cyclic and linear bradykinin analogues: implications for b2 antagonist design

K.H. HsiehJ.M. Stewart

Authors' af®liations:

K.H. Hsieh and J.M. Stewart, Department of

Biochemistry, University of Colorado School of

Medicine, Denver, CO 80262, USA.

Correspondence to:

Kun-hwa Hsieh PhD

Box 2498, College Station

Pullman

WA 99165 2498

USA

Fax: 509 3346619

Dates:

Received 30 June 1998

Revised 10 August 1998

Accepted 9 January 1999

To cite this article:

Hsieh, K.H. & Stewart, J.M. Cyclic and linear bradykinin

analogues: implications for B2 antagonist design.

J. Peptide Res., 1999, 54, 23±31.

Copyright Munksgaard International Publishers Ltd, 1999

ISSN 1397±002X

Cyclic and linear bradykininanalogues: implications for B2

antagonist design

Key words: bradykinin antagonists; bradykinin conformations;

constrained aromatic amino acids; cyclic bradykinin analogs;

extended vs. folded arginine; structure±activity relationship

Abstract: Bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)

antagonists are potentially useful for treating in¯ammation,

pain and severe trauma. To identify what chemical features

might promote effective antagonism, we replaced Arg1 and Pro7

with structurally constrained and proteolytic-resistant residues,

such as Bip (biphenylalanine), Dip (diphenylalanine) or 2Ind

(indane amino acid). To determine which BK folding might lead

to favourable interactions with receptors, the effects of cyclo(3,8)

vs. cyclo(5,8) analogues were compared. The resulting BK

analogues were examined for their agonistic and antagonistic

activities in guinea pig ileum, rat uterus and depressor assays.

The results suggest that co-planarity of the residue-7 side chain

with its backbone NH is important for potent agonism as well as

antagonism, and a D-directed side chain is crucial for

antagonism. For residue-1 an L-orientation is important, and

Dip1 may mimic a folded Arg1 side chain to elicit agonistic

activities, with Bip1 mimicking an extended Arg1 side chain to

elicit inhibitory activities. However, ileal and uterine receptors

appear to prefer differently folded BK. For ileum, a BK

conformation in which residues-3 and -8 are proximal to each

other, but apart from residue-5, led to improved pA2.

Abbreviations: Acpc, aminocyclopentanecarboxylic acid; Aib, 2-

aminoisobutyric acid; BK, bradykinin; CDF, D-p-

chlorophenylalanine; DCC, dicyclohexylcarbodiimide; DL-Bip, the

racemic b-(4-biphenyl)alanine (C6H5-C6H4)CH2CH(NH2)COOH; DL-

Dip, the racemic b,b-(diphenyl)alanine (C6H5)2CHCH(NH2)COOH;

GPI, guinea pig ileum; HMWK, high-molecular weight

kininogen; HOBt, 1-hydroxybenzotriazole; Hyp, hydroxyproline;

Igl, a-(2-indanyl)glycine; 2Ind, the achiral 2-amino-indan-2-

23

The kallikrein±kinin system is involved in regulating

vascular tone and electrolyte transport and in mediating

in¯ammation, pain and plasma extravasation (1±7). The

components for generating bradykinin (BK, Arg-Pro-Pro-

Gly-Phe-Ser-Pro-Phe-Arg) and the lysine-containing kallidin

(Lys0-BK) are present in plasma and tissues. In human

plasma, respectively, 0.9 and 2.7 mm of high- and low-

molecular weight kininogens (HMWK and LMWK) are

present, and both are effective inhibitors of cysteine

proteinases such as cathepsin L and H (8). The HMWK

circulates as a complex with plasma prekallikrein, which

upon contact activation gives kallikrein, whose proteolysis

of HMWK generates BK (9). Tissue kallikreins differ from

plasma kallikrein in molecular weight, isoelectric pH,

substrate speci®city (LMWK and HMWK) and the formation

of Lys0-BK (4, 10). More than 25 different tissue kallikrein

genes may exist in mouse, and rat pancreatic kallikrein

mRNA could cross-hybridize with mRNA from rat kidney,

spleen and submaxillary gland, but not with that from

intestine, lung or liver (4). Aside from the complexities of

HMWK vs. LMWK and plasma vs. tissue kallikreins, at least

two types of receptors exist for bradykinin (3). The B2

receptors are widely distributed throughout mammalian

tissues, including rat uterus and guinea pig ileum, and

display a nanomolar af®nity for BK and kallidin (11±13). The

B1 receptors are weakly expressed on a small number of

tissues, but can be induced by surgical trauma or in¯amma-

tion and display a nanomolar af®nity for [des-Arg9]-kallidin,

but a micromolar af®nity for [des-Arg9]-BK and an even

lower af®nity for BK (2, 14±19).

By coupling to different signal transduction mechanisms,

B2 receptors mediate a wide spectrum of effects. At 10-10 m,

BK increased vascular permeability, induced hypotension

and bound to sensory neurones for nociception (7, 17, 20). At

10-9 m, BK elicited bronchoconstriction (21). At 10-8 m, BK

induced contraction of smooth muscles, including rat

uterus, guinea pig ileum and lung strip (20±22). At 10-7 m,

BK stimulated secretion of mucin from airway submucosal

gland and interleukin from spleen cells (23, 24). The diverse

effects are a concern in B2 inhibitor design. Another problem

is the short inhibitory duration, due to kininases and

endopeptidase inactivation of BK peptides (2, 13, 25).

Because of their ¯exibility, linear BK peptides can fold

into multiple conformations. If assuming three possible

states for each ¯exible dihedral angle in the backbone,

318 con®gurations would be possible for a 9-residue peptide

(26). The various BK species may interact with different

receptors, thereby contributing to diverse B2 effects. One

approach to improve receptor-selectivity is to enhance the

desired peptide conformations by imposing conformational

constraints such as cyclization (27, 28).

