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Received Date : 27-May-2014 Revised Date : 01-Dec-2014 Accepted Date : 01-Dec-2014 Article type
: Research Article
Drug resistance reversal potential of ursolic acid derivatives against nalidixic acid and multidrug resistant Escherichia coli Synergistic antibacterial potentials of triterpenoids
Gaurav Raj Dwivedi#1, Anupam Maurya#2, Dharmendra Kumar Yadav3, Feroz Khan3, Mahendra P. Darokar1*, Santosh Kumar Srivastava2* Molecular Bioprospection Department; 2 Medicinal Chemistry Department; 3 Metabolic & Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, India 1
#both the authors contributed equally
*Address of Corresponding author: *Dr. Santosh Kumar Srivastava Medicinal Chemistry Department E-mail: [email protected] *Mahendra P Darokar Molecular Bioprospection Department E-mail: [email protected] Central Institute of Medicinal & Aromatic Plants (Council of Scientific & Industrial Research) P.O.-CIMAP, Kukrail Picnic Spot Road, Lucknow-226015 (U.P.) India Tel.: +91-522-2718581; Fax: +91-522-2342666 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12491 This article is protected by copyright. All rights reserved.
Abstract
Accepted Article
As a part of our drug discovery programme, ursolic acid (UA) was chemically transformed into six semi-synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with conventional antibiotic nalidixic acid (NA) against the nalidixic acid sensitive (NASEC) and nalidixic acid resistant (NAREC) strains of Escherichia coli. Although UA and its all semi-synthetic derivatives did not show antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration (MIC) of NA up to eight folds. The 3-O-acetyl-urs-12-en-28-isopropyl ester (UA4) and 3-O-acetyl-urs-12-en-28-n-butyl ester (UA-5) derivatives of UA reduced the MIC of NA by eight fold against NAREC and four and eight folds against NASEC, respectively. The UA-4 and UA-5 were further evaluated for their synergy potential with another antibiotic tetracycline (TET) against the multidrug resistant clinical isolate of E. coli (MDREC-KG4). The results showed that both these derivatives in combination with TET reduced the cell viability in concentration dependent manner by significantly inhibiting efflux pump. This was further supported by the in silico binding affinity of UA-4 and UA-5 with efflux pump proteins. These UA derivatives may find their potential use as synergistic agents in the treatment of multidrugresistant Gram negative infections.
Keywords: Triterpenoids; Ester analog; Nalidixic acid; Tetracycline; MDR, Non antibiotics.
Introduction
In developing countries, infectious diseases are one of the major causes of morbidity and mortality. The extensive use of antibiotics has raised serious health concern throughout the world due to development of resistance in bacteria against many classes of antibiotics [1]. The overexpression of transporters is often associated with multidrug resistance phenomenon that recognizes and efficiently expels a broad range of structurally unrelated compounds from the cells [2]. An additional burden of infection is represented by multidrug resistant organisms (MDRO), which ultimately result in inferior treatment by the latest generation antibiotics [3]. On the other hand, severity of infections is increased by MDRO, both in the hospital and community [4]. Among the Gram-negatives, drug resistant E. coli has become the most common cause of infections among many life-threatening diseases such as infections of the bloodstream [5]. U.S. National Healthcare Safety Network indicated that Gram-negative bacteria are responsible for more than 30% of the hospital-acquired infections of which E. coli predominates in case of urinary tract infections [6]. Similarly, reports from Europe, Asia pacific, and India emphasize that multidrug resistant E. coli is of global problems [7-9]. But due to vast structural diversity, the plant secondary metabolites viz. terpenoids, alkaloids, glycosteroids, flavonoids, polyphenols, tetralones and saponins etc., have emerged as alternate antimicrobial agents (10). It has also been observed that few of these plant secondary metabolites act as bioenhancers. Although bioenhancers do not possess antibacterial activity of their own, but they have the ability to enhance bioactivity of antibiotics by many folds (11-13). Thus, these bioenhancers will drastically reduce the dosage of antibiotics, which will significantly delay the resistance development process against the antibiotics. In this way these bioenhancers will enhance the life span of newly developed as well as of existing antibiotics (13, 14). Hence, this synergistic combination therapy may be used as an alternative to monotherapy for treating patients with invasive and MDR infections (15, 16).
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Ursolic acid, a pentacyclic triterpenoids, was isolated from the leaves of E. tereticornis. It possesses a wide range of biological activities such as ant-inflammatory, hepatoprotective, analgesic, antimicrobial, antimycotic, virostatic, immunomodulatory, diuretic, anti- spasmodic, anti-atherosclerotic, anti-tumor and anti HIV [17, 18]. Apart from above biological activities, ursolic acid when fed to rats at 5mg/kg bodyweight was able to cause infertility by inhibiting spermatogenesis [19, 20]. Further, ursolic acid was able to induce cell death in endothelial cells when the concentration exceeded 12.5uM, and the mechanism of death was apoptosis related [21]. In continuation of our work on synergistic potential of some natural product and their derivatives (13, 22-24), the present study describes drug resistance reversal potential of ursolic acid derivatives in combination with nalidixic acid and tetracycline. The most active derivatives of UA were further investigated in detail using efflux pump inhibition assay, ATPase inhibition assay, real time expression analysis, in-silico docking and ADMET analysis.
Material and Methods
General experimental procedures The NMR spectra were recorded on Avance 300 MHz spectrometer (Bruker). The 1H and 13C NMR chemical shifts were referenced to the solvent peaks. MS were recorded on hyphenated LC-PDA-MS (Prominence LC and mass MS-2010EV, Shimadzu). The compounds were visualized on TLC plates (silica gel 60F254, Merck) by spraying with vanillin-sulfuric acid reagent and heating the plate at 100°C for 5 minutes. Antibacterial agents The conventional antibiotics nalidixic acid and tetracycline were purchased from Sigma (purity ≥98%). Ursolic acid (UA) was isolated in ≥ 95% purity by fast centrifugal partition chromatography (FCPC) and characterized on the basis of its spectroscopic data [20]. Further, ester derivatives of ursolic acid were prepared according to the procedure given below. The purity of semi-synthetic derivatives was ≥95% (HPLC).
Chemical derivatization of ursolic acid (UA) Acetylation of ursolic acid Ursolic acid (1.0 g) was dissolved in dry pyridine (5 mL). To this solution acetic anhydride (1.5 equivalent) and catalytic amount of dimethyl amino pyridine (DMAP) were added and the reaction mixture was kept overnight (scheme 1). Progress of the reaction was monitored next morning by TLC [Hexane: EtOAc (7:3)], which confirmed completion of the reaction. Finally reaction was worked up by adding crushed ice and extracting the mixture with chloroform (3-4 times). Chloroform extract was then dried over anhydrous sodium sulphate and solvent was removed under vacuum which yield the crude acetylated product (1.097g).
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36. Livermore D.M. (2009) Has the era of untreatable infections arrived? J Antimicrob Chemother; 64:S1: i29–i36. 37. Infectious Diseases Society of America. (2010) The 10 x ‘20 Initiative: pursuing a global commitment to develop 10 new antibacterial drugs by 2020. Clin Infect Dis; 50: 10811083. 38. Spellberg B., Bartlett J.G., Gilbert D.N. (2013) The future of antibiotics and resistance. N Engl J Med; 368: 299-302. 39. Hassani M. (2014) The Crisis of Resistant Gram-Negative Bacterial Infections: Is there any Hope for ESKAPE? Clin Res Infect Dis; 1: 1005. 40. Li H.Z., Nikaido H. (2004) Efflux-mediated drug resistance in bacteria. Drugs; 64:159– 204. 35. 41. Garvey M.I., Piddock L.V.J. (2008) The efflux pump inhibitor reserpine selects multidrug resistantStreptococcus pneumoniae strains that overexpress the ABC transporters PatA and PatB. Antimicrob. Agents Chemother; 52: 1677-85. 42. Khan I.A., Mirza Z.M., Kumar A., Verma V., Qazi G.N. (2006) Piperine, a phytochemical potentiator of ciprofloxacin against Staphylococcus aureus. Antimicrob Agents Chemother; 50:810-812. 43. Kalia N.P., Mahajan P., Mehra R., Nargotra A., Sharma J.P., Koul S., Khan I.A. (2012) Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus. J Antimicrob Chemother: 67:2401-2408. 44. Viveiros M., Dupont M., Rodrigues L., Couto I., Davin-Regli A. (2007) Antibiotic stress, genetic response and altered permeability of E. coli. PLoS ONE; 2: e36. 45. Viveiros M., Jesus A., Brito M., Leandro C., Martins M., Ordway D.(2005) Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes. Antimicrob Agents Chemother; 49: 3578–3582. 46. Lubelski J., Konings W.N., Driessen A.J.M. (2007) Distribution and Physiology of ABCTypeTransporters contributing to Multidrug Resistance in Bacteria. Microbiol Mol Biol Rev; 71: 463-476. 47. Kobayashi N., Nishino K., Yamaguchi A. (2001) Novel Macrolide-Specific ABC-Type EffluxTransporter in Escherichia coli. J Bacteriol; 183: 5639-5644. 48. Martins M., Couto I., Viveiros M., Amaral L. (2010) Identification of efflux-mediated multidrug resistance in bacterial clinical isolates by two simple methods. Methods Mol Biol; 642: 143-57. 49. Marquez B. (2005) Bacterial efflux systems and efflux pumps inhibitors. Biochimie; 87: 1137–1147. 50. Viveiros M., Martins M., Couto I. (2008) New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps. Curr Drug Targets; 9: 760-78. 51. Hurdle J.G., O’Neill A.J., Chopra I., Lee R.E. (2011) Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nat Rev Microbiol; 9:62–75.
