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  • K. KrauseL.F. PinedaR. PeteranderlS. Reissmann

    Authors affiliations:

    K. Krause and L.F. Pineda, Institut fur Biochemie

    und Biophysik, Friedrich-Schiller-Universitat

    Jena, Philosophenweg 12, D-07743 Jena,

    Germany.

    R. Peteranderl, Institut fur Organische Chemie

    und Biochemie, Technische Universitat

    Munchen, Lichtenbergstrae 4, D-85747

    Garching, Germany.

    S. Reissmann, Institut fur Biochemie und

    Biophysik, Friedrich-Schiller-Universitat Jena,

    Philosophenweg 12, D-07743 Jena, Germany.

    Correspondence to:

    Luis Felipe Pineda De Castro

    Institut fur Molekulare Biotechnologie e.V.

    Postfach 100813

    D-07708 Jena

    Germany

    Tel.: 49-3641-656491

    Fax: 49-3641-609818

    E-mail: pineda@imb-jena.de

    Dates:

    Received 9 April 1999

    Revised 25 May 1999

    Accepted 24 July 1999

    To cite this article:

    Krause, K., Pineda L.F., Peteranderl, R. & Reissmann, S.

    Conformational properties of a cyclic peptide bradykinin

    B2 receptor agonist by experimental and theoretical

    methods.

    J. Peptide Res., 2000, 55, 6371

    Copyright Munksgaard International Publishers Ltd, 2000

    ISSN 1397002X

    Conformational properties ofa cyclic peptide bradykinin B2receptor antagonist usingexperimental and theoreticalmethods

    Key words: Molecular dynamics simulations; NOE; peptide

    hormones; simulated annealing

    Abstract: The solution conformation of the cyclic peptide J324

    (cyclo0,6-[Lys0,Glu6,D-Phe7]BK), an antagonist targeted at the

    bradykinin (BK) B2 receptor, has been investigated using

    experimental and theoretical methods. In order to gain insight

    into the structural requirements essential for BK antagonism, we

    carried out molecular dynamics (MD) simulations using simulated

    annealing as the sampling protocol. Following a free MD

    simulation we performed simulations using nuclear Overhauser

    enhancement (NOE) distance constraints determined by NMR

    experiments. The low-energy structures obtained were compared

    with each other, grouped into families and analyzed with

    respect to the presence of secondary structural elements in their

    backbone. We also introduced new ways of plotting structural

    data for a more comprehensive analysis of large conformational

    sets. Finally, the relationship between characteristic backbone

    conformations and the spatial arrangement of specific

    pharmacophore centers was investigated.

    Abbreviations: BK, bradykinin; DMSO, dimethylsulfoxide; MD,

    molecular dynamics; NOE, nuclear Overhauser effect; RMS, root

    mean square; RMSD, root mean square deviation; SA, simulated

    annealing; SPL, SYBYL programming language; TPPI, time

    proportional phase incrementation.

    Bradykinin (BK) is a peptide hormone with the sequence

    RPPGFSPFR, found in tissues and plasma. It is implicated in

    numerous pathophysiological processes, for example per-

    ipheral pain and inflammation. Owing to these observa-

    tions, great efforts have been undertaken in order to develop

    63

  • BK antagonists as potential therapeutic agents. In the past a

    large number of peptide antagonists has been synthesized by

    various groups (1), while peptide design has gone through

    several iterations.

    In the last three decades several groups have begun

    attempts to describe the conformation of BK and some of its

    analogs. After initial studies focused on the conformation of

    agonistic BK analogs and partial sequences, the situation

    changed with the discovery of the first BK antagonist. The

    goal of these investigations was to describe the bioactive

    conformation(s) of BK antagonists and define the differences

    between the agonists and antagonists. Whereas, in the past,

    conformational analysis was performed by means of con-

    ventional chemical and physical methods, such as thin film

    dialysis, H-exchange, ORD-, CD-, Raman-, ESR- and

    fluorescence spectroscopy, more recently NMR spectro-

    scopy combined with molecular modeling has been increas-

    ingly applied (2).

    The results of those numerous investigations were rather

    insufficient for an accurate description of the bioactive

    conformations. Thus, different conformational shapes have

    been proposed for agonists and antagonists, e.g. random

    conformations (3, 4), conformations stabilized by up to three

    hydrogen bonds (5) or quasicyclic structures (6, 7) have been

    proposed for agonists. As a result of studies on antagonists

    containing different amino acid substitutions at position 7

    and additional replacements at other sequence positions,

    turn structures have been postulated, either in the N-

    terminal sequence (8), the C-terminal sequence (912) or

    both (13, 14).

