Elsevier

Bioorganic Chemistry

Volume 104, November 2020, 104244
Bioorganic Chemistry

Structure based drug discovery and in vitro activity testing for DNA gyrase inhibitors of Salmonella enterica serovar Typhi

https://doi.org/10.1016/j.bioorg.2020.104244Get rights and content

Abstract

The emerged resistance in Typhoidal Salmonella has limited the treatment options for typhoid fever. In this scenario, there is a need to find alternate treatment modalities against this pathogen. Amongst the therapeutic agents currently being used to treat enteric fever, quinolones have enjoyed considerable success since past three decades. These drugs act upon DNA gyrase and the acquired resistance is due to mutations at Ser83 and Asp87 of gyrase A subunit. In the present study DNA gyrase enzyme was targeted to seek out potential new inhibitors which are not affected by these mutations. Molecular modelling and docking studies were performed in Schrödinger’s molecular modelling software. Homology model of DNA gyrase-DNA complex was built using templates 1AB4 and 3LTN. Molecular dynamic simulations were performed in SPC solvent for 100 ns. Total 17,900,742 drug like molecules were downloaded from ZINC library of chemical compounds. The Glide XP score of the compounds ranged from −5.285 to −13.692. All the ligands bound at the four base pair staggered nick in the DNA binding groove of DNA gyrase enzyme with their aromatic rings intercalating between the bases of two successive nucleotides stabilized by π - π stacking interactions. The binding pocket of DNA gyrase B comprising conserved residues Lys 447, Gly 448, Lys 449, Ile 450, Leu 451, Gln 465 and Val 467 interacts with the ligand molecules through van der Waals interactions. The MIC (minimum inhibitory concentration), MBC (minimum bactericidal concentration) and IC50 of the tested compounds ranged from 500 to 125 mg/L, 750 to 500 mg/L and 100 to 12.5 mg/L, respectively. The selected hits bind to quinolone binding pocket, but their mode of binding and conformation is different to fluoroquinolones, and hence, their binding is not affected by mutations at Ser83 or Asp87 positions. These lead compounds can be further explored as a scaffold to design inhibitors against DNA gyrase to bypass quinolone resistance.

Introduction

Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A, is a major public health problem in low resource countries. The International Vaccine Institute estimated that there were 11.9 million enteric fever illnesses and 129,000 deaths in low- and middle-income countries in 2010 [1], [2]. Although the disease is manageable with suitable antibiotic treatment but emergence of antimicrobial-resistance has posed many challenges in the selection of suitable treatment regimen [3].

Antimicrobial resistance to anti-typhoidal antibiotics started to emerge in the years following their use in the treatment of typhoid fever [1]. Due to the development of multidrug resistant strains (MDR) with resistance to traditional first-line drugs such as ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol; fluoroquinolone or an extended-spectrum cephalosporin became the main treatment options for enteric fever [4], [5]. The reports on fluoroquinolone-resistant S. Typhi and S. Paratyphi A, especially from developing countries, started to emerge in 1990′s [6], [7]. The resistance is mainly due to the mutations at Ser73 or Asp 87 positions of gyrase A subunit of DNA gyrase enzyme [8]. These days cephalosporin and azithromycin are the clinical mainstay for treatment but there are some reports on resistance to these drugs [9], [10]. In a recent study, an outbreak of extensively drug resistant (XDR) strains of S. Typhi having resistance to chloramphenicol, fluoroquinolones and cephalosporins has been reported from Pakistan [11].

This continuing emergence and development of resistance to existing antimicrobial agents have created the need for new compounds that retain activity against these resistant strains. The discovery of new antimicrobial agents employs different strategies to kill bacterial pathogens, among which the inhibition of DNA gyrase is an attractive approach [12].

Bacterial DNA gyrase is a type II topoisomerase that enables the cells to overcome topological barriers encountered during unwinding of double stranded DNA during replication, transcription, recombination and repair. The active form of enzyme exists as a heterotetramer (A2B2) consisting of two subunits of each gyrase A (gyrA) and gyrase B (gyrB) [13]. The enzyme introduces negative supercoils in DNA molecule by causing a double stranded break in one segment of DNA called as G segment, crossing another segment (T segment) through the gap and then resealing the G segment.

