Identification of boronic acids as antagonists of bacterial quorum sensing in Vibrio harveyi

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Biochemical and Biophysical Research Communications 369 (2008) 590–594 www.elsevier.com/locate/ybbrc

Identification of boronic acids as antagonists of bacterial quorum sensing in Vibrio harveyi Nanting Ni a, Han-Ting Chou b, Junfeng Wang a, Minyong Li a, Chung-Dar Lu b, Phang C. Tai b, Binghe Wang a,* a

Department of Chemistry, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302-4098, USA b Department of Biology, Georgia State University, Atlanta, GA 30302-4010, USA Received 2 February 2008 Available online 22 February 2008

Abstract Bacterial quorum sensing plays a very important role in the regulation of biofilm formation, virulence, conjugation, sporulation, and swarming mobility. Inhibitors of bacterial quorum sensing are important research tools and potential therapeutic agents. In this paper, we describe for the first time the discovery of several boronic acids as single digit micromolar inhibitors of bacterial quorum sensing in Vibrio harveyi. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Quorum sensing; Autoinducer-2 (AI-2); Boronic acids; Inhibitors

Bacterial quorum sensing is defined as the regulation of gene expression in response to changes in the number and/ or species present in a community and in environment [1]. Bacteria regulate quorum sensing through the secretion and detection of molecules called autoinducers (AI). Often, Gram-negative bacteria use acylated homoserine lactones (AHL) as the autoinducers, collectively known as AI-1 [1], while Gram-positive bacteria normally use autoinducing peptides (AIP) to communicate [2]. Autoinducer-2 (AI-2), however, mediates quorum sensing in both Grampositive and Gram-negative bacteria and is therefore referred to as a universal autoinducer [1–12]. Because of the involvement of bacterial quorum sensing in pathologically relevant events such as biofilm formation and bacterial virulence [13], quorum sensing inhibitors have the potential to be used in antimicrobial therapy as adjuvant. In addition, quorum sensing inhibitors are also useful research tools for understanding the functional roles of bacterial quorum sensing. Therefore, there has been a great

*

Corresponding author. Fax: +1 404 413 5543. E-mail address: [email protected] (B. Wang).

0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.061

deal of activities in developing quorum sensing inhibitors [14–17]. However, most of the activities have been focused on antagonizing the AI-1 and AIP mediated pathways. There have been a few reports of AI-2 analogs as agonists and partial agonists [18–21]. There are several reports of AI-2 antagonists/inhibitors with IC50 in the high micromolar range [22–27]. Herein, we describe our studies of boronic acids as potential AI-2 antagonists using V. harveyi as the model organism, which produces bioluminescence upon autoinducer stimulation [28]. We have identified several single digit micromolar inhibitors, which are much more potent than what have been described in the literature. The AI-2 molecule can exist in different forms in equilibrium and can complex with boric acid with complex F being the biologically active form in V. harveyi (Scheme 1) [29]. Binding of AI-2 (F) to the LuxP receptor in V. harveyi triggers a cascade of events that lead to quorum sensing and bioluminescence [29]. This boric acid complex (F) is very unique in that it is a rare boron-containing compound known to be active in a normal biological process. AI-2 binds with LuxP with the boron atom in its tetrahedral anionic form [29]. Presumably, the stabil-

N. Ni et al. / Biochemical and Biophysical Research Communications 369 (2008) 590–594

HO

591

OH B

O OH O

OH CH3 O

HO

H2 O

HO HO HO

O

O

D CH3 OH O

DPD A HO

H2O

C

O

O -2H2O +B(OH)4 HO

CH3 O

HO

B OH

OH CH3 O

HO HO HO

F AI-2 V. harveyi

CH3 OH O

E AI-2 S. typhimurium

Scheme 1. DPD exists in different forms.

