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

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