As BK peptides appeared as random coils in water, but

assumed folded structures when exposed to membrane-like

environments (29±31), two questions arise. How is BK folded

into various receptor-bound species, and which conforma-

tion may selectively interact with what receptor? Elucida-

tion of the latter relationship may permit imposition of the

appropriate conformations for selective biological effects,

and thus more speci®c B2 inhibitors. An example is the

cyclization of the eNH2 of Lys1 with the COOH of Arg9.

This folded cyclo(1,9)[Lys1]-BK peptide was highly hypoten-

sive, but elicited little (2.5%) vascular permeability and no

myotropic effect (32). More importantly, the lack of a

COOH-terminus hindered kininase inactivation, and ex-

tended the depressor duration from minutes for BK to hours

for cyclo(1,9)[Lys1]-BK.

Of the folded structure of BK, conformational studies of

Hoe-140 (D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]-BK)and B-9340 (D-

Arg0-[Hyp3,Thi5,D-Igl7,Oic8]-BK) indicated that in

membrane-like micelles, each B2 antagonist consisted of

two b-turns comprising residues 2±5 and 6±9. However, the

two structures differ. Folding of Hoe-140 was strengthened by

hydrogen bonds between the carbonyl of D-Tic7 with the

amide of Gly4, and between the carbonyl of Oic8 with the

hydroxyl of Hyp3. These bonds bring Oic8 near Hyp3, with

Thi5 on the opposite end (29). Folding of B-9340 was

strengthened by a salt bridge between the guanidine of Arg1

with thecarboxylofArg9, andby ahydrogenbondbetweenthe

carbonyl of Ser6 with the guanidine of Arg9. These bonds bring

Oic8 near Thi5, with Hyp3 on the opposite end (30).

In contractile assays, [DPhe7]-BK and [D-NMF7]-BK showed

200±10 000-fold selectivity for uterus over ileum (22),

suggesting signi®cant differences between these B2 receptors.

To determine whether the Hoe-140 and B-9340 conforma-

tions may resemble receptor-bound BK on guinea pig ileum or

rat uterus, we compared the activities of [D-Phe7]-BK

analogues cyclized at positions 3 and 8 vs. 5 and 8, and

examined how Arg1 and Pro7 might contribute to BK±receptor

interaction.

carboxylic acid, C6H4(CH2)2C(NH2)COOH; LMWK, low-molecular

weight kininogen; LS, guinea pig lung strip; Meb,

p-methylbenzyl; Nal, b-(2-naphthyl)alanine; NMF,

N-methylphenylalanine; Oic, (3aS,7aS)-octahydroindole-2-

carboxylic acid; DPhe, 2,3-dehydro-phenylalanine; RU, rat uterus;

Thi, b-(2-thienyl)alanine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-

carboxylic acid.

Hsieh and Stewart . Cyclic and linear bradykinin analogues

24 | J. Peptide Res. 54, 1999 / 23±31

Results and Discussion

Guinea pig ileum receptors prefer a B2 antagonist, whose

residue-8 is near residue-3 but away from residue-5

The proposed conformations for Hoe-140 and B-9340 (29, 30)

differ in the spatial relationship between the crucial X7-Oic8

with residues 3 and 5. In Hoe-140, Oic8 was adjacent to Hyp3,

but apart from Thi5. In B-9340, Oic8 was adjacent to Thi5.

To test which BK folding may be preferred by ileal and

uterine receptors, we replaced Phe8 and Pro3 or Phe5 with

Cys residues whose cyclization gave, respectively, the Hoe-

140- and B-9340-mimicking B-4992 and B-4990 (Table 1). To

provide a linear counterpart for comparison, Met-containing

B-4978 and B-4976 were prepared. By replacing Cys with

Met, spontaneous Cys-oxidation to inadvertently generate

the cyclic peptides was avoided.

Contractile assays of the linear analogues showed that

Met3,8- or Met5,8-containing B-4978 and B-4976 displayed

antagonistic activity on ileum, and agonistic activity (0.05±

0.08%) on uterus (Table 1). These pro®les closely parallel

those for their [D-Phe7]-BK precursor, which was an

antagonist (pA2 5.0) on ileum and an agonist (1% activity)

on uterus (33). Interestingly, Cys3,8 cyclization led to an

improved pA2 of 5.8 on ileum for B-4992, whereas Cys5,8

cyclization led to the loss of inhibitory activity for B-4990.

The opposite effects of cyclization suggest that ileal

receptors prefer a folded BK, in which residue-8 is in close

proximity to residue-3, but away from residue-5. This Hoe-

140-like conformation would bring residue-2 near residues 5

and 6 (29), which may explain the considerable ileal pA2 of

6.6 and 6.3 reported for Cys2,5- and Cys2,6-cyclized BK (34),

despite the steric perturbation introduced by such disul-

phides at sites so near the crucial residue-7.

On the other hand, neither Cys3,8 nor Cys5,8 cyclization

led to antagonism on uterus. Instead, cyclization of Cys0-

[Cys6,D-Phe7]-BK was shown to convert an inactive linear

analogue into an effective cyclo(0,6) antagonist with a pA2 of

7.15 on uterus (35). Together with this ®nding, our results

suggest that uterine receptors prefer an N-terminal folded

antagonist. Such a conformation may explain the similar

pA2 exhibited by the linear as well as cyclized Lys0-[Glu6]-

BK (35), due to the ability of the linear analogues to form a

Lys/Glu salt bridge, thereby assuming a folded structure

similar to that of their lactam-bridged counterparts.

