Toward an Understanding of Human Parathyroid Hormone Structure and Function
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PTH and Its Receptor Family
Parathyroid hormone (PTH) is synthesized as a 115 amino acid precursor and secreted as an 84 amino acid polypeptide that regulates extracellular calcium homeostasis via actions directly on kidney and bone and indirectly on the intestine by facilitating calcium absorption.
PTH and a related molecule, parathyroid hormone-related protein (PTHrP), act on cells via a common G protein-coupled, seven-transmembrane helix receptor (PTH/ PTHrP or PTH1 receptor).
PTH has both anabolic and catabolic effects on the skeleton.
Persistent elevation of plasma PTH causes predominately increased bone resorption, whereas intermittently administered PTH results in enhanced bone formation.
The mechanism by which PTH exhibits its dual effects is not fully known.
PTH interacts with the PTH1 receptor to stimulate adenylyl cyclase (AC)(5) and phospholipase C (PC) pathways.
The naturally occurring hPTH(1-37) fragment as well as hPTH(1-34) maintains the full spectrum of bone-relevant activities of the intact 1-84 hormone.
Studies, both in vitro and in vivo, have shown that hPTH(1-34) has the same biological activities as the intact hormone in eliciting cAMP responses and in stimulating bone formation.
Thus hPTH(1-34) has all the structural elements necessary for binding and activation of the PTH1 receptor. Once daily subcutaneous administration of hPTH(1-34) stimulates bone formation and increases bone mass in patients with osteoporosis and in ovariectomized monkeys.
Consequently, hPTH(1-34) represents a novel class of therapeutics for the treatment of osteoporosis.
Truncation and mutagenesis studies on PTH(1-34) have revealed that the N-terminal region of the peptide is critical for full activation of receptor signaling, while the N-terminal truncated peptide PTH (3-34) is a partial agonist, and the further shortened peptide PTH (7-34) becomes a low affinity antagonist.
Residues 17 to 31, near the C-terminus of PTH (1-34), are required for high affinity receptor binding. PTHrP is over-expressed in certain tumors and causes the syndrome of malignancyassociated humoral hypercalcemia.
Under physiological conditions, PTHrP is produced locally in a wide variety of tissues and is involved in cell growth, differentiation, and development of the skeleton. There are six identical amino acids in the first 13 amino acids in the known PTH and PTHrP sequences.
Like PTH, PTHrP binds to the same G protein-coupled receptor, and its N-terminal fragment PTHrP(1-34) has many functions that mimic those of full-length PTHrP, PTH(1-34) and PTH(1-84).
In addition, NMR studies indicate that hPTH(1-34) and hPTHrP(1-34) have similar three-dimensional structures.
The recently identified PTH2 receptor natural ligand, tuberoinfundibular peptide 39 (TIP39), has been characterized structurally as a PTH homolog.
The human PTH2 receptor shares 70% sequence similarity with the human PTH1 receptor.
The PTH1 receptor is activated by PTH and PTHrP but not by TIP39, while the rat PTH2 receptor is activated by PTH and TIP39 and responds very weakly to PTHrP.
Further studies demonstrated that TIP39 and its truncated analogs bind to the PTH1 receptor but its N-terminal domain failed to stimulate cAMP accumulation; therefore, they function as antagonists at the PTH1 receptor (21).
NMR studies indicate that TIP39, similar to hPTH(1-34), contains two stable α-helices at the N and C termini separated by a flexible hinge region.
TIP39 is in a somewhat extended conformation; the N-terminal helix shares a high structural and sequence homology with hPTH(1-34).
The differences are the lengths and amphipathic character of the helices as well as the location of the flexible hinge region.
Comparison of PTH, PTHrP and TIP39 as well as their binding differences with the PTH1 and PTH2 receptors provides a starting point to study the structure-function relationships between the ligands and receptors.
Studies such as mutagenesis in both ligand and receptors, creation of receptor chimeras, and cross-linking between ligand and receptors have been performed extensively in the PTH family.
Biophysical techniques such as circular dichroism (CD), nuclear magnetic resonance (NMR), x-ray crystallography and molecular modeling provide information on PTH structure and its possible interactions with receptors for cell signaling.
PTH Structural Determination
Various methods have been used to determine the structure of PTH, including dark-field electron microscopy, fluorescence spectroscopy, CD, NMR spectroscopy22-29 and X-ray crystallography. Results from these diverse approaches have not yet yielded a consistent structure for this peptide.
