Paul
Tyson
a,
Abdolreza
Hassanzadeh‡
a,
Mohd. Nizam
Mordi§
a,
David G.
Allison
a,
Viter
Marquez¶
b and
Jill
Barber
*a
aSchool of Pharmacy and Pharmaceutical Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester, UK M13 9PT. E-mail: jill.barber@manchester.ac.uk; Fax: +44-161-275-2396; Tel: +44-161-275-2369
bMax-Planck-Institut-für Molekulare Genetik, Ihnestr. 73, 14195, Berlin, Germany
First published on 3rd March 2011
COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compoundErythromycin B, an acid-stable co-metabolite of the important antibiotic COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A, differs from COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A only in the absence of a hydroxyl group at C12, yet it has never been licensed for clinical use. We describe an NMR-based analysis of the conformation of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B, both free in aqueous solution and when weakly bound to bacterial ribosomes and show that it is conformationally similar to COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A. The antibacterial activity of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B is shown to be similar to that of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A, but after acid-treatment, resembling exposure to the stomach, erythromycin B substantially retains antibacterial activity, whereas COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A does not.
In common with other macrolide antibiotics, COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B shows a broad range of activity against Gram-positive bacteria and limited activity against Gram-negative bacteria. It exerts its antibacterial activity by binding to the 50S subunit of the bacterial ribosome, thereby inhibiting microbial protein synthesis.4,5 Against a range of both Gram-negative and Gram-positive bacteria COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B is reported to have comparable6 or slightly lower7 potency than COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A.
COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compoundErythromycin A is more abundant and therefore easier to isolate than COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B. Presumably because of the success of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A, COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B has been largely neglected by the pharmaceutical industry. Recently, however, we have highlighted a number of advantages of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B over COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A. Firstly, it is much more stable to acid, because it is unable to cyclize in a 12,9-direction; erythromycin A undergoes facile formation of the spiro-6,9:12,9-cyclized anhydroerythromycin A, which has little or no antibacterial activity.7 The 2′-esters of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B are also much more stable to acid than those of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A. This finding is potentially important in paediatric medicine, in which 2′-esters serve as pro-drugs. Finally, we have been able to derivatize COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B to give pro-pro-drugs, for example erythromycin B enol ether ethyl succinate (3), which are almost insoluble in the medicine bottle and therefore unable to hydrolyse to yield the vile-tasting erythromycin free base. Compound 3 is activated in two stages, as shown in Scheme 1, to yield COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B.8
![]() | ||
Scheme 1
Activation of erythromycin B enol ether ethyl succinate to COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounderythromycin B. |
These findings suggest that COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B has potential as an independent entity in paediatric medicine, and prompted us to return to the fundamental chemistry of this drug, in particular to determine its conformation in free aqueous solution and when weakly bound to bacterial ribosomes.
It has previously been postulated that COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A and some of its derivatives bind to the ribosome of Escherichia coli in a two stage process. A strong binding interaction has been detected by equilibrium dialysis and related techniques,9–11 and more recently characterized in detail by X-ray crystallography.12 A weak interaction has been characterized as a fast exchange process by NMR spectroscopy.13,14 Unlike the strong interaction, the weak interaction appears to be magnesium independent.15 It has also been shown that for COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A, the E. coliribosome tautomerically recognizes and preferentially binds to the 9-ketone form of the drug.14
We now report the full assignments of the 1H and 13C NMR spectra of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B in organic and aqueous solutions. These assignments have allowed us to determine the conformational mix of the molecule in free aqueous solution and when bound (weakly) to the bacterial ribosome.
The one-dimensional 1H spectrum (Fig. 1 Supplementary Data†) showed immediately that, in aqueous solution, COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B exists overwhelmingly as a single isomer. Both erythromycins A and C (a biosynthetic precursor of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A in which C8′′ bears OH instead of OCH3) form mixtures of 9-ketone and 12,9-hemiacetal in aqueous solution, and show characteristic doubling of each signal in the 1H NMR spectrum. The lack of the 12-OH prevents this in erythromycin B; the formation of the 6,9-hemiacetal is perfectly possible, but the presence of a single set of signals in the 1H NMR spectrum indicate that it is unfavoured.
