as the lowest concentration preventing visible growth [15] . Cytotoxicity was assessed using the MTT colorimetric assay. HepG2 and HEK293 cells were seeded at 10,000 cells per well in 96-well plates and allowed to attach overnight. Test compounds were added at concentrations ranging from 200 to 0.1 micrograms per milliliter. Following 48-hour incubation, MTT solution was added, and formazan crystals were dissolved in dimethyl sulfoxide for absorbance measurement at 570 nanometers. Halfmaximal inhibitory concentrations were calculated from dose-response curves using non-linear regression [16] . Time-kill kinetics were performed for the lead compound against S. aureus ATCC 29213. Log-phase cultures were exposed to compound at one, two, and four times the minimum inhibitory concentration. Aliquots were removed at 0, 2, 4, 6, 8, 12, and 24 hours, serially diluted, and plated for colony enumeration. Bactericidal activity was defined as greater than three-log reduction from initial inoculum. DNA gyrase inhibition was assessed using a commercial supercoiling assay kit. Membrane permeability was evaluated by propidium iodide uptake measured by flow cytometry [17] . Screening of the aminobenzothiazole library revealed considerable variation in antibacterial potency and cytotoxicity profiles (Table 1). Minimum inhibitory concentrations against methicillin-sensitive S. aureus ranged from 0.125 to 4.0 micrograms per milliliter. ABX-105 emerged as the most potent compound with activity fourfold greater than vancomycin against both sensitive and resistant staphylococcal strains. Cytotoxicity testing identified compounds with favorable selectivity, with ABX-105 demonstrating the highest selectivity index of 997.

Results

Comprehensive interpretation

Extended spectrum testing of ABX-105 demonstrated potent activity against all Gram-positive organisms tested, including drug-resistant strains (Table 2, Figure 2). Activity against Gram-negative bacteria was substantially reduced, consistent with the outer membrane barrier limiting compound access. Time-kill analysis confirmed concentration-dependent bactericidal activity with rapid killing kinetics at supra-MIC concentrations (Figure 4). Mechanistic studies supported a dual mode of action involving DNA gyrase inhibition and membrane perturbation (Figure 3).

Discussion

This investigation identified ABX-105 as a promising antibacterial candidate from a focused library of aminobenzothiazole derivatives.

The compound demonstrated potent activity against clinically important Gram-positive pathogens including methicillin-resistant S. aureus, combined with an exceptionally favorable selectivity profile. These characteristics position ABX-105 as a viable lead for further optimization and preclinical development targeting serious staphylococcal infections. The selectivity index of 997 calculated for ABX-105 substantially exceeds typical thresholds for antibacterial development candidates. This favorable margin reflects the combination of potent antibacterial activity and low mammalian cytotoxicity, suggesting the compound targets bacterial-specific processes with minimal off-target effects on host cells. Such selectivity is essential for achieving therapeutic concentrations without dose-limiting toxicity in vivo.

Acknowledgement

Acknowledgements Funding sourcesThis research received support from the Westbrook Institute of Medical Research internal discovery fund and the Belgian Foundation for Antimicrobial Research.

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