DESIGN OF ENZYME INHIBITORS AS DRUGSBy : Ms.Tabhitha K
Guide : Mr. Sampath Ayyappa G, M.Pharm
ENZYMES - INTRODUCTION
ENZYME INHIBITION
• Enzyme inhibitors can be grouped into two general categories:
1. Reversible inhibitors
2. Irreversible inhibitors
• Reversible enzyme inhibitors can be classified into three
categories:
1. Competitive reversible inhibitors
2. Non-competitive reversible inhibitors and
3. Uncompetitive reversible inhibitors
• There are four different approaches to the design of competitive
reversible inhibitors:
1. Simple competitive inhibition
2. Alternative substrate inhibition
3. Transition state analog inhibition and
4. Slow, tight – binding inhibition
E + I E I
.
.
.
+ S -S
EEE S P P+
K on
K off
Kinetic scheme for competetive enzyme inhibition
DESIGN OF SIMPLE COMPETITIVE INHIBITORS
Arg Val Tyr Ile His Pro Phe His Leu Val Ile His Asn ...
Angiotesinogen (human liver)
renin
Arg Val Tyr Ile His Pro Phe Leu His
Angiotensin I
ACE /
His Val Ile Asn ...
Leu His
Arg Val Tyr Ile His Pro Phe
Angiotensin II
aminopeptidase N aminopeptidase A
aminopeptidase N
Asp
Asp
Asp
Arg Asp Asp
Arg Val Tyr Ile His Pro Phe Val Tyr Ile His Pro Phe Arg
Angiotensin IV Angiotensin III
Arg Val Tyr Ile His Pro Phe Leu His ACE
Leu His
aminopeptidase AAsp
Renin-angiotensin system
(proteolytic enzyme)
(hydrolysis)
(decapeptide)
C-terminal
(dipeptide)dipeptidyl carboxypeptidase I
(lungs, blood vessels)
(octapeptide)
(hexapeptide)
CH
CH2
CNH
CCH
NH
ORO
O
CO
OCH
CH2
C
CH2O
O
Zn2+
+S1'
S1"
-----
-----
---- -----
----------
----------
---substrate
(R)-2-benzylsuccinicacid
Hypothetical active site of carboxypeptidase A
carboxypeptidase A
NH
CH
C
R2'
NH
O
CH
C
R1' O
NH
CH
C
R1"
NH
O
CH
R2"
COO-
-----
Zn2+
OH-
Function of the Zn(II) cofactor in ACE catalysis
CH
CH2
CNH2
O
OCC
HNH
R O
O
CO
OCH
CH2
C
CH2O
O
Zn2+
+
S1'
S1"
-----
---
-----
products of hydrolysis
(R)-2-benzylsuccinicacid
The collected products hypothesis of enzyme inhibition
NH
SH
O
CH3
CO2HCaptopril
O
NH
O ONC
HNH
CH
R2'R3'
O
O ONC
H
R2'
OCH2
O
O
O ONC
H
R2'
OCH2
S
Zn2+
------------
-------
--------
------
----
--
--------
-------
-------
-------
-------
-------
---------
--
-------
-------
-------
-------
+B
HS1
S1'S2'
substrate
carboxyalkylproline
mercaptoalkylproline
ACE
Hypothetical binding of carboxyalkylprolineand mercaptoalkylproline derivatives to ACE
Z nII
S 1
S 1 'S 2 '
O
NH
OCH3
O
ON
CH2
CH
NH
H
B
H +
S u b s tr a te ( p ep tid e)
ACE
O
ONH
CH2
CH
OOCH3
ONNH2
H
OCH3
N
CH2
CH2
NH
HO
O
OO
Product
Enalaprilat
Hypothetical interactions of enalaprilat with ACE
CO2H
CH2
CH2 H
NH
NH
O
CH3
HOOC
EnalaprilatCO2H
CH2
CH2 H
