Peptoid−Peptide Hybrids as Potent Novel Melanocortin Receptor

May 25, 2005 - The length of the functional group of Trp was more important for MC3 and MC5 than for MC4 receptor binding. In general, the potency of ...
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Articles Peptoid-Peptide Hybrids as Potent Novel Melanocortin Receptor Ligands John A. W. Kruijtzer,‡ Wouter A. J. Nijenhuis,§ Nienke Wanders,§ Willem Hendrik Gispen,§ Rob M. J. Liskamp,*,‡ and Roger A. H. Adan*,§ Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands, and Department of Pharmacology and Anatomy, Rudolf Magnus Institute of Neuroscience, University Medical Center, P.O. Box 85060, 3508 AB Utrecht, The Netherlands Received December 7, 2004

All possible peptoid-peptide hybrids of an MC4 receptor agonist were synthesized and investigated on cells expressing different melanocortin (MC) receptor subtypes and for rat grooming behavior. In general, receptor selectivity remained while affinity and potency were decreased. The length of the functional group of Trp was more important for MC3 and MC5 than for MC4 receptor binding. In general, the potency of the peptoid-peptide hybrids to increase rat excessive grooming behavior correlated well with MC4 receptor pharmacology. Introduction “Peptoids” represent a class of peptidomimetics that, since their introduction more than a decade ago,1,2 have undergone tremendous developments. They are excellently suitable for transformation of peptides consisting of several amino acid residues into mimics retaining the biological activity of the original peptides while diminishing some of their disadvantages such as sensitivity to proteases.1,3-6 Despite the absence of crucial peptide features including chirality (with the exception of proline) and amide N-H’s, impressive examples of biologically active peptoids have been described.1,5-26 These include peptoid peptidomimetics that can serve as molecular transporters by enabling or enhancing the cellular uptake of agents7 and a tripeptoid library that was used for the identification of melanocyte-stimulating hormone (MSH) and gastrin-releasing peptide (GRP)/bombesin receptor ligands.8 The melanocortin-4 receptor (MC4-r) is an excellent drug target for obesity. Mutations of MC4-r in humans and mice have been associated with the development of obesity. MC4-r antagonists increase food intake and body weight, and MC4-r agonists have been shown to decrease food intake and body weight in various obesity models.9 Most available MC4-r agonists are peptides. To understand better the structural requirements for receptor activation, we generated peptoid-peptide hybrids. Haskell-Luevano et al.10 have described the synthesis and activity of peptoids and several peptidepeptoid hybrids based on the Ac-His-Phe-Arg-Trp-NH2 tetrapeptide sequence as melanocortin agonists. Recently we described the discovery and in vivo evaluation * To whom correspondence should be addressed. For R.M.J.L: phone, +31 30 2537396; fax, 31 30 2536655; e-mail, R.M.J.Liskamp@ pharm.uu.nl. For R.A.H.A.: phone, + 31 30 2538517; fax, +31 30 2539032; e-mail, [email protected]. ‡ Utrecht University. § University Medical Center.

of melanocortin-4-receptor-selective ligands, i.e., Ac-NleGly-Lys-D-Phe-Arg-Trp-Gly-NH2 (1) and Ac-Nle-GlyLys-D-Nal(2)-Arg-Trp-Gly-NH2.27 Here, we report the systematic transformation of the former heptapeptide ligand to all possible combinations of the corresponding peptoid-peptide hybrids and show their potency and selectivity in vitro on MC receptors expressed on cell lines. Using the grooming assay in rats as a bioassay for MC4 receptor activity, we demonstrate that these ligands display in vivo efficacy. Results Despite the very convenient “submonomer” approach of Zuckerman et al.,28 we have focused on the “monomer” approach in order to avoid a large excess of peptoid building blocks in solid-phase synthesis approaches.1,5,29 Moreover, progress of the solid-phase synthesis can be monitored, which is crucial for the quality of the end product, especially when it consists of several peptoid residues. Therefore, Fmoc-protected N-substituted glycine building blocks were developed, which can be used together with regular Fmoc amino acid derivatives in commercial (robot) synthesizers to prepare peptoidpeptide hybrids.1,5,29,30 All peptoid-peptide hybrids were prepared by solidphase synthesis using Fmoc amino acids for introduction of amino acid residues and using Fmoc peptoid monomers (Figure 1) for the peptoid residues at the appropriate positions in the peptoid-peptide hybrids derived from 1 (Ac-Nle-Gly-Lys-D-Phe-Arg-Trp-Gly-NH2, Figure 2). All hybrids were based on this potent and selective agonist for the hMC4 receptor,27 also denoted as (Nle4, Gly5, Lys6, D-Phe7) R-MSH(4-10). A peptoid-peptide hybrid library containing 32 ligands based on this ligand was thus synthesized. The structures of the peptide and representative peptoid-peptide hybrids are shown in Figure 2. Ligand 1 contains two Gly residues, and at all other positions, peptoid residues were introduced systematically using the monomers shown in Figure 3.