Rat uterus receptors can accommodate a B2 antagonist with

elongated aromaticity at residue-1

Due to its very basic (pKa 13.2) side chain which is charged

at physiological pH, Arg1 can facilitate BK diffusion out of

lipophilic membrane receptors, and provide a trypsin- and

Table 1. Agonistic and antagonistic activities of bradykinin analogues on guinea pig ileum and rat uterus

Agonistic activitya (%) Antagonistic activityb (pA2)

Compound

no. BK structure Ileum Uterus Ileum Uterus

BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)100 100

Cyclic vs. linear BK

B-4978

B-4992

B-4976

B-4990

[Met3,8,Thi5,D-Phe7]-BK

cyclo(3,8)[Cys3,8,Thi5,D-Phe7]-BK

[Met5,8,D-Phe7]-BK

cyclo(5,8)[Cys5,8,D-Phe7]-BK 0.13

0.05

Enhanced

0.08

NDe

Ic

5.8

5.7

Position 1 modi®ed BK

B-4980

B-4984

B-4988

[D-Arg1,Hyp3,D-Phe7]-BK

[2Ind1,Hyp3,D-Phe7]-BK

[DL-Dip1,Hyp3,D-Phe7]-BK

0

0

2.4

0

0

0.8

B-4986 [DL-Bip1,Hyp3,D-Phe7]-BK I 5.4

Position 7 modi®ed BK

B-4994 [2Ind7]-BK 1.3 2.9

a. The pD2 values for BK were 7.4 on guinea pig ileum and 7.9 on rat uterus (33). b. The [D-Phe7]-BK had a pA2 of 5.0 on ileum, but 1%agonistic activity on uterus (33). The [Hyp3,D-Phe7]-BK elicited a greater pA2 of 5.6 on ileum, with less agonistic activity (0.4%) on uterus(13). The [Thi5,8,D-Phe7]-BK showed a 20-fold improvement over [D-Phe7]-BK, and gave the respective pA2 of 6.3 and 6.4 on ileum anduterus (41). c. I indicates very weak antagonism, where less than a 50% reduction was produced. d. Enhanced response to BK. e. ND, notdetermined.


J. Peptide Res. 54, 1999 / 23±31 | 25

aminopeptidase-susceptible site for BK inactivation. As

these effects contribute to a short duration of action, Arg1

replacement by lipophilic and proteolytic-resistant residues

is desirable.

The Arg1 side chain appears important for BK activities.

Studies have shown that replacement of Arg1 by Lys, Orn or

citrulline decreased activities and receptor binding by 50±

1000-fold (36, 37), in contrast to the four-fold lower activity

due to NH2-terminus deletion (38). In general, replacement

of Arg1 by d-amino acids in [D-Phe7]-BK led to inactive Ki

values of . 1 mm for ileum and uterus receptors. Among the

inactive substituents were d-p-chlorophenylalanine in [D-

CDF1,D-Phe7]-BK, d-naphthylalanine in [D-Nal1,D-Phe7]-

BK and d-tyrosine in [D-Tyr1,Hyp3,D-Phe7]-BK (39).

Although [D-Nal1,Thi5,8,D-Phe7]-BK showed a marginal

af®nity for rat uterus (Ki of 248 nm vs. 0.16 nm for BK), it

did not bind appreciably to guinea pig ileum or neuroblas-

toma cells (7, 39).

To elucidate how B2 receptors may interact with Arg1, we

introduced into position-1 constrained residues, which are

highly proteolytic-resistant and lipophilic. These included

the racemic biphenylalanine (DL-Bip) and diphenylalanine

(DL-Dip), and the achiral indane amino acid (2Ind). In

comparison with Arg, whose side chain can assume an

extended or folded conformation, Bip and Dip are isomeric to

each other and each side chain contains two phenyl rings

constrained in, respectively, an extended or folded structure

(40). In spatial orientation, the rigid indane rings occupy a

space between those of d- and l-directed side chains.

Furthermore, Bip and Dip derivatives were resistant to

acylase and carboxypeptidase cleavage, and a single injection

of Bip- or 2Ind-containing angiotensin was effective for

1±2 h (40).

Unlike their [Hyp3,D-Phe7]-BK prototype, which dis-

played an antagonistic pA2 of 5.6 on ileum and 0.4%

agonistic activity on uterus (13), neither D-Arg1-containing

B-4980 nor 2Ind1-containing B-4984 was active (Table 1). On

uterus, [DL-Dip1,Hyp3,D-Phe7]-BK (B-4988) resembles the

prototype, and showed 0.8% agonistic activity. In contrast,

the closely related [DL-Bip1,Hyp3,D-Phe7]-BK (B-4986) was

an antagonist (pA2 5.4). Interestingly, the constrained

features in position 1 of B-4988 and B-4986 led to parallel

ileal and uterine pro®les, instead of the mixed agonism±

antagonism for the [Hyp3,D-Phe7]-BK precursor.

The inactivity of D-Arg1- and 2Ind1-containing analogues

suggests that the agonistic or antagonistic activity of DL-

Dip1- and DL-Bip1-containing diastereomers resulted from

their l-directed components.

More importantly, Dip may mimic the folded side chain of

Arg1 to elicit BK agonism, whereas the elongated aromati-

city of Bip may mimic an extended Arg1 to promote

antagonism. As uterine and ileal receptors can accommo-

date a residue-1 without the guanidine function, the Arg1±

Arg9 salt bridge is not critical. Nevertheless, the weak pA2

(, 5) for Bip-containing B-4986 on ileum suggests that the

Arg1 guanidine may contribute to receptor binding.

A residue-7 side chain perpendicular to the peptide backbone is

not conducive to B2 antagonism

Binding studies show that Arg9 is crucial for high-af®nity

binding to B2 receptors (3, 36, 41), whose activation is

Table 2. The structural characteristics of residue-7 in bradykinin agonists and antagonists

Side-chain coplanar with

Residue-7 aNH- aCOOH Side-chain orientation Biological pro®le

Pro Yes No L Bradykinin

Agonists

Acpc No No Between L and D Weak agonist (5% on cat ileum)

Aib Can be Can be L and D Potent agonist (418% on ileum)

2Ind No No Between L and D Weak agonist

D-NMF Can be Can be N-methyl and D Potent agonist (136% on uterus)

DPhe Yes Yes Neither L nor D Weak agonist

Antagonists

D-Hyp ether Yes No D Potent inhibitor

D-Igl Can be Can be D Potent inhibitor

D-Phe Can be Can be D Inhibitor

D-Tic Yes No D Potent inhibitor


26 | J. Peptide Res. 54, 1999 / 23±31

mediated by Pro7. Consequently, how the side chain of

residue-7 is orientated towards the backbone and indirectly,

Arg9, can have a major impact on BK activity.

