Repurposing Potential of Diminazene Aceturate as an Inhibitor of the E. coli DNA Gyrase B

Varsha Dwivedi1, Archana Ayyagari2, Rakhi Chandran3, Prerna Diwan1, Sanjay Gupta4 and Vandana Gupta1* 1Department of Microbiology, Ram Lal Anand College, University of Delhi, New Delhi 110021, India 2Department of Microbiology, Swami Shraddhanand College. University of Delhi, New Delhi 110036, India 3Department of Biotechnology and Microbiology, School of Sciences, Noida International University,Yamuna Expressway, Gautam Budh Nagar, UP-203201, India 4Independent Scholar (former Head and Professor, Department of Biotechnology, Jaypee Institute of Information Technology, Noida, Sector 62, UP, India


INTRODUCTION
E. coli is a gram negative bacterium, normally a commensal inhabiting the human colon, and has indeed proved to be a great experimental organism of choice for all microbiology as well as gene cloning experiments for long. However, quite a few of its strains are known to cause various intestinal as well as extraintestinal diseases, owing to possession of a handful of virulence factors in some of its serotypes, which infl uence a number of metabolic processes [1]. Some of the problems caused by Indiscriminate and improper usage of antibiotics has already resulted in an alarming multiple drug resistance among the pathogenic bacteria [2][3][4][5] and has become a global public health concern. Among many mechanisms of MDR, the predominant one is the plasmid-mediated synthesis of Extended-Spectrum Beta Lactamases (ESBLs), which breakdown the beta lactam antibiotics, including all the three generations of cephalosporins, penicillins and aztreonam particularly witnessed in E. coli and other Gram-ve EKAPE pathogens Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species [6,7].
Considering the newly emerging and life threatening AMR E. coli, there is an urgent requirement of newer strategies, such as repurposing of older drugs, re-evaluation of many abandoned compounds using modern chemical synthesis methods, tools and technologies [5]. Another potential approach could be to explore unusual targets within the pathogen system, such as topoisomerases. DNA gyrase or topoisomerase type 2 is an important enzyme that controls DNA supercoiling as needed for processes like replication and transcription, and it is reported to be the target of some successful antibacterials [8][9][10][11]. A functional DNA gyrase is heterotetramer (A 2 B 2 ), consisting of 2 subunits each of gyr A and gyr B, with 875 and 804 amino acids respectively [8]. GyrA creates single stranded nicks in the DNA and reseals them, whereas GyrB serves to provide energy for this process through ATP hydrolysis [9,10], which is essential for the unwinding of the DNA. Hence, DNA gyrases serve as the nanomachines that maintain the DNA in its appropriate topology throughout its replication and transcription [11]. Aminocoumarin antibiotics, quinolones and fl oroquinolones are the well-known catalytic inhibitors of the DNA gyrase [12][13][14][15][16][17][18][19]. An elegant evidence was provided by Gilbert and Maxwell [12] that a 24 kDa sub-domain located near the Nterminal of DNA gyrase B enzyme possessed the binding site towards coumarin antibiotics.
They also suggested that the interaction of coumarins with the protein was predominantly hydrophobic in nature [14].
For carrying out meaningful and productive research on these lines, it is of paramount importance to explore the specifi c residues of Gyrase B which play a crucial role in maintaining its functionality and stability, and also to know which compounds disturb the same. This kind of approach would make it possible to target DNA gyrase of the drug resistant, pathogenic E. coli, that has become resistant to older antibiotics targeting their cell wall, nucleic acid or protein synthesis etc.
Compounds belonging to three families namely quinolones such as norfl oxacin, cyclothialidines and caumarins (eg. Clorobiocin and novobiocin) have been reported to inhibit gyrB (14). A number of other compounds are also reported in the literature, which inhibit Gyrase B.
Important amino acids for the ATP hydrolysis in E. coli gyrB as reported in the literature include Tyr5, Ile10, His38, Glu42, Asn46, Glu50, Asp73, Arg76, Gly77, I78, P79, K103, Arg136, Thr165, Asp426 and Lys447 [26][27][28]. Tyr5 and Ile10 help the process of dimerization in gyrB so to increase binding affi nity and also to double the length of DNA site bound protein as well as to increase binding specifi city. Some dimer interface residues are important and mutations of these residues result in loss of enzyme catalysis [27]. Glu42 helps in hydrolysis process and His38 in ATPase reaction, acting to orient and polarize glutamate residue in gyrase B [10]. Substitutions with Alanine at Glu42, Asn46, Glu50, Asp73, Arg76, Gly77, Ile78 resulted in decreased or not demonstrable ATPase activity, signifying their importance in ATP hydrolysis [26]. Pro79 and Lys103 are reported to be important in the coupling of ATP hydrolysis and DNA unwinding and mutations in Asp73, Gly77, Ile78, and Thr165 lead to resistance to novobiocin [28]. Lastly, Asp426 and Lys447 are described to be the part of a quinolone-binding pocket of gyrB [26].

PHARMACOLOGY | THERAPEUTICS
possibly through the activation of angiotensin converting enzymes 1-7 as studied in the rats appears promising [30,31]. It upturns ACE2 and AT 2 receptor expression in the kidney cells in the type1 diabetes rat model and inhibits nephropathy [32]. It has also been reported to completely inhibit the topoisomerase I in Caenorhabditis elegans, though at high concentration of 125 μM [33] and was reported to dock successfully with Chikungunya Virus RNA polymerase [34]. To further support its therapeutic potential in diff erent conditions Oliveira, et al. [35] reported Computer-Aided Drug Discovery (CADD) is one of the most powerful approach to investigate drugs that would act upon the novel targets of pathogenic bacteria [36][37][38]. Correctly termed "Drug Repurposing", this strategy is indeed proving to be very productive, quicker, and more practical towards fi ghting the menace of multiple drug resistant pathogenic bacteria in general [39]. This in silico study was undertaken to analyse the interactions of the GyrB subunit towards a number of inhibitors, in an attempt to fi nd a potential lead against MDR E. coli.

Receptor preparation
Target protein and important residues were described through the thorough literature search.  (Figure 1a). An 'A' here with the residues signifi es the chain A recognized in the PDB structure. Three water molecules (HOH1001, HOH1066, HOH1160) with at least 2 interactions were included in the receptor and were made freely rotatable.

Ligand selection, docking and lead selection
Docking protocol as per Ghildiyals, et al. [34] was followed for the initial screening of molecules with some

Re ining docking
The autodetected docking pocket was altered by removing certain non-conserved residues and to take account of some more of the crucial conserved residues, without upsetting the core pocket's integrity. We used PyMOL for visualizing        conserved nature of the selected important residues and also the other interacting residues ( Figure 5). This signifi es our results as diminazene aceturate is predicted to have broad spectrum activity against gyrB including that of EKAPE gram negatives pathogens that are posing utmost challenges in the healthcare facilities because of their multidrug resistance.

CONCLUSION AND FUTURE ENHANCE-MENT
Outcomes from our study specify the repurposing potential of a very simple molecule like diminazene aceturate as a DNA gyraseB antagonist. Our results indicate that Dwivedi