10.1021/jm0490033 CCC: $30.25 © 2005 American Chemical Society Published on Web 05/25/2005

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Figure 1. Structures of the used peptoid monomers.

Figure 2. Melanocortin-4-receptor peptide ligand 1, its “complete” peptoid derivative (3), peptide-peptoid hybrid ligand 4, peptidepeptoid derivatives (17, 26, 36, and 38) with NTrp or NhTrp peptoid residues. Lower part: side chains and the corresponding peptoid residue abbreviations.

The resulting peptoid-peptide hybrids were analyzed for activation of and binding to the MC3, MC4, and MC5 receptors. First, the activity of these peptide-peptoid hybrids was determined in a reporter gene assay measuring MC receptor activity (Figure 3).

Two general trends are apparent from this library. First, introduction of peptoid monomers resulted in peptoid-peptide hybrid ligands with an increase in EC50 values of 1-2 orders of magnitude for every additional peptoid monomer that was introduced into the core

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Figure 3. Activation potencies of peptide-peptoid hybrids (containing NhTrp). The -log(EC50) values (M) with 95% confidence ranges are displayed for peptoid-peptide hybrid ligands on the MC3, MC4, and MC5 receptors expressed on 293 HEK cells. Missing bars indicate that for the receptor of interest, ligands showed no or an incomplete dose-response curve at 30 µM. The shaded symbols indicate amino acids residues. In the black symbols an “N” denotes a peptoid residue and “Nh” denotes the “homo” variant of the peptoid residue.

sequence (positions 6-9). However, selectivity for the MC4 receptor usually remained. Second, replacement of the D-Phe residue on position 7 by the corresponding peptoid monomer (NPhe) strongly increased the EC50 values of the resulting ligands. This was not unexpected in view of the importance of the chirality of the Phe residue for potency of MC receptor ligands,39 and a NPhe residue is not chiral. Sequential replacement of one amino acid residue in 1 by the corresponding N-substituted glycine (peptoid) residue resulted in peptoid-peptide hybrids 3-7, which had a lower activity than the “all”-peptide ligand 1. When all combinations of two amino acids in Ac-NleGly-Lys-D-Phe-Arg-Trp-Gly-NH2 were substituted by peptoid residues (8-17), there was a trend toward higher EC50 values compared to introduction of one peptoid residue except when one of the two peptoid

monomers was introduced at position 4 (thus, outside the 6-9 core sequence). However, a satisfactory degree of activity was retained if amino acids on both positions 8 and 9 (numbering as in R-MSH) were substituted by peptoid residues (as in 17). The EC50 value of this ligand was similar for the MC4 receptor but was lower for the MC3 and -5 receptors compared to the derivatives in which either residue 8 or 9 (6 and 7, respectively) was replaced by a peptoid residue. Replacement of three or four amino acid residues led to hybrids 18-32 displaying further increased EC50 values, and the all-peptoid ligand 2 containing five peptoid residues was devoid of activity. Exceptions were 26 and 27 in which, in addition to residues 8 and 9 (as in 17), residue 4 (as in 26) or residue 6 (as in 27) was substituted by (three) peptoid residues. These results stress the importance of keeping the distance between

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Figure 4. Activation potencies of peptoid-peptide hybrids with “literal” translation of Trp into NTrp. The -log(EC50) values (M) with 95% confidence ranges are displayed of peptoid-peptide hybrid ligands on the MC3, MC4, and MC5 receptors expressed on 293 HEK cells. Missing bars indicate that for the receptor of interest, ligands showed no or an incomplete dose-response curve at 30 µM.

