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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5472_Библиотеки_им_академика_М_И_Перельмана.pdf
Biotechnology in Drug Discovery
235
CURRENT TRENDS IN MODERN PHARMACEUTICAL ANALYSIS
FOR DRUG DISCOVERY
Successful drug discovery and development depends on close interactions between various
disciplines with inputs from biotechnology, biomedical engineering, proteomics and genomics
inter alia. Currently, drug discovery efforts are being revolutionized by high-input technologies,
combinatorial chemistry, informatics, genomics, proteomics and miniaturization. Today,
pharmaceutical analysis is the backbone of the whole drug discovery and development process. It
is used to provide accurate and precise data, not only supporting drug discovery and development,
but also postmarket surveillance, high-throughput analysis, structural analysis, purity analysis and
quantitative estimation.
The rational approach to drug design has been adopted in developing specific inhibitors of the
human cellular enzyme and purine nucleoside phosphorylase (PNP). The free purine released can
then be used in the biosynthesis of new nucleic acids within the cell.
Purine base
Sugar + Pi-Free Purine + Sugar P
RECENT PHARMACOKINETIC ADVANCES IN DRUG DISCOVERY
AND DEVELOPMENT
Pharmacokinetics (PK) is one of the major crucial areas to evaluate whether promising clinical
candidates can be developed into marketable drugs. This is also one of the important considerations
that accounts for the costly and high late-stage failure rates in drug development. So far, poor PK
profiles have accounted for around 40% attrition in late stage of drug development, which in
turn had substantial effect on R&D costs. Therefore, the working culture in the pharmaceutical
industry today has shifted to conducting PK investigations at a much earlier stage in drug
discovery. In addition to the application of chemometrics in the early stages of drug development
in silico technology has emerged as a new and promising tool for early ADME–Tox investigations.
Commercial ADME–Tox software is now available to screen compounds in the early stage of
drug development. However, more work needs to be performed to refine current in silico models,
because these predictive models are based on limited PK and toxicity data sets. Another exciting
novel innovation for ADME studies in early clinical drug development is human microdosing,
using two ultrasensitive analytical techniques, accelerator mass spectrometry (AMS) and positron
emission tomography (PET).
NATURAL PRODUCT DRUG DISCOVERY
Natural products are important sources of new structures, leading to drugs in all major disease
areas. Finding new habitats of fungi, bacteria or plants and identifying new living organisms have
been a continuous challenge. Traditionally, drug discovery programmers within the pharmaceutical
industry relied on screening various biological specimens for potential drugs. The examples include
digoxin and digitoxin, as well as morphine, codeine, taxol, etc. Plants are the sources of drugs as
Textbook of Pharmaceutical Biotechnology
236
they produce a vast array of novel bioactive molecules, many of which probably serve as chemical
defences against infection. If an interesting activity is described, larger quantities of the plant material
are collected, from which chemists purify and characterize the active principles. Microorganisms,
mainly bacteria and fungi, have also proved to be rich sources of bioactive molecules, such as
antibiotics, anticancer and anti-infective agents.
Testing and identifying biologically active natural products from plant extracts or microbes
in today’s high-throughput screening (HTS) environment pose a challenge to sample preparation
and single component identification. An enhancement of productivity levels in the drug discovery
process has been achieved with the implementation of library chemistry and HTS technologies.
Despite these efforts, the numbers of new chemical entities reaching the market have not increased.
Only one drug originated from a de novo combinatorial chemistry approach. However, the natural
products remain an important source of structures contributing to mostly semisynthetic or synthetic
drugs used in wide spectrum of human ailments.
Natural products have been the single most productive sources of leads for the development
of drugs. Natural products are important sources of unsurpassed structural diversity and functional
density to identify screening hits. The therapeutic effects of natural product-derived drugs are
predominantly achieved in antibiotic therapies, immunoregulation and oncology. It is less likely
to identify potent natural products against molecular targets of human diseases in indications, such
as cardiovascular or nervous systems. Those targets are generally not related to the biological
environment of the producing organism. However, higher hit rates are generally obtained for natural
product libraries in HTS campaigns in comparison to medchem or combichem libraries.
Around one hundred new natural products are in clinical development, particularly as anticancer
agents immunomodulators, hepatoprotectives and anti-infectives. Application of molecular
biological technique is increasing the availability of novel compounds that can be conveniently
produced in bacteria or yeasts. The combinatorial chemistry approaches are being based on natural
product scaffolds to create screening libraries that closely resemble drug-like compounds. Various
screening techniques have been developed to improve the ease with which natural products can be
used in drug discovery campaigns and data mining. The virtual screening techniques are also being
successfully applied to databases of natural products.