    The conformation of the linear nonapeptide changes

    through interaction with its receptor, a large membrane-

    bound protein. Two approaches to characterizing the

    receptor-bound conformation have been used considering

    this conformational change and the lack of an isolated and

    crystallized hormone receptor complex, suitable for X-ray

    analysis. Ottleben et al. (15) used a monoclonal antibody to

    mimic the receptor-binding area. The conformation of

    antibody-bound BK was studied after labeling with 13C

    and 15N by NMR spectroscopy. Nevertheless, this model fits

    the binding requirements of the receptor to only a limited

    extent. Thus, the C-terminal Arg is not necessary for

    binding to the antibody, but is essential for binding to the B2

    receptor. Therefore, the conformation found seems to

    correlate only partially with the receptor-bound conforma-

    tion. A second approach was used by Kyle et al. (16) to

    describe the receptor-bound conformation. Based on the

    results of structural homology modeling and computer

    simulation, the authors generated a model of the ligand

    bound to the rat B2 receptor, which, even though it is highly

    speculative, is supported by mutagenesis data. Therefore,

    conformational analysis of the hormonereceptor complex

    remains a great challenge for biochemistry and also a

    prerequisite for the real rational design of potent agonists

    and antagonists.

    Most conformational studies were performed on linear

    agonists and antagonists, i.e. on peptides with high

    conformational flexibility. In some analogs this flexibility

    was reduced through the introduction of constrained

    nonproteinogenic amino acids (17, 18) or by cyclization

    between the N- and C-termini (19, 20). We attempted to

    reduce the conformational flexibility by intramolecular

    cyclization of the side chains. Owing to conformational

    constraints, the solution conformation of a biologically

    active cyclic peptide is expected to be close to the bioactive

    conformation. For this reason we synthesized a series of BK

    antagonists with lactam bridges in either the N- or C-

    terminal part of the molecule. These bridges were built

    between the side chains of Lys and Glu residues added to or

    inserted into the sequence (21). A second series contained

    backbone cyclisized (22) analogs with lactam bridges built

    between peptide bonds modified with aminoalkyl and

    carboxyalkyl residues (23), respectively.

    In spite of the intramolecular cyclization, many of the

    analogs are still too flexible for conformational analysis by

    NMR spectroscopy and exist in solution as an equilibrium of

    many conformers. Few peptides fulfilled both requirements:

    biological potency and sufficiently reduced flexibility. In

    this paper we report the results of our first conformational

    studies on cyclic BK analogs. We started with the analog

    cycloo,6-[Lys0,Glu6,d-Phe7]BK (J324), a first-generation

    cyclic antagonist with a lactam bridge between the side

    chains of a Lys residue that had been introduced as an N-

    terminal extension and Glu residue that replaced the Ser

    residue at position 6 in the native sequence. This antagonist

    shows only modest antagonistic activity in the isolated rat

    uterus assay (pA2 = 5.78 u 0.02). Nevertheless, its activityis higher than that of the corresponding linear peptide (rat

    uterus: no activity; guinea-pig ileum: pA2 = 4.90 u 0.32;21).

    Preliminary studies by means of energy minimization

    calculations and MD simulations of J324, as well as of a

    second antagonist that contains a disulfide bridge in the N-

    terminal region, showed that a b-turn in this segment of the

    peptide appears to be a defining structural feature, although

    it is not the sole determinant of its antagonistic activity (21,

    24).

    Krause et al . Conformational properties of bradykinin B2 receptor agonist

    64 | J. Peptide Res. 55, 2000 / 6371

  • An additional approach towards the elucidation of the

    essential structural requirements for BK antagonism

    focused, not on the backbone conformation, but rather on

    the spatial arrangement of functional groups and their

    specific properties (charge, hydrophobicity, aromaticity,

    etc.). This led to the development of three-dimensional

    pharmacophore models with the help of expert systems such

    as Catalyst (25), and consensus MD simulations. Based on

    these models, several proprietary three-dimensional data-

    bases were screened for new potential BK antagonists (26).

    As a direct result of these studies, a novel class of nonpeptide

    lead structures for BK B2-receptor anta