Orphanin FQ/nociceptin receptor binding studies.

A review of the binding studies performed on the receptor (ORL) for Orphanin FQ/Nociceptin is presented. Binding studies have been conducted using a variety of receptor sources: cell lines expressing the cloned receptor, cell lines endogenously expressing the receptor, and brain and other tissue from several different species. Binding studies of opioids, new ligands and antagonists at the ORL receptor are briefly discussed. Saturation, competition and binding kinetic experiments, and the effects of buffer composition are reviewed. There are numerous instances of conflicting data in published reports on OFQ; the basis for these disparities is as yet undetermined. This review endeavors to compile the results and conditions employed in binding studies as an aid to current and new researchers in this field. In an attempt to explain binding disparities, we have determined that Orphanin/Nociceptin binds to glass fiber filtermats in a "specific" manner; these new data are presented.

New opioid peptides, peptidomimetics, and heterocyclic compounds from combinatorial libraries.

Here we review the use of combinatorial libraries in opioid receptor assays. Following a brief description of the history of the combinatorial field, methods for the generation of synthetic libraries and the deconvolution of mixture-based libraries are presented. Case studies involving opioid assays used to demonstrate the viability of combinatorial libraries are described. The identification of new opioid peptides from combinatorial libraries is reviewed. The peptides found are composed of L-amino acids, D-amino acids, or L-, D-, and unnatural amino acids, and range from tetrapeptides to decapeptides. Likewise, new opioid compounds identified from peptidomimetic libraries, such as peptoids and alkylated dipeptides, and those identified from acyclic (e.g., polyamine, urea) and heterocyclic (e.g., bicyclic guanidine) libraries, are reviewed.

Selective ligands for the mu, delta, and kappa opioid receptors identified from a single mixture based tetrapeptide positional scanning combinatorial library.

A combinatorial library of 6,250,000 tetrapeptides in the mixture based positional scanning format was screened in binding assays for the three opioid receptors, mu, delta, and kappa. Three different binding profiles were found. Individual peptides were synthesized representing all possible combinations of the active amino acids identified from the screening data. New, highly active peptides selective for each of the three receptors were chosen. This study demonstrates the power of mixture-based combinatorial libraries to identify distinctly different ligands for closely related receptors.

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 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]GTPgammaS 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]GTPgammaS 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 GTPgammaS 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.

Libraries from libraries: generation and comparison of screening profiles.

A positional scanning tetrapeptide library was chemically modified through alkylation and/or reduction of the amide bonds, thus generating three new combinatorial libraries with physico-chemical properties very different from the parent peptide library ('libraries from libraries'). Specific results were obtained with each of these libraries upon screening in kappa-opioid receptor binding and microdilution antimicrobial assays, illustrating the potential of the 'libraries from libraries' concept for the efficient generation of a variety of chemically diverse combinatorial libraries.

Orphanin FQ: receptor binding and analog structure activity relationships in rat brain.

A tritiated form of orphanin FQ (a heptadecapeptide also known as Nociceptin) has been prepared. This radioligand (33 Ci/mmole) was used to develop a radioreceptor assay using rat brain homogenates. Binding was observed to be saturable, and analyses of the binding data indicate the presence of a single binding site with a dissociation constant of 5 +/- 1.1 nM and Bmax of 535 +/- 85 fmoles/mg protein. Thirty-four analogues of orphanin FQ, including a complete alanine "scan" of orphanin FQ, and truncation analogues from both the N- and C- terminals were synthesized and tested. The data obtained indicate that the N-terminus plays a more critical role in binding than the C-terminus and that residues 1, 2, 4, and 8 are essential for binding.

Six highly active mu-selective opioid peptides identified from two synthetic combinatorial libraries.

Two synthetic combinatorial libraries (SCLs) were prepared, each composed of 52,128,400 L-amino acid hexapeptides, one with and the other without an N-terminal acetyl moiety. The two libraries were used in conjunction with an iterative selection process to identify individual peptides capable of inhibiting the binding of the mu-selective opioid peptide [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin to rat brain homogenates. As reported previously, when using the nonacetylated SCL the first five residues identified corresponded exactly to methionine- and leucine-enkephalin, both of which are endogenous opioid peptides. The iterative identification process has now been completed for two additional mixtures found to have activity in the initial screening of this SCL. Two new series unrelated to the enkephalins have been identified: YPFGFO-NH2 and WWPKHO-NH2 (where O = one of the 20 L-amino acids). Individual peptides from each of these were found to be agonists at the mu receptor and have high affinity (IC50 values of the most active peptides were 10-15 nM) and selectivity for the mu receptor. In addition to the acetalins (described previously), two new series have now been identified from the acetylated library: Ac-FRWWYO-NH2 and Ac-RWIG-WO-NH2 (IC50 values of the most active peptides were 5-30 nM). Ac-FRWWYM-NH2 was determined to be an agonist at the mu receptor, whereas Ac-RWIGWR-NH2 was found to be an antagonist at this receptor.

