Enzyme Enzyme Inhibitor Distribution of marketed drugs by biochemical target class. Importance of Enzyme inhibition Types of Enzyme inhibition Some examples of Enzyme Inhibitors used as Therapeutic tool Case Study – 1 Lovastatin Induced Control of Blast Cell Growth in an Elderly Patient with Acute Myeloblastic Leukemia Case Study – 2 Small Molecule-Based Enzyme Inhibitors in the Treatment of Primary Hyperoxalurias Enzymes Enzymes are the substance that increases the rate of a reaction. Reactants binds to the enzyme and products are released. Enzymes can accelerate reactions as much as 10^16 overall of uncatalyzed reaction. Specificity of an enzyme towards its substrate is control by its structure. This unique fit of substrate with enzyme controls the selectivity of substrate for its product formation. ENZYME INHIBITORS Enzyme inhibitors are molecules that reduce the catalytic activity of enzymes. The chemical substance which can react in place of substrate with the enzyme but is not transferred into products and block the active site of the enzyme temporarily or permanently is called enzyme inhibitor. Reducing of effective enzymatic activity or complete blocking of enzyme may cause either complete death of cell or modifications in the pathways. Distribution of marketed drugs by biochemical target class. Importance of enzyme inhibition For understanding the regulation of enzyme activity within the living cells. Useful in elucidating the cellular metabolic pathways by causing accumulation of intermediates. Identification of catalytic/functional groups at the active site of enzyme. Provide information about substrate specificity of the enzyme. Useful to study the mechanism of catalytic activity. Enzyme inhibitors have therapeutic applications. Drugs are competitive or suicide in inhibitors. Types of enzyme inhibition Reversible inhibition Competitive inhibition Non – competitive inhibition

1. ENZYME INHIBITORS
AS THERAPEUTIC TOOLS
Prepared by: Tosim Mulani
M. Pharm Pharmaceutical Chemistry 1st sem
SPER-JH

2. CONTENT
1. Introduction
1. Enzyme
2. Enzyme Inhibitor
2. Distribution of marketed drugs by biochemical target class.
3. Importance of Enzyme inhibition
4. Types of Enzyme inhibition
5. Some examples of Enzyme Inhibitors used as Therapeutic tool
6. Case Study – 1 Lovastatin Induced Control of Blast Cell Growth in an
Elderly Patient with Acute Myeloblastic Leukemia
7. Case Study – 2 Small Molecule-Based Enzyme Inhibitors in the
Treatment of Primary Hyperoxalurias

3. Enzymes
• Enzymes are the substance that increases the rate of a reaction.
• Reactants binds to the enzyme and products are released.
• Enzymes can accelerate reactions as much as 10^16 overall of
uncatalyzed reaction.
• Specificity of an enzyme towards its substrate is control by its
structure.
• This unique fit of substrate with enzyme controls the selectivity of
substrate for its product formation.

4. ENZYME INHIBITORS
• Enzyme inhibitors are molecules that reduce the catalytic activity
of enzymes.
• The chemical substance which can react in place of substrate with
the enzyme but is not transferred into products and block the
active site of the enzyme temporarily or permanently is called
enzyme inhibitor.
• Reducing of effective enzymatic activity or complete blocking of
enzyme may cause either complete death of cell or modifications
in the pathways.

5. Distribution of marketed drugs by
biochemical target class.

6. Importance of enzyme inhibition
• For understanding the regulation of enzyme activity within the living
cells.
• Useful in elucidating the cellular metabolic pathways by causing
accumulation of intermediates.
• Identification of catalytic/functional groups at the active site of
enzyme.
• Provide information about substrate specificity of the enzyme.
• Useful to study the mechanism of catalytic activity.
• Enzyme inhibitors have therapeutic applications. Drugs are competitive
or suicide in inhibitors.

7. Types of enzyme inhibition
1. Reversible inhibition
• Competitive inhibition
• Non – competitive inhibition
2. Irreversible inhibition
• Suicide inhibition
3. Allosteric inhibition

8. Reversible inhibition
• Combine non covalently with the enzyme.
• Rapid dissociation of E-I complex.
• Can be readily removed by dialysis.
• Fully active Enzyme can be recovered after removal of inhibitor.
• Reversible inhibition are further classified as:
• Competitive inhibition
• Uncompetitive inhibition
• Non competitive inhibition

9. 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.
Reversible inhibition

10. NON COMPETITIVE INHIBITION
• 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 .
Reversible inhibition

11. IRREVERSIBLE INHIBITOR
• Bind to enzymes very tightly through
covalent or non-covalent bonds.
• Combine with the functional groups of
the amino acids in the active site,
irreversibly
• Irreversible inhibition occurs when an
inhibited enzyme does not regain
activity on dilution of the enzyme-
inhibitor complex.
• Slow dissociation of EI complex

