A Novel Conformation in a Highly Potent, Constrained Gonadotropin-Releasing Hormone Antagonist Josep Rizo, ² R. Bryan Sutton, ² Joshua Breslau, ² Steven C. Koerber, ‡ John Porter, ‡ Arnold T. Hagler, § Jean E. Rivier,* ,‡ and Lila M. Gierasch* ,², | Contribution from the Department of Pharmacology, UniVersity of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines BouleVard, Dallas, Texas 75235-9041, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037, and Biosym Technologies, Inc., 9685 Scranton Road, San Diego, California 92121 ReceiVed September 19, 1995 X Abstract: Through design, synthesis, and biological testing of constrained gonadotropin releasing hormone (GnRH) antagonists, we are studying the structural requirements for biological activity. Here we describe the conformational analysis in solution of a highly potent, dicyclic GnRH antagonist, dicyclo(4-10/5,5′-8)[Ac-D-2Nal 1 ,D-pClPhe 2 ,D- 3Pal 3 ,Asp 4 ,Glu 5 (Gly),D-Arg 6 ,Dbu 8 ,Dpr 10 ]GnRH (1), using NMR spectroscopy. The dicyclic part of this molecule adopts a preferred conformation containing a type II  turn around residues 5-6, nested with a type I′  turn around residues 6-7, and a type II -turn-like structure involving residue 9 and the side chain of residue 10, which is stabilized by hydrogen bonds between Leu 7 NH/Asp 4 CO, Dbu 8 NH δ /Glu 5 CO, and Dpr 10 NH γ /Dbu 8 CO. This is a novel conformation that had not been observed previously in any constrained GnRH antagonist and is remarkably different from that found for another dicyclic (4-10/5-8) GnRH antagonist with very similar sequence, dicyclo(4- 10/5-8)[Ac-D-2Nal 1 ,D-pClPhe 2 ,D-Trp 3 ,Asp 4 ,Glu 5 ,D-Arg 6 ,Lys 8 ,Dpr 10 ]GnRH (2) (Bienstock et al. J. Med. Chem. 1993, 36, 3265-3273). The conformation of 2 contains a type II′  turn around residues 6-7, which had been proposed to be essential for GnRH activity. These results are important for our general understanding of polypeptide conformation, since they show that the dicyclo(4-10/5-8) backbone can adopt more than one family of conformations despite its dicyclic nature, and from the point of view of the design of GnRH antagonists, since they suggest that the presence of a turn around residues 6-7, rather than the type of  turn, may be necessary for biological activity. Naturally occurring peptides regulate a large diversity of biological functions and thus are logical targets for the design of drugs with clinical applications. Drug design based on such peptides is commonly hindered by difficulties in obtaining information on the structural requirements for biological activ- ity: The peptides are usually flexible and unstructured in solution, while direct determination of receptor bound confor- mations is hampered because most receptors are integral membrane proteins. A powerful strategy to overcome these problems involves the use of covalent constraints designed to induce particular conformational features that are suspected to be important for biological activity. 1 The constraints can increase biological activity by reducing the entropy loss upon receptor binding, if they preinduce correct conformations in solution. In addition, such conformations can then be studied directly in the constrained analogs, in the absence of receptor. Even if the conformations forced by the constraints are not optimal for biological activity, the increased likelihood that the structures adopted by the constrained analogs in solvent are analogous to their receptor-bound structures makes conforma- tional analyses in solution more meaningful. The results of such analyses can thus be used to refine putative binding conforma- tions, to suggest alternative or additional constraints, and, in general, to rationalize the observed activities on structural grounds. 1d Key considerations in the strategy outlined above are as follows. (i) To what extent do the constraints introduced limit the conformational possibilities of the resulting analogs? (ii) Do the conformations in solution of the constrained analogs indeed correspond to their biologically active conformations? (iii) Do analogous constraints always result in the same preferred conformations? To shed light on these questions, we have analyzed the conformation in solution of a highly potent, dicyclic antagonist of gonadotropin-releasing hormone (GnRH), dicyclo- (4-10/5,5′-8)[Ac-D-2Nal 1 ,D-pClPhe 2 ,D-3Pal 3 ,Asp 4 ,Glu 5 (Gly),D- Arg 6 ,Dbu 8 ,Dpr 10 ]GnRH (1) (Figure 1, Table 1). Here we describe the results of this analysis, and compare them with those obtained previously 2 for an equipotent GnRH antagonist incorporating the same type of constraints, dicyclo(4-10/5-8)- [Ac-D-2Nal 1 ,D-pClPhe 2 ,D-Trp 3 ,Asp 4 ,Glu 5 ,D-Arg 6 ,Lys 8 ,Dpr 10 ]- GnRH (2). GnRH is a linear decapeptide hormone involved in the regulation of ovulation and spermatogenesis. 3 Both 1 and 2 were designed in the context of ongoing efforts in our ² University of Texas Southwestern Medical Center at Dallas. ‡ The Salk Institute. § Biosym Technologies, Inc. | Present address: Department of Chemistry, Lederle Graduate Research Center, University of Massachusetts, Amherst, MA 01003-4510. 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