Structure±activity relationship studies indicate that Pro7

replacement by a d-amino acid, such as D-Hyp7 ether, D-

Igl7, D-Phe7 or D-Tic7 is essential to B2 antagonism (1, 20,

42, 43). Nevertheless, [D-NMF7]-BK was an agonist with

136% activity on uterus and 0.4% activity on ileum (22). In

addition, Pro7 replacement by achiral substituents led to a

highly active [Aib7]-BK with 418% activity on ileum (vs. less

than 1% activity for its [Ala7]- or [D-Ala7]-BK counterpart),

but a marginally active [Thi5,8,DPhe7]-BK with 1.5% activity

of [Thi5,8]-BK (22, 33).

To examine how the side-chain orientation may affect B2

activity, we introduced into position 7 a rigid homologue of

D- and L-Phe, the achiral 2Ind. Like D-Igl, 2Ind contains a

bicyclic indane. Unlike D-Igl, the 2Ind side chain is

perpendicular to both NH2 and COOH (40). Structurally,

2Ind can be viewed as an Aib fused to a benzene ring, which

occupies a space between those of the d- and l-orientated

side chains. Thus, 2Ind retains some structural features of

the Aib7 agonist and the D-Igl7 antagonist, but differs

markedly in spatial orientation (Table 2).

In contrast to the very potent [Aib7]-BK, [2Ind7]-BK is a

weak agonist (B-4994, Table 1). Because expansion of the

Aib7 methyl groups into 2Ind7 will greatly increase the

volume taken up by the indane rings, it is possible that such

expansion may intrude on a sterically sensitive receptor

region to cause a low [2Ind7]-BK activity. However, Pro7

replacement by Acpc7, a cyclopentane amino acid resem-

bling Pro in size but with a 2Ind-like orientation, led to a

weak agonist (5% activity) on cat ileum (19). Similarly, the

change in orientation converted the potent D-Igl7 antagonist

into a weak 2Ind7 agonist, despite the similarity in size and

structure. Taken together, the side-chain orientation is

important for B2 agonism as well as antagonism.

Table 2 summarizes the residue-7 orientations for B2

agonists and antagonists. As part of the cyclic ring, the Pro

side chain is co-planar with the imino NH-group, but

perpendicular to the COOH. Because of the two methyl side

chains in Aib, its NH2 is co-planar with one methyl, with

the COOH being co-planar with the other methyl. Cycliza-

tion of the Aib methyl groups gives Acpc and 2Ind and alters

the spatial relationship of the side chains, which are

perpendicular to both NH2 and COOH. As a result of the

Ca = Cb bond in DPhe, its aromatic Cc is co-planar with both

NH2 and COOH. D-Hyp and D-Tic resemble D-Pro, with

their side chains being co-planar with the NH group and

directed at an angle about 120 degrees from that of l-

orientated Pro. The side chains of D-Igl and D-Phe are

similarly orientated, and can be co-planar with either NH2

or COOH.

The low activities of the 2Ind7 and DPhe7 analogues

suggest that neither a perpendicular nor a planar side chain

can mimic the receptor-bound arrangement of BK. Instead,

comparison of the native Pro7 with the inhibitory D-Tic7

and D-Hyp7 suggests that effective BK agonism as well as

antagonism arises from the common feature of a residue-7

side chain co-planar with the backbone NH, but perpendi-

cular to COOH (Table 2). Accordingly, 2Ind7 is incapable of

co-planarity with NH, and showed little activity. In

contrast, the closely related D-Igl7 can assume such a

conformation to elicit B2 antagonism (20), for which a

d-orientation is crucial.

Conclusions

Bradykinin antagonists are potentially useful for elucidating

kallikrein±kinin pathology and for clinical treatment of

severe trauma and in¯ammation (1, 2). An example is the

effectiveness of BK antagonists in urate-induced pain and

antigen-induced arthritis, that reveals kallikrein activation

in arthritic joints (7, 17). However, signi®cant improvement

of the selectivity and half-life of BK antagonists is needed if

they are to become therapeutically useful.

As introduction of conformational restraint can increase

the half-life and/or receptor-selectivity of bradykinin,

angiotensin and opioid peptides (27, 28, 32, 40), it is

important to determine how rigidi®cation and cyclization

of different BK side chains may affect B2 activity. For drug

design of BK antagonists, two questions are of interest. What

features may elicit potent antagonism, and which confor-

mations may lead to receptor-selectivity? To answer these

questions, the marginally active [D-Phe7]-BK prototype can

readily reveal both major and minor improvements.

Taken together with reported ®ndings, our results suggest

the general requirement of a d-residue7, preferably with a

side chain co-planar with the backbone NH, for potent B2

antagonism. To de®ne the BK conformation for ileal vs.

uterine receptors, [D-Phe7]-BK was cyclized into folded

structures mimicking those found in physical studies (29,

30). The six-fold more effective pA2 for cyclic B-4992 than

linear B-4978 on ileum, but not for cyclic B-4990 than linear

B-4976 (Table 1), suggests that ileal receptors prefer the Hoe-

140-like conformation, in which residues 3 and 8 are near to

each other, but apart from residue-5. On the other hand, an

elongated biphenylalanine side chain in position 1 (B-4986)


J. Peptide Res. 54, 1999 / 23±31 | 27

promoted antagonistic effect on uterus, and folding of the N-

terminal region of [D-Phe7]-BK was shown (35) to result in a

500-fold greater activity on uterus than ileum (respective

pA2 of 7.15 vs. 4.43).