12. • Since these covalent changes are relatively stable, an enzyme that has
been poisoned” by an irreversible inhibitor remains inhibited even after
removal of the remaining inhibitor from the surrounding medium.
• Irreversible inactivation by covalent bonding of inhibitor and enzyme.
• Inhibitors are usually toxic substances like Organo Phosphate poisons.
• Eg: Iodoacetate; inhibitor of papain and glyceraldehyde 3 phosphate
dehydrogenase.
• Diisopropyl flurorphoshate (DFP) binds with enzymes containing serine at
the active site like serine proteases, acetylcholine esterase.
IRREVERSIBLE INHIBITOR

13. SUICIDE INHIBITION
• The original 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 and thus causes suicide inhibition.
• Eg. ALLOPURINOL, Inhibitor of xanthine oxidase gets converted to alloxanthine
which is more effective inhibitor of xanthine oxidase.
• 5-FLUOROURACIL gets converted to fluorodeoxyuridylate which inhibited
enzyme thymidylate synthase, and this nucleotide synthesis.
IRREVERSIBLE INHIBITOR

14. ALLOSTERIC INHIBITION
• Mixed kind of inhibition when the inhibitor
binds to the enzyme at a site other than the
active site but on a different region in the
enzyme molecule called allosteric site.
• Allosteric inhibition does not follow the
Michaelis-Menten hyperbolic kinetics.
Instead it gives a sigmoid kinetics Allosteric
inhibitors shift the substrate saturation
curve to the right. However as opposite to
inhibitors, the presence of activators shifts
the curve to the left.

15. Enzyme inhibitors used in the treatment of
bacterial, fungal, viral and parasite diseases
INHIBITOR ENZYME INHIBITED CLINICAL USE
Trimethoprim,
Methotrexate
Dihydrofolate reductase Antibacterial
D-Cycloserine Alanine racemase Antibacterial
Terbinafine,
Naftifine
Fungal squalene-epoxidase Antifungal
Cytosine arabinoside DNA, RNA polymerase Antiviral
Acyclovir,
Vidarabine
Viral DNA polymerase Antiviral
Alpha-difluoromethyl
ornithine
Ornithine decarboxylase Antiprotozoal

16. Examples of Enzyme Inhibitors used in the
Treatment of Cancer
TYPES OF CANCER ENZYME INHIBITED INHIBITOR
Benign prostatic hyperplasia Steroid 5 alpha reductase Finasteride
Estrogen mediated breast
cancer
Aromatase Aminoglutethimide
Colorectal cancer Thymidylate synthase 5 - Fluorouracil
Small- cell lung cancer Topoisomerase II Etoposide
Hairy – cell leukaemia Adenosine- deaminase Pentostatin

17. Example of Enzyme Inhibitors used in various
Human Disease States
CLINICAL USE ENZYMES INHIBITED INHIBITOR
Epilepsy GABA transaminase Gama vinyl GABA
Antidepressants MAO Tranylcypromine
Antihypertensive ACE Captopril, enaprilate
Cardiac disorders ATPase Cardiac glycoside
Gout Xanthine oxidase Allopurinol
Ulcer ATPase Omeprazole

18. Lovastatin Induced Control of Blast Cell Growth
in an
Elderly Patient with Acute Myeloblastic Leukemia
CASE STUDY - 1

19. Introduction
ACUTE MYELOID LEUKAEMIA (AML)
• It is type of cancer of the blood and
bone marrow with excess immature
white blood cells.
• AML progresses rapidly, with myeloid
cells interfering with the production of
normal white blood cells, red blood cells
and platelets.

20. Introduction
LOVASTATIN
• It is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase (HMG-CoA reductase), an enzyme that catalyzes
the conversion of HMG-CoA to mevalonate.
• Mevalonate is a required building block for cholesterol
biosynthesis and lovastatin interferes with its production by
acting as a reversible competitive inhibitor for HMG-CoA,
which binds to the HMG-CoA reductase.
• This inhibition by a statin may result in decreased levels of
mevalonate and its downstream products, which affects
critical cell functions such as membrane integrity, cell
signaling, protein synthesis and cell cycle progression. The
effect of statins on these processes and consequently on
tumor cells, may therefore be able to control tumor
initiation, growth and metastasis.
Lovastatin

21. Introduction to Case study – 1
• Present therapeutic regimens for the
treatment of patients with AML are toxic and
often ineffective.
• The targeting of HMG-CoA reductase, the rate
limiting enzyme of de novo cholesterol
synthesis, represents a potential novel
therapeutic approach in the treatment and
control of AML.
• Lovastatin inhibition of resume function
induced a significant apoptotic response in
majority of AML samples tested.

