A Carbocyclic Curcumin Inhibits Proliferation of Gram-Positive

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A carbocyclic curcumin inhibits proliferation of Gram positive bacteria by targeting FtsZ Paul W. Groundwater, Rajeshwar Narlawar, Vivian Wan Yu Liao, Anusri Bhattacharya, Shalini Srivastava, Kishore Kunal, Munikumar Reddy Doddareddy, Pratik M Oza, Ramesh Reddy Mamidi, Emma Claire Louise Marrs, John David Perry, David E Hibbs, and Dulal Panda Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00879 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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Biochemistry

A carbocyclic curcumin inhibits proliferation of Gram positive bacteria by targeting FtsZ

Paul W. Groundwater,1* Rajeshwar Narlawar,1 Vivian Wan Yu Liao,1 Anusri Bhattacharya,2 Shalini Srivastava2, Kishore Kunal,2 Munikumar Doddareddy,1 Pratik M. Oza,1 Ramesh Mamidi,1 Emma C. L. Marrs,3 John D. Perry,3 David E. Hibbs,1 and Dulal Panda2*

1

Faculty of Pharmacy, Bank Building, Science Road, The University of Sydney, Sydney NSW

2006, Australia 2

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay,

Mumbai 400076, India 3

Microbiology Department, Freeman Hospital, High Heaton, Newcastle upon Tyne, NE7 7DN,

United Kingdom *Corresponding authors: [email protected]; [email protected]

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ACS Paragon Plus Environment

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Abstract Inhibition of FtsZ assembly has been found to stall bacterial cell division. Here, we report the identification of a potent carbocyclic curcumin analogue (2d) that inhibits Bacillus subtilis 168 cell proliferation by targeting the assembly of FtsZ. 2d also showed potent inhibitory activity (MICs 2-4 mg/L) against several clinically important species of Gram positive bacteria, including methicillin-resistant Staphylococcus aureus. Further, 2d displayed a significantly reduced inhibitory effect on human cervical cancer cells in comparison to bacterial cells. Using live cell imaging of GFP-FtsZ by confocal microscopy, 2d was found to rapidly perturb the cytokinetic FtsZ rings in Bacillus subtilis cells. The immunofluorescence imaging of FtsZ also showed that 2d destroyed the Z-ring in bacteria within 5 minutes. Prolonged treatment with 2d produced filamentous bacteria, but 2d had no detectable effect either on the nucleoids or on the membrane potential of bacteria. 2d inhibited FtsZ assembly in vitro whereas it had minimal effects on the tubulin assembly. Interestingly, 2d strongly enhanced the GTPase activity of FtsZ, while it reduced the GTPase activity of tubulin. Furthermore, 2d bound to purified FtsZ with a dissociation constant of 4.0 ± 1.1 µM and the binding of 2d altered the secondary structures of FtsZ. The results together suggested that the non-natural curcumin analogue 2d possesses powerful antibacterial activity against important pathogenic bacteria and the evidence indicates that 2d inhibits bacterial proliferation by targeting FtsZ.

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Introduction Filamentous temperature-sensitive mutant Z (FtsZ), the prokaryotic counterpart of tubulin, drives the cell division process in bacteria;1,2 its assembly and disassembly mechanism regulates the formation and functioning of the dynamic Z-ring at the mid-cell position, the pre-requisite for efficient cell division in bacteria. The disruption of Z-ring formation by external agents has been found to have a lethal effect on cell growth.3,4 In addition, the perturbation of FtsZ assembly induces filamentation in bacterial cells, leading to cell death. Several studies have identified small molecule inhibitors which directly target FtsZ assembly in cells and, in turn, arrest bacterial proliferation.3-6 In addition, the broad conservation of FtsZ among prokaryotes and its constant intracellular concentration make it an attractive target for antibacterial drug discovery programs.7-9 Curcumin 1a (Figure 1), which can be extracted from the rhizomes of Curcuma longa, is a naturally occurring polyphenolic compound exhibiting a wide range of biological activities.10 Apart from being an important constituent of many diets, curcumin has widespread potential biological applications  it has been reported to exhibit antibacterial, anticancer, antiinflammatory, and anti-oxidant properties.11-14 In addition, curcumin has been found to have inhibitory effects against various viruses and is also active against non-microbe mediated diseases such as Alzheimer’s disease, Parkinson’s disease, irritable bowel disease (IBD), and psoriasis.15-18 Curcumin 1a has been found to be effective against both Gram positive and Gram negative bacteria, such as Staphylococcus aureus, Enterococcus faecalis, Staphylococcus intermedius, Bacillus subtilis, Escherichia coli, Salmonella typhimurium, and Sarcina lutea.19 It has also been shown to exhibit synergistic effects when used in combination with certain antibiotics, e.g. 3


