In silico Screening of Approved Drugs to Describe Novel E. coli DNA Gyrase A Antagonists

Rakhi Chandran1, Archana Ayyagari2, Prerna Diwan3, Sanjay Gupta4 and Vandana Gupta3* 1Department of Biotechnology and Microbiology, School of Sciences, Noida International University, Yamuna Expressway, Gautam Budh Nagar, UP-203201, India 2Department of Microbiology, Swami Shraddhanand College, University of Delhi, New Delhi-110036, India 3Department of Microbiology, Ram Lal Anand College, University of Delhi, New Delhi-110021, India 4Independent Scholar (former Head and Professor, Department of Biotechnology, Jaypee Institute of Information Technology, Sector 62, Noida, UP-201309, India)


INTRODUCTION
Escherichia coli, a common intestinal pathogen, is known to cause gastroenteritis and a variety of extra-intestinal diseases, such as Urinary Tract Infections (UTIs), meningitis among newborns, colisepticemia, and skin and soft tissue infections [1,2]. E. coli infection is also reported to be responsible for several post-operative abscesses and other complications such as neonatal sepsis [3,4]. It has been developing more and more resistance towards the available antibiotics. Extended- Spectrum -Lactamase (ESBL) producing E. coli (ESBL-Ec) has recently gained much importance as a common cause of contagious nosocomial and community acquired infections in India and abroad, because it resists treatment with almost all the -lactam antibiotics including three generations of cephalosporins [5]. Multiple surveys recorded the highest rate of ESBL-Ec in India (80%), followed by China (60%), and less than 30% in East and Southeast Asia. In Europe, Australia and North America it ranges between 5-10%. Further resistance to the advanced antibiotics like carbapenems due to the production of carbapenemases/ New Delhi Metallo -lactamases (NDM) among these pathogens has rendered treatment of such infections extremely challenging. During the last decade, the preventive measures followed to curb such infections have not proved to be adequate to prevent the rapid spread of resistant Gram-Negative Bacteria (GNB), particularly extended-spectrum -lactamase producing Escherichia coli [6,7]. Approximately 67% of E. coli isolates from extra-intestinal infections are reported to be multidrug-resistant, of which up to 85% producing ESBL and 6% producing NDMs. These enzymes are responsible for inactivating -lactam and carbapenem antibiotics commonly used in treating E. coli infections [1].
A single Extra-Intestinal Pathogenic strain Escherichia coli (ExPEC) clone, named Sequence Type (ST) 131, is the cause of millions of drug-resistant infections annually [1,8]. NDM was fi rst described by Yong, et al. [9] in a Swedish national who fell ill with Klebsiella infection, acquired in New-Delhi.
Similar trends were subsequently reported for other gramnegative pathogens including Proteus vulgaris, Serratia marcescens, Enterococcus, etc. Coupled with the increment in antibiotic-resistant bacteria, the extremely slow speed of newer approved antibiotics for treatment, several infectious diseases are not being addressed successfully [10].
These astonishing global health threats call for urgent accelerated research into fi nding novel and more eff ective therapeutic options for such infections. Available options for the same encompass either newer antibacterials or newer strategies to target such drug-resistant microorganisms.
Given this challenging scenario, there is an urgent need to look for either newer target components of the bacterial cells or fi nd novel inhibitors of the older ones.
Conventionally, all anti-infection agents are used to target certain pathways within the pathogenic bacteria, such as cell wall production, nucleic acid synthesis, protein synthesis, and folate synthesis [11]. However, over a span of time, indiscriminate, excessive, and improper usage of these has led to astonishing unresponsiveness of these bacteria towards the same antibiotics, translating into the emergence of drug resistant mutant bacteria [12,13]. This very fact necessitates a paradigm shift in our focus towards unconventional targets presents within bacterial machinery.
DNA gyrase is one such important enzyme that introduces negative supercoils in DNA, classifi ed as topoisomerase type 2 that controls DNA topology in proper form during its replication and transcription as well as during cell division.
The native DNA gyrase (370kD protein) comprises two types of subunits gyrase A (gyrA) and gyrase B (gyrB) with 875 and 804 residues respectively [14]. Its active form is made up of a hetero-tetramer complex A 2 B 2. It possesses various molecular interfaces named N-gate, DNA gate, and C-gate which assist in strand passage and DNA binding in a specifi c manner. GyrA function is to break and re-join DNA and GyrB function is to hydrolyze ATP to provide energy for the DNA unwinding. DNA gyrase is known to be targeted by catalytic inhibitors such as aminocoumarins, or 'poison' such as quinolones. Several citations suggest that the known inhibitors of DNA gyrase mostly dock onto amino acids located near the amino-terminal of the gyrA. Gepotidasin, a novel inhibitory antibiotic is demonstrated to interact with the complete E. coli DNA gyrase nucleoprotein complex [15]. Similarly inhibition of gyrA by 7-oxy-4methyl coumarinyl amino alcohol derivatives 17 and 18 [16], 4,5,6,7-tetrahydrobenzeno (1,2-d) thiazole-2,6 diamine, 2-(2-aminothiazol-4-yl) acetic acid and benzol (1,2,-d) thiazole-2,6-diamine) [17], fl uoroquinolones with alkylamine, alkylpnthalimide, and alkylphenyl groups introduced at N-1 position [18] and 4,5,6,7 tetrahydrobenzo(d)thiazole [19] has been reported.
Alanine substitutions at Asp87 and Ser83 lead to DNA cleavage, DNA supercoiling, and resistance to diff erent quinolones [14,20,21], Lys42 stabilize interaction with ciprofl oxacin [14], His45 and Arg91 are involved in binding to nalidixic acid [22], deletion of Tyr122 leads to the change in the conformation of the active site of enzyme aff ecting its interaction with neighboring amino acids and binding of ciprofl oxacin to gyrase [23] while Asp424 is crucial to the DNA cleavage at the active site of gyrA and is involved in a conformational change, while others are either active site residues or are involved in binding to simocyclinone D8 [14].
The in silico research is very promising in this direction and also is much quicker. Further, to make it more rewarding and fruitful for successfully dealing with the emergence of AMR in the bacterial pathogens, these studies could be combined with the stimulating possibility of repurposing the already available drugs, indicated for other ailments [24]. Various computational tools may be used for in silico screening of libraries of these approved drugs. Scaff old hopping of the top leads from each protein without altering the ADME properties signifi cantly helps in further optimizing these leads. Molecular docking paves the way quite eff ectively towards establishing a quantitative structure-activity relationship between the antibacterial and the target enzyme of infecting pathogens [25]. concerning their active sites and inhibitor binding residues, and an attempt was made to explore the potential of existing FDA Approved drugs to be repurposed as DNA gyrase inhibitors. Such a piece of work certainly holds a paramount potential for competent healthcare and well-being of the society at large, and therefore, is worth attempting.