In part, this uncertainty arises from the flexible nature of most small peptides in solution, as well as different experimental conditions such as peptide concentration, solvent conditions, pH, temperature and differences in methods used for data interpretation.
There is general agreement that PTH(1-34) and PTHrP(1-34) have an N-terminal helix and a C-terminal helix, which vary in length and stability, depending on the specific experimental conditions, and are connected by a highly flexible mid-region.
The C-terminal helix is more stable than the N-terminal helix. In aqueous solution, PTH(1-34) and PTHrP(1-34) form fewer and less stable secondary structural elements than under membrane-mimicking conditions such as in dodecylphosphocholine micelles, palmitoyloleoylphosphatidylserine vesicles, or in the presence of a secondary structure-inducing solvent such as trifluoroethanol (TFE).
Here we shall describe our x-ray crystallographic studies on hPTH(1-34) and give a brief review of recent NMR and molecular modeling results.
X-Ray Crystallography
Human PTH(1-34) (Forteo, Eli Lilly and Company,
Indianapolis, IN, U.S.A.) was expressed in E. coli and subsequently purified and refolded by reverse phase and cation exchange chromatography.
For phase determination, selenomethionine hPTH(1-34) was synthesized on an ABI-430A peptide synthesizer using Boc seleno-L-methionine.
hPTH(1-34) could be readily crystallized in many different forms under slightly different solvent conditions. The best crystals were grown at 20°C by the hanging drop vapor diffusion method by mixing 20 mg ml-1 of hPTH (1-34) in 20% glycerol, at 1:1 ratio (v/v), with a solution containing 2.5 M ammonium sulfate, 5% isopropanol and 0.1 M sodium acetate buffer, pH 4.5.
Crystals appeared overnight and continuously grew to 0.6 x 0.2 x 0.1 mm3 in a week.
Ultra-high 0.9 Å resolution data were collected by a Mar CCD detector at the Industrial Macromolecule Crystallography Association beam line ID-17 in Argonne National Laboratories.
The crystals belong to haxagonal space group P65 with unit cell dimensions of a= 30.18 Å and c=110.44 Å.
Three data sets were collected for a selenomethionine hPTH (1-34) crystal at wavelengths of 0.9795, 0.97936 and 0.9840 Å for multiwavelength anomalous dispersion (MAD) phasing calculations.
The structure was solved by the program SOLVE with the MAD data. The polypeptide chain was fitted to the electron density using the program O.31
The model was refined to 2.0 Å resolution using the MAD data, and to 0.9 Å resolution using the native data in X-PLOR9832 by simulated annealing.
The model was then further refined in SHELX 9733 by the conjugate-gradient algorithm with riding hydrogens. Six-parameter anisotropic temperature factors for all non-H atoms were included in and after anisotropic refinement. Sequential model building processes were performed in QUANTA (Molecular Simulations, Inc) against 2Fo-Fc and Fo-Fc maps. The final R factor for all data was 13.7%, R-free was 14%. The final structure contains 660 non-hydrogen peptide atoms and 104 water molecules.
All residues are in the most favorable conformation in Ramachandran plot.
The crystal structure of hPTH(1-34) is a slightly bent helix. The bend is located between residues 12 and 21 with a bending angle of 15° between the N-terminal helix (residues 3-11) and the C-terminal helix (residues 21-33). Hydrogen bonds between the side chains of Asn16 and Glu19, Ser17 and Arg20, and a salt bridge between Glu22 and Arg25 were observed.
Although hPTH(1-34) is a continuous helix, residues 6-20 and residues 21-33 form two amphiphilic helices with their hydrophobic sides facing in different directions.
Thus, the hydrophobic residues of hPTH(1-34) form a twisted belt from the N-terminus to the C-terminus with the crossing-point near residue Arg20.
Gly12 is a conserved residue in all the known PTH and PTHrP species. Despite the flexible nature of glycine, Gly12 is in a strict helical conformation in the crystal structure.
Substitution of Gly12 with Ala, a helix promotor, in [Tyr34]hPTH(1-34)NH2 was well tolerated; while substitution with Pro, a helix breaker, decreased receptor binding affinity by 840 fold and adenylyl cyclase-stimulating activity by 3500 fold.34 Together, these findings indicate that the helical conformation around Gly12 may play an essential role for full biological activity. hPTH(1-34) crystallizes as a dimer in the hexagonal space group P65.
