FULL PAPER DOI: 10.1002/ejoc.201301773 Structural Prerequisites for Receptor Binding of Helicokinin I, a Diuretic Insect Neuropeptide from Helicoverpa zea Chien Tran Van, [a] Tino Zdobinsky, [b] Guiscard Seebohm, [c] Dirk Nennstiel, [d] Oliver Zerbe,* [e] and Jürgen Scherkenbeck* [a] Keywords: Synthetic methods / Amino acids / Peptidomimetics / Insect neuropeptides / Helicokinin I In insects essential physiological processes such as muscle activity or water balance are controlled by neuropeptides. However, owing to their metabolic instability and adverse physicochemical properties peptides are unsuited as crop protection agents. Helicokinin I, a diuretic neuropeptide of cotton pest Helicoverpa zea represents a most promising tar- get for the design of neuropeptide mimetics. Several helicok- inin analogues containing scaffolds with varying rigidity and Introduction Neuropeptides control specific physiological processes in insects. [1] Thus, insect neuropeptides and their receptors present promising targets for a new generation of insectici- dal agents that offer levels of selectivity and environmental compatibility that are absent from conventional insecti- cides. [2] Despite of all progress in insect-neuropeptide re- search a major obstacle remains to be solved. Peptides are metabolically unstable and display physicochemical and pharmacological properties that render them unsuitable for any application as crop-protection agents. Nevertheless, their short sequences and established structure-activity rela- tionships make insect neuropeptides prime candidates for the design of peptide mimetics with improved drug proper- ties. The large family of myokinins appears most promising as lead structures for peptidomimetic approaches owing to their short chain lengths of 6–13 residues and their potent diuretic activity with EC 50 values for receptor activation in the low nanomolar range. [3,4] Recently, the helicokinins [a] Institute of Organic Chemistry, University of Wuppertal, Gaußstraße 20, 42119 Wuppertal, Germany http://www.bioorganik.uni-wuppertal.de/ [b] Department of Organic Chemistry, Weizmann Institute of Science, 234 Herzl Street, 76100 Rehovot, Israel [c] Department of Myocellular Electrophysiology, University of Münster, Albert-Schweitzer Campus 1, 48149 Münster [d] Bayer CropScience AG, Alfred-Nobel-Straße 50, 40789 Monheim, Germany [e] Institute of Organic Chemistry, University of Zürich, Winterthurerstraße 190, 8057 Zürich, Switzerland http://www.chem.uzh.ch/zerbe/index.html Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.201301773. © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2014, 2714–2725 2714 different orientations of the N- and C-terminal peptide chains were synthesized and tested for receptor binding. Additional conformational analyses by NMR spectroscopy in a mem- brane-mimicking environment together with MD simulations provide a deeper insight into the structural requirements for receptor binding and explain the remarkable activity of a macrocyclic helicokinin I derivative. have also been reported to inhibit weight gain by larvae of the tobacco budworm (Heliothis virescens), a serious agri- cultural pest. [5] One strategy to find analogues of a biologically active peptide comprises high-throughput screenings of large com- pound collections. Following this approach ten thousand compounds were tested for agonistic activity on the diuretic helicokinin receptor from Heliothis virescens . Surprisingly, not a single hit was found during this mass screening cam- paign, calling into questions, once again, the value of this strategy. [6] Obviously, the helicokinin receptor only tolerates very limited structural variations of the natural peptide mo- tif. Therefore, a rational strategy holds much more promise to identify active peptidomimetic analogues of helicoki- nin I. [7] The ideal basis for the rational design of peptidomimetics is of course the knowledge of the receptor-bound neuropep- tide conformation. Unfortunately, this remains a challenge because neuropeptides bind to membrane-bound G pro- tein-coupled receptors, which are notoriously difficult to crystallize. [8,9] In fact, there have been no reports describing the conformation of a receptor-bound insect neuropeptide. As a consequence only indirect information on biologically active conformations of insect neuropeptides is available. Several efforts to get a better insight into the conforma- tional prerequisites for receptor binding of kinins have been undertaken. For instance, macrocyclic analogues of the ach- etakinin core (Phe-Phe-Pro-Trp-GlyNH 2 ) were prepared in order to freeze potentially relevant conformations into a small, conformationally restricted structure that can easily be analyzed by conventional NMR spectroscopy. Based on those cyclic neuropeptide analogues together with peptido- mimetic approaches and molecular dynamic (MD) simula-