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In-silico Druggability Studies of 4-hydroxy-α-tetralone and its Derivatives with RND Efflux pump of E. coli Pharmaceutical and Biosciences Journal

The compound 4-hydroxy-α-tetralone ( 1 ) has been reported to possess potent anti-tubercular, anti-diabetic and anti-leishmanial activities. In our earlier studies the compound 1 and its various semi-synthetic derivatives showed potent bioenhancing activity in combination with nalidixic acid (NAL) and tetracycline (TET) reducing the minimum inhibitory concentration (MIC) of antibiotics up to 8 folds by inhibition of ABC efflux pump. However, in gram negative bacteria, resistance nodulation division (RND) family are considered as major efflux pump responsible for multidrug resistance (MDR). Hence, the current study was carried out to access the in-silico docking potential of compound 1 and its cinnamoyl ( 1a ), 3, 4, 5-trimethoxybenzoyl ( 1b ) derivatives against RND efflux pump target proteins AcrA, AcrB, TolC of E. coli . The docking study showed that the test compounds have good binding affinity with target proteins. The derivative 1a showed highest interaction with AcrA followed by AcrB showing binding energies -8.7 and -8.2 kcal/mol respectively. The low molecular weight ≤500, high hydrogen bonding, high log p value (>1) with hydroxy, methoxy and aromatic group of ligands make these compounds as effective efflux pump inhibitor. In drug likeliness studies, these compounds pass the safety criteria with enhanced bioavailability and absorption, less acute oral toxicity, less hepatotoxicity. This study promises that the compound 1 and its derivatives ( 1a & 1b ) might be RND efflux pump inhibitors providing the initial platform for development of safer and cost-effective antibacterial drug to manage MDR infections.


Introduction
The drug discovery and development programme is complicated process involving multidisciplinary, multi-sector cooperation and regulations of the United States Food and Drug Administration (USFDA) which ultimately makes it time consuming and expensive. In general, the process of drug discovery involves target identification, validation, target and cell-based screening, lead compound identification its optimization, preclinical and clinical trials, FDA approval and marketing 1,2 .
In recent years the computer aided drug design (CADD) has become a powerful tool which certainly has accelerated the drug discovery process by minimizing the time as well as reducing the cost 3,4 . The druggability of the lead molecule can be assessed in silico by homology modeling, docking, multi-target searching & design, pharmacophore development, conformation generation and quantitative structure activity relationship (QSAR) for the optimization of novel molecules with affinity to a target by the pharmacokinetics, absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization of phytomolecules 5 plants being used in folklore remedy continue to be of great interest in drug discovery because of better biologically relevant chemistry, hence lesser side effects 8 . The plant based bioactive leads find either direct use as drug or as novel chemophore which may be converted into druggable compounds by chemical transformation 9,10 . However, the recent reports on emerging and sustained resistance to antibiotics and depleted pipeline of new antibacterial are creating a major health issue worldwide 11,12 .
Over the years, continued selective pressure by different antibacterial drugs has resulted in emergence of new strains of organisms that are multidrug resistance (MDR) 13 . In view of drug resistance and lack of novel antibiotics, WHO listed 12 human pathogenic bacteria (dirty dozen) including Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species which urgently require novel antibiotics 14 . In spite of recent advances in therapy, the diseases caused by them cause over 50% of deaths in hospital settings 15,16 . Moreover, the economic impact of 'superbug' outbreaks could top $100 trillion; low-income countries would suffer disproportionately 17 . WHO estimates the global typhoid fever disease burden at 11-20 million cases annually, resulting in about 128,000-161,000 deaths per year 18 .
The resistance among common pathogens causing community and hospital-associated infections has increased significantly worldwide, though regional patterns of resistance vary 15,19,20 .
Several drug resistance mechanisms such as target alteration, drug inactivation, decreased permeability and increased efflux has been reported, among these multidrug efflux pumps are supposed as key players for MDR 21,22 . These active efflux pumps of pathogenic bacteria extrude structurally unrelated group of drugs. In prokaryotes, five different types of efflux pumps are reported namely major facilitator (MF), multidrug and toxic efflux (MATE), resistance-nodulation-division (RND), small multidrug resistance (SMR) and ATP binding cassette (ABC) [23][24][25] . Amongst these, RND efflux pumps extrude wide range of compounds 18 . In the post genomic era, these efflux pumps are promising drug target 26 . However, there is major gap in developing strategies to control the spread of drug resistance caused by efflux pumps.
The compound 4-hydroxy-α-tetralone is a marker compound of genus Ammannia and has been reported to possess potent antitubercular, anti-diabetic and anti-leishmanial activities [27][28][29] . In our earlier studies 4-hydroxy-α-tetralone and its various semisynthetic derivatives showed potent bioenhancing activity in combination with nalidixic acid (NAL) and tetracycline (TET) reducing the minimum inhibitory concentration (MIC) of antibiotics up to 8 folds by inhibition of ABC efflux pump 29,30 .
However, in gram negative bacteria, RND family is considered as major efflux pump responsible for MDR. This prompted us to carry out the in-silico docking studies of 4-hydroxy-α-tetralone and its semi-synthetic derivatives with efflux pump proteins to assess statistical parameters for screening test set of RND efflux pump of E. coli.