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were incubated at 37°C for 24 hours. The MIC was recorded as the last dilution without any turbidity as per CLSI guidelines. Time kill studies The time kill study of tetracycline alone and in combination with UA and its most potent derivatives (UA-4 and UA-5) against MDREC-KG4 strain was conducted at one, two and four concentrations of MIC using earlier described method of Eliopoulus and Moellering (30). Each analysis was carried out in triplicate with a control without test sample. Time kill curves were derived by plotting log10 CFU/mL against time (h). Time kill kinetics was also studied in combinations with TET and test compounds at the reduced concentrations at which maximum synergy was observed. Ethidium bromide efflux studies The fluorometric determination of ethidium bromide (EB) efflux was performed as per reported method (31). To get metabolically active cells, the culture of MDREC-KG4 was grown in 10 mL MHB (pH 7.3±0.2) with optical density (OD) of 0.6 at 600nm. The cells were collected by centrifugation at 16,060 × g for 3 min and washed with phosphate buffer saline. Ethidium bromide (25μg/mL) wad added in the bacterial suspension and incubated for 60 min at 25°C in the absence/presence of UA-4 and UA-5 at their minimum effective concentrations. The EBloaded bacterial suspension were centrifuged at 16,060 × g for 3 min, the supernatant discarded and the pellet re-suspended in cold PBS (1×). The tubes then placed on ice. Aliquots of 0.095 mL of the bacterial suspension was distributed to 0.3 mL 96 well plates. Loss of fluorescence was recorded for 30min at a regular interval of 1 min at the excitation and emission wavelength of 530nm and 585nm respectively using spectrofluorometer (FLUO star omega, BMG Labtech Germany). ATPase inhibitory activity The bacterial membrane protein was isolated by the method described earlier (13, 24, 32). ATPase assay was carried out in a 96 micro plate format using QuantichromTM ATPase Assay Kit (BioAssay Systems, USA). Initially, optimal enzyme concentration was determined by a series of dilutions of enzyme (membrane protein containing ATPase) in assay buffer. Enzyme and inhibitor were incubated first for a certain period of time, before adding the substrate. Reactions were set up to 40μL containing 20μL Assay Buffer, 5μL enzyme (20μg μL-1), 5μL Inhibitor, 10μL (4mM ATP) and control with no inhibitor in separate wells. At the end of reaction, 200μL reagent was added and incubated for 30 min at room temperature. The improved malachite green reagent forms a stable dark green color with liberated inorganic phosphate(Pi), which was measured spectrophotometrically on a plate reader (620nm) (33). qRT– PCR analysis Different efflux pump genes of resistance nodulation division family (RND) and ATP binding cassettes super family (ABC) were used to find out the transcriptional profile in presence of UA4 and UA-5. All the genes were analyzed in treated and non-treated cells of MDREC-KG4. Cells were grown to mid-log phase in the presence of sub-inhibitory concentration (1/4 MIC) of tetracycline, UA-4 and UA-5 alone and in combination. The real-time quantification of the RNA template was analyzed by SYBR GreenER qPCR supermix (Invitrogen, Carlsbad, CA, USA) using 7900HT fast real time PCR system (Invitrogen, Carlsbad, CA, USA). Observations were
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recorded in terms of LogRQ after normalization of indigenous gene coding for Dglyceraldehyde-3-phosphate-dehydrogenase (gapdh) (13, 24).
In silico molecular modeling and docking parameters Since no crystallographic data for YOJI protein of E. coli was available in the Protein Data Bank (PDB), hence a homology model was developed for it. For this purpose, simulated annealing and energy minimization approaches were used to build the 3D model of YojI protein by employing GENO3D. Query protein sequence of YojI protein with length of 251 amino acids was retrieved from GenBank database (NCBI, USA). Homologous template protein sequences with known 3D structure were identified through Basic Local Alignment Search Tool (BLAST) program. Template protein maltose/maltodextrin transport ATP-binding protein (acc.No. NP_142200.1) showed 32.9% sequence identity with YojI a multidrug ATP binding cassettes (ABC) transporter protein (acc. No. YP_490449.1). Template-query protein sequence validation was done by multiple sequence alignment method through CLUSTALW, while the statistical verification of generated model was evaluated by PROCHECK, and model comparisons in three-dimensional form were performed by RasMol-3D viewer. Finally, after structural and energy minimization evaluation, Model_2 was identified as more accurate than others with maximum homology to the known 3D structure. Best model based on highest number of residues present in the allowed region of Ramachandran plot was selected. Molecular docking study of ursolic acid and its derivatives UA-4 and UA-5 was performed with multidrug ABC transporter ATP-binding protein (YOJI), AcrB (PDB: 1OY8), TolC (PDB: 1EK9), AcrA (2F1M), MacB (3FTJ) receptor by using the Sybyl-X v2.0 molecular modeling & drug discovery software (Tripos International, USA). The Tripos force field with a distance-dependent dielectric and Powell gradient algorithm with a convergence criterion of 0.001 kcal mol−1 was used for optimization. Partial atomic charges were assessed by using Gasteiger-Hückel method. Further geometry optimization was done through MOPAC-6 package using the semi-empirical PM3 Hamiltonian method. During docking procedure all parameters were assigned to their default values (24, 34, 35).
Results and Discussion
Although, before 2010 there were many antibiotics for Gram-positive bacteria in the drug discovery pipeline, but none was for Gram-negative bacteria (GNB) (36). But with the change in policy and initiatives taken by many regulatory agencies such as FDA, Infectious Diseases Society of America (IDSA), and the European Medicines Agency, now there is some hope for new antibiotics against MDR-GNB (37-39). Multi drug resistant organisms acquire high degree of resistance through the involvement of ATP-dependent and ATP-independent efflux pumps (1, 3, 40). Hence, it would be of paramount importance to search for those agents, which can reverse the drug resistance by inhibiting these efflux pumps in microbes. This will reduce selection pressure of antibiotics on the microbes and improve efficacy of the existing and novel antibiotics too (13, 14, 22, 23, 24, 41). As a the part of our drug discovery program, ursolic acid (a triterpenoid isolated from the leaves of E. tereticornis) and its semi-synthetic analogs were studied for their multidrug resistance reversal potential against the Gram-negative bacteria. Antibacterial activity evaluation of UA against the nalidixic acid-sensitive (NA SEC) and nalidixic acid-resistant (NAREC) strains of E. coli showed that it does not possess antibacterial activity (MIC1000 µg/mL) of its own. But when given in combination with nalidixic acid, UA (10 µg/mL) reduced MIC of NA by two folds. This synergistic effect of UA prompted us to
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study the synergistic potential of UA derivatives. For this purpose ursolic acid was first converted into 3-O-acetyl ursolic acid (UA-1) and tested with NA against the two E. coli strains NASEC and NAREC. The results showed four folds reduction in the MIC of NA. Since, UA-1 was lipophilic derivative of UA; this prompted us to prepare some other lipophilic ester derivatives of UA-1. Hence, methyl (UA-2), ethyl (UA-3), isopropyl (UA-4), n-butyl (UA-5) and amyl (UA-6) ester derivatives of UA-1 were prepared and evaluated for their drug resistance reversal potential against the NASEC and NAREC and the results are presented in Table 1. From the Table 1, it is evident that conversion of ursolic acid acetate (UA-1) in to one and two carbon methyl (UA-2) and ethyl (UA-3) ester derivatives did not show any effect on activity, but further increase in ester carbon chain to three carbon isopropyl (UA-4) and four carbon n-butyl (UA-5) derivatives increased the drug reversal potential four folds to that of UA and two folds to that of UA-1 against NAREC. It was interesting to note that UA-4 more effective against NAREC, while UA-5 was equally effective against both NASEC and NAREC. Further increased in ester chain by five carbons as in UA-6 decreased the activity. On comparing our results with known drug reversal agent reserpine (2, 42), the two derivatives UA-4 and UA-5 showed four times better activity than the reserpine (Table 1).
Further, drug reversal potential of UA and its various derivatives in different concentrations were tested on another antibiotic tetracycline against the MDREC-KG4. For this purpose MDREC-KG4 was treated with TET at one, two and four fold concentrations of MIC, which reduced the viability of E. coli significantly (Figure 1a).
