Table Of ContentENZYME INHIBITION
AND BIOAPPLICATIONS
Edited by Rakesh Sharma
Enzyme Inhibition and Bioapplications
Edited by Rakesh Sharma
Published by InTech
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Copyright © 2012 InTech
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First published May, 2012
Printed in Croatia
A free online edition of this book is available at www.intechopen.com
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Enzyme Inhibition and Bioapplications, Edited by Rakesh Sharma
p. cm.
ISBN 978-953-51-0585-5
Contents
Preface IX
Section 1 Basic Concepts 1
Chapter 1 Enzyme Inhibition: Mechanisms and Scope 3
Rakesh Sharma
Section 2 Applications of Enzyme Inhibition 37
Chapter 2 Cytochrome P450 Enzyme Inhibitors from Nature 39
Simone Badal, Mario Shields and Rupika Delgoda
Chapter 3 Pharmacomodulation of
Broad Spectrum Matrix Metalloproteinase
Inhibitors Towards Regulation of Gelatinases 57
Erika Bourguet, William Hornebeck,
Janos Sapi, Alain Jean-Paul Alix and Gautier Moroy
Chapter 4 Non-Enzymatic
Glycation of Aminotransferases
and the Possibilities of Its Modulation 85
Iva Boušová, Lenka Srbová and Jaroslav Dršata
Chapter 5 Inhibition of Nitric Oxide
Synthase Gene Expression: In vivo Imaging
Approaches of Nitric Oxide with Multimodal Imaging 115
Rakesh Sharma
Chapter 6 Transcriptional Bursting in the Tryptophan Operon of
E. coli and Its Effect on the System Stochastic Dynamics 179
Emanuel Salazar-Cavazos and Moisés Santillán
Chapter 7 Mechanisms of Hepatocellular Dysfunction
and Regeneration: Enzyme Inhibition by
Nitroimidazole and Human Liver Regeneration 195
Rakesh Sharma
VI Contents
Chapter 8 Reversible Inhibition of
Tyrosine Protein Phosphatases by Redox Reactions 253
Daniela Cosentino-Gomes and José Roberto Meyer-Fernandes
Chapter 9 Feasible Novozym 435-Catalyzed
Process to Fatty Acid Methyl Ester Production
from Waste Frying Oil: Role of Lipase Inhibition 277
Laura Azócar, Gustavo Ciudad, Robinson Muñoz,
David Jeison, Claudio Toro and Rodrigo Navia
Chapter 10 Urease Inhibition 303
Muhammad Raza Shah and Zahid Hussain Soomro
Preface
Enzyme is a protein molecule exhibiting specific activity and binding affinity with its
substrate molecule to complete enzyme reaction or biocatalytic reaction. Substrate
analogues can inhibit the enzyme reaction and act as enzyme inhibitor. Enzyme
inhibition (Enz-ai-m ie-ni-hi-bi-son) means reducing or blocking an enzyme action on
specific location of enzyme active site by specific substrate or analogue so called
enzyme inhibitor. In modern times, most of the pharmaceutical as well as nutriceutical
compounds are marketed as enzyme inhibitors and such inhibitors exhibit their
specific action in enzyme inhibition inside cells, bacteria, virus, animal plants and
human body. The action of enzyme inhibitors in drug discovery has become a
fundamental approach to pharmacology at any pharmaceutical industry, university
research lab or drug research center. The present issue has been compiled from
various data sources with aim of incorporating a wide range of basic concepts and
applied enzyme inhibition evaluation methods in drug discovery. It is aimed at those
who are embarking on drug discovery research projects, immobilized enzyme solid
state devices as well as relatively experienced pharmacologists, biochemists and
pharmacy scientists who might wish to develop their experiments further to the
advanced level. While it is not possible to detail and include every possible technique
related with enzyme inhibition evaluation in drug design by using specific inhibitors
at specific metabolic mechanism(s), the present issue attempts to provide working tips
with examples and analysis relevant to a wide range of more commonly available
enzyme inhibition techniques.
The methods and concepts described in this book are aimed at giving the reader a
glimpse of some existing enzyme inhibition studies and also methods with context of
each enzyme inhibition method applied for, as well as providing some basis of
familiarizing oneself with these biochemical methods. While enzyme inhibition has
been used as major approach in drug design in the research and industry over last two
decades, it was only later part of 20th century that it has become a major part of so
many applications in biochemical engineering, biomedical engineering of miniatured
clinical chemistry devices in microbiological, bacteriological, immunological,
hormonal testing, nanotechnology, physiological monitoring in health science, plant
science and environment research work. This is, at least in part, due to the continued
development of new solid state polymer platform, pure enzymes and specific
X Preface
inhibitors available, better understanding of enzyme inhibition mechanisms and
precise detection methods, new awareness of drug discovery and design, with
scanning and monitoring accessories. Thanks to the continued joint efforts of
governmental, industrial and academic institutions globally to promote the need of
new generations of drugs or enzyme inhibitors and new mechanisms of enzyme
inhibition. Regardless of the enzymes or drugs and their brands that are used, one
should always be able to understand and justify the use of right inhibitor or drug
action on enzyme of right choice to drug design for specific study. With this aim,
different approaches of enzyme inhibition methods are presented in separate chapters
on the use of different enzyme inhibitors. For learners, basic concepts, mechanistic
issues, limitations in drug testing, skepticism in enzyme inhibition approach and drug
variability due to non-specific analogues of enzyme inhibitors or substrates is
presented with a working enzyme inhibition protocols for drug design and analysis of
their inhibitory action.
