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Antimicrobial Agents and Chemotherapy, August 2005, p. 3367-3372, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3367-3372.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Fluorescence Polarization Method To Characterize Macrolide-Ribosome Interactions

Kang Yan,^1* Eric Hunt,² John Berge,² Earl May,¹ Robert A. Copeland,¹ and Richard R. Gontarek¹

Department of Enzymology and Mechanistic Pharmacology, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426,¹ Department of Medicinal Chemistry, GlaxoSmithKline, New Frontiers Science Park, Harlow, CM19 5AW, United Kingdom²

Received 12 March 2005/ Returned for modification 2 April 2005/ Accepted 7 May 2005

Top Abstract Introduction Materials and Methods Results and Discussion Conclusions. References

 
A fluorescence polarization assay is described that measures the binding of fluorescently labeled erythromycin to 70S ribosomes from Escherichia coli and the displacement of the erythromycin from these ribosomes. The assay has been validated with several macrolide derivatives and other known antibiotics. We demonstrate that this assay is suitable for determining the dissociation constants of novel compounds that have binding sites overlapping those of macrolides. This homogeneous binding assay provides a valuable tool for defining structure-activity relationships among compounds during the discovery and development of new ribosome-targeting drugs.

Top Abstract Introduction Materials and Methods Results and Discussion Conclusions. References

 
Macrolide antibiotics comprise a large group of clinically useful compounds, characterized by having a 14-, 15-, or 16-membered lactone ring with two or more sugar groups attached. Of particular importance are the 14-membered macrolide erythromycin and its newer-generation derivatives clarithromycin, roxithromycin, and azithromycin (a 15-membered macrolide), which are valuable therapeutic agents for the treatment of community-acquired respiratory tract infections. These antibiotics selectively inhibit bacterial protein synthesis by binding reversibly to the ribosome. They have been shown to interact with domain V of 23S rRNA near the peptidyl transferase center (8). Recent structural work (12, 21) has confirmed that these antibiotics exert their inhibitory effect by blocking the entrance to the polypeptide exit tunnel on the 50S ribosomal subunit and thus preventing the extrusion of nascent polypeptides. Sixteen-membered macrolides, such as tylosin, spiramycin, and carbomycin, also bind so as to block the peptide exit tunnel, but in addition these antibiotics have a mycaminose-mycarose disaccharide side chain that protrudes towards the peptidyl transferase center and more directly inhibit the peptidyl transferase reaction (12).

Since the introduction of erythromycin nearly 50 years ago, antibacterial resistance to macrolides has been an increasingly persistent threat to public health. One major resistance mechanism is a form of target modification, wherein the dimethylation of A2058 of the 23S rRNA by erm gene-encoded ribosomal methylases results in cross-resistance to macrolides, to the structurally related lincosamides, and to group B streptogramins (commonly referred to as the MLS_B phenotype). The emergence of these resistant bacterial pathogens has been of particular concern and has fueled the search for newer and more potent antibiotics with activity against these organisms.

For years the interaction of erythromycin and other macrolides with the bacterial ribosome has been the focus of intense investigation, and a number of experimental approaches have been developed to characterize the equilibria of binding and the kinetics of these drugs with their ribosomal target. In many of these studies, the binding of macrolides to ribosomes was investigated using a radiolabeled antibiotic as a ligand (6, 7, 10, 11, 19, 22). Since binding was measured by filtration, RNA footprinting, and peptidyl transferase activity, these approaches were low throughput and could not provide the rapidity and sensitivity needed to efficiently explore the mechanistic details of the bimolecular interactions. Ribosome-macrolide binding has also been characterized by measuring changes in fluorescence intensity, which could be either quenched or enhanced upon binding of a fluorescent derivative to ribosomes (3, 9, 17). Although this method does not require separation of unbound and free ligand, the measurement may not be suitable for compound screening due to the potential interference from highly absorbing or fluorescing compounds.

