Pharmacological evaluation of synthetic galloyl catechin analogues with anti-staphylococcal properties

Antibiotics are among the most beneficial drugs ever introduced but their use is compromised by the emergence and spread of antibiotic resistant bacteria. The need for new treatments for infection has never been greater but society has been slow to respond and we are now facing a situation where superbugs such as MRSA frequently cause difficult-to-treat, life-threatening infections and where colonisation of our hospitals by these bacteria is increasingly likely to result in the cancellation of major surgery. The problem is of worldwide importance and, in the USA, MRSA now kills more patients than the AIDS virus. We have been researching ways to reverse antibiotic resistance in MRSA, making it again sensitive to inexpensive antibiotics such as methicillin and oxacillin. These antibiotics prevent bacteria from making the rigid cell wall that they need to survive; MRSA subverts this action by altering the way it makes its wall. We have found that a constituent of Japanese green tea called epicatechin gallate, or ECg, interferes with the MRSA-subverting machinery and converts the bacteria to methicillin sensitivity. We investigated the main target for ECg in MRSA and obtained strong evidence that it exerts its resistance modifying effect by binding to the MRSA membrane. MRSA and other staphylococci are dangerous pathogens because, not only are they resistant to our best antibiotics, they also excrete a range of toxins that can damage the host, cause symptoms of disease and circumvent the bodys immune defences. These proteins must be transported across the membrane and out of the cell in order to damage the patient: we have found that ECg prevents this transport process, effectively disarming the pathogen and raising the possibility that it could be used as a therapeutic in its own right. Unfortunately, ECg has a number of properties that could prevent its therapeutic use - it is broken down to inactive compounds in the body and its potency is only moderate - but we have designed ways to engineer the molecule to improve its attraction as a drug. We therefore propose to enhance the potential of ECg through rational design and chemical synthesis of novel analogues and to determine their properties in the laboratory and in models of infection. We hope that these studies will enable us to incorporate key design features into a new drug candidate that we can take forward to the next stage of development.

Galloyl catechins, in particular (-)-epicatechin gallate (ECg), a polyphenol abundant in green tea, have the capacity to inhibit the expression of mecA-mediated beta-lactam drug resistance in methicillin-resistant Staphylococcus aureus (MRSA) and to prevent the secretion from staphylococci of key virulence determinants such as alpha-toxin and coagulase. If such changes were to be elicited at the site of infection, they could form the basis of a novel therapy for staphylococcal infections. Galloyl catechins have little or no intrinsic anti-staphylococcal activity but modify the pathogenic bacterial phenotype by intercalation into the cytoplasmic membrane: alterations in the biophysical properties of the phospholipid bilayer result in changes to the cell wall architecture and to the orderly secretion of proteins. Our previous work has shown that degradation of ECg in biological milieu can be prevented by replacement of a hydrolytically susceptible ester linkage with a stable amide and that membrane penetration can be enhanced by removal of hydroxyl functions on one of the two ECg pharmacophores; molecules incorporating these features display modulating activity equal to or greater than the native molecule. We propose to explore the pharmacodynamic and pharmacokinetic properties of a range of synthetic ECg analogues designed on the basis of our insights into the structure ? activity relationships of this group of molecules. The design and synthesis of bioactive and stable compounds provides the opportunity to evaluate the efficacy of the most potent analogues in animal models of systemic and soft tissue infection both in combination with beta-lactams and as stand-alone potential therapeutics. We propose to synthesise 30 - 40 target chemical structures, examine their capacity to increase susceptibility to beta-lactams and to reduce qualitatively and quantitatively the secretion of extracellular proteins associated with virulence. Estimation of the degree of penetration into lipid vesicles that mimic the staphylococcal cytoplasmic membrane will further aid the selection of promising lead structures. Determination of peak plasma concentration, plasma half life and ?area under the curve? will guide the development of dosing schedules for therapeutic experiments in murine neutropenic thigh and peritonitis infection models. This study will contribute to our core aim: to identify a potent ECg analogue that will be suitable for evaluation in human trials.