The full assignments of the 1H and 13C spectra of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B and a description of the assignment process are given in the Supplementary Data.†
Many previous studies of the conformations of macrolide antibiotics have made use of the 1H nuclear Overhauser effect (NOE) NMR experiment as the main means of obtaining conformational data.19–21 However, this method can be problematic when studying molecules of an intermediate size in aqueous solutions. At operating frequencies of 500–600 MHz, NOEs for erythromycins are close to zero and either slightly positive or negative. These effects make even semi-quantitative analysis of NOESY spectra unreliable when interpreting cross-peaks in terms of internuclear distances. We therefore chose to use the rotating frame Overhauser effect experiment,22 in which all the Overhauser signals are positive. When the spin-lock field and mixing time are adjusted accurately, artefacts are minimized and good data are obtained.
The ROESY spectrum of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B in phosphate buffered D2O (mixing time 250 ms) is shown in Fig. 1A. The cross-peaks were designated very small, small, medium or large and the corresponding connectivity table is shown in Table 2 (top) of the Supplementary Data.† Known internuclear distances (e.g. H2“proR-H2”proS, H4–H317) were used to predict internuclear distances for very small (3.5 ± 1.5 Å), small (2.5 ± 1.0 Å), medium (2.3 ± 0.5 Å) and large (2.0 ± 0.4 Å) cross-peaks. Medium H4–H11, H5–H318 and H8–H318 cross-peaks suggest that the folded-out conformation is highly represented in the solution, and H3–H8, H3–H11 and H316–H317 signals (indicative of the folded-in conformation) are absent. A small H8–H11 cross-peak suggests that the 8-endo-confimation might be represented. Curiously, no H315–H316 cross-peak was seen, although this had been found in folded-out COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compoundclarithromycin and COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A.19
![]() | ||
Fig. 1 500 MHz NMR spectra of 4 mM COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounderythromycin B in aqueous solution; A: ROESY spectrumB: TRNOESY spectrum in the presence of 0.48 μM E. coliribosomes. |
The experiment was repeated with a mixing time of 500 ms, with essentially identical results.
The NMR data therefore strongly suggest that COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B exists exclusively or almost exclusively in the folded-out conformation in aqueous solution. Conformational flexibility leads to small amounts of the 8-endo-folded-out conformer, but we see no direct evidence for the presence of the folded-in conformer.
Conformational searching is, at its best, satisfyingly rigorous, with all conformational space being explored. Macrolides are, however, rather large for this type of search and a 10,000 step calculation does not necessarily achieve an endpoint of a global minimum found 20 times. This calculation found no structure in which H8 approached H11, despite the presence of an H8–H11 crosspeak in the ROESY spectrum.
We therefore constrained the H8–H11 distance to 2.5 ± 0.5 Å and recalculated. The global minimum obtained actually had slightly lower energy than the folded-out global minimum (144 vs. 149 kJ mol−1), but was clearly a folded-in structure. This structure has an H3–H8 distance of 2.3 Å and an H4–H18 distance of 2.2 Å, but neither pair of hydrogens gives rise to a crosspeak in the ROESY spectrum.
A third calculation involved constraining both H8–H11 and H4–H11 to 2.5 ± 0.5 Å. This yielded the expected 8-endo-folded-out conformer with an energy of 158 kJmol−1, 9 kJ mol−1 above that of the folded-out conformer and consistent with the presence of this conformer at low abundance in aqueous solution.
The three global minima are shown in Fig. 2 and the full details of these structures are given in the Supplementary Data.†
![]() | ||
Fig. 2 Results of conformational searches on erythromycin B A: folded-out conformer B 8-endo-folded-out conformer C folded-in conformer. |
The conformation of the ligand bound in a fast exchange process to bacterial ribosomes may be determined using the transferred NOESY (TRNOESY) experiment. In this experiment the ligand is present in a large excess over ribosomes, and equilibrium exists between free and bound ligand. There is a very rapid build-up of NOEs in the bound state and this NOE information is transferred to the free state when the ligand molecule dissociates from the ribosome. Because NOE build-up in the free state is very slow, or even absent, it is relatively easy to choose a mixing time such that all the observable information pertains to the bound ligand. The resulting NOESY spectrum has chemical shifts due to the free drug, but NOE information due to the bound drug. The method is prone to spin-diffusion artefacts but these can be almost eliminated by the use of fully deuteriated ribosomes and an appropriate mixing time.