Fluorescence polarization (FP) is a quantitative, solution-based, homogeneous assay format which measures the rate of molecular rotation of a fluorescently labeled ligand. FP is related to the molecular sizes and thus the differing rotational properties of small versus large molecules; the smaller the size and the faster the rotation, the lower the FP value (13, 23). Since the bacterial ribosome has a huge molecular mass (2.5 million daltons), its binding to a small fluorescently labeled compound will result in a significant change in FP values. This phenomenon provides a large signal window and high sensitivity for studying ribosome-ligand interactions. In addition, because FP is a ratiometric measurement, it is less sensitive to sources of interference caused by fluorescence quenching or light scattering. Recently, an ultrahigh-throughput screening method using a fluorescently labeled antibiotic to measure equilibrium binding to bacterial ribosomes was described (25). In the present report, we have extended this FP approach to characterize the binding of fluorescently labeled erythromycin to Escherichia coli ribosomes and we compare the data to those obtained using a radiolabeled erythromycin ligand. Using a series of known compounds which bind to the ribosome, we have validated the assay and have established its use to rank potencies (dissociation constants [K_Ds]) of any compounds that compete with erythromycin. This sensitive and robust FP-based displacement assay is suitable for quantitative assessment of protein synthesis inhibitors that target the bacterial ribosome; therefore, it will be of great utility in determining structure-activity relationships (SAR) during the discovery and development of new ribosome-targeting drugs.

Top Abstract Introduction Materials and Methods Results and Discussion Conclusions. References

 
Reagents. Bacterial ribosomes were extracted from an E. coli strain (MRE600) sensitive to erythromycin (24). The final concentration of ribosomes was determined at A₂₆₀ (optical density of 1; concentration at A₂₆₀, 25 pmol). [³H]erythromycin (80 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc. 4,4-Difluoro-3a,4a-diaza-s-indacene (BODIPY)-erythromycin was synthesized in-house. Under an atmosphere of argon, 0.5 ml of 9-(S)-erythromycylamine A (17 mg), which was synthesized from erythromycin (5) and dissolved in tetrahydrofuran, was mixed with 0.5 ml of BODIPY succinimidyl ester (10 mg; Molecular Probes, Inc.) in tetrahydrofuran. After stirring at room temperature for 24 h, the solvent was evaporated. The residue was then purified by reverse-phase chromatography and eluted with aqueous acetonitrile containing 0.5% trifluoroacetic acid. BODIPY-erythromycin was obtained as an orange trifluoroacetate salt (3.7 mg), and its structure was confirmed with both nuclear magnetic resonance and liquid chromatography-mass spectrometry.

FP. E. coli ribosomes were incubated at 37°C for 15 min and then diluted in a binding buffer (20 mM HEPES [pH 7.5], 50 mM NH₄Cl, 10 mM MgCl₂, 0.05% Tween 20). For equilibrium binding, 4 µl of BODIPY-erythromycin at five different concentrations (5 to 200 nM) was mixed with 36 µl of ribosomes in a series of concentrations (0.05 to 1,400 nM) in a 96-well plate (Corning round-bottom black plate) and incubated at room temperature for 2 h. For ligand displacement, BODIPY-erythromycin (5.5 nM) was preincubated with the ribosomes (37.8 nM) for 30 min. Then 4 µl of a compound, diluted in 10% dimethyl sulfoxide, was mixed with 36 µl of the above-described ligand-ribosome mixture in the 96-well plate and incubated at room temperature for 2 h. FP values (mP) were measured using an LJL Analyst (excitation at 485 nm and emission at 530 nm). Background levels (nonspecific binding) were determined by displacement of the ligand with 10 µM unlabeled erythromycin.

