A Computational Approach on the Anti-biofilm Effect of Ocimum sanctum Bio-compounds Against ptk of Acinetobacter baumannii

Introduction: Acinetobacter baumannii is a gram negative coccobacilli often considered as a nosocomial pathogen and as an opportunistic pathogen in immunocompromised patients. It is considered to be multi-drug resistant and a potent bacteria forming vital biofilms. Ptk which is protein tyrosine kinase is a protein coding gene involved with the synthesis of capsular polysaccharide. Ocimum sanctum is a perennial plant belonging to the Lamiaceae family. Tulsi and holy basil are the common names of this plant. In-silico docking approach method is much more convenient and cost effective to assess the bioactive properties of the natural drugs against any target ligands. Aim: The aim of the study to assess the inhibitory effect of Ocimum sanctum bio-compounds against ptk of Acinetobacter baumannii using a computational approach. Materials and Methods: Retrieval of the structure of ptk was followed by Ligand preparation and optimisation. Further drug likeliness was assessed using Molinspiration parameters, docking simulations and visualisation for the binding energy and hydrogen bonds. Original Research Article Kamalli et al.; JPRI, 33(58A): 91-103, 2021; Article no.JPRI.74341 92 Results: Among the bio compounds of O.sanctum, benzofuran is selected as an active inhibitory compound with -11.12 as its binding energy showing a high affinity. Conclusion: The findings of the present study documents benzofuran as the promising candidate to design novel drugs from O.sanctum and to target the ptk of A.baumannii. However further experimental validation must be done to observe its efficacy and safety in the treatment of nosocomial infections caused by A.baumannii.


INTRODUCTION
Acinetobacter baumannii is gram negative coccobacilli and may be considered as a hospital derived nosocomial pathogen and sometimes can be considered as an opportunistic pathogen in immunocompromised patients. A.baumannii is considered to be among the top six drug resistant microbes [1]. The antimicrobial effect of different molecules can be used for targeting various nosocomial diseases [2]. These molecules also find insight in implications to enhance immunity [3]. Many modifications were recently found involved in the regulation of such as inflammatory and antitumor immune responses, antiviral immunity [4]. Variations in the human genetic system are proven to affect disease progression and prognosis of the diseases [5]. Exosomal microRNAs were found to be a promising tool in diagnosis of various systemic conditions [6]. Removal of pathogens from the site of infection remains a confusing task which requests the use of antibiotics [7]. A.baumannii is considered to be multi-drug resistant and a dangerous bacteria forming biofilm. A.baumannii models a unique property to maintain and exhibit a multidrug-resistant phenotype, further leading to complicating treatment [8]. The natural habitat of the microbe is still not known. Prolonged hospital stay, weak immune system, chronic lung diseases, illness that requires use of hospital catheters and ventilators, forms some of the base etiological factors for the disease caused by A.baumannii. Its ability to survive on the artificial surfaces and resistance to desiccation and hospital environment is suspected to be favourable for the growth of A.baumannii due to constant use of antibiotics. Open wounds, catheters and breathing tubes pave the way for the entry of the microbe. Symptoms of this infection include pneumonia, meningitis, necrotising fasciitis and UTI infections. Previous studies stress the fact that molecular mechanisms guide the antimicrobial potential of drugs against complex pathogens [9]. There are a wide range of virulence factors exhibited by A.baumannii such as phospholipases, outer membrane proteins, lipopolysaccharides, hemolytic factors, elastases and many more amongst which ptk gene is taken into an account. Ptk gene is taken as a gene of interest as it is a potent virulence factor of A.baumannii. The ptk gene which is protein tyrosine kinase is a protein coding gene involved with the synthesis of capsular polysaccharide. It is concentrated in local adhesions between cells growing in an extracellular matrix. Biofilm formation is one the important features of A.baumannii due to the existing niche and the chemical nature of antimicrobial agents. A.baumannii shows a variety of molecular mechanism actions which includes such as mutations, membrane permeability variations [10]. The microbe has stealthily entered the oral cavity and acts as a potential pathogen by expressing various virulence factors [11].
As Siddha and Ayurveda are a vital part of Indian medicine, an arena of natural plants and herbs are used as antimicrobial agents against many microbes. These natural plants and herbs can be converted into pharmaceuticals and commercialized as they are easily available in abundant quantities. In a similar manner, here Ocimum sanctum commonly known as tulsi, the queen of herbs is considered to be the herb of interest. Ocimum sanctum is a perennial plant belonging to the Lamiaceae family. Tulsi and holy basil are the common names of this plant. It is considered for its aroma, traditional medicinal properties. It is a many branched subshrub with green leaves and strongly scented. This plant is widely used in day to day practice because of its easy availability. It is useful in the treatment of many diseases such as bronchitis, malaria, skin diseases and many more. Lately, it is also suggested for possessing antifertility, anticancer, antifungal, antimicrobial actions. The chemical constituents of tulsi consist of oleanolic acid, ursolic acid, eugenol, linalool, caryophyllene. The benzene extracts of various parts of the plant is useful in curing various ailments and eugenol which is one of the main chemical components acts on the immune system [12]. Tulsi essential oil acts as a valuable topical antimicrobial agent for management of many skin diseases [13]. Experimental validation can be time consuming, expensive and requires a lot of sources. This study is thus achieved with a computational approach for identifying each compound-ligand interaction and it is made easier. In-silico docking approach method is much more convenient and cost effective. The purpose of this practice is to give a tinge of how docking works to identify small flexible molecules to enormous protein structures. This method is extremely useful for finding potential binding sites and to discover novel molecules that possess the capacity to bind to a known site. Virtual screening and docking are employed in order to discover new medicines. The knowledge and expertise gained from the previous literature have been incorporated in the study design of this investigation. In-silico based computational approaches holds promising for the detection of bio-active compounds from that are efficient against drug resistant strains [14], from natural sources [15], synthetic disinfectants [15], modelling of novel proteins [16] and from marine sources [17,18] as well. A.baumannii is selected as our study strain as it is considered as a major nosocomial pathogens causing reclacitrant infections and is often multi-drug resistant ]19,20]. In-silico based efficacy studies and homology modelling of the biocompound structures holds promising in determining the invitro studies [14,15]. Our team has extensive knowledge and research experience that has translate into high quality publications [16][17][18][19][20] The aim of this study was to identify the inhibitory effect of Ocimum sanctum bio-compounds against ptk of Acinetobacter baumannii using a computational approach [21,22].