22. Case study – 1
• In phase I trial which do not include AML
patients, employed Lovastatin at
concentration 20-50 times the
hypercholesterolemia dose and achieved
serum levels in the 4microM range, with
no significant adverse side effects.
• At these concentration, AML cells are
clearly responsive to the
differentiative and apoptotic effects
of Lovastatin.

23. Case study – 1
• Consolidation therapy consisted of daunorubicin 45 mg/m2 on day
1 and ara-C 100 mg/m2/d by continuous infusion for 5 days. The
complete remission lasted for 9 months.
• At the time of relapse the hemoglobin was 80, the white blood
count 16.8x109/L and 15x109/L blast cells.
• As the patient’s cells proved to be sensitive in culture to
lovastatin, the patient was offered this drug.

25. Case study – 1
• The patient was started on lovastatin at a dose of 40 mg by mouth
twice a day.
• Three days later the total white blood count had risen to 34x109/L
with a blast count of 31x109/L.
• At that time the dose of lovastatin was increased to 40mg by
mouth four times a day (day 12 of relapse).
• At this dose, the white blood count was not appreciably higher
four days later, and over the ensuing weeks the white blood cell
count fell to the range of 10x109/L with 8x109/L blast cells.

26. Case study – 1
• The patient remained on lovastatin for a period of 54 days.
• During that time the patient did not complain of any nausea or
muscle pain, two of the major complaints of higher dose
lovastatin.
• The drug was discontinued because of the development of
ulcerative skin lesions.
• Following lovastatin withdrawal the white blood cell and blast
counts remained low for approximately three months.
• The only other drug the patient was on, during this time was
trimethopridsulfa as antibacterial prophylaxis.

27. Conclusion of Case study – 1
• In-vitro sensitivity assays may predict patient’s AML cells responsiveness
to Lovastatin.
• In this case study, we found that patient’s leukemic Blast cells was
reduced by Lovastatin at double the usual recommended dose for
hypercholesterolemia.
• Side effects were minimal and effect of the drug on blast count
persisted once the drug was discontinued.
• This study identified HMG-CoA reductase as a potential therapeutic
target of AML and illustrates the potential for Lovastatin to provide a
novel means of the controlling leukemic cell growth in patient.

28. Small Molecule-Based Enzyme Inhibitors in
the Treatment of Primary Hyperoxalurias
CASE STUDY - 2

29. Introduction
• Primary hyperoxalurias (PHs) are a group of inherited alterations
of the hepatic glyoxylate metabolism.
• PHs classification based on gene mutations parallel a variety of
enzymatic defects, and all involve the harmful accumulation of
calcium oxalate crystals that produce systemic damage.
• Until recently, treatments were limited to palliative measures and
kidney/liver transplants in the most severe forms.
• Lumasiran, a siRNA product against glycolate oxidase, which has
become the first effective therapy to treat PH1.

30. Introduction
• Primary hyperoxaluria (PH) is a rare disease of liver metabolism
that results in excess oxalate production and urine excretion
(hyperoxaluria).
• This severe disease is caused by genetic changes that alter
glyoxylate and hydroxyproline metabolism resulting in
overproduction of oxalate by the liver.
• Other situations in which elevated oxalate in the urine is due to
excessive intake or absorption of oxalate or its precursor are
known as secondary hyperoxalurias.

33. Case Study - 2
• PH1 patients typically yield oxalate
excretion > 1 mmol/1.73 m2 per
day (normal range < 0.5 mmol).
• The increased urinary excretion of
oxalate results in urinary CaOx
supersaturation, which leads to
crystal aggregation, urolithiasis,
and/or nephrocalcinosis.
• Most frequently in PH1, oxalate
nephropathy results in end stage
renal disease (ESRD)
Types of Primary
Hyperoxaluria
Mutated Gene
PH 1
AGXT gene (coding for
alanine: glyoxylate amino-
transferase, AGT)
PH 2
GRHPR gene (coding for
glyoxylate/hydroxypyruva
te reductase, GRHPR)
PH 3
HOGA1 gene (coding 4-
hydroxy-2-oxoglutarate
aldolase 1, HOGA1).

