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 An Innovative Approach to New Antimicrobial Drugs SUMMARY Resistance to antibiotics is a growing problem. This project will build on our extensive previous work on dihydrofolate reductase (DHFR), a validated antibiotic drug target (dihydrofolate analogues trimethoprim, brodimoprim, iclaprim and tetroxprim). We will use a new approach to determine the molecular characteristics of compounds that might bind to this enzyme in a completely unprecedented the way in that the inhibitors will not be substrate or cofactor analogues but reflect the local characteristics of the transition state of the reaction. They will therefore be of much higher affinity than conventional anti-­‐DHFR drugs and hence be efficient at much reduced concentrations with reduced side effects. Molecules matching the predicted characteristics will then be synthesised and tested for activity against the target enzyme. This will provide a pool of lead compounds for further development towards clinically useful drugs. SIGNIFICANCE This project will address the problem of resistance to antibiotics through the design of transition state analogues for DHFR, a known and validated drug target. These transition state analogues will combine characteristics of both substrate (dihydrofolate) and cofactor (NADPH) of the reaction but still be small enough to act as conventional drugs. They will have significantly increased affinity for their target and thereby not only provide new antibiotics but due to the lower concentrations required for inhibition reduce the emergence of drug resistance and the severity of side effects when used in patients. We will use kinetic isotope effects to create a map of the transition state to aid the design process, and will synthesise candidate inhibitors and test them in vitro and in vivo. Market Research commissioned by the Cardiff’s Research, Innovation and Enterprise Services (RIES) with Evolution Bioscience Ltd has indicated that the Bioscience industry has significant interest in the development of our new approach to drug discovery. DHFR is also a drug target for anticancer compounds (e.g. methotrexate) and to treat rheumatoid arthritis (e.g. methotrexate) and malaria (e.g. pyrimethamine and cycloguanil), but our approach will first be focused on the establishment of development in the antibacterial category. BACKGROUND The Allemann group has studied enzymes from the DHFR family for many years in pursuit of a deeper understanding of the mechanisms that control enzyme action; they are recognised leaders in the field.1–11 DHFR catalyses the conversion of dihydrofolate to tetrahydrofolate, which in turn is required for synthesis of DNA and a number of amino acids. DHFR is therefore a vital biosynthetic enzyme and a target for agents such as the antibacterial trimethoprim, the antimalarial pyrimethamine or methotrexate, a clinically highly successful anticancer drug. DHFR has recently attracted renewed attention to overcome resistance now seen against existing drugs and to reduce side-­‐effects by increasing selectivity. This recent work has however made obvious the need for novel approaches such as the one described here that increase the chemical space covered by DHFR inhibitors. Enzymes bind and stabilise the transition state of a reaction. This work will use highly sensitive isotope effect methods in combination with computational and modelling techniques to create a map of the transition state of the DHFR catalysed reaction, which will be used to inform the design of transition state analogues that act as novel DHFR inhibitors. This approach has recently been applied to purine nucleotide phosphorylase and has allowed the design of potent inhibitors with specificity for the Plasmodium falciparum enzyme over the human enzyme based on transition-­‐state differences inferred from the isotope effects. The transition state analogues generated from these studies had astonishingly high binding affinity for the P. falciparum enzyme, binding to the enzyme a million-­‐fold more tightly than its natural substrate.12 INNOVATION The methodology described in this proposal is novel and has not been applied to DHFR or indeed any antimicrobial drug targets before. DHFR is a known drug target but all established inhibitors have been the result of modifications to existing drug scaffolds rather than truly novel compounds, and in any case all existing clinical and preclinical DHFR inhibitors are folate analogues rather than transition state analogues. There is an urgent need for new DHFR targeting drugs, as shown by our recent market research, and this is underlined by the recent resurgence of activity in this field in the pharmaceutical industry. Our work will provide a new class of DHFR inhibitors with improved selectivity over other folate-­‐binding enzymes. No