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Covalent Interactions Between Quinols and the Thioredoxin Active Site: a Proposed New Mechanism

Covalent Interactions Between Quinols and the Thioredoxin Active Site: a Proposed New Mechanism. Gareth Hall 2 nd Year Ph.D. X-Ray Crystallography. Summary of Present Results. X-Ray Crystallography:  Unable to achieve drug bound protein structure - reversible Micheal addition?

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Covalent Interactions Between Quinols and the Thioredoxin Active Site: a Proposed New Mechanism

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  1. Covalent Interactions Between Quinols and the Thioredoxin Active Site: a Proposed New Mechanism Gareth Hall 2nd Year Ph.D. X-Ray Crystallography

  2. Summary of Present Results • X-Ray Crystallography:  Unable to achieve drug bound protein structure - reversible Micheal addition? - mass spec. demonstrates binding • Insulin reduction assays:  Lineweaver Burke plot shows competitive inhibition - recovery of activity? • NMR:  Presence of at least 2 species after addition of AW464 - is compound being altered?

  3. TrxR PMX 464 Thioredoxin-S2 (inactive) Thioredoxin-(SH)2 (active) Proposed New Mechanism Driving Forces – Formation of Intramolecular disulphide bond and aromatisation of ring

  4. AW464 Proving the Hypothesis • Need to determine an experiment to detect the formation of the phenol variant of AW464 • Quinols have a conjugated ketone system  UV spectroscopy to measure changes in the α,β-unsaturated ketone Quinones expected to have absorption maxima of ~ 242 and 281nm (Williams and Flemming, 1997)

  5. UV Spectrum of AW464

  6. UV Spectrum of AJM478

  7. UV Spectrum of HTrx, TCEP and AW464

  8. AJM478 peak? UV Spectrum of HTrx, TCEP and AW464 – removal of AW464 peak

  9. Imperfect Substrate vs. Irreversible Inhibitor • Data suggests that AW464 could be acting as an imperfect substrate • Both cysteine residues present in the thioredoxin active site are required to catalyse the reaction of AW464 • Even if the phenol is not being formed by the reaction, something is definitely happening to the drug • Consider ways to alter the chemical structure to prevent the turnover of the compound

  10. Future Work • Lots more data to collect – validate hypothesis • Mass Spectroscopy – extract product (phenolic form) from the reaction • NMR – try and determine if the phenol is responsible for the peaks • Look at other proteins containing a dithiol active site – and even others that don’t, but are redox enzymes, to determine benzothiazole activity • UV spectroscopy method is a fairly quick and easy assay to carry out to test the effect of AW464 on other proteins

  11. Acknowledgments I would like to thank: Dr. Jonas Emsley Dr. Charlie Laughton Prof. Malcolm Stevens Dr. Manish Shah Dr. Tracey Bradshaw Dr. Andrew McCarroll Dr. Jenny Cookson Dr. Andrew Westwell Dr. Huw Williams Mr. Charlie Matthews Everyone in the X-ray crystallography lab. for their support University of Nottingham for scholarship And finally you all for listening

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