IN-SILICO DOCKING STUDIES OF PHYTO-LIGANDS AGAINST E. COLI PBP3: APPROACH TOWARDS NOVEL ANTIBACTERIAL THERAPEUTIC AGENTHTML Full Text
IN-SILICO DOCKING STUDIES OF PHYTO-LIGANDS AGAINST E. COLI PBP3: APPROACH TOWARDS NOVEL ANTIBACTERIAL THERAPEUTIC AGENT
- Sahare and A. Moon *
Department of Biochemistry, RTM Nagpur University, Nagpur 440033, Maharashtra, India.
ABSTRACT: Emergence of antibiotic resistance has become a serious problem worldwide. The extensive and uncontrolled use of antibiotics increases the number of multi drug resistant (MDR) bacterial strains. The major mechanisms that bacteria evolve to develop resistance against β-lactam class of antibiotics are the production of β-lactamases and expression of low affinity penicillin binding proteins (PBPs). These PBPs are involved in the final stages of peptidoglycan synthesis. Hence, PBPs are the primary targets for developing antibacterial agents as its inhibition leads to irregularities in cell wall structure and eventually cell death. Bioactive phytochemicals from medicinal plants have been proven to reveal important pharmacological and therapeutic properties for developing novel antibacterial agents. The current study epitomizes the binding of phytochemicals to the transpeptidation (TP) domain of PBP3 of E. coli, a major causative agent of bacterial infection. The phytochemicals exhibiting antibacterial activities were selected as ligands for docking studies employing Schrӧdinger Suite (Maestro, LLC, New York, NY, 2015). Amongst all the phytochemicals studied, chlorogenic acid (CGA) was found to have highest affinity towards PBP3 TP domain. The bocillin binding assay corroborated the docking analysis. The blocking of active site of PBPs by phytochemicals opens up new avenues for screening and developing new phytochemical-based therapeutic antibacterial agents
Penicillin Binding Protein (PBP), Chlorogenic acid, Antibiotic Resistance, Phytochemicals, Molecular Docking, Bocillin Assay
INTRODUCTION: Bacterial murein sacculus, also known as peptidoglycan orthe cell wall, represents essential cell stress-bearing and shape-maintaining entity 1. The synthesis of this murein sacculus is catalysed by various penicillin binding proteins (PBPs) 2. One of them is PBP3; a membrane bound mono functional protein with C-terminal domain having transpeptidase activity 3, 4, encoded by ftsI gene and is essential for cell division 3, a process that involves more than 10 essential cell division (Fts) proteins which assemble to form a ring-like structure at the division site 4 via its N-terminal region and dependent on the presence of FtsW 5.
All β-lactam antibiotics share the same mechanism to inhibit peptidoglycan synthesis by binding to transpeptidase domain of PBPs. They form a stable covalent adduct with the active site serine residue of PBPs 6. So, PBPs remain attractive targets for developing novel antibacterial agent. But widespread use of β-lactam antibiotics has led to the worldwide appearance of drug-resistant bacterial strains through expression of low affinity PBPs.
Due to emergence of multi drug resistance in bacteria, there is an urgent need to search for new antibacterial agents. Medicinal plants reveal important pharmacological activities that can be applied to developing novel therapeutic agents. The phytoconstituents purified from plants were investigated for their antibacterial efficacy and potent antibacterial agents were docked in silico with the PBP3 from E. coli to analyse their interactions with active site residues of transpeptidase domain. The phytochemicals analysed were chlorogenic acid, catechol, ellagic acid, gallic acid, naringenin, pyrogallol, quercetin, resorcinol, salicylic acid, squalene, theophylline, tannic acid and vanillin. Further characterization of their interaction provided catechol, chlorogenic acid, gallic acid, salicylic acid and vanillin as potent antibacterial molecule. Overall results open new avenues to design and synthesize phytochemical based antibacterial agents to fight against serious bacterial infections.
The aim of this study was to identify the mutations in PBP3 from multidrug resistant E. Coli and analysing the effects of the mutation(s), on the binding efficiency of antibiotics as well as phytochemicals to the active site of PBP3 in wildtype and mutant forms.
Since, the multidrug resistant E. coli were isolated, identified and characterized from uropathological samples from UTI infected patients, hence, no ethical approval was required for this study.