the side chains of Trp and Arg residues in the hybrids similar to that in the “all”-peptide ligand 1 in order to attain a satisfactory degree of potency. Since it was possible to transform residues 8 and 9 to peptoid residues and retain the biological activity to a large extent, it was decided to have a closer look at the pharmacophore tryptophane side chain to investigate if it would be possible to approach the biological activity of the peptide ligand more closely. So far, when a Trp residue was replaced by the corresponding peptoid residue, a “homo” variant (NhTrp) was used in which two carbon atoms were present between the amide nitrogen atom and the indol aromatic nucleus (Figure 1). A more “literal” translation of an amino acid-Trp residue to a peptoid-NTrp residue would be just one carbon atom between the amide nitrogen and the indole aromatic nucleus corresponding to the one-carbon atom between the R-carbon atom and the aromatic nucleus in Trp. For this purpose the required peptoid monomer (NTrp) was synthesized and subsequently incorporated, leading to peptidepeptoid hybrids 33-40. Introduction of the “NTrp” residue as in 33, 34, and 35 compared to the “NhTrp” containing derivatives 7, 11, and 14, respectively, did not affect the EC50 value on the MC4 receptor, but EC50 values of the MC3 and MC5 receptors decreased, thereby indicating an increased activation potency (Figure 4). Thus, the presence of the NhTrp residue resulted in a higher selectivity for MC4 than the presence of NTrp. It seems that 37 is roughly equally inactive as 20. Again, if the changes in peptide-peptoid hybrids also involve residue 8, then the activity was clearly diminished as was the case in 38 compared to 26, in 39 compared to 27, and in 40 compared to 30. Because the ligands with a homo-NTrp (“NhTrp”) residue were more selective than ligands with the “literal” translation of Trp (NTrp), the other important pharmacophore residue, Arg, was translated into a homopeptoid residue (NhArg) to see if it would be possible to increase the affinity/activity and selectivity. For this purpose the required peptoid monomer FmocNhArg(Boc)2-OH (Figure 1) was synthesized and subsequently incorporated into the most active peptide-

peptoid hybrids (6, 17) leading to peptide-peptoid hybrids 41 and 42. Unfortunately, upon comparison of 6 with 41, there was no decrease in EC50 values for the MC4 receptor with a NArg residue incorporated. However, the activity for the MC3 receptor was lost, whereas the activity on the MC5 receptor was increased. Thus, an increased selectivity between MC4 and MC3 receptor was observed. These results were in line with the observations of Holder et al.,40 who found that modification of the Arg residue in a tetrapeptide into a homoArg residue resulted in an increased selectivity (56-fold) between MC4 and MC3 receptors without increased activity for the MC4 receptor. When 42 is compared with 17, both the activation of MC3 and MC4 decreased slightly with introduction of NhArg compared to NArg, with no effect on MC5. All compounds that activated MC receptors were tested in a binding assay. The IC50 values (Figure 5)) correlated well with the EC50 values. Peptide-peptoid hybrids not shown in Figure 5 did not displace [125I]-NDP-MSH binding at a concentration of 30 µM. These included compounds that did not show any activity in the reporter gene assay. In general, the IC50 values were similar to or slightly higher than the EC50 values as measured in the reporter gene assay. Exceptions were 17, 36, and 42 in which Arg and Trp were both replaced by peptoid residues. For these compounds the IC50 value for the MC5 receptor were higher than the EC50 value for this receptor. Similarly in 41 a higher activity was found for the MC5 receptor than was expected based upon binding. Other exceptions were 35 and 37, in which IC50 values for all tested MC receptors were higher than EC50 values. To test the in vivo efficacy of these ligands, those ligands that activated the MC4 receptor were tested in a rat grooming assay. Figure 6 shows a clear relationship between the in vitro efficacy (EC50) of ligands, compared to the in vivo induction of grooming behavior. Hybrid 26 induced more excessive grooming than expected based upon its activation potency in vitro. Discussion Introduction of peptoid monomers in the MC4 receptor ligand 1 resulted in a decreased activation potency,

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Figure 5. Affinities of peptoid-peptide hybrids. The -log(IC50) values (M) with 95% confidence ranges are displayed of peptoidpeptide hybrid ligands on the MC3, MC4, and MC5 receptors expressed on 293 HEK cells. Missing bars indicate that for the receptor of interest, ligands showed no or partial displacement of radiolabel at 30 µM.

