Molecular Insights into 14-Membered Macrolides Using the MM-PBSA Method Wai Keat Yam † and Habibah A. Wahab* ,†,‡ Pharmaceutical Design and Simulation (PhDS) Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia, and Division for Advanced Drug Delivery, Malaysian Institute of Pharmaceuticals and Nutraceuticals, Malaysian Ministry of Science, Technology and Innovation, SAINS@USM 10, Persiaran Bukit Jambul, 11900 Bukit Jambul, Pulau Pinang, Malaysia Received September 24, 2008 Erythromycin A and roxithromycin are clinically important macrolide antibiotics that selectively act on the bacterial 50S large ribosomal subunit to inhibit bacteria’s protein elongation process by blocking the exit tunnel for the nascent peptide away from ribosome. The detailed molecular mechanism of macrolide binding is yet to be elucidated as it is currently known to the most general idea only. In this study, molecular dynamics (MD) simulation was employed to study their interaction at the molecular level, and the binding free energies for both systems were calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The calculated binding free energies for both systems were slightly overestimated compared to the experimental values, but individual energy terms enabled better understanding in the binding for both systems. Decomposition of results into residue basis was able to show the contribution of each residue at the binding pocket toward the binding affinity of macrolides and hence identified several key interacting residues that were in agreement with previous experimental and computational data. Results also indicated the contributions from van der Waals are more important and significant than electrostatic contribution in the binding of macrolides to the binding pocket. The findings from this study are expected to contribute to the understanding of a detailed mechanism of action in a quantitative matter and thus assisting in the development of a safer macrolide antibiotic. INTRODUCTION Erythromycin A (ERYA) and roxithromycin (ROX) are prominent macrolide antibiotics introduced years ago and are clinically effective in the treatment of diseases caused by a wide range of bacteria. ERYA is one of the most important and effective macrolide antibiotics that is still widely in use today to treat infections caused by common bacterial pathogens and some nontypical pathogens. 1-3 It belongs to the first generation of the 14-membered ring macrolide and is produced by Saccaropolyspora erythraea. 4 ROX, on the other hand, is a second generation, semisyn- thetic macrolide antibiotic derived from ERYA. 5 It has a broader antibacterial spectrum and has a longer half-life and better absorption 1 than ERYA. It was also shown that ROX has higher inhibitory activity against Gram negative bacteria. 6 Both macrolides shared a similar chemical structure with a common 14-membered lactone ring, a desosamine sugar branched from position 5 of the lactone ring and cladinose sugar branched from the C-3 position of the lactone ring (Figure 1). However, the major difference of both macrolides is that ROX has an etheroxime chain sprouting at the C-9 position of the lactone ring. The two macrolides have a similar mechanism of action, i.e. they inhibit the bacterial protein synthesis by selectively binding to the 50S large ribosomal subunit. The crystal structure showed that both macrolides interact exclusively with Domain V of the 23S rRNA, at the entrance of the peptide exit tunnel in the peptidyl transferase center, 7 while other experimental studies revealed that these macrolides also interact with the Helix 35 of Domain II. 6,8-12 Besides that, our previous MD studies also demonstrated a possible interaction with the Domain IV. 13 These macrolides showed high drug affinity and specificity to the ribosome with K d in the nanomolar range as measured by equilibrium dialysis, 14 footprinting protection experiments, 6,9,15 and binding kinetics 16,17 studies. The actual inhibition mode of ERYA and ROX to the bioactivity of ribosome is so far understood to the most general idea only. Therefore, to enable a rational approach for antibiotic development, a comprehensive understanding and elucidation of the drug inhibition at the molecular level is desired. Here we attempt to address these issues by employing molecular dynamics (MD) simulation, complemented with energetic analysis using the MM-PBSA (molecular mechan- ics Poisson-Boltzmann surface area) method 18,19 for the complexes of the 50S ribosomal subunit and macrolides in aqueous solution. MD simulations of a large ribosomal subunit complexed with ERYA and ROX systems were performed for 2.5 ns. Preliminary MD results on a large ribosomal subunit complexed with the ERYA system has been reported, 13 and a further energetic analysis on this system is reported here. The MM-PBSA method was used to calculate the binding free energies of both systems comprising ERYA and ROX with the binding pocket in the 50S ribosomal subunit. The estimated binding free energies are slightly overestimated compared to the experimental * Corresponding author phone: (604)653-2206, (604)653-4533; fax: (604)642-7504; e-mail: habibah@ipharm.gov.my, habibahw@usm.my. † Universiti Sains Malaysia. ‡ Malaysian Ministry of Science, Technology and Innovation. J. Chem. Inf. Model. 2009, 49, 1558–1567 1558 10.1021/ci8003495 CCC: $40.75  2009 American Chemical Society Published on Web 05/26/2009 Downloaded by UNIV SAINS MALAYSIA on June 29, 2009 Published on June 22, 2009 on http://pubs.acs.org | doi: 10.1021/ci8003495