DESIGN OF ENZYME

INHIBITORS AS DRUGS
By : 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
 Kon
E + I .
E I
Koff
+S -S

E .S E .P E + P

Kinetic scheme for competetive enzyme inhibition

• 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
 DESIGN OF SIMPLE COMPETITIVE
INHIBITORS
Asp Arg Val Tyr Ile His Pro Phe His Leu Val Ile His Asn ...
Angiotesinogen (human liver)

renin (proteolytic enzyme)

Val Ile His Asn ...
(hydrolysi s)

Asp Arg Val Tyr Ile His Pro Phe His Leu
Angiotensin I (decapeptide)

(lungs, blood vessel s)

C-terminal His Leu
(dipeptide)
ACE /
dipeptidyl carboxypeptidase I

Asp Arg Val Tyr Ile His Pro Phe aminopeptidase A

Asp
Angiotensin II (octapeptide)

aminopeptidase N aminopeptidase A
Asp Arg Asp

aminopeptidase N ACE
Val Tyr Ile His Pro Phe Arg Val Tyr Ile His Pro Phe Arg Val Tyr Ile His Pro Phe His Leu
Arg His Leu
Angiotensin IV Angiotensin III
(hexapeptide)

Renin-angiotensin system
 2+
Zn
- -
--- OH
-
R2' O R1' O R1" O R2"
-
N C C N C C N C C N C COO
H H H H H H H H

Function of the Zn(II) cofactor in ACE catalysis

S1"
---
--

S1' 2+
Zn
-- --
-- +
--
-- --
R O CH2 - carboxypeptidase A
O
N C C N C C substrate
H H H H O
---

--- ---
--- ---
-- --
-- --
CH2
O O
C C C C (R)-2-benzylsuccinic
O H2 H O acid

Hypothetical active site of carboxypeptidase A

 S1"

---
--
S1'
--- 2+
---
Zn +
--
R O CH2 O
products of
N C C H2N C C hydrolysis
H H O H O

CH2
O O (R)-2-benzylsuccinic
C C C C
O H2 H acid
O
The collected products hypothesis of enzyme inhibition
 B S2'
S1 2+ S1'
Zn --- +--
--- --- --- H ---
ACE
--- --- --- --- - --
--
- - - - -
R3' O R2' O O
N C N C N substrate
H H H H --- --- O
--- --- --- ---
--- --- ---
- - - - ---
-
O R2' O O
C C carboxyalkylproline
--- N --
O H2 H --- --- O
--- --- -- --- ---
H3C ---
- --- --
-- -- ---
H - -
HS N --- R2' O
- O
mercaptoalkylproline
S C C N
O H2 H O
CO2H
Captopril Hypothetical binding of carboxyalkylproline
and mercaptoalkylproline derivatives to ACE
 S1
Hypothetical interactions of enalaprilat with ACE

B S2'
S1'
ZnII H + ACE
CH2 CH3
O O O
H
N C N N Substrate (peptide)
H H H O

CH2 CH3 O
O O
N C
H
N
Product
H2N
H H
O O

O O
CH2 CH3 O O
H
C N N Enalaprilat
H2 H O
 CH2 CH2
H2C H3C esterase H2C CH3
H H
H H
N N
H2CH3COOC N HOOC
N
H H
O O
CO2H CO2H
Enalapril (prodrug) Enalaprilat

CH2 NH2

H2C (CH2)4
CO2H
H
R N
N
H
O
CO2H
Lisinopril
DESIGN OF ALTERNATIVE SUBSTRATE
INHIBITORS
NH2

H2N N H2N SO2NH2

N SO2NH2
Sulfanilamide
Prontosil

dihydropteroate diphosphate + p-amino benzoic acid

dihydropteroate synthase sulfonamides

dihydropteroate

dihydrofolate

dihydrofolate reductase trimethoprim

tetrahydrofolate
Biosynthesis of bacterial folic acid
 H
H2N N N
O O
N O P O P O
N
O O
OH
dihydropteroate diphosphate

O H2N SO2NH2
dihydropteroate synthase H2N
O sulfanilamide
PABA
H
H2N N N
H
H2N N N
N
N O N
OH N N
H
O OH N SO2NH2
dihydropteroate H

produces tetrahydrofolate for the biosynthesis of purines inhibits tetrahydrofolate biosynthesis

DESIGN OF TRANSITION STATE ANALOGS
2+
Zn 2+ **1
Zn
2+
R1' O Zn
R1" R1' O R1"
N C C NC NC C NC R1' O R1"
H H HH HH HH NC C NC
O O HH HH
H H O H
H H
H B
B B +

-O
Ph O
CH3O
CH2 C
C C N C C Pro
H2 H H
Enalaprilat
2+ **2
2+ Zn
Zn
R1' O R1"
R1' O R1"
-H NC NC C HN C
NC C 2 HH H
HH H O
O H
H
HB B
+

-O
Ph O
CH3O
CH2 C
C C N C C Pro
H2 H H
Enalaprilat
Hypothetical mechanism for ACE catalyzed peptide hydrolysis
 DESIGN OF MULTISUBSTRATE ANALOGS
2 -OOC PO3
2 -OOC ** -OOC
PO 3
O O H
+ H2N H2N N
NH2 COO-
O NH2 COO- O COO -
O
NH2
carbamoyl L-aspartic 2 - N-carbamoyl-
phosphate acid PO 3
-OOC L-aspartate
CH2

O N COO -
H
PALA
N-phosphonoacetyl
-L-aspartate

Hypothetical mechanism for the reation catalyzed by aspartate transcarbamylase

DESIGN OF AFFINITY LABELING AGENTS
O H b
H
H N CO2H
Me
a S Me
O N
H
R
O H
H
Me Nb CO2H
H Me
O Na
H
Peptidoglycan

Comparison of the structure of penicillins with acyl D-alanyl-D-alanine

 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.
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