| Summary: | <p>TRPA1 is a thermosensitive, calcium permeable ion channel present in small diameter neurons present in most organs including the skin, lungs and trachea. TRPA1 is activated by small-molecule ligands, notably electrophiles including α,β-unsaturated aldehydes, tear gases, allyl isothiocyanate, iodoacetamide, and formaldehyde (HCHO). Deletion of TRPA1 in mice causes loss of sensitivity to HCHO. Modulation of TRPA1 activity is of interest as a target for pain treatment. GRC-17536 is in clinical trials, however, the mechanism by which it acts as a TRPA1 antagonist is unknown. The mechanism by which channel opening results from electrophile (e.g. HCHO) binding to cysteine residues within TRPA1 is unclear. The work described in this thesis attempted to explore these mechanisms.</p>
<p>The reaction of HCHO with biological nucleophiles is of interest from disease and regulatory perspectives. The reaction of cysteine containing peptides with HCHO was shown to involve selective formation of an S-hydroxymethyl adduct. The stability of these adducts was explored through the addition of the ubiquitous cellular nucleophile glutathione (GSH), which was found to remove HCHO from the adducts. A recombinant protein model of the TRPA1 electrophile binding domain was designed, purified and its reactions with electrophiles studied.</p>
<p>To investigate the binding site of GRC-17536, a series of photolabile ligands based on GRC-17536 were designed and synthesised. A structure-activity relationship was constructed from the compounds’ ability to inhibit cinnamaldehyde evoked TRPA1 activity. Binding of a compound showing the most potent inhibition (3-60) was studied by cryo-electron microscopy (cryo-EM). From a cryo-EM structure of 3-60 bound to TRPA1, it was possible to propose modifications to the ligand which would improve binding and pharmacokinetic properties of theophylline-based ligands such as GRC-17536.</p>
<p>TRPA1 shows a “U”-shaped sensitivity towards agonists under changing oxygen concentration. It is controversially reported that this is a result of hydroxylation of the channel by PHD2, a hydroxylase involved in the hypoxic response. Peptides corresponding to the ankyrin loops of TRPA1 were synthesised and screened against FIH, a different HIF-hydroxylase, the results reveal that TRPA1 ankyrin 8 is a substrate of FIH at the peptide level.</p>
<p>The combined results provide new insight into the reaction of HCHO with cysteine residues involved in signalling pathways and open up new avenues for modulating the activity of TRPA1.</p>
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