Then the bactericidal activity of TET was evaluated at low concentration in combination with UA, UA-4 and UA-5 (Figure 1b) and the results are presented in Table 2. From the Table 2 it is evident that UA, UA-4 and UA-5 reduced the MIC of tetracycline by 2, 8 and 8 folds respectively. The MICs of UA, UA-4 and UA-5 were also deduced through checkerboard assay and found 50, 50 and 25µg/mL respectively (Table 2). Similar bioenhancing potential of some natural compounds have also been reported earlier (13, 22-24, 25, 42, 43), which are in full agreement with our present findings.
In order to understand the possible mechanism of action of UA derivatives, they were subjected to fluorescence based ethidium bromide efflux assay using multidrug resistant strain MDREC-KG4 of E. coli. As shown in Figure 2, significant decrease in fluorescence was observed in non treated control cells. While in presence of UA-4 and UA-5 the loss of fluorescence was significantly reduced, reflecting a strong interference with ethidium bromide efflux by these compounds. Accumulation and efflux of ethidium bromide are good indicators of the involvement of efflux pumps in the resistance mechanism, particularly in Gram negative bacteria such as E. coli (44, 45).
Further, to understand whether these compounds interfere with ATP dependent efflux pump, they were evaluated for ATPase inhibitory activity in MDREC. It was found that UA-4 and UA-5 significantly inhibited ATPase activity (Fig. 3) in terms of liberated inorganic
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phosphate (Pi) indicating involvement of these compounds in the inhibition of ATP dependent efflux pumps. Among 74 ATP dependent transporters known so far in E.coli, 69 belong to ABC family efflux pumps while others are nominal such as P type ATPase, F type ATPases and H+ATPase (41). The presence of higher number of ABC family efflux pumps in E. coli encouraged us to search ATPase inhibitors having similar properties like ouabain, reserpine and phenothiazine (46-48).
The effect of UA-4 and UA-5 on the expression of efflux pump genes especially RND tripartite complex (acrA, acrB and tolC), multidrug ABC transporter ATP binding protein (yojI) and macB were evaluated using MDREC-KG4. As it is evident from the Figure 4, up regulation of efflux pump encoding genes was observed when the cells were treated with ¼ concentration of tetracycline MIC. While, different level of expression of these genes was found upon the treatment of MDREC by UA-4 and UA-5. In the presence of noxious agents, efflux pump genes are over-expressed (44, 45, 48). This information led us to explore and analyze the interplay between the major efflux pump systems present in E. coli. In this study the expression of RND efflux pump genes especially acrA, acrB and tolC, which play a key role in the high degree of multidrug resistance in E. coli was significantly down regulated. In E. coli, ABC type of efflux pumps are also known to be responsible for high degree of multi drug resistance and interestingly, UA-4 and UA-5 were able to reduce the expression level of efflux pump genes of this family, this enhances use of these derivatives as efflux pump inhibitor/modulators (48).
Exploration of binding site interacting residues through molecular docking In order to further support our above observations, molecular docking of ursolic acid derivatives was carried out to explore the binding site interacting residues of drug resistant efflux pump proteins of E. coli. In this study ursolic acid and its derivatives, UA-4 and UA-5 showed good binding affinity with multidrug ABC transporter ATP-binding protein (YojI), AcrB(PDB:1OY8), TolC(PDB:1EK9), AcrA(PDB:2F1M), MacB (PDB:3FTJ) receptor protein as indicated by the Sybyl Surflex-Dock scores (Table3 and Table S1) similar to control reserpine. In docking pose of compound UA-4 with YOJI protein, the chemical nature of binding site residues within a radius of 4Å from bound nucleophilic (polar, hydrophobic) e.g., SER-78 (Serine), aromatic (hydrophobic) e.g., PHE-81(Phenylalanine), TRP-85 (Tryptophan), LEU-86, (Leucine), acidic (polar, negative charged) e.g., GLU-94, GLU-144 (Glutamic acid), basic (polar, hydrophobic and positive charged) e.g., ALA-79, ALA-139, ALA-140, ALA-141, ALA-143, (Alanine), and ARG-74, ARG-122, ARG-146(Arginine), LEU-107 (Leucine), VAL-80 (Valine), LYS-96 (Lysin), and ILE-123 (Isolecine) thus bound ET-1d showed good binding affinity as indicated by total score of 7.2716 and formed two hydrogen bond with ARG-122 of length 1.8, which may lead to more stability and activity (Fig. 1A). On the other hand, docking results for control reserpine showed low binding affinity as indicated by total score of 6.5866 (Table 3).
Similarly, docking pose of UA-5 respectively with TolC, AcrB and YOJI protein the chemical nature of binding site residues within a radius of 4Å from bound THR-100, GLN-103, THR-104, LEU-107, ASN-220, LEU-221, SER-222, LEU-223, ALA-319, SER-322, VAL-323, GLN-325, THR-326, SER-329, SER-330, ASN-333, ASN-399, SER-402, ALA-403and ASN-
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177 showed good binding affinity as indicated by total score of 6.5935 and formed three hydrogen bond with THR-326, SER-329 and GLN-325 of length 2.5, 2.4 and 2.5 (Fig. 1B); ILE18, LEU- 21, ALA-22, GLY-23, LEU-25, ALA-26, LYS-29, ALA-381, ALA-384, ALA-385, PHE-386, GLY-387 showed low binding affinity as indicated by total score of 3.7193 and formed a hydrogen bond with LYS-29 of length 1.8 (Fig. 1C) and ARG-74, PHE-77, SER-78, ALA-79, VAL-80, PHE-81, TRP-85, LEU-86, GLU-94, LYS-96, LEU-107, ARG-122, ILE123, ALA-137, ALA-138, ALA-139, ALA-140, ALA-141, ALA-143, GLU-144and ARG-146 showed strong binding affinity as indicated by total score of 6.8942 and formed three hydrogen bond with ARG-146 and ARG-122 of length 2.1, 1.8 and 1.8 and strong hydrophobic interaction which may lead to more stability and activity. However, docking results for control compound reserpine showed low binding affinity as indicated by total score of 5.6579, 3.8169 and 6.5866 (Fig. 5 and Table 3).
On the basis of all above results, it may be concluded that ursolic acid derivatives clearly pass all the litmus tests of therapeutic strategy hypothesis according to which, molecules that target the cell membrane or cell walls are most likely to synergize with conventional antibiotics or antiseptics by inhibiting the efflux pumps, increasing cellular permeability and weakening the cell envelope (51).
Conclusions It is for the first time, drug resistance reversal potential of UA derivatives have been proved by inhibition of efflux pump, which may be useful in: (i) lowering the dose of antibiotics; (ii) reducing the drug resistance development frequency; and (iii) increasing the efficacy of antibiotics against multidrug resistant E. coli strains. These results may be of great help in the development of inexpensive and dose economic antibacterial drug formulations from a very common and widely distributed herb, E tereticornis
Acknowledgement
Authors are thankful to the National Gene Bank of Medicinal and Aromatic Plants at CSIRCentral Institute of Medicinal and Aromatic Plants, Lucknow, India for providing plant material. Financial support from CSIR Network project BSC 0203 is gratefully acknowledged. Two of us (GRD and AM) are grateful to CSIR for providing Research Fellowships. Competing interests: There is no conflict of interest.