MATERIALS AND METHOD:
Isolation and identification of β-lactam resistant E.coli isolates:
Twenty four E. coli isolates were obtained from 100 UTI (urinary tract infection) patients by streaking uropathological samples on LB agar plate (Himedia DT001) and then identified by Gram’s staining and various biochemical tests(Himedia KBM001, KB002, and KB003) 7.
The antibiotic sensitivity for these isolates was checked with following β-lactam antibiotics using 4X-concentration of the reported MIC (minimum inhibitory concentration) by Kirby-Bauer method 8. The β-lactam antibiotics used were Cefpodoxime (16µg), Cephanexin (4.0µg), Cefuroxime (16µg), Cefixime (4.0µg), Ceftazidime (2.0µg), Cefazoline (4.0µg), Cefotaxime (32µg), Ceftriaxome (2.0µg), Cefaclor (32µg), Feropenem (8.0µg) and Cefepime (32µg). The β-lactam resistant E. coli strains were selected for further molecular analysis.
Amplification and Sequencing of PBP3:
The bacterial cells were pelleted by centrifugation at 3000rpm for 5mins and washed with milliQ water. The pellet was suspended in water and boiled for 10 min at 1000C. This lysate was centrifuged and the supernatant was used directly as a template for PCR amplification.
The primer was designed using NCBI primer blast tool (www.ncbi.nlm.nih.gov) for amplification of PBP3 (encoded by ftsI gene) was Forward (5’- ACT GGT GCT GGA GCG AGA TGC-3’) and Reverse (5’- AGC TAC AAA GAG ATC GCC CG -3’). Thermal cycler (Peq Lab) was used to amplify ftsI under following conditions: Initial denaturation at 95ºC for 5min, followed by 30 cycles of 1min denaturation at 94ºC, 1 min of annealing at 58.2ºC and 1.5min at 72ºC and finally 10min at 72ºC 9.
PCR amplicons were detected electrophoretically in 1% agarose gel using 1X TAE, visualized by staining with 0.5µg/ml ethidium bromide, examined in UV light and photographed by Molecular Imager Gel Doc XR (Biorad Laboratories).The purified PCR products were sequenced for mutational analysis.
Purification and identification of phytochemicals:
The antibacterial activity was studied for the extracts of following ten medicinal plants, obtained by 50% aqueous-methanol (v/v) solution with cold maceration; Andrographis paniculata (Ap), Astercantha longifolia (Al), Bixa orellana (Bo), Gardenia resinifera (Gr), Pongamia pinnata (Pp), Psoralea corylifolia (Pc), Sphaeranthus indicus (Si), Solanum trilobatum (St), Soyamida febrifuga (Sf) and Thespesia populnea (Tp). All these extracts showed significant antibacterial activity, hence were utilized for identification of phytochemicals by HPLC 8.
Antibacterial activity of purified phytochemicals against E. coli isolates:
The phytochemicals identified and purified were betulinic acid, chlorogenic acid, catechol, ellagic acid, gallic acid, naringenin, pyrogallol, quercetin, resorcinol, salicylic acid, squalene, theophylline, tannic acid and vanillin. These purified phytochemicals were studied for their antibacterial activity against E. coli 8.
Purification of PBP3
The PCR amplicon was purified by QIA quick purification kit (Qiagen) and cloned into pCR 2.1-TOPO vector. These were digested with XhoI and Bam HI and cloned into the expression vector pET15b (Novagen) at the same restriction sites. BL21 (DE3) E. coli host cells were transformed with expression vectors. These cells were grown at 370C till OD600 reached 0.6. 1mM IPTG was used as an inducer. The bacterial cells were pelleted down at 5000rpm/10min/40C.
These cells were suspended in 20mMTris (pH 8.0) and 300mMNaCl and broken by sonication. Recombinant protein containing an N-terminal (His)6 purified by nickel chelation chromatography in the presence of 1 mM DDM. The N-terminal (His)6 tag was removed by thrombin cleavage (Sigma–Aldrich) at room temperature overnight. PBP3 was further purified by Superdex 200 size-exclusion column (GE Life Sciences). Peak fractions were collected and were exchanged to PBS using Amicon Ultra filter units (Millipore).