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cefixime, vancomycin, tetracycline, and cefotaxime. In contrast, the effect of curcumin was antagonistic on the activity of nalidixic acid.20 Curcumin also disrupts the SOS response and inhibits UV-induced mutagenesis in S. typhimurium and E. coli.21 Curcumin 1a is also known to inhibit mammalian cell proliferation and to inhibit cellular microtubules.22,23 Since FtsZ has structural similarity to tubulin, the effect of curcumin on the assembly of FtsZ was monitored in a separate study.24 Curcumin was found to inhibit the proliferation of B. subtilis 168 and E.coli K12 cells by targeting the assembly of FtsZ; in vitro, it inhibited FtsZ assembly by enhancing the GTPase activity of FtsZ.24 Although extensive studies were performed in order to establish curcumin as a potent antibacterial drug, its fate met with very little success, with the primary reasons being its low bioavailability, solubility, instability in aqueous solutions, slow cellular uptake, and rapid cellular metabolism.25 Moreover, the half maximal inhibitory (IC50) concentrations of curcumin against bacterial (IC50 ~17 ± 3 µM for B. subtilis 168 cells) and mammalian (IC50 ~13.8 ± 0.7 for HeLa and 12 ± 0.6 µM for MCF-7 cells, respectively) cells were found to be similar.22,24 We therefore sought to design more potent analogues of curcumin, with increased antibacterial efficacy and enhanced physicochemical properties, and we synthesized analogues 1-3 by utilizing a range of substituted aldehydes 4 and diketones 5 in the synthetic procedure (Figure 2). A series of non-natural curcumin analogues 1-3 was synthesized (Figure 2) via either a slight modification of the procedure reported by Pabon26 or a new microwave method. In the former, a mixture of acetylacetone 5a (R1 = R2 = H), acetylcyclohexanone 5b (R1,R2 = (CH2)3) or 2acetylcyclopentanone 5c (R1,R2 = (CH2)2) and boric anhydride was heated at 75 °C, in anhydrous ethyl acetate, to form a complex as a white precipitate which facilitates the subsequent 1,5-bisaldol condensation with various benzaldehydes 4 in the presence of tri-n-butyl borate and n4


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butylamine, to give the non-natural curcumins 1-3 (Figure 1) in 10-39 % yield. In the microwave method, a mixture of the 1,3-diketone 5, benzaldehyde 4, and boric anhydride was heated at 65 °C, in DMF for 5 minutes in the microwave reactor, then n-butylamine was added and the mixture heated for a further 5 minutes at 65 °C in the microwave reactor. The solid product precipitated after pouring the mixture into acetic acid and stirring for 1 hour, and the purified analogues were obtained in 20-69% yield. The purity of all the compounds was determined by HPLC. Amongst the derivatives of curcumin synthesized, derivative 2d showed remarkably increased antibacterial activity in comparison to curcumin; 2d perturbed the formation of the Z-ring in B. subtilis 168 cells and induced filamentation in these cells. Live cell imaging of GFP-FtsZ showed that the Z-rings were destroyed within 5 min of 2d treatment. Similarly to curcumin, 2d also enhanced the GTPase activity of FtsZ and inhibited the assembly of FtsZ in a concentrationdependent manner, suggesting that it targets FtsZ. In addition, 2d was found to be several fold less cytotoxic to mammalian cell proliferation in comparison to the bacterial cells. This study reports the discovery of a novel non-natural curcumin analogue (2d) with improved efficacy and potential as a lead molecule for the development of FtsZ targeted antibacterial agents.

EXPERIMENTAL PROCEDURES The synthesis and characterization of all novel compounds is included in the supplementary information. Preparation of non-natural curcumin derivative stocks The synthesized non-natural curcumin derivatives were dissolved in 100% DMSO and stored at – 20°C. In all experiments, the final concentration of DMSO was kept below 0.1%. Screening  effect on proliferation of B. subtilis 168 cells 5


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A culture of B. subtilis 168 cells grown overnight was diluted (O.D.600 = 0.1) in LB medium and allowed to grow for 4 h in the absence and presence of 15 µM of all the non-natural curcumin derivatives in a 96-well plate at 37 °C. The absorbance of each bacterial culture was measured at 600 nm using a 96 well Plate Reader (Spectra max, M2) and the corresponding blanks were subtracted from the respective data sets. Those non-natural curcumin derivatives, which inhibited >80% bacterial cell proliferation were selected for further investigation and again tested on the proliferation of B. subtilis 168 cells according to the same procedure as described above. Each experiment was repeated three times. Determination of half maximal inhibitory concentration (IC50) values for non-natural curcumin derivatives in B. subtilis 168 cells B. subtilis 168 cells (O.D.600 = 0.1) were grown at 37 °C for 4 h without and with different concentrations (1, 2, 5, 7, 10 and 15 µM) of compounds 1e, 1k, 1p, 2d, 3b, and

3d,

respectively. The absorbance of each of the reaction sets was determined and the percentage inhibition of cell proliferation was calculated for each of the compounds. The concentration of the compounds at which 50% inhibition of cell growth occurs was determined to be their IC50 value. Visualization of cell morphology Selected compounds (1e, 1k, 1p, 2d, 3b, and 3d) at their IC50 values were incubated with B. subtilis 168 cells in LB medium for 4 h at 37 °C. Cells were obtained by centrifugation at 25 °C, re-suspended in PBS (pH 7.4) and were fixed using 2.8% formaldehyde and 0.04% glutaraldehyde. The cells were examined using a fluorescence microscope (Eclipse TE2000-U microscope; Nikon (Tokyo, Japan) at 60× under the differential interference contrast mode. The

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length of the bacterial cells was measured using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, U.S.A.). Purification of Bacillus subtilis FtsZ The Bacillus subtilis FtsZ gene cloned in pET16 vector was transformed in E. coli BL21 (DE3) pLysS cells. FtsZ was purified as described previously27and stored at -80 °C. The FtsZ concentration was determined using the Bradford method with BSA as a standard.28 The concentration of the protein was finally adjusted using a correction factor of 1.2 for the FtsZ/BSA ratio.29 Purification of tubulin Tubulin was purified from fresh goat brain tissues as described previously.30 The concentration of tubulin was also estimated by the Bradford method.28 Effect of non-natural curcumin derivatives on the GTPase activity of FtsZ The effect of non-natural curcumin derivatives on the GTPase activity of FtsZ was examined using malachite green ammonium molybdate.31,32 Briefly, FtsZ (6 µM) was incubated without and with 15 µM solutions of the selected compounds (1e, 1k, 1p, 2d, 3b, and 3d) in buffer A (25 mM PIPES, 5 mM MgCl2, 50 mMKCl, pH 7.2) on ice for 15 min. Polymerization was initiated by adding 1 mM GTP to each of the reaction mixtures, which were further incubated at 37 °C for 10 min. The reaction was quenched by the addition of 7 M perchloric acid (10% v/v) and then malachite green-ammonium molybdate solution was added and further incubated at 25 °C for 30 min. The number of moles of inorganic phosphate released was determined by measuring the absorbance at 650 nm and quantifying from a standard phosphate curve. The experiment was performed two times.