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21. Messner B., Zeller I., Ploner C., Frotschnig S., Ringer T., Steinacher-Nigisch A., Ritsch A., Laufer G., Huck C., Bernhard D. (2011) Ursolic acid causes DNA-damage, p53mediated, mitochondria- and caspase-dependent human endothelial cell apoptosis, and accelerates atherosclerotic plaque formation in vivo. Atherosclerosis, 2: 402–408 22. Upadhyay H. C., Dwivedi G. R., Darokar M. P., Chaturvedi V., Srivastava S. K. (2012) Bioenhancing and Antimycobacterial Agents from Ammannia multiflora. Planta Medica; 78:79-81. 23. Gupta V.K., Verma S., Pal A., Srivastava S.K. (2013) In vivo efficacy and synergistic interaction of 16α-hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide, a clerodane diterpene from Polyalthia longifolia against methicillin-resistant Staphylococcus aureus. Appl Microbiol Biotechnol; 97:9121-9131. 24. Dwivedi G.R., Upadhyay H.C., Yadav D.K. (2013) Drug resistance reversal potential of a natural product 4-hydroxy-α-tetralone and its derivative against multidrug resistant Escherichia coli (MDREC). Chem Biol Drug Design; doi: 10.1111/cbdd.12263. 25. Kumar S. (1976) Properties of adenyl cyclase and cyclic adenosine 3′,5′-monophosphate receptor protein deficient mutants of Escherichia coli. J Bacteriol; 125:545–555. 26. Dwivedi G.R. (2013) Studies on efflux pumps for high throughput screening of phytomolecules combating bacterial multidrug resistant infections. PhD Thesis. Faizabad, India: Dr. R. M. L. Avadh University. 27. Clinical Laboratory Standards Institute (CLSI) (2012) Performance standards for antimicrobial susceptibility testing. 22nd Informational Supplement testing M100-S15. CLSI, Wayne, PA 28. Stermitz F.R., Lorenz P., Tawara J.N., Zenewicz L.A., Lewis K. (2000) Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5*methoxyhydnocarpin, a multidrug pump inhibitor. Proc Natl Acad Sci;97:1433–1437. 29. Eliopoulos G.M., Wennersten C.B. (2002) Antimicrobial activity of quinupristin– dalfopristin combined with other antibiotics against vancomycin resistant enterococci. Antimicrob Agents Chemother; 46:1319–1324. 30. Eliopoulus G.M., Moellering R.C.J. (1996) Antimicrobial combinations. In: Lorian V., editor. Antibiotics in Laboratory Medicine, 4th edn. Baltimore: Williams & Wilkins, p. 330–336. 31. Viveiros M., Rodrigues L., Martins M., Couto I., Spengler G., Martins A., Amaral L. (2010) Evaluation of efflux activity of bacteria by a semi-automated fluorometric system. Methods Mol Biol; 642:159-172. 32. Suzuki Y., Ueno S., Ohnuma R., Koyama N. (2005) Cloning, sequencing and functional expression in E. coli of the gene for a P-type Na+- ATPase of the facultative anaerobic alkaliphile, Exiguobacterium auranticum. Biochim Biophys Acta; 1727: 162-168. 33. Glavinas H., Méhn D., Jani M., Oosterhuis B., Herédi-Szabó K. (2008) Utilization of membrane vesicle preparations to study drug-ABC transporter interactions. Expert Opin Drug Metab Toxicol; 4:721-732 34. Yadav D.K., Ahmad I., Shukla A., Khan F., Negi A.S., Gupta A. (2014). QSAR and docking studies on Chalcone derivatives for anti-tubercular activity against M. tuberculosis H37Rv. Journal of Chemometrics. DOI: 10.1002/cem.2606. 35. Yadav D.K., Khan F. (2013) QSAR, docking and ADMET studies of camptothecin derivatives as inhibitors of DNA topoisomerase-I. J of Chemomatrics; 27:21–33.
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36. Livermore D.M. (2009) Has the era of untreatable infections arrived? J Antimicrob Chemother; 64:S1: i29–i36. 37. Infectious Diseases Society of America. (2010) The 10 x ‘20 Initiative: pursuing a global commitment to develop 10 new antibacterial drugs by 2020. Clin Infect Dis; 50: 10811083. 38. Spellberg B., Bartlett J.G., Gilbert D.N. (2013) The future of antibiotics and resistance. N Engl J Med; 368: 299-302. 39. Hassani M. (2014) The Crisis of Resistant Gram-Negative Bacterial Infections: Is there any Hope for ESKAPE? Clin Res Infect Dis; 1: 1005. 40. Li H.Z., Nikaido H. (2004) Efflux-mediated drug resistance in bacteria. Drugs; 64:159– 204. 35. 41. Garvey M.I., Piddock L.V.J. (2008) The efflux pump inhibitor reserpine selects multidrug resistantStreptococcus pneumoniae strains that overexpress the ABC transporters PatA and PatB. Antimicrob. Agents Chemother; 52: 1677-85. 42. Khan I.A., Mirza Z.M., Kumar A., Verma V., Qazi G.N. (2006) Piperine, a phytochemical potentiator of ciprofloxacin against Staphylococcus aureus. Antimicrob Agents Chemother; 50:810-812. 43. Kalia N.P., Mahajan P., Mehra R., Nargotra A., Sharma J.P., Koul S., Khan I.A. (2012) Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus. J Antimicrob Chemother: 67:2401-2408. 44. Viveiros M., Dupont M., Rodrigues L., Couto I., Davin-Regli A. (2007) Antibiotic stress, genetic response and altered permeability of E. coli. PLoS ONE; 2: e36. 45. Viveiros M., Jesus A., Brito M., Leandro C., Martins M., Ordway D.(2005) Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes. Antimicrob Agents Chemother; 49: 3578–3582. 46. Lubelski J., Konings W.N., Driessen A.J.M. (2007) Distribution and Physiology of ABCTypeTransporters contributing to Multidrug Resistance in Bacteria. Microbiol Mol Biol Rev; 71: 463-476. 47. Kobayashi N., Nishino K., Yamaguchi A. (2001) Novel Macrolide-Specific ABC-Type EffluxTransporter in Escherichia coli. J Bacteriol; 183: 5639-5644. 48. Martins M., Couto I., Viveiros M., Amaral L. (2010) Identification of efflux-mediated multidrug resistance in bacterial clinical isolates by two simple methods. Methods Mol Biol; 642: 143-57. 49. Marquez B. (2005) Bacterial efflux systems and efflux pumps inhibitors. Biochimie; 87: 1137–1147. 50. Viveiros M., Martins M., Couto I. (2008) New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps. Curr Drug Targets; 9: 760-78. 51. Hurdle J.G., O’Neill A.J., Chopra I., Lee R.E. (2011) Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nat Rev Microbiol; 9:62–75.
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Figure Captions Scheme1. Semi-synthesis of ursolic acid derivatives. Figure 1: Time–kill curves of MDREC-KG4 showing the dose dependent bactericidal effect of (a) Tetracycline (b) Tetracycline in combination with UA derivatives Figure 2: Inhibition of ethidium bromide efflux by UA derivatives
Figure 3: ATPase inhibitory activity of UA derivatives. Figure 4: Efflux pump expression analysis of UA derivatives. Figure 5: In silico molecular docking studies elucidating the (A) interaction of UA-4 in the binding site of YOJI (B) ) interaction of UA-5 in the binding site of TolC, (C) AcrB and (D) YOJI. The docking studies were carried out using SYBYL-X 2.0.
Table 1. In vitro drug resistance reversal activity of ursolic acid derivatives Compound code NAL UA UA-1 UA-2 UA-3 UA-4 UA-5 UA-6 Reserpine
MIC of compounds (µg/mL) CA8000 DH5α 6.25 100 1000 1000 1000 1000 1000 1000 1000 1000 500 500 1000 1000 1000 1000 500 500
MIC of Nalidixic acid in presence of compounds (10µg/mL) CA8000 F.R. DH5α F.R. 6.25 100 3.125 2 50 2 1.56 4 25 4 1.56 4 25 4 1.56 4 25 4 1.56 4 12.5 8 0.78 8 12.5 8 1.56 4 25 4 3.125 2 50 2
NAL = nalidixic acid; CA8000 & DH5α = drug sensitive & drug resistant strains of E. coli respectively; F.R. = fold reduction;
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Table 2. Reduction in the minimum inhibitory concentrations of TET in combination with derivatives UA, UA-4 and UA-5 against MDRECKG4 Combinations Agents
MIC (Alone) (µg/mL)
Plant compounds (µg/mL)
MIC of tetracycline with/without plant compounds against MDREC-KG4
TET UA
800 1000
UA-4
500
UA-5
1000
RES
800
UA (100) UA (50) UA (25) UA (12.5) UA (6.25) UA (3.125) UA (1.56) UA (0.78) UA-4 (100) UA-4 (50) UA-4 (25) UA-4 (12.5) UA-4 (6.25) UA-4 (3.125) UA-4 (1.56) UA-4 (0.78) UA-5 (100) UA-5 (50) UA-5 (25) UA-5 (12.5) UA-5 (6.25) UA-5 (3.125) UA-5 (1.56) UA-5 (0.78) RES (100) RES (50) RES (25) RES (12.5) RES (6.25) RES (3.125) RES (1.56) RES (0.78)
400/800 400/800 800/800 800/800 800/800 800/800 800/800 800/800 100/800 100/800 200/800 200/800 200/800 200/800 400/800 400/800 100/800 100/800 100/800 200/800 200/800 200/800 200/800 400/800 400/800 400/800 400/800 800/800 800/800 800/800 800/800 800/800
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Fold Reduction in TET
2 2 No No No No No No 8 8 4 4 4 4 2 2 8 8 8 4 4 4 4 2 2 2 2 No No No No No
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Table 3. Comparison of docking score and binding site residues of compounds against drug efflux pump proteins.