The purified PBP3 was assayed for its binding with phytochemicals in presence of bocillin. Bocillin is a fluorescent analogue of penicillin V and it binds to the transpeptidase domain of all PBPs. In the current study, binding efficiency of catechol, chlorogenic acid (CGA), gallic acid, salicylic acid and vanillin was analysed to TP domain of PBP3. The purified PBP3 was incubated with 20, 50 and 100µM of respective phytochemicals in bocillin binding assay 10. CGA showed the highest binding efficiency to TP domain of PBP3.
AMES test for CGA mutagenicity testing:
AMES test was performed to check the mutagenicity of CGA with Bio Era’s kit method (BTK 150905, India) containing mutant Salmonella typhimurium oxotrophic bacterial cell. These bacteria contain mutation in a gene required for histidine synthesis. This mutation prevents translation of functional enzyme and thus cell cannot synthesize histidine. These are histidine-dependent mutants because they depend on external source of histidine in medium.
- typhimurium was revived in 10ml of sterile LB broth (2gm of dehydrated LB broth powder in100ml distilled water) and incubated at 370 in incubator for 48 hours. Minimal agar plates were prepared by dissolving 1.2gm of dehydrated media in 400ml distilled water; pH was adjusted to 7.0 with 1N NaOH and then added 2gm agar powder. This was sterilised and poured in petriplates. The soft agar was prepared by adding 0.1gm of agar powder in 10ml minimal media and sterilised.
10mg of chlorogenic acid was dissolved in 1ml sterile distilled water. The concentration of CGA tested for mutagenicity varied from 10µg to 1mg. In 1ml of soft agar, added required concentration of CGA and 50µl of bacterial culture (McFarland’s turbidity standard of 0.5 which in turn was equal to 1.5×108cells/ml), 200µl of this mixture was spotted on minimal agar plate. The plates were incubated at 370 for 72 hrs. The number of visible colonies was counted.
Cytotoxicity testing for CGA:
Reduction of 3-(4,5-dimethylthiazol-2-yl) – 2,5 – diphenyltetrazolium bromide (MTT) by CPR (a microsomal enzyme, NADPH-cytochrome P450 reductase) was used in measuring the cell cytotoxicity. Blood lymphocytes were used for cytotoxicity assay (HiSep LSM). The reduced was measured spectroscopically at 610nm. The concentrations of CGA tested for mutagenicity were 10µg and 1mg. The detailed methodology has been described by Sung-Kun Yim et. al. 11.
In Silico studies:
The computational studies were carried out using Windows 8.1 professional, 64 bit Intel core M-5Y10c CPU at 1.00 GHz, 4 GB RAM) using the Schrödinger 2015 suite (Maestro 10.1, Schrödinger, LLC, New York, NY, 2015).Ligand preparation:
The phytochemicals showing prominent antibacterial activity against MDR E. coli (chlorogenic acid, catechol, ellagic acid, gallic acid, naringenin, pyrogallol, quercetin, resorcinol, salicylic acid, squalene, theophylline, tannic acid and vanillin) were sketched in maestro 10.1 (Maestro, Schrödinger, LLC, New York, NY, 2015), and prepared by ligprep (LigPrep, version 3.3, Schrödinger, LLC, New York, NY, 2015) with OPLS 2.1 force field to rectify the molecular geometries and get least energy conformations.
Selection and preparation of PBP3 structure;
- coli PBP3 is encoded by ftsI gene and plays an important role in catalysing the cell wall peptidoglycan during cell division. PBP3 protein structure was downloaded from protein data bank (PDB ID:4BJP, http://www.rcsb.org/pdb/. The PBP3 structure crystallised by Souvage et. al. 2014 was selected for analysis as it belongs to the same species of bacteria i.e E. coli with the resolution 2.50A0 12.
The protein was prepared and minimised by protein preparation wizard with OPLS 2005 force field in maestro 10.1 (Maestro, Schrödinger, LLC, New York, NY, 2015). The sitemaps were generated by grid based method (Glide, sitemap, Schrödinger, LLC, New York, NY, 2015) to locate primary ligand binding site in the receptor. The sitemaps were analysed by Site Score, D score, balance of hydrophobic-hydrophilic characters of the site and H-bond donor/acceptor ratio of ligand. The grid was specified by Glide receptor grid generation using centroid of crystal native ligand for ligand docking. The reported conserved region in the active site of penicillin binding proteins is Gly-Ser-X-X-Lys-Pro. The catalytically important active Serine in PBP3 is S(307). This information was used to locate and confirm the active site region and set up a grid for ligand docking.