Figure 6. Induction of rat grooming behavior by melanocortin peptoid-peptide hybrids. Melanocortin peptoid-peptide hybrids were administered intracerebroventricularly with 3 µg of compound, and grooming behavior was scored. The Y-axis indicates the average grooming score (n ) 6) obtained by the compound. The X-axis indicates the activation potency (EC50 value in nM) in vitro on the rat MC4 receptor expressed on 293 HEK cells.

with a greater loss in potency when more peptoid building blocks were introduced. Holder et al.40 also found that tetrapeptide-peptoid hybrids displayed lower potency but only measured ligand activation potency. Here, we show that similar to the potencies (EC50 values), the affinities (IC50 values) of the compounds are also reduced. Thus, the decreased potency is probably a result of decreased affinity. This is not entirely

unexpected because the flexibility of the peptides containing peptoid residues is increased. The resulting greater conformational freedom will lead to an increased entropy loss upon binding and therefore reduction of affinity. However, there were a few discrepancies between activation potency and affinity. Introduction of peptoid residues at both Arg and Trp positions (17 and 36) resulted in a discrepancy between binding and potency for the MC5 receptor. Affinity was lower than expected on the basis of the activation studies. This suggests that either the relative position or increased conformational space due to the increased flexibility of the backbone or the side chains of Arg and Trp of melanocortin compounds is essential for activating the MC5 receptor but not for binding to this receptor. Be this as it may, the data indicate that the backbone is less important, since apparently the backbone N-H’s , which are absent in the peptoid residues, play no significant role. These findings may be helpful for the design of an MC5 receptor agonist. Since a compound that does not activate a receptor may still be an antagonist, ligands from the peptoidpeptide hybrid library that did not show activation of MCRs were tested for binding to these receptors at a single high dose (30 µM). Since no displacement of [125I]NDP-MSH could be detected, loss of activation was most likely due to decreased or lost affinity for the receptors and not to antagonistic properties of the ligands.

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The more “literal” translation NTrp (such as in 33) resulted in a smaller decrease in affinity and potency than NhTrp (such as in 7) for the MC3 and MC5 receptors but not for the MC4 receptor. Thus, the distance of the functional group of Trp to the backbone is more critical for the MC3 and MC5 receptors compared to the MC4 receptor. Deviation of length of the Arg side chain as is present in peptides by introduction of a homo-Arg side chain in a peptoid residue had little effect. This too may be helpful for the design of MC4 receptor selective ligands. Although with the introduction of peptoid residues activation potency in vitro decreased, one may expect that stability is increased. Therefore, the compounds that had the highest in vitro potency on the MC4 receptor were tested in a rat grooming assay. It was demonstrated before that melanocortin-induced excessive grooming behavior in rats is most probably mediated via the MC4 receptor.41 The data in Figure 6 show that there is a clear relationship between activation potency of the MC4 receptor in vitro and induction of rat grooming behavior. However, 26 elicited a higher grooming score than expected based upon activation potency in vitro. 26 has three peptoid residues, and from the group of hybrids with three peptoid residues, it has the highest affinity and activation potency in vitro. Although some compounds with one or two peptoid residues displayed a higher or similar activation potency on the MC4 receptor in vitro (for instance 4 and 8), 26 was more potent in inducing excessive grooming behavior. Since 26 has three peptoid residues, this suggests that increased stability due to higher resistance to degradation contributed to increased potency in vivo. However, as an alternative explanation for differences between the in vitro and in vivo potencies, we cannot exclude that one of the compounds acted on a receptor or protein other than an MC receptor, which may have affected the grooming response. For determining EC50 values in vitro, we used a sensitive reporter gene assay in which all agonists behaved as full agonists. Less sensitive but possibly biologically more relevant assays may indicate differences in agonist efficacies of the ligands that were characterized here. Therefore, we cannot exclude that differences in efficacies of the ligands contribute to the differences in potencies to induce grooming behavior in the rat. Although introduction of peptoid moieties in an MC4 receptor peptide resulted in a decrease of affinity, many of the compounds were still active at submicromolar concentrations. In addition, receptor selectivity was maintained to a considerable extent. Fortuitously, this was even the case in some peptide-peptoid residues containing up to three peptoid residues. Finally, the data also suggest that bioavailability was increased when three (approximately half of the compound) of seven amino acids were replaced by peptoid residues.