Screening of Natural Products
Ethnobotany is the study of the use of plants in the society, and ethnopharmacology is an area
inside ethnobotany, which is focused on medicinal uses of plants. Both can be used in selecting
starting materials for future drugs. A collection of plant, animal and microbial samples from rich
ecosystems may give rise to novel biological activities. One example of a successful use of this
strategy is the screening for antitumour agents by the National Cancer Institute, United States,
started in the 1960s.
Two main approaches exist for the finding of new bioactive chemical entities from natural sources:
(1) random collection and screening of material and (2) exploitation of ethnopharmacological
knowledge in the selection. The former approach is based on the fact that only a small part of
Biotechnology in Drug Discovery
237
biodiversity of the planet has ever been tested for pharmaceutical activity, and organisms living in
a species-rich environment need to evolve defensive and competitive mechanisms to survive.
Current research in drug discovery from medicinal plants involves a multifaceted approach
combining botanical, phytochemical, biological and molecular techniques. Medicinal plant or herb
drug discovery continues to provide new and important leads against various pharmacological
targets, including cancer, HIV/AIDS, Alzheimer’s disease, malaria and pain. Several natural product
drugs of plant origin have either recently been introduced into the US market, including arteether,
galantamine, nitisinone and tiotropium, or are currently involved in late-phase clinical trials. As part
of National Cooperative Drug Discovery Group (NCDDG) research project, numerous compounds
from tropical rainforest plant species with potential anticancer activity have been identified.
Artemisinin, an antimalarial agent from sweet wormtree Artemisia annua, used in Chinese
medicine since 200 BC is one drug used as a part of combination therapy for multiresistant
Plasmodium falciparum. Several compounds mainly from plants used as dietary supplements have
been extracted for their use as chemoprotective agents.
Structural Elucidation
The elucidation of the chemical structure of a natural product is critical to avoid the rediscovery
of a chemical agent that is already known for its structure and chemical activity. The UV-visible
infrared spectroscopy, high-performance liquid chromatography (HPLC) and elemental analysis are
useful techniques for establishing quality profiles of chemical moieties. Mass spectrometry (MS),
often used to determining structure is an important technique by which individual compounds
are identified based on their mass/charge ratio, after ionization. The chemical compounds usually
exist in nature as mixtures. Hence, the combinations of liquid databases of mass spectra for known
compounds are available. Nuclear magnetic resonance (NMR) spectroscopy is another important
technique for determining chemical structures of natural products. NMR yields information about
individual hydrogen and carbon atoms in the structure, allowing detailed reconstruction of the
molecule’s architecture.
QUESTIONS
1. Write a note on the design of drug discovery system for biotechnological production.
2. Write a note on current trends in modern pharmaceutical analysis for drug discovery.
3. Explain about natural product drug discovery.
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INTRODUCTION
Enzyme is a biocatalyst that accelerates biochemical reactions. The concept of biocatalysts is very
wide, and it includes pure enzyme, crude cell extract, viable plant and animal cells, microbial cells
and intact nonviable microbial cells. The sources of enzymes used in commerce are microorganisms,
higher plants and animals. Most important plant enzymes are papain, proteases, amylases and
soybean lipoxygenase, whereas commonly used animal enzymes are lipase, trypsin, rennet, etc.
The enzyme papain, extracted from papaya fruit, is a meat tenderizer, and pancreatic protease is
used in leather softening and manufacture of detergents.
The enzyme-containing detergents have been known since 1913, but their use was limited
because of their instability in detergent formulations. In 1965, a new stable enzyme (e.g. protease)
was introduced for application in detergent production. The first commercial process was developed
for production of fructose from glucose in the 1970s through the isomerization of glucose. Malt
is used as the source of enzymes in brewing industry. The production of primary and secondary
metabolites by microorganisms is possible only due to involvement of various enzymes. They are of
two types: (1) extracellular (secreted outside the cell) and (2) intracellular enzymes (secreted within
cell). There is a wide range of extracellular enzymes produced by pathogenic and saprophytic
microorganisms, such as cellulase, polygalacturonase, polymethyl galacturonase, pectin methyl
esterase, etc. These enzymes help in establishment in host tissues or decomposition of organic
substrates. The intracellular enzymes such as asparaginase, invertase and uric oxidase are of high
economic value and difficult to extract as they are produced inside the cell. They can be obtained
Immobilization of Enzymes
17
CHAPTER
Introduction
History of Enzymes
Enzyme Structure
Enzyme Kinetic
Bioproduction of Enzymes
Enzyme Production
Enzyme Immobilization
Immobilization of Plant Cells
Enzyme Engineering
Applications of Enzymes
Biosensors
Questions
Textbook of Pharmaceutical Biotechnology
240
by breaking the cells by means of a homogenizer and extracting them through the biochemical
processes. The process of enzyme purification is difficult, as the cell debris and nucleic acid are not
easily removed.