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Triton X-100 (0.01 % v/v) was used to solubilize the colloidal aggregates of FtsZ targeting agents if formed under the assembly conditions.33 Therefore, the effect of 2d on the GTPase activity of FtsZ was studied in the presence of 0.01% (v/v) Triton X-100 as described previously.33,34 Different concentrations (5, 10, 15, 20, 25, 30 and 50 µM) of 2d were incubated with 0.01% (v/v) Triton X-100 for 30 min in buffer A at 37 °C. Further, the mixture was centrifuged and the supernatant collected. The supernatant was mixed with FtsZ in buffer A containing 0.01 % (v/v) Triton X-100. The concentration of Triton X-100 in the reaction mixture was 0.01% (v/v). Then, 1 mM GTP was added to the reaction mixtures and the GTPase activity was determined after 10 min of assembly at 37 ºC as described previously. The concentration of 2d required to increase the GTPase activity by 50% (EC50) was determined by fitting the percentage increase in the GTPase activity in a sigmoidal curve (log of 2d vs % increase in GTPase activity) using OriginPro8.The effect of 2d on the GTPase activity of tubulin was also determined. Tubulin (10 µM) was polymerized in PEM buffer (50 mM PIPES, 3 mM MgCl2 and 1 mM EGTA) with 0.7 M glutamate and 1 mM GTP in the absence and presence of different concentrations (5, 10, 15, 20, 25, 30 and 50 µM) of 2d. The reaction was quenched after 10 minutes of polymerization by addition of 4% perchloric acid. The moles of inorganic phosphate released were calculated by using the malachite green-ammonium molybdate colorimetric assay as described above. Light scattering assay The assembly kinetics of FtsZ in the absence and presence of 2d was monitored by 90° light scattering assay in a temperature-controlled spectroflourimeter FP-6500 JASCO (Tokyo, Japan).35 Briefly, FtsZ (6 µM) was incubated without and with different concentrations of 2d (5, 10 and20 µM) in buffer A and the extent of assembly was monitored by the addition of 1 mM 8


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GTP to the reaction sets. The light scattering intensity of the blanks was monitored and subtracted from each of the corresponding reaction sets. Similarly, tubulin (10 µM) was incubated in PEM buffer (50 mM PIPES, 3 mM MgCl2 and 1 mM EGTA) with 0.7 M glutamate without and with different concentrations of 2d (5, 10, 15 and 20 µM). Polymerization was initiated by the addition of 1 mM GTP to each of the reaction mixtures and the polymerization profile was monitored by measuring absorbance at 350 nm using spectra max, M2. Sedimentation assay FtsZ (6 µM) was incubated without and with 2d (5, 10 and 15 µM) in buffer A and polymerized with the addition of 1 mM GTP at 37 °C for 10 min. The polymerized protein was separated by ultracentrifugation at 88760×g for 30 min at 30 °C. The pellet obtained was re-suspended and equal volume of supernatant and pellet was analyzed on the SDS-PAGE. The amount of FtsZ polymerized was estimated by subtracting the supernatant concentration from the total protein concentration. Determination of the dissociation constant (Kd) of the interaction between 2d and FtsZ 2d exhibited weak fluorescence at 540 nm on excitation at 375 nm. However, the fluorescence intensity of 2d was enhanced upon binding to FtsZ (Figure S1A). Therefore, the increase in the fluorescence intensity of 2d upon binding to FtsZ was used to determine the affinity of interaction of 2d and FtsZ. FtsZ (2 µM) was incubated without and with different concentrations (0.5, 1, 2, 3, 5, 7, 9 and 10 µM) of 2d for 10 min at 25 °C. Fluorescence spectra were recorded in the wavelength range of 470-600 nm in a fluorescence spectrometer (JASCO, FP-6500, Tokyo, Japan) using 375 nm as the excitation wavelength. To ensure reduced inner filter effects, the fluorescence measurements were taken using a cuvette of path length 0.3 cm. FtsZ did not 9


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show any fluorescence in this wavelength range. The fluorescence spectra of blanks were recorded (Figure S1B) and subtracted accordingly from the corresponding reactionsets. The change in the fluorescence ∆F obtained was further corrected for the inner filter effect using the formula Fcorrected= Fobserved× antilog (λex+ λem/2), where, Fcorrected is the fluorescence of 2d after inner filter corrections, Fobserved is the observed fluorescence of 2d and λex and λem are the absorbance of 2d at its excitation and emission wavelength, respectively. The corrected fluorescence was then fitted to the formula, ∆F =