His14 and Ser17 from both molecules are located at the crossing-point of the X-shaped dimer. The Nδ of His14 from one molecule forms a hydrogen bond with Nδ of His14 from another molecule, while Ser17 from one molecule packs against the imidazole ring of His14 from the other molecule. Within the dimeric interface, hydrophobic interactions extend from the crossing-point toward the N- and C-termini. Residues Leu7, Leu11 and Leu15 of the N-terminal amphiphilic helix from one molecule are in van de Waals contact with residues Leu24, Val21 and Met18 of the C-terminal amphiphilic helix of the other molecule.
There is no evidence of well-ordered dimerization of PTH under physiological conditions; therefore, the dimer formation is most likely the result of crystal packing artifacts under specific solvent condition.
The monomer structure was used for molecular modeling.
NMR Studies
NMR and x-ray crystallography are in many respects complementary.
X-ray crystallography deals with the structure of proteins in the crystalline state, while NMR determines structure in solution.
X-ray structures are determined at different levels of resolution.
At high resolution, most atomic positions can be determined to a high degree of accuracy.
NMR has the advantage to determine structures in near-physiological solution. In NMR, a COSY (correlation spectroscopy) experiment gives peaks between hydrogen atoms that are covalently connected through one or two other atoms.
An NOE (nuclear overhauser effect) spectrum, on the other hand, gives peaks between pairs of hydrogen atoms that are close together in space even if they are quite distant in primary sequence. To determine the three-dimensional structure of proteins, combined multidimensional NMR experiments including double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), NOE spectroscopy (NOESY) have been used to obtain a list of distance constraints between atoms in the molecule, from these data a set of three-dimensional structures that satisfy these constraints are calculated. NMR studies of hPTH(1-84) defined three helices between Ser3 to Asn10, Ser17 to Lys27, and Asp30 to Leu37.
In the C-terminus, a less well-defined helix between Asn57 to Ser62 and series of loose turns were detected.
In contrast to the hPTH(1-34) structure, the intact hormone shows a number of long-range NOEs, specially between helix 1 and helix 2.
Several of the NMR studies have been interpreted to show a “U or V-shaped” tertiary structure with the N- and C-terminal helices interacting with each other to form a hydrophobic core. However the majority of the NMR analyses of PTH and PTHrP do not provide evidence of long-range interactions between the two helices.
NMR studies of hPTH(1-34) in near-physiological solution revealed a relatively extended two-helical component structure with the absence of any tertiary interactions between the two helices proposed in the U-shaped model.
Extensive NMR studies have been carried out on PTH and PTHrP in different solvent environments. In general, NMR studies show that PTH(1-34) and PTHrP(1-34) form an N-terminal helix and a C-terminal helix connected by a highly flexible region in solution.
The highly flexible region in the NMR structures (residues 10-20) is found to form a regular helix in the crystal structure.
Evidence from several NMR studies on PTH(1-34) and PTHrP(1-34) concluded that the helical content increases with increasing TFE concentration or conditions that mimic the membrane environment.
In 70% TFE, the N- and C-terminal helices (residues 3-13 and 15-29) of PTH(1-34) were very regular with a short discontinuity at residue 14, NMR structures of PTHrP(1-34) in 50% TFE also showed two well-defined helices (residues 3-12 and 17-33).
Our crystal structure is similar to the NMR structures determined in high concentrations of TFE or under membrane-mimicking conditions.
This similarity is not surprising because hPTH(1-34) in the crystal has very limited solvent exposure. The solvent content of the hPTH(1-34) crystal is less than 30% with extensive hydrophobic protein-protein interactions.
In fact, the crystal structure might represent the conformation of PTH (1-34) when it is close to its membrane receptor as proposed for the NMR structures under high concentrations of TFE or membrane-mimicking conditions.
Molecular Modeling
Currently, there is no satisfactory method to directly determine the PTH-PTH receptor complex structure. Molecular modeling provides an alternative method to explore the ligand-receptor binding mode.
PTH1 and PTH2 receptors belong to a GPCR family from which members have not yet been crystallized.
The crystal structure of rhodopsin has provided the first three-dimensional model in atomic resolution for the GPCR families. Although construction of realistic models of certain GPCRs like PTH receptors with large extra-cellular domain remains challenging, the crystal structure of rhodopsin does provide a general starting point for modeling GPCRs, specifically in the seven-transmembrane (TM) helix region. We undertook a molecular modeling with the program QUANTA using the Protein Design tools.
