Design and Development of Potential Flavonoid Moiety for Pbp2a Inhibition for Mrsa Therapy-A Computational Technique

1Department of Pharmaceutical Chemistry and Analysis, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Amrita Institute of Medical Sciences, AIMS Health Science Campus, AIMS Ponekara P O, Kochi-682041. 2Computational Chemistry Group (CCG), Amrita Molecular Modeling and Synthesis Research lab,Amrita School of Engineering, Coimbatore, Amrita Vishwa Vidyapeetham, India. *Corresponding Author E-mail : n_krishnan@cb.amrita.edu

Methicillin-Resistant Staphylococcus aureus is a commonly known bacterium that leads to several infections in humans. It is the major cause of multidrug-resistant infections with significant morbidity and mortality. Based on the reports of 'Centers for disease control and prevention (CDC)' in 2011, 80,000 invasive infections and 11,000 deaths were reported in the United States. An estimated 2 billion people carry staphylococcus aureus; about 53 million people are thought to carry MRSA 1 .
The common mechanisms used by bacteria to minimize the effects of antibiotics include changes in the antibiotic molecule, reduced antibiotic penetration and efflux, changes in the target site and resistance due to global cell adaptations, antibiotic sequestration, etc. The most important mechanism behind the antibiotic resistance is often acquired by the transfer of resistance-conferring genes between bacteria facilitated by a conjugative plasmid 2,3 .
Staphylococcus aureus is a gram-positive bacterium that commonly seen inside the nose and on human skin. These bacteria are resistant to numerous antibiotics. Methicillin is an antibiotic widely uses against Staphylococcus aureus. Once the bacteria develop a self-resistance to methicillin, the resistant bacteria is known as MRSA. Mainly all antibiotics are resistant to all microorganisms. The mecA gene, which is encoded with penicillin-binding protein (PBP2a) and is with reduced affinity for ß-lactam antibiotics, acquires methicillin resistance. The mecA gene, found in bacterial cells, is responsible for developing resistance to penicillin related antibiotics.
In the 'mec operator' is keeping mecA, mecI, mecRI and mecR2 4 , out of which, the mecA gene, encoding 'penicillin-binding protein 2a (PBP2a)', acquires Methicillin-Resistant Staphylococcus Aureus (MRSA) infection. The inhibition of penicillin-binding protein 2a (PBP2a) has been identified as a potential therapeutic technique to overcome MRSA. This could be possible by using the antibiotic along with a supporting natural product such as flavonoid, which would inhibit the PBP2a activity 5 .
The pathophysiology of methicillin resistance includes the transcription of the mecA gene into the cell wall producing resistance and leading into the degradation of cell membrane. So, the transcription of the mecA gene will produce resistance against the drug.
MRSA is the main carrier of the mecA gene, which can undergo horizontal gene transfer into the host species 6 through a mobile genetic element, Staphylococcal Cassette Chromosome (SCC) mec . The protein targets of PBP2a of Staphylococcus aureus does not have any crystalized structure in the database. However, the protein models can be generated through 'homology modeling' by taking one of the biosynthesized proteins as the template 7,8 . The interaction between the target protein models and potential drug molecules can be studied through 'docking studies' [9][10][11][12][13] . Further, pharmacokinetic and pharmacodynamic parameters can also be predicted to make screening of the molecules identified.
In this work, the structural protein model molecules of the mecA gene and their interactions with selected flavonoids have been carried out. This would provide an insight into finding a suitable and potential flavonoid moiety for the treatment of MRSA infection.

Materials and Methods
The mecA gene protein sequence has been identified using BLAST search and is further subject to homology modelling using 'Swiss Model' by taking 1MWT as the template [14][15][16] . The 'protein model' characterization has been carried out to study the quality of the models.

results and discussions
The protein models have been designed and developed using homology modelling using the biosynthetic protein, 1MWT as the template. The model protein showed, 96% sequence similarity towards the template protein. The structure of 1MWT and the model protein are shown in Fig 1. An interaction study has been carried out with the ligand molecules and the modelled protein structure. It has been observed that the amino acids GLU33, SER 76, ARG 36 and Val 64 were found to be interacting with the ligand molecules Fig 2 as expected through the mechanism of MRSA 19 . The phenolic hydroxyl groups in position 7 and the -OH group at position 3 on the C-ring of Hesperetin are found to be interacting site of the flavonoid moiety. Among the flavonoid molecules used for the analysis, Hesperetin (Fig 3) is found to be most interacting with the model protein. In fact, this molecule has been identified as more interacting than the standard drug, linezolid used as the control molecule Table 1.
The drug-likeness studies suggest Kaempferol and Hesperetin as potential flavonoids with favourable number of 'hydrogen bonding acceptors and donors Table 2.
All the molecules are keeping good absorption excepting isoquercetin. Hesperain and Kaempferol showed good solubility and less blood-brain barrier penetration. However, these molecules are expected to be more hepatotoxic than the control drug (Table 3).

conclusion
The extensive research into the interactions of flavonoid compounds with the penicillin-binding proteins of many species has shown the variety of the sensitivity of individual PBPs. The 'penicillin-binding proteins (PBP)' can be potential drug targets. The protein models have been generated through homology modelling and are used for studying the possibility of using flavonoids along with Methicillin for controlling MRSA. While studying the interaction between these molecules and the model protein molecules, Hesperetin is found to be most favourable potential molecule. The interacting aminoacids present in the protein models are found to be in the expected binding site. Hence, Hesperetin ((S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one) can be tried as a flavonoid to be used along with methicillin for MRSA therapy subject to further invitro-invivo evaluations.

acknowledgeMent
The author thankful to Department of Pharmaceutical Chemistry and Analysis, Amrita School of Pharmacy, Amrita Molecular Modeling And Synthesis (AMMAS) resaech lab for their support in completing this work.