Investigating binding affinity with and sequence recognition by peptidylglycine a-amidating monooxygenase
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2014
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Morris, Kelly Maree
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This thesis presents studies conducted to elucidate structural features of substrates and inhibitors of the enzyme peptidylglycine alpha-amidating monooxygenase (PAM) that may be important for drug design. Given the focus on applicability to drug design, studies focussed on subtypes of PAM from human cancer cell lines. Chapter 1 of this Thesis describes the development of a cultured cell assay suitable for monitoring PAM activity from human carcinomas and quantifying enzyme inhibition. These studies identified sources of human PAM and lead to the later development by another member of the Easton group of another, more sensitive, cultured cell assay. Having identified cellular sources of PAM, cellular and secreted enzyme subtypes were extracted from two vastly different cancer cell lines for use in further PAM studies. A range of PAM substrate and inhibitor peptidomimetic analogues of the prohormone of thyrotropin releasing hormone as well as prooxytocin were synthesised and assayed with two enzyme subtypes. These studies demonstrated that the potency of both substrates and inhibitors can be increased through substitution of a penultimate amino acid with phenylalanine and that such alteration of peptidomimetic glycolate inhibitors results in a loss of specificity for cellular enzyme. These studies also demonstrated that PAM recognises peptide sequences and that this sequence recognition extends beyond four amino acids from the C terminus. Potent inhibitors with binding affinities for PAM in the low micromolar range were developed. The N-terminal amide of the penultimate residue was shown, using assays with cellular and secreted subtypes from two enzymes, to decrease inhibitor potency. It also eliminates specificity, probably as a result of forcing inhibitors to adopt an alternative binding mode. Conversely, it was found that substitution of the N-terminal nitrogen of a penultimate residue with a polar substituent resulted in improved binding to PAM. Indeed the improvement in binding affinity resulting from substitution at the amide nitrogen was found to be greater than analogous substitution at the alpha- position. Such substitution did however result in a loss of specificity. An olefinic fatty acid was shown to be a potent and selective inhibitor of PAM from two cell lines. This inhibitor had a binding affinity in the low micromolar range.
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Thesis (PhD)
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