Selective Ligands for the μ, δ, and κ 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, μ, δ, and κ. 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. 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, μ, δ, and κ. 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. Synthetic combinatorial libraries have gained widespread acceptance for the rapid identification of new drug leads. Complete combinatorial libraries contain all possible arrangements of building blocks used in their synthesis. Peptides were the first compound class to be used in constructing such libraries (1Houghten R.A. Pinilla C. Blondelle S.E. Appel J.R. Dooley C.T. Cuervo J.H. Nature. 1991; 354: 84-86Crossref PubMed Scopus (1139) Google Scholar, 2Lam K.S. Salmon S.E. Hersh E.M. Hruby V.J. Kazmierski W.M. Knapp R.J. Nature. 1991; 354: 82-84Crossref PubMed Scopus (1707) Google Scholar, 3Geysen H.M. Rodda S.J. Mason T.J. Mol. Immunol. 1986; 23: 709-715Crossref PubMed Scopus (426) Google Scholar). This laboratory first demonstrated the application of soluble mixture based combinatorial libraries through the identification of the peptide Ac-DVPDYA-NH2 from a library of 52 millionN-acetylated hexapeptides. The sequence corresponds to the antigenic determinant recognized by the antibody 17DO9 (1Houghten R.A. Pinilla C. Blondelle S.E. Appel J.R. Dooley C.T. Cuervo J.H. Nature. 1991; 354: 84-86Crossref PubMed Scopus (1139) Google Scholar). Concurrently, a second combinatorial library containing 52 million non-acetylated hexapeptides was subsequently used to identify ligands for the opioid receptors, which were closely related to the natural ligands methionine- and leucine-enkephalin (YGGFM, YGGFL) (4Houghten R.A. Dooley C.T. Biomed. Chem. Lett. 1993; 3: 405-412Crossref Scopus (55) Google Scholar). In later studies, six distinctly hexapeptide sequences that specifically bound to the μ opioid receptor were identified from the acetylated and nonacetylated libraries (5Dooley C.T. Kaplan R.A. Chung N.N. Schiller P.W. Bidlack J.M. Houghten R.A. Pept. Res. 1995; 8: 124-137PubMed Google Scholar). The combinatorial libraries described above are all mixture-based. Many questions have arisen concerning the use of mixture-based libraries. For example, how many different families of compounds for a particular target exist and can they be identified in a given library? What are the chances of missing the most active compound? What other compounds are present or could be identified from the data that are not immediately obvious? Are similar building blocks universally replaceable? Peptides, because of their ease of synthesis, represent the most convenient compound class for use in the study of mixture-based combinatorial libraries. By achieving an appreciation for the behavior of large mixtures through the use of peptide libraries, we believe these same principles can be applied to virtually all other compound classes (i.e. heterocycles and other small molecules). The opioid receptors represent a convenient system to investigate the power of combinatorial libraries to identify distinctly different ligands for related receptors. There are three primary opioid receptors: mu (μ), delta (δ), and kappa (κ). All three receptors have recently been cloned, and they belong to the seven-transmembrane G-protein-coupled family of receptors and have approximately 60% amino acid sequence homology. Screening of the same combinatorial library in separate assays selective for each of the three receptors provides not only new ligands for these receptors but yields insights into the ability of combinatorial libraries to discriminate between closely related receptors. A combinatorial library of 6,250,000 tetrapeptides, made using 50 different amino acids, was prepared in the positional scanning format (6Pinilla C. Appel J.R. Blanc P. Houghten R.A. BioTechniques. 1992; 13: 901-905PubMed Google Scholar, 7Dooley C.T. Houghten R.A. Life Sci. 1993; 52: 1509-1517Crossref PubMed Scopus (177) Google Scholar). The use of positional scanning synthetic combinatorial libraries (PS-SCLs) 1The abbreviations used are: PS-SCLs, positional scanning synthetic combinatorial libraries; DAMGO, [3H-d-Ala2,MePhe4,Gly5-ol]enkephalin; DSLET, [d-Ser2,Leu5,Thr6]enkephalin; Boc, butoxycarbonyl; d-Cha,d-cyclohexylalanine; l-Cha,l-cyclohexylalanine; d-Nle,d-norleucine; l-Nal, 2-l-naphthylalanine; d-Nal, 2-d-naphthylalanine; d-Nve,d-norvaline; l-Nve, l-norvaline; y,d-tyrosine; w, d-tryptophan; r,d-arginine; f, d-phenylalanine; l,d-leucine; aAba, l-α-aminobutyric acid. 1The abbreviations used are: PS-SCLs, positional scanning synthetic combinatorial libraries; DAMGO, [3H-d-Ala2,MePhe4,Gly5-ol]enkephalin; DSLET, [d-Ser2,Leu5,Thr6]enkephalin; Boc, butoxycarbonyl; d-Cha,d-cyclohexylalanine; l-Cha,l-cyclohexylalanine; d-Nle,d-norleucine; l-Nal, 2-l-naphthylalanine; d-Nal, 