Compound
PDB ID/
Total
Target
score*
Biding pocket residue in (4Å)
protein
Lengt
Amino acid
No. of
h of
residue
Hydrogen
hydro
involved in
Bond (H)
gen
Docking
bond
interaction
(Å) 1EK9/ TolC
6.2536
-
-
-
-
-
-
ARG-74, SER-78, ALA-79, VAL-80, PHE-81,
1.8
ARG-122
1
TRP-85, LEU-86, GLU-94, LYS-96, LEU-107,
1.8
GLN-236, GLN-239, ALA-240, GLN-301, GLN304, ALA-305, ASN-308, ASP-23, ALA-26, ALA-27, LYS-30, GLU-33, GLN-87, ALA-90, GLN-94
1OY8/ AcrB
4.4674
UA-4
ILE-18, ILE-19,
ALA-22, LEU-25, ALA-26,
LYS-29, GLY-378, ALA-381, ALA-384, ALA385, LEU-480 Model/ YOJI
7.2716
ARG-122, ILE-123, ALA-139, ALA-140, ALA141, ALA-143, GLU-144, ARG-146 1EK9/ TolC
6.5935
THR-100, GLN-103, THR-104, LEU-107, ASN-
2.5
THR-326
220, LEU-221, SER-222, LEU-223, ALA-319,
2.4
SER-329
SER-322, VAL-323, GLN-325, THR-326, SER-
2.5
GLN-325
1.8
LYS-29
1
ARG-146
3
329, SER-330, ASN-333, ASN-399, SER-402, ALA-403, ASN-177 1OY8/ AcrB
3.7193
ILE-18, LEU-21, ALA-22, GLY-23, LEU-25, ALA-26, LYS-29, ALA-381, ALA-384, ALA-
UA-5
385, PHE-386, GLY-387 Model/ YOJI
6.8942
ARG-74, PHE-77, SER-78, ALA-79, VAL-80,
2.1
PHE-81, TRP-85, LEU-86, GLU-94, LYS-96,
1.8
LEU-107, ARG-122, ILE-123, ALA-137, ALA-
1.8
ARG-122
-
-
-
2.0
LYS-29
1
THR-48, ARG-74, PHE-77, SER-78, ALA-79,
2.1
ARG-122
2
PHE-81, TRP-85, LEU-86, GLU-94,
2.1
ARG-146
1.9
ASN-115
138, ALA-139, ALA-140, ALA-141, ALA-143, GLU-144, ARG-146 1EK9/ TolC
4.1761
THR-111, ASN-333, ALA-336, SER-337, SER340, ASN-388, ALA-389, ASN-392, ILE-395, ASN-396, ASN-399, THR-170, ASN-173, ASN174, ASN-177
1OY8/ AcrB
2.9236
ALA-22, LEU-25, ALA-26, LYS-29, PHE-380, ALA-381, VAL-382, ALA-384, ALA-385, PHE-
UA
386 Model/ YOJI
4.5955
LYS-96,
ARG-122, ILE-123, ALA-140, ALA-141, ALA143, GLU-144, ARG-146 Reserpine
1EK9/ TolC
5.6579
THR-104, LEU-107, ASN-108, THR-111, ASN-
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2
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115, GLN-325, THR-326, SER-329, SER-330,
2.1
ASN-333
-
-
-
LYS-75, PHE-77, SER-78,
2.7
ARG-146
-
ALA-79, PHE-81, TRP-85, LEU-86, GLU-94,
2.4
ALA-79
ASN-333, ILE-395, ASN-396, ASN-399, ASN173, ASN-177 1OY8/ AcrB
3.8169
ILE-19, ALA-22, LEU-25, ALA-26, LYS-29, LEU-30, ALA-381, VAL-382, ALA-384, ALA385, GLY-387, ALA-479, LEU-480, LEU-483
Model/ YOJI
6.5866
THR-48, ARG-74,
LYS-96, ARG-122, ILE-123, ALA-140, ALA141, ALA-143, GLU-144, ARG-146 *Surflex-Dock scores (total scores) are expressed in –log10 (Kd) units to represent binding affinities
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Received Date : 27-May-2014 Revised Date : 01-Dec-2014 Accepted Date : 01-Dec-2014 Article type
: Research Article
Drug resistance reversal potential of ursolic acid derivatives against nalidixic acid and multidrug resistant Escherichia coli Synergistic antibacterial potentials of triterpenoids
Gaurav Raj Dwivedi#1, Anupam Maurya#2, Dharmendra Kumar Yadav3, Feroz Khan3, Mahendra P. Darokar1*, Santosh Kumar Srivastava2* Molecular Bioprospection Department; 2 Medicinal Chemistry Department; 3 Metabolic & Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, India 1
#both the authors contributed equally
*Address of Corresponding author: *Dr. Santosh Kumar Srivastava Medicinal Chemistry Department E-mail: [email protected] *Mahendra P Darokar Molecular Bioprospection Department E-mail: [email protected] Central Institute of Medicinal & Aromatic Plants (Council of Scientific & Industrial Research) P.O.-CIMAP, Kukrail Picnic Spot Road, Lucknow-226015 (U.P.) India Tel.: +91-522-2718581; Fax: +91-522-2342666 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12491 This article is protected by copyright. All rights reserved.
Abstract
Accepted Article
As a part of our drug discovery programme, ursolic acid (UA) was chemically transformed into six semi-synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with conventional antibiotic nalidixic acid (NA) against the nalidixic acid sensitive (NASEC) and nalidixic acid resistant (NAREC) strains of Escherichia coli. Although UA and its all semi-synthetic derivatives did not show antibacterial activity of their own, but in combination, they significantly reduced the minimum inhibitory concentration (MIC) of NA up to eight folds. The 3-O-acetyl-urs-12-en-28-isopropyl ester (UA4) and 3-O-acetyl-urs-12-en-28-n-butyl ester (UA-5) derivatives of UA reduced the MIC of NA by eight fold against NAREC and four and eight folds against NASEC, respectively. The UA-4 and UA-5 were further evaluated for their synergy potential with another antibiotic tetracycline (TET) against the multidrug resistant clinical isolate of E. coli (MDREC-KG4). The results showed that both these derivatives in combination with TET reduced the cell viability in concentration dependent manner by significantly inhibiting efflux pump. This was further supported by the in silico binding affinity of UA-4 and UA-5 with efflux pump proteins. These UA derivatives may find their potential use as synergistic agents in the treatment of multidrugresistant Gram negative infections.
Keywords: Triterpenoids; Ester analog; Nalidixic acid; Tetracycline; MDR, Non antibiotics.
Introduction
In developing countries, infectious diseases are one of the major causes of morbidity and mortality. The extensive use of antibiotics has raised serious health concern throughout the world due to development of resistance in bacteria against many classes of antibiotics [1]. The overexpression of transporters is often associated with multidrug resistance phenomenon that recognizes and efficiently expels a broad range of structurally unrelated compounds from the cells [2]. An additional burden of infection is represented by multidrug resistant organisms (MDRO), which ultimately result in inferior treatment by the latest generation antibiotics [3]. On the other hand, severity of infections is increased by MDRO, both in the hospital and community [4]. Among the Gram-negatives, drug resistant E. coli has become the most common cause of infections among many life-threatening diseases such as infections of the bloodstream [5]. U.S. National Healthcare Safety Network indicated that Gram-negative bacteria are responsible for more than 30% of the hospital-acquired infections of which E. coli predominates in case of urinary tract infections [6]. Similarly, reports from Europe, Asia pacific, and India emphasize that multidrug resistant E. coli is of global problems [7-9]. But due to vast structural diversity, the plant secondary metabolites viz. terpenoids, alkaloids, glycosteroids, flavonoids, polyphenols, tetralones and saponins etc., have emerged as alternate antimicrobial agents (10). It has also been observed that few of these plant secondary metabolites act as bioenhancers. Although bioenhancers do not possess antibacterial activity of their own, but they have the ability to enhance bioactivity of antibiotics by many folds (11-13). Thus, these bioenhancers will drastically reduce the dosage of antibiotics, which will significantly delay the resistance development process against the antibiotics. In this way these bioenhancers will enhance the life span of newly developed as well as of existing antibiotics (13, 14). Hence, this synergistic combination therapy may be used as an alternative to monotherapy for treating patients with invasive and MDR infections (15, 16).
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Ursolic acid, a pentacyclic triterpenoids, was isolated from the leaves of E. tereticornis. It possesses a wide range of biological activities such as ant-inflammatory, hepatoprotective, analgesic, antimicrobial, antimycotic, virostatic, immunomodulatory, diuretic, anti- spasmodic, anti-atherosclerotic, anti-tumor and anti HIV [17, 18]. Apart from above biological activities, ursolic acid when fed to rats at 5mg/kg bodyweight was able to cause infertility by inhibiting spermatogenesis [19, 20]. Further, ursolic acid was able to induce cell death in endothelial cells when the concentration exceeded 12.5uM, and the mechanism of death was apoptosis related [21]. In continuation of our work on synergistic potential of some natural product and their derivatives (13, 22-24), the present study describes drug resistance reversal potential of ursolic acid derivatives in combination with nalidixic acid and tetracycline. The most active derivatives of UA were further investigated in detail using efflux pump inhibition assay, ATPase inhibition assay, real time expression analysis, in-silico docking and ADMET analysis.
Material and Methods
General experimental procedures The NMR spectra were recorded on Avance 300 MHz spectrometer (Bruker). The 1H and 13C NMR chemical shifts were referenced to the solvent peaks. MS were recorded on hyphenated LC-PDA-MS (Prominence LC and mass MS-2010EV, Shimadzu). The compounds were visualized on TLC plates (silica gel 60F254, Merck) by spraying with vanillin-sulfuric acid reagent and heating the plate at 100°C for 5 minutes. Antibacterial agents The conventional antibiotics nalidixic acid and tetracycline were purchased from Sigma (purity ≥98%). Ursolic acid (UA) was isolated in ≥ 95% purity by fast centrifugal partition chromatography (FCPC) and characterized on the basis of its spectroscopic data [20]. Further, ester derivatives of ursolic acid were prepared according to the procedure given below. The purity of semi-synthetic derivatives was ≥95% (HPLC).