Identi®cation of the active BK conformations for different

tissues can be useful. For example, different rank-orders

were reported for pain inhibition by D-Arg0-[Hyp3,Thi5,8,D-

Phe7]-BK (NPC-349), [D-Nal1,Thi5,8,D-Phe7]-BK (NPC-573)

and [Leu5,8,Gly6,D-Phe7]-BK (NPC-722). For BK-induced

vascular pain, the inhibitory rank-order of NPC-

722 . NPC-349 was observed and NPC-573 was inactive

(7). For urate-induced pain, the inhibitory rank-order of

NPC-349 . NPC-722 . NPC-573 was found. As the an-

algesic NPC-722 was also inhibitory on ileum (but not on

uterus), and the relatively inactive NPC-573 had little

af®nity for ileum (7, 39), pain receptors appear to resemble

ileal more than uterine receptors. Thus, it may be possible to

improve analgesic B2 inhibitors by combining pA2-enhan-

cing features, including D-Arg0, Hyp3 and Thi5 (7, 41), with a

guanidinated biphenylalanine at position 1 and a Hoe-140

conformation for increased af®nity and half-life.

In limited comparison of B2 activities on guinea pig ileum

(GPI), lung strip (LS) and rat uterus (RU), parallel results

were observed for ileum and lung, but not uterus (21, 22, 35).

Some examples are the disparate agonistic potencies (0.04%

on GPI, 1.4% on LS, vs. 136% on RU) for [D-NMF7]-BK, the

antagonistic (pA2 of 4.9 on GPI, and 4.5 on LS) vs. agonistic

activity (1.5%, RU) for [D-Phe7]-BK, and the inactivity (GPI

and LS) vs. antagonistic activity (pA2 of 6.2 on RU) for Lys0-

[Glu6,D-Phe7]-BK. However, cloning of B2 receptors from rat

uterus and human lung revealed an 81% overall identity for

the, respectively, 366- and 364-residue sequences (12). At the

present time, computational analysis still can not accurately

predict the conformations of proteins beyond the size of 20

to 40 residues (26). For this reason, further development of

constrained analogues can complement molecular model-

ling of BK and mutagenesis of B2 receptors (44) to

differentiate the detailed folding and shapes of various

receptors, thereby facilitating the design of receptor-

selective inhibitors.

It is worth noting that both ileal and uterine receptors can

accommodate the elongated side chain of Bip1, whose

considerable proteolytic-resistance and lipophilicity should

decrease BK degradation by aminopeptidase and promote BK

binding with lipoprotein receptors. In membrane vesicles

(25), BK was rapidly degraded by kininase II, whose cleavage

of the C-terminal dipeptide removed the crucial Arg9

binding-site. The small amount of [des-Arg1]-BK, [des-

Arg9]-BK and BK6±9 metabolites indicated a lesser degrada-

tion by aminopeptidase, carboxypeptidase and endopepti-

dase. As none of the analogues in this study contains the

kininase II-resistant, Pro-like Oic8, a prolonged in vivo effect

is neither expected nor found for Bip1-containing B-4986

(data not shown). Nevertheless, when other proteolytic-

resistant residues capable of replacing the ionizable Arg1,

Arg9 or the endopeptidase-susceptible Phe5 are identi®ed,

their combination should markedly reduce BK degradation,

in addition to suppressing BK departure from the lipophilic

membrane. The prolonged receptor blockade together with

improved receptor-selectivity may ®nally lead to therapeu-

tically useful BK antagonists.

Experimental Section

Materials

All chemicals were reagent grade. Synthesis of Boc-DL-Bip,

Boc-DL-Dip and Boc-2Ind has been reported (40). Brie¯y, DL-

Bip and DL-Dip were prepared through acetamidomalonate

condensation with the appropriate aralkyl halide, and 2Ind

was prepared through Strecker synthesis with 2-indanone

(45). Introduction of the Boc-group required treatment of the

tetramethylammonium salts of DL-Bip and DL-Dip in 1 : 4

of dimethylsulphoxide-t-butanol with di-t-butyl dicarbonate

(Boc2O) at 608C overnight (40). Boc-2Ind was prepared by

standard Boc2O addition to 2Ind in aqueous NaOH-t-butanol

at room temperature (40, 46). Other Boc-amino acids were

commercially available.

Peptide synthesis

Starting from Boc-Arg(Tos)-resin, standard solid-phase

synthesis (47, 48) was performed on a Beckman 900

automatic synthesizer. Side-chain protection included Boc-

Arg(Tos), Boc-Cys(Meb) and Boc-Ser(Bzl). No side-chain

protection was needed for Boc-Hyp. DCC coupling of Boc-

amino acid to the peptide±resin was monitored by the

ninhydrin test (49), and repeated when indicated. To avoid

excessive coupling, Boc-Gly was introduced as preactivated

HOBt ester (50). HF cleavage (08C, 60 min) in the presence of

anisole (51) gave the following peptides: Arg-Pro-Pro-Gly-

Met-Ser-D-Phe-Met-Arg (B-4976); Arg-Pro-Met-Gly-Thi-Ser-

D-Phe-Met-Arg (B-4978); D-Arg-Pro-Hyp-Gly-Phe-Ser-D-

Phe-Phe-Arg (B-4980); 2Ind-Pro-Hyp-Gly-Phe-Ser-D-Phe-

Phe-Arg (B-4984); DL-Bip-Pro-Hyp-Gly-Phe-Ser-D-Phe-Phe-

Arg (B-4986); DL-Dip-Pro-Hyp-Gly-Phe-Ser-D-Phe-Phe-Arg

(B-4988); Arg-Pro-Pro-Gly-Cys-Ser-D-Phe-Cys-Arg (B-4990

precursor); Arg-Pro-Cys-Gly-Thi-Ser-D-Phe-Cys-Arg (B-


28 | J. Peptide Res. 54, 1999 / 23±31

4992 precursor); and Arg-Pro-Pro-Gly-Phe-Ser-2Ind-Phe-Arg

(B-4994). After HF cleavage, Cys-containing peptides were

extracted from the resin with 80% acetic acid in water, and

the aqueous solutions (about 1 mg/mL) were oxidized (room

temperature, 24 h) with a 10-molar excess of I2 to give the

disulphide products.

