Virtual Screening to Identify the Protein Network Interaction of Theophylline in Red Complex Pathogens

Background: Plants, herbs and plant derived compounds have been used since decades in folk and traditional medicine. These compounds were found to be non-toxic and compatible with the human cells. Screening of this exhaustive collection of compounds seems to be a herculean task. Hence, virtual screening methods have been developed to predict the potential targets of these compounds enabling researchers to acquire preliminary data on the compound intended to be tested in vitro. Aim: The present study aims to identify protein targets of theophylline in red complex pathogens which are mainly associated with periodontitis. Methods: The STITCH v5.0 pipeline was mainly used to classify drug-protein interactions, while VirulentPred and VICMPred were used to determine the proteins' virulence properties and Original Research Article Amrithaashri et al.; JPRI, 33(64B): 374-381, 2021; Article no.JPRI.74463 375 functional classes. PSORTb v3.0 was used to determine the subcellular localization of viral proteins, and BepiPred v1.0 Linear Epitope Prediction was used to classify epitopes. Results: Proteins of red complex pathogens implicated in cellular process, metabolism, and virulence were identified to interact with theophylline. The virulent proteins attacked by the drugs were present in the cytoplasm, which would improve the drugs' antimicrobial efficacy even more. Finally, epitope prediction showed a number of epitopes in virulent proteins that can be targeted. Conclusions: Proteins involved in the cellular process, metabolism, and virulence of red complex pathogens targeted by theophylline were identified. To substantiate the true interactions between the drugs and the protein arsenal of pathogens, further in vitro experiments on a broad range of pathogens are required.


INTRODUCTION
Antimicrobial drug resistance has emerged as a great menace in the hospital settings. Novel drugs are being screened for their activity against microbial pathogens. One common and the most cost effective method of drug screening is using silico tools. Several studies have been designed previously to assess the role of bioactive compounds against common dental pathogens [1][2][3] In view of the facts, existing bioactive compounds are being tested to identify the potential targets in common pathogens. Theophylline is a bioactive component in black tea (Camellia sinensis). It has been found in green coffee beans and trace amounts were detected in cacao cotyledon (https://www.ncbi.nlm.nih.gov/books/NBK507021 /). Several existing antibiotics or non-steroidal anti-inflammatory drugs have been repurposed for use against dental pathogens [4]. Singh and team reported that theophylline possesses antifungal activity against Candida albicans.Theophylline has no discernible presystemic elimination, readily distributes into fatfree tissues, and is substantially processed in the liver. Renal excretion of unaltered theophylline in newborns is around 50% of the dosage, compared to around 10% in children older than three months and adults [5].
The mode of action was damage to the membrane exerted by the elevation in flippase activity which resulted in membrane fluidity. Theophylline relaxes the smooth muscle of the bronchial airways and pulmonary blood vessels, lowering airway reactivity to histamine, methacholine, adenosine, and allergens [6]. Theophylline inhibits type III and type IV phosphodiesterase (PDE), the enzyme responsible for breaking down cyclic AMP in smooth muscle cells, perhaps leading to bronchodilation. Theophylline also binds to the adenosine A2B receptor, preventing adenosineinduced bronchoconstriction [7]. Theophylline stimulates histone deacetylase in inflammatory situations to block transcription of inflammatory genes that require histone acetylation to initiate transcription. Another study conducted by Borkowski et al., 2016 demonstrated the cytotoxic effects of theophylline based ionic liquids towards Bacillus cereus and Escherichia coli. These results provide evidence on the use of theophylline as a potent antimicrobial drug. But the lacunae which is encountered is the information on the molecular targets [8]. The aim of this study is to identify protein network interaction of theophylline in red complex pathogens using virtual screening. Our team has extensive knowledge and research experience that has translate into high quality publications [9][10][11][12], other articles about cytotoxic effects like [13][14][15][16] studies are also done about antioxidant [17][18][19][20], other studies about antibacterial activity was also done [21][22][23][24], and also other studies about microbes were also done [25][26][27].