Chemical derivatization of ursolic acid (UA) Acetylation of ursolic acid Ursolic acid (1.0 g) was dissolved in dry pyridine (5 mL). To this solution acetic anhydride (1.5 equivalent) and catalytic amount of dimethyl amino pyridine (DMAP) were added and the reaction mixture was kept overnight (scheme 1). Progress of the reaction was monitored next morning by TLC [Hexane: EtOAc (7:3)], which confirmed completion of the reaction. Finally reaction was worked up by adding crushed ice and extracting the mixture with chloroform (3-4 times). Chloroform extract was then dried over anhydrous sodium sulphate and solvent was removed under vacuum which yield the crude acetylated product (1.097g).
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36. Livermore D.M. (2009) Has the era of untreatable infections arrived? J Antimicrob Chemother; 64:S1: i29–i36. 37. Infectious Diseases Society of America. (2010) The 10 x ‘20 Initiative: pursuing a global commitment to develop 10 new antibacterial drugs by 2020. Clin Infect Dis; 50: 10811083. 38. Spellberg B., Bartlett J.G., Gilbert D.N. (2013) The future of antibiotics and resistance. N Engl J Med; 368: 299-302. 39. Hassani M. (2014) The Crisis of Resistant Gram-Negative Bacterial Infections: Is there any Hope for ESKAPE? Clin Res Infect Dis; 1: 1005. 40. Li H.Z., Nikaido H. (2004) Efflux-mediated drug resistance in bacteria. Drugs; 64:159– 204. 35. 41. Garvey M.I., Piddock L.V.J. (2008) The efflux pump inhibitor reserpine selects multidrug resistantStreptococcus pneumoniae strains that overexpress the ABC transporters PatA and PatB. Antimicrob. Agents Chemother; 52: 1677-85. 42. Khan I.A., Mirza Z.M., Kumar A., Verma V., Qazi G.N. (2006) Piperine, a phytochemical potentiator of ciprofloxacin against Staphylococcus aureus. Antimicrob Agents Chemother; 50:810-812. 43. Kalia N.P., Mahajan P., Mehra R., Nargotra A., Sharma J.P., Koul S., Khan I.A. (2012) Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus. J Antimicrob Chemother: 67:2401-2408. 44. Viveiros M., Dupont M., Rodrigues L., Couto I., Davin-Regli A. (2007) Antibiotic stress, genetic response and altered permeability of E. coli. PLoS ONE; 2: e36. 45. Viveiros M., Jesus A., Brito M., Leandro C., Martins M., Ordway D.(2005) Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes. Antimicrob Agents Chemother; 49: 3578–3582. 46. Lubelski J., Konings W.N., Driessen A.J.M. (2007) Distribution and Physiology of ABCTypeTransporters contributing to Multidrug Resistance in Bacteria. Microbiol Mol Biol Rev; 71: 463-476. 47. Kobayashi N., Nishino K., Yamaguchi A. (2001) Novel Macrolide-Specific ABC-Type EffluxTransporter in Escherichia coli. J Bacteriol; 183: 5639-5644. 48. Martins M., Couto I., Viveiros M., Amaral L. (2010) Identification of efflux-mediated multidrug resistance in bacterial clinical isolates by two simple methods. Methods Mol Biol; 642: 143-57. 49. Marquez B. (2005) Bacterial efflux systems and efflux pumps inhibitors. Biochimie; 87: 1137–1147. 50. Viveiros M., Martins M., Couto I. (2008) New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps. Curr Drug Targets; 9: 760-78. 51. Hurdle J.G., O’Neill A.J., Chopra I., Lee R.E. (2011) Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nat Rev Microbiol; 9:62–75.
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Accepted Article
were incubated at 37°C for 24 hours. The MIC was recorded as the last dilution without any turbidity as per CLSI guidelines. Time kill studies The time kill study of tetracycline alone and in combination with UA and its most potent derivatives (UA-4 and UA-5) against MDREC-KG4 strain was conducted at one, two and four concentrations of MIC using earlier described method of Eliopoulus and Moellering (30). Each analysis was carried out in triplicate with a control without test sample. Time kill curves were derived by plotting log10 CFU/mL against time (h). Time kill kinetics was also studied in combinations with TET and test compounds at the reduced concentrations at which maximum synergy was observed. Ethidium bromide efflux studies The fluorometric determination of ethidium bromide (EB) efflux was performed as per reported method (31). To get metabolically active cells, the culture of MDREC-KG4 was grown in 10 mL MHB (pH 7.3±0.2) with optical density (OD) of 0.6 at 600nm. The cells were collected by centrifugation at 16,060 × g for 3 min and washed with phosphate buffer saline. Ethidium bromide (25μg/mL) wad added in the bacterial suspension and incubated for 60 min at 25°C in the absence/presence of UA-4 and UA-5 at their minimum effective concentrations. The EBloaded bacterial suspension were centrifuged at 16,060 × g for 3 min, the supernatant discarded and the pellet re-suspended in cold PBS (1×). The tubes then placed on ice. Aliquots of 0.095 mL of the bacterial suspension was distributed to 0.3 mL 96 well plates. Loss of fluorescence was recorded for 30min at a regular interval of 1 min at the excitation and emission wavelength of 530nm and 585nm respectively using spectrofluorometer (FLUO star omega, BMG Labtech Germany). ATPase inhibitory activity The bacterial membrane protein was isolated by the method described earlier (13, 24, 32). ATPase assay was carried out in a 96 micro plate format using QuantichromTM ATPase Assay Kit (BioAssay Systems, USA). Initially, optimal enzyme concentration was determined by a series of dilutions of enzyme (membrane protein containing ATPase) in assay buffer. Enzyme and inhibitor were incubated first for a certain period of time, before adding the substrate. Reactions were set up to 40μL containing 20μL Assay Buffer, 5μL enzyme (20μg μL-1), 5μL Inhibitor, 10μL (4mM ATP) and control with no inhibitor in separate wells. At the end of reaction, 200μL reagent was added and incubated for 30 min at room temperature. The improved malachite green reagent forms a stable dark green color with liberated inorganic phosphate(Pi), which was measured spectrophotometrically on a plate reader (620nm) (33). qRT– PCR analysis Different efflux pump genes of resistance nodulation division family (RND) and ATP binding cassettes super family (ABC) were used to find out the transcriptional profile in presence of UA4 and UA-5. All the genes were analyzed in treated and non-treated cells of MDREC-KG4. Cells were grown to mid-log phase in the presence of sub-inhibitory concentration (1/4 MIC) of tetracycline, UA-4 and UA-5 alone and in combination. The real-time quantification of the RNA template was analyzed by SYBR GreenER qPCR supermix (Invitrogen, Carlsbad, CA, USA) using 7900HT fast real time PCR system (Invitrogen, Carlsbad, CA, USA). Observations were
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recorded in terms of LogRQ after normalization of indigenous gene coding for Dglyceraldehyde-3-phosphate-dehydrogenase (gapdh) (13, 24).
In silico molecular modeling and docking parameters Since no crystallographic data for YOJI protein of E. coli was available in the Protein Data Bank (PDB), hence a homology model was developed for it. For this purpose, simulated annealing and energy minimization approaches were used to build the 3D model of YojI protein by employing GENO3D. Query protein sequence of YojI protein with length of 251 amino acids was retrieved from GenBank database (NCBI, USA). Homologous template protein sequences with known 3D structure were identified through Basic Local Alignment Search Tool (BLAST) program. Template protein maltose/maltodextrin transport ATP-binding protein (acc.No. NP_142200.1) showed 32.9% sequence identity with YojI a multidrug ATP binding cassettes (ABC) transporter protein (acc. No. YP_490449.1). Template-query protein sequence validation was done by multiple sequence alignment method through CLUSTALW, while the statistical verification of generated model was evaluated by PROCHECK, and model comparisons in three-dimensional form were performed by RasMol-3D viewer. Finally, after structural and energy minimization evaluation, Model_2 was identified as more accurate than others with maximum homology to the known 3D structure. Best model based on highest number of residues present in the allowed region of Ramachandran plot was selected. Molecular docking study of ursolic acid and its derivatives UA-4 and UA-5 was performed with multidrug ABC transporter ATP-binding protein (YOJI), AcrB (PDB: 1OY8), TolC (PDB: 1EK9), AcrA (2F1M), MacB (3FTJ) receptor by using the Sybyl-X v2.0 molecular modeling & drug discovery software (Tripos International, USA). The Tripos force field with a distance-dependent dielectric and Powell gradient algorithm with a convergence criterion of 0.001 kcal mol−1 was used for optimization. Partial atomic charges were assessed by using Gasteiger-Hückel method. Further geometry optimization was done through MOPAC-6 package using the semi-empirical PM3 Hamiltonian method. During docking procedure all parameters were assigned to their default values (24, 34, 35).