from indole-3-carboxaldehyde.31,32 Fmoc-NhArg(Boc)2-OH was synthesized in two steps from Fmoc-NLys(Boc)-OH involving guanylation of the amino group with N,N′-bis-Boc-1-guanylpyrazole.33,34 Peptoid-peptide hybrids were synthesized employing the “monomer approach”5 in which Fmoc-protected N-substituted glycine monomers (peptoid monomers) and Fmoc-protected amino acids are coupled. Binding Assay. Binding experiments on the human MC3 or MC4 receptors or the mouse MC5 receptors were carried out according to Oosterom et al.35 Activation assay. Activation of human MC3 or MC4 receptors (and rat MC4 receptor) or the mouse MC5 receptors was determined using LacZ as a reporter gene.36 Grooming Assay. Male Wistar rats weighing 200-240 g at the start of the study were used. Rat received an intracerebroventricular (icv) cannula as described by Brakkee et al.,37 and grooming assays were performed as described by Gispen et al.38

Experimental Section Synthesis of Peptoid-Peptide Hybrids. Required peptoid monomers, i.e., Fmoc-protected N-substituted glycines, Fmoc-NNle-OH, Fmoc-NLys(Boc)-OH, Fmoc-NArg(Boc)2-OH, and Fmoc-NhTrp(Boc)-OH, were synthesized as described earlier.1,29,30 Fmoc-Trp(Boc)-OH was synthesized in four steps

Acknowledgment. Financial support by NWOSTIGO is gratefully acknowledged. Supporting Information Available: Additional experimental data on the synthesis of peptoid-peptide hybrids, including yields and purity and mass data, full details of the binding, activation, and grooming assay, and a table containing the EC50 values of the compounds and their ranges. This material is available free of charge via the Internet at http:// pubs.acs.org.

References (1) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.; Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen, F. E.; Bartlett, P. A. Peptoids: a modular approach to drug discovery. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 93679371. (2) Kessler, H. Peptoids: Key compounds in a new path to pharmaceutically active substances. Angew. Chem., Int. Ed. Engl. 1993, 32, 543-544. (3) Miller, S. M.; Simon, R. J.; Ng, S.; Zuckermann, R. N.; Kerr, J. M.; Moos, W. H. Proteolytic studies of homologous peptide and N-substituted glycine peptoid oligomers. Bioorg. Med. Chem. Lett. 1994, 4, 2657-2662. (4) Miller, S. M.; Simon, R. J.; Ng, S.; Zuckermann, R. N.; Kerr, J. M.; Moos, W. H. Comparison of the proteolytic susceptibilities of homologous L-amino acid, D-amino acid, and N-substituted glycine peptide and peptoid oligomers. Drug Dev. Res. 1995, 35, 20-30. (5) Kruijtzer, J. A. W.; Hofmeyer, L. J. F.; Heerma, W.; Versluis, C.; Liskamp, R. M. J. Solid-phase syntheses of peptoids using Fmoc-protected N-substituted glycines, the synthesis of (retro)peptoids of leu-enkephalin and substance P. Chem.sEur. J. 1998, 4, 1570-1580. (6) Wang, Y.; Lin, H.; Tullman, R.; Jewell, C. F.; Weetall, M. L.; Tse, F. L. S. Absorption and disposition of a tripeptoid and a tetrapeptide in the rat. Biopharm. Drug Dispos. 1999, 20, 6975. (7) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. U.S.A. 2000, 1303-13008. (8) Heizman, G.; Hildebrand, P.; Tanner, H.; Ketterer, S.; Pansky, A.; Froidevaux, S.; Beglinger, C.; Eberle, A. N. Combinatorial peptoid library for the identification of novel MSH and GRP/ bombesin receptor ligands. J. Recept. Signal Transduction Res. 1999, 19, 449-466. (9) O’Rahilly, S.; Farooqi, I. S.; Yeo, G. S.; Challis, B. G. Minireview: human obesityslessons from monogenic disorders. Endocrinology 2003, 144, 3757-3764. (10) Holder, J. R.; Bauzo, R. M.; Xiang, Z.; Scott, J.; Haskell-Luevano, C. Design and pharmacology of peptoids and peptide-peptoid hybrids based on the melanocortin agonists core tetrapeptide sequence. Bioorg. Med. Chem. Lett. 2003, 13, 4505-4509. (11) Zuckermann, R. N.; Martin, E. J.; Spellmeyer, D. C.; Stauber, G. B.; Shoemaker, K. R.; Kerr, J. M.; Figliozzi, G. M.; Goff, D. A.; Siani, M. A.; Simon, R. J.; Banville, S. C.; Brown, E. G.; Wang, L.; Richter, L. S.; Moos, W. H. Discovery of nanomolar ligands for 7-transmembrane G-protein-coupled receptors from a diverse N-(substituted)glycine peptoid library. J. Med. Chem. 1994, 37, 2678-2685.