H2CH3COOC NH
NH
O
CH3
Enalapril (prodrug)
esterase
CO2H
CH2
CH2 CO2H(CH2)4
NH2
R NH
NH
O
Lisinopril
DESIGN OF ALTERNATIVE SUBSTRATE INHIBITORS
Biosynthesis of bacterial folic acid
dihydropteroate diphosphate
dihydropteroate synthase
dihydropteroate
dihydrofolate
dihydrofolate reductase
tetrahydrofolate
p-amino benzoic acid+
sulfonamides
trimethoprim
SO2NH2
NH2
NH2
N
N
Prontosil
NH2 SO2NH2
Sulfanilamide
N
N
N
NH
NH2
OH
O P O P O
O
O
O
O
N
N
N
NH
NH2
OH NH
O
O
NH2
O
O
dihydropteroate diphosphate
dihydropteroate synthase
PABA
dihydropteroate
NH2 SO2NH2
N
N
N
NH
NH2
OH NH
SO2NH2
sulfanilamide
produces tetrahydrofolate for the biosynthesis of purines inhibits tetrahydrofolate biosynthesis
DESIGN OF TRANSITION STATE ANALOGS
O
NH
CH
C NH
CH
R1' R1"
Zn2+
OH H
NH
CH
C NH
CH
R1' R1"O
Zn2+
OH H
B
B
Zn2+
OH
O
NH
CH
C NH
CH
R1' R1"
BH
+
Zn2+
O
NH
CH
C
R1' O
HB
HN CH
R1"
H
Zn2+
O
O
NH
CH
C
R1'
B+
NH2 CH
R1"-
**1
**2
OCH3CH2CH2
C NH
CCH
C
OO
Pro
Ph
-
H
OCH3CH2CH2
C NH
CCH
C
OO
Pro
Ph
-
Enalaprilat
Enalaprilat
Hypothetical mechanism for ACE catalyzed peptide hydrolysis
Hypothetical mechanism for the reation catalyzed by aspartate transcarbamylase
O
CH2
PO3
NH
COO
OOC--2
PALA
-
O NH2
O
PO3
COO
OOC
NH2NH
O
NH2
COO
OOC
+ -
- -
-
2
NH2
O
PO3
O COO
OOC
NH2
2 -
-
**
DESIGN OF MULTISUBSTRATE ANALOGS
carbamoylphosphate
L-aspartic acid
N-carbamoyl- L-aspartate
N-phosphonoacetyl -L-aspartate
DESIGN OF AFFINITY LABELING AGENTS
The Na to Nb distances (3.3 A0) and the Nb to carboxylate carbon
distances (2.5 A0) in both molecules are identical.
The Na to carboxylate carbon distance is 5.4 A0 in the penicillins
and 5.7 A0 in D-alanyl-D-alanine.
S
NH
MeMe
HO
H
N
R
O
H
CO2H
Me
NH
O
Me
N
Peptidoglycan
O
H
CO2HH
H
a
b
a
b
Comparison of the structure of penicillins with acyl D-alanyl-D-alanine
Acylation of peptidoglycan transpeptidase by penicillins
MECHANISM-BASED ENZYME INACTIVATORS
• Vigabatrin, an Anticonvulsant Drug
• 4-Amino-5-hexenoci acid (vigabatrin, (A), Sabril) is the first
rationally designated mechanism-based inactivator drug .
• In the case of normal substrate turnover, hydrolysis of the complex
of PLP and aminoacid gives pyridoxamine 5’-phosphate (PMP) and
the keto acid.
• The same hydrolysis could occur with vigabatrin and PLP complex
(E) to give the corresponding products, PMP and keto acid (D).
• However, the vigabatrin and PLP complex (E) is a potent
electrophile, a Michael acceptor, which can undergo conjugate
addition by an active site nucleophile (x-) and produce inactivated
enzyme (F or G).
Hypothetical mechanism for inactivation of GABA aminotransferase by Vigabatrin
THANK YOU