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Synthesis and opioid activity of conformationally constrained dynorphin A analogues
13 SYNTHESIS AND OPIOID ACTIVITY OF CONFORMATIONALLY CONSTRAINED DYNORPHIN A ANALOGUES Arttamangkul S, Murray TF, DeLander GE, Aldrich JV, College of Pharmacy, Oregon State University, Corvallis, OR 97331, U.S.A. ABSTRACT Cyclic analogues of dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) were synthesized and their opioid receptor affinity and opioid activity determined. Cyclic peptides constrained in both the "message" and "address" regions of Dyn A-(1-13)NH2 (1) were prepared in order to investigate possible biological conformations of different regions of the peptides. The design of the constraint was based upon Schwyzer's proposal that Dyn A-(l-13) adopts an u-helix when it binds to x opioid receptors in the lipid membrane (2). Molecular modeling with AMBER suggested that a four atom bridge between the c~ carbons of residues i (D-configuration) and i+3 (L-configuration) may be compatible with a helical structure. Therefore we synthesized a series of cyclo[D-Asp~,Dapi÷3]Dyn A-(1-13)NH2 analogues (Dap = c~,/~-diaminopropionic acid) with the lactam bridge between noncritical residues 2 and 5, 3 and 6, 5 and 8, or 6 and 9. Of these cyclic peptides, the [2,5] cyclic analogue exhibited the highest opioid receptor affinity and opioid activity. METHODS
Chemistry The cyclic peptide analogues were prepared by solid phase synthesis (3). N=-Fmoc amino acids were used for elongation of the peptide backbone on an MBHA (methylbenzhydrylamine) resin. The cyclizations were performed on the resin using HBTU (2-(1H-benzotriazol-l-yl)-l,l,3,3tetramethyluronium hexafluorophosphate), BOP (benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate) or PyBOP (benzotriazol-l-yl-oxy-tris-(pyrrolidino)phosphonium hexafluorophosphate). Using HBTU for the cyclization reactions resulted in a major side reaction which decreased the yields of the cyclic peptides. The phosphonium reagents BOP and PyBOP gave slower cyclization reactions but higher yields and purer crude cyclic peptides.
Opioid Receptor Binding Assays and Smooth Muscle Assays Opioid receptor binding assays were performed using rat r and # opioid receptors expressed in COS-7 (compounds 1 and 2; see Table 1) or CHO cells (compounds 3 - 4) (4). Binding affinities for K- and t~-receptors were determined by displacing [3H]diprenorphine and [3H]DAMGO, respectively, from membrane preparations. Incubations were performed for 90 min at 22°C in the presence of peptidase inhibitors, and nonspecific binding was determined in the presence of 10 t~M unlabelled Dyn A-(1-13)NH2 and DAMGO, respectively, for r and # receptors. The peptides were evaluated for opioid activity in the electrically stimulated guinea pig ileum (GPI) (5) generally in the presence of peptidase inhibitors. RESULTS AND CONCLUSIONS Conformationally constrained Dyn A-(1-13)NH2 analogues cyclized between the side chains of DAsp i and Dap i+3 exhibited significant differences in opioid receptor affinity and opioid activity depending upon the positions involved in the cyclization (Table 1). Compound 1, cyclized between residues 2 and 5, showed the highest binding affinity for both r and tz receptors and was a potent agonist in the GPI assay. Shifting the constraint to residues 3 and 6 gave compound 2, which exhibited very poor binding affinity as well as weak agonist activity. Compound 3, which was cyclized between residues 5 and 8, gave dissimilar results for binding to x receptors vs. opioid