Drug likeliness profiling
The drug likeliness profiling of the test molecules was studied by  33 . The secondary structural characteristics of the proteins were also studied to understand the factors involved in folding and functioning of proteins (Table 3). It was observed that α-helices are dominating the composition with respect to the random coils.

Analysis of proteins
TolC has the highest percentage of α-helices which might be a factor responsible for the extrusion of antibiotics at transmembrane regions of pathogenic microorganisms 34,35 .

Drug likeliness properties
It has been seen that most drug candidates fail clinical trials because of unsatisfactory pharmacokinetics. The in silico screening of drug likeliness properties of the lead molecules is largely helpful to overcome such problem. In our study, the parameters used for in silico screening were absorption, distribution, metabolism, excretion and toxicity (ADMET). The results indicated that the drug likeliness properties of these compounds are within the domain of applicability to be considered as potent drug candidates (Table 4).

Molecular docking studies
In gram negative bacteria, RND families are considered as major efflux pump responsible for MDR. The current study was carried out to access the in-silico docking potential of compound 1 and its semi-synthetic derivatives (1a & 1b) against RND efflux pump target proteins AcrA, AcrB, TolC of E. coli. The docking study showed that the test compounds have good binding affinity with target proteins ( Table 5).
The derivative 1a showed highest interaction with AcrA followed by AcrB with binding energies -8.7 and -8.2 kcal/mol respectively.
However, the binding affinity of 1a was less in case of TolC protein of E. coli. The interaction of amino acids with the ligands is given in table 6. The parent compound 1 and the derivative 1b showed same binding affinity with AcrA protein (binding energies -7.3 kcal/mol) while the binding affinity of 1b was more than parent compound 1 for the target protein AcrB.
From the results obtained it is noteworthy to observe that all the compounds are interacting well with amino acids of receptor proteins (Fig 2); however, due to presence of aromatic amino acids in receptor AcrB the ligands bind more efficiently with AcrB.
The protein AcrB has different group of amino acids which are helpful in interaction through H-bonding as well as hydrophobic interaction. However, both in-vitro and in-vivo validation of results are required to establish these molecules as novel drug resistance reversal agent against critical bacterial superbugs.

Conclusion
The docking studies of 4-hydroxy-α-tetralone (1) and its semi- Finally, our continued efforts on 4-hydroxy-α-tetralone and its semi-synthetic derivatives indicate that these compounds are able to inhibit a variety of efflux pumps responsible for drug resistance in critical superbug, creating a new platform for development of cost-effective antibacterial combinations for the management of MDR infections.

Conflict of interest
The authors declare that there are no conflicts of interest among us.

Acknowledgements
Authors thank to Director of their respective institutes for providing necessary facilities, support and co-operation in the study.