∆Fmax × L Kd + L

Where, ∆F is the change in fluorescence of 2d when it is in equilibrium with FtsZ, ∆Fmax is the maximum change in the fluorescence intensity of 2d when it is fully saturated with FtsZ and L is the concentration of 2d. The dissociation constant (Kd) was calculated using GraphPad Prism 5 software (Graph Pad Software, CA, USA). CD spectroscopy FtsZ (5 µM) was incubated without or with 10 and 20 µM of 2d in 10 mM phosphate buffer (pH 6.8) at 25 °C for 15 min. The far-UV CD spectra were monitored in the wavelength range of 200-260 nm in a JASCO J810 spectropolarimeter (Tokyo, Japan) using a cuvette of 0.1 cm pathlength. The CD spectra of the corresponding blanks were also taken and substracted from the respective data sets. The CD spectra were deconvoluted using CDNN software. Effect of 2d on the growth of B. subtilis 168 cells

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B. subtilis 168 cells were grown in the presence of different concentrations (0.2, 0.5, 0.7 1.0 and

1.5 µM) of 2d at 37 °C for 5 hr. After every 30 min interval, the absorbance of the culture was measured at 600 nm in a JASCO V-530 UV/Vis spectrophotometer. The absorbance of B. subtilis 168 cells in the absence and presence of varying 2d concentrations were then plotted

against time to represent the growth curve. Determination of the minimum inhibitory concentration of 2d in B. subtilis 168 cells Agar dilution method was used to find the minimum inhibitory concentration (MIC) of 2d on B. subtilis 168 cell proliferation.36 Bacterial culture containing 1 × 105 cells were spread onto Luria

agar plates containing different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5 µg/ml) of 2d and incubated at 37 °C for 16 h. The concentration at which no colony formation was observed was determined to be the MIC. Effect of 2d on the localization of Z ring and nucleoid segregation in B. subtilis 168 cells The effect of 2d on the localization of FtsZ was examined in a B. subtilis strain 2020 expressing GFP-FtsZ in casein hydrolysate media (CHB).37 GFP-FtsZ expressing B. subtilis strain 2020 cells was grown on pre-prepared agarose pad in the presence of either the vehicle or 6 µM 2d. Agarose pad (2% agarose in CHB) was prepared with help of GeneFrame® on a microscopy slide as recommended in the manufacturer protocol (Thermo Scientific). The agarose pad was transferred from a microscopy slide to a petri dish with glass coverslip bottom.38 The cells were observed at 37 °C under confocal laser scanning microscope (Zeiss, LSM-700). Further, the effect of 2d on the localization of the Z-ring and nucleoid segregation of B. subtilis 168 cells was observed using immnuoflourescence microscopy.35 B. subtilis 168 cells were grown till the OD600 reached to 0.2-0.3. Thereafter, the cells were incubated without and with 6 µM 2d for 5 min at 37 °C. The cells were harvested and fixed with 2.8% formaldehyde and 0.04% 11


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glutaraldehyde for 30 min at room temperature. The cells were permeablized and the nonspecific binding sites were blocked using 2% BSA-PBS. Cytoplasmic FtsZ was stained with a polyclonal FtsZ antibody developed in rabbit, followed by staining with a Cy3-conjugated goat anti-rabbit secondary antibody. DNA was stained with DAPI and images were taken at 60× magnification using confocal laser scanning microscope (Zeiss, LSM-700). Further, the effect of 2d on the Z-ring was analyzed by incubating B. subtilis 168 cells with1 µM 2d for 4 h and subsequently, staining the cells with FtsZ IgG and DAPI as described previously.34 The samples were visualized with a fluorescence microscope (TE2000-U). Effect of 2d on the membrane potential of B. subtilis 168 cells The membrane potential of B. subtilis 168 cells in the absence and presence of 2d was determined using fluorescence spectroscopy. 39,40 Briefly, a culture of B. subtilis 168 cells grown overnight was inoculated into fresh LB tubes and grown at 37 °C for 2 h. 1 × 105 cells were resuspended in filtered PBS and further incubated without or with 0.2 µM of carbonyl cyanide 3chlorophenylhydrazone (CCCP) or 2d (1 µM) at 25 °C for 30 min. Following the incubation, 12 µM 3,3′-diethyloxacarbocyanine iodide (DiOC2) was added to each of the reaction sets and further incubation was continued in dark for 30 min. Each of the reaction sets was then excited at 470 nm and the emission spectra (500-550 nm) were monitored using a fluorescence spectrometer (JASCO, FP-6500, Tokyo, Japan). Spectra of the respective blanks were also taken and subtracted from each of the data sets to obtain the corrected spectra. Effect of 2d on the proliferation of a range of Gram positive and negative organisms Broth micro dilution methodology was performed in accordance with ISO 20776-1:2006. Compound 2d was prepared as a 10,000 mg/L stock in DMSO and then diluted to 128 mg/L in Mueller-Hinton broth, from which two-fold serial dilutions were prepared in Mueller-Hinton 12


