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Rosas-Sandoval G, Ambrogelly A, Rinehart J, Wei D, Cruz-Vera LR, Graham DE, Stetter KO, Guarneros G, Söll D. (2002). Orthologs of a novel ... Gwen Harden.
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Natural Product Communications

Peptidyl-tRNA Hydrolase Screening Combined with Molecular Docking Reveals the Antibiotic Potential of Syzygium johnsonii Bark Extract

2011 Vol. 6 No. 10 1421 - 1424

Sarah M. Harrisa, Hana McFeetersa, Ifedayo V. Ogungbea, Luis R. Cruz-Verab, William N. Setzera, Betsy R. Jackesc and Robert L. McFeetersa,* a

Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA

b

Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, AL 35899, USA

c

School of Marine and Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia [email protected]

Received: May 24th, 2011; Accepted: June 20th, 2011

With the rapid rise of antibiotic resistance in pathogenic bacteria, the need for new antibacterial agents is overwhelming. Herein we report the limited screening of tropical plant extracts for inhibitory activity against the essential enzyme peptidyl-tRNA hydrolase (Pth). Initial screening was conducted through an electrophoretic mobility assay and Northern blot detection. The ability of Pth to cleave the peptidetRNA ester bond was assessed. The ethanol bark extract of Syzygium johnsonii showed strong inhibitory potential. Molecular docking studies point to Syzygium polyphenolics as the potential source of inhibition. This work is the forerunner of activity-directed isolation, purification, and structure elucidation of the inhibitory components from Syzygium johnsonii extracts and studies of compound interaction with Pth. Keywords: antibacterial, tropical plant extracts, Syzygium johnsonii, peptidyl-tRNA hydrolase, molecular docking, phytochemical screening.

Traditional natural products provide many new antimicrobial lead compounds. Terrestrial plants, which have a long history in medicine [1], are the origin of a large fraction of drugs on the market today. New plant species are potentially large reservoirs for antibacterial compounds. Targeting Staphylococcus aureus, we have screened a library of tropical plant extracts that have shown previous antibacterial activity [2] for inhibition of the essential enzyme peptidyl-tRNA-hydrolase (Pth). Pth cleaves the ester bond of peptidyl-tRNAs, separating the peptide moiety from the tRNA [3]. Accumulation of peptidyl-tRNAs is toxic for cells presumably by impairing the initiation of translation or slowing protein synthesis due to specific tRNA starvation [4]. Thus, it is necessary for cells to maintain Pth activity. Bacteria possess a single class of peptidyl-tRNA hydrolase with high primary sequence conservation among all known eubacterial genes. Therefore, an inhibitor for one bacterial Pth will likely inhibit many others (broad spectrum). Unlike bacteria, eukaryotes possess several enzymes with Pth-like activities [5]. Namely, the Pth2 family is structurally unrelated to bacterial Pth. These factors (essential enzymatic function, high degree of conservation in bacteria, no critical human homolog) make bacterial Pth a prime target for antibiotic development.

Members of a group of tropical bark extracts shown to have anti-S. aureus activity were screened for Pth inhibition. A list of extracts and their relative Pth activity is shown in Figure 1. Syzygium johnsonii bark extract showed the highest degree of Pth inhibition, completely abrogating peptidyl-tRNA cleavage. Extracts from Byrsonima crassifolia, Maurauria heterophylla, and Zanthoxylum procerum also showed strong inhibitory activity, though not as complete as S. johnsonii. Drypetes lasiogyna, Nectandra membranacea and Verbisina tubacensis extracts showed partial inhibition while Eugenia monteverdensis showed a complete lack of Pth inhibition. Syzygium johnsonii (F. Muell.) B. Hyland, Johnson’s satinash, rose satinash, is a small to medium-sized tree in rainforest along watercourses and ranges from central Queensland to north Queensland. Leaves are elliptic to obovate, 5-12 cm long, apex abruptly narrowed to the drawn out rounded tip, base cuneate, margins bent under, hairless, glossy; midvein sunken above, lateral veins 30-50 pairs, crowded rather indistinct on both surfaces; oil dots large and numerous, petiole 4-10 mm long. The fruit a berry, ellipsoid, 1-2.5 cm long, fleshy, purplish mauve, crowned by calyx [6].

1422 Natural Product Communications Vol. 6 (10) 2011

Harris et al.

Figure 1: Pth inhibition by bark extracts from tropical plant. From left to right: Pth inhibition relative to a control experiment with extracts showing inhibitory activity in black; list of plants used to prepare extracts; solvent used for extraction.

Figure 3: The crystal structure of M. smegmatis Pth (PDB: 3KK0) with the lowest-energy docked structures of the four strongest binding Syzygium tannins. -helices are shown in red, -sheets in blue, and tannins in yellow, green, pink and cyan.

Figure 2: Pth activity assay. Experimental data showing the activity assay for Pth mediated peptidyl-tRNA cleavage. In presence of Pth, the peptide portion of peptidyl-tRNA gets cleaved and the tRNA appears as a single band (lane 1). The addition of DMSO to the reaction mixture does not intefere with normal Pth activity (lane 2). In absence of Pth, peptidyltRNA runs as a smear due to the different sizes of peptides bound (lane 3). The remaining lanes show hypothetical data with lanes 4-6 and 8-9 having no inhibition and lane 7 with strong inhibtion towards Pth.