activity in GPI, similar to results reported for cyclic disufide analogues of Dyn A constrained through residue 5 (6). Compound 3 exhibited moderate affinity for x receptors, with 9-fold higher affinity for K than/~ receptors. In the GPI assay, however, compound 3 was a very weak agonist and was 33,000 times less potent than Dyn A-(1-13)NH2. In contrast, shifting the cyclic constraint to the 6 and 9 positions resulted in a compound with both moderate affinity for K and g receptors and modest agonist activity in the GPI. Table 1 Opioid receptor binding affinity and opioid activity of cyclic Dyn A analogues. Dyn A-(1-13)NH2 analogues
GPI IC5o (nM)
1. cyclo[D-Asp2,DapS]2. cyclo[D-Asp3,Dap6]3. cyclo[D-AspS,DapS]4. cyclo[D-Asp6,Dapg]Dyn A-(1-13)NH2
0.16 (0.12-0.21) 1100 (570-2300) 6300 (2400-17000) 105 (75-150) 0.19 (0.13-0.26)
K~ (nM)a x 0.15 (0.07-0.29) 520 (430-610) 9.52 (7.37-12.3) 3.57 (2.77-4.60) 0.31 (0.14-0.69)
#
K~ ratio K//z
0.64 (0.49-0.82) 970 (490-1900) 89 (72-110) 6.03 (4.07-8.86) 0.92 (0.57-1.48)
1/4.3 1/1.9 1/9.4 1/1.7 1/3.0
a: 95% confidence intervals shown in parentheses. In summary, the [2,5] cyclic Dyn A analogue exhibited the highest opioid receptor affinity and opioid activity, comparable to the parent linear peptide. The [5,8] cyclic analogue demonstrated the greatest selectivity for K receptors, but exhibited weak agonist activity in the GPI. ACKNOWLEDGEMENTS The authors thank D.G. Grandy for his generous gift of the cloned receptors. The authors also thank M. Knittel, B.G. Olenchek, V. Caldwell, B. Hettinger-Smith and J. Roth for performing pharmacological evaluations. This research was supported by NIDA grant R01 DA05195. REFERENCES 1. C. Chavkin and A. Goldstein (1981) Proc. Natl. Acad. Sci. USA 78, 6543-6547. 2. R. Schwyzer (1986) Biochemistry 25, 4281-4286. 3. S. Arttamangkul, T.F. Murray, G.E. DeLander, W.C. Johnson and J.V. Aldrich (in preparation). 4. J.R. Bunzow, G. Zhang, C. Bouvier, C. Saez, O.K. Ronnekleiv, M. Kelly and D.G. Grandy (1994) J. Neurochem. (in press). 5. S.C. Story, T.F. Murray, G.E. DeLander, J.V. Aldrich (1992) Int. J. Pept. Protein Res. 40, 89-96. 6. A.M. Kawasaki, R.J. Knapp, T.H. Kramer, W.S. Wire, O.S. Vasquez, H.I. Yamamura, T.F. Burks, V.J. Hruby (1990) J. Med. Chem. 33, 1874-1879.
Chemistry The cyclic peptide analogues were prepared by solid phase synthesis (3). N=-Fmoc amino acids were used for elongation of the peptide backbone on an MBHA (methylbenzhydrylamine) resin. The cyclizations were performed on the resin using HBTU (2-(1H-benzotriazol-l-yl)-l,l,3,3tetramethyluronium hexafluorophosphate), BOP (benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate) or PyBOP (benzotriazol-l-yl-oxy-tris-(pyrrolidino)phosphonium hexafluorophosphate). Using HBTU for the cyclization reactions resulted in a major side reaction which decreased the yields of the cyclic peptides. The phosphonium reagents BOP and PyBOP gave slower cyclization reactions but higher yields and purer crude cyclic peptides.
