Importance of Ala and ValZ in Signal

Transduction. SAR studies have implicated the C-terminus domain to be important for maintaining potent receptor-binding affinity. The more flexible N-terminus region appears to play a critical role in signal transduction. Deletion of the first two amino-terminal residues results in a loss of all, or virtually all, biological activity but not in a loss of binding affinity to the PTH1 receptor in vitro. Thus there appears to be an apparent structural separation of binding and activation regions in PTH. Deletion of Ala1 was accompanied by a marked decrease in adenylate cyclase activity, implying that the minimum sequence required for biological activity starts at the second amino acid.

4.1.4 importance of ArgZO and Argz5 in Re-ceptor/Ligand interactions. Early SAR studies had indicated that conserved structural modifications in the central portion of the active fragment of PTH-(1-34) were remarkable for their relative lack of effect on biological activity. To evaluate the biological role of Arg20 and Arg25 in PTH-(l-34), fragment-selective postsynthetic modifications of these two residues in hPTH-(l-34) active fragment were done by use of 1,2-cyclohexanedione (100).

reacts specifically, completely, and reversibly with the guanidino group of arginine, to give a single product, iV7,iV8-( 1,2-dihydroxycyclohex- l,2-ylene)argi-nine, [DHCH-Arg2^ DHCH-Arg^JhPTH-U-34) (103). This modified analog showed, at most, 16% of the activity of the unmodified hormone hPTH-( 1-34), based on in vitro renal adenylate cyclase assays using borate buffer. Reversal of arginine modification completely restores biopotency, indicating that the po tency decline observed after 1,2-cyclohexanedione treatment is not caused by nonspecific alterations of structure. The near total loss in biopotency of the modified hormone may be further explained by the increase in steric bulk of the Arg20,25 side chains or by a change in the formal charge of the guanido group from a + 1 to zero in a putative arginine-borate complex. Whether these two effects individually or in combination may cause a dramatic decrease in biopotency through adverse effects on hormone-receptor interactions or through the induction of a conformational change in the peptide is not clear. However, these results illustrate the importance of the positively charged guanidino group in Arg on binding and bioactivity.

4.1.5 Importance of Gly12 in Ligand-Recep-tor Actions. Glycine, the simplest amino acid, plays a very important role in proteins and peptides. The absence of a side-chain functional group renders Gly a high degree of conformational flexibility in peptides. In addition, this amino acid can also access the phi and psi space of d-amino acids. Hence, glycine is generally found in the region where proteins and peptides undergo reverse-turn structures (55, 104,105). Glycine is the 12th residue from the N-terminus in the PTH sequence (106, 107). This residue is highly conserved in PTH and PTHrP isolated from all species to date. Structural studies that use the Chou-Fasman analysis and both CD and NMR studies have predicted that the a-helical N-terminal domain is followed by a j3-turnat positions 12-15 in PTH and 9-12 in PTHrP. Thus, the biological consequences of single-residue changes at position 12 were assessed in vitro, to determine the role of this residue in hormone-receptor interactions and its contribution toward the bioactive conformation of the peptide. In the human hormone [Tyr34]hPTH-(l-34)-NH2 substitution of Gly12 by Ala12, d-Ala12 and Alb12 gave agonists with binding affinities similar to that of [Tyr34]hPTH-(l-34)-NH2 in bovine renal cells (Kh [Tyr34]hPTH-(l-34)-NH, = 0.7 d ; Kh for substituted analogs between 0.7 and 1.0 cOThe Pro12 analog binds approximately 840-fold less tightly than [Tyr34]hPTH-(l-34)-NH2. The iTm values for adenylate cyclase activity are also similar to

Table 2.4 Binding and Cyclase Activity of Selected Antagonist Analogs of PTH with Bone Derived ROS17/2.8 Cells


Cyclase Km (nM)


[Tyr34]hPTH-(7-34)NH2 (1) [d-Trp 12,Tyr34]hPTH-( 7-34)NH2 (2) [Trp12,Tyr34]bPTH-(7-34)NH2 (3) [Nle8'18,Tyr34]bPTH-(7-34)NH2 (4) [Nie8'18,D-Trp12,Tyr34]bPTH-(7-34)NH2 (5) [Nle8'18,Trp12fTyr34]bPTH-(7-34)NH2 (6)

700 120 400 960 180 410

2700 210 1360 1550 70 220

110 110 110 110

that of [Tyr ]hPTH-(l-34)-NH2 for the Ala12, d-Ala12, and Aib12 analogs, whereas the Km value for the Pro12 analog is 3500-fold lower (107).

To design potent and selective antagonists (108) of PTH or any other peptide ligand, it is necessary to identify the sites within the hormone where structural modifications can be made that maintain high receptor affinity, but without inducing agonist properties. In principle these goals can be achieved by two different routes: (1) stabilize the antagonist in a conformation favored by the receptor for molecular recognition but not transduction; and (2)introduce new structural moieties that will add binding elements that interact with the receptor at sites different from or in addition to those present in the native agonist. These binding elements should retain the "bioactive conformation" of the ligand required for receptor binding that does not lead to information transduction.

Previous SAR studies with human and bovine PTH have shown that removal of the first six amino-terminal residues gives an analog with potent antagonistic activity in vivo, such as [Tyr34]hPTH-(7-34) NH2 (109) (Table 2.4). Substitution of Gly12 in this fragment by Ala12 and d-Ala12 gives analogs that are about twice as potent as the parent peptide [Tyr34]hPTH-(7-34) NH2 in both binding and cAMP assays, whereas the Pro12 analog is approximately twofold less active vs. the parent peptide. Furthermore, replacement of Gly12 with D-Trp (Table 2.4) results in an antagonist that is approximately 10-fold more potent in the binding assay and 12-fold more potent at inhibiting agonist-induced (3 nM [Nles'18,Tyr34]bPTH-(1-34)NH2) cAMP activity (Table 2.4). In contrast, the L-Trp analog (110) failed to alter either binding or cAMP activity, suggesting that introduction of hydrophobic side-chain groups in the d-configuration at position 12 provides auxiliary hydrophobic interactions with the receptor, which promotes binding. A combination of bulky hydrophobic residues at position 12 together with the substitution of Met8 and! Met1' by Nle gave analogs having binding activity 10- to 15-fold more potent than that cF [Nle8,18 Tyr34]bPTH-(7-34)NH2. These results suggest that introducing amino acid residues with aliphatic and/or bulky hydrophobic side chains in either the d- or l-conformer can. improve antagonism in the 7-34 truncated series (Table 2.4).

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