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Volume 10(12)

Hypothesis

Computer aided screening of potent inhibitor compounds against inhibitor resistant TEM β-lactamase mutants from traditional Chinese medicine

Qifeng Zhu*, Yanxia Yin,Hanjie Liu & Jinhong Tian

College of Pharmaceutical Sciences, Southwest University, Chongqing, China; Qifeng Zhu - Email: qifeng900627@sina.com; *Corresponding author

Received October 23, 2014; Accepted November 12, 2014; Published December 31, 2014

Abstract:

Inhibitor-resistant TEM (IRT) type β-lactamase mutation is largely known. Therefore, it is of interest to identify new yet improved leads against IRT from traditional Chinese medicine. Hence, we screened more than 10,000 compounds from Chinese medicine (tcm@ database) with mutant molecular IRT models through docking techniques. This exercise identified compounds affeic acid, curcumin, salvianolic acid E, ferulic acid and p-coumaric acid with high binding score with the mutants. This was further validated in vitro where salvianolic acid E combined with cefoperazone and sulbactam effectively inhibit the R244S mutant.

Keywords: β-lactamase, mutants, docking, Chinese traditional medicines

Background:

β-lactam antibiotics are the most wieldy used in antibiotics because of their wide spectrum of efficiency, bactericidal activity, and low toxicity. But, the drastic emergence of resistance has become a serious problem [1]. The major resistance mechanism of bacteria is the production of β-lactamase, which can hydrolyze the amide bond of the β-lactam ring, leading to antibiotic inactivation [2]. Class A β-lactamases, which are considered to be responsible for many failures in the treatment of infectious diseases, are most widespread enzymes. TEM β-lactamase is one type of class A type β-lactamase, it is commonly found in Escherichia coli, which is one of the most common pathogens in community-acquired and nosocomial infections [3]. To overcome the problem of β-lactamase, one of the effective methods is the combination of β-lactamase inhibitor and β-lactam antibiotic. Beta-lactamase inhibitors can protect β-lactam antibiotics by inhibiting β-lactamase. Their antimicrobial activities are low, but they can be irreversible

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bound with the β-lactamase, in order to prevent the hydrolysis reaction of β-lactam antibiotics [4]. However, the emergence of inhibitor resistance strains aggravated the problem. Mutation in some positions can lead to high catalytic activity and resistance to β-lactamase inhibitors, turning the wild type β-lactamases into extended spectrum β-lactamase (Esbls) or inhibitor resistant β-lactamases(IRTs) [5, 6]. M69I, S130G and R244S are three clinical inhibitor resistant TEM β-lactamase mutants which have been reported [7, 8, 9]. Studies focused on them showed that Met69, Ser130 and Arg244 were three active residues close to Ser70, the active site of TEM-1 β-lactamase, and played very important roles in the hydrolysis reaction of antibiotics [10, 11]. Therefore, it is necessary for developing inhibitors against these resistant mutants. The study mainly focused to screen new compounds against IRTS based on docking studies. The compounds screened out from database were later used for in vitro studies.

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Figure 1: The accessible surface areas of wide type and three mutanted β-lactamases. Methodology: ligands were variable, finally the accessible surface area was Proteins preparation within radius 6.5 A. Results are Table 1 to Table 3 (see The crystal structure of tem-1 β-lactamase (pdbid: 1bt5) was supplementary material). downloaded from RSCB website [12], then the software

Discovery Studio 3.5 was used for design the mutants of tem-1 In vitro studies

β-lactamase. The crystal structure of M69I was constructed by The fabrication of inhibitor-resistant β-lactamase mutants the amino acid substitution Met-69→Ile, and the S130G was The mutation of the single amino acid would lead to the drug constructed by the amino acid substitution Ser-130→Gly, and resistance [15]. According to the mechanism, and the ampicillin

resistance genes from PAMP was used as template, the No.139-the R244S was constructed by the amino acid substitution Arg-244→Ser. Each structure was refined by removing the 141 loci of tem-1 β-lactamase are GCG, the No.141 locus G was heteroatoms. These structures of the proteins would be used as changed to T, it would lead the arginine of No.244 mutate into

serine, and this mutant protein was R244S. In a similar way, the drug targets in molecular docking.