2-d-naphthylalanine; d-Nve,d-norvaline; l-Nve, l-norvaline; y,d-tyrosine; w, d-tryptophan; r,d-arginine; f, d-phenylalanine; l,d-leucine; aAba, l-α-aminobutyric acid. enables the most active amino acids at each position of a peptide or non-peptide to be determined directly from the initial screening data. This information can then be used to synthesize highly active individual compounds. A PS-SCL of tetrapeptide amides used in the current study consists of four separate sublibraries, each having a single defined position (O) and three mixture positions (X) as follows:O 1 XXX-NH2,XO 2 XX-NH2,XXO 3 X-NH2, andXXXO 4-NH2. The defined positions of the mixtures making up each of the four separate sublibraries address a single position in the tetrapeptide. It should be noted that each of the four positional sublibraries are made up of the same 6,250,000 tetrapeptides. Screening the four sets of mixtures in the three separate opiate specific assays yielded information about the most important amino acids of each position in the tetrapeptide and led to the identification of three different series of active individual tetrapeptides selective for the μ, δ, and κ receptors. The PS-SCL used in this study is composed of 6,250,000 tetrapeptides and contains four sublibraries, in which one of the four positions is defined with a single amino acid (O) and the three remaining positions are a mixture of 50 different l-,d-, and unnatural amino acids (X). The tetrapeptides were synthesized using the solid phase simultaneous multiple peptide synthesis approach (8Houghten R.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5131-5135Crossref PubMed Scopus (1481) Google Scholar) on methylbenzhydrylamine polystyrene resin using t-butoxycarbonyl-protected amino acids. Mixture resins (X) were prepared using mixtures oft-butoxycarbonyl-protected amino acids at each coupling step. Each of the amino acids was present in a concentration that yielded close to equimolar coupling of each amino acid. The ratio of the concentrations of the individual amino acids used to yield this approximate equimolar coupling was pre-determined using reverse phase-high pressure liquid chromatography to compare mixture profiles relative to standard mixtures synthesized using the divide, couple, and recombine method (1Houghten R.A. Pinilla C. Blondelle S.E. Appel J.R. Dooley C.T. Cuervo J.H. Nature. 1991; 354: 84-86Crossref PubMed Scopus (1139) Google Scholar) as detailed in Ref. 9Ostresh J.M. Winkle J.H. Hamashin V.T. Houghten R.A. Biopolymers. 1994; 34: 1681-1689Crossref PubMed Scopus (140) Google Scholar. Coupling completion was determined using Kaiser's ninhydrin test (10Kaiser E.T. Colescott R.L. Blossinger C.D. Cook P.I. Anal. Biochem. 1970; 34: 595-598Crossref PubMed Scopus (3487) Google Scholar). Side chain deprotection and cleavage from the resin support were achieved using low hydrogen fluoride (11Tam J.P. Heath W.F. Merrifield R.B. J. Am. Chem. Soc. 1983; 105: 6442-6455Crossref Scopus (520) Google Scholar) and high hydrogen fluoride (12Houghten R.A. Bray M.K. DeGraw S.T. Kirby C.J. Int. J. Pept. Protein Res. 1986; 27: 673-678Crossref PubMed Scopus (145) Google Scholar) procedures. The 200 peptide mixtures were individually extracted with water, lyophilized, and resuspended in water at a final concentration of 10 mg/ml. Peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl chemistry on a COMPAS 242 multiple peptide synthesizer (Spyder Instruments, San Diego). The peptides were synthesized on TentaGel resin in polypropylene mesh packets using inclusion volume synthesis. Reagent and wash solutions were removed by centrifugation (13Pokorny V. Mudra P. Jehnicka J. Zenisek K. Pavlik M. Voburka Z. Rinnova M. Stierandova A. Lucka A.W. Eichler J. Houghten R.A. Lebl M. Epton R. Innovation and Perspectives in Solid Phase Synthesis-Peptides, Proteins and Nucleic Acids. Mayflower Worldwide Ltd., Birmingham, UK1994: 643-648Google Scholar, 14Eichler J. Houghten R.A. Lebl M. J. Pept. Sci. 1996; 2: 240-244Crossref PubMed Scopus (9) Google Scholar). Membrane homogenates were prepared from rat brains. Brains were homogenized in 40 ml of Tris-HCl buffer, 50 mm, pH 7.4, and for 10 The were resuspended in and at for 40 the were as the resuspended in of buffer, and the Membrane were prepared and used on the same Protein of the homogenates was determined by the method described by Anal. Biochem. PubMed Scopus Google Scholar). Each ml of 3 and and 50 Tris-HCl in a final volume of were for 1 at was used as a to a standard and The was by through on a The were subsequently with ml of 50 pH at was on a were homogenized as described above using 40 ml of 50 Tris-HCl buffer, C.T. Chung N.N. Schiller P.W. Bidlack J.M. Houghten R.A. 1994; PubMed Scopus Google 1 pH 7.4, were and as Each ml of 3 in a volume of were for at The was and as was used as a to a standard and and were homogenized in 40 ml of A. were and as Each ml of 3 in a volume of were for at The was and as was used as a to a standard and The was a from K. for The was from and been to the κ opioid receptor but not the μ or receptors Bidlack J.M. Biochem. 