Photochemical harpoons: covalent labels for multi-protein complexes

The identification of the biomolecular interaction partners of small bioactive molecules is a fundamental problem in drug discovery and cell biology. This thesis describes the development of fluorescent chemical probes to identify the biomolecular targets of the known organophosphate toxin, phenyl saligenin phosphate (PSP), and the cardioprotective agent diazoxide. PSP is an organophosphate toxin that irreversibly inhibits hydrolase enzymes such as trypsin and chymotrypsin along with the common organophosphate target acetylcholine esterase. PSP is also suspected of affecting many other cell functions and may interact with a large number of cellular proteins. In this work phenyl saligenin phosphate has been synthesised and its inhibitory effect on the action of transglutaminase 2 (TGase2) demonstrated. Analogues of PSP containing an attached dansyl amide fluorescent group have been prepared and incubated with purified enzymes trypsin, chymotrypsin and TGase2. SDS-PAGE analysis demonstrates effective fluorescent labelling and a covalent interaction between the toxin analogue and the enzymes. The KATP channel opener, diazoxide displays marked cardioprotective effects and is reported to bind to mitochondrial KATP channels. However, the molecular structure of these channels is still largely unknown.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

Nottingham Trent University

Year dc:date.issued

2013

Author dc:creator

• Smida, FA

Language dc:language

en