No.478-480 loci ACT were changed to ACC, leading to the

serine of No.130 was mutated into glycine, and this mutant Preparation of ligand library

It was reported that many compounds based herbal had high protein was S130G. And the No.661-663 loci CAT were changed medicinal values against many pathogens [13]. Hence, it was to AAT, leading to the methionine of No.69 was mutated into possible to find out the inhibitors against the IRTs by computer isoleucine, this Mutant protein was M69I. The mutated aided screening and the data would be useful to screen best plasmids were transformed into BL21 competent cells in order lead molecules. A library of about 10,000 compounds from 300 to get three mutated strains. different Chinese medicinea was prepared for molecular

docking. All the 3D structures of ligands were retrieved from The experiment of mutant strains resistant to enzyme Traditional Chinese Medicine Database@Taiwan inhibitors

The MIC was determined by the doubling dilution method. On (http://tcm.cmu.edu.tw/) [14].

the sterile microplate, 200ul LB liquid medium were added into

each hole, and the cefperazone-sulbactam was added into the Molecular docking

The software FlexX (a component of LeadIT) was used for first hole of each row with the final concentration of molecular dock in this study. In the docking, three mutants and 1024mg/ml, then stepwise double dilute it to the next holes of the tem-1 β-lactamase from wild type stain were treated as the row. At last, three mutant strains and the primordial strain receptor protein, which were docked with the ligand library were inoculated in the microplate respectively and the receptor respectively. The docking was semi-flexible, and microplate was incubated at 37℃ for 24 hours. Result is shown receptor residue bond angles were immutable, furthermore in Table 4 (see supplementary material).

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was determined by the doubling dilution method,but the Chinese medicinal ingredients were mixes into Cefperazone-Sulbactam with the final concentration of 20% respectively. The blank control groups without inoculation were seted up, as well as the control groups which added no Cefperazone-Sulbactam but only Chinese medicine ingredients. Result is shown in Table 5 (see supplementary material).

Invitro studies of combination of cefperazone-sulbactam and traditional Chinese medicine ingredient

Based on the docking studies, five Chinese herbal medicinal ingredients were identified for in vitro testing. The Chinese herbal medicinal ingredients used in the study are caffeic acid(ligand id:1066):, curcumin(ligand id:9863), Salvianolic acid E(ligand id:6013), ferulic acid(ligand id:3070) and p-coumaric acid(ligand id:1680) [16,17,18,19]. Similar to the above, the MIC Figure 2: The combination of antibiotics and Salvianolic acid E flight the R244S mutant. The MIC was determined by the doubling dilution method in the microplate. A row was the blank control, C row was Ampicillin, D row was Ampicillin and salvianolic acid E, E row was Cefperazone-Sulbactam, F row was Cefperazone-Sulbactam and Salvianolic acid E. The turbid holes indicate the growth of bacteria, and the limpid ones indicate the bacteria were killed. (Please cite figure 2 in main text) Figure 3: Binding of salvianolic acid E within the active site of R244S. (Please cite figure 3 in main text) ISSN 0973-2063 (online) 0973-84 (print) Bioinformation 10(12):746-752 (2014)

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Discussion:

Inhibitors-resistant mutants of TEM β-lactamase

In this study, three mutant strains which resistant enzyme inhibitors were constructed by site direct mutagenesis. Based on the experiment of mutant strains resistanted to enzyme inhibitors, the results showed that the sulbactam could effectively inhibit the activity of wild-type β-lactamase, leading to cefoperazone could inhibit the growth of bacteria with the concentration of 1ug/ml . It also showed that R244S mutant, S130G mutant and M69I mutant could certain resistant the inhibitor, sulbactam. In the experiment, R244S mutant had the best result of inhibitors-resistance, the MIC could reach 32ug/ml. The result of S130G mutant is worst, but its MIC could still reach 4ug/ml. And the MIC of M69I mutant is 8ug/ml. The results was consistent with previously reported [7, 8, 9], showed that mutation in these three positions could lead to resistance to β-lactamase inhibitors, turning the wild type β-lactamases into IRTs.