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Kruijtzer et al.

(12) Gibbons, J. A.; Hancock, A. A.; Vitt, C. R.; Knepper, S.; Buckner, S. A.; Brune, M. E.; Milicic, I.; Kerwin, J. F.; Richter, L. S.; Taylor, E. W.; Spear, K. L.; Zuckermann, R. N.; Spellmeyer, D. C.; Braeckman, R. A.; Moos, W. H. Pharmacologic characterization of CHIR 2279, an N-substituted glycine peptoid with highaffinity binding for alpha 1-adrenoceptors. J. Pharmacol. Exp. Ther. 1996, 277, 885-899. (13) Goodfellow, V. S.; Marathe, M. V.; Kuhlman, K. G.; Fitzpatrick, T. D.; Cuadrado, D.; Hanson, W.; Zuzack, J. S.; Ross, S. E.; Wieczorek, M.; Burkard, M.; Whalley, E. T. Bradykinin receptor antagonists containing N-substituted amino acids: in vitro and in vivo B(2) and B(1) receptor antagonist activity. J. Med. Chem. 1996, 39, 1472-1484. (14) Hamy, F.; Felder, E. R.; Heizmann, G.; Lazdins, J.; Aboul-El, F.; Varani, G.; Karn, J.; Klimkait, T. An inhibitor of the Tat/ TAR RNA interaction that effectively suppresses HIV-1 replication. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 3548-3553. (15) Tran, T. A.; Mattern, R. H.; Afargan, M.; Amitay, O.; Ziv, O.; Morgan, B. A.; Taylor, J. E.; Hoyer, D.; Goodman, M. Design, synthesis, and biological activities of potent and selective somatostatin analogues incorporating novel peptoid residues. J. Med. Chem. 1998, 41, 2679-2685. (16) Murphy, J. E.; Uno, T.; Hamer, J. D.; Cohen, F. E.; Dwarki, V.; Zuckermann, R. N. A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1517-1522. (17) Re´ve´sz, L.; Bonne, F.; Manning, U.; Zuber, J. F. Solid phase synthesis of a biased mini tetrapeptoid-library for the discovery of monodentate ITAM mimics as ZAP-70 inhibitors. Bioorg. Med. Chem. Lett. 1998, 13, 405-408. (18) Goodman, M.; Bhumralker, M.; Jefferson, E. A.; Kwak, J.; Locardi, E. Collagen mimetics. Biopolymers 1998, 47, 127-142. (19) Ng, S.; Goodson, B.; Ehrhardt, A.; Moos, W. H.; Siani, M.; Winter, J. Combinatorial discovery process yields antimicrobial peptoids. Bioorg. Med. Chem. 1999, 7, 1781-1785. (20) Nguyen, J. T.; Porter, M.; Amoui, M.; Miller, W. T.; Zuckermann, R. N.; Lim, W. A. Improving SH3 domain ligand selectivity using a non-natural scaffold. Chem. Biol. 2000, 7, 463-473. (21) Daelemans, D.; Schol, D.; Witvrouw, M.; Pannecouque, C.; Hatse, S.; Van Dooren, S.; Hamy, F.; Klimkait, T.; De Clercq, E.; Vandamme, A. M. A second target for the peptoid Tat/transactivation response element inhibitor CGP64222: inhibition of human immunodeficiency virus replication by blocking CXCchemokine receptor 4-mediated virus entry. Mol. Pharmacol. 2000, 57, 116-124. (22) Ruijtenbeek, R.; Kruijtzer, J. A. W.; van de Wiel, W.; Fischer, M. J. E.; Fluck, M.; Redegeld, F. A. M.; Liskamp, R. M. J.; Nijkamp, F. P. Peptoid-peptide hybrids that bind Syk SH2 domains involved in signal transduction. ChemBioChem 2001, 2, 171-179. (23) de Haan, E. C.; Wauben, M. H. M.; Grosfeld-Stulemeyer, M. C.; Kruijtzer, J. A. W.; Liskamp, R. M. J.; Moret, E. E. Major histocompatibility complex class II binding characteristics of peptoid-peptide hybrids. Bioorg. Med. Chem. 2002, 10, 19391945. (24) Peretto, I.; Sanchez-Martin, R. M.; Wang, X. H.; Ellard, J.; Mittoo, S.; Bradley, M. Cell penetrable peptoid carrier vehicles: synthesis and evaluation. Chem. Commun. 2003, 2312-2313. (25) Patch, J. A.; Barron, A. E. Helical peptoid mimics of magainin-2 amide. J. Am. Chem. Soc. 2003, 125, 12092-12093. (26) Biondi, L.; Giannini, E.; Filira, F.; Gobbo, M.; Marastoni, M.; Negri, L.; Scolaro, B.; Tomatis, R.; Rocchi R. Synthesis, conformation and biological activity of dermorphin and deltorphin I

