Molecular and Cellular Pharmacology
Structure–function relationship studies of PTH(1–11) analogues containing D-amino acids

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Abstract

Parathyroid hormone (PTH) is an 84-amino acid peptide hormone. Produced in the parathyroid glands, it acts primarily on bone and kidney to maintain extracellular calcium levels within normal limits. It has been shown that the 1–34 amino acid fragment of PTH is sufficient to bind and activate the PTH type-I receptor. Recent investigations focusing on the interaction of N-terminal fragments of PTH with PTH type-I receptor showed that certain modifications can increase signalling potency in peptides as short as 11 amino acids. To understand the role of the side chains of all the amino acid residues in PTH(1–11), we synthesized all-d PTH, three retro-inverso analogues of the most active modified PTH(1–11), H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2, and we substituted every l-AA of the latter with the corresponding d-AA, obtaining a library of PTH(1–11) analogues that were tested as agonists. The library was synthesized by SPPS, employing the Fmoc protocol. The biological tests showed that the activity of the d-Har11 analogue is of the same order of magnitude of that of the most active modified PTH(1–11). This behaviour is paralleled by an increase of the helical content on going from the d-Val2 to the d-Har11 analogue. This is in agreement with previous work where a correlation between activity and helical content has been demonstrated. The importance of a positively charged group in the C-terminal position is shown to be independent of the configuration of the Cα-carbon.

Introduction

In mammals, parathyroid hormone (PTH) (Kronenberg et al., 1997), an 84-amino acid hormone, plays a vital role in regulating the concentrations of ionized calcium and phosphate in blood and extracellular fluids. PTH-related protein (PTHrP) plays a critical role in the development of the fetal skeleton (Chorev and Rosenblatt, 1994). The biological actions of both these peptides are mediated by the PTH/PTHrP receptor (or PTH1 receptor) (Jüppner et al., 1991), a family B G protein-coupled receptor (Kronenberg et al., 1997) expressed on the surface of bone and kidney target cells. It has been shown that the first 34-amino acid fragment of PTH is sufficient for in vivo bioactivity, and to reproduce biological responses characteristic of the native intact PTH (Kronenberg et al., 1997). Clinical studies have demonstrated that PTH(1–34) is a powerful bone anabolic agent able to restore bone mineral density.

The prevailing view of the biologically relevant conformation of PTH(1–34) includes a flexible molecule with no tertiary structure (Gardella and Jüppner, 2001, Shimizu et al., 2002). NMR analyses of PTH(1–34) analogues in a variety of polar and non polar solvents suggest that the N-terminal portion of PTH, known to be responsible for receptor activation, contains a short α-helical segment from Ser3 to Lys13. In addition, there is a more stable C-terminal α-helical segment (from Arg20 to Val31), where the principal receptor binding domain is located. These two helices are separated by two hinge-like motifs located around positions 12 and 19 (Scian et al., 2006). Studies to reduce the peptide size have demonstrated that enhancement of α-helicity in the PTH(1–11) sequence results in potent PTH(1–11)NH2 analogues (Potts and Gardella, 2008, Tsomaia et al., 2004, Barazza et al., 2005). Based on mutagenesis studies and on the position and shape of the binding sites for residues in position 2, 5 and 8, high helicity has been suggested to be essential for receptor activation (Monticelli et al., 2002, Shimizu et al., 2000a, Shimizu et al., 2000b). Furthermore, location of residue 8 on the same face of the helix as Ile5, as well as the position of Val2 projecting toward the third extracellular loop (EC3) have been hypothesized to be fundamental requirements for the activation of PTH1 receptor (Shimizu et al., 2000a, Shimizu et al., 2000b).

Introduction of conformational constraints, such as α-amino isobutyric acid (Aib), into peptides can improve their activity and receptor binding selectivity (Hirschmann, 1991, Gante, 1994, Kessler et al., 1995).

To probe the effect of an enantiomeric topological disposition of the critical side chains in PTH, we synthesized first of all an analog of PTH(1–34) containing all d-amino acids. To investigate the importance of the relative position of specific side chains, we focused on the most active PTH(1–11) analogue, H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2 (Shimizu et al., 2001a, Shimizu et al., 2001b). Initially, we introduced retro-inverso modifications and consequently, we substituted every l-amino acid with the corresponding d-amino acid, obtaining a library of PTH(1–11) analogues that were tested as agonists (Table 1).

Replacement of each residue by its optical isomer provides useful information regarding possible turn positions as only certain turn types can accommodate either l or d-residues, and d-amino acids may confer resistance to proteolytic degradation. Another important advantage for peptide drug development is that analogues containing d-amino acids have been found to be significantly less immunogenic than those containing their l-amino acid counterparts (Gill et al., 1963, Borek et al., 1965, Quintana et al., 2007).

The replacement of Met with Nle is known to be well tolerated with no loss of binding affinity (Rosenblatt et al., 1976) and avoids methionine oxidation, which would result in a decrease of the biological response (Frelinger and Zull, 1984). The replacement of Leu11 with Arg11 enhanced autoactivation (Shimizu et al., 2000b) in the PTH1 receptor/[Arg11]PTH(1–11) tethered system, which lacks most of the extracellular N-terminal domain (N-ECD) of PTH1 receptor, thus leading to the hypothesis that neither the 1–181 N-ECD of the receptor nor the C-terminal portion of PTH(1–34) are essential for bioactivity. Moreover, the presence of Arg11/Har11 turned some analogues of PTH(1–11) into potential agonists (Shimizu et al., 2001a, Shimizu et al., 2001b), while PTH analogues truncated at position 10 displayed poor binding and low activity (Shimizu et al., 2004).

Section snippets

Synthesis

A general synthetic protocol for the solid-phase synthesis of allDPTH(1–34) and peptidic analogues of PTH(1–11)NH2 was used with an automated peptide synthesizer (model 348 Ω, Advanced ChemTech, Louisville, KY). The analogues were prepared using the Fmoc methodology with a Rink Amide MHBA Resin (Novabiochem) (0.73 mmol/g loading) as a solid support (Carpino et al., 1994) and using Fmoc (N-(9-fluorenyl)methyloxycarbonyl) main chain protecting group chemistry. All amino acids and reagents were

Results

The synthesized library of analogues of PTH(1–11)NH2 is summarised in Table 2.

An allDPTH(1–34) analogue was synthesized following general protocol for the solid-phase peptide synthesis. We used HBTU/DIPEA coupling reagents in 4 equivalents and we used double couplings only for the hindered residues. In the synthesis of retro-inverso analogues, the reversed direction of the peptide introduced a steric difficulty in the coupling of the first amino acid to the solid support. In fact, Aib is a

Discussion

Our first approach to address the pharmacological properties of the N-terminal PTH fragment was the use of retro-inverso modifications (Schumacher et al., 1996). In this method, the l-amino acids of a peptide are replaced with d-amino acids, and the direction of the peptide chain is reversed. The topology of the side chains is largely maintained and such structures are resistant to proteases. The retro-inverso modification does not introduce conformational restrictions relative to the native

Acknowledgements

The authors thank MIUR, Ministry of Education and University of Italy, for financial support and Prof. E. Ragazzi for precious assistance in pharmacological comments.

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