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broth using the Ericsson and Sherris method as recommended in the ISO standard. Aliquots of 100 µL were added to the wells of a 96-well flat-bottomed microtitre tray along with 5 µL of the inoculum. The inocula were prepared in sterile deionised water, from overnight cultures, to a 0.5 McFarland standard equivalent density and subsequently diluted 1 in 15 in Mueller-Hinton broth. After inoculation of microtitre wells, this gave a final bacterial concentration of approximately 5 x 105 CFU per mL. Using this methodology, 2d was tested against 17 strains acquired from the National Collection of Type Cultures (NCTC, London, UK) and the American Type Culture Collection (ATCC, Manassas, USA); at a concentration range between 0.125 and 128 mg/L. A collection of 37 distinct isolates of methicillin-resistant Staphylococcus aureus (MRSA), which included representative strains most frequently encountered in Europe was tested alongside MRSA NCTC 11939 at a concentration range of 0.5-32 mg/L of 2d. Growth controls (incorporating the corresponding amount of DMSO) and sterility controls were included. Trays were incubated for 18 hours at 37 ± 0.5 °C in air and read both visually and using spectrophotometry at 620 nm. Any wells that did not show detectable growth were subcultured (50 µL) onto Columbia agar plus 5% defibrinated horse blood to determine the minimum bactericidal concentration. Viable counts, prepared as recommended by ISO 20776-1:2006 and inoculated onto horse blood agar plates for overnight incubation, were utilized to allow calculation of minimum bactericidal concentrations. All tests were performed in duplicate on separate occasions. Effect of 2d on proliferation of HeLa cells Human cervical carcinoma cells (HeLa) were cultured in MEM medium supplemented with 10% FBS and 1% antibacterial and antifungal solution in a humidified 5% CO2 incubator at 37 °C. HeLa cells (1 × 105 cells/ml) were seeded in 96 well plates for 24 h and then, incubated with 13


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either vehicle or with different concentrations (2, 5, 10, 15, 20, 25, 30, 40, 50 and 70 µM) of 2d for 24 h. The effect of 2d on the proliferation of HeLa cells was determined by sulforhodamine B assay.41,42 These experiments were repeated three times.

RESULTS Screening of non-natural curcumin derivatives against B. subtilis 168 cell proliferation In this study, using a cell proliferation assay as an initial screen, we evaluated the effect of the non-natural curcumin derivatives on the growth of B. subtilis cells. Several of the non-natural curcumin derivatives efficiently inhibited the growth of B. subtilis 168 cells (Tables S2 and S3); six of the compounds tested (at 15 µM) inhibited cell proliferation by ≥ 80%. Next, we determined the half-maximal proliferation inhibitory concentration (IC50) of these active compounds against B. subtilis 168 cells (Table 1). These compounds displayed antibacterial activity which is superior to that of curcumin 1a (Tables S1 and S2), with 2d exhibiting the greatest antibacterial activity (IC50, 1.2 ± 0.5 µM). FtsZ has previously been shown to be one of the primary targets for curcumin;24 it is the major cell division protein in bacteria and compounds targeting FtsZ assembly have been shown to induce elongation of the bacterial cells due to their inability to separate from the septum.3,4 We therefore wished to examine whether any of the 6 most potent non-natural curcumin derivatives could inhibit bacterial cytokinesis by monitoring bacterial cell length in the presence of the IC50 concentrations of these compounds (Table 1). Interestingly, 2d was found to elongate the cell length of B. subtilis 168 cells significantly (14-fold) at its IC50 concentration, in comparison to the control (Figures 3A and B), thus implying that this compound might target FtsZ assembly in vitro. In addition, 1e and 3b were also found to increase cell length, but had much weaker effects

on cell elongation than 2d (Table 1). 14


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Cyclohexanone derivative 2d enhances the GTPase activity of FtsZ Since analogues 1e, 2d, and 3b induced the elongation of B. subtilis 168 cells, we next examined their effect on the assembly of purified FtsZ in vitro. FtsZ is a GTPase, and GTP hydrolysis governs the assembly dynamics of FtsZ;1 with most of the small molecule inhibitors targeting FtsZ assembly inducing a change in its GTP hydrolysis rate.3 In this respect, the non-natural curcumin derivatives were assessed for their effect on the GTPase activity of FtsZ at the test concentration of 15 µM (Tables 1). While 2d strongly increased the GTPase activity of FtsZ, 1e had no effect, and 3b weakly inhibited the GTPase activity of FtsZ. Since it potently inhibited bacterial proliferation, greatly increased bacterial cell length, and also increased the GTPase activity of FtsZ, analogue 2d was selected for further studies. 2d inhibits the assembly of FtsZ The effect of 2d on the assembly kinetics of FtsZ was evaluated by a 90° light scattering assay. 2d inhibited the assembly of FtsZ in a concentration-dependent manner (Fig. 4A). For example, the inhibition of the assembly of FtsZ in the presence of 5, 10 and 20µM 2d was determined to be 53 ± 9, 60 ± 5 and 70 ± 10%, respectively. In addition, the effect of 2d on the amount of FtsZ polymerized was determined by sedimentation (Fig. 4B and C). 2d was found to reduce the amount of polymerized FtsZ in a concentration dependent manner (Figure 4C). For example, 15 µM 2d inhibited the amount of polymerized FtsZ by 66%. Further, the effect of 2d on the assembly of tubulin was analyzed (Fig. 4D). In the concentration range tested, 2d had no discernable inhibitory effect on the assembly of purified tubulin (Fig. 4D). 2d enhances the GTPase activity of FtsZ The effect 2d on the GTPase activity of FtsZ was determined by incubating 2dwith 0.01% (v/v) Triton X-100 to avoid colloidal aggregation of 2d.33 The GTPase activity of FtsZ was found to 15