Molecular docking

Virtual screening of chemical databases is an efficient method of computer aided drug design [20]. In this study, we used virtual screening in order to find out new compounds with high affinity against three IRTs from traditional Chinese medicine ingredients. According to the results of molecular docking, caffeic acid, curcumin, salvianolic acid E, ferulic acid and p-coumaric acid have high binding score with the mutants. Hence, these five Chinese medicine ingredients were chosen as inhibitors for in vitro study.

Combination of antibiotic and Chinese herbal medicinal ingredients

The invitro studies of five Chinese medicine ingredients aimed to verify the accuracy of molecular docking. The results showed that using the Chinese medicinal ingredients alone could not inhibit the growth of mutated E. coli in vitro. But the combination of cefperazone-sulbactam and Salvianolic acid E could inhibit the growth of R244S mutated strain effectively (Figure 1). The MIC of cefperazone-sulbactam reduced from 32ug/ml to 1ug/ml by using salvianolic acid E.

Experiments show that salvianolic acid E did not have antibacterial activity, but it could reduce the MIC of cefperazone-sulbactam when it was used in conjunction with cefperazone-sulbactam. This illustrates that the mechanism of salvianolic acid E was binding with β-lactamase as enzyme inhibitor, protecting cefperazone from hydrolysis, to achieve the purpose of inhibiting bacterial growth. This is consistent with the results of molecular docking. The other Chinese herbal medicinal ingredients such as caffeic acid, curcumin and so on, also had high affinity for these β-lactamase mutants, but they

did not show this feature, the reason of that remained to be further in-depth study.

Conclusion:

Computer aided modeling followed by invitro validation show that the ingredient of traditional Chinese medicine salvia, salvianolic acid E inhibits the r244s β-lactamase mutant. The MIC of cefoperazone significantly reduced, and drug-resistant mutant strains of Escherichia coli growth has been effectively suppressed by combination of cefperazone-sulbactam and salvianolic acid E. This finds application in the development of compounds to combat inhibitor resistant strains.

Acknowledgement:

The authors thankfully acknowledge all colleagues at SWU College of Pharmaceutical Sciences for their support and help.

References:

[1] Shakil S et al. J Biomed Sci. 2008 15: 5 [PMID: 17657587] [2] Page MI, Curr Pharm Des. 1999 5: 5 [PMID: 10539995]

[3] Ding J et al. Diagn Microbiol Infect Dis. 2013 76: 532 [PMID:

23726651]

[4] Drawz SM et al. Clin Microbiol Rev. 2010 23: 160 [PMID:

20065329]

[5] Coutinho HD et al. Chemotherapy. 2008 : 328 [PMID:

18698137]

[6] Eumkeb G et al. Phytomedicine. 2010 18: 40 [PMID:

21036573]

[7] Poirel L et al. Antimicrob Agents Chemother. 2004 48: 4528

[PMID: 15561821]

[8] Kaye KS et al. Antimicrob Agents Chemother. 2004 48: 1520

[PMID: 15105100]

[9] Goussard S & Courvalin P, Antimicrob Agents Chemother.