The transferred NOESY spectrum of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B (4 mM) in the presence of E. coli deuteriated ribosomes (0.48 μM – sufficient to cause an approximate doubling of the low frequency H315 linewidth) is shown in Fig. 1B. The mixing time was optimized empirically at 75 ms. Table 2 (bottom) of the Supplementary Data shows the connectivity table derived from these data. Signals were designated very small, small, medium or large on the basis of the size of the cross-peak, the abundance of information making a semi-quantitative analysis possible. Known internuclear distances were again used to predict the internuclear distances for very small (3.5 ± 1.5 Å), small (2.5 ± 1.0 Å) medium (2.3 ± 0.5 Å) and large (2.0 ± 0.4 Å) cross peaks.
More cross peaks were seen in the TRNOESY than in the ROESY spectrum of the free drug. This reflects the relative sensitivities of the two techniques. The maximum TRNOE has a relative intensity of −1, whereas the ROE has +½. The NOE build-up is very much faster in the TRNOESY than in the ROESY experiment, because of the very large size of the drug-ribosome complex. Most of the extra peaks in the TRNOESY spectrum were consistent with the structure of the free drug, with internuclear distances of 3–4 Å.
In the TRNOESY spectrum the H11–H8 signal is stronger than in the ROESY spectrum and signals corresponding to H3–H8 and H4–H18 are still absent, suggesting strongly that the 8-endo-folded-out conformer is present at low abundance and the folded-in conformer undetectable.
Three control experiments were carried out in order to control for non-specific binding to large molecules in the solution. Erythromycin B (4 mM) was incubated in the presence of 0.96 μM ribosomes and treated with Rnase-1-AS for four hours at 30 °C. The resulting TRNOESY spectrum (not shown) is essentially blank, containing only the cross-peaks seen in the NOESY spectrum of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B with the same mixing time.
To control for non-specific binding we have generally prepared ribosomal “core” particles from 50S ribosomal subunits. The subunits are treated with 2 M lithium chloride solution, as previously described,24 to remove 12 outer proteins, including L15.25Protein L15 has been shown, remarkably, to bind to COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A in isolation, in the absence of other ribosomal components.26 It is also essential for the binding of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin to reconstituted ribosomes.26 Fig. 3 in the Supplementary Data shows the NOESY spectrum obtained when COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B (4 mM) was incubated with 0.96 μM LiCl treated 50S cores (a doubling of the molar amount of ribosomes used above, to compensate for the reduction in molecular weight). This spectrum again shows very few cross-peaks, illustrating clearly that the binding to whole ribosomes (or to 50S subunits13) that we observe in the TRNOESY experiment is specific, and not the result of random interactions with large macromolecular complexes.
The preparation of ribosomal core particles is a demanding and time-consuming procedure, requiring density gradient ultracentrifugation. We sought to simplify our control studies by treating whole ribosomes with COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compoundlithium chloride. Surprisingly, this was unsuccessful. Whereas a single treatment with 1.3 M or 2 M LiCl was sufficient to prevent 50S subunits from binding in the NMR experiment, we typically required four treatments to prevent binding to 70S particles. Excellent TRNOESY spectra were obtained when 70S ribosomes were treated with COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compoundlithium chloride only once or twice. 30S ribosomal subunits do not bind to macrolide antibiotics in the NMR experiment, whilst 50S subunits do.13 It seems therefore that the 30S subunits protect the 50S subunits from loss of the proteins involved in weak binding.
Finally, we used 30S ribosomal subunits in place of whole ribosomes. Macrolide antibiotics are known to bind to the 50S subunit of the ribosome, and binding to the 30S subunit was undetectable.