Homology model preparation for mutant PBP3:
The sequencing analysis has shown the alteration of N(104)I in PBP3 in the form of a point mutation in E. coli. The homology model for mutant PBP3 was built in Prime (Prime, Homology model, structure prediction wizard, Schrödinger, LLC, New York, NY, 2015) by using wild type structures of respective proteins with 99% homology. The built model was validated by Ramachandran plot. The homology modelling includes protein structure prediction from the template, alignment and model building. The built models were again prepared in protein preparation wizard (Maestro 10.1, Schrödinger, LLC, New York, NY, 2015) and site map were generated to predict the ligand binding site to compare with the wild type data in further calculations. The MM-GBSA method was used for predicting binding energy in protein-ligand complex.
Molecular docking was carried out by glide docking (Glide, version 6.0, Schrödinger, LLC, New York, NY, 2015)using an extra precision mode, which utilizes as an input the prepared ligand and generated grid. The ligand sampling was selected as flexible for docking against the biological target. Here the interaction pattern between ligands and protein, glide energy and docking score were studied. It was observed that among all the tested phytochemicals, chlorogenic acid showed most prominent results.
RESULTS AND DISCUSSION:
Selection of β-lactam resistant E. coli isolates:
We have found that 26% of the UTI patients were infected with multi drug resistant E. coli. The other predominated bacteria isolated were Pseudomonas aeruginosa (36%) and Enterococcus faecalis (35%). E. coli were found completely resistant to Cefpodoxime (16µg), Cephalexin (4.0µg), Cefuroxime (16µg) and Cefazoline (4.0µg). Also, they showed emerging resistant towards Cefixime (4.0µg), Ceftazidime (2.0µg) and Ceftriaxome (2.0µg).
The PCR amplification of ftsI gene in E. coli was standardised at 58.20C primer annealing temperature. The amplification was seen in nineteen E. coli isolates out of twenty four. The E. coli strains numbers that showed amplifications were 1, 2, 4, 5, 6, 11, 12, 13, 14,1 5, 16, 17, 18, 19, 20, 21, 22, 23 and 24 (data shown in Fig.1).
FIG. 1: AMPLIFICATION OF PBP-3 (FTSI GENE) FROM E. COLI ON 1%AGAROSE GEL
Mutation in PBP-3 of E. Coli:
The point mutation observed in PBP-3 gene was N(104)I. At position 104, Aspargine (N) is replaced by Isoleucine (I). These sequence data have been submitted to NCBI gene bank as shown in Table 1.
TABLE 1: ACCESSION NUMBERS OF FTSI GENE
|S. No.||E. coli MDR isolate number||Accession Number|
EC: E. coli
Purified phytochemicals by HPLC:
The phytochemicals identified by HPLC were tannic acid, ellagic acid, gallic acid, quercetin, chlorogenic acid, naringenin, theophylline, betullinic acid, resorcinol, catechol, salicylic acid, vanillin, squalene and pyrogallol 8.
Antibacterial activity of phytochemicals:
Though MDR E. coli isolates have emerged resistant against commonly prescribed β-lactam antibiotics, they are still susceptible to many selected phytochemicals. These phytochemicals have shown significant antibacterial activity against MDR isolates. The antibacterial potency of these purified phytochemicals has been analysed, as described in Table 2.
TABLE 2: ZONE OF INHIBITION (MM) FOR RESPECTIVE PHYTOCHEMICALS AGAINST MDR E. COLI
|S.No.||Phytochemical (1mg)||Antibacterial activity|
|MDR E. coli isolates|
The crystal structure of wild type PBP3 was downloaded from PDB. The homology model for mutant PBP3 was prepared based on wild type sequence as template and both wild type as well as mutant PBP3 structures were validated by Ramachandran plot, as shown in the following Fig.2.
FIG. 2: RAMACHANDRAN PLOT OF WILD TYPE AND MUTANT PBP3 RESPECTIVELY
Effect of N(104)I mutation on active site of PBP3
It was found that N(104)I mutation is not in the active site cavity of PBP3 but still have significant impact on active site. The mutation has increased the site score, size, drug ability score and volume as observed from sitemap analysis. Moreover, ligand interaction surface has increased as evidenced from increased hydrogen bond donor/acceptor area, hydrophobic as well as hydrophilic area of the active site. The data is shown in following Table 3, Fig.3 and Table 3.