Filter binding assay. [³H]erythromycin was diluted in 10% dimethyl sulfoxide. After incubation at 37°C for 15 min, E. coli ribosomes were diluted in binding buffer (20 mM HEPES [pH 7.5], 50 mM NH₄Cl, 10 mM MgCl₂, 0.05% Tween 20) to a final concentration of 3 nM in the assay mixture. Ten microliters of [³H]erythromycin was mixed with 90 µl of ribosomes in a 96-well plate and then incubated at room temperature for 2 h. The free and bound ligands were then separated through a filter plate (UniFilter GF/B; Perkin-Elmer) by using a cell harvester. The filter plate was quickly washed three times through the cell harvester by using the binding buffer. Each of the wash steps lasted less than 5 s. The plate was allowed to dry at 50°C for 30 min. After the addition of 50 µl of MicroScint-20 to the plate, radioactivity was measured using a TopCount (Perkin-Elmer). Nonspecific binding was determined by assessing the displacement of [³H]erythromycin with 10 µM unlabeled erythromycin.

Data analysis. For the equilibrium binding using either BODIPY-erythromycin or [³H]erythromycin as the ligand, the binding data were fit to the following quadratic equation with consideration of ligand depletion:

(1)

where K_D is the equilibrium dissociation constant, [R] is the ribosome concentration, [L] is the ligand concentration, and [RL] is the concentration of the ribosome-ligand complex (4). For homologous competition, the binding data were globally fit to a homologous competition model:

(2)

where B_max is the maximum binding of the radiolabeled ligand, K_D is the equilibrium dissociation constant, and NS is nonspecific binding (18). The K_D of BODIPY-erythromycin was confirmed by titrating the concentrations of BODIPY-erythromycin with a fixed concentration of ribosomes and [³H]erythromycin. The binding data were fit to a cubic equation (GraFit) that solves K_D for a single binding site with two competitive ligands (1):

(3)

(4)

(5)

(6)

where [A]₀ is the concentration of ribosomes, [B]₀ is the concentration of a compound (BODIPY-erythromycin), [C]₀ is the concentration of [³H]erythromycin, K_D^AB is the dissociation constant of the BODIPY-erythromycin-ribosome complex, and K_D^AC is the dissociation constant of the [³H]erythromycin-ribosome complex. In the absence of a competitive ligand B, the concentration of the [³H]erythromycin-ribosome complex ([AC]₀) formed is given by equation 1. The above-described method was also used to determine the K_Ds of unlabeled compounds in the displacement assay using BODIPY-erythromycin as a ligand.

Filter binding assays to determine the K_Ds of [³H]erythromycin and BODIPY-erythromycin were performed several times to estimate standard errors. The K_Ds given in the text are the means plus standard errors. In the FP displacement assay, the K_Ds were obtained either from two experiments (n = 2) or from a single experiment (n = 1).

Top Abstract Introduction Materials and Methods Results and Discussion Conclusions. References

 
Equilibrium binding using BODIPY-erythromycin as a ligand. BODIPY-erythromycin (Fig. 1) was prepared using standard N-hydroxysuccinimidyl ester chemistry as detailed in Materials and Methods. The time course for reaching equilibrium was investigated by monitoring the binding of ribosomes (30 nM) to BODIPY-erythromycin (5 nM) over 2 h. The binding reached equilibrium in approximately 20 min (data not shown). During the time course, the total fluorescence intensity levels remained similar, suggesting that no significant quenching was caused by formation of the ligand-ribosome complex. Measurement of the equilibrium dissociation constant was then performed by titrating E. coli ribosomes in a range of concentrations from at least 100-fold below to 100-fold above the estimated K_Ds at different concentrations of BODIPY-erythromycin (0.1 to 20 nM). The binding reaction was allowed to proceed at room temperature for 2 h, which allowed sufficient time to reach equilibrium. Because the binding affinity of BODIPY-erythromycin for ribosomes is estimated to be in the nanomolar range (which is similar in magnitude to the concentration of the ligand), formation of the ligand-ribosome complex may lead to significant free-ligand depletion. Due to this tight binding behavior, the binding data (mP) were transformed and fit to a quadratic equation that considers ligand depletion. Experimentally, the lowest ligand concentration showing a response was limited to 1 nM by the sensitivity of the instrument. A typical binding isotherm obtained at a 5 nM ligand concentration is shown in Fig. 2. The apparent K_Ds were determined at several ligand concentrations. Since the binding data were fit to a quadratic equation, we expected that the K_D would be independent of ligand concentration. However, the measured K_Ds appeared to increase linearly (from 4.7 to 19 nM) with ligand concentrations (data not shown). With the increase of ligand concentrations, the observed K_Ds also increased. The reason for this is unclear.

