J. Med. Chem. 1989,32,597-601 of 5 mg/ kg given intravenously 40 min apart. Arterial blood pressure was taken at 1-, 3-, and 5-h i n t e ~ &after dosing to a s w drug effects. All animals were habituated to the test procedure through their long history of testing. To investigate the mechanism of antihypertensive action, male SHR of the Okamoto-Aoki strain (Taconic Farms, Germantown, NY) of approximately 350 g body weight were restrained in a supine position with elastic tape (Elastikon, Johnson & Johnson, New Brunswick, NJ). The area a t the base of the tail was locally anesthetized by subcutaneous infiltration with 2% procaine. The ventral caudal artery was isolated and a cannula of PE 10 and P E 20 fused tubing was passed into the lower abdominal aorta. The cannula was secured, heparinized (1000 IU/mL), and sealed and the wound closed. A second cannula was introduced into the lateral caudal vein for the intravenous administration of challenge substances. Control responses to the challenges were recorded prior to drug administration, after which each animal received
597
either vehicle (10 mL/kg) or compound under study orally. One hour later, the challenges were repeated. The animals were tilted 7 5 O for 30 s for effect on B P and the challenges administered intravenously. The challenges consisted of epinephrine (Epi) 1 and 2 pg/kg, norepinephrine (NE) 1 pg/kg, isoproterenol (Iso) 1pg/kg, acetylcholine (ACh) 2 pglkg, angiotensin I1 (Angio) 0.2 pg/kg, tyramine (Tyr) 250 pg/kg, and l,l-dimethy1-4-phenylpiperazinium iodide (DMPP) 25 pg/kg. All challenges were calculated as the free base. Each challenge concentration was adjusted such that the volume required was 0.5 mL/kg body weight and followed by 0.1 mL of 0.9% sodium chloride to wash any residual agent from the cannula.
Acknowledgment. We thank Drs. J. James, R. Ryall, and group for microanalysis, J. Baldoni and staff for spectral analysis, and Dr. J. Emma and group for diuretic testing.
Synthesis and Hypertensive Activity of Neuropeptide Y Fragments and Analogues with Modified N- or C-Termini or D-Substitutionst J. H. Boublik, N. A. Scott,* M. R. Brown,* and J. E. Rivier* Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, P.O. Box 85800,Sun Diego, California 92138-9216, and University of California Sun Diego Medical Center, Dickinson Street, Sun Diego, California 92103. Received June 3, 1988 Porcine neuropeptide Y (NPY), NPY fragments, and analogues with D-&a", Ala9, D-Alaa, and Met" substitutions or modifications to the C- or N-termini were synthesized. The synthesis and purification of these peptides was achieved by using routine laboratory strategies and techniques. The ability of these peptides to alter mean arterial pressure (MAP)and heart rate (HR) in conscious rats was monitored for 15 min following intraarterial administration. Potencies and efficacies of these peptides relative to NPY were determined by comparison of dose-response curves. Administration of 40 pg/kg NPY resulted in a rapid, though short-lived, rise in mean arterial pressure from a basal value of 107.0 2.6 to 157 i 5.5 mmHg (means sem, n = 13). The EDm ( M E ) for this response was 3.04 0.88 pg/kg. Peptide YY (PYY) elicited a response that was similar in magnitude but with an EDm ( M E , n = 3) of 0.76 0.24 pg/kg while porcine pancreatic polypeptide (pPP) was inactive when tested a t 40 pg/kg ( n = 4). Relative potencies for [Ac-Tyr'INPY, [ A c - D - T ~ ~ ~ I N[des-amino-Tyr'INPY, PY, and [Me-Tyr'INPY ranged from 1.1to 2.2. Potencies relative to NPY for D-substitutions at positions 2-6 and 8-13 inclusive ranged from 0.1 to 1.0. Analogues with D-substitutions at positions 1-3 exhibited an extended duration of action. Analogues with D-substitutions a t Y 10-fold less potent than NPY, suggesting positions 33-35 inclusive were inactive at 40 pg/kg, and [ D - T ~ I N P was that the integrity of the C-terminal region is critical to the overall biological action of NPY. This conclusion is supported by studies with C- and N-terminal deletion peptides. NPY2-= showed full intrinsic activity at 40 pg/kg and retains 40% of the hypertensive potency of NPY. There was a sequential decrease in efficacy upon further N-terminal deletion. In contrast to the finding with NPY2-%,modification of the C-terminus either from the native carboxamide to the free carboxylic acid or by deletion of the C-terminal residue resulted in analogues which were inactive a t 40 pg/kg. These data indicate that an essentially full-length, C-terminally amidated NPY structure is required for the hypertensive activity observed in conscious rats upon intraarterial administration of NPY and NPY analogues.