In order to identify potential phytochemical inhibitors of Pth, a molecular docking study was carried out using the Syzygium phytochemicals listed in the Dictionary of Natural Products [7]. Syzygium tannins and other polyphenolic compounds were found to be the strongest docking compounds (Table 1). Key molecular contacts between the Syzygium polyphenolics and E. coli Pth were His 113, Leu 95, Gly 111, Gly 112, and Lys 142, while M. smegmatis important contacts were His 115, Asn 116, His 22, Gly 114, and Asp 95 (Figure 3), and M. tuberculosis key contacts were His 115, Asn 116, His 22, Val 150, and Asp 95. Thus the compounds make contacts in the previously proposed enzyme active site [8]. Tropical rainforest floras are poorly characterized and contain numerous unexplored and pharmacologically interesting phytochemicals. The findings reported here suggest that Syzygium johnsonii bark extract contains several potential lead compounds for development of a new antimicrobial agent. Thus the potential for finding Pth inhibitors from this extract is promising. Bioactivity-

directed isolation, purification, and structure elucidation of the inhibitory components from S. johnsonii are currently underway in our laboratories, and future studies will involve specific interactions of active components with Pth and Pth specific bacterial growth inhibition. Experimental Pth preparation: The Pth gene from E. coli was cloned into a pKQV4 vector with an N-terminal hexahistidine tag. The 24 kDa recombinant protein was expressed in shake flasks with LB media at 37°C. When the cell density reached an OD600 of 0.8, protein expression was induced with 1.0 mM isopropyl -D-1-thiogalactopyranoside. Cells were harvested by centrifugation after 4 hours and stored frozen. Cells were then resuspended in a buffer of 50 mM sodium phosphate, 300 mM NaCl, 2 mM DTT, pH 7.4 and lysed with lysozyme and mechanical disruption via sonication. After centrifugation, the clarified supernatant was subjected to metal chelation chromatography resulting in >90% pure Pth. Fractions containing Pth were pooled and dialyzed against a buffer of 10 mM Tris acetate, pH 8.0 in 30% glycerol and DEPC treated water. The protein was stored at -80°C until further use. Peptidyl-tRNA generation: A bacterial strain with temperature sensitive Pth mutant was used to produce peptidyl-tRNA [9]. At 30°C, functioning Pth allowed for normal growth and cell proliferation. Shifted to 42°C, Pth is no longer functional allowing for the accumulation of peptidyl-tRNAs. Bacterial cultures in LB media were

Pth inhibition by Syzygium johnsonii

Natural Product Communications Vol. 6 (10) 2011 1423

Table 1: Docking energies (kJ/mol) of Syzygium phytochemicals with bacterial peptidyl-tRNA hydrolase.

Ligand Eugenol galloyl glucoside S. aromaticum tannin C S. aromaticum tannin G S. aromaticum tannin D S. aromaticum tannin F 2'-C-Methylmyricetin galloyl rhamnoside S. aromaticum tannin A S. aromaticum tannin E Isobiflorin 6'-galloyl Myricetin 3-robinobioside Syzygiol B Syzygiol S. cumini lignan C S. aromaticum tannin B Syzygiresinol A Rhamnocitrin S. cumini lignan A S. cumini lignan B S. levinei glucoside Syzyginin B Stercurensin Ellagic acid rhamnoside Aurentiacin Syzygiol A Myrigalone H 3-Galloylglucose Uvangoletin S. samarangense chalcone A Samarangenin A Glucosyltrihydroxyacetophenone Methylhexanoylphloroglucinol Orsellinic acid glucoside Syzalterin 7-Hydroxy-5-methoxy-6,8-dimethylflavanone Samarangenin B Ellagic acid acetylrhamnoside Isobiflorin Biflorin Cuminiresinol Platanic acid Epifriedelin Oleanolic acid

2PTH E. coli X-ray -134.8 -137.4 -152.6 -137.3 -136.3 -132.6 -126.6 -125.9 -123.2 -112.4 -115.7 -101.8 -94.6 -121.8 -97.6 -108.9 -100.9 -102.4 -129.8 -130.4 -105.0 -98.4 -106.7 -104.6 -100.2 -107.5 -102.6 -101.1 -68.8 -95.4 -94.7 -88.1 -85.6 -78.3 -86.0 -101.9 -93.9 -104.4 -83.5 -28.6 -46.8 -51.2

