This document discusses approaches to rational design of enzyme inhibitors. It begins by explaining how natural products have historically been used as medicines by targeting enzymes. The concept of the "magic bullet" introduced targeted chemotherapy. Modern strategies use assays, crystal structures, site-directed mutagenesis, and molecular docking. The document then classifies enzymes and their inhibition mechanisms, including reversible competitive, uncompetitive, and non-competitive inhibition as well as irreversible inhibition. It discusses using enzyme inhibitors in medicine and research by targeting specific enzymes.

1. APPROACHES TO THE RATIONAL DESIGN
OF ENZYME INHIBITORS
Submitted by :Manju Abraham
Department : Pharma chemistry.
1st year

2. CONTENTS
• Introduction
• Definition and description of enzyme
• International classification of enzymes
• Enzyme inhibition
• Classification of enzyme inhibitors
• Mechanism of enzyme inhibition
• Enzyme inhibitors in medicine
• Enzyme inhibitors in basic research

3. INTRODUCTION
 Historically , compounds that were extracted from natural
products have been used as medicinal agents .
 Subsequently , they have been shown to have their
therapeutic effect by targeting certain systemic enzymes .
 e.g. bark of the willow tree (anti-pyretic and analgesic
effects )
 Another e.g. is physostigmine , an inhibitor of
acetycholinesterase .
 In 20th century , the concept of the “MAGIC BULLET”having
selective toxicity , was introduced by Ehrlich as a rational
approach to chemotherapy .

4.  The discovery in 1935 of the antibacterial activity of the
azodye prontosil by Domagk , and subsequent explanation
in 1940 by Woods of its metabolic reduction to
sulfanilamide --------finally paved the way .
 These strategies use :
1. Sophisticated assays ,
2. enzyme crystal structures and active site environments ,
3. site-directed mutagenesis experiments of catalytic
residues of enzymes , and
4. molecular docking experiments employing computers .

5. ENZYMES
Enzymes are soluble ,colloidal ,organic catalysts ,formed by
living cells specific in action ,protein in nature ,inactive at
zero degree celsius and destroyed by moist heat at 100
degree celsius .
DESCRIPTION :
 Enzymes are the specialised proteins which catalyze various
biochemical reactions .
 The concept of enzyme inhibition is routinely utilized to
affect biosynthesis and metabolic pattern of various
hormones ,autocoids , and neurotransmitters .
 In 1926 , J.B.Sumner have isolated urease .

6.  Each enzyme is assigned with ,
1. a recommended name ,
2. a systemic name ,
3. a classification number .

7. INTERNATIONAL CLASSIFICATION OF ENZYMES
1) Oxido-reductases :
Enzymes which catalyze oxidoreductions between two
substrates e.g. A and B.
Areduced + Boxidised = Aoxidised + Breduced
classic e.g. Include succinic dehydrogenase , cytochrome
oxidase .
2) Transferases :
They catalyze the transfer of some group or radical ,R,
from one molecule A ,to another B. e.g. transfer of a
phosphate between ATP and a sugar molecule.

8. 3) Hydrolases :
These enzymes have the ability to induce hydrolysis of esters ,acid
anhydrides, glycosidic bonds, peptide bonds and other
bonds , e.g. esterases.
4) Lyases:
Enzymes which catalyze the formation or addition of double bonds .
e.g. hydratase .
5) Isomerases :
Enzymes catalyzing interconversion of optical, geometrical , or
positional isomers .
e.g .mutarotation of glucose by mutases .
6) Ligases :
Enzymes which catalyze joining of two substrates at the expense of
energy , also referred to as synthetases.

9. ENZYME INHIBITION
o Inhibition of a biochemical pathway will be most effective if
the drugs act on the rate-limiting step .
e.g. the conversion of tyrosine to dopa .
o The rate limiting step in a biochemical pathway is not always
possible to block .
o This results into an increase in the turnover in the sequence
of steps located before the enzyme blocked .
o The net outcome is an accumulation of the intermediate
product (substrate ).
o As a consequence ,the inhibitor will be displaced from the
enzyme surface by the substrate thus accumulated.

