Elsevier

Peptides

Volume 20, Issue 7, August 1999, Pages 795-801
Peptides

Original Articles
Structure-activity studies on cysteine-substituted neurokinin A analogs☆

https://doi.org/10.1016/S0196-9781(99)00064-9Get rights and content

Abstract

A complete series of analogs of tyrosine modified neurokinin A ([Tyr1]-NKA or [Tyr0]-NKA) has been synthesized by substituting each natural residue with l-Cys. These analogs were tested for their ability to bind recombinant neurokinin-2 (NK-2) receptor. Substitution of Phe6 with Cys completely abolished binding of the analog to the receptor. Substitution of residues in the carboxyl-terminal region of the peptide (Met10, Leu9, Gly8, Val7) and Asp4 with Cys gave reductions in binding affinity of between 23- and 250-fold. Molecular dynamics simulations of these analogs suggest that changes in peptide structure and flexibility are not large contributors to the losses in receptor binding affinity. Reductions in binding affinity are therefore more confidently ascribed to losses of peptide-receptor interactions.

Introduction

Peptide hormones and neurotransmitters constitute a large class of compounds that serve as chemical messengers of intercellular communication. The mammalian neurokinins—substance P (SP), neurokinin A (NKA), and neurokinin B (NKB)—are neuropeptides of 10–11 amino acid residues in length. They share a common C-terminal sequence Phe-X-Gly Leu-Met [23], wherein X is typically an aromatic or branched aliphatic amino acid. These peptides are widely distributed throughout the nervous system and are associated with a variety of activities such as smooth muscle contraction, salivation and depolarization of central neurons. SP, NKA, and NKB bring about their effects through activation of the G-protein-coupled receptors neurokinin-1, neurokinin-2, and neurokinin-3, respectively [2], [8].

The wide spectrum of action of NKA has stimulated many structure-activity relationship investigations [4], [9], [17], [18], [19], [21], [22]. Studies of fragments have shown that the C-terminal residues are more important for interaction with receptor than the N-terminal residues. Deletion of a single residue from the C-terminus is enough to abolish biologic activity and removal of any more eliminates binding to receptor [4]. In contrast, deletion of the N-terminal tripeptide yields the fragment NKA4–10 that binds to the NK-2 receptor even more tightly than the full length peptide [7]. The minimum known sequence to maintain some biologic activity is the C-terminal hexa-peptide NKA5–10 whereas N-terminal fragments have no biologic activity. Substitution or modification of residues of NKA with natural or unnatural amino acids have shown that residues in the consensus C-terminal sequence are essential for activity toward all three type of receptors, whereas other positions can apparently tolerate substitutions [17], [19], [21].

The study of the relationship between chemical structure and pharmacological properties of neuropeptides not only enables us to determine the most biologically important residues but also aids prediction of interactions of the peptide with a putative receptor binding site [11], [16]. The presence of specific structural features in the peptide structure can be used to design new analogs sharing those specific three-dimensional properties and any biologic specificity associated with them.

Molecular dynamics (MD) attempts to describe the motion of the atoms as a function of time in a molecular system and has frequently been used to study peptides [14]. The complementary technique of essential dynamics (ED) analysis [1] can be used to determine the most significant concerted motions within a MD trajectory. This powerful new combination of techniques has been applied to a variety of proteins of all sizes [6], [10], [15].

In this study, we have gained insight into the mechanism of action of NK-2 agonist by structure-activity studies of receptor–peptide interaction in combination with molecular and essential dynamics analysis of the peptide structure in solution.

Section snippets

Materials

2-[125I]Iodohistidyl neurokinin A (2200 Ci/mmol, radiochemical purity > 99%) was obtained from DuPont/NEN. pFastBac1 baculovirus expression vector, CELLFECTIN™, Escherichia coli DH10BAC cells and TC-100 insect medium were purchased from Life Technologies.

Synthesis, purification and quality assessment of neurokinin A analogs

Peptides were synthesized on a MilliGen/Biosearch 9050 PepSynthesizer (PerSeptive Biosystems) by using solid-phase 9-fluorenylmethoxycarbonyl (FMOC) chemistry. C-terminally activated FMOC-amino acid OPfp-esters were used (except for serine and

Binding potencies of neurokinin A analogs

The wide spectrum of NKA action, and the potential usefulness of its receptor NK-2 as a target for antiasthmatic drugs, has stimulated many structure-activity relationship studies [4], [9], [17], [18], [19], [21], [22]. In the present study, we apply a systematic substitution of tyrosine modified neurokinin A ([Tyr1]-NKA or [Tyr0]-NKA) with l-Cys. The resulting analogs were tested for ability to bind NK-2 receptor to assess the role of each NKA residue in the interaction with NK-2 receptor.

Discussion

The replacement of His1 and Lys2 with Cys lead to analogs with affinities comparable to NKA, showing that these amino acids probably do not interact directly with the receptor and that their substitution does not perturb the bioactive conformation. This is in agreement with the finding that deletion of the first three N-terminal residues yields the fragment NKA4–10, that is more selective than the full-length peptide for the NK-2 receptor. Substitution of residues in positions 3, 4, and 5 shows

Acknowledgements

We thank B.L. de Groot for his support during the computational analysis.

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  • Mutations in the extracellular amino-terminal domain of the NK2 neurokinin receptor abolish cAMP signaling but preserve intracellular calcium responses

    2002, Journal of Biological Chemistry
    Citation Excerpt :

    Therefore, binding to R0 or to R1 could not be kinetically resolved. The cysteine substitution of Asp4 of the NKA analogs (Tyr1-NKA and Tyr0-NKA) was described to reduce 23-fold the affinity for the human NK2 receptor compared with NKA (33). By competition experiments with [3H]SR48968, we have determined a 9-fold loss of affinity of TRC4-NKA for the Y-NK2wt receptor compared with NKA or TR1-NKA.

☆ This work was supported by the Commission of European Communities.

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