TABLE 3: SITEMAP COMPARISON BETWEEN WILDTYPE AND MUTANT ACTIVE SITE
|Active site||Site Score||Size||D score||Volume|
FIG. 3: SURFACE ANALYSIS OF ACTIVE SITE OF PBP3 IN WILDTYPE AND MUTANT PROTEIN RESPECTIVELY
TABLE 4: ACTIVE SITE SURFACE ANALYSIS
|PBP3 Active Site Surface||Surface Area|
|Wild type PBP3||Mutant PBP3|
|H-bond acceptor (red)||423.012||618.982|
|H-bond donor (blue)||428.698||1089.330|
TABLE 5: COMPARATIVE ANALYSIS OF DOCKING DATA BETWEEN WILD TYPE AND MUTANT PBP3
|Ligands||Wildtype PBP3||Mutant PBP3|
|Glide score||Binding energy||Glide score||Binding energy|
Binding energy: Kcal/mol
From the above table for docking analysis, it is clear that, the N(104)I point mutation in PBP3 has lead to noticeable changes in respect to its binding affinity towards tested β-lactam antibiotics as well as towards phytochemicals.
The interactions of bocillin, penicillin, cephalosporin, chlorogenic acid, quercetin, ellagic acid and gallic acid with the residues of ligand binding pocket of wildtype and mutant PBP3 is shown in the following Fig.4.
FIG. 4: DOCKED POSE OF LIGANDS TO LIGAND BINDING POCKET OF WILDTYPE AND MUTANT PBP3 RESPECTIVELY
The interactions of various ligands with active side residues of PBP3 are shown in the following Table 6.
TABLE 6: INTERACTION OF LIGAND WITH ACTIVE SITE RESIDUES OF WILDTYPE AND MUTANT PBP3
|Ligand||Interaction of ligand with active site residues of wild type PBP3||Interaction of ligand with active site residues of mutant PBP3|
|Bocillin||LYS 358 (H-bond, 1.7856A0)
TYR 348 (H-bond, 2.05977A0)
SER 307 (H-bond, 2.76096A0)
|GLY 542(H-bond, 2.06613A0)
THR 497 (H-bond, 2.55801A0)
THR 497 (H-bond, 2.17004A0)
LYS 499 (Salt bridge, 3.60317A0)
LYS 499(H-bond, 2.17102A0)
|Penicillin||SER 359 (H-bond, 2.09756A0)
TYR 419 (H-bond, 1.75585A0)
|GLY 542 (H-bond, 2.39749A0)
THR 497 (H-bond, 2.26763A0)
THR 497 (H-bond, 2.36757A0)
|Cephalosporin||THR 497 (H-bond, 2.09298A0)
TYR 419 (H-bond, 2.09932A0)
LYS 499 (H-bond, 1.91674A0)
LYS 499 (Salt bridge 3.37512A0)
LYS 310 (Salt bridge, 3.03825A0)
|GLY479 (H-bond, 1.68185A0)
GLY 543 (H-bond, 1.99558A0)
GLY 542 (H-bond, 2.02225A0)
THR 497 (H-bond, 2.16351A0)
THR 497 (H-bond, 1.9157A0)
SER 307 (H-bond, 2.17677A0)
LYS 358 (Salt bridge, 3.27551A0)
LYS 358 (H-bond, 2.22045A0)
|Chlorogenic acid||LYS 310 (H-bond, 2.43818A0)
SER 307 (H-bond, 1.86255A0)
TYR 419 (H-bond, 2.08417A0)
GLU 340 (H-bond, 1.98435A0)
LYS 342 (H-bond, 1.82931A0)
|ASN 361 (H-bond, 2.11181A0)
ASN 361 (H-bond, 1.8853A0)
THR 497 (H-bond, 1.85426A0)
THR 497 (H-bond, 2.03069A0)
TYR 419 (H-bond, 2.44813A0)
ASP 534 (H-bond, 1.93849A0)
ASP 534 (H-bond, 1.97045A0)
SER 307 (H-bond, 1.89537A0)
LYS 499 (H-bond, 2.58246A0)
|Quercetin||THR 497 ((H-bond, 1.93998A0)
SER 307 (H-bond, 2.33022A0)
TYR 419 (H-bond, 1.87225A0)
SER 359 (H-bond, 2.289A0)
THR 495 (H-bond, 1.81335A0)
|THR 495 ((H-bond, 1.79042A0)
SER 359 (H-bond, 2.02285A0)
GLY 543 (H-bond, 2.59896A0)
ASN 361 (H-bond, 2.12148A0)
LYS 499 (H-bond, 1.94126A0)
|Ellagic acid||ASN 361 (H-bond, 2.76686A0)
LYS 342 (H-bond, 1.86866A0)
GLU 340 (H-bond, 2.38452A0)
|SER 359 (H-bond, 2.19023A0)
ASN 361 (H-bond, 2.51006A0)
LYS 499 (H-bond, 2.02554A0)
|Gallic acid||SER 307 (H-bond, 2.01539A0)
THR 497 (H-bond, 1.97068A0)
PHE 417 (H-bond, 2.49363A0)
TYR 419 (π-π stacking, 5.47751A0)
|ASN 361 (H-bond, 2.39468A0)
ASN 361 (H-bond, 2.64108A0)