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FIG. 1. Chemical structure of BODIPY-erythromycin used for ribosome binding experiments. NMe2, N-dimethyl; OMe, O-methyl.

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FIG. 2. Equilibrium binding of BODIPY-erythromycin to bacterial ribosomes in an FP assay. Equilibrium dissociation constant was measured by titrating E. coli ribosomes at a fixed concentration of BODIPY-erythromycin. The binding data (mP) were transformed and fit to a quadratic equation. A representative binding isotherm is shown. RL complex, ribosome-ligand complex.

One explanation for the observed ligand concentration dependence of the K_D was that the BODIPY fluorophore influences the binding of erythromycin to the ribosomes. This possibility was excluded by using BODIPY or BODIPY-cystine as a ligand, both of which did not show specific binding to the ribosome (data not shown). Another possibility is heterogeneity of the ribosome preparation. Since ribosomes may not be homogenous in assembly, purity, and activity, it is possible that there is more than one population of ribosomes, with one of them having a higher-affinity binding site than the others. At a lower concentration of BODIPY-erythromycin, binding would be preferential to the higher-affinity site, and at a higher concentration of the ligand, binding would occur at both of the sites in the ribosomes, which could result in the observed K_D dependence on ligand concentration.

Equilibrium binding using [³H]erythromycin as a ligand. Because the affinity of BODIPY-erythromycin appeared to be ligand concentration dependent, a filtration binding assay was established to measure the true affinity of the ribosome-ligand interaction using [³H]erythromycin as a ligand. Two approaches were taken to measure the equilibrium dissociation constant for the binding of [³H]erythromycin to ribosomes. First, we measured the dissociation constant by using cold ligand competition, which has the advantages of using lower concentrations of radioligand with lower nonspecific binding. In this experiment, the concentration of [³H]erythromycin was constant and [³H]erythromycin competed with unlabeled erythromycin in a range of concentrations from 300-fold below to 500-fold above the estimated K_D for binding to E. coli ribosomes. Binding data (cpm) for ribosomes with two concentrations of the radioligand were globally fit to a homologous binding model with depletion (Prism; GraphPad), resulting in a K_D of 8.95 ± 1.26 nM (n = 4), which is comparable with published data (K_D, 8 to 36 nM) (6, 7, 19, 22). Figure 3A shows a representative set of binding isotherms at equilibrium.

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FIG. 3. Measurement of dissociation constants in a filter binding assay. The dissociation constants of both [3H]erythromycin and BODIPY-erythromycin were measured in a filter binding assay using [3H]erythromycin as a ligand. (A) Measurement of KD of [3H]erythromycin by cold ligand competition. The binding data were globally fit to a homologous binding model with ligand depletion. (B) Measurement of KD of [3H]erythromycin by saturation binding. The binding data were fit to a quadratic equation. (C) Determination of KD of BODIPY-erythromycin in competition with [3H]erythromycin. The binding data were fit to a cubic equation that solves KD for a single binding site with two competitive ligands.

The equilibrium dissociation constant was also measured by conventional saturation binding. Saturation binding was studied at a fixed concentration of ribosomes (3 nM) and a series of [³H]erythromycin concentrations ranging from 50-fold below to 40-fold above the anticipated K_D. The binding data were fit to a quadratic equation (GraFit), resulting in a K_D of 5.54 ± 0.76 nM (n = 5), which is comparable to the results from the cold ligand competition (K_D = 8.95 nM). Figure 3B illustrates a representative saturation binding isotherm at equilibrium. Due to the limited capacity of the filter plates, we were unable to address whether the K_D was ligand concentration dependent as observed in the fluorescence-based displacement assay. Instead, we asked whether the affinity could be affected by the ribosome concentrations used in the assay. For this purpose, the saturation binding was performed at different ribosome concentrations ranging from 1 to 27 nM. The K_Ds derived from these experiments (n = 4) ranged from 5 to 8 nM and did not display any systematic dependence on ribosome concentrations (data not shown). The average K_D (6 nM) from these experiments was taken to represent the true dissociation constant.