The seven transmembrane helical domains of the PTH1 receptor were first determined by several programs provided by the ExPASy Molecular Biology Server.
The crystal structure of bacteriorhodopsin at 1.9 Å (PDB code: 1QHJ) was used as a template for the topological orientation and arrangement of the transmembrane helices. Sequences of the TM helices for the PTH1 receptor and bacteriorhodopsin were aligned, and then homology modeling was carried out to create the TM helices of the PTH1 receptor.
The conformations of the intracellular and extracellular loops were constructed in QUANTA using the fragment database-searching algorithm. For the N-terminal receptor region 168-198, the NMR structure determined in a lipid environment (PDB code: 1BL1) was incorporated in the model.
This was accomplished by aligning the membrane embedded helix (residues 190-196) with the beginning of transmembrane helix 1.
The full-length PTH1 receptor contains residues 1-593, our model is only partial, containing residues 168-469.
One hPTH(1-34) monomer, derived from the crystal structure, was docked onto the receptor using two constraints based on cross-linking studies.
Energy minimization was applied to the complex of hPTH(1-34) and residues 168-198 of the receptor using the default setting in QUANTA until no significant changes were observed.
The hPTHrP(1-34) model was produced by homology modeling using the crystal structure of hPTH(1-34) as a template.
The model of hPTHrP(1-34) binding to the PTH1 receptor was created similarly to the hPTH(1-34)-receptor complex. Previous studies on PTH- or PTHrP-receptor interactions have suggested that the juxtamembrane region of the transmembrane helices and extracellular loops (especially the third loop) of the PTH1 receptor interact with the amino-terminus of PTH or PTHrP agonists (the so-called “activation domain”) to induce second messenger signaling; the amino-terminal extracellular region of the receptor interacts with the carboxyl-terminal region (residues 15-34) of either PTH or PTHrP during ligand binding.
Results from photoaffinity cross-linking by p-benzoylphenylalanine and site-directed mutagenesis identified two contact points in the PTH(1-34):PTH1 receptor complex, Ser1 of hPTH(1-34) to Met425 of the receptor and Lys13 of hPTH(1-34) to Arg186 of the receptor.
Our model of hPTH(1-34) bound to the PTH1 receptor was created by incorporating these restraints. In our model, the N-terminal region of hPTH(1-34), responsible for its agonist activity, binds to a pocket consisting of the extracellular portions of TM3, TM4 and TM6 and the second and third extracellular loops of the receptor. The middle region of hPTH(1-34) is sandwiched between the first extracellular loop and the N-terminal extracellular region of the receptor adjacent to TM1.
The C-terminal region of hPTH(1-34) forms extensive interactions with the putative binding domain of the PTH1 receptor.
This interface consists of hydrophobic interactions (residues Leu24, Trp23 and Leu28 of hPTH(1-34), and Phe173, Leu174 of the receptor), and hydrophilic interactions between Arg20 of hPTH(1-34) and Glu180, Glu177 of the receptor, as well as Lys27 of hPTH(1-34) and Glu169 of the receptor.
Recent photoaffinity cross-linking studies reveal that residues 23, 27, 28 of native PTHrP are indeed near regions of the amino-terminal extracellular domain of the PTH1 receptor.
Site-directed mutagenesis in the C-terminal region of hPTH(1-34) have suggested that Leu24 and Leu28 are intolerant to mutation.
When Leu24 and Leu28 are substituted by Glu, the receptor binding affinities were decreased by 4000 and 1600 fold respectively.
A less dramatic reduction of receptor binding affinity (40 fold) is observed when Val31 is replaced by Glu. In contrast, replacement of Asp30 by Lys has no effect on receptor binding.
In our model, Leu24 and Leu28 of hPTH(1-34) are located at the center of the hydrophobic interface while Val31 is located at the end of the hydrophobic patch. Asp30 is exposed to solvent; therefore, the lysine mutant at this position would not be likely to change the receptor binding affinity.
The hydrophilic interaction between Lys27 of hPTH(1-34) and Glu169 of the PTH1 receptor may be less important for binding than other interactions because a variety of mutations were tolerated at Lys27. Several other models have been proposed in the literature for the binding of hPTH(1-34) to the PTH1 receptor, utilizing the NMR structure of hPTH(1-34) with Nand C-terminal helices connected by a flexible loop.