Fluoroquinolones are the class of most successful inhibitors of DNA gyrase enzyme and have a good history as therapeutic agents for treatment of typhoid fever [1]. The fluoroquinolones bind to the DNA gyrase in the quinolone binding pocket (QBP) and arrest the supercoiling process by inhibiting the rejoining of DNA strands. QBP consists of a cavity formed by region of DNA gyrase A, DNA gyrase B and aromatic bases of cleaved DNA, complexed with the heterotetramer. Fluoroquinolone binding is basically stabilized by stacking interaction of quinolone ring in between the aromatic bases of DNA and the quinolone carboxylate group interacts with Ser83. Asp87 forms a coordinate bond with Mg2+ and water molecule to stabilize the drug-gyrase complex. Thus, the fluoroquinolone resistance is most commonly associated with amino acid substitutions at Ser83 and Asp87 in gyrA subunit [8], [13].

A number of inhibitors like quinazolinediones are known to bind at the same site in a different manner, without interacting with these mutated hotspot residues [14]. The reported structure of Streptococcus pneumoniae DNA gyrase complexed with DNA and a molecule of quinazolinedioneyl}-1-cyclopropyl-6-fluoro-8-methylquinazoline-2,4(1H,3H)-dione also known as PD0305970 (PDB ID: 3LTN) clearly elucidates this diverse mode of binding[15].

The present study was designed to identify novel drug-like molecules that target the cleavage complex of DNA gyrase enzyme. In an effort to discover novel inhibitors that would act on S. Typhi DNA gyrase, high throughput virtual screening strategy was employed. A three-dimensional (3D) structure of target molecule is required to determine binding mode and activity of the compound. Since the three-dimensional structure for S. Typhi DNA gyrase is not available, it was built adopting homology modelling approach using a suitable 3D template structure from Streptococcus pneumoniae topoisomerase IV (PDB ID: 3LTN). The model complex consisting of DNA gyrase A, DNA gyrase B and DNA was built and subsequently used as a receptor for molecular docking based virtual screening of in silico library. ZINC library of chemical compounds was screened to find the potential inhibitors binding at the quinolone binding pocket of DNA gyrase. The identified hits that yielded higher binding affinity (calculated) were tested experimentally for their inhibitory potential against supercoiling activity followed by antibacterial assays to determine their MICs. This study has successfully identified inhibitors of S. Typhi DNA gyrase which will not be affected due to the mutations conferring resistance to fluoroquinolones through the structure-based drug design approach.

Section snippets

Homology modeling of DNA gyrase

The two DNA gyrase subunits, A and B, were modeled separately. Homology model for breakage reunion domain (amino acid 30–522) of DNA gyrase subunit A was constructed using template from DNA gyrase of E. coli (PDB ID: 1AB4). The sequence similarity was 98% in the region of interest (Supplementary Fig.S1). Homology model was also generated for C-terminal TOPRIM domain of gyrase B (residue 408–628) using the ParE subunit for S. pneumoniae (PDB ID: 3LTN). The sequence similarity between the

Discussion:

The treatment of enteric fever continues to pose major therapeutic challenges due to the emergence of antimicrobial resistance in Salmonella enterica serovars Typhi and Paratyphi A, two major pathogens causing enteric fever [1], [4]. Extensively drug resistant S. Typhi strains have been reported with antimicrobial resistance to recommended antityphoidal agents. The emergence and development of bacterial resistance to existing antibacterial agents have created the need for new antimicrobial

Modeling of three-dimensional structure of DNA gyrase

The three-dimensional (3D) structure of a protein is the pre-requisite for screening of chemical library using structure based molecular docking approach. Therefore, the 3D model structure of gyrA and gyrB complexed with DNA was built using molecular modeling approach.

The model was generated separately for N-terminal of gyrase A and C-terminal TOPRIM domain of gyrase B subunits, and then the subunits were merged with DNA to form the structure of core complex of DNA gyrase. The homology model

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors thank Indian Council of Medical Research, New Delhi for financial support.

Funding

This work was partially supported for funding by Indian Council of Medical Research, New Delhi. (Grant No: ISRM/12(41)/2019)

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