Minimum Inhibitory Concentrations (μg mL−1) | ||||||
---|---|---|---|---|---|---|
S. aureus | S. pyogenes | H. influenzae | ||||
U | P | U | P | U | P | |
Ery A | 0.9 | >8 | 0.2 | 1 | 2.2 | >4 |
Ery B | 0.7 | 1.4 | 0.2 | 0.3 | 2.3 | 3.6 |
Clari | 0.6 | 0.9 | 0.2 | 0.3 | 0.6 | 1.8 |
The nature of the NMR-detected binding remains unclear. Recent kinetic analysis suggests that COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin (COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A in their study) binds to bacterial ribosomes in a two-stage process28 suggesting that the first stage of this binding is detected by NMR. This suggestion does, however, require that a single ribosome can bind two molecules of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin simultaneously, one detectable by crystallography, the other by NMR. We are inclined to the view that the transferred NOESY experiment detects binding weakened by partial denaturation of the ribosome. This suggestion is (to a degree) supported by the observation that the weak binding cannot readily be eliminated by LiCl treatment of whole ribosomes.
COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compoundErythromycin B remains the Cinderella of the macrolide family, unlicenced for human or veterinary use. Yet, unlike COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A, it is acid-stable. Unlike COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compoundclarithromycin, it can be readily esterified at the 2′-position to give pro-drugs with acceptable solubility for paediatric use. We have described some simple chemistry that allows the preparation of taste-free pro-pro-drugs of COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B.8 No other macrolide available at this time supports this chemistry. We and others have shown that COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B has similar in vitro antibacterial properties to COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A and COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compoundclarithromycin.6,7 In the present paper we have shown that COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B is conformationally similar to COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin A both in aqueous solution and when bound to bacterial ribosomes, and that the drug retains antibacterial activity after acid treatment mimicking the stomach.
The Supplementary Data describes the protocols for the assignment of signals
Monte Carlo searches using AMBER and MM2 force fields were then carried out to obtain global minimum structures, with water solvation. The cross peaks from the ROESY spectra were designated as very small (3.5 ± 1.5 Å), small (2.5 ± 1.0 Å), medium (2.3 ± 0.5 Å) or large (2.0 ± 0.4 Å), the magnitude of the constraints being determined empirically by referring to known interatomic distances within the molecule. The distance constraints from each ROESY and TRNOESY spectra were then compared with both global minimum COMPOUND LINKS
Read more about this on ChemSpider
Download mol file of compounderythromycin B conformers. Where the global minima did not satisfy all the NMR data, attention were turned to the higher energy structures and if no structure satisfying all the NMR data was found a constrained local minimization (or a restrained Monte Carlo search where appropriate) was performed to obtain conformers satisfying constraints which were not satisfied with the global minima.
The 50S ribosomal subunits were prepared as described,24 and were used to prepare ribosomal core particles by treatment with 2 M LiCl solution.24 The core particles were stored in phosphate buffered D2O at −80 °C for no longer than 2 weeks, and were defrosted no more than once.
Footnotes |
† Electronic supplementary information (ESI) available: The full assignments of the 1H and 13C spectra of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounderythromycin B and a description of the assignment process. 500 MHz NMR spectra of 4 mM COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounderythromycin B in aqueous solution: ROESY spectrum connectivity table. Transferred NOESY spectrum of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounderythromycin B (4 mM) in the presence of E. coli deuteriated ribosomes (0.48 μM) mixing time 75 ms, connectivity table. Folded-out, 8-endo-folded-out and folded-in conformers of erythromycin B: full details. NOESY spectrum obtained when COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounderythromycin B (4 mM) was incubated with 0.96 μM LiCl treated 50S ribosomal core particles. Broth microdilution Minimum Inhibitory Concentrations: Protocol. See DOI: 10.1039/c0md00251h |
‡ Present address: Pharmaceutics Research Centre, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. |
§ Present address: Centre for Drug Research, Universiti Sains Malaysia, 11800, Penang, Malaysia. |
¶ Present address: Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377 München, Germany. |
This journal is © The Royal Society of Chemistry 2011 |