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

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 molecules

In 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.

Members of the radical SAM (S-adenosyl methionine) enzyme family catalyse a wide variety of chemically challenging biotransformations, often at unreactive centres. These include methylation of a range of substrates, of which one is the 23S RNA component of the bacterial ribosome. The radical SAM enzyme Cfr catalyses methylation at the 8-position of a specific nucleotide base (A2503) resulting in resistance to a range of antibiotics that inhibit translation by binding to the large ribosomal subunit. Most significantly, these include linezolid, the first oxazolidinone antibiotic in clinical use and a key agent for treatment of infections by multi-resistant Gram-positive bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Transferrable linezolid resistance was previously unknown; however Cfr is now beginning to disseminate amongst clinical MRSA strains.
Radical-SAM-catalysed methyl transfer remains incompletely understood. Accordingly, we propose to develop spectroscopic (fluorescence and absorbance-based) and biochemical methods with which to study the Cfr-catalysed rRNA methylation reaction. Using these we will screen directed libraries of small-molecule compounds with the aim of identifying Cfr inhibitors and investigate the relationship between substrate (SAM and rRNA) binding and the oxidation state of the enzyme to obtain new mechanistic information. We will test the ability of the inhibitors we identify to restore the effectiveness of linezolid against Cfr-expressing S.aureus strains, and initiate a programme of crystallisation experiments with the aim of determining crystal structures of Cfr:RNA and Cfr:inhibitor complexes. Our results will identify routes towards inhibiting a new and challenging mechanism of antibiotic resistance in Gram-positive bacteria and provide new information on how radical-SAM enzymes catalyse unfavourable chemical reactions.

This proposal seeks to investigate RNA modification by the Cfr radical SAM methyltransferase, a cause of resistance to multiple antibiotics in Gram-posive bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) that cause healthcare-associated infections. The main outputs of this research programme will be:

i) a new assay for investigating interaction of the Cfr protein with RNA and with small molecule ligands that disrupt this process
ii) comprehensive characterisation of the relationship between SAM binding, RNA binding and turnover and the redox potential of the Cfr Fe-S cluster
iii) application of the assay developed in (i) above to identify small molecules able to disrupt Cfr activity in vitro
iv) high-resolution crystal structures of complexes of the Cfr protein bound to RNA and/or to small molecule ligands identified in iii) above

This work will benefit the academic community by providing new information on how radical SAM enzymes such as Cfr catalyse methyl transfer reactions and how this process may be monitored and inhibited. Although it is now appreciated that radical SAM enzymes catalyse a wide variety of biotransformations, including reactions that are involved in synthesis of industrially or medicinally important secondary metabolites such as antibiotics, the UK has relatively few groups active in this area. Our work will thus help to build UK capacity in this rapidly growing field.

The project also carries implications for both industrial/pharmaceutical research and for public health. The cfr gene is now spreading in S. aureus and MRSA strains, protecting host organisms from five antibiotic classs including recently developed agents such as the oxazolidinones (linezolid/Zyvox) and pleuromutilins (retapamulin/Altabax). The impact of these infections is enormous- the cost of healthcare-associated infections to the N.H.S. has been estimated at £1 billion, and MRSA is implicated in over 1 000 deaths, per annum. Linezolid is a key antibiotic for systemic treatment of pneumonia and skin infections by antibiotic-resistant Gram-positive bacteria, while retapamulin is an important topical treatment for these organisms. Dissemination of cfr threatens the continued effectiveness of these important antibacterial agents. Our work will develop tools suitable for identifying Cfr inhibitors by screening compound libraries, and will therefore inform the studies of both academic and industrial researchers investigating antibacterial drug development.