Microbial enzymes have the following two distinct advantages over the animal and plant
enzymes:
1. They can be produced on large scale within limited space, and their production is economical
with respect to time. The amount produced depends on type of microbial strain used, size of
fermenter and growth conditions. It can be easily extracted and purified.
2. The technical advantages associated in production of enzymes using microorganism are given
below.
a. They can grow in a wide range of environmental conditions.
b. They are capable of producing a wide variety of enzymes.
c. They show genetic flexibility, and they can be genetically manipulated to the yield of
enzymes.
d. They have short generation times.
At present, more than 2000 enzymes have been isolated and characterized, of which about 1000
enzymes are recommended for various applications. Amongst them, around 50 microbial enzymes
have industrial applications.
In recent years, industrial production of enzymes has considerably increased. The total global
production of enzymes was estimated around 65,000 tonnes with a value of about US$ 4 u 10
8
around a decade ago. Now the cost is expected to be doubled.
HISTORY OF ENZYMES
As early as the late 18th and early 19th centuries, the digestion of meat by stomach secretions and
the conversion of starch to sugar by plant extracts and saliva were known. However, the mechanism
by which this occurred had not been identified.
In the 19th century, while studying the fermentation of sugar to alcohol by yeast, Louis Pasteur
came to the conclusion that this fermentation was catalysed by a vital force contained within the
yeast cells called ferments that were thought to function only within living organisms.
In 1877, German physiologist Wilhelm Kühne first used the term enzyme, which comes from
Greek word enzymos. The word enzyme was used later to refer to nonliving substances such as
pepsin, and the word ferment was used to refer to chemical activity produced by living organisms.
In 1897, Eduard Buchner submitted his first paper on the ability of yeast extracts that lacked
any living yeast cells to ferment sugar. He named the enzyme that brought about the fermentation
of sucrose zymase. In 1907, he received the Nobel Prize in chemistry for his biochemical research
and his discovery of cell-free fermentation. Following Buchner’s example, enzymes are usually
named according to the reaction they carry out. Typically, to generate the name of an enzyme, the
Immobilization of Enzymes
241
suffix -ase is added to the name of its substrate (e.g. lactase is the enzyme that cleaves lactose) or
the type of reaction (e.g. DNA polymerase for DNA polymers).
Many early workers noted that enzymatic activity was associated with proteins, but several
scientists (such as Nobel laureate Richard Willstätter) argued that proteins were merely carriers
for the true enzymes and that proteins per se were incapable of catalysis. However, in 1926,
James B. Sumner showed that the enzyme urease was a pure protein and crystallized it. Sumner
did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes
was definitively proved by Northrop and Stanley (1930), who worked on the digestive enzymes
pepsin, trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in
chemistry.
This discovery that enzymes could be crystallized eventually allowed their structures to be
solved by X-ray crystallography. This was first done for lysozyme, an enzyme found in tears, saliva
and egg whites that digest the coating of some bacteria. The structure was solved by a group led by
David Chilton Phillips in 1965. This high-resolution structure of lysozyme marked the beginning
of the field of structural biology and the effort to understand how enzymes work at an atomic level
of detail.
ENZYME STRUCTURE
The enzymes are proteins, and their function is determined by their complex structure. The reaction
takes place in a small part of the enzyme called the active site, while the rest of the protein acts as
scaffolding. This is shown in this diagram of a molecule of the enzyme trypsin, with a short length
of protein being digested in its active site. The amino acids around the active site attach to the
substrate molecule and hold it in position while the reaction takes place. This makes the enzyme
specific for one reaction only, as other molecules would not fit into the active site (Fig. 17.1).
Figure 17.1 Structure of enzyme.
Holoenzyme
Apoenzyme
Active site
Cofactor
Protein
chain
Textbook of Pharmaceutical Biotechnology
242
Many enzymes need cofactors or coenzymes to work properly. These can be metal ions
(such as Fe
2+
, Mg
2+
, Cu
2+
) or organic molecules (such as haem, biotin, FAD, NAD or coenzyme
A). Many of these are derived from dietary vitamins. The complete active enzyme with its
cofactor is called a holoenzyme, while just the protein part without its cofactor is called the
apoenzyme.
