Angiotensin converting enzyme (ACE) ACE is a zinc-containing dipeptidyl carboxypeptidase, which converts angiotensin I to angiotensin II

Angiotensin II is an octapeptide that contracts blood vessels and increases salt and water reabsorption in the kidney (mainly by stimulating the release of aldosterone), resulting in the increase of blood pressure

angiotensinogen

angiotensin I

angiotensin II

renin

ACE

vasoconstriction aldosterone release

captopril

INCREASE IN BLOOD PRESSURE

enalaprilatlysinopril

Inhibitors of ACE, like captopril, enalaprilat and lisinopril are important antihypertensive drugs

captoprilenelaprilatlisinopril

H

H

H

H

HH

H

HNNN

NNN

H2N

O

O

O

O

O

O

COOHNN

N

O

O

O

COOH

NH

HN NH2

OH

N NH

NHN

41

23 5

6

78

910

ACE

A II

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

A I1N9U1N9V

Solution structures of angiotensin I & II by NMR

captopril

HS N

CO2HO

CH3

H

NN

CO2HO

OHO

NH2

H

enalaprilat

lisinopril

CH3

NN

CO2HO

OHO

Captopril, enalaprilat and lisinopril are antihypertensive drugs used to lower high blood pressure in hypertensive individuals. Their mechanism of action is inhibition of angiotensin converting enzyme (ACE)

Chemistry of captopril

HS N

COOHO

CH3

N

COOHO

CH3

H2N

captopril

L-Ala L-Pro (S)-Ala (S)-Pro

C N C

COOHO

CH3

H2N

CH2CH2

CH2

H

H

pyrrolidine

carboxyl

methyl

(mercaptoor thiol) amide

sulfhydryl

Captopril was developed (by Ondetti and Cushman at Squibb), using rational design, based on (1) the analogy of its target ACE to carboxypeptidase A, a much studied enzyme, and (2) the structure of its inhibitor, benzylsuccinate

Carboxypeptidase A (CPA)A proteolytic enzyme with a catalytic zinc at the active siteSelectively cleaves at a C-terminal aromatic residue, e.g., phenylalanine

a b

ba

= XbKi

aKiabKi

Zn

Benzylsuccinate

Arg145

CPA

1CBX

H

Zn+2

CO2

O

O

Zn+2

CO2N

O

peptide

OH

H

C-terminal

Zn+2

O

peptide O CO2H2N

products still bound to the active site

benzylsuccinate (bi-product i nhibitor)

E N Z Y M E

E N Z Y M E

E N Z Y M E

substrate

phenylalanine

terahedral intermediate

E N Z Y M E

C-terminal

Zn+2

CO2N

O

peptide

HO

H

C-terminal

E N Z Y M E

phosphonate(transition-sta te analog)

peptide

Zn+2

CO2OP

OO

CH2

7CPA

Arg145

phosphonate

Zn

Based on the potent CPA inhibitory activity of benzylsuccinic acid, succinyl-proline was designed as an inhibitor of ACE - it had modest activity (Ki 0.3 mM) The CH3-group (Ala side-chain) was attached that improved the Ki (0.02 mM). The COOH-group was replaced by an SH-group for stronger binding to the active site Zn leading to captopril with a superior Ki-value of 2 x 10-8 M (0.00002 mM) and potent in vivo activity. Enalaprilat (Ki-value of 1.2 x 10-8 M ) was subsequently developed to replace the oxidizable SH-group with a COOH. In addition, an NH- and a phenylethyl-group were inserted to compensate for the loss of the strong Zn-S bond

Zn+2

CO2

O

O

benzylsuccinate (bi-product i nhibitor)

Enzyme (CPA)

Enzyme (ACE)

H

enalaprilat

Zn+2

N

CO2O

CH3

N

O

O

Ki = 1.2 x 10-8 M

Ki = 2 x 10-5 M

Enzyme (ACE)

Ki = 2 x 10-8 M

N

CO2O

CH3

Zn+2

S

captopril

succinyl-Pro

Zn+2

N

CO2O

O

O

Ki = 3 x 10-4 M

Enzyme (ACE) Enzyme (ACE)

Zn+2

N

CO2O

O

O

CH3

Me-succinyl-Pro

Complex of captopril with angiotensin converting enzyme

1UZF

Captopril bound to the active site of ACE

• The thiol (SH) group of captopril makes direct interaction with Zn2+

• The carbonyl group forms strong hydrogen bonding with two histidines, His 513 and His353

• One oxygen of proline carboxylate group is H-bonded to Tyr 520, Gln 281 and Lys 511

HS

CH3

O

N

COOH

1UZF

Ki = 2 x 10-8 M

• Side effects of captopril lead to the design of enalaprilat• The carboxylate group introduced to replace the SH-group binds to Zn2+ as

well as to Glu 384 and Tyr 523• The carbonyl group attached to proline forms strong H-bonds with two

histidines, His 513 and His 353, similarly to captopril• One oxygen of proline carboxylate group is H-bonded to Tyr 520, Gln 281

and Lys 511, similarly to captopril• The carbonyl oxygen of Ala 354 forms a H-bond to the new NH-group • The phenylethyl side-chain introduced occupies a hydrophobic pocket

lined by Phe 512 and Val 518

Enalaprilat bound to the active site of ACE

1UZE

N

CH3

O

N

COOH

HO O

H

Ki = 1.2 x 10-8 M

In lisinopril the alanine of enalaprilat is replaced by lysine. The positively charged side-chain of lysine binds to Glu 162 as well as toAsp 377 via a bridging water molecule (green)

Lisinopril bound to the active site of ACE

1O86

N

O

N

COOH

HO O

H

NH2

Ki = <1 x 10-9 M