The most predictive model will likely be based on a combination of all the mutagenesis, cross-linking and structural experimental data available.
In the PTHrP:PTH1 receptor model (not shown here), residues Arg20, Phe23, Leu24, Ile28 and Ile31 of hPTHrP(1-34) form similar interaction with receptor as the corresponding residues of hPTH (1-34).
Residue Leu27 in hPTHrP, which is lysine in hPTH, is included in the extensive hydrophobic interface.
hPTH and hPTHrP(1-34) share eight identical amino acids in the region 1-13, but only three identical amino acids in the region 14-34. However, the C-termini of both peptides form similar amphiphilic helices that are proposed to be responsible for high affinity receptor binding.
When residues 22-31 were substituted with a model amphiphilic sequence (ELLEKLLEKL) in the PTHrP analog RS-66271, it demonstrated high in vivo bone anabolic efficacy.
CD and NMR studies confirmed that RS-66271 exists in a helical conformation from residues 16 to 32.
Our models for the interaction of PTH and PTHrP to the PTH1 receptor support the hypothesis that the amphiphilic helices at the C-terminal regions of the PTH and PTHrP(1-34) are responsible for high affinity peptide-receptor interaction.
Structural Based Design of PTH Analogs
Stabilizing α-Helical Conformation
Lactam bridges were introduced at different locations along the peptide to connect the side chains at i and i+4 positions in an effort to stabilize a helical conformation and identify the bioactive conformation of PTH and PTHrP. Structural and functional studies have suggested that increasing helical content by such conformational constraints may increase biological potency, but this result is highly sensitive to the constrained positions.
Condon, S.M. et al, reported that adenylyl cyclase- stimulating activity in ROS 17/2.8 cells was increased when a lactam bridge was introduced between residues 14 and 18 or 18 and 22 of hPTH (1-31)NH2, but decreased when the lactam bridge was introduced between residues 10 and 14.
In PTHrP, when lactamization was introduced between residues 13 and 17, adenylyl cyclase-stimulating activity was also increased. However, a lactam bridge introduced between residues 26 and 30 resulted in 400 times lower binding affinity and 30 times lower adenylyl cyclase-stimulating activity. Interestingly, the lactam-containing structures of hPTH(1-31) and hPTHrP(1-34) by NMR were both in extended helical conformations, similar to our crystal structure of hPTH(1-34).
In the crystal structure of hPTH(1-34), the three well tolerated lactam bridges (residues 13-17, 14-18 and 18-22) are located on either the convex or concave sides of the arc formed by the slightly bent helix and are in the mid-region of the molecule.
Thus, it appears that enhancing helical structure in this flexible region of the peptide increases the biological activity of PTH and PTHrP. The poorly tolerated bridges (residues 10-14 and 26-30) are located on the sides of the hPTH(1-34) helical arc.
In these cases, the decreased biological activities may be caused by twisting the helical arc sideways or interfering directly with the ligand-receptor interaction.
Thus, rigidity in the middle region of hPTH(1-34), as well as the bending direction of the helix appears to have significant functional effects. Therefore, the extended helical conformation observed in the crystal structure may well represent an active receptor-binding conformation of hPTH(1-34).
This led us to propose that hPTH(1-34) could be in a flexible conformation in solution as would occur in the extracellular space, but a regularized, slightly bent helical conformation is likely to be induced when the peptide approaches the hydrophobic membrane before receptor binding.
Substitution of Key Amino Acids
Extensive amino acid scanning has been done in PTH(1-34), especially in the 1-14 activation domain. In hPTH(1-34), substitution of Gly1 for Ser reduced phospholipase C activity but did not affect adenylyl cyclase activity, while removal of either Gly1 or the alpha-amino group eliminated phospholipase C activity completely.
Randomly mutated codons for amino acids 1-4 in hPTH(1-34) demonstrated that Val2 and Glu4 are important determinants of receptor binding and activation.
Studies with a series hybrid analogs containing both PTH and PTHrP sequences demonstrated that residue 5 (Ile in PTH and His in PTHrP) is the specificity “switch” between the PTH1 and PTH2 receptors, switching residues at 5 and 23 in PTHrP to PTH residues yielded a ligand that avidly bound the PTH2 receptor and fully stimulate cAMP formation in contrast to the totally inactive native PTHrP on the PTH2 receptor.
