Abstract
Fifteen hexapeptides having high affinity for the opioid-like receptor ORL1 were identified from a combinatorial library containing more than 52 million different hexapeptides. The five compounds with the highest affinity were characterized further by use of a variety of in vitro models. Binding studies indicated that these five peptides have affinity for ORL1 in the nanomolar range, similar to the recently discovered endogenous ligand called nociceptin and orphanin FQ (N/OFQ). The activity of these compounds was investigated in three different assays: stimulation of [35S]GTPγS binding and inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells transfected with ORL1, and inhibition of electrically induced contractions in the mouse vas deferens. In each assay, the five hexapeptides acted as partial agonists. The EC50 values for stimulation of [35S]GTPγS binding and inhibition of cAMP accumulation were in the range of that for N/OFQ, but maximal effects ranged from 70 to 90% of N/OFQ in the cAMP assay, and 30 to 60% of N/OFQ in the GTPγS assay. The positive hexapeptides identified were found to have minimal structural similarity to N/OFQ. The peptides are positively charged, which could enable them to bind to the negatively charged second extracellular loop thought to be a likely binding site for N/OFQ.
Footnotes
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Send reprint requests to: Dr. Lawrence Toll, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025.
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↵1 This work was supported by National Institute on Drug Abuse grants DA06682 (LT) and DA09410 (RAH), and Houghten Pharmaceuticals Inc., San Diego, CA.
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↵2 To whom correspondence concerning the combinatorial chemistry should be addressed: Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121.
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↵3 To whom correspondence concerning the pharmacological evaluation should be addressed: SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025.
- Abbreviations:
- N/OFQ
- Nociceptin, Orphanin FQ
- SCL
- synthetic combinatorial libraries
- PS-SCL
- Positional Scanning SCL
- CHO
- Chinese hamster ovary
- MVD
- mouse vas deferens
- DPDPE
- [d-Pen2-d-Pen5]enkephalin
- HEPES
- N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid
- Received March 20, 1997.
- Accepted July 14, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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