BINDING AND IN-VITRO ACTIVITIES OF PEPTIDES WITH HIGH-AFFINITY FOR THE NOCICEPTIN ORPHANIN FQ RECEPTOR, ORL1/

Fifteen hexapeptides having high affinity for the opioid-like receptor ORL1 were identified from a combinatorial library containing more tha n 52 million different hexapeptides. The five compounds with the highe st affinity were characterized further by use of a variety of in vitro models. Binding studies indicated that these five peptides have affin ity for ORL1 in the nanomolar range, similar to the recently discovere d endogenous ligand called nociceptin and orphanin FQ (N/OFQ). The act ivity of these compounds was investigated in three different assays: s timulation of [S-35]GTP gamma S binding and inhibition of forskolin-st imulated 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 part ial agonists. The EC50 values for stimulation of [S-35]GTP gamma S bin ding 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 GTP gamma S assay. The positive hexapeptides identified were found to have minimal structural similari ty to N/OFQ. The peptides are positively charged, which could enable t hem to bind to the negatively charged second extracellular loop though t to be a likely binding site for N/OFQ.