Depending on its separation by different solvents on thin-

layer chromatography (TLC), each peptide was puri®ed by

countercurrent distribution (CCD) in the solvent giving the

best TLC separation. B-4976, B-4978 and B-4988 were

puri®ed by CCD in 8 : 1 : 2 : 9 of n-butanol±pyridine±acetic

acid±water (respective K-value of 0.59, 1.19 and 3.54) until

homogeneous to TLC. B-4980 and B-4984 were puri®ed by

CCD in 1 : 1 of n-butanol±aqueous 1% CF3COOH (respec-

tive K-value of 3.08 and 8.09). B-4986 was puri®ed by CCD

in 4 : 1 : 5 of n-butanol±acetic acid±water (K-value of 4.25).

B-4990 and B-4992 were puri®ed by CCD in 1 : 1 of n-

butanol-3% CF3COOH (respective K-value of 0.45 and 1.89).

B-4994 was puri®ed by two CCD separations (1 : 1 of n-

butanol-1% CF3COOH, followed by 4 : 1 : 5 of n-butanol±

acetic acid±water; respective K-value of 5.45 and 0.19). After

CCD separation, the appropriate fractions were combined,

evaporated to dryness, gel-®ltered on a column (2 3 45 cm)

of Sephadex G-25 and lyophilized from acetic acid.

The composition of peptides was veri®ed by amino acid

analysis, and mass spectrometry (MALDI-MS) indicated

correct masses for all BK analogues. The MALDI-MS

analyses enabled identi®cation of the appropriate cyclo(3,8)-

and cyclo(5,8)-BK, and ensured the absence of unoxidized

precursors or intermolecular disulphide products in B-4990

and B-4992. As acetylation of Arg side chain can occur

during capping of Boc-Arg(Tos)-resin by acetic anhydride and

triethylamine (52), the correct masses also ensure an

unmodi®ed Arg9 for binding to B2 receptors and suggest

that, during peptide synthesis, either a low level of

acetylation or HF cleavage of the acetyl group from

guanidine, or CCD separation of the Arg9-modi®ed side

product might have occurred.

Contractile assays on isolated rat uterus and guinea pig ileum

These assays were conducted according to reported proce-

dures (20, 33). Brie¯y, up to 20 mg/mL (about 10-5 m) of

bradykinin analogues were examined on both tissues, and

agonistic activities were determined by comparing their

dose±response curves with that for BK, whose ED50 was

reported to be 4 3 10-8 m (pD2 7.4) on ileum, and 1.2 3 10-8

m (pD2 7.9) on uterus (33). Inhibitory pA2 was determined

from the concentrations of antagonists that reduced the

response of a double ED50 dose of BK to that of an ED50 dose.

Rat depressor assay

Intra-carotid artery (i.a.) vs. intrajugular vein (i.v.) adminis-

tration of bradykinin analogues was examined according to

reported procedures (20, 33). Due to the abundance of

kininase II (angiotensin converting enzyme) in pulmonary

vasculature, about 98% of intravenously administered BK

was inactivated in a single passage through the lung (33). By

determining the different abilities of BK analogues to affect

mean rat pressure by i.a. vs. i.v. route, the level of pulmonary

inactivation of BK analogues could be estimated.

The [Met5,8,D-Phe7]-BK and [2Ind7]-BK were weak ago-

nists with depressor effect (data not shown). The

[Met3,8,Thi5,D-Phe7]-BK, cyclo(5,8)[Cys5,8,D-Phe7]-BK and

[DL-Bip1,Hyp3,D-Phe7]-BK were weak BK antagonists. The

cyclo(3,8)[Cys3,8,Thi5,D-Phe7]-BK was inactive. None of

these analogues showed a decreased pulmonary inactivation

or a prolonged in vivo effect.

Acknowledgments: This work was supported by a grant from

the National Institutes of Health (HL-26284 to JMS). Synthesis of

unusual amino acids was supported by HL-32264 (to KHH). We

thank F. Shepperdson for conducting the bioassays, V. Sweeney

for amino acid analyses and Cortech, Inc. for MALDI-MS

analyses. The preprints and discussion with Dr S. Reissmann

are most helpful to the preparation of this manuscript by KHH.

References

1. Stewart, J.M. (1995) Bradykinin antagonists:

development and applications. Biopolymers

37, 143±155.

2. Marceau, F. (1995) Kinin B1 receptors: a

review. Immunopharmacology 30, 1±26.

3. Farmer, S.G. & Burch, R.M. (1992)

Biochemical and molecular pharmacology of

kinin receptors. Annu. Rev. Pharmacol.

Toxicol. 32, 511±536.

4. Fuller, P.J. & Funder, J.W. (1986) The cellular

physiology of glandular kallikrein. Kidney

Int. 29, 953±964.

5. Sharma, J.N. (1988) Interrelationship between

the kallikrein±kinin system and

hypertension: a review. Gen. Pharmac. 19,

177±187.

6. Bhoola, K.D., Figueroa, C.D. & Worthy, K.

(1992) Bioregulation of kinins: kallikreins,

kininogens and kininases. Pharmacol. Rev.

44, 1±80.


J. Peptide Res. 54, 1999 / 23±31 | 29

7. Steranka, L.R., Manning, D.C., DeHaas, C.J.,

Ferkany, J.W., Borosky, S.A., Connor, J.R.,

Vavrek, R.J., Stewart, J.M. & Snyder, S.H.

(1988) Bradykinin as a pain mediator:

receptors are localized to sensory neurons,

and antagonists have analgesic actions. Proc.

Natl Acad. Sci. USA 85, 3245±3249.