Opioid Receptor Binding Assays and Smooth Muscle Assays Opioid receptor binding assays were performed using rat r and # opioid receptors expressed in COS-7 (compounds 1 and 2; see Table 1) or CHO cells (compounds 3 - 4) (4). Binding affinities for K- and t~-receptors were determined by displacing [3H]diprenorphine and [3H]DAMGO, respectively, from membrane preparations. Incubations were performed for 90 min at 22°C in the presence of peptidase inhibitors, and nonspecific binding was determined in the presence of 10 t~M unlabelled Dyn A-(1-13)NH2 and DAMGO, respectively, for r and # receptors. The peptides were evaluated for opioid activity in the electrically stimulated guinea pig ileum (GPI) (5) generally in the presence of peptidase inhibitors. RESULTS AND CONCLUSIONS Conformationally constrained Dyn A-(1-13)NH2 analogues cyclized between the side chains of DAsp i and Dap i+3 exhibited significant differences in opioid receptor affinity and opioid activity depending upon the positions involved in the cyclization (Table 1). Compound 1, cyclized between residues 2 and 5, showed the highest binding affinity for both r and tz receptors and was a potent agonist in the GPI assay. Shifting the constraint to residues 3 and 6 gave compound 2, which exhibited very poor binding affinity as well as weak agonist activity. Compound 3, which was cyclized between residues 5 and 8, gave dissimilar results for binding to x receptors vs. opioid
activity in GPI, similar to results reported for cyclic disufide analogues of Dyn A constrained through residue 5 (6). Compound 3 exhibited moderate affinity for x receptors, with 9-fold higher affinity for K than/~ receptors. In the GPI assay, however, compound 3 was a very weak agonist and was 33,000 times less potent than Dyn A-(1-13)NH2. In contrast, shifting the cyclic constraint to the 6 and 9 positions resulted in a compound with both moderate affinity for K and g receptors and modest agonist activity in the GPI. Table 1 Opioid receptor binding affinity and opioid activity of cyclic Dyn A analogues. Dyn A-(1-13)NH2 analogues
GPI IC5o (nM)
1. cyclo[D-Asp2,DapS]2. cyclo[D-Asp3,Dap6]3. cyclo[D-AspS,DapS]4. cyclo[D-Asp6,Dapg]Dyn A-(1-13)NH2
0.16 (0.12-0.21) 1100 (570-2300) 6300 (2400-17000) 105 (75-150) 0.19 (0.13-0.26)
K~ (nM)a x 0.15 (0.07-0.29) 520 (430-610) 9.52 (7.37-12.3) 3.57 (2.77-4.60) 0.31 (0.14-0.69)
#
K~ ratio K//z
0.64 (0.49-0.82) 970 (490-1900) 89 (72-110) 6.03 (4.07-8.86) 0.92 (0.57-1.48)
1/4.3 1/1.9 1/9.4 1/1.7 1/3.0
a: 95% confidence intervals shown in parentheses. In summary, the [2,5] cyclic Dyn A analogue exhibited the highest opioid receptor affinity and opioid activity, comparable to the parent linear peptide. The [5,8] cyclic analogue demonstrated the greatest selectivity for K receptors, but exhibited weak agonist activity in the GPI. ACKNOWLEDGEMENTS The authors thank D.G. Grandy for his generous gift of the cloned receptors. The authors also thank M. Knittel, B.G. Olenchek, V. Caldwell, B. Hettinger-Smith and J. Roth for performing pharmacological evaluations. This research was supported by NIDA grant R01 DA05195. REFERENCES 1. C. Chavkin and A. Goldstein (1981) Proc. Natl. Acad. Sci. USA 78, 6543-6547. 2. R. Schwyzer (1986) Biochemistry 25, 4281-4286. 3. S. Arttamangkul, T.F. Murray, G.E. DeLander, W.C. Johnson and J.V. Aldrich (in preparation). 4. J.R. Bunzow, G. Zhang, C. Bouvier, C. Saez, O.K. Ronnekleiv, M. Kelly and D.G. Grandy (1994) J. Neurochem. (in press). 5. S.C. Story, T.F. Murray, G.E. DeLander, J.V. Aldrich (1992) Int. J. Pept. Protein Res. 40, 89-96. 6. A.M. Kawasaki, R.J. Knapp, T.H. Kramer, W.S. Wire, O.S. Vasquez, H.I. Yamamura, T.F. Burks, V.J. Hruby (1990) J. Med. Chem. 33, 1874-1879.