Advanced Methods for Studying Structure and Interactions of Macrolide Antibiotics

Abstract

Macrolide antibiotics are macrocyclic compounds that are clinically used and prescribed for the treatment of upper and lower respiratory tract infections. They inhibit the synthesis of bacterial proteins by reversible binding to the 23S rRNA at or near the peptidyl transferase center. However, their excellent antibacterial profile was largely compromised by the emergence of bacterial resistance. Today, fighting resistance to antibiotics is one of the greatest challenges in medicinal chemistry. Considering various physicochemical properties of macrolides, understanding their structure and interactions with macromolecular targets is crucial for the design of new antibiotics efficient against resistant pathogens. The solid-state structures of some macrolide-ribosome complexes have recently been solved, throwing new light on the macrolide binding mechanisms. On the other hand, a combination of NMR spectroscopy and molecular modeling calculations can be applied to study free and bound conformations in solution. In this article, a description of advanced physicochemical methods for elucidating the structure and interactions of macrolide antibiotics in solid state and solution will be provided, and their principal advantages and drawbacks will be discussed.

Keywords: NMR spectroscopy; X-ray crystallography; biochemical and fluorescence methods; biomolecular targets; cryo-electron microscopy; macrolide antibiotics; macrolide interactions; molecular dynamics simulations; structure characterization.

Figure 1

Representatives of four macrolide generations: (a) Erythromycin A; (b) Azithromycin; (c) Telithromycin; (d) Solithromycin with atom numbering of the macrolactone ring.

Figure 2

Basic principles of: (a) X-ray crystallography; (b) Cryogenic electron microscopy (Cryo-EM); (c) NMR spectroscopy.

Figure 3

Crystal structures of (a) Erythromycin (PDB ID: 1YI2) [11,29]; (b) Azithromycin (PDB ID: 1M1K) [27,30] bound to the H. marismortui 50 S subunit.

Figure 4

Telithromycin bound to the E. coli ribosome. A comparison of the conformations reported for telithromcyin bound to the ribosome. 23S rRNA for E. coli is shown in gray. Telithromycin models from H. marismortui (gold), D. radiodurans (cyan), and E. coli (pink) are shown. Nitrogens in the alkyl–aryl arm are also shown for reference; reproduced with permission by PNAS [12].

Figure 5

NMR experiments frequently used for studying the structure and interactions of macrolides.

Figure 6

Nuclear Overhauser effect spectroscopy (NOESY) spectrum of 4″-aminopropyl azithromycin in (a) acetonitrile-d₃ prior to addition of the E. coli ribosome compared with (b) NOESY (red and pink signals) and transferred nuclear Overhauser effect spectroscopy (trNOESY) spectrum (blue and green signals) of the same compound in Tris-d₁₁ buffer (pH 7.4) prior to and after addition of the E. coli ribosome, respectively. The spectra were recorded at 25 °C and 600 MHz.

Figure 7

Binding of 4″-aminopropyl azithromycin to the E. coli ribosome studied by STD: (a) Proton spectrum prior to the ribosome addition; (b) Off-resonance spectrum after the ribosome addition; (c) Difference spectrum after the ribosome addition; (d) Chemical structure and atom numbering of 4″-aminopropyl azithromycin. The spectra were recorded at 25 °C and 600 MHz in Tris-d₁₁ buffer (pH 7.4).

Figure 8

Macrolide binding to bacterial ribosome studied by: (a) Footprinting; (b) Toeprinting; (c) Fluorescence assays.