To determine the true K_D of the BODIPY-erythromycin-ribosome complex, a competitive binding experiment was performed using [³H]erythromycin. In these experiments, a range of BODIPY-erythromycin concentrations was mixed with 10 nM [³H]erythromycin and 3 nM ribosomes. The binding was allowed to occur for 2 h at room temperature, and the bound and free ligands were separated by filtration. Since the true K_D of the [³H]erythromycin-ribosome complex is known, the binding data were fit to a cubic equation (GraFit) that solves K_D for a single binding site with two competitive ligands (Fig. 3C). The results of this experiment yielded an estimate of the K_D of 20.3 ± 1.0 nM (n = 3), which we assume represents the true potency of the BODIPY-labeled erythromycin for ribosome binding. Knowing the true K_D of the BODIPY-labeled ligand provides a constant in the data fitting for compounds that compete with erythromycin in ribosome binding in the FP-based displacement assay, making the assay suitable for tracking the binding affinities of protein synthesis inhibitors that target the bacterial ribosome.

Behavior of known macrolides and selected antibiotics in the BODIPY-erythromycin displacement assay. To determine the utility of the displacement assay for identifying ribosome ligands that compete with erythromycin, two groups of antibiotics with known mechanisms of action were tested in competition with BODIPY-erythromycin in the displacement assay. The first group of antibiotics were macrolides/ketolides that bind to the 50S subunit of the bacterial ribosome at a site that is known to overlap with the erythromycin binding site. These protein synthesis inhibitors include the 14- and 15-membered macrolides (clarithromycin and azithromycin) that bind at the entrance of the peptidyl transferase cavity, 16-membered macrolides (tylosin, kitasamycin, josamycin, and rokitamycin) that bind at the polypeptide exit tunnel with the longer sugar chain extended towards the peptidyl transferase center, and a ketolide (telithromycin) with the cladinose sugar replaced by a keto functionality. As expected, all of these compounds effectively displaced BODIPY-erythromycin with K_Ds between 2 and 22 nM (Table 1). Another group of antibiotics which is not expected to have binding sites that overlap with that of erythromycin was tested, including chloramphenicol, tetracycline, mupirocin, ciprofloxacin, rifamycin, cerulenin, and ampicillin. Consistent with the known modes of action of these compounds, none of them was able to displace the BODIPY-erythromycin (Table 1).

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TABLE 1. Assay validation with known antibioticsa

Characterization of macrolides derivatives using the BODIPY-erythromycin displacement assay. The rapid and sensitive FP assay described in this paper provides a powerful tool to track the binding potencies of protein synthesis inhibitors whose binding sites may overlap with that of erythromycin (and those of other macrolides) on bacterial ribosomes. The utility of this assay was demonstrated using a collection of 17 known derivatives of erythromycin A. These compounds were tested in the ribosome binding assay in a 96-well-plate format, and all were found to compete with the fluorescently labeled erythromycin in ribosome binding. The binding affinities (K_Ds) of these compounds, summarized in Table 2, could be quickly determined when the displacement data were fit to a cubic equation. The K_D of erythromycin was determined to be about 4 nM, in agreement with its potencies (6 to 9 nM) measured in the above-described filter binding assay and with previously reported values (6, 7, 19, 22).