There are two methionine residues in hPTH(1-34) that can be oxidized.
Oxidation of Met 8 was reported produce a partial agonist with greatly reduced potency; oxidation of Met 18 full agonist with slightly reduced potency. Thus,
Met 8 is important in receptor binding and activation.
Systematic site-directed mutagenesis at multiple sites has been used to create more than 50 hPTH(1-34) analogs.
A highly active combination variant has been identified with six substitutions (K13S, E19S, V21Q, E22S, K27Q, D30N) and is 15 times more active for adenylyl cyclase-stimulation.
A variety of different unnatural amino acids have also been introduced into the PTH sequence to search for a more potent or signaling pathway specific agonist.
Combinations of cyclization and substitution with either natural or unnatural amino acids may yield agonists with greatly enhanced potency.
Minimizing Active PTH Length
It is well established that the hPTH(1-37) and hPTH(1-34) fragments maintain the full spectrum of bone-relevant activities of the intact 1-84 hormone.
A slightly shorter peptide, hPTH(1-31)NH2, appears to have nearly equipotent anabolic effects on the skeletons to hPTH(1-34), and predominantly stimulates adenylyl cyclase activity.
The reported NMR structure of hPTH(1-31)NH2 shows a V-shaped two helical structure.
Deletion of less structured residues His32, Asn33, and Phe34 as well as capping and specific hydrophobic interactions between the end of the N-terminal helix and the beginning of the C-terminal helix stabilize the helical conformation.
A lactam bridged analog (Leu27-cyclo(Glu22-Lys26)hPTH(1-31)NH2 demonstrated six times more AC-stimulating activity and slightly enhanced bone anabolic effects in ovariectomized rats.
To probe the role of residue 19 in the N-terminal domain’s binding, a series of hPTH (1-20) analogues have also been made and show biological activity.
Glu19 is conserved in all PTH sequences; in all PTHrP sequences residue 19 is Arg.
NMR studies indicate that PTH (1-20) is an extended α-helix from residue 4 to 19. Interestingly, mutation of Glu19 to Arg19 resulted in a more stably extended α-helix with enhanced biological activity. Arg 20 is conserved in both PTH and PTHrP families.
Substitutions of Arg 20 with a series of unnatural amino acids decrease the AC-stimulating activity.
Hydrogen bonding, hydrophobicity of the Arg side chain, and stabilization of α-helix in this region are all important for the interaction of the peptide with receptor.
Gardella and colleagues further shortened the active PTH analogs to 14 and 11 residues. PTH(1-14) and PTH(1-11) can stimulate the cAMP-signaling response, although they exhibit extremely weak binding affinities to the PTH1 receptor.
Specific substitutions with natural amino acids at positions 3, 11, 14 greatly enhance the potency in cell-based signaling assays.
Substitutions of residue 11 in PTH(1-11)NH2 with unnatural amino acids to increase the length and polarization of the side chain resulted in full cAMP agonists.
Substitutions of residues 1 and 3 with the sterically hindered and helix-promoting amino acid α-aminoisobutyric acid (Aib) in PTH(1-14) and PTH(1-11) analogs resulted in peptides that are highly active in bone cells.
These studies indicate that the α-helical conformation is important for receptor activation and cell signaling and only 11 amino acids are sufficient.
NMR data of hPTH(1-14) shows a typical helical conformation from 3-11 in aqueous solution.
Functional studies further demonstrated that residues Gln6 and Asn10 play a direct role for intramolecular side chain interactions with the receptor.
The findings are consistent with our model that shows residues 6, 10 along with residues 1, 3, 14 in the same face of the helix are involved in ligand-receptor interation. hPTH(1-34) was approved as an agent for the treatment of osteoporosis with the unique pharmacologic activity to build new bone.
However, the requirement for subcutaneous injection may limit its use in some patients.
The ultimate goal is to create a potent PTH analog or small molecule mimic that can be delivered by the oral or pulmonary routes.
The X-ray and NMR structures of hPTH (1-34), combined with the accumulated biochemical data have allowed modeling of the interactions of hPTH and hPTHrP with the PTH1 receptor.
These findings provide a conceptual starting point for unraveling the ligand-receptor recognition mechanism and, consequently, to guide structure-based design of novel PTH analogs and mimics.
