Dr Andy Holt
Associate Professor
Education:
BSc (Hons), Pharmacology, University of Dundee, 1989
MPhil, Pharmacology, Queens' College, University of Cambridge, 1990
PhD, Pharmacology, Queens' College, University of Cambridge, 1993
Teaching: PMCOL201, PMCOL343, PMCOL337, PMCOL508
Research: Molecular pharmacology and kinetic analyses of amine oxidases and other enzymes
Research Interests / Laboratory Techniques
Metabolic reactions in the body can proceed billions of times faster when catalysed by an enzyme; enzymes therefore provide real-time regulation of tissue concentrations of reactants (substrates) and products. An imbalance between bioactive substrates and products can cause, or exacerbate Holt-Figurethe symptoms of, many disease conditions. It is often possible to correct such an imbalance and improve a patient's health by administering a drug which alters enzyme activity. For example, atorvastatin (Lipitor), which blocks the activity of HMG-CoA reductase, reduces formation of cholesterol and lowers plasma cholesterol levels. Most drugs that act on enzymes are, like atorvastatin, inhibitors of enzyme activity.
Development of novel drugs that target enzymes requires an in-depth understanding of enzyme mechanism and structure. In the Holt laboratory, we use a variety of approaches to study how enzyme proteins catalyse reactions and how, as well as where, drugs interact with enzyme proteins to alter catalytic rate. Conventional radiochemical and spectroscopic methods allow quantification of reaction velocities, while absorbance and fluorescence platereader approaches facilitate large-scale studies of steady-state catalysis. Stopped-flow spectroscopy and competition radioligand binding approaches allow us to determine on- and off-rates and calculate ligand affinities for enzymes. Equations are developed to describe drug-enzyme interactions, with results of regression analyses providing information on the mechanism, affinity and binding site location for inhibitor drugs.
Our main interests lie in the study of amine oxidases; inhibitors can act as antidepressants, antiParkinsonian agents, smoking cessation therapies, anti-inflammatory agents and may reduce vascular damage in diabetes. Human enzymes are expressed in yeast and purified by column chromatography prior to kinetic and structural analyses. Introduction of point mutations at specific sites prior to expression can provide useful information on molecular mechanisms. We also collaborate on studies with ATPases, matrix metalloproteinases and cytochromes P450
Selected Recent Publications
Fatehi M, Carter CR, Youssef N, Hunter BE, Holt A and Light PE. (2015) Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity. Biosci Rep 35(4) pii: e00238 PMID: 26181369.
Narang D, Kerr PM, Lunn SE, Beaudry R, Sigurdson J, Lalies MD, Hudson AL, Light PE, Holt A and Plane F. (2014) Modulation of Resistance Artery Tone by the Trace Amine beta-Phenylethylamine: Dual Indirect Sympathomimetic and alpha1-Adrenoceptor Blocking Actions. J Pharmacol Exp Ther 351 (1):164-171. PMID: 25118217.
Zhurova M, Olivieri A, Holt A, Acker JP. (2014) A method to measure permeability of red blood cell membrane to water and solutes using intrinsic fluorescence. Clin Chim Acta 431:103-10. PMID: 24522163.
Kozuska JL, Paulsen IM, Belfield WJ, Martin IL, Cole DJ, Holt A, Dunn SM (2013) Impact of intracellular domain flexibility upon properties of activated human 5-HT receptors. Br J Pharmacol 171(7):1617-28. PMID: 24283776.
Foot JS, Deodhar M, Turner CI, Yin P, van Dam EM, Silva DG, Olivieri A, Holt A, McDonald IA (2012) The discovery and development of selective 3-fluoro-4-aryloxyallylamine inhibitors of the amine oxidase activity of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1). Bioorg Med Chem Lett. 22(12), 3935-40. PMID: 22595173.
Ali MA, Stepanko A, Fan X, Holt A and Schulz R (2012) Calpain inhibitors exhibit matrix metalloproteinase-2 inhibitory activity. Biochem Biophys Res Commun 423:1-5. PMID: 22575511.
Oivieri A, Degenhardt OS, McDonald GR, Narang D, Paulsen IM, Kozuska JL, Holt A (2012) On the disruption of biochemical and biological assays by chemicals leaching from disposable laboratory plasticware. Can J Physiol Pharmacol. 90(6), 697-703. PMID: 22509735.
Fatehi M, Raja M, Carter C, Soliman D, Holt A, Light PE (2012) The ATP-sensitive K(+) channel ABCC8 S1369A type 2 diabetes risk variant increases MgATPase activity. 61(1), 241-9. PMID: 22187380.
Ramsay RR, Olivieri A, Holt A (2011) An improved approach to steady-state analysis of monoamine oxidases. 118(7), 1003-19. PMID: 21643793.
Kachalova G, Decker K, Holt A, Bartunik HD (2011) Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase family. Proc. Nat. Acad. Sci. (USA), 108(12), 4800-4805. PMID: 21383134.
Bonivento D, Milczek EM, McDonald GR, Binda C, Holt A, Edmondson DE, Mattevi A. (2010) Potentiation of ligand binding through cooperative effects in monoamine oxidase B. J. Biol. Chem., 285(47), 36849-36856. PMID: 20855894.
McDonald GR, Olivieri A, Ramsay RR, Holt A (2010) On the formation and nature of the imidazoline I2 binding site on human monoamine oxidase-B. Pharmacol. Res., 62, 475-488. PMID 20832472.
Holt A (2009) Membrane-bound copper amine oxidases. In: Floris G, Mondovi B (eds.) Copper amine oxidases: structures, catalytic mechanisms and role in pathophysiology. Taylor and Francis, Boca Raton, Florida, Ch. 6, pp. 69-86.
McDonald GR, Hudson AL, Dunn SM, You H, Baker GB, Whittal RM, Martin JW, Jha A, Edmondson DE, Holt A (2008) Bioactive contaminants leach from disposable laboratory plasticware. Science, 322, 917. PMID 18988846.