10. 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 .

11.  Various clinically used enzyme inhibitors may be broadly
categorised in to :
a) Reversible enzyme-inhibitors , and
b) Irreversible enzyme-inhibitors .
REVERSIBLE ENZYME- INHIBITORS
Inhibitors may further be categorised into ,
1. Competitive inhibitor
2. Uncompetitive inhibitor
3. Non-competitive inhibitor

12. COMPETITIVE INHIBITION :
• The inhibitor reacts reversibly with an enzyme to form an
enzyme-inhibitor complex .
• The inhibitor must possess structural similarity with the natural
substrate to act as competitive enzyme inhibitor .
UNCOMPETITIVE INHIBITION :
• The inhibitor combines with enzyme-substrate complex rather
than with the free enzyme to give inactive enzyme inhibitor
complex.

13. NON-COMPETITIVE INHIBITOR :
• A non-competitive inhibitor can combine with either the
free enzyme or the enzyme-substrate complex , interfering
with the action of both .
• Deforms the shape of enzyme .
• The altered shape and conformation of the enzyme slow
down both , the rates of formation and dissociation of
enzyme-substrate complexes .

14. • DRAWBACKS :
 Loss of potency .
The reasons behind this loss in potency include
a) Because of invivo metabolism ,
b) The rate-controlling step in the metabolic chain need not
to be always catalysed by the target enzyme .
a) A required concentration of the drug does not always
built up at target sites .

15. MECHANISMS OF ENZYME INHIBITION
• The different ways of enzyme inhibition are as follows :
1. The blockade of an enzyme , catalysing a particular
biochemical reaction leads to accumulation of the
substrate. This results into a desired biological response .

16. 2. The inhibition of an enzyme that catalyses the rate-
limiting step decreases the production of a metabolite .
(Multistep biochemical pathway)
3. Most of the enzymes need co-factors to catalyse the
biochemical pathway . Inhibitors can be developed
selectively for the co-factor involved .

17. 4. Two inhibitors may be employed simultaneously to achieve
a great therapeutic effects .
e.g. co-trimoxazole ( trimethoprim and sulphamethoxazole ).
5. If the metabolism of the end-product is minimised , the
accumulated end-product decreases the activity of an
enzyme on its substrate .

18. 6. Inhibition of metabolizing target enzyme permits higher
plasma levels as well as an increase in the plasma half-life
of the drug .
e.g. clavulanic acid inhibits beta-lactamase enzymes .
7. Multisubstrate analogs :
A multisubstrate compound consists the features of
binding sites of two or more different substrates in the
same molecule .
e.g. pyridoxyl alanine has more binding ability towards
pyruvate transaminase than pyridoxal and alanine .

19. Pyridoxal alanine

20. 9. Transition state analogs :
An enzyme interacts with a substrate to form enzyme
substrate complex through a tetrahedral transition state .
A transition state analog binds more tightly to an enzyme
than it does its substrate .
e.g. penicillin is a transition state analog which binds to the
peptidoglycan transpeptidase .

21. PENICILLINS
10. Suicide enzyme inhibitors ( Kcat inhibitors ) :
Irreversible enzyme inhibitor which utilizes highly
electrophilic species such as alpha-halogenated carbonyl
compounds or other strong alkylating agents which inturn
irreversibly reacts with a nucleophile group on the
enzyme.

22. Suicide enzyme inhibitors are the compounds that possess
latent reactive functional groups which are unmasked by
the catalytic action of the enzyme.
e.g. Inhibition of aldehyde dehydrogenase by
cyclopropanone , a metabolite of coprine .

23. 11. Active site directed irreversible inhibitors :
This category of inhibitors are designed by two important
aspects :
a) 1st approach :
A section of the reactive part of the enzyme may be
incorporated into the inhibitor to recognize and to direct
the inhibitor molecule to the enzyme’s active site .
b) 2nd approach :
A strong nucleophile is placed in the inhibitor molecule
which is expected to interact with an electrophile moiety
located near the active enzyme site .