1999 43: 1657 [PMID: 9925535]

[10] Thomas VL et al. Biochemistry. 2005 44: 9330 [PMID:

15981999]

[11] Thomas VL et al. J Mol Biol. 2010 396: 47 [PMID: 19913034] [12] Maveyraud L et al. J Biol Chem. 1996 271: 10482 [PMID:

8631844]

[13] Liu IX et al. J Pharm Pharmacol. 2000 52: 361 [PMID:

10757427]

[14] Chen CY et al. PLoS One. 2011 6: e15939 [PMID: 21253603] [15] Pierce KE et al. J Mol Diagn. 2013 15: 291 [PMID: 23518216] [16] Yagi A et al. Planta Med. 19 55: 51 [PMID: 2717690]

[17] Suksamrarn et al. Chem Pharm Bull. 2005 53: 1327 [PMID:

16204994]

[18] González AG et al. J Nat Prod. 2003 66: 793 [PMID:

128284]

[19] Banskota AH et al. Planta Med. 2003 69: 500. [PMID:

12865966]

[20] Baig MH et al. Bioinformation 2012 8: 1225 [PMID: 23275724]

Edited by P Kangueane

Citation: Zhu et al. Bioinformation 10(12): 746-752 (2014)

License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium,

for non-commercial purposes, provided the original author and source are credited

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Supplementary material:

Table 1: The top 10 docking results of ligands docked with M69I. Ligand id Score match lipo ambig clash rot 3461 -38.0729 -43.8594 -5.9867 -9.0790 2.8521 12.6000 6013 -33.2017 -45.1958 -12.1111 -10.3221 3.8272 25.2000 2216 -29.4123 -34.4233 -2.1872 -4. 2.1527 4.2000 1879 -27.0653 -35.6529 -3.8365 -7.0511 2.8752 11.2000 3421 -26.01 -37.6213 -2.6193 -6.1883 1.0100 14.0000 2695 -25.1269 -24.8539 -9.0500 -7.3946 3.7716 7.0000 1903 -23.9318 -30.5255 -4.4616 -5.0443 2.2996 8.4000 -23.6351 -36.0236 -1.6046 -5.2103 1.2034 12.6000 1941 -21.9863 -35.4600 -2.9998 -5.4638 1.1373 15.4000 1680 -21.1125 -23.1296 -2.7826 -2.6377 0.6374 1.4000

Table 2: The top 10 docking results of ligands docked with R244S. Ligand id Score match lipo ambig clash rot 6013 -33.08 -41.8441 -15.1835 -19.4142 12.7530 25.2000 3467 -26.3783 -36.5709 -4.4271 -5.9692 3.9888 11.2000 3460 -26.0524 -32.5309 -5.0422 -7.4187 3.7395 9.8000 3461 -24.3798 -36.9619 -5.9988 -7.3360 7.9169 12.6000 2695 -21.7085 -21.9024 -6.4866 -7.9611 2.2415 7.0000 -21.2277 -31.8608 -3.4952 -5.9134 2.0417 12.6000 1879 -20.2515 -30.0084 -4.0625 -4.18 1.4092 11.2000 1066 -19.8870 -21.2996 -3.2711 -6.1131 2.5968 2.8000 3070 -19.2058 -21.8566 -5.4698 -5.4406 5.3612 2.8000 1028 -19.0708 -25.7814 -2.8802 -4.0446 1.2355 7.0000

Table 3: The top 10 docking results of ligands docked with S130G. Ligand id Score match lipo ambig clash rot 2216 -30.5969 -37.31 -2.96 -4.8227 4.7185 4.2000 3461 -28.9950 -44.2409 -2.9714 -6.7375 6.98 12.6000 6013 -28.5353 -42.3716 -10.99 -9.6868 3.8880 25.2000 1879 -27.5260 -38.60 -2.7796 -5.4456 2.7631 11.2000 1680 -25.7674 -26.9908 -3.1570 -3.2357 0.8161 1.4000 3070 -25.7660 -28.9473 -4.5676 -3.62 3.1752 2.8000 1066 -25.4115 -27.7853 -2.9621 -3.6165 0.7523 2.8000 3467 -24.6374 -32.3822 -4.5707 -6.8795 2.5950 11.2000 3460 -23.2770 -32.7962 -4.2271 -5.9397 4.4861 9.8000 7001 -22.1122 -28.7722 -2.9580 -5.0786 0.66 8.4000

