RAPID REVERSIBLE ENZYME INHIBITORS ACE, GLYCINAMIDE RIBONUCLEOTIDE TRANSFORMYLASE.pptx

RAPID REVERSIBLE ENZYME INHIBITORS ACE, GLYCINAMIDE RIBONUCLEOTIDE
TRANSFORMYLASE
SUBJECT-ADVANCES IN MEDICINAL CHEMISTRY
(PHSC CC-2202 )
SUBMITTED BY :
MANISH KUMAR
MPHARM 1ST
YEAR
(Y24254052)
SUBMITTED TO :
Prof. ASHMITA GAJBHIYE
Dr. MUKESH K KUMAWAT
DEPARTMENT OF PHARMACEUTICAL SCIENCES
Dr. HARISINGH GOUR VISHWAVIDYALAYA
SAGAR (M.P.)- 470003,INDIA
(A CENTRAL UNIVERSITY)

CONTENTS
• Introduction
• Classification
• Classification Of Non-Covalent Inhibitor.
• Inhibition Of Acetylcholinesterase
• Glycinamide ribonucleotide transformylase

GENERAL CONCEPT OF ENZYME INHIBITION
 The body is composed of thousands of different enzymes, many of them acting in concert to maintain
homeostasis.
 Although disease states may arise from the malfunctioning of a particular enzyme, or the introduction of
a foreign enzyme through infection by microorganisms, inhibiting a specific enzyme to alleviate a
disease state is a challenging process.
 Most bodily functions occur through a cascade of enzymatic systems, and it becomes extremely difficult
to design a drug molecule that can selectively inhibit an enzyme and result in a therapeutic benefit.
 However, to address this problem, the basic mechanism of enzyme action needs to be understood.
 Once knowledge of a particular enzymatic pathway is determined and the mechanism and kinetics are
worked out, the challenge is then to design a suitable inhibitor that is selectively used by the enzyme
causing its inhibition. As outlined earlier, enzymes (E) represent the best known biochemical catalysts,
because they are uniquely designed to carry out specific biochemical reactions in a highly efficient
manner.

CLASSIFICATION
 Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a
decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature.
There are three broad categories of enzyme inhibition
 1 . Reversible inhibition.
 2. Irreversible inhibition.
 3. Allosteric inhibition

l. Reversible inhibition
 The inhibitor binds non-covalently with enzyme and the enzyme inhibition can be reversed if the
inhibitor is removed
 The reversible inhibition is further sub-divided into:
l. Competitive inhibition.
ll. Non-competitive inhibition.

l. Competitive inhibition : The inhibitor (I) 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
E
ES
EI
E + P
+S
+I
Enzyme and Substrate Complex

Example of enzyme with their respective substrate and competitive inhibitors
ENZYME SUBSTRATE INHIBITOR(S) SIGNIFICANCE OF INHIBITORS
Xanthine Hypoxanthine Allopurinol Used in the control of gout to reduce
oxidase production of uric acid
Monoamine Catecholamine Ephedrine Useful for elevating catecholamine
Oxidase (epinephrine) amphetamine level
Dihydrofolate Dihydrofolic acid methotrexate Employed in the treatment of leukemia.
reductase
Dihydropteroate PABA sulfonamide Prevent bacterial synthesis of folic acid
synthase
HMG CoA HMG CoA Lovastatin, Inhibit cholesterol biosynthesis
reductase compactin

ll. 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.
 Acetazolamide Carbonic anhydrase
 Disulfiram Aldehyde dehydrogenase
 Omeprazole Proton Pump ATPase
 Digoxin Sodium Potassium ATPase
 Theophylline Phosphodiesterase
ENZYME ENZYME INHIBITED

Irreversible inhibition
 The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible.
 These inhibitors are usually toxic poisonous substances.
 lodoacetate combines with sulfhydryl (-SH) groups at the active site of these enzymes and makes
them inactive.
 DFP irreversibly binds with enzymes containing serine at the active site, e.g. serine proteases,
acetylcholine esterase.
 The penicillin antibiotics act as irreversible inhibitors of serine - containing enzymes, and block
the bacterial cell wall synthesis. lrreversible inhibitors are frequently used to identify amino acid
residues at the active site of the enzymes, and also to understand the mechanism of enzyme
action.

Non-covalent Enzyme Inhibition
• Non-covalent interactions play an essential role in the structure-based design of new substituent
with specific properties.
• They have been recognized in different fields related to chemical pharmacological, biological,
physical, and materials sciences.
• Non-covalent interactions like hydrogen bonding, hydrophobic interactions, van Waals
interactions, electrostatic interaction and salt bridges, has been the main focus in designing and
improving drugs.
• Understanding these interactions and their physical basis is of significant interest in improving
the currently available drug design strategies.
• n spite of their importance in many scientific domains, controversy on properties, nature, and
importance of hydrogen bonds are increasing.
• van der Waals interactions are responsible for the consistency between molecules of solids and
nonpolar liquids.