An all D-amino acid opioid peptide with central analgesic activity from a combinatorial library.

A synthetic combinatorial library containing 52,128,400 D-amino acid hexapeptides was used to identify a ligand for the mu opioid receptor. The peptide, Ac-rfwink-NH2, bears no resemblance to any known opioid peptide. Simulations using molecular dynamics, however, showed that three amino acid moieties have the same spatial orientation as the corresponding pharmacophoric groups of the opioid peptide PLO17. Ac-rfwink-NH2 was shown to be a potent agonist at the mu receptor and induced long-lasting analgesia in mice. Analgesia produced by intraperitoneally administered Ac-rfwink-NH2 was blocked by intracerebroventricular administration of naloxone, demonstrating that this peptide may cross the blood-brain barrier.

Acetalins: opioid receptor antagonists determined through the use of synthetic peptide combinatorial libraries.

A synthetic peptide combinatorial library made up of 52,128,400 hexapeptides, each having an acetyl group at the N terminus and an amide group on the C terminus, was screened to find compounds able to displace tritiated [D-Ala2,MePhe4,Gly-ol5]enkephalin from mu opioid receptor binding sites in crude rat brain homogenates. Individual peptides with mu receptor affinity were found using an iterative process for successively determining the most active peptide mixtures. Upon completion of this iterative process, the three peptides with the highest affinity were Ac-RFMWMT-NH2, Ac-RFMWMR-NH2, and Ac-RFMWMK-NH2. These peptides showed high affinity for mu and kappa 3 opioid receptors, somewhat lower affinity for delta receptors, weak affinity for kappa 1 receptors, and no affinity for kappa 2 receptors. They were found to be potent mu receptor antagonists in the guinea pig ileum assay and relatively weak antagonists in the mouse vas deferens assay. These peptides represent a class of opioid receptor ligands that we have termed acetalins (acetyl plus enkephalin).

The use of positional scanning synthetic peptide combinatorial libraries for the rapid determination of opioid receptor ligands.

The application of a new synthetic peptide combinatorial library (SPCL) is described. This library, termed a positional scanning SPCL, contains six positional SPCLs, each of which contains all possible hexameric combinations of 18 of the 20 natural L-amino acids (18(6) = 34,012,224 peptides). Each positional SPCL (O1XXXXX-NH2, XO2XXXX-NH2, XXO3XXX-NH2, XXXO4XX-NH2, XXXXO5X-NH2, and XXXXXO6-NH2) was used to determine the most active amino acid for the six positions of a hexamer. Combinations of these amino acids were used to synthesize 24 individual peptides, which were then tested for activity. The most active peptide found corresponded to a hexameric analogue of methionine-enkephalin. Results obtained in this study are compared to those obtained using the SPCL described earlier (1) (O1O2XXXX-NH2), and the subsequent iterative process.

The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides.

The systematic preparation of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal peptide sequences has been achieved through the screening in solution of SPCLs, each element of which is composed of more than 100,000 nonsupport-bound peptides in equimolar representation, along with an iterative synthesis and screening process. Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay. This same library was also used to develop highly potent antimicrobial peptides in bacterial growth inhibition assays. A separate non-acetylated SPCL was used to screen and identify high affinity peptide ligands using an opiate radio-receptor binding assay.

Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery.

Existing methods for the synthesis and screening of large numbers of peptides are limited by their inability to generate and screen the requisite number (millions) of individual peptides and/or their inability to generate unmodified free peptides in quantities able to interact in solution. We have circumvented these limitations by developing synthetic peptide combinatorial libraries composed of mixtures of free peptides in quantities which can be used directly in virtually all existing assay systems. The screening of these heterogeneous libraries, along with an iterative selection and synthesis process, permits the systematic identification of optimal peptide ligands. Starting with a library composed of more than 34 million hexa-peptides, we present here the precise identification of an antigenic determinant recognized by a monoclonal antibody as well as the straightforward development of new potent antimicrobial peptides.