analogues containing N-alkylglycine in place of residues in position 1, 3, 5 and 6. J. Pept. Sci. 2003, 9, 638-648. Nijenhuis, W. A. J.; Kruijtzer, J. A. W.; Wanders, N.; Vrinten, D. H.; Garner, K. M.; Schaaper, W. M. M.; Meloen, R. H.; Gispen, W. H.; Liskamp, R. M. J.; Adan, R. A. H. Discovery and in vivo evaluation of new melanocortin-4 receptor-selective peptides. Peptides 2003, 24, 271-280. Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. Method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase synthesis. J. Am. Chem. Soc. 1992, 114, 10646-10647. Kruijtzer, J. A. W.; Liskamp, R. M. J. Synthesis in solution of peptoids using Fmoc-protected N-substituted glycines. Tetrahedron Lett. 1995, 36, 6969-6972. Kruijtzer, J. A. W.; Synthesis of Peptoid Peptidomimetics. Ph.D. Thesis, Utrecht University, The Netherlands, 1996. Wolman, Y. Simple synthesis of 1-tert-butyloxycarbonyl-3formylindole. Synthesis 1975, 732. Ten Kortenaar, P. B. W.; Van Dijk, B. G.; Peeters, J. M.; Raaben, B. J.; Adams, P. J. H. M.; Tesser, G. I. Rapid and efficient method for the preparation of Fmoc-amino acids starting from 9-fluorenylmethanol. Int. J. Pept. Protein Res. 1986, 27, 398-400. Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Urethane protected derivatives of 1-guanylpyrazole for the mild and efficient preparation of guanidines. Tetrahedron Lett. 1993, 34, 33893392. Wu, Y.; Matsueda, G. R.; Bernatowicz, M. An efficient method for the preparation of ω,ω′-bisurethane protected arginine derivatives. Synth. Commun.1993, 23, 3055-3060. Oosterom, J.; Nijenhuis, W. A.; Schaaper, W. M.; Slootstra, J.; Meloen, R. H.; Gispen, W. H.; Burbach, J. P.; Adan, R. A. Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors. J. Biol. Chem. 1999, 274, 16853-16860. Chen, W.; Shields, T. S.; Stork, P. J.; Cone, R. D. A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. Anal. Biochem. 1995, 226, 349-354. Brakkee, J. H.; Wiegant, V. M.; Gispen, W. H. A simple technique for rapid implantation of a permanent cannula into the rat brain ventricular system. Lab. Anim. Sci. 1979, 29, 78-81. Gispen, W. H.; Wiegant, V. M.; Bradbury, A. F.; Hulme, E. C.; Smyth, D. G.; Snell, C. R.; de Wied, D. Induction of excessive grooming in the rat by fragments of lipotropin. Nature 1976, 264, 794-795. Sawyer, T. M.; Sanfilippo, P. J.; Hruby, V. J.; Engel, M. H.; Heward, C. B.; Burnett, J. B.; Hadley, M. E. 4-Norleucine, 7-Dphenylalanine-alpha-melanocyte-stimulating hormone: a highly potent alpha-melanotropin with ultralong biological activity. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 5754-5758. Holder, J. R.; Xiang, Z.; Bauzo, R. M.; Haskell-Luevano, C. Structure-activity relationships of the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 at the mouse melanocortin receptors. Part 3: modifications at the Arg position. Peptides 2003, 24, 73-82. Adan, R. A.; Szklarczyk, A. W.; Oosterom, J.; Brakkee, J. H.; Nijenhuis, W. A.; Schaaper, W. M.; Meloen, R. H.; Gispen, W. H. Characterization of melanocortin receptor ligands on cloned brain melanocortin receptors and on grooming behavior in the rat. Eur. J. Pharmacol. 1999, 378, 249-258.

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