8. Muller-Esterl, W. (1987) Novel functions of

the kininogens. Semin. Thromb. Hemost. 13,

115±126.

9. Van Iwaarden, F. & Bouma, B.N. (1987) Role

of high molecular weight kininogen in

contact activation. Semin. Thromb. Hemost.

13, 15±24.

10. Mannhalter, C.H. (1987) Biochemical and

functional properties of Factor XI and

prekallikrein. Semin. Thromb. Hemost. 13,

25±35.

11. McEachern, A.E., Shelton, E.R., Bhakta, S.,

Obernolte, R., Bach, C., Zuppan, P., Fujisaki,

J., Aldrich, R.W. & Jarnagin, K. (1991)

Expression cloning of a rat B2 bradykinin

receptor. Proc. Natl Acad. Sci. USA 88, 7724±

7728.

12. Hess, F.J., Borkowski, J.A., Young, G.S.,

Strader, C.D. & Ransom, R.W. (1992) Cloning

and pharmacological characterization of a

human bradykinin (BK-2) receptor. Biochem.

Biophys. Res. Commun. 184, 260±268.

13. Stewart, J.M. & Vavrek, R.J. (1990) Kinin

antagonists: design and activities. J.

Cardiovasc. Pharmacol. 15 (Suppl. 6), S69±

S74.

14. Menke, J.G., Borkowski, J.A., Bierilo, K.K.,

MacNeil, T., Linemeyer, D.L. & Hess, F.J.

(1994) Expression cloning of a human B1

bradykinin receptor. J. Biol. Chem. 269,

21583±21586.

15. Pesquero, J.B., Pesquero, J.L., Oliveira, S.M.,

Roscher, A.A., Metzger, R., Ganten, D. &

Bader, M. (1996) Molecular cloning and

functional characterization of a mouse

bradykinin B1 receptor gene. Biochem.

Biophys. Res. Commun. 220, 219±225.

16. Regoli, D., Marceau, F. & Barabe, J. (1978) De

novo formation of vascular receptors for

bradykinin. Can. J. Physiol. Pharmacol. 56,

674±677.

17. Cruwys, S.C., Garrett, N.E., Perkins, M.N.,

Blake, D.R. & Kidd, B.L. (1994) The role of

bradykinin B1 receptors in the maintenance

of intra-articular plasma extravasation in

chronic antigen-induced arthritis. Br. J.

Pharmacol. 113, 940±944.

18. Khsar, S.G., Miao, F.J.P. & Levine, J.D. (1995)

In¯ammation modulates the contribution of

receptor-subtypes to bradykinin-induced

hyperalgesia in the rat. Neurosci. 69, 685±

690.

19. Park, W.K., St-Pierre, S.A., Barabe, J. &

Regoli, D. (1978) Synthesis of peptides by the

solid-phase method. III. Bradykinin:

fragments and analogs. Can. J. Biochem. 56,

92±100.

20. Stewart, J.M., Gera, L., Hanson, W., Zuzack,

J.S., Burkard, M., McCullough, R. & Whalley,

E.T. (1996) A new generation of bradykinin

antagonists. Immunopharmacology 33, 51±

60.

21. Reissmann, S., Greiner, G., Seyfarth, L.,

Pagelow, I., Werner, H., Vietinghoff, G.,

Boeckmann, S., Schulz, E., Wartner, U. &

Gera, L. (1996) A new type of bradykinin B2

receptor antagonists: bradykinin analogs with

N-alkyl amino acids at position 2.

Immunopharmacology 33, 73±80.

22. Reissmann, S., Schwuchow, C., Seyfarth, L.,

De Castro, L.F.P., Liebmann, C., Pagelow, I.,

Werner, H. & Stewart, J.M. (1996) Highly

selective bradykinin agonists and antagonists

with replacement of proline residues by N-

methyl-d- and l-phenylalanine. J. Med.

Chem. 39, 929±936.

23. Nagaki, M., Shimura, S., Irokawa, T., Sasaki,

T., Oshiro, T., Nara, M., Kakuta, Y. &

Shirato, K. (1996) Bradykinin regulation of

airway submucosal gland secretion: role of

bradykinin receptor subtype. Am. J. Physiol.

270, L907±L913.

24. Pagelow, I., Werner, H. & Reissmann, S.

(1995) Effects of bradykinin and bradykinin

analogues on spleen cells of mice. Eur. J.

Pharmacol. 279, 211±216.

25. Schafer, G., Nau, R., Cole, T. & Conlon, M.

(1986) Speci®c binding and proteolytic

inactivation of bradykinin by membrane

vesicles from pig intestinal smooth muscle.

Biochem. Pharmacol. 35, 3719±3725.

26. Berendsen, H.C. (1998) A glimpse of the Holy

Grail? Science 282, 642±643.

27. Mosberg, H.J., Hurst, R., Hruby, V.J., Gee, K.,

Yamamura, H.I., Galligan, J.J. & Burks, T.F.

(1983) Bis-penicillamine enkephalins possess

highly improved speci®city toward d opioid

receptors. Proc. Natl Acad. Sci. USA 80,

5871±5874.

28. Schiller, P.W., Nguyen, T.M.D., Maziak,

L.A., Wilkes, B.C. & Lemieux, C. (1987)

Structure±activity relationships of cyclic

opioid peptide analogues containing a

phenylalanine residue in the 3-position. J.

Med. Chem. 30, 2094±2099.

29. Guba, W., Haessner, R., Breipohl, G., Henke,

S., Knolle, J., Santagada, V. & Kessler, H.

(1994) Combined approach of NMR and

molecular dynamics within a biphasic

membrane mimetic: conformation and

orientation of the bradykinin antagonist Hoe

140. J. Am. Chem. Soc. 116, 7532±7540.

30. Sejbal, J., Cann, J.R., Stewart, J.M., Gera, L. &

Kotovych, G. (1996) An NMR, CD, molecular

dynamics, and ¯uorometric study of the

conformation of the bradykinin antagonist B-

9340 in water and in aqueous micellar

solutions. J. Med. Chem. 39, 1281±1292.