*
*
*
*
Neuropeptide Y (NPY; structure shown below) is a 36 amino acid, C-terminally amidated peptide that was first isolated from porcine brain by Tatemoto et a1.'S2 in 1982
using a chemical method for the detection of peptide ami d e ~ .NPY ~ has 69% sequence homology with peptide YY (PYY)4 and 50% homology with porcine pancreatic polypeptide (PPP).~
'Abbreviations: The abbreviations for the amino acids are in accord with the recommendations of the IUPAC-IUB Joint Commission on Biochemical nomenclature (Eur.J . Biochem. 1984, 138, 9-37). The symbols represnt the L-isomer except when indicated otherwise. In addition: NPY, neuropeptide tyrosine; PYY, peptide tyrosine tyrosine; pPP, porcine pancreatic polypeptide; MAP, mean arterial pressure; HR, heart rate; Me-Tyr, N-methyltyrosine; Ac-Tyr, N-acetyltyrosine; des-amino-Tyr, 4-hydroxyphenylpropanoic acid; BOC, tert-butoxycarbonyl; MBHA, 4-methylbenzhydrylamine, CM, chloromethyl; Tos, p toluenesulfonyl; OcHx, cyclohexyl ester; 2ClZ, 2-chlorobenzyloxycarbonyl; Bzl, benzyl ester; SBrZ, 2-bromobenzyloxycarbonyl; DMF, dimethylformamide; TFA, trifluoroacetic acid; EDT, ethanedithiol; TEA, triethylamine; GRF, growth hormone releasing factor; CRF, corticotropin-releasing factor; NE, norepinephrine; HPLC, high-performance liquid chromatography; C18,octadecyl; TEAP, triethylammonium phosphate; PE, polyethylene. *University of California San Diego Medical Center.
5 10 Tyr-Pro-Ser- Lye-Pro-Asp-Asn-Pro-Gly-Glu15 20 Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr25 30 Tyr-Ser-Ala-Leu-Arg-His-Tyr-IleAsn-Leu35
Ile-Thr-Arg-Gln-Arg-Tyr-NH2
(1) Tatemoto, K.; Carlquist, M.; Mutt, V. Nature 1982, 296,
659-660. (2) Tatemoto, K. Proc. Natl. Acad. Sci. U.S.A. 1982, 79,
5485-5489. (3) Tatemoto, K.; Mutt, V. R o c . Natl. Acad. Sci. U.S.A. 1978,75,
4115-4119. (4) Tatemoto, K. Proc. Natl. Acad. Sci. U.S.A. 1982, 79,
2514-2518.
0022-2623/89/ 1832-0597$01.50/0 0 1989 American Chemical Society
598 Journal of Medicinal Chemistry, 1989, VoE. 32, No. 3
NPY possesses potent vasoconstrictor properties and increases blood pressure when injected into animak6p7 NPY and norepinephrine (NE) are contained in and coreleased from sympathetic nerves;s however the NPY-induced vasoconstriction is independent of the release and action of NE.9 To date, structure-function studies on NPY1*'4 have been limited to the measurement of activities and potencies of fragments. In the present study we have sought to define the domains responsible for bioactivity by the synthesis of NPY analogues with D-substitutions or deletions in the N- and C-terminal regions, and several analogues with modified N- or C-termini. The analogues were administered intraarterially to conscious rats to determine their effects on mean arterial pressure and heart rate.