ENZYME KINETIC
Kinetics is that branch of enzymology that deals with the factors that affect the rate of enzyme-
catalysed reactions. Enzyme kinetics is the study of the chemical reactions that are catalysed by
enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions
of the reaction investigated. Studying an enzyme’s kinetics in this way can reveal the catalytic
mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or
a poison might inhibit the enzyme.
The kinetic studies on enzymes that only bind one substrate, such as triose-phosphate isomerase,
aim to measure the affinity with which the enzyme binds this substrate and the turnover rate.
When enzymes bind multiple substrates, such as dihydrofolate reductase enzyme kinetics can also
show the sequence in which these substrates bind and the sequence in which products are released.
Examples of enzymes that bind a single substrate and release multiple products are proteases,
which cleave one protein substrate into two polypeptide products.
Importance of Enzyme Kinetics
1. It is essential for detailed study of an enzyme.
2. Influence of various factors on enzyme limits can be reflected in enzyme kinetics.
3. Catalytic function can be studied by measurement of the rate of the catalysed reaction.
4. It helps in understanding various biological phenomenon.
5. It helps define how an enzyme works in chemical terms and how it functions in the cell.
6. If factors are analysed properly, it is possible to totally understand the nature of enzyme-
catalysed reaction.
Factors Affecting Enzyme Reaction Velocity
The factors influencing enzyme reaction velocity are listed below.
1. Enzyme concentration
2. Substrate concentration
3. Temperature
4. pH
5. Inhibitors
6. Activators
Immobilization of Enzymes
243
Effect of enzyme concentration on rate of enzyme reaction (Fig. 17.2): The rate of enzyme
(E)-catalysed reaction is proportional to the enzyme concentration (provided S is saturating E).
v [E]; v = k [E]
As E increases rate of reaction increases in a linear manner. However, some deviations occur, which
are as follows:
Upward curve: In the beginning the rate is low, but as E concentration is increased rate increases.
This is due to presence of a dissociable activator or coenzyme in the enzyme preparation. Binding
of substrate A
E + A A EA
BE ABA makes E active (e.g. some proteases).
This is due to presence of highly toxic impurity in the reaction mixture (not in E solution). So, when
E is in small amount it is inhibited, but as its concentration is increased, it overcomes the toxic
impurity and rate increases.
Downward curve: This is more commonly observed. As E concentration is increased beyond a
certain point, the rate decreases. This may be due to the following:
1. A limitation in the capacity of the method of estimation. This is not a true decrease, but occurs
as the assay method cannot give higher reading (e.g. in spectrophotometer the maximum OD
is 2.0).
2. Substrate may be used up.
3. Presence of a reversible inhibitor in the enzyme preparation.
E + I
@
A
EI
Where E = enzyme and I = inhibitor.
As E concentration increases, I increases and inhibits E.
Figure 17.2 Effect of enzyme concentration on rate of enzyme reaction.
a[E]
[E]
[V]
Textbook of Pharmaceutical Biotechnology
244
Effect of substrate concentration on the rate of enzyme: It is the most important factor in
determining velocity of enzyme reaction.
1. At low substrate concentration [S], rate of reaction is low and a straight line is obtained.
As S concentration is increased a mixed-order reaction is obtained and the curve reaction is
obtained.
2. As S is increased further, the rate does not change and becomes constant. This is because
E active sites are all filled and E is saturated with S. At this point, the velocity is equal to
maximum velocity (V
max
).
3. The S concentration at half V
max
(V
max
/2) is called Michaelis constant (K
m
). This is a constant
for an E and a specific substrate. It gives the affinity between E and S.
4. High K
m
indicates low affinity while low K
m
indicates high affinity.
5. However, some E do not obey Michaelis–Menten kinetics and do not give a hyperbolic curve,
but give a sigmoidal curve. These are allosteric enzymes. These are regulatory enzymes and
have quaternary structure.
6. When [S] is plotted versus V, the saturation curve is sigmoidal. This indicates cooperative
binding of S to multiple sites. Binding of one site affects binding at other sigmoidal curve
cooperative effect.
Allosteric E has multiple binding sites.
1. Active sites binds S and converts to product [P].
2. Modulatory site binds S and other modulatory molecules and this binding affects the activity
of active site.
Modulators may be +ve modulators ABactivity, –ve modulators A?activity.
Effect of temperature on enzyme-catalysed reactions (Fig. 17.3): For example, human
E: ~25–37°C.
Figure 17.3 Effect of temperature on enzyme-catalysed reactions.
Zero-order reaction
K
m
V
max
Mixed-order reaction
First-order reaction