Phenylpropanoids inhibit protofilament formation of Escherichia coli cell division protein FtsZ Shanmugam Hemaiswarya, Rohini Soudaminikkutty, Mohana Lakshmi Narasumani and Mukesh Doble Correspondence Mukesh Doble mukeshd@iitm.ac.in Received 19 January 2011 Accepted 1 April 2011 Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India The earliest step in cell division in bacteria is the assembly of FtsZ, an essential cell division protein, into a ring at the division site. FtsZ has GTPase activity and can assemble in vitro to form protein filaments. The present work involved the study of eight phenylpropanoids (cinnamic, p-coumaric, caffeic, chlorogenic, ferulic, 3,4-dimethoxycinnamic and 2,4,5-trimethoxycinnamic acids and eugenol) as inhibitors of Escherichia coli FtsZ. Phenylpropanoids make up the majority of our diet and act as antibacterial agents. Polymerization and GTPase inhibition assays showed that chlorogenic and caffeic acids were the most active amongst these (IC 50 of 70 and 106 mM, respectively). Circular dichroism studies indicated that chlorogenic acid perturbed the protein conformation and electron microscopy showed distorted filaments. Bacillus subtilis 168 cells treated with the phenylpropanoids were longer when compared to the control. The highest binding energy was observed between chlorogenic acid and the homology modelled E. coli FtsZ, which was consistent with the experimental results. A strong negative correlation was observed between binding energy and inhibition of the polymerization ability. 3D-Quantitative structure– activity relationship studies using GTPase activity indicated that the presence of more hydrophilic groups around the 39- and 49-carbon increased the activity. The effect of stress-induced formation of cell filamentation has to be understood before confirming the role of phenylpropanoids as FtsZ inhibitors. INTRODUCTION Antimicrobial resistance is a global concern due to its increased prevalence. The major issues are the reduced susceptibility to currently available antimicrobial agents and shortage of newly approved compounds. A growing number of Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae, etc.) and Gram-negative (Pseudomonas aerugi- nosa, Klebsiella pneumoniae, Escherichia coli, etc.) pathogens are responsible for infection in both the community and health-care settings (Furtado & Nicolau, 2010). Around 19– 60 % of Staphylococcus aureus isolates from ocular infections have been shown to be resistant to macrolides, penicillin and fluoroquinolones (McDonald & Blondeau, 2010). The overall levels of antibiotic resistance can be dramatically decreased by focusing on the overuse of antibiotics, the pharmacokinetic and pharmacodynamic properties of different drug formulations, and use of proper hygiene and protective barriers. There is also a need for the introduction of new antimicrobial agents with novel targets. FtsZ is a bacterial cytoskeleton protein which assembles into a protofilament in a GTP-dependent manner. This forms a dynamic Z-ring at the mid-cell position. A study done in E. coli showed that 12 different proteins congregate at the Z-ring in a sequentially dependent pathway, causing assembly of the septal ring that guides the synthesis of the circumferential septum (Buddelmeijer & Beckwith, 2002; Bernhardt & de Boer, 2003; Romberg & Levin, 2003; Schmidt et al., 2004). The septal ring constricts in concert with septal progression, culminating in complete disassem- bly as soon as it matures. Inhibition of this central protein perturbs the formation of the Z-ring and bacterial cytokin- esis (Margolin, 2005). FtsZ has become an attractive target, due to its evolutionary distance from eukaryotic tubulin. There are many efforts to identify inhibitors of FtsZ that do not target eukaryotic tubulin. Interestingly, most of the agents that target tubulin/microtubule, including paclitaxel, vinblastine and colchicine, do not affect the dynamics of FtsZ assembly (Jaiswal et al., 2007), indicating that the latter can be a selective antibacterial target. PC190723, a synthetic compound (substituted benzamide and thiazolopyridine moieties linked with an ether bond), has been found to kill meticillin-resistant Staphylococcus Abbreviations: CD, circular dichroism; QSAR, quantitative structure– activity relationship. Figures showing phenylpropanoid-induced filament formation in B. subtilis 168, the interaction of E. coli FtsZ with chlorogenic acid and a contribution plot of hydrophobic interactions are available as supple- mentary data with the online version of this paper. Journal of Medical Microbiology (2011), 60, 1317–1325 DOI 10.1099/jmm.0.030536-0 030536 G 2011 SGM Printed in Great Britain 1317