Abstract
The Phe1 cyclic tetrapeptide Phe-c[d-Cys-Phe-d-Pen]NH2(Et) (JH-54) has been shown previously to exhibit high affinity and selectivity for the μ-opioid receptor. To examine the role of the Phe1 residue in the unexpected high affinity of this peptide, 11 analogs of JH-54 have been synthesized and evaluated for opioid ligand binding and for efficacy using the [35S]GTPγS assay. Alteration of the bridging groups between the d-Cys2 andd-Pen4 residues of JH-54 from dithioether to disulfide revealed the importance of the relative position of the aromatic rings of the first and third residues in determining μ- and δ-affinities. The one carbon distance between the α carbon and phenyl ring in the N-terminal residue was critical. Additional steric bulk in the N-terminal Phe1 residue was accommodated without large reductions in affinity in two naphthyl analogs, but not with 3,3-(diphenyl)alanine. Conformational restriction of the Cα-Cβ and/or Cβ-Cγ bonds had little effect on affinities in two peptides with 2-amino-2-carboxytetralin in position 1, but it abolished activity in an isoquinoline analog and differentially altered activity in four phenylproline1-containing peptides. Most surprisingly, replacement of the Phe1 aromatic ring with cyclohexyl resulted in a peptide of moderate affinity (Ki = 32.5 nM) and potency (EC50 = 58.8 nM). Thus, the tyrosylpara-hydroxyl substituent and even aromaticity in the N-terminal amino acid of these tetrapeptides are shown to be important, but not critical, features for μ-opioid receptor affinity, agonist potency, and efficacy.
Footnotes
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Send reprint requests to: Dr. John R. Traynor, Department of Pharmacology, University of Michigan, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail: jtraynor{at}umich.edu or Dr. Henry Mosberg, College of Pharmacy, University of Michigan, CC Little Bldg., Ann Arbor, MI 48109-1065. E-mail: him{at}umich.edu
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↵1 This work was supported by National Institute of Health Grants DA03910 and DA00254.
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↵2 These authors contributed equally to this work.
- Abbreviations:
- DPDPE
- [d-Pen2,d-Pen5]-enkephalin
- DAMGO
- [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin
- RP-HPLC
- reverse-phase high-performance liquid chromatography
- TLC
- thin-layer chromatography
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- TFA
- trifluoroacetic acid
- Atc
- 2-amino-2-carboxytetralin
- Cha
- cyclohexylalanine
- Dip
- 3,3-(diphenyl)alanine
- Hfe
- homophenylalanine
- 1-Nal
- 3-(1-naphthylalanine)
- 2-Nal
- 3-(2-naphthylalanine)
- Pen
- penicillamine (3,3-(dimethyl)cysteine)
- Pgl
- phenylglycine
- c-PhPro
- cis-3-phenylproline
- t-PhPro
- trans-3-phenylproline
- Tic
- 1,2,3,4-tetrahydroisoquinoline 3-carboxylic acid
- Rf
- retardation factor
- Et
- –S–CH2–CH2–S–
- Received April 28, 2000.
- Accepted August 15, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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