Results and Discussion
Although, before 2010 there were many antibiotics for Gram-positive bacteria in the drug discovery pipeline, but none was for Gram-negative bacteria (GNB) (36). But with the change in policy and initiatives taken by many regulatory agencies such as FDA, Infectious Diseases Society of America (IDSA), and the European Medicines Agency, now there is some hope for new antibiotics against MDR-GNB (37-39). Multi drug resistant organisms acquire high degree of resistance through the involvement of ATP-dependent and ATP-independent efflux pumps (1, 3, 40). Hence, it would be of paramount importance to search for those agents, which can reverse the drug resistance by inhibiting these efflux pumps in microbes. This will reduce selection pressure of antibiotics on the microbes and improve efficacy of the existing and novel antibiotics too (13, 14, 22, 23, 24, 41). As a the part of our drug discovery program, ursolic acid (a triterpenoid isolated from the leaves of E. tereticornis) and its semi-synthetic analogs were studied for their multidrug resistance reversal potential against the Gram-negative bacteria. Antibacterial activity evaluation of UA against the nalidixic acid-sensitive (NA SEC) and nalidixic acid-resistant (NAREC) strains of E. coli showed that it does not possess antibacterial activity (MIC1000 µg/mL) of its own. But when given in combination with nalidixic acid, UA (10 µg/mL) reduced MIC of NA by two folds. This synergistic effect of UA prompted us to
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study the synergistic potential of UA derivatives. For this purpose ursolic acid was first converted into 3-O-acetyl ursolic acid (UA-1) and tested with NA against the two E. coli strains NASEC and NAREC. The results showed four folds reduction in the MIC of NA. Since, UA-1 was lipophilic derivative of UA; this prompted us to prepare some other lipophilic ester derivatives of UA-1. Hence, methyl (UA-2), ethyl (UA-3), isopropyl (UA-4), n-butyl (UA-5) and amyl (UA-6) ester derivatives of UA-1 were prepared and evaluated for their drug resistance reversal potential against the NASEC and NAREC and the results are presented in Table 1. From the Table 1, it is evident that conversion of ursolic acid acetate (UA-1) in to one and two carbon methyl (UA-2) and ethyl (UA-3) ester derivatives did not show any effect on activity, but further increase in ester carbon chain to three carbon isopropyl (UA-4) and four carbon n-butyl (UA-5) derivatives increased the drug reversal potential four folds to that of UA and two folds to that of UA-1 against NAREC. It was interesting to note that UA-4 more effective against NAREC, while UA-5 was equally effective against both NASEC and NAREC. Further increased in ester chain by five carbons as in UA-6 decreased the activity. On comparing our results with known drug reversal agent reserpine (2, 42), the two derivatives UA-4 and UA-5 showed four times better activity than the reserpine (Table 1).
Further, drug reversal potential of UA and its various derivatives in different concentrations were tested on another antibiotic tetracycline against the MDREC-KG4. For this purpose MDREC-KG4 was treated with TET at one, two and four fold concentrations of MIC, which reduced the viability of E. coli significantly (Figure 1a).
Then the bactericidal activity of TET was evaluated at low concentration in combination with UA, UA-4 and UA-5 (Figure 1b) and the results are presented in Table 2. From the Table 2 it is evident that UA, UA-4 and UA-5 reduced the MIC of tetracycline by 2, 8 and 8 folds respectively. The MICs of UA, UA-4 and UA-5 were also deduced through checkerboard assay and found 50, 50 and 25µg/mL respectively (Table 2). Similar bioenhancing potential of some natural compounds have also been reported earlier (13, 22-24, 25, 42, 43), which are in full agreement with our present findings.
In order to understand the possible mechanism of action of UA derivatives, they were subjected to fluorescence based ethidium bromide efflux assay using multidrug resistant strain MDREC-KG4 of E. coli. As shown in Figure 2, significant decrease in fluorescence was observed in non treated control cells. While in presence of UA-4 and UA-5 the loss of fluorescence was significantly reduced, reflecting a strong interference with ethidium bromide efflux by these compounds. Accumulation and efflux of ethidium bromide are good indicators of the involvement of efflux pumps in the resistance mechanism, particularly in Gram negative bacteria such as E. coli (44, 45).
Further, to understand whether these compounds interfere with ATP dependent efflux pump, they were evaluated for ATPase inhibitory activity in MDREC. It was found that UA-4 and UA-5 significantly inhibited ATPase activity (Fig. 3) in terms of liberated inorganic
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phosphate (Pi) indicating involvement of these compounds in the inhibition of ATP dependent efflux pumps. Among 74 ATP dependent transporters known so far in E.coli, 69 belong to ABC family efflux pumps while others are nominal such as P type ATPase, F type ATPases and H+ATPase (41). The presence of higher number of ABC family efflux pumps in E. coli encouraged us to search ATPase inhibitors having similar properties like ouabain, reserpine and phenothiazine (46-48).
The effect of UA-4 and UA-5 on the expression of efflux pump genes especially RND tripartite complex (acrA, acrB and tolC), multidrug ABC transporter ATP binding protein (yojI) and macB were evaluated using MDREC-KG4. As it is evident from the Figure 4, up regulation of efflux pump encoding genes was observed when the cells were treated with ¼ concentration of tetracycline MIC. While, different level of expression of these genes was found upon the treatment of MDREC by UA-4 and UA-5. In the presence of noxious agents, efflux pump genes are over-expressed (44, 45, 48). This information led us to explore and analyze the interplay between the major efflux pump systems present in E. coli. In this study the expression of RND efflux pump genes especially acrA, acrB and tolC, which play a key role in the high degree of multidrug resistance in E. coli was significantly down regulated. In E. coli, ABC type of efflux pumps are also known to be responsible for high degree of multi drug resistance and interestingly, UA-4 and UA-5 were able to reduce the expression level of efflux pump genes of this family, this enhances use of these derivatives as efflux pump inhibitor/modulators (48).
Exploration of binding site interacting residues through molecular docking In order to further support our above observations, molecular docking of ursolic acid derivatives was carried out to explore the binding site interacting residues of drug resistant efflux pump proteins of E. coli. In this study ursolic acid and its derivatives, UA-4 and UA-5 showed good binding affinity with multidrug ABC transporter ATP-binding protein (YojI), AcrB(PDB:1OY8), TolC(PDB:1EK9), AcrA(PDB:2F1M), MacB (PDB:3FTJ) receptor protein as indicated by the Sybyl Surflex-Dock scores (Table3 and Table S1) similar to control reserpine. In docking pose of compound UA-4 with YOJI protein, the chemical nature of binding site residues within a radius of 4Å from bound nucleophilic (polar, hydrophobic) e.g., SER-78 (Serine), aromatic (hydrophobic) e.g., PHE-81(Phenylalanine), TRP-85 (Tryptophan), LEU-86, (Leucine), acidic (polar, negative charged) e.g., GLU-94, GLU-144 (Glutamic acid), basic (polar, hydrophobic and positive charged) e.g., ALA-79, ALA-139, ALA-140, ALA-141, ALA-143, (Alanine), and ARG-74, ARG-122, ARG-146(Arginine), LEU-107 (Leucine), VAL-80 (Valine), LYS-96 (Lysin), and ILE-123 (Isolecine) thus bound ET-1d showed good binding affinity as indicated by total score of 7.2716 and formed two hydrogen bond with ARG-122 of length 1.8, which may lead to more stability and activity (Fig. 1A). On the other hand, docking results for control reserpine showed low binding affinity as indicated by total score of 6.5866 (Table 3).
Similarly, docking pose of UA-5 respectively with TolC, AcrB and YOJI protein the chemical nature of binding site residues within a radius of 4Å from bound THR-100, GLN-103, THR-104, LEU-107, ASN-220, LEU-221, SER-222, LEU-223, ALA-319, SER-322, VAL-323, GLN-325, THR-326, SER-329, SER-330, ASN-333, ASN-399, SER-402, ALA-403and ASN-
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177 showed good binding affinity as indicated by total score of 6.5935 and formed three hydrogen bond with THR-326, SER-329 and GLN-325 of length 2.5, 2.4 and 2.5 (Fig. 1B); ILE18, LEU- 21, ALA-22, GLY-23, LEU-25, ALA-26, LYS-29, ALA-381, ALA-384, ALA-385, PHE-386, GLY-387 showed low binding affinity as indicated by total score of 3.7193 and formed a hydrogen bond with LYS-29 of length 1.8 (Fig. 1C) and ARG-74, PHE-77, SER-78, ALA-79, VAL-80, PHE-81, TRP-85, LEU-86, GLU-94, LYS-96, LEU-107, ARG-122, ILE123, ALA-137, ALA-138, ALA-139, ALA-140, ALA-141, ALA-143, GLU-144and ARG-146 showed strong binding affinity as indicated by total score of 6.8942 and formed three hydrogen bond with ARG-146 and ARG-122 of length 2.1, 1.8 and 1.8 and strong hydrophobic interaction which may lead to more stability and activity. However, docking results for control compound reserpine showed low binding affinity as indicated by total score of 5.6579, 3.8169 and 6.5866 (Fig. 5 and Table 3).
