01.Rational Design of Enzyme inhibition.pptx

Rational Design of Enzyme Inhibitors
Presenting by:
Mr. Purushotham K N
Assistant Professor
Dept of Pharmaceutical Chemistry
SAC College of Pharmacy, B.G.Nagara
2021-2022
Dept of Pharmaceutical Chemistry, Sri Adichunchanagiri College of Pharmacy, 1

Contents
• Introduction to Enzyme
• Enzyme Kinetics
• Principles of Enzyme inhibitors
• Enzyme inhibitors in Medicine
• Enzyme inhibitors in Basic Research
• Rational design of Non-Covalently Binding Enzyme
• Rational design of Covalently Binding Enzyme
• Reference
2
Dept of Pharmaceutical Chemistry, Sri Adichunchanagiri College of Pharmacy,

Introduction
What is ENZYME?
• Enzymes are protein in nature, soluble, colloidal, organic catalysts, formed by
living cells specific in action, inactive at 0°C and destroyed by moist heat at
100°C.
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Description
• Enzymes are the specialised proteins which catalyse various biochemical
reactions.
• The concept of enzyme inhibition is routinely utilized to affect biosynthesis
and metabolic pattern of various hormones, autocoids, and neurotransmitters.
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Dept of Pharmaceutical Chemistry, Sri Adichunchanagiri
College of Pharmacy,
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Classification Reaction Catalyzed Typical Reaction Ex.,
Oxidoreductases
To catalyse oxidation/reduction reactions;
transfer of H & O atoms or electrons from
one substance to another.
AH + B → A + BH (Reduced)
A + O → AO (Oxidized)
Dehydrogenase,
Oxidase
Transferases
Transfer of functional group from one
substance to another.
The group may be methyl-, acyl-, amino-,
or phosphate group.
AB+C → A+BC Transaminase,
Kinase
Hydrolases
Function of two products from a substrate
by hydrolysis.
AB + H2O → AOH + BH Lipase, Amylase,
Peptidase
Lyases
Non-Hydrolytic addition or removal of
groups from substrates. C-C, C-N, C-O or
C-S bonds may be cleaved.
RCOCOOH → RCOH +CO2
[X-A-B-Y] → [A=B + X-Y]
Decarboxylase
Isomerases
Intra-Molecular rearrangement, i.e.,
Isomerization changes within a single
molecule.
AB → BA Isomerase, Mutase
Ligases
Join together two molecules by the
synthesis of new C-O, C-S, C-N or C-C
bonds with a simultaneous breakdown of
ATP.
X + Y + ATP → XY +ADP + Pi Synthetase

How Enzyme Catalyse Reaction ?
• Enzymes provide a reaction surface and a suitable environment
• It bring reactants together and position them correctly so that they easily attain
their transition state configurations.
• It weaken bonds in the reactants.
• It may participate in the reaction mechanism
• It form stronger interactions with the transition state than with the substrate or
the product.
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Enzyme Kinetics
• Enzyme kinetics is the study of the chemical reactions that are catalyzed by
enzymes.
• In enzyme kinetics, the reaction rate is measured and the effects of varying the
conditions of the reaction is investigated.
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Michaelis – Menten Equation
• It explain how an enzyme can cause kinetic rate enhancement of a reaction and
why the rate of a reaction depends on the concentration of enzyme present.
k1 k2
A + E ⇌ B → C + E
k-1
𝒅 𝑪
𝒅𝒕
=
𝑲𝟐
[𝑨]
𝑲𝑴+[𝑨}
𝑬 𝟎
Where KM is Michaelis Constant
KM =
𝐾2+𝐾1
𝐾1
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• KM is the substrate concentration required to reach half- maximal velocity
(Vmax/2).
• KM is a measure of a substrate's affinity for the enzyme.
• Considering the total enzyme concentration the maximal rate, that the enzyme
can attain is Vmax.
• Vmax is equal to the product of the catalytic rate constant (Kcat) and the
concentration of the enzyme.
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Enzyme
Activity
• pH
• Temperature
• Enzyme Concentration
• Time
• Product Concentration
• Effect of Radiation