Study Design
The present study follows an observational study design which aims to screen for the interaction of theophylline in red complex pathogens. The interaction was analysed using STITCH v.5 pipeline [28]. The functional class of proteins identified were assessed using VICMPred [29] and VirulentPred softwares [30]. The microbial pathogens Treponema denticola ATCC 35405, Tannerella forsythia ATCC 43037, Porphyromonas gingivalis ATCC 33277 are the strains of red complex pathogens that are included in the present study.

Prediction of Protein-Drug Interactions
To predict the interactions between proteins and chemicals STITCH database (Version 5; 2016) is used. The repertoire of proteins which interacts with T. forsythia, P. gingivalis and T. denticola and were further used for predicting virulence [28].

Virulence Prediction
For the identification of virulence factors the software used was VICMpred [29] and VirulentPred [30] pipelines. There are two groups of virulence factors that were screened using the VirulentPred tool based on amino acids that are virulent and avirulent. VICMpred groups proteins are classified into four major classes: proteins involved in metabolism, information storage, virulence and cellular processes. The overall accuracy of VirulentPred servers and VICMpred were 86% and 70.75%, respectively.

Prediction of Subcellular Localization of the Virulent Proteins
The novel drug targets plays an important role in an antimicrobial drug which targets the virulent protein. The subcellular localization of proteins aids in designing using the Computational prediction. The great interest is that cell surface proteins can be used in making vaccines. An algorithm which assigns a probable localization site to a protein from an amino acid sequence is pSORTb V3.0 [31].

Prediction of B-cell Epitopes in the Virulent Proteins
For the prediction of B-cell epitopes from a protein sequence the server BepiPred-2.0 was used. It employs the Random Forest algorithm, which discriminates between epitopes and nonepitope amino acids determined by its crystal structures. To be part of an epitope the residues with scores above the threshold (>0.5) [32,33].

RESULTS AND DISCUSSION
Numerous proteins were identified in the red complex pathogens being targeted by theophylline. Proteins identified in P. gingivalis are mostly involved in cellular processes. The proteins belonging to three functional classes except for virulence factors were identified in T. denticola. The proteins of T. forsythia were mostly avirulent and of the functional class cellular process and metabolism. Only one protein was identified as a virulence factor viz., hypothetical protein [BFO_2902]. The subcellular location of type I phosphodiesterase-nucleotide pyrophosphatase, and cardiolipin synthase were found in the cytoplasmic membrane. The acetyl hydrolase identified in T. denticola was found to be the virulent protein. Polymer mixing is a simple yet appealing way for obtaining polymer physical and mechanical characteristics that are blended. In this research, three different types of blend hydrogels were created by physically mixing two distinct natural polymers, and a model drug, theophylline (TPH), was adsorbed into these hydrogels for drug release investigations, comparatively this study is based on the effect of the red complex pathogens.A cost-effective method to fast-track development of drugs is drug repurposing. Some of the drugs have been already tested against dental pathogens using in silico procedures [34]. These drugs when used with other existing antibiotics act synergistically to enhance the therapeutic spectrum. Biofilm formers are especially important in dental settings because the combination of pathogens are found to do more harm to the host tissues than when they are seen individually This complex demonstrates how interlocking structural motifs may be used to provide a highly selective ligand-binding site with high affinity and molecular selectivity, same with the red complex pathogens. Targeting biofilm formers renders pathogens more vulnerable to antibiotic treatment and immune responses of the host. Theophylline, a xanthine analog, causes abnormal cells [35]. There are not many reports which can relate to the antimicrobial effect of theophylline against dental pathogens. In this context, the present study has identified targets of theophylline which are potential virulent proteins of the red complex pathogens. Further experimental analysis is required to establish this fact.

CONCLUSION
The research discovered molecular targets of theophylline on red complex pathogens, which must be verified in order to validate the essential mechanism caused by the drugs in physiological conditions. To the best of our knowledge, this is the first research of its kind that seeks to understand the molecular targets of theophylline in red complex pathogens. Further, in vitro and in vivo tests can be used to determine the drug's dose, minimum inhibitory concentration and minimum bactericidal concentration.

CONSENT AND ETHICAL APPROVAL
It is not applicable.

ACKNOWLEDGEMENT
The author would like to thank the faculty members of the department of Microbiology, Saveetha Dental College and Hospital, Chennai for helping out with research.