Table 4: Doubling dilution method to determine the MIC. The parent strain has no resistance to Cefperazone-Sulbactam, but all the mutant strains showed high resistance to it. Strains Cefperazone-Sulbactam ug/mL 2048 1024 512 256 128 32 16 8 4 2 1 primordial strain - - - - - - - - - - - - R244S - - - - - - + + + + + + S130G - - - - - - - - - + + + M69I - - - - - - - - + + + + blank control - - - - - - - - - - - -

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Table 5: Combination of antibiotic and Chinese herbal medicinal ingredients against the mutant strains, the MIC was determined by the doubling dilution method. The holes which the bacterial have grown were marked as “+”, and the holes which the bacteria which the bacterial could not grow were marked as “-“. Drugs against M69I strain

Cefperazone-Sulbactam

combination of Cefperazone-Sulbactam and caffeic acid

combination of Cefperazone-Sulbactam and curcumin

combination of Cefperazone-Sulbactam and concentration ug/mL 2048 - - - 1024 - - - 512 - - - 256 - - - 128 - - - - - - 32 - - - 16 - + - 8 + + + 4 + + + 2 + + + 1 + + + Salvianolic acid E

combination of Cefperazone-Sulbactam and p-coumaric

combination of Cefperazone-Sulbactam and ferulic acid

blank control

Drugs against S130G strain

Cefperazone-Sulbactam

combination of Cefperazone-Sulbactam and caffeic acid

combination of Cefperazone-Sulbactam and curcumin

combination of Cefperazone-Sulbactam and Salvianolic acid E

combination of Cefperazone-Sulbactam and p-coumaric

combination of Cefperazone-Sulbactam and ferulic acid

blank control Drugs against R244S strain

Cefperazone-Sulbactam

combination of Cefperazone-Sulbactam and caffeic acid

combination of Cefperazone-Sulbactam and curcumin

combination of Cefperazone-Sulbactam and Salvianolic acid E

combination of Cefperazone-Sulbactam and p-coumaric

combination of Cefperazone-Sulbactam and ferulic acid

blank control Drugs against R244S strain

Cefperazone-Sulbactam

combination of Cefperazone-Sulbactam and caffeic acid

combination of Cefperazone-Sulbactam and curcumin

combination of Cefperazone-Sulbactam and Salvianolic acid E

combination of Cefperazone-Sulbactam and p-coumaric

combination of Cefperazone-Sulbactam and ferulic ISSN 0973-2063 (online) 0973-84 (print)

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- - - - - - - - - - - - - - - - - - -

-

-

-

-

-

Cefperazone-Sulbactam ug/mL 2048 1024 512 256 128 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - concentration ug/mL

2048 1024 512 256 128 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - - - - - - - - - - - - concentration ug/mL

2048 1024 512 256 128 - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - + -

-

-

-

-

-

751

- + + + + + - - + + + + - - + + + + -

-

-

- - - 32 16 8 4 2 1 - - - + + + - - - + + + - - - + + + - - - + + + - - - + + + - - - + + + - - - - - - 32 16 8 4 2 1 + + + + + + + + + + + + + + + + + + - - - - - - + + + + + + + + + + + + - - - - - - 32 16 8 4 2 1 + + + + + + + + + + + + + + + + + + - - - - - - + + + + + + +

+

+

+

+

+

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acid

blank control Drugs caffeic acid curcumin Salvianolic acid E p-coumaric ferulic acid blank control

- 2048 + + + + + -

- 1024 + + + + + -

- 512 + + + + + -

- 256 + + + + + -

- 128 + + + + + -

- + + + + + -

- 32 + + + + + -

- 16 + + + + + -

- 8 + + + + + -

- 4 + + + + + -

- 2 + + + + + -

- 1 + + + + + -

concentration ug/mL

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