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increase in the presence of 2d (Fig. 5A). For example, the amount of Pi released/per mol of FtsZ after 10 min of assembly was found to increase by 83 ± 11 and 232 ± 23% in the presence of 10 and 50 µM 2d, respectively, in comparison to the control. The fitting of the data into a sigmoidal curve provided an EC50 value to 12.9 ± 0.6 µM (Figure 5B).The data indicated that 2d inhibits the assembly of FtsZ, in association with increasing the GTPase activity of the protein. In the absence of Triton-X 100, 2d was also found to increase the GTPase activity of FtsZ in a concentration dependent manner. Though 2d enhanced the GTPase activity of FtsZ, it weakly inhibited the GTPase activity of tubulin (Figure 5C). For example, the GTPase activity of tubulin was reduced by 40 ± 6%in the presence of 50 µM 2d (Figure 5D). The results indicated that 2d exhibits contrasting effects on the GTPase activity of tubulin and FtsZ. 2d interacts with FtsZ and perturbed its secondary structure Since 2d was found to affect FtsZ assembly and polymer morphology, we next determined the affinity of the interaction of 2d with that of FtsZ. When a fixed concentration of FtsZ was incubated with varying concentrations of 2d, the fluorescence intensity of 2d increased in a concentration-dependent manner (Figure 6A). The change in the fluorescence of 2d when fitted into a binding isotherm gave a dissociation constant of 4.0 ± 1.1 µM, indicating that 2d interacted with FtsZ with a modest affinity (Figure 6B). The affinity of the interaction of the parent compound curcumin with that of FtsZ was determined to be 7.3 ± 1.8 µM.24 Further, 2d altered the far-UV circular dichroism spectra of FtsZ (Figure 6C). An analysis of the data indicated that 2d increased the random coil and α-helical contents of FtsZ while it reduced the βturn content of the protein suggesting that 2d altered the secondary structure of FtsZ. 2d inhibits the proliferation of B. subtilis 168 cells

16


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Biochemistry

The dose dependent effect of 2d on B. subtilis 168 cell proliferation indicated that the growth of the bacterial cells was inhibited in a concentration-dependent manner (Figure S2); the estimation of the minimum inhibitory concentration (MIC) was performed using the standard agar dilution method.36 The results indicated that, while 80% inhibition of bacterial cell growth occurred in the presence of 0.32 ± 0.04 µg/ml of 2d, the number of colony forming units was reduced in the presence of higher 2d concentrations. Finally, the concentration of 2d at which 100% inhibition of bacterial cell growth occurred was determined to be 0.7 µg/mL (2 µM) (Table S3). Interestingly, 2d did not arrest the growth of E. coli cells, an initial indication that this compound is not effective against Gram negative bacteria. 2d perturbs the Z-ring formation in B. subtilis 168 cells but does not affect nucleoid segregation Using GFP-FtsZ expressing B. subtilis strain 2020, the effect of 2d on the localization of the Zring in live bacteria was analyzed in real time by confocal microscopy. GFP-FtsZ was found to localize at the center of the cells (Fig. 7A). A distinct Z-ring was found in the vehicle treated control cells and no discernable change in the Z-ring was observed at 5 min. In the cells treated with 2d, a Z-ring was seen in the beginning (zero min) of the experiment and no Z-ring was found at 5 min (Figure 7A). Only diffused fluorescence signals were observed in the cells treated with 2d (Figure 7A). The finding suggests that treatment with 2d rapidly perturbs the Z-ring in bacteria. Further, the effects of 2d on the localization of the Z ring and nucleoid of B. subtilis 168 cells were probed by immunofluorescence microscopy (Figure 7B). The majority of the control cells showed distinct Z-rings localized at the septum. The treatment of B. subtilis 168 cells with 6 µM 2d for 5 min destroyed the Z-rings, with cells treated with 2d showing dispersed FtsZ staining 17


Biochemistry

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throughout their length. 2d had no effect on the nucleoids (Figure 7B), however, and these results support the finding that 2d can rapidly disassemble the Z-ring. In a separate experiment, B. subtilis 168 cells were also incubated with 1 µM 2d for 4 h, leading to an increase in the

length of the bacterial cells. In the vehicle-treated control cells, typical Z-rings were observed in the middle of the cells while no defined Z-ring was found in the treated cells. The FtsZ-staining was dispersed throughout the length of bacteria in the treated cells, showing that 2d perturbed the localization of the Z-ring in B. subtilis 168 cells (Figure 7C). 2d does not perturb the membrane potential of B. subtilis 168 cells Perturbed membrane potential in cells also leads to the inhibition of bacterial cell proliferation.43 The effect of 2d on the membrane potential of B. subtilis 168 cells was thus determined using a fluorescent probe, DiOC2. Monomeric DiOC2 exhibits green fluorescence,39 but there is a bathochromic shift in the emission spectrum (to longer wavelength [red]) on self-association of the dye as a result of an increased membrane potential.39 CCCP is a small molecule known to perturb the membrane potential in cells and is largely used as a positive control. 40 As expected, a large increase in the fluorescence spectra of DiOC2 was observed in the presence of CCCP, However, no enhancement of fluorescence was observed in the presence of 2d (Figure S3),suggesting that 2d did not affect the membrane potential of B. subtilis 168 cells. Effect of 2d on mammalian cells FtsZ is a distant prokaryotic homolog to tubulin in eukaryotes.44 Since, 2d targeted FtsZ assembly in bacterial cells, we also determined its effect on mammalian cell proliferation. The results of the SRB assay indicated that 2d inhibited the proliferation of HeLa cells, with an IC50 value of 37 ± 5 µM (Figure S4). Consistent with a previous report,22 curcumin was found to inhibit the proliferation of HeLa cells with an IC50 of 17 ± 4 µM. These results therefore indicate 18


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Biochemistry

that the cytotoxicity of 2d against mammalian cells is greatly reduced in comparison to its effect upon bacterial cells. Effect of 2d on the proliferation of a range of Gram positive and Gram negative organisms Having demonstrated the effects of the cyclohexyl-containing analogue 2d on FtsZ, we next investigated the effect of this compound upon the proliferation of both Grampositive and Gramnegative organisms. As was predicted by the initial testing in E. coli cells, it can be seen from the data presented (Table 2) that this analogue is a selective inhibitor of the growth of Grampositive organisms. The MIC and MBC for all Gram negative organisms were greater than 128mg/l, while the greatest effects were observed against S. epidermidis (MIC 2 mg/l, MBC 4 mg/l), S. aureus (MIC 2 mg/l, MBC 4 mg/l) and S. pyogenes (MIC 2 mg/L, MBC2 mg/l). On extended testing, 2d displayed good activity against 37 MRSA strains with both an MIC50 and an MIC90 of 2 mg/l, and an MBC50 of 8 mg/l (Table 3).