In chapter 1, the authors have introduced the basic concepts on enzyme, enzyme
reaction, inhibitors and types of inhibition with a handful established applications in
drug discovery, immobilized enzyme engineering, and biosensing. Major three basic
types of enzyme inhibition kinetics is highlighted for beginners. Examples of substrate
analogues and their enzyme inhibition behaviour are illustrated with color schemes.
Application of immobilized enzyme on chips for environment monitoring and
biosensor development is quite intriguing for engineers, scientists and industrialists.
In chapter 2, authors overviewed isolates from Pepermia amplexicaulis and Spathelia
sorbifolia plants have examined for CYP inhibitions to exhibit antiprotozoal,
chemopreventive and anti-cancer activity. Other plant sources of tea and fruits of
Rhytidophyllum tomentosa, Psidium guajava, Symphytium officinale, Momordica charantia
showed inhibition properties of these teas against a panel of CYP450 enzymes in order
to assess the potential for drug interactions with co-medicated pharmaceuticals. The
chapter highlights the potential of a few natural products emanating from the
Caribbean: chromene amides isolated from Amyris plumieri, quassinoids isolated from
Picrasma excelsa, anhydrosorbifolin isolated from Spathelia sorbifolia and 5-Hydroxy-2,7-
dimethyl-8-(3-methyl-but-2-enyl)-2-(4-methyl-penta-1,3-dienyl)-chroman-6-carboxylic
acid isolated from Peperomia amplexicaulis. New information is presented on bioactive
screens against CYP enzymes in the presence of five aqueous infusions of popularly
used herbs; Rhytidophyllum tomentosa, Psidium guajava, Symphytium officinale, Momordica
charantia and Picrasma excelsa. Authors emphasized search of CYPs 1A1 and 1B1 activity
inhibitors as chemoprotectors such as CA1 and quassin for CYP 1A1; and
anhydrosorbifolin and 5-Hydroxy-2,7-dimethyl-8-(3-methyl-but-2-enyl)-2-(4-methyl-
penta-1,3-dienyl)-chroman-6-carboxylic acid for CYP 1B1 in search of safer herbal
remedies in co-adiministration of medicines in cancer prevention. In chapter 3, authors
described pharmacomodulation in metalloprotease enzymes by inhibitors. Authors
aim to achieve better metallloprotease (MMP) enzyme inhibitors with good MMP-2
selectivities to increase hydrophobicity and rigidity with the dehydro and didehydro
analogues and synthesized (analogue 2a-d and 3a-h). Authors displayed sevral
Preface XI
examples of inhibitors including pharmacomodulation of galardin®, a powerful broad
spectrum MMPI, hydrazide and sulfonylhydrazide-type functions as potential Zinc
Binding Compounds for gelatinase enzyme inhibition, double-headed elastase-MMP
inhibitor (d-hEMI) able to block elastase and MMP activities, with display of
structural-functional models in the last sections. Authors speculate to develop hybrid
nanoprobes build from MMPI and fluorescent nanocrystal quantum dots (QDs).
Design and chemical synthesis of derivatives of galardin®, selective inhibitors of MMP-
2, tagging with QDs. In hope of photo- and chemical stability of QDs, it is possible that
apporoach will enable long-term spatiotemporal tracking of the process of inhibition
of MMP-2 enzymes to offer better understanding of physiological process of invasion
of melanoma. In chapter 4, authors introduce the readers with nonenzymatic glycation
of aminotransferases and modulation of these enzymes as model protein. Further
authors emphasized the significance of advanced glycation end-products with
introduction of structure, targeting with aim to evaluate potential antiglycation
activity of two mitochondrial antioxidants, α-phenyl N-tert-butyl nitrone (PBN) and N-
tert-butyl hydroxylamine (NtBHA). In next section, authors established the effect of
natural compound on fructose induced AST glycation, basic techniques to determine
primary amino groups, enzyme molecular charge of AST modulated by fructose by
electrophoresis. Authors attempted to search the compounds with antioxidant and
potential antiglycating activities in an illustrated description with a perspective of
their use as remedies against diabetic complications. In chapter 5, author introduces
the readers with basic mechanism of reduced nitric oxide synthase gene expression
and applications of nitric oxide synthase (NOS) inhibition and approaches of nitric
oxide (NO) content by using less known multimodal imaging. Nitric oxide imaging
techniques utilize mapping NO in tissue using NO specific imaging contrast agents
sensitive to fluorescence, magnetic resonance and electron spin resonance. A handful
account is presented on NOS expression inhibitors, possibility of dithiacarbamates,
paramagnetic complexes for bioimaging of NO. For learners, MRI protocol is given as
example. In the light of recent developments in multimodal bioimaging of NO/NOS
expression by bioluminescence, fluorescence techniques, a handful information is
given in cells, animals, plants and humans body in different diseases including
endothelial cell injury, apoptosis, renal, liver, lung, muscle, brain, inflammation,
bones, retina with emphasis on multimodal techniques of calcium, ion channels, iron
bound complexes. Author speculated the future possibility of NO/NOS bioimaging by
combined radioimaging techniques and it remains to see if real-time imaging becomes
routine modality. In chapter 6, transcriptional bursting in E.coli tryptophan operon is
introduced to explain stochastic dynamics of the tryptophan synthase enzyme
feedback inhibition regulatory mechanism at various levels of Trp operon genes.
Authors have described in chapter various components of trp operon structure, model
development, parameter estimation, and numerical methods with results on stochastic
stimulations to evidence the least response time with inhibition-less strain. Authors
further investigated that the repressor-operator interaction stimulates transcriptional
bursting which shows several dynamic effects on transcriptional bursting possibly by
a feedback enzyme-inhibition regulatory mechanism. In chapter 7, a hepatocellular