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TABLE 2. Comparison of ribosome binding potencies and relative antibacterial activities of various derivatives of erythromycin Aa

Erythromycin is composed of three elements: a 14-membered lactone ring, a desosamine sugar at C-5, and a cladinose sugar at C-3 (Fig. 4). The derivatives of erythromycin A listed in Table 2 cover a range of antibacterial potencies and structural diversities in the C-6 to C-12 region of the lactone ring. In particular, for this set of compounds, the nature of the substituent at C-9 significantly affects ribosome binding affinity. All the erythromycin 9-oxime ethers, BRL-47939ER, BRL-47364ER, BRL-42859ER, BRL-47937ER, BRL-47362ER, BRL-47950ER, BRL-38180ER, and BRL-47938ER, bind to ribosomes with high affinities; the most potent analogue in this set is the oxime ether 11,12-carbonate BRL-47939ER, which has 10-fold-higher binding affinity than erythromycin. Erythromycylamine (BRL-42852ER) also has high binding affinity, whereas the corresponding alcohol, 9-dihydro-erythromycin A (BRL-36473ER), is almost 20-fold less potent than erythromycin. Derivatives of 9-dihydro-erythromycin such as the ether BRL-38206ER, the 9,11-methylene acetal BRL-38189ER, and the 11,12-cyclic acetal BRL-38197ER also have poor binding affinities, whereas the 9,11-ethylidene acetal derivative BRL-38178ER is considerably more potent than erythromycin. The acid degradation products of erythromycin, BRL-46357ER and BRL-46355ER, and the internal 6,9-acetal BRL-38175ER have moderate to poor affinities. The potencies in ribosome binding are well correlated with the antibacterial activities. This further validates the BODIPY-erythromycin displacement assay for determining the affinities of unlabeled compounds that compete with BODIPY-erythromycin, thus making the assay suitable for convenient ranking of the potencies (K_Ds) of novel protein synthesis inhibitors.

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FIG. 4. Structures of erythromycin A and its representative derivatives. The C-9 substituent plays a significant role in both ribosome binding and antibacterial activities. NMe2, N-dimethyl; OMe, O-methyl. Numbers indicate the carbon position in the lactone ring.

Top Abstract Introduction Materials and Methods Results and Discussion Conclusions. References

 
FP has been applied to ultrahigh-throughput screening for identifying novel protein synthesis inhibitors that target bacterial ribosomes (25). Our work further extended this approach to drive SAR for a subclass of protein synthesis inhibitors by developing an FP displacement assay using a fluorescently labeled macrolide antibiotic (erythromycin) as a ligand. A critical parameter in ranking potencies of compounds for the bacterial ribosome is the true K_D of BODIPY-erythromycin; therefore, a filter binding assay using radiolabeled erythromycin was used to support the FP assay development. The K_Ds of both erythromycin and BODIPY-erythromycin determined in the filter binding assay in turn facilitated the FP assay development. Our results show that the binding of BODIPY-erythromycin to the ribosomes was reversible, reproducible, and sensitive. The assay was further validated with known antibiotics. The potency (K_D) of unlabeled erythromycin determined in this assay was consistent with the results from in-house studies and published literature. Using 17 erythromycin derivatives, we demonstrated that potencies (K_Ds) of unlabeled compounds that compete with BODIPY-erythromycin could be rapidly profiled and well correlated with their antibacterial activities, making this assay suitable for supporting lead optimization in SAR efforts.

 
We thank Christine Voigt for protocols and advice on E. coli ribosome preparation, Symon Erskine for helpful discussions regarding FP and the use of the LJL Analyst, David Rominger for assistance in setting up the filter binding assay, and Zhihong Lai for advice on K_D determination. We also acknowledge David Pompliano and Allen Oliff for their support of this work.

 
* Corresponding author. Mailing address: 1250 South Collegeville Road, Collegeville, PA 19426. Phone: (610) 917-7282. Fax: (610) 917-7901. E-mail: kang.2.yan{at}gsk.com.

Top Abstract Introduction Materials and Methods Results and Discussion Conclusions. References

 

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Antimicrobial Agents and Chemotherapy, August 2005, p. 3367-3372, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3367-3372.2005
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