- 5 views
- 10 downloads
The interaction of phenelzine with human monoamine oxidase B: characterization and consequences
-
- Author / Creator
- Held, Morganne
-
Phenelzine (PLZ) is a monoamine oxidase (MAO) inhibitor developed in the 1960s which has been used clinically for psychiatric conditions such as depression and anxiety disorders. However, despite over 60 years of research, the mechanism by which PLZ exerts its effects on MAO has yet to be satisfactorily described. The work presented in this thesis has investigated the mechanism of inhibition of human MAO-B by PLZ and discussed these findings in the context of two mechanisms: the forked mechanism and the stepwise mechanism.
First of all, the formation of β-phenylethylidenehydrazine (PEH) from the metabolism of PLZ by MAO-B was examined through high pressure liquid chromatography-mass spectrometry
(LC-MS) analysis of deuterated incubations of MAO-B and PLZ. This provided evidence for the intramolecular rearrangement of the diazene intermediate β-phenylethyldiazene (PEDz) to PEH in
bulk solvent, contrary to the forked mechanism which states that inactivation of MAO-B occurs in situ.
Secondly, the role of oxygen in the covalent modification of the flavin cofactor of MAOB by PEDz was examined through inclusion of the redox dye 2,6-dichlorophenolindophenol (DCPIP) in anaerobic incubations of MAO-B and PLZ. These results suggest that oxygen may be
necessary for both re-oxidation of the flavin cofactor and radicalization of PEDz to form a phenylethyl radical.
Chapter 4 further examined the dissociation of PEDz from the active site, building on the indirect evidence detailed in Chapters 2 and 3, through use of the free radical scavenger ascorbate and chelating agent diethylenetriaminepentaacetic acid (DTPA). These findings suggest a stabilizing effect for PEDz by ascorbate, preventing loss of the inhibiting compound to PEH formation before rebinding to and inhibiting oxidized enzyme.
Finally, the characteristics of irreversible inhibition of MAO-B by a range of PLZ
concentrations were compared by utilizing a membrane particle centrifugation protocol which was optimized in Chapter 5. At a single concentration of MAO-B, inhibition by PLZ differed significantly, and in an unexpected manner, in half-life and extent of inactivation depending on whether the concentration of PLZ was in the low-mid micromolar range, or at a super-saturating mid-millimolar concentration. While increased half-lives at high PLZ concentrations were consistent with dissociation of PEDz from the reduced active site and with competition between
PEDz, PEH, and PLZ for reassociation, the magnitude and nature of the effect implicated a role for inter-subunit communication. Results are consistent with the idea that the redox state of one monomer may be sensed by the opposing monomer through movement of gating loops, and that this communication may be disrupted by binding of PLZ at high concentrations to a site distinct from the active site - possibly the I2 site in the entrance channel of the inactivated monomer.
The findings in this thesis suggest that the mechanism by which MAO is inhibited by PLZ is rather more complex than first thought. Indeed, elucidation of the complete mechanism may provide much insight into the role of gating loops in communication within the dimer, information invaluable for development of novel drug therapies targeting dimeric flavin-containing amine oxidases. -
- Subjects / Keywords
-
- Graduation date
- Spring 2022
-
- Type of Item
- Thesis
-
- Degree
- Master of Science
-
- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.