• The design of enzyme inhibitors is one of the most captivating research topics in medicinal chemistry.
• Covalent inhibitors provide the opportunity of combining concepts of chemical reactivity and mechanisms
of organic reactions with the structural features required for optimal molecular recognition in order to obtain
the appropriate reactivity and selectivity profiles towards the desired enzyme target.
• A look at drug approvals in recent years suggests that covalent drugs will continue to make an impact on
human health for years to come.
• The toxicity, high potencies and prolonged effects of covalent drugs result in less-frequent drug dosing.
• There are several examples of covalent inhibitors that are widely used drugs, including acetylsalicylic acid
(an active ingredient of aspirin), orlistat (anti-obesity drug) and ampicillin (antibiotic). Overall, nearly 30%
of the enzymes are irreversibly inhibited via covalent modification and highlights the therapeutic potential of
covalent inhibitors.

 The van der Waals interactions, on the other hand, result from a temporary
random fluctuation in the circulation of the electrons of atom, which give an
upsurge to a transient differing circulation of electrons, a transient electric
dipole.
Classification Of Non-Covalent Inhibitor.
• Rapid reversible inhibitors.
• Tight, slow, slow-tight binding inhibitors.
• Multi substrate analogs.
• Transition-state analogs.

INHIBITION OF ACETYLCHOLINESTERASE
• Acetylcholinesterase (AChE) is the enzyme that catalyzes the catabolism of the neurotransmitter
acetylcholine to acetate and choline.
• Thus, inhibition of AChE would lead to increased concentrations of acetylcholine in both of the cholinergic
muscarinic and nicotinic synapses resulting in a prolonged cholinergic action.
• Reversible AChEIs are those compounds that are substrates for and react with AChE to form an acylated
enzyme, which is more stable than the acetylated enzyme but still capable of undergoing hydrolytic
regeneration or those that bind to AChE with greater affinity than acetylcholine but do not react with the
enzyme as a substrate.

• They are also used in open-angle glaucoma to decrease intra ocular pressure by stimulating contraction of the
ciliary muscle and sphincter of the iris. This facilitates outfl ow of aqueous humor via the canal of Schlemm.
• The Anti-Ches are mostly ester carbamic acid or derivatives of phosphoric acid.
• Some anti-ChEs like edrophonium, tacrine, donepezil and galantamine.
Anticholinesterases
R1 O C
O
R2
R3
HN
PH R1
R2
R3
O
CARBAMATES ORGANOPHOSPHATES
Reversible Irreversible
Carbamates Non-carbamates
• Physostigmine
• Neostigmine
• Pyridostigmine
• Rivastigmine
• Edrophonium
• Tacrine
• Donepezil
• Galantamine
Carbamate Organophosphate
• Carbaryl
• Propoxur
• Dyflos(DFP)
• Echothiophate
• Melathion

• AChE has an anionic active site that can bind the positively charged quaternary ammonium group of the
choline functionality and an active esteric site that contains a nucleophilic Ser-CH2-OH group that is
involved in the hydrolysis of the ester bond .
• The hydrolysis mechanism involves attack of the nucleophilic Ser-CH2-OH group on the ester carbonyl
group of acetylcholine to form a tetrahedral intermediate that collapses, releasing choline and forming an
acetylated Ser-CH2-O-AChE complex, which subsequently hydrolyzes with water to release AChE, acetic
acid (as acetate ion), and choline.
• Physostigmine has been used in the treatment of glaucoma.
• It is an alkaloid with a carbamate moiety that resembles the ester linkage of acetylcholine.
• Being an alkaloid, it is protonated at physiologic pH and, thus, can bind to the anionic site of AChE.

• Similar to the mechanism for the hydrolysis of acetylcholine by AChE, the Ser-CH2-OH group of
AChE can attack the carbamate carbonyl group of physostigmine, and in the process, the Ser-CH2
OH group is carbamylated.
H3C O CH2 CH2 N(CH3)3
O
Ethylene
group
Acyloxy
group Quaternary ammonium
group
HO
COOH
NH2
HO
NH2
HO
NH(CH3)3
O
N(CH3)3
O
H3C
Serine decarboxylase
Choline N-methyl
transferase
Acetyl-S-coA
Choline acetultransferase

Structure Activity Relationship
N CH2 C
H
O C CH3
O
Methacholine
N CH2
H2
C O C NH2
O
Carbachol
N CH2 C
H
O C NH2
O
Bethanechol

REVERSIBLE ACETYLCHOLINESTERASE INHIBITORS
A. Reversible anticholinesterase. All these drugs are structurally resemble to cholinesterase enzyme and have greater
affinity for the active sites which results into a temporary inhibition of the enzyme.
Hence, they are termed as reversible anticholinesterases.
1. Physostigmine.
2.Neostigmine
3.Pyridostigmine
4.Edrophonium
O
N
H
O
N
N
N+
O
N
O
N
O
O
N+
N+
HO