preconceptions of what a DHFR inhibitor should look like will be used, but we will instead generate the information necessary to design and synthesise such molecules, which will therefore have completely novel chemical structures, significantly increased affinities for their target. This will lead to drugs that are effective at much reduced concentration and hence with reduced side effects and limited emergence of resistance. RESEARCH OBJECTIVES AND APPROACH 1. Measurement of various kinetic isotope effects (KIEs) for the reactions catalysed by human and E. coli DHFRs. This will require the production of DHFR substrates (NADPH and dihydrofolate) with suitably positioned isotopic labels. We have already cloned the genes encoding all enzymes required for the biosynthesis of both molecules and have developed a procedure that allows the one pot in vitro synthesis of NADPH and dihydrofolate from commercially available, labelled precursors in high yield. This will allow us to produce labelled DHFR substrates and NADPH. KIEs will be measured using either mixed-­‐isotope radiolabelling and scintillation counting, or NMR methodology. 2. Creation of transition state maps for the reaction catalysed by the two DHFRs. This will use computational chemistry packages to determine the electrostatic and geometric parameters required to produce the observed pattern of KIEs. 3. Optimisation of compounds using state of that art molecular modelling techniques (AB). Using a structure-­‐based approach, we will examine the possible binding modes and protein/ligand interactions of the novel compounds to better understand the key structural features needed for optimal activity and selectivity. The results obtained will support the design of the novel DHFRs inhibitors. 4. Synthesis of molecules approximating the transition state maps. As potential antibiotics, these molecules will match the E. coli DHFR map and avoid overlap with the human DHFR map, creating selectivity for the bacterial enzyme over the human one. Although our market research indicates that anticancer drug discovery is not such an attractive market, due to the relative crowding in that area, compounds retaining high affinity for human DHFR could in future be taken forward as potential anticancer drugs. 5. Testing of these molecules for in vitro inhibition of human and E. coli DHFRs. This will require kinetic studies of the two enzymes in the presence of the potential inhibitors, and will assess binding and the degree of selectivity for bacterial vs mammalian DHFRs. Successful candidate molecules may then be further used for in vivo inhibition studies against bacterial species to determine their activity spectrum and hence their likely clinical usefulness. LITERATURE 1. Guo, J., Luk, L. Y. P., Loveridge, E. J. & Allemann, R. K. Thermal Adaptation of Dihydrofolate Reductase from the Moderate Thermophile Geobacillus stearothermophilus. Biochemistry in press, DOI: 10.1021/bi500238q (2014). 2. Luk, L. Y. P. et al. Unraveling the role of protein dynamics in dihydrofolate reductase catalysis. Proc. Natl. Acad. Sci. U.S.A. 110, 16344–16349 (2013). 3. Ruiz-­‐Pernia, J. J. et al. Increased Dynamic Effects in a Catalytically Compromised Variant of Escherichia coli Dihydrofolate Reductase. J. Am. Chem. Soc. 135, 18689–18696 (2013). 4. Loveridge, E. J., Behiry, E. M., Guo, J. & Allemann, R. K. Evidence that a `dynamic knockout’ in Escherichia coli dihydrofolate reductase does not affect the chemical step of catalysis. Nat. Chem. 4, 292–297 (2012). 5. Loveridge, E. J. et al. The Role of Large-­‐Scale Motions in Catalysis by Dihydrofolate Reductase. J. Am. Chem. Soc. 133, 20561–20570 (2011). 6. Loveridge, E. J., Tey, L. H. & Allemann, R. K. Solvent Effects on Catalysis by Escherichia coli Dihydrofolate Reductase. J. Am. Chem. Soc. 132, 1137–1143 (2010). 7. Loveridge, E. J., Evans, R. M. & Allemann, R. K. Solvent Effects on Environmentally Coupled Hydrogen Tunnelling During Catalysis by Dihydrofolate Reductase from Thermotoga maritima. Chem. -­‐ a Eur. J. 14, 10782–10788 (2008). 8. Allemann, R. K., Evans, R. M. & Loveridge, E. J. Probing coupled motions in enzymatic hydrogen tunnelling reactions. Biochem. Soc. Trans. 37, 349–353 (2009). 9. Allemann, R. K. et al. Protein motions during catalysis by dihydrofolate reductases. Philos. Trans. R. Soc. B-­‐Biological Sci. 361, 1317–1321 (2006). 10. Swanwick, R. S., Maglia, G., Tey, L. & Allemann, R. K. Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase. Biochem. J. 394, 259–265 (2006). 11. Maglia, G. & Allemann, R. K. Evidence for environmentally coupled hydrogen tunneling during dihydrofolate reductase catalysis. J. Am. Chem. Soc. 125, 13372–13373 (2003). 12. Ho, M. C. et al. Four generations of transition-­‐state analogues for human purine nucleoside phosphorylase. Proc. Natl. Acad. Sci. U. S. A. 107, 4805–4812 (2010).