Study Setting
This is an observational in-silico study done in the Department of Microbiology, Saveetha Dental College and Hospital. Institutional approval for the research was obtained (SRB approval number: IHEC/SDC/UG-1907/21/158).

Retrieval of Structure of ptk Gene
The sequence of PTK from Acinetobacter baumannii was retrieved from NCBI database and the Biovia discovery studio visualiser was used to view the three dimensional structure of ptk gene [23]. The structure was not available in protein databank. Thus it was modeled using Swissmodel server using the template 3LA6 -A Chain.

Ligand Preparation and Optimisation
The structures of the bio-active derivatives of Ocimum sanctum were retrieved from the Pubchem database. The generated 3D structures were then optimised. 2D structure was drawn and optimized using ACD Chemsketch and saved in .mol format and converted to .pdb format using Open Label molecular converter tool.

Mol-inspiration Assessment of the Molecular Properties of the Selected Compounds
The counts of hydrogen bond acceptors and donors in correlation to the membrane permeability and bio-availability of the compounds.
The n-violation values of bioactive compounds are 0 satisfying Lipinski's Rule of 5. TPSA is a very useful descriptor used to characterize drug absorption and bioavailability, permeability through Caco-2 cells and transport across blood brain barriers. The characteristics of absorption, distribution, metabolism and elimination of the bio compounds of Ocimum sanctum were further analysed on the basis of ''The Lipinski's rule of five".

Docking Simulations
The Auto Dock tool was used for docking analysis to interpret the affinity between biocompounds of Ocimum sanctum against ptk of A. baumannii.

Docking Visualisation
Using Biovia Discovery Studio Visualizer, the hydrogen bond interaction between biocompounds of Ocimum sanctum against ptk of A. baumannii were visualised. Further docking score assessments, binding affinities, molecular dynamics and energy simulations, the relative stabilities hydrogen interactions 2D diagram between ptk gene and bio compounds were evaluated.

Structural Retrieval of ptk from A. baumannii
The 3D structure of ptk gene was retrieved from Biovia discovery studio visualiser (Fig. 1). The sequence of PTK from Acinetobacter baumannii was retrieved from NCBI database and its sequence Id was A0A171EWN0. The structure of PTK was not available in the PDB database.
The modeled structure was found to be highly plausible as it had 44.53% sequence identity with that of the template. Moreover, the Ramachandran plot also showed 89.5% of residues in most favored regions and with no residues in disallowed regions (Fig. 2).

Structural Retrieval of the Ligands from Ocimum sanctum Bio Compounds
Chemsketch was used for retrieving the 2D, 3D structures, its SMILES format of the ligands from Ocimum sanctum as shown in (Table 1).

