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Enzyme dysfunction, an essential catalyst for the smooth running of biochemical reactions and the maintenance of vital processes, is at the root of many pathologies that have paved the way for the development of numerous drugs.
Aimed at pharmacists, biologists, biochemists, doctors, veterinarians, medicinal chemists and students from a wide range of disciplines, Enzymes and Drugs brings together, for the first time, extensive documentation highlighting the relationships between a large number of drugs and enzymes. The book also highlights new prospects for therapeutic discoveries offered by enzyme targeting.
Numerous applications have been developed thanks to strategies for studying enzyme inhibition or activation, as well as the development of allosteric effectors, presented with their advantages and disadvantages. Various rare diseases, known as "orphan" diseases, have resulted from enzyme deficiency or absence. For their treatment, the introduction of substitute enzymes has led to major therapeutic advances.
Serge Kirkiacharian holds a doctorate in physical sciences. He is a pharmacist, professor emeritus of therapeutic chemistry, hospital practitioner and honorary head of department at Paris Hospitals. His work at the CNRS, France, and in academia focuses on the development of bioactive molecules and natural products. He has also won multiple awards for his work.
Julien Dumond holds a doctorate in virology and enzymology. As a team leader in pharmaceutical companies and a professor of biology and bioengineering, his research focuses on the development of enzymatic assays of pharmaceutical interest, as well as premarketing studies for drugs.
Preface ix
Introduction xi
Chapter 1 Enzyme Kinetics, Inhibition and Activation 1
1.1 Michaelis and Menten theory 2
1.2 Irreversible inhibitors 3
1.3 Reversible inhibitors in a Michaelian system 4
1.3.1 Reversible competitive inhibitors 4
1.3.2 Reversible non-competitive inhibitors 7
1.3.3 Reversible uncompetitive inhibitors 11
1.4 Allostery: inhibitors and activators 13
1.4.1 Allosteric enzyme kinetics 14
1.4.2 Mechanism of allosteric enzyme regulation 16
1.5 References 17
Chapter 2 Targeted Viral and Microbial Enzymes 19
2.1 Viral targets 20
2.1.1 Herpes group viruses: DNA-dependent DNA polymerases (EC 2) 20
2.1.2 Influenza virus: exo-alpha-sialidase or neuraminidase (EC 3) 21
2.1.3 HIV protease (EC 3) 22
2.1.4 HIV reverse transcriptase (RNA/DNA-dependent DNA polymerase -- EC 2) 26
2.1.5 HIV integrase (EC 2) 29
2.1.6 Hepatitis C virus (HCV): RNA-dependent RNA polymerase (NS5B -- EC 2) and viral protease (NS3-4A -- EC 3) 30
2.2 Bacterial targets 31
2.2.1 Specific target (action mainly focused on the Mycobacterium tuberculosis species) 31
2.2.2 General actions 33
2.3 Fungal targets 64
2.3.1 1,3-beta-glucan synthase (EC 2) 64
2.3.2 Squalene mono-oxygenase (EC 1) 65
2.3.3 14-sterol demethylase (EC 1) 65
2.3.4 Thymidylate synthase (EC 2) 66
2.4 Parasite targets 66
2.4.1 Ornithine decarboxylase (EC 4) 66
2.4.2 Heme polymerase (EC 2) 67
2.5 References 67
Chapter 3 Targeted Human Enzymes 71
3.1 Treatment via effectors 71
3.1.1 Ophthalmology 71
3.1.2 Neurology 73
3.1.3 Metabolism and endocrinology 79
3.1.4 Cardiovascular and immunology 94
3.1.5 Oncology 115
3.1.6 Phosphatome and Kinome 131
3.2 Enzyme replacement therapy 143
3.2.1 Gout 143
3.2.2 Acute leukemia and non-Hodgkin's lymphoma 144
3.2.3 Hypophosphatasia 144
3.2.4 Chronic obstructive pulmonary disease 144
3.3 References 144
Chapter 4 What are the New Targets? 151
4.1 Inhibition and activation strategies: advantages, disadvantages and current status 151
4.1.1 Irreversible inhibitors 152
4.1.2 Competitive inhibitors 153
4.1.3 Non-competitive inhibitors 155
4.1.4 Uncompetitive inhibitors 156
4.1.5 Allosteric effectors 156
4.1.6 Other strategies 157
4.2 Exogenous and endogenous targets 157