Irreversible cholinesterase inhibitors
1. Pralidoxime: The molecule pralidoxime is a useful antidote for intoxication with cholinesterase
inhibitors such as the organophosphates. The molecule removes the inhibitor from the active
site in the form of an oxime phosphonate.
2. Echothiopate: Echothiopate is a long acting irreversible anti-AChE drug that is used in the treatment of
glaucoma.
3. Parathion: Parathion is used as an agricultural insecticide. It is especially used for controlling
aphids, spider mites, and scale insects.
4.Malathion: Malathion is another effective pesticide, which is more effective on insects
than on humans because it requires biotransformation to the phosphate form, which can only
be carried out by insects.
N C=C OH
CH3
NH (CH2)2 S P
OC2H5
OC2H5
H3C
H3C
H3C
S
P
O
O
O
N+
-
O
O

Glycinamide ribonucleotide transformylase
• Glycinamide ribonucleotide transformylase is one of the most important trifunctional enzymes involved in
purine synthesis.
• GARFT is an essential step in the synthesis of purine nucleotides, and a target for blocking the proliferation
of malignant cells.
• is a folate-dependent enzyme central to the de novo purine biosynthetic pathway.
• GARTfase utilizes the cofactor (6R)-N10
-formyltetrahydrofolic acid (10-formyl-THF) to transfer a formyl
group to the primary amine of its substrate, β-glycinamide ribonucleotide (β-GAR).
• his one carbon transfer provides the C-8 carbon of the purines and is the first of two formyl transfer reactions
enlisted in the biosynthesis of purines,

• Purines are synthesized via two principal routes: the de novo and salvage pathways.
• The de novo purine synthesis pathway is a metabolically costly process (6 ATP molecules per molecule of
purine synthesized) that involves 10 catalytic steps to assemble the purine ring from carbon and nitrogen
moieties donated by amino acids (e.g., glutamine, aspartate, glycine) and one-carbon units.
• This pathway is highly regulated through multiple mechanisms, including transcriptional, post-transcriptional,
feedback inhibition, or organization into multi-enzyme assemblies .
• The de novo purine synthesis is also controlled by pro-growth signaling pathways, including mTORC1
signaling,
• MAPK/ERK signaling, and MYC transcription factor,15
which stimulate this pathway to support cell growth.
• Inhibitors of folate dependent enzymes including GARTfase have provided important compoundIns for cancer
chemotherapy as a result of their inhibition of the biosynthesis of nucleic acid precursors.

• GARTfase as a useful anticancer target emerged with the discovery of the first potent and selective
inhibitor, 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF).
Drugs That inhibit Glycinamide ribonucleotide :
• Pemetrexed
• Methotrexate
• Lometrexol

Pemetrexed
• Pemetrexed, a chemotherapy drug, works by inhibiting multiple enzymes involved in folate
metabolism and DNA synthesis, specifically thymidylate synthase (TS), dihydrofolate reductase
(DHFR), and glycinamide ribonucleotide formyltransferase (GARFT), thereby disrupting cell
replication and growth.
• GARFT is involved in the synthesis of purines, another building block of DNA and
RNA. Pemetrexed inhibits GARFT, leading to a decrease in purine synthesis and, consequently,
DNA and RNA synthesis.
• The drug appear to be effective against a range of tumor including , mesothelioma, NSCLC
(Non-Small Cell Lung Cancer), colorectal cancer, bladder cancer.
N
H
O
OH
O
OH
O
HN
H
N
N
O
H2N

Methotrexate
• These antifolates were found to inhibit dihydrofolate reductase (DHFR), which generates
tetrahydrofolate (THF) from dihydrofolate and therefore maintains a cellular supply of this important
coenzyme.
• Methotrexate, a nonselective antifolate and cytotoxic agent, suffers from toxicity, ineffectiveness
against many types of human cancer and the development of tumor cell resistance.
• methotrexate inhibits GART, an enzyme crucial for the conversion of glycinamide ribonucleotide
(GAR) to formyl-glycinamide ribonucleotide (FGAR).
• By inhibiting GART, methotrexate disrupts the de novo purine biosynthesis pathway, leading to a
decrease in purine nucleotides, which are essential for DNA and RNA synthesis.

N
1
6
7
N
8
8a
N
1
2
H2N
N
3
4
NH2
4a
N
5
4
3
2
1
6
5
O
N
H
2
3
4
5
OH
O
1
OH
O
Methotrexate
Lometrexol:
• Is a folate analog and antimetabolite, inhibits de novo purine synthesis by targeting glycinamide
ribonucleotide formyltransferase (GARFT), an enzyme crucial in the purine biosynthesis pathway,
leading to tumour cell growth inhibition and apoptosis.
N
N
H
N
H
8
H2N
O
1
O
HN
O OH
OH
O

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3. seventh edition, Wolters Kluwer | Lippincott Willians & Wilkins, Page no-(320-325).
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References:

RAPID REVERSIBLE ENZYME INHIBITORS ACE, GLYCINAMIDE RIBONUCLEOTIDE TRANSFORMYLASE.pptx

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