DISCUSSION In this study, we report the synthesis and biochemical activity of 2d, a non-natural curcumin derivative. 2d inhibited the proliferation of B. subtilis 168 cells with improved efficacy over its parent compound curcumin. The prolonged incubation of bacteria with 2d produced filamentous phenotype suggesting that 2d inhibits bacterial cell division process. Live cell imaging using GFP-FtsZ showed that 2d perturbed Z-ring formation. Immunostaining of FtsZ and the staining of the DNA suggested that 2d could rapidly destroy (within 5 min) the Z-ring without affecting the nucleiods and bacterial cell length. In vitro 2d inhibited the assembly of FtsZ and enhanced its GTPase activity. 2d bound to FtsZ with a modest affinity and the binding induced conformational changes in FtsZ. These results together strongly suggest that 2d inhibits bacterial cell proliferation by inhibiting FtsZ assembly. 2d was found to only inhibit the cell growth of 19


Biochemistry

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Page 20 of 39

Gram positive organisms, and was most potent against the staphylococcal and streptococcal species tested, suggesting that it might act as a lead for the development of anti-MRSA agents. The toxicity of 2d against mammalian cells was significantly reduced in comparison to that againstbacterial cells, suggesting the selectivity of this compound as an antibacterial agent. The cytotoxicity of curcumin in mammalian cells (IC50: HeLa; 13.8 ± 0.7 and MCF-7; 12 ± 0.6 µM)

22, 23

was found to be similar to that of the bacterial cells (IC50 in B. subtilis 168; 17 ± 3

µM).24 Interestingly, 2d was found to display much stronger anti-proliferative effects against bacterial cells (IC50, 1.2 ± 0.5 µM) compared to that of the mammalian cells (IC50, HeLa; 37 ± 5 µM).As FtsZ shares some structural similarities with tubulin, small molecules targeting FtsZ assembly have also been found to target microtubules in the mammalian cells. In this respect, compounds exhibiting a marked difference in their cytotoxicity towards mammalian and bacterial cells are considered to be suitable as FtsZ inhibitors. Thus, 2d qualifies as an attractive small-molecule inhibitor targeting FtsZ assembly. The non-natural curcumin derivatives synthesized and tested here all retain the 1,3-diketo functionality of the parent compound. The most potent compound 2d is structurally dissimilar to curcumin, with major differences being the lack of the methoxy groups on the aromatic rings and the substitution of a cyclohexyl group at the core. Several studies have reported the synthesis of curcumin derivatives with improved efficacy.45 The assembly dynamics of FtsZ are regulated by its GTPase activity;1 2d was found to enhance the GTPase activity of FtsZ in a concentration-dependent manner; similar results were exhibited by its parent compound, curcumin.23 2d inhibits the assembly of FtsZ most likely by increasing the GTP hydrolysis rate. The binding of 2d to FtsZ perturbed the secondary structure of FtsZ so the altered conformation of FtsZ-curcumin may also inhibit the assembly of FtsZ. It is also 20


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Biochemistry

possible that the presence of the bulky cyclohexane moiety may offer steric hindrance to the association of FtsZ monomers, thus leading to the inhibition of assembly. In conclusion, we have synthesized a non-natural curcumin analogue 2d that potently inhibits bacterial cell division by targeting FtsZ assembly. The antibacterial potency of 2d is several folds higher than curcumin, while its effect on mammalian cells is greatly reduced in comparison to that of curcumin. In addition, the cyclohexyl-containing analogue 2d is a selective inhibitor of Gram positive cell growth, suggesting that it has potential as a promising anti-Gram positive agent.

SUPPLEMENTARY INFORMATION The synthesis, characterization and purity of all novel compounds are included in the supplementary information, along with data from the biological testing (Figures S1-4 and Tables S1-3).

ACKNOWLEDGEMENTS The work was partly supported by a grant from the Department of Science and Technology, Government of India (to DP). VWYL acknowledges the receipt of a John A. Lambert Research Scholarship from the University of Sydney.

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Biochemistry

Table 1.

Determination of IC50 of non-natural curcumin derivatives on B.

subtilis 168 cell proliferation and their effect on the length of B. subtilis 168 cells and on the GTPase activity of FtsZ.

Compound

IC50 (µM)

Cell length (µm)

GTPase activity (15 µM of each analogue)*

Control

N/A

3.02 ± 0.6

1e

11 ± 3

10.9 ± 13.3

2.5 % increase

1k

4.5 ± 1.2

6.8 ± 2.3

28 % decrease

1p

12.5 ± 4.5

7.1 ± 2.4

10 % increase

2d

1.2 ± 0.5

43.8 ± 20.5

86 % increase

3b

12.7 ± 2.3

15.1 ± 17.2

18 % decrease

3d

10.5 ± 0.7

4.2 ± 1.6

19 % decrease

1a

17 ± 4

8.6 ± 5.3

11% increase

* average of two independent sets

27


Biochemistry

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Table 2.

