Superactive analogs of glucagon having an amidated carboxy terminal and no tryptophan or methionine
1
$46 SUPERACTIVE ANALOGS OF GLUCAGON HAVING AN AMIDATED CARBOXY TERMINAL AND NO TRYPTOPHAN OR METHIONINE. William A. Mu~phT~ Valentine A. Lance~ and David H. COy, Peptide Research Labs, Department of Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA. Structure-activity studies with glucagon have for the most part been based on semisynthetic approaches which produced analogs with diminished or no activity compared to the native glucagon molecule. We have recently reported the solid-phase synthesis of glucagon which allows analog studies on the entire 29 amino acid sequence. This work resulted in D-Phe-4-glucagon which was 6 times more active than glucagon in elevating blood glucose levels in the rat (J. Med. Chem. 27:310 (1984)). We have extended this work to include analogs with modifications in both the amino and COOH-terminal regions. The analogs were injected subcutaneously into fed, pentobarbital-anesthetized rats from which blood samples were withdrawn 15 min after peptide injection. Blood glucose levels were determined and compared to glucagon stimulated levels using a 4-point assay to determine relative potency. Substitution of serine in position 2 by its D isomer (D-Ser-2-glucagon) increased activity twofold. Substitution of glutamine in position 3 by its D isomer (D-Gln-3-glucagon) or replacement of threonine in position 7 by D-threonine (D-Thr-7-glucagon) resulted in analogs with approximately the same potency as glucagon. Unexpectedly, substitution of phenylalanine for tryptophan in position 25 coupled with replacement of methionine by leucine in position 27 and amidation of the carboxy terminus (Phe-25, Leu-27-glucagon-amide) resulted in an analog twice as active as glucagon itself. Addition of the D-Phe-4 modification to this analog increased the potency to levels at least as high as the D-Phe-4 analog itself. The data are in direct contrast to previous semisynthetic work which indicated that a free carboxy terminus and an indole ring in position 25 were required for full biological activity. This work was funded by NIH grant AM-30167. COMPARATIVE STRUCTURAL REQUIREMENT5 OF GRF ANALOGS FOR OCCUPANCY OF GRF AND VIP RECEPTORS AND COUPLING TO ADENYLATE CYCLASE IN RAT ADENOPITUITARY, LIVER AND PANCREAS. P. ROBBERECHT~ M. WAELBROECK t D. COY+: P. DE NEEF T 3 . - C . CAMUS AND 3. CHRISTOPHE. Department of Biochemistry and ~utrition, Medical School, Universit~ Libre de Bruxelles, Brussels, Belgium and Department of Medicine, Tulane University, New Orleans, LA, USA. The ability of synthetic GRF(1-29)-NH 2 analogs to stimulate adenylate cyclase was investigated in membranes from adenopituitary, liver and pancreas in rat. In adenopituitary, GRF and its analogs interacted with GRF receptors only, whereas in liver and pancreas they interacted with rIP receptors. The C-terminal moiety of GRF was required for GRF receptor recognition as synthetic helodermin (1-35)-NH2, a parent peptide with ~ amino~cids common to GRF in the 1-10 sequence, and the synthetic analog (His~,D-Ala)-GRF(1-9)VIP(lO-28) stimulated pituitary adenylate cyclase through the occupancy of VIP receptors only. When GRF analogs occupied GRF or VIP receptors, the N-terminal part of the molecule appeared to be critical for adenylate cyclase activation. This was established using 30 analogues mono-, bi-, or trisubstit~ted in ~ositions 1 to I0. The major findings were : a) the discovery of (N-Ac-Tyr , D-Arg )-FIRF(1-29)-NH 2 as an antagonist of s~imulated~ituita~y adenylate cyclase; b) the discovery of the (N-Ac-TYR 1, D-Phe=)-,(D-Ala ~, D-Asp', 8 27 1 2 3 27 1 2 7 D-Asn , NLeu )-,(His , D-Ala , D-Ser , NLeu )-, and (His , D-AIa , D-Thr , NLeu27)- derivatives of GRF(1-29)-NH 2 as specific antagonists of VIP receptors in the rat pancreas; c) the importance of the free NH 2 function on amino acid residue in position one for activation of adenylate cyc~ase through VIP receptors (but not through GRF receptors) as acetylation of Tyr ~ or His ~ decreased the intrinsic activity of the peptides; d) iodination of Tyr I decreased the potency and efficacy of GRF in all systems tested.
Transcript of Superactive analogs of glucagon having an amidated carboxy terminal and no tryptophan or methionine
$46
SUPERACTIVE ANALOGS OF GLUCAGON HAVING AN AMIDATED CARBOXY TERMINAL AND NO TRYPTOPHAN OR METHIONINE. William A. Mu~phT~ Valentine A. Lance~ and David H. COy, Peptide Research Labs, Department of Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA.