31. Kyle, D.J., Blake, P.R., Smithwick, D., Green,

L.M., Martin, J.A., Sinsko, J.A. & Summers,

M.F. (1993) NMR and computational

evidence that high-af®nity bradykinin

receptor antagonists adopt C-terminal b-

turns. J. Med. Chem. 36, 1450±1460.

32. Chipens, G.I., Mutulis, F.K., Katayev, B.S.,

Klusha, V.E., Misina, I.P. & Myshlyiakova,

N.V. (1981) Cyclic analogue of bradykinin

possessing selective and prolonged biological

activity. Int. J. Peptide Protein Res. 18, 302±

311.

33. Vavrek, R.J. & Stewart, J.M. (1985)

Competitive antagonists of bradykinin.

Peptide 6, 161±164.

34. Chakravarty, S., Wilkins, D. & Kyle, D.J.

(1993) Design of potent, cyclic peptide

bradykinin receptor antagonists from

conformationally constrained linear peptides.

J. Med. Chem. 36, 2569±2571.

35. Seyfarth, L., De Castro, L.F.P., Liepina, I.,

Pagelow, I., Liebmann, C. & Reissmann, S.

(1995) New cyclic bradykinin antagonists

containing disul®de and lactam bridges at the

N-terminal sequence. Int. J. Peptide Protein

Res. 46, 155±165.

36. Odya, C.E., Goodfriend, T.L. & Pena, C.

(1980) Bradykinin receptor-like binding

studied with iodinated analogues. Biochem.

Pharmacol. 29, 175±185.

37. Schroeder, E. (1970) Structure±activity

relationships of kinins. Handb. Exp.

Pharmacol. 25, 324±350.

38. Stewart, J.M. (1979) Chemistry and biological

activity of peptides related to bradykinin.

Handb. Exp. Pharmacol. 25(Suppl.), 227±272.

39. Farmer, S.G., Birch, R.M., DeHaas, C.J., Togo,

J. & Steranka, L.R. (1989) [Arg1-D-Phe7]-

substituted bradykinin analogs inhibit

bradykinin- and vasopressin-induced

contractions of uterine smooth muscle. J.

Pharmacol. Exp. Ther. 248, 677±681.

40. Hsieh, K.H., LaHann, T.R. & Speth, R.C.

(1989) Topographic probes of angiotensin and

receptor: potent angiotensin II agonist

containing diphenylalanine and long-acting

antagonists containing biphenylalanine and

2-indan amino acid in position 8. J. Med.

Chem. 32, 898±903.

41. Braas, K.M., Manning, D.C., Perry, D.C. &

Snyder, S.H. (1988) Bradykinin analogues:

differential agonist and antagonist activities

suggest multiple receptors. Br. J. Pharmacol.

94, 3±5.


30 | J. Peptide Res. 54, 1999 / 23±31

42. Lembeck, F., Griesbacher, T., Eckhardt, M.,

Henke, S., Breipohl, G. & Knolle, J. (1991)

New, long-acting, potent bradykinin

antagonists. Br. J. Pharmacol. 102, 297±304.

43. Kyle, D.J., Martin, J.A., Burch, R.M., Carter,

J.P., Lu, S., Meeker, S., Prosser, J.C., Sullivan,

J.P., Togo, J., Noronha-Blob, L., Sinsko, J.A.,

Walters, R.F., Whaley, L.W. & Hiner, R.N.

(1991) Probing the bradykinin receptor:

mapping the geometric topography using

ethers of hydroxyproline in novel peptides. J.

Med. Chem. 34, 2649±2653.

44. Herzig, M.C.S., Nash, N.R., Connolly, M.,

Kyle, D.J. & Leeb-Lundberg, L.M.F. (1996)

The N terminus of bradykinin when bound to

the human bradykinin B2 receptor is adjacent

to extracellular Cys20 and Cys277 in the

receptor. J. Biol. Chem. 271, 29746±29751.

45. Greenstein, J.P. & Winitz, M. (1961)

Chemistry of the Amino Acids. Wiley, New

York.

46. Hsieh, K.H., Jorgensen, E.C. & Lee, T.C.

(1979) Angiotensin II analogues. 12. Role of

the aromatic ring of position 8 phenylalanine

in pressor activity. J. Med. Chem. 22, 1038±

1044.

47. Marshall, G.R. & Merri®eld, R.B. (1965)

Synthesis of angiotensins by the solid-phase

method. Biochemistry 4, 2394±2401.

48. Stewart, J.M. & Young, J.D. (1984) Solid

Phase Peptide Synthesis, 2nd edn. Pierce

Chemical Company, Rockford.

49. Kaiser, E., Colescott, R.L., Bossinter, C.D. &

Cook, P.I. (1975) Color test for detection of

free terminal amino groups in the solid-phase

synthesis of peptides. Anal. Biochem. 34,

595±598.

50. Konig, W. & Geiger, R. (1970) Eine neue

Methode zur Synthese von Peptiden:

aktivierung der Carboxylgruppe mit

Dicyclohexylcarbodiimid unter Zusatz von 1-

Hydroxy-benzotriazolen. Chem. Ber. 103,

788±798.

51. Sakakibara, S., Shimonishi, Y., Kishida, Y.,

Okada, M. & Sugihara, H. (1967) Use of

anhydrous hydrogen ¯uoride in peptide

synthesis. I. Behavior of various protective

groups in anhydrous hydrogen ¯uoride. Bull.

Chem. Soc. Jpn 40, 2164±2167.

52. Hsieh, K.H., DeMaine, M.M. & Gurusidaiah,

S. (1996) Side reactions in solid-phase peptide

synthesis and their applications. Int. J.

Peptide Protein Res. 48, 292±298.


J. Peptide Res. 54, 1999 / 23±31 | 31

cyclic and linear bradykinin analogues: implications for b2 antagonist design

Documents