MATERIALS AND METHODS
To accomplish the stated goals, an extensive literature search was carried out and many target proteins were

RESULTS AND DISCUSSION
The crystal structure of the DNA gyrA N-terminal domain 4CKL is a 55kD dimer with the co-crystallized ligand SD8. The structure is resolved at 2.05 Å. As the interface between Lys42 forms polar contacts and helps in protein stability in association with ciprofl oxacin a fl uoroquinolone gyrase inhibitor and mutation lys42Ala also results in resistance to SD8. His45 and Arg91 are involved in the interaction with nalidixic acid and SD8 [14,26]. Whereas Ser83, Ala84, and Asp87 are involved in drug binding and are considered as hot spots for quinolone resistance mutations [14,20,21]. Alanine substitution at these positions leads to DNA cleavage, DNA supercoiling, and resistance to diff erent quinolones, hence implying the importance of these residues in the functioning of gyrA [20,21,26].
In silico studies revealed that the docking site created   Table 1: Residues of the docking pocket and the signifi cance of crucial residues in the pocket [14].      the aminocoumarin moiety of the reference ligand SD8 also binds. None of the selected leads docked at the pocket at the interface of the two monomers in this 55kD N-terminal domain homodimer of gyrA where the polyketide moiety of SD8 was reported to be binding [14]. The binding site and affi nity of polyketide moiety change if the 55kD partial gyrA protein is replaced with 59kD partial gyrA protein, whereas the binding of aminocaumarin moiety remains essentially the same [14]. This signifi es our results as in native tetrameric gyrA there is a high probability of the retention of the integrity of the aminocaumarin binding pocket, where all our lead molecules are apparently binding. Moreover, as the aminocaumarin moiety of SD8 interacted with Lys42, His45, Arg91, and Ser172, the leads molecules identifi ed in our study also interacted with at least two to all four of these residues along with other crucial residues, signifying the distinction of our study.

Residues involved in quinolone/
In comparison to the docking score of the reference ligand SD8 which is calculated to be -15.16 using Flex-X, lead molecules identifi ed in our study demonstrated much lower scores and hence stable docking. The not so good docking score with the reference ligand could be attributed to the stearic bumps observed when we analyzed the co-crystallized structure 4CKL and the top scored docking pose we obtained with Flex-X (Figure 4a  Further, to target ESBL-Ec, which are resistant to most of the generations of cephalosporins by virtue of production of ESBL, that can cleave the six-carbon beta-lactam ring in cephalosporins, we suggest detailed QSAR analysis of the selected leads. The leads can be optimized by fragment building and/or replacement to enhance their bioactivity and resistance to cleavage by ESBL. The addition of active groups from cephalosporins to non-cephalosporin leads, and also to the fl uoroquinolones and vice versa to create a new library of chemically synthesizable molecules, and validation of these hybrid molecules will possibly lead to a newer class of gyrA inhibitors. Alternatively, simple in vitro validation using antibiotic sensitivity assays could be planned for diff erent combinations of lead drugs to formulate newer concoctions in a pursuit to combat the drug-resistant E. coli.

CONCLUSION AND FUTURE ENHANCE-MENT
CADD in conjunction with drug repurposing has already resulted in the worthwhile possibility of second medical use for many drugs, as is quite evident from the trends towards the discovery of eff ective therapeutics to SARS-CoV-2 to curtail the ongoing COVID-19 pandemic (reviewed by Singh and Gupta, unpublished) [28]. In this research, 12 repurposed drugs with reasonably good binding scores are reported. Out of these, six are cephalosporins and the rest are rather simple molecules like the antibiotic azlocillin, tetrahydrofolate, ATP, 1,4-dihydroNAD, and leucal. Hence, this study has widened the scope of developing promising leads in signifi cantly less time and cost for the most part anti E. coli compounds. Accurate in vitro studies could be planned in the future based on CADD studies and their outcome may be expected to be fruitful to mankind.

ACKNOWLEDGEMENT
This research did not receive any specifi c grant from funding agencies in the public, commercial, or not-for-profi t sectors but we would like to acknowledge research grant No. RLA-202 (2013-14) received under the innovation project scheme of the University of Delhi for the purchase of docking software and other utilities. We acknowledge Ram Lal Anand College for providing all the support required in carrying out this work.