Materials and methods Reagents. All the boronic acids samples were provided by Frontier Scientific, Inc., Combi-Blocks, Inc., Optima Chemical, and AsymChem. The MM32 strain of V. harveyi was purchased from ATCC (#BAA-1211). MM32 test. Experiments were conducted by following literature procedures [3]. Specifically, MM32 bacteria were streak-seeded on fresh LM plates and then cultured in the presence of kanamycin 50 lg/mL and chloramphenicol 10 lg/mL. Colonies appeared after overnight incubation at 30 °C. A single colony was picked from the LM plate. Then this strain was grown for 16 h with aeration (175 rpm) at 30 °C in 2 mL of Autoinducer Bioassay (AB) medium [30] with antibiotics (kanamycin 50 lg/mL and chloramphenicol 10 lg/mL). Then the solution was diluted to OD600 0.7 and the bacteria preinoculum was grown in AB-Fe medium with 1.2 mM of iron to an OD600 of 1.0–1.1 with shaking at 30 °C 175 rpm for 1–1.5 h [31]. The resulting inoculum culture was then diluted 5000-fold in fresh AB medium. Solutions of the test compounds in AB medium were prepared in 96-well plates. To these solutions, freshly synthesized DPD solution (pH 7) was added for a final concentration of 5 lM. Boric acid was added to give a final concentration of 1 mM (The optimal DPD and boric acid concentrations were determined based on the literature [3] precedents as well as our own experimental confirmation). After addition of bacteria in AB medium, the micro plates were covered with a non-toxic plate sealer and incubated at 30 °C with aeration for 3–4 h. Light production was measured every half hour using a Perkin-Elmer luminescence microplate reader. Cell growth test. Bacteria were grown for 16 h with aeration (175 rpm) at 30 °C in 2 mL of AB medium with antibiotics (kanamycin 50 lg/mL and chloramphenicol 10 lg/mL). Then this bacterial culture was diluted 100-fold with 20 mL AB medium in a 250 mL flask and incubated at 30 °C (175 rpm). The OD600 values were determined every 20 min. The doubling time was calculated based on the OD600 values.

Results and discussion As described earlier, quorum sensing in V. harveyi triggers bioluminescence [28]. Therefore, quorum sensing studies can follow the effect of individual chemicals on luminescence intensity. For the initial studies, we chose the MM32 strain of V. harveyi for screening the anti-quorum sensing effect of various boronic acids. MM32 lacks the LuxN receptor needed to respond to autoinducer AI1 [32] and the LuxS enzyme needed to synthesize DPD (Scheme 1). Thus the MM32 strain of V. harveyi produces no endogenous AI-2 signal. Consequently, bioluminescence was measurable only following the addition of DPD, which was synthesized by following literature procedures [33]. In order to search for the optimal experimental conditions, we first tested the concentration effect of added DPD on bioluminescence production (Fig. 1). Concentration-dependent luminescence production was observed. From Fig. 1, it can be seen that maximal luminescence was observed within a wide concentration range of about 6–100 lM. The regions between 0–6 lM and between

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Luminescence

ization of the negative charge is through interactions with the receptor binding site. Indeed, in the receptorAI-2 complex, boron is next to two arginine residues (215 and 310), which can afford significant stabilization through ionic interactions. Based on the concept of molecular mimicry, we envisioned that boronic acids could serve as excellent candidates for binding to LuxP because of the structural similarities between a boric acid complex (F) and the boronic acid functional group. Therefore, we screened a series of about 50 boronic acid compounds for their ability to inhibit AI-2 mediated quorum sensing in V. harveyi.

10000 1000 100 10 1 0.1

1

10

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Concentration of DPD (μM) Fig. 1. DPD concentration-dependent luminescence intensities (400, 200, 100, 50, 25, 12.5, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2 lM) in the MM32 strain of V. harveyi.

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100–400 lM were especially sensitive to concentration variations. Therefore, for our studies, we chose to use 5 lM of DPD so as to stay in a ‘‘sensitive region.” We also observed that DMSO, which was used to solubilize the boronic acids in making the stock solutions, had a significant effect on the bioluminescence production by V. harveyi. Therefore, one has to be very careful in controlling a consistent DMSO (or other organic solvent) concentration in preparing the final test solution. Particularly important is to maintain consistent DMSO concentration even when the boronic acid concentration changes. This requires the preparation of the boronic acid stock solution at different concentrations. In all our experiments, the final DMSO concentration was no more than 1%. For the screening effort, we collected about 50 compounds (Fig. 2). The effect of these boronic acids on bacterial quorum sensing was examined by following literature procedures [3]. Fig. 3 shows a typical set of data reflecting the concentration-dependent luminescence intensity changes with added boronic acids. Out of the 50 compounds tested, 5 boronic acids (1–5) showed significant inhibitory activities with IC50 in the single digit micromolar range. These compounds are 3-fluoro4-methylphenylboronic acid (1, IC50: 9 ± 5 lM), 4(methoxycarbonyl)phenylboronic acid (2, IC50: 5 ± 2 lM), 4-(benzyloxy)phenylboronic acid (3, IC50: 4 ± 1 lM), 2-fluoro-4-methylphenylboronic acid (4, IC50: 4 ± 1 lM) and 4-cyanophenylboronic acid (5, IC50: 6 ± 4 lM) (Fig. 2). Seventeen of these boronic acids (6–22) showed no/minimal inhibitory activities at below 100 lM concentrations (Fig. 2). These were considered inactive and we did not pursue them any further. From such results, we were able to make a few general structure–activity relationship observations. First, the

