Discovery of a linear lead antagonist to the insect pheromone biosynthesis activating neuropeptide (PBAN) Irina Zeltser a , Chaim Gilon a , Orna Ben-Aziz b , Irit Schefler b , Miriam Altstein b, * a Dept. of Organic Chemistry, The Hebrew Univ. of Jerusalem, Jerusalem 91904, Israel b Dept. of Entomology, The Volcani Center, Bet Dagan 50250, Israel Received 28 January 2000; accepted 29 June 2000 Abstract We report the discovery of a linear lead antagonist for the insect pheromone biosynthesis activating neuropeptide (PBAN) which inhibits sex pheromone biosynthesis in the female moth Heliothis peltigera. Two approaches have been used in attempting to convert PBAN agonists into antagonists. The first involved omission of the C-terminal amide and reduction of the sequence from the N-terminus in a linear library based on PBAN 1–33NH 2. The second involved replacement of L amino-acids by the D hydrophobic amino acid D-Phe in a linear library based on PBAN28 –33NH 2. Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of one compound out of the D-Phe library (Arg-Tyr-Phe-D-Phe-Pro-Arg-Leu-NH 2 ) which inhibited sex pheromone production by 79 and 64% at 100 pmol in two moth colonies and exhibited low agonistic activity. Omission of the C-terminal amide in PBAN 1–33NH 2 and its shorter analogs did not lead to the discovery of an antagonistic compound. © 2000 Elsevier Science Inc. All rights reserved. Keywords: PBAN; Pheromone biosynthesis; Insect neuropeptides; Heliothis peltigera; Neuropeptide antagonists 1. Introduction The pheromone biosynthesis activating neuropeptide (PBAN) is an important neuropeptide that mediates some of the key functions in insects. PBAN was first reported in 1984, as the neuropeptide that regulates sex pheromone production in female moths [51] and its amino acid se- quence was revealed in 1989 from Helicoverpa zea (Hez- PBAN; PBAN 1–33NH 2 ) [52]. Since then, six other PBAN molecules have been isolated from five additional moth species, and their entire primary structures have been de- termined [9,16,29,32,33,42] and c-DNA and genes have been cloned [9,14,16,29 –31,40]. PBAN molecules were found to be C-terminally amidated neuropeptides consisting of 33–34 amino acids, and comparison of their primary structures revealed that they share a high degree of homol- ogy and an identical pentapeptide C-terminal sequence (Phe-Ser-Pro-Arg-Leu-NH 2 ) which composes the active core required for its biologic activity [2,3,5,6,34,49,53,55]. Since 1984, the presence of PBAN-like activity has been demonstrated in a variety of moths as well as other non- Lepidopteran species, and its mode of action has been stud- ied extensively (for review see [19,56]). Further studies on the regulation of sex pheromone bio- synthesis in moths have revealed that additional neuropep- tides isolated from various insects, all of which share the common C-terminal pentapeptide of PBAN (Phe-Xxx-Pro- Arg-Leu-NH 2 ; Xxx = Ser, Gly, Thr, Val), have the ability to evoke sex pheromone biosynthesis upon injection into female moths [1,18,37,38,64]. Among these peptides are the pyrokinins (Lem-PK, Lom-PK-I and Lom-PK-II) and the locustamyotropins (Lom-MT-I to IV) (myotropic peptides isolated from the Madeira cockroach Leucophaea maderae (Fabricius) and the migratory locust, Locusta migratoria [47,63]; pheromonotropin (Pss-PT) an 18-amino acid pep- tide isolated from Pseudaletia (Mythimna) separata (Walk- er) [44] and diapause hormone (Bom-DH) isolated from the silkworm, Bombyx mori (L.) [28]. These peptides have recently been designated the pyrokinin/PBAN family. In addition to their ability to stimulate sex pheromone biosyn- thesis in moths, members of this family have been found to  Contribution from the Institute of Plant Protection, Agricultural Re- search Organization, The Volcani Center, Bet Dagan, Israel. No. 507/00 series. * Corresponding author. Tel.: +972-3-968-3710; fax: +972-3-968- 3835. E-mail address: vinnie2@netvision.net.il (M. Altstein). Peptides 21 (2000) 1457–1465 0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0196-9781(00)00298-9