On the basis of all above results, it may be concluded that ursolic acid derivatives clearly pass all the litmus tests of therapeutic strategy hypothesis according to which, molecules that target the cell membrane or cell walls are most likely to synergize with conventional antibiotics or antiseptics by inhibiting the efflux pumps, increasing cellular permeability and weakening the cell envelope (51).
Conclusions It is for the first time, drug resistance reversal potential of UA derivatives have been proved by inhibition of efflux pump, which may be useful in: (i) lowering the dose of antibiotics; (ii) reducing the drug resistance development frequency; and (iii) increasing the efficacy of antibiotics against multidrug resistant E. coli strains. These results may be of great help in the development of inexpensive and dose economic antibacterial drug formulations from a very common and widely distributed herb, E tereticornis
Acknowledgement
Authors are thankful to the National Gene Bank of Medicinal and Aromatic Plants at CSIRCentral Institute of Medicinal and Aromatic Plants, Lucknow, India for providing plant material. Financial support from CSIR Network project BSC 0203 is gratefully acknowledged. Two of us (GRD and AM) are grateful to CSIR for providing Research Fellowships. Competing interests: There is no conflict of interest.
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Figure Captions Scheme1. Semi-synthesis of ursolic acid derivatives. Figure 1: Time–kill curves of MDREC-KG4 showing the dose dependent bactericidal effect of (a) Tetracycline (b) Tetracycline in combination with UA derivatives Figure 2: Inhibition of ethidium bromide efflux by UA derivatives
Figure 3: ATPase inhibitory activity of UA derivatives. Figure 4: Efflux pump expression analysis of UA derivatives. Figure 5: In silico molecular docking studies elucidating the (A) interaction of UA-4 in the binding site of YOJI (B) ) interaction of UA-5 in the binding site of TolC, (C) AcrB and (D) YOJI. The docking studies were carried out using SYBYL-X 2.0.
Table 1. In vitro drug resistance reversal activity of ursolic acid derivatives Compound code NAL UA UA-1 UA-2 UA-3 UA-4 UA-5 UA-6 Reserpine
MIC of compounds (µg/mL) CA8000 DH5α 6.25 100 1000 1000 1000 1000 1000 1000 1000 1000 500 500 1000 1000 1000 1000 500 500
MIC of Nalidixic acid in presence of compounds (10µg/mL) CA8000 F.R. DH5α F.R. 6.25 100 3.125 2 50 2 1.56 4 25 4 1.56 4 25 4 1.56 4 25 4 1.56 4 12.5 8 0.78 8 12.5 8 1.56 4 25 4 3.125 2 50 2
NAL = nalidixic acid; CA8000 & DH5α = drug sensitive & drug resistant strains of E. coli respectively; F.R. = fold reduction;
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Table 2. Reduction in the minimum inhibitory concentrations of TET in combination with derivatives UA, UA-4 and UA-5 against MDRECKG4 Combinations Agents
MIC (Alone) (µg/mL)
Plant compounds (µg/mL)
MIC of tetracycline with/without plant compounds against MDREC-KG4
TET UA
800 1000
UA-4
500
UA-5
1000
RES
800
UA (100) UA (50) UA (25) UA (12.5) UA (6.25) UA (3.125) UA (1.56) UA (0.78) UA-4 (100) UA-4 (50) UA-4 (25) UA-4 (12.5) UA-4 (6.25) UA-4 (3.125) UA-4 (1.56) UA-4 (0.78) UA-5 (100) UA-5 (50) UA-5 (25) UA-5 (12.5) UA-5 (6.25) UA-5 (3.125) UA-5 (1.56) UA-5 (0.78) RES (100) RES (50) RES (25) RES (12.5) RES (6.25) RES (3.125) RES (1.56) RES (0.78)
400/800 400/800 800/800 800/800 800/800 800/800 800/800 800/800 100/800 100/800 200/800 200/800 200/800 200/800 400/800 400/800 100/800 100/800 100/800 200/800 200/800 200/800 200/800 400/800 400/800 400/800 400/800 800/800 800/800 800/800 800/800 800/800
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Fold Reduction in TET
2 2 No No No No No No 8 8 4 4 4 4 2 2 8 8 8 4 4 4 4 2 2 2 2 No No No No No
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Table 3. Comparison of docking score and binding site residues of compounds against drug efflux pump proteins.
Compound
PDB ID/
Total
Target
score*
Biding pocket residue in (4Å)
protein
Lengt
Amino acid
No. of
h of
residue
Hydrogen
hydro
involved in
Bond (H)
gen
Docking
bond
interaction
(Å) 1EK9/ TolC
6.2536
-
-
-
-
-
-
ARG-74, SER-78, ALA-79, VAL-80, PHE-81,
1.8
ARG-122
1
TRP-85, LEU-86, GLU-94, LYS-96, LEU-107,
1.8
GLN-236, GLN-239, ALA-240, GLN-301, GLN304, ALA-305, ASN-308, ASP-23, ALA-26, ALA-27, LYS-30, GLU-33, GLN-87, ALA-90, GLN-94
1OY8/ AcrB
4.4674
UA-4
ILE-18, ILE-19,
ALA-22, LEU-25, ALA-26,
LYS-29, GLY-378, ALA-381, ALA-384, ALA385, LEU-480 Model/ YOJI
7.2716
ARG-122, ILE-123, ALA-139, ALA-140, ALA141, ALA-143, GLU-144, ARG-146 1EK9/ TolC
6.5935
THR-100, GLN-103, THR-104, LEU-107, ASN-
2.5
THR-326
220, LEU-221, SER-222, LEU-223, ALA-319,
2.4
SER-329
SER-322, VAL-323, GLN-325, THR-326, SER-
2.5
GLN-325
1.8
LYS-29
1
ARG-146
3
329, SER-330, ASN-333, ASN-399, SER-402, ALA-403, ASN-177 1OY8/ AcrB
3.7193
ILE-18, LEU-21, ALA-22, GLY-23, LEU-25, ALA-26, LYS-29, ALA-381, ALA-384, ALA-
UA-5
385, PHE-386, GLY-387 Model/ YOJI
6.8942
ARG-74, PHE-77, SER-78, ALA-79, VAL-80,
2.1
PHE-81, TRP-85, LEU-86, GLU-94, LYS-96,
1.8
LEU-107, ARG-122, ILE-123, ALA-137, ALA-
1.8
ARG-122
-
-
-
2.0
LYS-29
1
THR-48, ARG-74, PHE-77, SER-78, ALA-79,
2.1
ARG-122
2
PHE-81, TRP-85, LEU-86, GLU-94,
2.1
ARG-146
1.9
ASN-115
138, ALA-139, ALA-140, ALA-141, ALA-143, GLU-144, ARG-146 1EK9/ TolC
4.1761
THR-111, ASN-333, ALA-336, SER-337, SER340, ASN-388, ALA-389, ASN-392, ILE-395, ASN-396, ASN-399, THR-170, ASN-173, ASN174, ASN-177
1OY8/ AcrB
2.9236
ALA-22, LEU-25, ALA-26, LYS-29, PHE-380, ALA-381, VAL-382, ALA-384, ALA-385, PHE-
UA
386 Model/ YOJI
4.5955
LYS-96,
ARG-122, ILE-123, ALA-140, ALA-141, ALA143, GLU-144, ARG-146 Reserpine
1EK9/ TolC
5.6579
THR-104, LEU-107, ASN-108, THR-111, ASN-
This article is protected by copyright. All rights reserved.
2
Accepted Article
115, GLN-325, THR-326, SER-329, SER-330,
2.1
ASN-333
-
-
-
LYS-75, PHE-77, SER-78,
2.7
ARG-146
-
ALA-79, PHE-81, TRP-85, LEU-86, GLU-94,
2.4
ALA-79
ASN-333, ILE-395, ASN-396, ASN-399, ASN173, ASN-177 1OY8/ AcrB
3.8169
ILE-19, ALA-22, LEU-25, ALA-26, LYS-29, LEU-30, ALA-381, VAL-382, ALA-384, ALA385, GLY-387, ALA-479, LEU-480, LEU-483
Model/ YOJI
6.5866
THR-48, ARG-74,
LYS-96, ARG-122, ILE-123, ALA-140, ALA141, ALA-143, GLU-144, ARG-146 *Surflex-Dock scores (total scores) are expressed in –log10 (Kd) units to represent binding affinities
This article is protected by copyright. All rights reserved.
Accepted Article This article is protected by copyright. All rights reserved.
Accepted Article This article is protected by copyright. All rights reserved.
Accepted Article This article is protected by copyright. All rights reserved.