Inhibitors
• Inhibitors are chemicals that reduce the rate of enzymic reactions.
• The are usually specific and they work at low concentrations.
• They block the enzyme but they do not usually destroy it.
• Many drugs and poisons are inhibitors of enzymes in the nervous system.
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Enzyme Inhibitors
Enzyme inhibitors are molecules that reduce the catalytic activity of
enzymes. Reducing of effective enzymatic activity or complete blocking of
enzyme may cause either complete death of cell either modifications in the
pathways.
Application: Drugs with cause complete inactivation of enzymes from essential
pathways will cause cell death and therefore such drugs can be used as an
antibiotics.
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Classification of Enzyme Inhibitors
The inhibition of a suitably selected target enzyme leads to build up in
concentration of substrates and a corresponding decrease in the concentration of
the metabolites.
• Important parameters for selecting an enzyme inhibitor are:
1. Biochemical environment of the target enzyme,
2. Specificity of action,
3. The time period for which an enzyme is blocked.
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Enzyme
Inhibitors
Allosteric
Reversible
Competitive
Non-Competitive
Uncompetitive
Irreversible
Suicide
Inhibitors
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Reversible Inhibition
• Inhibitor binds to Enzyme reversibly through weak non- covalent interactions.
• An Equilibrium is established between the free inhibitor and EI Complex and
is defined by an equilibrium constant (Ki)
E + I ⇌ EI
• Reversible Inhibitors depending on concentration of E, S and I, show a
definite degree of inhibition which is reached fairly rapidly and remains
constant when initial velocity studies are carried out.
• The reversible inhibition is further sub-divided into:
I. Competitive Inhibition
II. Non-competitive Inhibition
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1. Competitive inhibition
• The inhibitor which closely resembles the real substrate (S) is regarded as a
substrate analogue.
• The inhibitor competes with substrate and binds at the active site of the enzyme
but does not undergo any catalysis.
• As long as the competitive inhibitor holds the active site, the enzyme is not
available for the substrate to bind. During the reaction, ES and El complexes
are formed.
• The relative concentration of the substrate and inhibitor and their respective
affinity with the enzyme determines the degree of competitive inhibition. The
inhibition could be overcome by a high substrate concentration.
• In competitive inhibition, the KM value increases whereas Vmax remains
unchanged.
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Example for Competitive Inhibition
• Competitive inhibition accounts for the antibacterial action of sulphanilamide
which is a structural analogue of PABA.
PABA Sulphanilamide
• Sulphanilamide inhibits the bacterial enzyme dihydropteroate synthetase which
catalyses the incorporation of PABA into 7,8 dihydro-pteroic acid.
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2. Non-competitive inhibition
• The inhibitor binds at a site other than the active site on the enzyme surface.
This binding impairs the enzyme function.
• The inhibitor has no structural resemblance with the substrate. However, there
usually exists a strong affinity for the inhibitor to bind at the second site. In
fact, the inhibitor does not interfere with the enzyme-substrate binding. But the
catalysis is prevented, possibly due to a distortion in the enzyme conformation.
• The inhibitor generally binds with the enzyme as well as the ES complex.
• For non-competitive inhibition, the KM value is unchanged while Vmax is
lowered.
• Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non-competitively inhibit the
enzymes by binding with cysteinyl sulfhydryl groups.
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25

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Rate of
Reaction
Substrate Concentration

Irreversible Inhibition
• Inhibitor binds at or near the active site of the enzyme irreversibly, usually by
covalent bonds, so it can't dissociate from the enzyme.
• No equilibrium exits
E + I ΕΙ
• Effectiveness of I is expressed not by equilibrium constant but by a velocity
constant, which determines the fraction of the enzyme inhibited in a given
period of time by a certain concentration of the I.
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Suicide inhibition:
• Suicide inhibition is a specialized form of irreversible inhibition. In this case,
the original inhibitor (the structural analogue/competitive inhibitor) is
converted to a more potent form by the same enzyme that ought to be inhibited.
• The so formed inhibitor binds irreversibly with the enzyme. This is in contrast
to the original inhibitor which binds reversibly.
• A good example of suicide inhibition is allopurinol an inhibitor of xanthine
oxidase, gets converted to alloxanthin, a more effective Inhibitor of this
enzyme.
• The use of certain purine and pyrimidine analogues in cancer therapy. is also
explained on the basis suicide inhibition. For instance, 5- fluorouracil gets
converted to fluoro-deoxy-uridylate which inhibits the enzyme thymidylate
synthase, and thus nucleotide synthesis.
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Allosteric Inhibition
• When an inhibitor binds to the enzyme, all the active sites of the protein
complex of the enzyme undergo conformational changes so that the activity of
the enzyme decreases.
• In other words, an allosteric inhibitor is a type of molecule which binds to the
enzyme specifically at an allosteric site.
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Enzyme Inhibition by drugs
• Enzymes are the natural targets for development of pharmacologic agents.
Many of the drugs used in the treatment of diseases act as enzyme inhibitors.
• Cholesterol lowering statin drugs (lovastatin) inhibit the enzyme HMG CoA
reductase.
• Drugs (tenofovir, emtricitabine) employed to block HIV replication inhibit the
enzyme viral reverse transcriptase.
• Hypertension is often treated by the drugs (captopril, enalapril) which inhibit
angiotensin converting enzyme (ACE).
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Enzyme Inhibitors in Medicine
• A selective inhibitor may block either a single enzyme or a group of enzymes.
This will results in either a decrease in the concentration of enzymatic products
or an increase in the concentration of enzymatic substrates.
• The effectiveness of an enzyme inhibitor as a therapeutic agent will depend on:
a. The potency of the inhibitor
b. Its specificity
c. The choice of a metabolic pathway
d. The inhibitor or derivative possessing appropriate pharmacokinetic characteristics.
• Low dosage and high specificity combine to reduce the toxicity problems.
• High specificity can avoid depletion of the inhibitor concentrations in the host
by non-specific pathways.
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• Some enzyme inhibitors used in treatment of bacterial, fungal, viral and parasite
diseases.
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Clinical Use Enzyme Inhibited Inhibitor
Anti-bacterial Dihydrofolate reductase Trimethoprim, Methotrexate
Anti-bacterial Alanine racemase D-cycloserine
Anti-fungal Fungal squalene-expoxidase Terbinafine, Naftifine
Anti-viral DNA, RNA polymerases Cytosine arabinoside
Anti-viral Viral DNA polymerase Acyclovir, Vidarabine
Anti-protozoal Ornithine decarboxylase
Alpha-difluoromethyl
Ornithine

