Unravelling a Novel Mode of Multiple Antibiotic Resistance: Mechanism and Inhibition of Radical-SAM RNA Methyltransferases

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Many chemical reactions are difficult to accomplish in the laboratory. However, in many cases biological systems have evolved specialised mechanisms that enable them to carry out these intrinsically difficult processes. Consequently the range of chemical structures able to be produced in nature is considerably greater than can currently be produced in the laboratory. As many such structures have useful properties, such as pharmaceutical activity, understanding how these are made in nature will expand our ability to make new molecules with a wide range of potential applications.It is now clear that nature achieves many of these intrinsically unfavourable reactions through the action of specific biological catalysts- enzymes. In particular, a large group known as the radical SAM enzymes are responsible for catalysing a diverse range of these "difficult" chemistries. Here we propose to study one such enzyme, known as Cfr, that introduces a specific modification to the bacterial ribosome. The ribosome is the component of the bacterial cell that is responsible for synthesising proteins and as such is essential to the viability of the bacterium. Many antibiotics act by poisoning the bacterial ribosome, but the Cfr enzyme causes a specific modification to the ribosome that makes Cfr-containing bacteria resist their activity. In particular, Cfr makes bacteria resistant to linezolid, an antibiotic that is particularly important as it represents a last line of defence against many bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) that are no longer easily treatable with other types of drugs. Cfr is beginning to spread through the bacterial population, potentially threatening our ability to use linezolid to treat serious infections. However, many aspects of the way that Cfr modifies the ribosome remain to be investigated. Improving our understanding of how enzymes like Cfr function may both permit us to develop drugs that block their activity, and enable us to exploit their ability to perform "difficult" chemical reactions to synthesise new and potentially useful moleculesIn this proposal, we will develop tools to investigate the activity of Cfr. We will use these to obtain fundamental information about how Cfr recognises a specific portion of the ribosome and how this site is selectively modified. We will also use the methods that we develop to screen a limited selection of synthetic molecules with the intention of identifying some that are able to block Cfr activity. We will establish, at a near atomic level of detail, how the molecules we identify are bound by Cfr, and how Cfr recognises the ribosome. The information we obtain will identify strategies for countering the activity of Cfr, that may prolong the therapeutically useful lifetime of linezolid, The application will also strengthen the UK knowledge and skills base with respect to the radical SAM enzyme family. Radical SAM enzymes are attracting increasing attention, due to the extraordinary range of reactions that they can catalyse, but there remain relatively few UK research groups active in this area. Our proposal will build capacity in an area of growing clinical and biotechnological relevance.