Identification of secondary metabolites from Crescentia cujete as promising antibacterial therapeutics targeting type 2A topoisomerases through molecular dynamics simulation

Highlights

• The in silico effect of C. cujete was evaluated against type IIA topoisomerases (topo2As).

• Cistanoside D, chlorogenic acid, xycaine, and naringenin elicited the best effect.

• The effects were attributed to the molecular interactions with the active site amino acids of the enzyme.

• The findings would guide structural modification of the compounds as novel inhibitors of topo2As.

Abstract

The potential of fluoroquinolones as remarkable antibacterial agents evolved from their ability to generate ‘poison’ complexes between type IIA topoisomerases [topo2As (DNA gyrases and topoisomerases IV)] and DNA. However, the overuse of fluoroquinolones coupled with chromosomal mutations in topo2As has increased incidence of resistance and consequently undermined the application of the currently available fluoroquinolones in clinical practice. In this study, the molecular mechanism of interaction between the secondary metabolites of Crescentia cujete (an underutilized plant with proven anti-bacterial activity) and topo2As was investigated using computational methods. Through molecular docking, the top five compounds with the best affinity for each topo2A were identified and subjected to molecular dynamics simulation over a period of 100 ns. The results revealed that the identified compounds had higher binding energy values than the reference standards against the topo2As except for topoisomerase IV ParC, and this was consistent with the results of the structural stability and compactness of the resulting complexes. Specifically, cistanoside D (−49.18 kcal/mol), chlorogenic acid (−55.55 kcal/mol), xylocaine (−33.08 kcal/mol), and naringenin (−35.48 kcal/mol) had the best affinity for DNA gyrase A, DNA gyrase B, topoisomerase IV ParC, and topoisomerase IV ParE, respectively. Of the constituents of C. cujete evaluated, only apigenin and luteolin had affinity for all the four targets. These observations are indicative of the identified compounds as potential inhibitors of topo2As as evidenced from the molecular interactions including hydrogen bonds established with the active site amino acids of the respective targets. This is the first in silico report on the antibacterial effect of C. cujete and the findings would guide structural modification of the identified compounds as novel inhibitors of topo2As for further in vitro and in vivo assessments.

Introduction

Fluoroquinolones remain one of the most potent bactericidal antibiotics against both Gram-negative and Gram-positive strains [1]. This broad-spectrum activity of the fluoroquinolones has been lucidly demonstrated against the DNA gyrase and topoisomerase IV of bacteria. These enzymes constitute the type IIA topoisomerases (topo2As) essential for chromosomal supercoiling during bacterial DNA synthesis and thus, remain important therapeutic targets for the development of potent fluoroquinolone-like antibacterial drugs [2]. The DNA gyrase (Gyr) is subdivided into GyrA and GyrB, while topoisomerase IV comprises the ParC and ParE subunits [2]. Over the years, new fluoroquinolones have been developed through structural modification of the existing ones [3,4]. While this strategy has yielded more potent drugs with improved antibacterial activity, inherent adverse effects such as dizziness, headache, nausea, risks of mental health, low blood sugar adverse reaction, liver failure, and even death, have undermined their applications in clinical practice [[3], [4], [5]]. Consequently, several fluoroquinolones such as trovafloxacin and grepafloxacin have been minimally used and/or withdrawn from the market [5]. Furthermore, the overuse of the currently available fluoroquinolones such as ciprofloxacin, ofloxacin, and novobiocin, coupled with chromosomal mutations that have resulted in structural alterations in topo2As as potential targets have further limited the use of fluoroquinolones [5,6]. Hence, the identification of new antibacterial agents against topo2As remains imperative. Interestingly, plants and plant-derived compounds have been used in the treatment of human diseases including bacterial infections [7,8]. Hence, exploring plant-derived compounds as novel antibacterial therapeutics could be a viable strategy to identify potent and less toxic drug candidates against bacterial topo2As.

Crescentia cujete L. is an underutilized plant with antibacterial activities against several multidrug-resistant bacteria strains [[9], [10], [11]]. Its ethanolic leaf extract has been demonstrated against Staphylococcus aureus, Bacillus megaterium, Bacillus cereus, Bacillus subtilis, Shigella dysenteriae, and Bacillus cereus with minimum inhibitory concentrations ranging between 2.5 and 4.5 mg/L [12]. Similarly, Parvin et al. [11] investigated the antibacterial activity of the ethanol and chloroform extracts of its leaf and stem bark against S. aureus and Escherichia coli and reported that the chloroform extract was more effective against the tested organisms, with E. coli (29 mm) being the most susceptible at 200 g/disk of the extract. While the antibacterial activities of the different extracts of C. cujete have been attributed to its diverse bioactive metabolites, the associated mechanism of action and the actual metabolite(s) implicated in the process remain elusive and have not been clearly identified [9,12].

The traditional method of drug discovery, which is mainly based on identifying drug candidates using the conventional trial and error screening process, is usually time-consuming and costly with a high risk of failure rate [13]. However, through computational methods such as the use of advanced computer-aided molecular screening tools, lead compounds from plant secondary metabolites with promising antibacterial activities have been identified [14,15]. Also, with better understanding of essential biomolecular processes provided by more advanced computer modeling tools, such as molecular dynamics (MD) simulation, insight on the associated mechanisms of action of therapeutic metabolites have been deciphered [16,17]. Molecular dynamics simulation provides useful information on protein folding, conformational changes, and appreciation of how atoms move at picosecond temporal resolution when ligands interact with bacterial therapeutic targets [16,17]. Hence, in this study, computational methods were adopted to establish the molecular interactions between the lead metabolites from C. cujete and active site amino acid residues of topo2As as antibacterial targets (Fig. 1). Exploring pharmacologically important plants in this manner may help identify potent and less toxic inhibitors of topo2As that could be developed as novel antibacterial therapeutics.

DNA gyrase and topoisomerase IV are found in both Gram-positive and Gram-negative bacteria, with each playing essential roles in replication [2]. More specifically, DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the main therapeutic targets in Gram-negative and Gram-positive bacteria, respectively [18,19]. Unfortunately, due to mutations in these four targets/subunits (GyrA and GyrB, ParC and ParE), varying degrees of resistance to fluoroquinolones have been reported [18]. For instance, in S. aureus, a low level of resistance to fluoroquinolones is due to mutations in ParC subunit [19] and the resistance has been shown to increase if the mutation affects both the ParC and GyrA subunits [19]. Therefore, in this study and for the first time, the susceptibility of the topo2A subunits to lead metabolites from C. cujete was undertaken to identify novel drug candidates as potential inhibitors of topo2As.