1995; PubMed Scopus Google Scholar). The were in with pH and containing 50 and in at were in containing 10 acid for in to the as described J. Google Scholar). were prepared for use in the assays as described Bidlack J.M. J. 1993; Google Scholar). the initial centrifugation at 200 for at the were resuspended in 40 2 pH were at 200 and then homogenized in with of a were at for at by in The concentration was determined by the method of Anal. Biochem. PubMed Scopus Google Scholar) using as at a concentration of were at were in a final volume of of 40 containing of 1 50 50 3 and and were at final concentrations as in the and were used to μ, δ, and κ opioid receptors, The was by the of of at the was by the of 40 of and then the were at for at in a The of present in of the to the by of was determined by the use of a This which a in a binding is based on the method of PubMed Scopus Google Scholar) and was used with the from was used of the in the was into ml of and of the was to the The final were in 10 ml of The tetrapeptide PS-SCL was screened in each of the three separate opioid receptor binding assays described Each mixture tetrapeptides The mixtures were screened at a concentration at this mixture concentration each peptide was present at a concentration of Each of the 200 mixtures for each of the four making up the library was screened for ability to binding of the to The library was screened at a concentration many mixtures of binding in the initial were subsequently for all mixtures that of binding from each of the four positions The most active mixture from the series in which the first position was defined was This mixture was active the second most active mixture and active the most active mixture There was a in between the most active and active mixture of mixtures to be the most active in the initial of the tetrapeptide PS-SCL at the μ Each one of the four positional (i.e. position one 1 the of the mixture for the μ receptor as of the S.E. acids defined in the mixtures (O) are on the the mixtures from which amino acids were to individual were for mixtures in which the second position was defined The most active mixtures were all defined with acids. mixtures a between the most and active for the first was the most active mixture found. The and most active mixtures were a in and and were for mixtures in which the position was defined of the mixtures and all six acids in the defined The three most active mixtures were for mixtures in which the position was defined The most active mixture The second most active mixture was active the most active and the most active mixture was active the most active The amino acids to individual peptides are in for these peptides in the μ receptor binding are given in All of the peptides were to have high for the μ receptor were Three of the amino acids in the most active peptide were the most active amino acids for their particular in the position was the second most active amino acid for that The four acids for the second position were to be (i.e. peptides which only by the amino acid at this position similar all four amino acids in the position yielded active with small amino acid and were active with and were to be at the The of active peptides identified at the μ receptor was acid with small all of the peptides synthesized were to be peptides are to be identified from this The of peptide at or κ of peptide at the μ of the peptides are in 3 A. All peptides were to be μ of the were The most peptide was in All peptides with at the second position μ peptides were active at the receptor at the κ acids for synthesis of individual compounds for the μ of individual tetrapeptides, 1 2 in a new for individual peptides identified from the tetrapeptide PS-SCL in an selective for the μ amino acids defined at each position of the library and the single or are as w, d-tryptophan; y,d-tyrosine; l-α-aminobutyric l-norvaline; 2-l-naphthylalanine; at the μ receptor of tetrapeptides, representing all possible combinations of the amino acids are are detailed Peptides are by The amino acids defined at each position of the library and the single or are as w, d-tryptophan; y,d-tyrosine; l-α-aminobutyric l-norvaline; 2-l-naphthylalanine; in a new of individual tetrapeptides, 1 2 The at the μ receptor of tetrapeptides, representing all possible combinations of the amino acids are are detailed Peptides are by an initial screening in a receptor binding using as the tetrapeptide PS-SCL was screened at a concentration were for mixtures that of binding for positions 1 and 2 and of binding for positions 3 and The most active of the 10 mixtures at position 1 The most active mixture was approximately active the second most active the active