34. • Glycolate oxidase (GO; EC 1.1.3.15) is a FMN-dependent flavoenzyme
that belongs to the α-hydroxy acid oxidase family.
• GO is present both in mammals and plants and is localized in the
peroxisome.
• Human glycolate oxidase (hGO) is mainly expressed in liver and
catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and
from glyoxylate to oxalate, contributing to stone formation in PHs.
• In order to determine the effect of the oral administration of Goi’s to
animals, the GO inhibitory activity of pyrrole derivatives was explored.
Case Study - 2

35. • Established GO,I’s share structural features including a polar head and a side chain.
• The polar head, which bears an acidic functionality, enters the active site and mimics the
substrate interactions with the cited key amino acids, whilst the side chain, whether
aromatic or aliphatic, hangs from the polar head and remains in the access channel of the
enzyme establishing hydrophobic interactions and causing a disorder that prevents the
adoption of the closed state.
• Besides, the stabilization of the ternary complex enzyme-cofactor-substrate is enhanced by
the presence of flat, electron rich fragments in the polar head that establish π-π interactions
with FMN flavin ring.
• In the polar head, the presence of a protonated heteroatom located in β with respect to the
acidic function has shown to be beneficial. Different polar heads have already been explored
so far, including α-hydroxy acids, α-keto acids, oxamates and salicylic acids.
Case Study - 2

37. • Thus, a library of 4-substituted 3-hydroxy-1H-pyrrole-2,5-dione
derivatives was prepared and tested for pig liver GO inhibition,
showing a competitive inhibition pattern for this enzyme.
• Compound 4-(40-bromo[1,10-biphenyl]-4-yl)-3-hydroxy-1H-
pyrrole-2,5-dione was identified as the most potent hit within
this set (IC50 = 87 nM for pig GO and IC50 =110 nM for hGO).
• Furthermore, its administration to rats fed with ethyleneglycol
resulted in a significant reduction in urinary oxalate.
Case Study - 2

38. • Molecular modeling and docking studies on
sGO and hGO were performed with the
flavonoids quercetin and kaempherol.
• In in-vitro experiment, flavonoids: quercetin
behaved as a noncompetitive inhibitor of
sGO (Ki = 0.56 µM and IC50 = 0.22 µM), and
conversely, kaempherol was a competitive
inhibitor of the enzyme (Ki = 0.37 µM and
IC50 = 0.3 µM).
• Thus, these compounds were established as
promising leads for future drug optimization.
Case Study - 2
Quercetin
Kaempherol

39. • COLOSTIMETHATE sodium have been
identified as novel and more potent
Goi’s, with IC50 value of 2.3 μM and
showing a mixed linear inhibition
pattern of hGO.
• A cell-based assay with CHO-GO cells
useful for high-throughput screening of
compound libraries was also developed
and optimized to test the activity of
colistimethate sodium in cell culture,
providing an IC50 value of 8.3 μM.
Case Study - 2

40. • The SALICYLIC ACID polar head that bears free
carboxy and hydroxy functionalities interacts at the
binding region 1 and needs to be substituted on C5.
• On the other hand, electron withdrawing
substituents are preferred in the aryl or heteroaryl
hydrophobic tail that interacts with the second
binding region, and a flexible linker can be
introduced to space the polar head and the
hydrophobic tail, therefore allowing a better
accommodation of the molecule.
Case Study - 2

41. • This constitutes an unprecedented activity for salicylates, and
their easy one-step synthesis along with their drug-like structure
makes them promising candidates for drug development.
• Inherent to their salicylic structure, these compounds could exert
a possible favorable anti-inflammatory activity or, on the other
hand, a possible renal toxicity due to cyclooxygenase inhibition.
These are issues under evaluation for these compounds
Case Study - 2

42. CONCLUSION
• The development of small molecule inhibitors designed against key enzymes of
glyoxylate metabolism is on the focus of Case Study.
• In Primary Hyperoxalurias, effective enzymatic targets have been determined
and characterized for drug design and interesting inhibitory activities have been
achieved both in vitro and in vivo.
• Quercetin and Kaempherol were established as promising leads for future drug
optimization for Primary Hyperoxalurias.
• Colistimethate sodium gave the promising result in cell culture.
• Salicylic acid derivatives found to have good accommodation and also help to
control inflammatory responses.

43. References
1. Salido. Sofia; G. Monica; Small Molecule based enzyme inhibitors
in the treatment of primary Hyperoxalurias; JPM; 11,
https://doi.org/10.3390/jpm11020074
2. MARK D. MINDEN, JIM DIMITROULAKOS, DANA NOHYNEK and
LINDA Z. PENN, Lovastatin Induced Control of Blast Cell Growth
in an Elderly Patient with Acute Myeloblastic Leukemia;
Leukaemia and lymphoma, 2001, 40, 659 to 652.
3. U. Satyanarayan, Chakrapani; Textbook of Biochemistry, 2013,
Pg. 76 to 98.

44. THANK YOU