Conformational and biological characterization of human parathyroid hormone hPTH(1-34) analogues containing beta-amino acid residues in positions 17-19

The N-terminal 1-34 fragment of parathyroid hormone (PTH) elicits the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(18) upon biological function, we synthesized and characterized the following human (h) PTH(1-34) analogues containing beta-amino acid residues: [analogues: see text]. Biological activity and binding affinity of analogue I are one order of magnitude lower than those of the parent compound. In analogue II, both binding affinity and biological activity are partially recovered. Analogues III and V have no binding affinity and very low biological activity. Both bioactivity and binding affinity are partially recovered in analogue IV. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, 2D-nuclear magnetic resonance and molecular dynamics calculations. The results confirmed the presence in all analogues of two helical segments located at the N-terminal and C-terminal sequences. The insertion of beta-amino acid residues around position 18 does not cause appreciable conformational differences in the five analogues. The differences in biological activity and binding affinity among the five analogues cannot be related to structural differences in the membrane mimetic environment reported in this study. Our results stress the importance of the side-chain functionalities in the sequence 17-19 for biological function.     

Effects of bone in vitro of bovine parathyroid hormone and synthetic fragments representing residues 1-34, 2-34 and 3-34.

The biological activities of bovine parathyroid hormone (BPTH) and fragments comprising portions of its amino-terminal sequence have been compared in three different assay systems using embryonic rat bone in vitro. Whereas the 3-34 fragment was without significant activity the 1-34 fragment caused all the actions characteristic of BPTH 1-84, extending to bone previous evidence that the amino-terminal residues are sufficient for expression of the biological effects of intact parathyroid hormone. However, the relative potencies of the fragment and the intact hormone were different in the various systems. BPTH 1-34 showed relatively low osteolytic activity and induced anabolic effects in both osteoblasts and cartilage cells of cultivated embryonic mouse radii which were not evoked by the intact hormone. Further work is required to determine the mechanisms responsible for these interesting alterations in relative potency of fragment and native hormone.     

Structure-function studies of analogues of parathyroid hormone (PTH)-1-34 containing beta-amino acid residues in positions 11-13

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.     

Differential effects of parathyroid hormone responsive cultured human cells on biological activity of parathyroid hormone and parathyroid hormone inhibitory analogues

We have employed parathyroid hormone (PTH) responsive human cells cultured from dermis or giant cell tumors of bone (GT) to evaluate the biological properties of a newly developed in vivo PTH inhibitor, [Tyr34]bPTH-(7-34)-amide (PTH-Inh). Short periods of incubation of cells from dermis or GT with maximal stimulatory concentrations of PTH in the presence of increasing concentrations of PTH-Inh resulted in a dose-dependent inhibition of the adenosine cyclic 3',5'-phosphate (cAMP) response (Ki = 3 X 10(-7) M and 4.2 X 10(-7) M for GT and dermal cells, respectively). In both cell cultures, PTH-Inh alone did not increase cAMP levels, and in desensitization experiments, preincubation with PTH-Inh alone did not desensitize cells to PTH. Hence, the analogue displayed no agonist properties. Unexpectedly, when PTH-Inh was incubated with dermal cells in the presence of PTH, the PTH-Inh failed to block desensitization, suggesting a loss of biological effectiveness of the inhibitor. When medium containing PTH-Inh alone was removed from dermal cells and tested for inhibition of the acute PTH response in untreated cells, there was apparent loss of inhibitory efficacy (t1/2 = 20 h). In contrast, incubation of native PTH or bPTH-(1-34) with cells did not affect the biological activity of these ligands. Unlike the dermal cells, the PTH-Inh did block desensitization to PTH in GT, and there was no loss of inhibitor efficacy when medium containing PTH-Inh was incubated with GT (48 h) and then tested in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.     

Two-dimensional 1H-NMR study of the 1-34 fragment of human parathyroid hormone

The complete assignment of resonances in the proton nmr spectrum of the 1-34 amino acid fragment of human parathyroid hormone [hPTH(1-34)], determined using a combination of one- and two-dimensional nmr techniques at 500 MHz, is described. In particular, homonuclear Hartmann-Hahn experiments, recorded in H2O and D2O, are used to resolve ambiguities in the connectivities between the highly overlapped resonances in the aliphatic region of the spectrum. One-dimensional multiple quantum filtering experiments are used to identify serine and phenylalanine spin systems. Analyses of the through-bond and through-space connectivities in the alpha H-NH fingerprint regions of the correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra lead to the assignment of resonances to specific amino acid residues in the polypeptide. Examination of the observed NOE cross peaks indicates that hPTH(1-34) has no detectable secondary structural elements in aqueous solution.     