mixtures identified were similar to in the μ receptor they were the mixture was active in the μ the receptor mixtures with defined amino acids in the second position were to be similar to identified in the μ were determined for The most active mixtures The 10 mixtures in which the position was defined were to be active of the remaining three and mixture was to have an The four most active mixtures There was in between the most active mixtures at the the 10 mixtures between and were for mixtures in which the position was the in the μ receptor the most active amino acids at the position were and at this position were as was in the μ receptor and 2 The amino acid combinations for the synthesis of individual peptides are in Peptides that in the are in three of the peptides were to have 10 The of the peptides is in 3 of the peptides in the μ in the of This is not as many of the mixtures with defined amino acids for the peptides were active in the μ receptor It is not that the most peptide 2 in in the this amino acid was first in the receptor screening and in the μ receptor Many of the peptides were virtually at the κ receptor at the concentration data are by an in 3 acids for synthesis of individual compounds for the l-Nve, of individual tetrapeptides, 2 3 2 in a new for individual tetrapeptides from PS-SCL using a selective for representing all possible combinations of the amino acids were were are detailed The most active peptides are by for in a new of individual tetrapeptides, 2 3 2 representing all possible combinations of the amino acids were were are detailed The most active peptides are by For the κ the library was screened at an initial concentration of in homogenates using as were subsequently for mixtures which of binding for positions The most active mixtures in the not to in the μ and receptor The mixtures in which the first position was defined in from to The four most active mixtures acids at this position and mixtures were in which the second position was for the first acids were The most active was active the second most active The most active mixtures for the position of the mixtures at this position were to have of The mixture at this position all other mixtures acids. mixtures were for the was in the receptor amino acids were to have the in the position and The most active mixture The amino acids for inclusion in the synthesis of individual peptides are in four peptides were and their are given in of the peptides 50 of which were 10 The most active peptide was to be were at the first were at the second and were at the of the peptides in the position This the of family of κ ligands in the The active peptides were highly selective for the κ receptor 3 The most κ selective peptides and of acids for synthesis of individual compounds for the κ of individual tetrapeptides, 2 2 3 2 in a new for individual tetrapeptides from the PS-SCL using a κ selective for at the κ receptor of representing all possible combinations of the amino acids are are detailed Peptides are by for in a new of individual tetrapeptides, 2 2 3 2 The at the κ receptor of representing all possible combinations of the amino acids are are detailed Peptides are by in of Life Sci. 1991; PubMed Scopus Google Mol. 1983; 23: Google Scholar). using or was used to a peptide was an opioid or The opioid receptors on the in the of 10 acid are approximately of the μ and of the J. Google Scholar). M. The contains only the κ receptor A of to of the was as of an opioid that the of was by an opioid was used for μ receptors and was used for κ receptors. ability to the of was similar to that of of was by but not the that the in was by μ receptors. The most active peptides for the μ and κ assays have been for ability to All peptides were to be at their The of was similar to the standard that their at the receptor is similar to standard of in or for with high for μ and κ receptors were for as by of in containing large of μ or κ receptors. are detailed Peptides were at a final concentration of A and a were used as of was to be by the opioid or the that the was by opioid for abbreviations in a new with high for μ and κ receptors were for as by of in containing large of μ or κ receptors. are detailed Peptides were at a final concentration of A and a were used as of was to be by the opioid or the that the was by opioid receptors. The opiate receptor used in the current study have been described in (4Houghten R.A. Dooley C.T. Biomed. Chem. Lett. 1993; 3: 405-412Crossref Scopus (55) Google Scholar, 7Dooley C.T. Houghten R.A. Life Sci. 1993; 52: 1509-1517Crossref PubMed Scopus (177) Google Scholar, C.T. Chung N.N. Schiller P.W. Bidlack J.M. Houghten R.A. 1994; PubMed Scopus Google Scholar, C.T. Chung N.N. Schiller P.W. Houghten R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). In this laboratory combinatorial libraries for the identification of opioid ligands have on