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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
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