4.2.1 Exogenous targets 158
4.2.2 Endogenous targets 162
4.3 Rare diseases and enzymes 166
4.3.1 Lysosomal storage diseases 167
4.3.2 Other rare genetic diseases linked to one or more enzymes 176
4.4 References 176
Chapter 5 Which "New" Drugs are We Moving Towards Now? 181
5.1 Chemistry 181
5.1.1 Combinatorial chemistry 181
5.1.2 Vectorization 182
5.2 Biology 183
5.2.1 Inducers and repressors 184
5.2.2 Antibodies 184
5.3 Genetics 186
5.3.1 Gene therapy 186
5.3.2 Antisense strategies 193
5.4 References 194
Conclusion 197
Index 201
Biological reactions are essentially catalyzed by enzymes. Some enzymes function solely on the basis of their protein structure, in other words, the spatial arrangement of their amino acids. Others need to be coupled to an atom or molecule in order to act. These are enzymes linked to functional co-factors, which may be ionized atoms, small molecules or coenzymes.
In any chemical reaction where a reactant (R) must form a product (P), the R molecules must have enough stored energy to reach an activation state, also known as a transition state. The reaction rate constant k is related to the free energy of activation (Ea) under standard conditions by Arrhenius' semi-empirical law:
where A is the Arrhenius constant, R is the ideal gas constant (J.K-1.mol-1) and T is the temperature in kelvin (K).
The velocity of a chemical reaction in which R is transformed into P is proportional to the concentration of R molecules in the transition state. The activation energy, at a given temperature, indicates the energy required to drive one mole of R to the transition state.
An enzymatic catalyst can be used to lower the activation energy and optimize the chemical reaction. As enzymes are biological macromolecules, they are sensitive to pH and temperature.
To monitor an enzymatic reaction in which a substrate (S) is transformed into a product (P), units of enzymatic activity are used. The most common unit is the amount of enzyme required to transform one micromole of substrate per minute at 25°C. Specific activity corresponds to enzyme activity per milligram of protein (access to the enzyme's degree of purity). Finally, specific molar activity is the amount of substrate transformed in one minute by one mole of enzyme.
The most useful and common kinetics observed in enzymology are briefly presented here. Specific kinetics obtained for complex enzymatic systems, such as the proteasome (Stein et al. 1996), are not discussed.
Enzymatic kinetics is no exception to the rules of classical chemical kinetics. However, there is one specific feature to bear in mind: the limited concentration of the biological catalyst can be saturated by the reaction substrate.
By plotting vi (initial reaction velocities) as a function of the different substrate concentrations used (graph vi = f ([S]) (Figure 1.1)), it is possible to accurately determine the parameters of the Michaelis constant KM and the maximum velocity Vmax.
Figure 1.1. Graph vi = f ([S]) for at least 10 different substrate concentrations. A hyperbolic curve is obtained within the framework of Michaelian kinetics
A hyperbolic curve is obtained within the framework of Michaelian kinetics. At low substrate concentrations, the reaction rate is proportional to the substrate concentration. Under these conditions, the reaction is pseudo-first order. Then, as the substrate concentration continues to rise, the reaction rate increases again, but no longer proportionally. Finally, when the enzyme is completely saturated with substrate, a plateau is reached. The enzyme is operating at full capacity, and the system is at Vmax.
Vmax is the maximum vi obtained under the conditions of the experiment. The KM (Michaelis constant) represents the substrate concentration for which vi = Vmax / 2. The KM is used to approximate the affinity of the substrate for the enzyme.