3KK0 M. smegmatis X-ray -125.3 -100.8 -101.6 -101.5 -126.5 -100.2 -100.8 -110.4 -102.5 -105.2 -109.1 -91.5 -104.6 -59.6 -103.5 -99.5 -101.8 -97.0 -100.7 -67.8 -98.3 -94.4 -92.8 -100.7 -99.9 -101.9 -99.7 -91.7 -72.5 -78.8 -92.6 -84.4 -97.6 -96.5 -70.1 -45.2 -87.3 -77.9 -72.3 -66.5 -14.9 -11.5

grown at 30°C until reaching an OD600 of 0.4. The temperature was then shifted to 42°C for 1 hour. After brief cooling on ice, the cells were spun down and the pellets were stored at -80°C. Frozen cell pellets were resuspended in a buffer of 0.3 M NaOAc, pH 4.5, 10 mM EDTA and tRNA extracted via phenol/chloroform extraction as described elsewhere [10]. For long term storage, pellets of precipitated tRNA were stored at -80°C. Pth inhibition assay: Tropical plant extracts from our collection were screened for Pth inhibition using a gel migration assay to detect the cleavage of peptidyl-tRNAs. Briefly, recombinant Pth (0.8 g) and peptidyl-tRNAs (10 g) were incubated with plant extracts (1 L of 1% solution). In the control experiment, DMSO was used

2JRC M. tuberculosis NMR #1 NMR #13 -93.1 -97.0 -102.6 -120.7 -93.5 -96.7 -99.6 -94.4 -85.6 -89.4 -92.3 -90.9 -92.7 -98.2 -88.0 -101.5 -76.0 -91.1 -85.6 -78.6 -61.3 -77.2 -70.3 -91.3 -88.5 -84.6 -85.7 -79.3 -79.1 -83.6 -63.2 -96.3 -68.4 -92.9 -75.8 -70.7 -84.6 -19.8 -93.3 -51.5 -63.6 -81.7 -72.9 -77.1 -71.8 -74.3 -61.5 -69.2 -55.4 -83.4 -66.8 -59.9 -46.1 -87.8 -67.9 -69.9 -85.5 -97.7 -76.2 -67.4 -56.3 -70.9 -63.0 -73.5 -54.8 -81.0 -55.7 -91.1 -65.2 -72.3 -71.5 -76.5 -72.5 -37.5 -69.9 -14.2 -56.1 -60.9 -55.7 -56.6 -69.9 -62.1 -26.6 -63.2

2Z2K M. tuberculosis X-ray -101.5 -87.2 -102.3 -112.0 -91.4 -99.5 -88.0 -80.3 -97.7 -91.1 -81.3 -85.7 -63.7 -88.9 -66.7 -59.9 -59.1 -76.0 -85.7 -76.1 -70.4 -73.8 -69.3 -76.0 -72.1 -73.1 -71.7 -72.9 -69.3 -70.8 -71.5 -70.8 -54.8 -50.3 -73.9 -71.6 -72.4 -63.3 -53.2 -37.4 -39.3 -52.5

AVERAGE -110.3 -109.7 -109.3 -109.0 -105.8 -103.1 -101.3 -101.2 -98.1 -94.6 -88.9 -88.1 -87.2 -87.1 -86.1 -85.6 -84.6 -84.4 -84.1 -83.8 -83.8 -83.3 -83.0 -82.4 -82.2 -81.9 -81.6 -80.7 -78.8 -77.7 -77.2 -76.0 -74.8 -74.4 -73.5 -73.4 -72.7 -65.9 -65.2 -49.0 -46.6 -41.0

instead of the plant extract. The 20 L reaction took place in a buffer composed of 10 mM Tris acetate, pH 8.0, 10 mM magnesium acetate and 20 mM ammonium acetate in DEPC treated water and was allowed to run for 30 minutes. It was quenched by adding ethanol for tRNA precipitation and the obtained tRNA was applied on a 45 cm 40% polyacrylamide gel. The tRNA was transferred to a nylon membrane and probed with a 32P anti-tRNALys probe. The amount of free tRNA and peptidyl-tRNA was qualitatively gauged from visual analysis of film exposures. Pth with an equal amount of DMSO instead of extract served as a positive control. For a negative control, an equal amount of peptidyl-tRNA without Pth was run. The activity of each extract tested was normalized to the positive control (100%) on each gel run.

1424 Natural Product Communications Vol. 6 (10) 2011 Molecular Docking: Protein-ligand docking studies were carried out based on the crystal structures of Pth from Escherichia coli (PDB 2PTH) [11], Mycobacterium smegmatis (PDB 3KK0) [12], and Mycobacterium tuberculosis (PDB 2Z2K) [8], as well as two NMR structures from M. tuberculosis (PDB 2JRC) [4]. All solvent molecules and the co-crystallized ligands were removed from the structures. Molecular docking calculations for all compounds with each of the proteins were undertaken using Molegro Virtual Docker v. 4.3 [13], with a sphere large enough to accommodate the cavity centered on the binding sites of each protein structure in order to allow each ligand to search. Different orientations

Harris et al. of the ligands were searched and ranked based on their energy scores. The RMSD threshold for multiple cluster poses was set at < 1.00Å. The docking algorithm was set at maximum iterations of 1500 with a simplex evolution population size of 50 and a minimum of 30 runs for each ligand. Acknowledgments – L. R. C.-V. and R. L. M. thank the UAH Office of the Vice President of Research for financial support under the internal URII program. S. M. H. is grateful for summer research support and travel funds from the UAH Chemistry Department Research Experiences for Undergraduates program.

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