Results and Discussion The methodology used for the synthesis of NPY and the NPY analogues resulted in crude materials with a single major peak on analytical HPLC. When comparing the quality of these crude materials with those for other classes of peptides in this size range (20-40 residues, i.e. CRF, GRF) generated under similar conditions, we conclude that NPY is an easily attainable target for solid-phase peptide synthesis. The crude peptides were purified by preparative HPLC in two steps to yield the highly purified peptides. The analytical techniques used for the characterization of NPY and its analogues included analytical HPLC in two buffer systems and amino acid analyses. NPY was further characterized by Edman microsequencing and mass spectrometry. Results from these studies confirmed the identity of the intended structures. While this characterization was acceptable for fragments and non-D-substitutions which differed in composition (and usually retention time) from the standard native peptide against which they were compared, D-substituted analogues had the same composition and often similar retention time to that of the native peptide. It is unlikely that the samples of D-analOgUeS that were prepared contained appreciable amounts of the native (all-L) NPY as optical purities of the BOC-amino acid starting materials were checked prior to their use in the syntheses. Also, the amount of racemization seen in solid-phase synthesis, under the coupling conditions used here, has been shown to be very low (97 -58.2 3.04 (0.88) 1.0 2 [ Ac-Tyr') 5.1 @ 33.6 >95 -57.2 1.72 (0.60) 1.8 3 [ Me-Tyr'] 3.6 @ 35.4 >95 -54.2 1.48 (0.48) 2.1 4 [des-amino-Tyr'] 3.7 @ 36.0 >97 -58.0 2.88 (0.88) 1.1 5 [~-Tyr'] 4.4 @ 33.6 >98 -67.7 1.40 (0.40) 2.2 6 [Ac-~-Tyr'] 4.9 @ 33.6 >95 -62.8 2.84 (1.84) 1.1 7 [D-PrO'] 4.4 @ 33.6 >98 -46.6 8.25 (1.72) 0.4 8 [D-Ser3] 4.4 @ 33.6 >98 -47.8 11.36 (4.48) 0.3 9 [ D-LyS4] 4.2 @ 33.6 >98 -53.7 18.84 (4.72) 0.2 10 [D-PrO'] 4.0 @ 33.6 >95 -53.9 28.24 (8.84) 0.1 11 [D- Asp6] 4.1 @ 33.6 >95 -55.9 12.00 (4.76) 0.3 12 [D-PrO'] 3.7 @ 33.6 >98 -53.9 5.52 (1.24) 0.6 13 [Ala9] 4.1 @ 33.6 >98 -56.7 4.28 (1.36) 0.7 14 [D-Ala9] 4.1 @ 33.6 >95 -64.3 5.76 (1.04) 0.5 15 [D-Glu''] 3.7 @ 33.0 >98 -58.0 2.92 (0.72) 1.0 16 [D- Asp"] 3.4 @ 33.0 >98 -52.2 15.04 (2.32) 0.2 17 [ D-Ala12] 3.4 @ 34.2 >95 -45.0 11.56 (3.84) 0.3 18 [ D-PrO'3] 3.8 @ 33.0 >98 -52.1 16.16 (3.68) 0.2 19 [Met"] 4.4 @ 33.6 >98 -60.9 4.12 (1.72) 0.7 20 [~-Arg'~] 3.4 @ 33.0 >98 -65.0 4.36 (1.36) 0.7 21 [D-Arg33] 3.4 @ 36.6 >98 -58.4 >400 22 [~-Gln~'] 3.5 @ 36.0 >98 -55.6 >400 23 [~-Arg~~] 3.8 @ 35.4 >98 -54.5 >400 24 [~-Tyr~~] 3.4 @ 35.4 >98 -59.7 23.72 (7.72) 0.1 25 NPY2-36 4.0 @ 35.4 >98 -59.4 7.72 (3.64) 0.4 26 NPY8-36 3.7 @ 35.4 >98 -45.3 >400 27 NPYll-36 3.8 @ 34.8 >98 -43.2 >400 28 NPY14-36 4.0 @ 31.2 >95 -40.1 >400 3.5 @ 19.8 >95 -44.2 >400 29 NPY26-36 30 NPY 1-14-OH 3.7 @ 24.6 >98 -125.4 >400 31 NPY 1-za-OH 3.3 @ 10.8 >95 -88.9 >400 32 NPY1-36-0H 3.9 @ 36.0 >98 -61.1 >400 33 NPYy-qrOH 3.4 @ 35.4 >98 -55.7 >400 " Isocratic retention times in minutes at the specified % MeCN. Consensus of values from three analytical determinations. Determined a t 25 "C in 1.0 M AcOH (c -0.5). dED,'s from dose-response curves f SE in pg/kg (n 1 3). eRelative potency: ED,(NPY)/ED,(analogue). ~~~~
Table 11. AMAP (Measured a t 1 min, Means f SEM, n 2 3) for NPY Fragments at 40 and 400 Ng/kg NPY /fragment 40 clg/kg 400 wglkg" NPY 50 f 6 ND NPYZ-36 49 f 14 ND NPY8-36 20 f 5 ND NPY11-36 15 f 5 ND NPY14-36