• Some enzyme inhibitors used in various human disease states
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Clinical Use Enzyme Inhibited Inhibitor
Epilepsy GABA transaminase Gama-vinyl GABA
Anti-depressant MAO Tranylcypromine, Phenelzine
Anti-hypertensive ACE Captopril, Enalapril
Cardiac disorders ATPase Cardiac glycosides
Gout Xanthine Oxidase Allopurinol
Ulcer ATPase Omeprazole

• Some enzyme inhibitors used in treatment of cancer
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Type of Cancer Enzyme Inhibited Inhibitor
Being prostatic hyperplasia Steroid 5 alpha-reductase Finasteride
Estrogen mediated breast
cancer
Aromatase Aminoglutethimide
Colorectal Cancer Thymidylate Synthetase 5-fluorouracil
Small-call Lung cancer,
Non-Hodgkin’s lymphoma
Topoisomerase II Etoposide
Hairy-cell leukemia Adenosine-deaminase Pentostatin

Enzyme inhibitors in basic research
• Enzyme inhibitors have found a multitude of uses:
1. As useful tools for the elucidation of structure and function of enzymes.
2. As probes for chemical and kinetic processes and in the detection of short-
lived reaction intermediates.
3. Product inhibition patterns provide information about an enzymes kinetic
mechanism and the order of substrate binding.
4. Covalently binding enzyme inhibitors have been used to identify active-site
amino acid residues.
5. Reversible enzyme inhibitors are used to facilitate enzyme purification.
6. Immobilized enzyme inhibitors can also be used to identify their intracellular
targets whereas irreversible inhibitors can be used to localize and quantify
enzymes in-vivo
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Rational design of non-covalently binding enzyme
inhibitors
• This class of inhibitors binds to the enzyme's active site without forming a
covalent bond.
• Therefore the affinity and specificity of the inhibitor for the active site will
depend on a combination of the electrostatic and dispersive forces, and
hydrophobic and hydrogen-bonding interactions.
• To understand the design concepts of the various types of non-covalently
binding enzyme inhibitors, a basic knowledge of the binding forces between an
enzyme's active site and its inhibitors is required.
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Forces involved
in an
inhibitor/Substat
e Binding
Ionic
(electrostatic)
interactions
Hydrogen
bonding
Ion dipole
and dipole-
dipole
interaction
Hydrophobic
interaction
Van der
waals
interaction
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Rational design of covalently binding enzyme
inhibitors
• The targets for these inhibitors are the chemically reactive groups found within
the enzyme's active site These groups, in the majority of cases, are
nucleophiles.
• In some cases the -NH, and -COOH groups of the enzyme's N- and C-termini,
respectively, are also active site nucleophiles, whereas enzymic cofactors may
also provide targets for covalently binding inhibitors.
• Arginine is the only common amino acid that has an electrophilic side chain
and it also can be modified with suitable nucleophilic agents.
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Targets for covalently binding enzyme inhibitors
Nucleophiles such as –OH group of
Serine, Threonine, Tyrosine
-SH group of Cysteine
-COOH groups of aspartic acid
glutamic acid residues
Imidazole ring of Histidine
ɛ amino group of lysine
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Reference
• Textbook of Organic Chemistry of the Drug Design and Drug Action
by Richard B Silverman 2nd Edition.
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01.Rational Design of Enzyme inhibition.pptx

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