Structure-activity studies with glucagon have for the most part been based on semisynthetic approaches which produced analogs with diminished or no activity compared to the native glucagon molecule. We have recently reported the solid-phase synthesis of glucagon which allows analog studies on the entire 29 amino acid sequence. This work resulted in D-Phe-4-glucagon which was 6 times more active than glucagon in elevating blood glucose levels in the rat (J. Med. Chem. 27:310 (1984)). We have extended this work to include analogs with modifications in both the amino and COOH-terminal regions. The analogs were injected subcutaneously into fed, pentobarbital-anesthetized rats from which blood samples were withdrawn 15 min after peptide injection. Blood glucose levels were determined and compared to glucagon stimulated levels using a 4-point assay to determine relative potency. Substitution of serine in position 2 by its D isomer (D-Ser-2-glucagon) increased activity twofold. Substitution of glutamine in position 3 by its D isomer (D-Gln-3-glucagon) or replacement of threonine in position 7 by D-threonine (D-Thr-7-glucagon) resulted in analogs with approximately the same potency as glucagon. Unexpectedly, substitution of phenylalanine for tryptophan in position 25 coupled with replacement of methionine by leucine in position 27 and amidation of the carboxy terminus (Phe-25, Leu-27-glucagon-amide) resulted in an analog twice as active as glucagon itself. Addition of the D-Phe-4 modification to this analog increased the potency to levels at least as high as the D-Phe-4 analog itself. The data are in direct contrast to previous semisynthetic work which indicated that a free carboxy terminus and an indole ring in position 25 were required for full biological activity. This work was funded by NIH grant AM-30167.
COMPARATIVE STRUCTURAL REQUIREMENT5 OF GRF ANALOGS FOR OCCUPANCY OF GRF AND VIP RECEPTORS AND COUPLING TO ADENYLATE CYCLASE IN RAT ADENOPITUITARY, LIVER AND PANCREAS. P. ROBBERECHT~ M. WAELBROECK t D. COY+: P. DE NEEF T 3.-C. CAMUS AND 3. CHRISTOPHE. Depar tment o f B i o c h e m i s t r y and ~ u t r i t i o n , Med ica l Schoo l , U n i v e r s i t ~ L i b r e de B ruxe l l es , Brussels, Belgium and Department of Medicine, Tulane Un i ve r s i t y , New Orleans, LA, USA.
The a b i l i t y of syn the t i c GRF(1-29)-NH 2 analogs to s t imu la te adenylate cyclase was i nves t i ga ted in membranes from adenop i t u i t a r y , l i v e r and pancreas in ra t . In adenop i t u i t a r y , GRF and i t s analogs i n te rac ted wi th GRF receptors only , whereas in l i v e r and pancreas they i n te rac ted wi th r IP receptors . The C- terminal moiety of GRF was requ i red fo r GRF receptor recogn i t i on as syn the t i c helodermin (1-35)-NH2, a pa ren t p e p t i d e w i t h ~ a m i n o ~ c i d s common to GRF in the 1-10 sequence, and the s y n t h e t i c ana log (H is~ ,D-A la ) -GRF(1 -9 )V IP( lO-28 ) s t imu la ted p i t u i t a r y adenylate cyclase through the occupancy of VIP receptors only . When GRF analogs occupied GRF or VIP receptors, the N-terminal part of the molecule appeared to be critical for adenylate cyclase activation. This was established using 30 analogues mono-, bi-, or trisubstit~ted in ~ositions 1 to I0. The major findings were : a) the discovery of (N -Ac -Ty r , D-Arg )-FIRF(1-29)-NH 2 as an a n t a g o n i s t of s ~ i m u l a t e d ~ i t u i t a ~ y adenylate cyc lase; b) the d iscovery of the (N-Ac-TYR 1, D-Phe=)-, (D-Ala ~, D-Asp',
8 27 1 2 3 27 1 2 7 D-Asn , NLeu ) - , ( H i s , D-Ala , D-Ser , NLeu ) - , and (H is , D-AIa , D-Thr , NLeu27)- d e r i v a t i v e s of GRF(1-29)-NH 2 as s p e c i f i c antagonis ts of VIP receptors in the ra t pancreas; c) the importance of the f ree NH 2 func t ion on amino acid residue in p o s i t i o n one fo r a c t i v a t i o n of adenylate cyc~ase through VIP receptors (but not th rough GRF r e c e p t o r s ) as a c e t y l a t i o n o f Tyr ~ or His ~ decreased the i n t r i n s i c a c t i v i t y of the pept ides; d) i o d i n a t i o n of Tyr I decreased the potency and e f f i cacy of GRF in a l l systems tes ted.