the μ have a as first to new ligands and of the opioid receptors, and second to the use of combinatorial libraries made up of large The ability to identify highly active individual ligands using and positional scanning was demonstrated for the μ receptor (4Houghten R.A. Dooley C.T. Biomed. Chem. Lett. 1993; 3: 405-412Crossref Scopus (55) Google Scholar, 7Dooley C.T. Houghten R.A. Life Sci. 1993; 52: 1509-1517Crossref PubMed Scopus (177) Google from which the C.T. Chung N.N. Schiller P.W. Houghten R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar) and an all acid C.T. Chung N.N. Schiller P.W. Bidlack J.M. Houghten R.A. 1994; PubMed Scopus Google were found. recently we have used the μ receptor to how a of different sequences could be identified from such libraries. In the present the same PS-SCL containing 6,250,000 tetrapeptides was screened in receptor assays for the μ, δ, and κ opioid receptors. The screening data using this same library in three closely related receptors yielded three different binding The mixtures screened were to have the in the μ receptor relative to their at the and κ receptors. The of active peptides identified for the μ receptor was was the only amino acid for the first position to the for this amino acid. The acids at the position were or the amino acids or This peptide is similar to the of and described S. S. A. P. Biochem. Res. 1991; PubMed Scopus Google Scholar). The peptide was identified from a tetrapeptide library using an C.T. Houghten R.A. Peptides of the Scholar) but was not identified was not in this The most peptides in this study at the second position and are similar to peptides P.W. Chung N.N. C. J. Chem. PubMed Scopus Google There are other tetrapeptides with high for the μ receptor that were not identified in this Nature. PubMed Scopus Google Scholar) and (5Dooley C.T. Kaplan R.A. Chung N.N. Schiller P.W. Bidlack J.M. Houghten R.A. Pept. Res. 1995; 8: 124-137PubMed Google Nature. PubMed Scopus Google Scholar) have been identified using a tetrapeptide library made up only of acids or amino acids were at each are on that the peptides to be synthesized the of amino acids at each In all three the of amino acids for the synthesis of individual peptides was the of combinations with the of amino acids at each position (i.e. tetrapeptides for 3 amino acids are at each tetrapeptides amino acids are at each and tetrapeptides amino acids are at each This in a in the identification of active This can be in the current in which active sequences for the μ receptor were identified the same library was screened the It should be noted that these sequences have been identified from the μ screening data a of combinations been to a of amino acids is In the amino acids at each one to between the in and the between similar amino acids which are not The of the combinations identified from the data for the were to be active in the μ receptor A for peptides was as can be in which the at the μ and receptors of of the peptide There is between in amino acids and of at position 1 by but of by at position 2 in a of at position 3 in of a of at position 1 and at position 3 the a This the in making on the of amino acids of similar of amino acid on for amino acid on a peptide sequence peptide or for in a new amino acid on a peptide sequence peptide or The between the peptides to have μ and receptors to a between the receptors, as to the peptides to be active at the κ is to to μ receptors, but we have C.T. Chung N.N. Schiller P.W. Bidlack J.M. Houghten R.A. 1994; PubMed Scopus Google a highly selective μ in the in to the from binding to μ receptors. the library was screened using as a and library profiles were The sequences from screening the tetrapeptide PS-SCL in an selective for the κ receptor are the most of this are peptides to have at κ receptors and not have been identified by A is not The sequence to acids at all four peptide synthesized at position 3 The first position an or an but this be to all similar amino acids is not Peptides with 10 were with in the that only in the position were active with peptides identified for the kappa receptor were highly A study the synthesis of individual tetrapeptides based on the data in this study is In this highly active individual compounds and highly and compounds were identified from a large mixture-based positional scanning combinatorial All of the most active peptides were to be It to be determined these peptides are of the but is that the of acids in their sequences their The κ peptides identified for as κ compounds have into in as having in for C. 1996; PubMed Scopus Google Scholar). the other these κ peptides not the they be in the of V. Life Sci. 1995; PubMed Scopus Google Scholar). This study not only the power of the positional scanning for the rapid identification of new ligands but how ligands be identified for closely related receptors. and for and for