THR 497 (H-bond, 1.962720)
THR 497(H-bond, 2.489270)
From the docking analysis of phytochemicals, in comparison to standard antibiotics, as antibacterial agents, and also through analysis of their interaction with active site residues, it is clear that chlorogenic acid, ellagic acid, quercetin, naringenin, gallic acid, pyrogallol, catechol, resorcinol, vanillin, salicylic acid and theophylline are able to bind the active site of PBP3.
Bocillin binding assay: The docking analysis showed the highest affinity of CGA towards TP domain as compared to penicillin, bocillin, naringenin, quercetin, salicylic acid and theophylline. To corroborate this docking data, the binding analysis of bocillin in presence and absence of CGA was performed. The bocillin binds to TP domain of purified PBP3 and gives fluorescence in UV light but the incubation of protein with CGA prevents the binding of bocillin and decreases the fluorescence, as described in Fig.4.
FIG. 5: INTERACTION OF CHLOROGENIC ACID TO THE ACTIVE SITE OF PBP3
FIG. 6: CGA BINDING DECREASES BOCILLIN FLUORESCENCE
Mutagenicty and cytotoxicity of CGA: CGA did not show any indication for mutagenic and cytotoxic effect at tested concentration.
FIG.7: CYTOTOXIC ASSAY FOR CGA
CONCLUSION: PBP3 has only one active site, TP domain. The docking analysis showed decreased binding affinity after mutation towards penicillin (glide score -6.74 to -4.44), methicillin (glide score decreased from -6.87 to -4.33), mezlocillin (from -6.72 to -6.49) and ampicillin (from -6.59 to -4.49) but increased with amoxicillin (-5.23 to -5.63). This shows that these antibiotics are binding with less affinity to the mutated PBP3 and could be less effective as antibacterials except amoxicillin. But, the glide score for cephalosporin has increased from (-7.28) to (-8.78). So, cephalosporin can still be used as a potent antibacterial agent.
Among all the phytochemicals tested, the top three molecules found to bind the active site of wild type PBP3 were chlorogenic acid (-9.04), quercetin (-8.05) and ellagic acid (-6.49). Moreover, chlorogenic acid and quercetin were found to have higher affinity than cephalosporin (-7.28). Although, in mutated PBP3, the affinity for these three phytochemicals has been found to decrease, but gallic acid (-7.01) has showed good affinity for mutated protein.
The phytochemicals; chlorogenic acid, quercetin and ellagic acid have not only showed promising results in blocking the active site of PBP3, but also, they have showed significant efficacy in inhibiting β-lactamase activity (unpublished data). The following patents have been filed and published in Indian Patent Journal with following application numbers 4342/MUM/2015, 4621/MUM/2015 and 4622/MUM/2015.
ACKNOWLEDGMENTS: UGC Financial support scheme for the project 42-645/2013 (SR) is gratefully acknowledged. Also Schrödinger support in evaluating the data is greatly acknowledged by the authors.
CONFLICT OF INTEREST: No conflict of interest
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How to cite this article:
Sahare P and Moon A: In-Silico Docking Studies of Phyto-Ligands against E. Coli PBP3: Approach Towards Novel Antibacterial Therapeutic Agent. Int J Pharm Sci Res 2016; 7(9): 3703-11.doi: 10.13040/IJPSR.0975-8232.7(9).3703-11.
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