The equation of the resulting equilateral hyperbola is:
An effector modifies the rate of a reaction by combining with the enzyme alone [E] and/or with the enzyme/substrate complex [ES]. Some effectors are inhibitors, slowing down the rate of reaction, while others are activators, speeding it up. This book is dedicated to effectors. These "classical" characterizations are extremely important for understanding the mechanism of action of molecules marketed for the treatment of disease. In 2002, 47% of therapeutic molecules targeted enzymes (Hopkins and Groom 2002). In 2011, the estimate was 29% (Rask-Andersen et al. 2011).
These derivatives bind covalently to the active site. Once bound, the irreversible inhibitor renders the enzyme permanently non-functional by preventing substrate transformation.
The reaction is as follows:
By plotting 1 / kobserved, with the kobserved deduced from assays carried out with different inhibitor concentrations, as a function of 1 / [I], Ki (inhibition constant) and kinactivation (kinetic constant of inactivation) can be deduced.
The standard equation of the line is:
Figure 1.2. Irreversible inhibition graph: 1 / kobserved = f (1 / [I]) for at least three different inhibitor concentrations. A straight line is obtained. Ki and kinactivation can be deduced
These inhibitors (I) combine with the free enzyme (E) and compete with the substrate (S) at the enzyme's active site.
These inhibitors bind reversibly to the catalyst, forming an EI complex similar to the ES complex. The reaction is as follows:
In the presence of a competitive inhibitor, the Michaelis and Menten equation becomes:
This is equivalent to having a constant KM increased by a factor (1 + [I] / KI) without any change in Vmax (Figure 1.3).
Figure 1.3. Graph vi = f ([S]) obtained for an inhibitor-free control and three effector concentrations. Competitive inhibition
Figure 1.4. Graphs by Lineweaver and Burk, Eadie Hofstee and Dixon. Competitive inhibition
The Lineweaver and Burk, Eadie Hofstee and Dixon representations for a competitive inhibitor are shown in Figure 1.4.
The equations of the lines are shown below.
The particular characteristics of the lines for these graphs (competition (converging lines), slopes of the lines in each case) give precise access to the kinetic parameters (Vmax, Ki and KM).
These inhibitors (I) can bind to the free enzyme (E) or to the enzyme that has bound the substrate (ES complex).
These inhibitors combine reversibly with the catalyst outside its active site. The reaction is as follows:
The equations of the curves are given below. The particular characteristics of the lines obtained for the relative graphs (parallelism, competition (converging lines), slopes of the lines according to each case) give precise access to the kinetic parameters (Vmax, Ki and KM).
Figure 1.5A. Graphs vi = f ([S]) and Lineweaver and Burk. Absolute non-competitive inhibition
Figure 1.5B. Eadie Hofstee and Dixon representations. Absolute non-competitive inhibition
Figure 1.5 shows the vi = f ([S]) graph and the Lineweaver and Burk, Eadie Hofstee and Dixon representations for an absolute non-competitive inhibitor (which binds with the same affinity to the free enzyme and to the enzyme-substrate complex with Ki = Ki').
These inhibitors (I) only bind to the enzyme that has bound the substrate (ES complex). The result is an ESI complex. The inhibitor does not compete with the substrate, and its action requires prior binding of the substrate to the enzyme. A conformational change reveals the inhibitor's binding site. The reaction is as follows:
Figure 1.6 shows the vi = f ([S]) graph and the Lineweaver and Burk, Eadie Hofstee and Dixon representations for an uncompetitive inhibitor. The equations of the curves are shown below. The particular characteristics of the lines obtained for the relative graphs (parallelism, competition (converging lines), slopes of the lines according to each case) give precise access to the kinetic parameters (Vmax, Ki and KM).
Figure 1.6A. Graphs vi = f ([S]) and Lineweaver and Burk. Uncompetitive inhibition
Figure 1.6B. Eadie Hofstee and Dixon representations. Uncompetitive...
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