Crystal structure of human parathyroid hormone 1-34 at 0.9-A resolution

The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.     

Sulfur-free parathyroid hormone analogues containing D-amino acids: biological properties in vitro and in vivo

Three sulfur-free analogues of bovine parathyroid hormone (bPTH) containing D-amino acids were synthesized by the solid-phase method and their biological properties compared in an in vitro bioassay (rat renal adenylate cyclase assay), a receptor assay for parathyroid hormone (PTH) (canine renal membranes), and an in vivo bioassay (chick hypercalcemia assay). The analogue [Nle8,Nle18,D-Tyr34]-bPTH-(1-34)-amide, which was found to be more than 4 times as potent in vitro as unsubstituted PTH, is the most potent analogue of PTH yet synthesized. The enhanced potency was largely attributable to increased affinity for the PTH receptor. In vivo, however, this analogue was only one-third as potent as bPTH-(1-34). Cumulative evidence suggests that the nearly 15-fold decline in the relative potency when the compound was assayed in vivo is due to the substitution of norleucine for methionine. The other analogues, [D-Val2,Nle8,D-Tyr34]bPTH-(1-34)-amide and [D-Val2,Nle8,Nle18,D=Tyr34]bPTH-(2-34)-amide, were only weakly active in vitro and in vivo, indicating that substitution with D-amino acids at the NH2 terminus of PTH causes markedly diminished receptor affinity. In fact, the placement of a D-amino acid at the NH2 terminus is more deleterious to biological activity than is omission of amino acids at positions 1 and 2.     

Expression-level dependent activation of recombinant human parathyroid hormone/parathyroid hormone-related peptide receptor: effect of human parathyroid hormone (1-34), (1-31), and (28-48)

A stable recombinant chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10(5) to I.9 x 10(6) receptors per cell. The affinity of the receptors for hPTH-(1-34) was independent of the receptor number per cell (Kd approximately = 8 nmol/1). The induction of cAMP by hPTH-(1-34) is maximal in clones expressing >2x10(5) receptors per cell and Ca++ signals were maximal in cell lines expressing >1.4x10(6) receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca++ are independent and Ca++ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(1-31) showed also an increase in intracellular Ca++. Even in cell lines expressing more than 10(6) receptors per cell the Ca++/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.     

Minimization of parathyroid hormone. Novel amino-terminal parathyroid hormone fragments with enhanced potency in activating the type-1 parathyroid hormone receptor

The amino-terminal and carboxyl-terminal portions of the 1-34 fragment of parathyroid hormone (PTH) contain the major determinants of receptor activation and receptor binding, respectively. We investigated how the amino-terminal signaling portion of PTH interacts with the receptor by utilizing analogs of the weakly active fragment, rat (r) PTH(1-14)NH(2), and cells transfected with the wild-type human PTH-1 receptor (hP1R-WT) or a truncated PTH-1 receptor which lacked most of the amino-terminal extracellular domain (hP1R-delNt). Of 132 mono-substituted PTH(1-14) analogs, most having substitutions in the (1-9) region were inactive in assays of cAMP formation in LLC-PK1 cells stably expressing hP1R-WT, whereas most having substitutions in the (10-14) region were active. Several substitutions (e.g. Ser(3) --> Ala, Asn(10) --> Ala or Gln, Leu(11) --> Arg, Gly(12) --> Ala, His(14) --> Trp) enhanced activity 2-10-fold. These effects were additive, as [Ala(3),(10,12),Arg(11), Trp(14)] rPTH(1-14)NH(2) was 220-fold more potent than rPTH(1-14)NH(2) (EC(50) = 0.6 +/- 0.1 and 133 +/- 16 micrometer, respectively). Native rPTH(1-11) was inactive, but [Ala(3,10), Arg(11)]rPTH(1-11)NH(2) achieved maximal cAMP stimulation (EC(50) = 17 micrometer). The modified PTH fragments induced cAMP formation with hP1R-delNt in COS-7 cells as potently as they did with hP1R-WT; PTH(1-34) was 6,000-fold weaker with hP1R-delNt than with hP1R-WT. The most potent analog, [Ala(3,10,12),Arg(11), Trp(14)]rPTH(1-14)NH(2), stimulated inositol phosphate production with hP1R-WT. The results show that short NH(2)-terminal peptides of PTH can be optimized for considerable gains in signaling potency through modification of interactions involving the regions of the receptor containing the transmembrane domains and extracellular loops.     