Luminescence

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B(OH)2

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N H

(HO) 2B

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HCl H 2N

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6 COOH

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HCl H 2N

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(HO) 2B

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B(OH) 2

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B(OH)2

B(OH) 2 HOOC

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13 B(OH) 2

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O

(HO)2 B

NH 2 H2O

O 17

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15

B(OH) 2

Br

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active boronic acids were all phenylboronic acids with minimal substitutions except at the para-position. Second, boronic acids directly attached to a sp3 carbon are generally not active (e.g., 16, 18, 21, and 22). One reason could be the relatively high pKa of such alkylboronic acids compared with vinyl or arylboronic acids, which are considered electron-withdrawing groups. Since the boron atom of the DPD–boric acid complex exists in the anionic tetrahedral form, boronic acids with low pKa represent the best chance for molecular mimicry because of their high tendency to exist in the anionic tetrahedral form [34]. Third, boronic acids with additional ionizable functional groups tend to be less active [34]. For example, boronic acids 11, 14 and 15 have a carboxyl group and boronic acids 8 and 12 have an amino group, all of which are ionizable under normal physiological conditions. None of these boronic acids has significant activities below 100 lM.

NC 1

400

Fig. 3. Concentration-dependent inhibition of V. harveyi luminescence by boronic acids. Boronic acid 1, IC50 = 9 ± 5 lM; boronic acid 2, IC50 = 5 ± 2 lM.

B(OH) 2

MeO

300

Concentration of boronic acids (μM)

B(OH) 2

B(OH) 2

B(OH)2

50000

20

B(OH) 2 nC 5 H9 21

B(OH) 2 22

Fig. 2. Boronic acids with IC50 in the single digit micromolar range (1–5) and boronic acids with no/minimal inhibitory effects (6–22).

N. Ni et al. / Biochemical and Biophysical Research Communications 369 (2008) 590–594

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Time (min) Fig. 4. Growth curve for blank (doubling time: 80 min), 1 at 20 lM (doubling time: 81 min), 2 at 10 lM (doubling time: 103 min), 3 at 10 lM (doubling time: 86 min), 4 at 10 lM (doubling time: 73 min), and 5 at 15 lM (doubling time: 80 min).

With those compounds that show single digit micromolar activities, we also examined their effect on bacteria growth in order to eliminate general toxicity reflected in retarded bacterial growth as the reason for reduced bioluminescence production. Fig. 4 shows that none of these boronic acids (1–5) exhibited significant inhibition of bacterial growth when compared with the control group (no boronic acid). Therefore, no general cytotoxicity was observed at the concentrations tested for those boronic acids that showed significant quorum sensing inhibition. In conclusion, several phenylboronic acids were found to exhibit AI-2 inhibition effect with IC50 values in the single digit micromolar range. Some initial structure–activity relationship was drawn. This represents the first example that boronic acids are found to be quorum sensing antagonists. The potency observed was also much higher than all the AI-2 antagonists reported. These boronic acids will be very useful tools for research and good leads for further structural optimization. Acknowledgments Financial support from the Molecular Basis of Disease program at GSU, Georgia Cancer Coalition, and Georgia Research Alliance is gratefully acknowledged. We thank Frontier Scientific, Inc., Combi-Blocks, Inc. Optima Chemical, and AsymChem for providing boronic acid samples to us. Appendix A. Supplementary data Supporting Information Available: Procedures and biological data. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2008.02.061. References [1] A. Camilli, B.L. Bassler, Bacterial small-molecule signaling pathways, Science 311 (2006) 1113–1116.

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