24. 12. Quinone inhibition of enzymes :
Quinones can oxidise two SH-groups to form an S-S
linkage .

25. ENZYME INHIBITORS IN MEDICINE
• A selective inhibitor may block either a single enzyme or a
group of enzymes .
• This will result 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 a derivative possessing appropriate
, pharmacokinetic characteristics .

26. • 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 .
• Good bioavailability of the drug is also important .
• Two types of approaches to facilitate the transport of
enzyme inhibitors into the cell include :
1. physical approaches ,
2. chemical approaches .

27. {Examples of enzyme inhibitors used in the treatment of
bacterial,fungal ,viral and parasite diseases }
CLINICAL USE ENZYME INHIBITED INHIBITOR
Antibacterial Dihydrofolate reductase Trimethoprim ,
methotrexate
Antibacterial Alanine racemase D-cycloserine
Antifungal Fungal squalene - Terbinafine , naftifine
expoxidase
Antiviral DNA, RNA polymerases Cytosine arabinoside
Antiviral Viral DNA polymerase Acyclovir ,vidarabine
antiprotozoal Ornithine decarboxylase Alpha-difluoromethyl
ornithine

28. • Beta – lactamases are bacterial enzymes which is an
important target for drug design .
• Inhibitors of beta-lactamases include clavulanic acid and
sulbactam (penicillanic acid sulfone ) .
• Act to prevent the bacterial degradation of penicillins and
cephalosporins by beta-lactamases , thereby extending their
life-time and effectiveness .

29. EXAMPLES OF ENZYME INHIBITORS USED IN THE
TREATMENT OF CANCER
TYPE OF CANCER ENZYME INHIBITED INHIBITOR
Benign prostatic Steroid 5 alpha-reductase Finasteride
hyperplasia
Estrogen mediated breast Aromatase Aminoglutethimide
cancer
Colorectal cancer Thymidylate synthase 5-fluorouracil
Small-cell lung cancer , Topoisomerase ii Etoposide
non-Hodgkin’s lymphoma
Hairy-cell leukemia Adenosine-deaminase pentostatin

30. • SIDE EFFECTS :
1. Rapidly dividing normal cells, such as hair follicles , the
cells lining the GIT , and the bone marrow cells involved
in the immune system are also significantly affected .
2. The resultant nausea ,hair loss , and susceptibility to
infection means that this type of chemotherapy is seldom
employed as a first line defence against cancer .

31. EXAMPLES OF ENZYME INHIBITORS USED IN VARIOUS HUMAN
DISEASE STATES
CLINICAL USE ENZYME INHIBITED INHIBITOR
Epilepsy GABA transaminase Gama- vinyl GABA
Antidepressant MAO Tranylcypromine ,
phenelzine
Antihypertensive ACE Captopril, enalaprilat
Cardiac disorders -ATPase Cardiac glycosides
Gout Xanthine oxidase Allopurinol
Ulcer , - ATPase omeprazole

32. • A variety of diseases may be treated by the inhibition of
an individual enzyme or by using enzyme inhibitors to
regulate the metabolite concentration in the body .
• For e.g. an imbalance of the two neurotransmitters ,
glutamate and gama-aminobutyric acid , is responsible for
the convulsions observed in epileptic seizure .
• This led to the development of the GABA-T inhibitor ,
vigabatrin .

33. • The statins , a group of serum cholesterol lowering drugs
, are inhibitors of HMG-CoA reductase .
• Catalyses the irreversible conversion of HMG-CoA to
mevalonic acid (rate-determining step ) .
• Inhibitors such as simvastatin----------hyperlipidemia and
hypercholesteremia .

34. 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 .

35. 6. Immobilized enzyme inhibitors can also be used to
identify their intracellular targets whereas irreversible
inhibitors can be used to localize and quantify enzymes
invivo .

36. REFERENCES
1. Drug design , by Dr.V.M.Kulkarni and Dr.K.G.Bothara ,4th
edition , page no:25-31 .
2. Medicinal chemistry and drug discovery ,by Burger , 6th
edition ,1st volume , page no :716-720.

37. THANK YOU