Drug properties by Molinspiration Assessments
The bioactivity score prediction, molecular weight, hydrogen bonds, and rotatable bonds of essential compounds of Ocimum sanctum against ptk gene of A. baumannii towards drug likeness was assessed and tabulated (Table 2).

Docking Analysis for the Drug-ligand Interactions Against ptk of A. baumannii
The bond interactions between the essential compounds from Ocimum sanctum and ptk gene of A. baumannii are shown in (Fig. 3). The ptk gene interactions with compounds from Ocimum sanctum are shown in (Table 3). The number of hydrogen bonds, torsional energy and overall docking scores between the ligands and the drugs were evaluated (Table 4). The docking energies and interactions between the ptk gene and the Ocimum sanctum biocompounds were evaluated based on the Hydrogen bonds interactions, van der Waals interactions, π-r interactions/ amide-π stacked interactions/ πcation interactions, alkyl/π-alkyl interactions and π-sulfur interaction.

DISCUSSION
A.baumannii has the ability to form biofilm by four steps which is attachment to the surface followed by formation of micro colony, maturation and colonisation. PTK is a potent virulence factor of A.baumannii. Previous research studies lack specific documentations on PTK. Computational based approaches seem to be of higher value in assessing the drug ligand interactions based docking studies. In a previous study, csgA protein of A.baumannii and the anti-biofilm activity of A.indica was assessed and predictions of epitope peptides against the virulent protein of A.baumanni has also been assessed [24,25]. However no earlier studies had documented the inhibitory effect of Ocimum sanctum biocompounds on ptk of A.baumannii.
Selection of essential compounds from Ocimum sanctum was based on references from earlier literatures [12]. Eugenol which is an active and main constituent was found to occupy the major percentage of therapeutic use of Tulsi [26]. The phenolic compounds of the herb were wellestablished with the help of spectroscopic methods leading to the identification of new compounds which had antioxidant and cyclooxygenase inhibitory activity [27]. The pharmacological actions of Ocimum sanctum can be converted into standardized medicinal products which can be commercialised [28].
A.baumannii being a multidrug resistant and A.baumannii being a multidrug resistant and invasive pathogen, there are enormous possibilities for bio-compound interaction with the same that can be converted into drugs of pharmaceutical use [29]. Carbapenemases which is one of the enzyme components of A.baumannii was discovered to increase and transform the species which paves way for identification of more potential medicines [30]. Routine therapy enables the application of only fewer antibiotics as the species develops resistance towards many drugs. A.baumannii exhibited resistance by both phenotypic and genotypic characterisation methods [31].
In overall docking energies, ceftazidime has the highest number of hydrogen bonds and has highest avidity, however violating the Lipinsky's rule. Estragole has the lowest number of hydrogen bonds which shows that it has low avidity. The binding energy of ligands were analysed in the overall docking energies. The compound which possesses more negative value is said to have more affinity. In that case, amongst the bio-compounds of O.sanctum, benzofuran which is an active compound has -11.12 binding energy which shows that it has highest avidity. Citral has -5.18 binding energy which shows it has low avidity.
When the molecular weights of all the compounds were taken into account, ceftazidime had the highest molecular weight with 546.59 whereas methyl-eugenol had the lowest molecular weight with 18.47. Remaining compounds were found to have molecular weight ranging between 140 to 375. Resistance to ceftazidime is common among the clinical strains [32], thus suggesting the natural bio-compounds as the alternative source for treatment.
The TPSA value which is Topological Polar Surface Area acts as a major factor in deciding the importance of a bio-compound as it evaluates the oral bioavailability of drugs. The value should be <140Å. In the present study, it is notable that almost 6 out of 7 compounds have TPSA value less than 140 Å where ceftazidime has 191.23 Å. In the study by Sivaharini et al., caffeic acid had TPSA value < 140 Å and it was found to have best oral bioavailability [33]. Computational based approaches thus hold good to predict the oral availability of the compounds to be designed as novel drugs [34].

CONCLUSION
The present study is undertaken to evaluate the inhibitory effect of the bio-compounds selected from O.sanctum. Computational approach on the same, documents the promising inhibitory effect of benzofuran that can efficiently target the ptk of A.baumannii. However further experimental validation must be done to observe its efficacy and safety in the treatment of nosocomial infections caused by A.baumannii.

NOTE
The study highlights the efficacy of "Siddha and Ayurveda" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

FUNDING SOURCE
The authors are grateful supported by Saveetha Institute of Medical and Technical Sciences