Page 28 of 39

Minimum inhibitory concentration (MIC) and minimum bactericidal

concentration (MBC) for 2d against 17 isolates of clinically important bacteria. Organism and strain

MIC (mg/l)

MBC (mg/ml)

Acinetobacter baumanniiATCC 19606

>128

>128

BurkholderiacepaciaATCC 25416

>128

>128

Enterobacter cloacae NCTC 11936

>128

>128

Escherichia coli NCTC 10418

>128

>128

Klebsiella pneumoniae NCTC 9528

>128

>128

Providencia rettgeriNCTC 7475

>128

>128

Pseudomonas aeruginosa NCTC 10662

>128

>128

Salmonella typhimurium NCTC 74

>128

>128

SerratiamarcescensNCTC 10211

>128

>128

SerratiamarcescensNCTC 10211

>128

>128

Enterococcus faecalisNCTC 775

4

>128

Enterococcus faeciumNCTC 7171

4

>128

Listeria monocytogenes NCTC 11994

4

4

Staphylococci epidermidis NCTC 11047

2

4

Staphylococcus aureus NCTC 6571

2

4

Staphylococcus aureus (MRSA)NCTC 11939

2

4

Streptococcus pyogenes NCTC 8306

2

2

Gram negative

Gram positive

28


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Biochemistry

Table 3.

Minimum inhibitory concentration (MIC) and minimum bactericidal

concentration (MBC) distributions and descriptive statistics for 2d against 37 MRSA strains.

MIC (mg/l)

Number of isolates MIC

MBC

1

2

―

2

35

2

4

―

11

8

―

6

16

―

7

32

―

9

> 32

―

2

Parameter

mg/l

Range

1-2

2 - >32

MIC50 / MBC50

2

8

MIC90 / MBC90

2

32

Mode

2

4

29


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Page 30 of 39

Figure legends Figure 1: Curcumin 1a and analogues. Figure 2: Synthesis of curcumin analogues. Figure 3: 2d induced elongation of B. subtilis 168 cells. (A) Differential interference contrast images of B. subtilis 168 cells in the absence (i), and presence of 2d (1.2 µM) (ii). Scale bar is 5 µm. (B) Histogram showing the cell length distribution of B. subtilis 168 cells in the absence and presence of 2d. Figure 4: Effect of 2d on the assembly of FtsZ and tubulin. (A) FtsZ (6 µM) was polymerized without (■) and with 5 (●), 10 (▲) and 20 (▼) µM 2d in 25 mM PIPES, 50 mM KCl, 5 mM MgCl2 and 1 mM GTP at 37 °C. The assembly kinetics was monitored by 90° light scattering. The experiment was repeated thrice. (B) FtsZ (8 μM) was polymerized in the absence and presence of different concentrations (5, 10 and 15 μM) of 2d. The supernatant and the pellet were analyzed on SDS-PAGE. The experiment was performed three times. (C) Quantification of the amount of FtsZ pelleted in absence and in the presence of 2d. (D) Tubulin (10 µM) was incubated without and with varying concentrations of 2d and polymerized at 37 °C. Shown are the assembly kinetics of tubulin in the absence (○) and the presence of 5 (▲), 10 (▼), 15 (□) and 20 µM (×) of 2d at 350 nm. Figure 5: Effect of 2d on the GTPase activity of FtsZ and tubulin. (A) FtsZ (6 µM) was incubated with varying concentrations of 2d and the moles of Pi released was determined after 10 min of assembly in the presence of 0.01% v/v Triton X-100. The experiment was performed three times. (B) The increase in the GTPase activity of FtsZ with increasing concentration of 2d was fitted in a sigmoid curve (C) The moles of Pi released per mole of tubulin in absence and presence of varying 2d concentrations are shown. (D) The decrease in the GTPase activity of 30


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Biochemistry

tubulin was plotted against increasing concentrations of 2d. The experiment was performed three times. Error bar represents S.D.

Figure 6: 2d interacted with FtsZ in vitro and perturbed its secondary structure. (A) FtsZ (2 μM) was incubated without (■) and with 0.5 (○), 1 (▲), 2 ( ), 3 (♦), 5 (►), 7 (◄), 9 (×) and 10 (□)μM 2d. Shown are the fluorescence spectra of FtsZ in the presence of varying 2d concentrations. (B) The changes in the fluorescence intensity of FtsZ-2d were plotted against varying 2d concentrations. (C) FtsZ (5 µM) was incubated without (■) and with 10 (●) and 20 (▲) µM 2dand the far-UV CD spectrum was measured. The experiment was performed three times.

Figure 7: 2d perturbed the formation of Z-ring in B. subtilis 168 cells. (A) Effect of 2d on the localization of GFP-FtsZ in live B. subtilis cells. GFP-FtsZ expressing B. subtilis strain 2020 cells was incubated either with the vehicle or with 6 µM 2d. The images were taken immediately (zero min) and at 5 min of the addition of the compound. Scale bar is 1 µm. (B). Effect of short exposure of 2d on FtsZ and nucleoids in B. subtilis 168. The cells were incubated without and with 6 µM 2d for 5 min at 37 °C. FtsZ was stained using anti-FtsZ antibody and DNA was stained using DAPI. Scale bar is 1 µm. (C) B. subtilis168 cells were incubated without or with 1 µM2d for 4 h and stained with FtsZ IgG and DAPI. Scale bar is 5 µm.

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Biochemistry

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Figure 1

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Biochemistry

Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6 A)

B)

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Figure 7

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Table of Contents Graphic

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A Carbocyclic Curcumin Inhibits Proliferation of Gram-Positive

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