Action of insulin analogues on cultured human fibroblasts reflects biological potency.

The biological sensitivity of cultured human skin-derived fibroblasts was examined in terms of the ability of insulin and insulin analogues to stimulate the uptake of alpha-aminoisobutyrate in these cells. The relative biological activity measured: insulin=desalanine insulin > proinsulin > desoctapeptide insulin parallels exactly the relative activity of these compounds on fat and muscle preparations both $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$. Inactive insulin analogues do not stimulate alpha-aminoisobutyrate uptake. It is concluded that the chemical specificity of human fibroblast insulin receptors is retained in culture and that the biological responsiveness (alpha-aminoisobutyrate uptake) of such cells to insulin should prove useful for comparative studies of receptors obtained from different individuals.     

Anabolic effects of recombinant human parathyroid hormone (1 - 84) and synthetic human parathyroid hormone (1 - 34) on the mandibles of osteopenic ovariectomized rats with maxillary molar extraction.

In rodent osteoporosis models such as ovariectomized (OVX) rats, intermittently administered human parathyroid hormone (hPTH) has an anabolic effect in vertebrae and long bones. In the present experiments, subcutaneously injected hPTH(1 - 34) or hPTH(1 - 84) dose- and time-dependently increased bone mineral density (BMD) as measured by dual energy X-ray absorptiometry in mandibles, L2 to L4 vertebrae and femurs of such rats. The highest dose (15.9 nmol/kg, s. c.) of either peptide given four times weekly for 10 weeks completely reversed the effects of overiectomy on BMD. Significant elevation in lumbar BMD after 10 weeks was observed with hPTH(1 - 34) or hPTH(1 - 84) at 1.1 nmol/kg, whereas hPTH(1 - 34) at 1.1 and 4.2 nmol/kg significantly increased BMD of the whole bone and the metaphysis of the femur and the diaphysis of the bone, respectively. In contrast, significant effects of hPTH(1 - 84) administration on BMD increase in the femur were observed at 4.2 and 15.9 nmol/kg in the whole bone and the metaphysis, and in the diaphysis, respectively. Maxillary molar extraction left mandibular BMD in rats with intact ovaries unchanged, but significantly decreased mandibular BMD in OVX rats. Administration of hPTH(1 - 84) for 10 weeks in OVX rats without or with extraction significantly increased BMD in the mandibular molar region at doses of 15.9 and 4.2 nmol/kg, respectively, indicating that efficacy was increased by extraction. A significant BMD increase in the molar region in OVX rats with extraction occurred at only 1.1 nmol/kg of hPTH(1 - 34) and 4.2 nmol/kg of hPTH(1 - 84). Also, BMD of the ramus region was increased by administration of both peptides to a lesser extent than that of the molar region in these rats. Thus, intermittent administration of hPTH, especially hPTH(1 - 34), has an anabolic effect on bone, particularly alveolar bone. Such treatment may increase alveolar bone mass in postmenopausal women with osteoporosis.     

Preparation and characterization of a novel recombinant human parathyroid hormone (1-34) analog (Gly1-Gln26-rhPTH(1-34)) with enhanced biological activity

A recombinant human parathyroid hormone (rhPTH) fragment (Gly1-Gln26-rhPTH(1-34)) which contains two amino acids substitutions (Gly1 and Gln26) was acquired through Escherichia coli expression system using a soluble fusion protein strategy. The soluble fusion protein MBP-Gly1-Gln26-rhPTH(1-34) was harvested after purification by Phenyl-Sepharose F.F and Q-Sepharose F.F chromatographies. Following tobacco etch virus (TEV) protease cleavage and further purification by SP-Sepharose F.F chromatography, 30.8 mg/L Gly1-Gln26-rhPTH(1-34) without tag was obtained with high purity up to 99%. Cyclic AMP (cAMP) stimulation assay suggested that Gly1-Gln26-rhPTH(1-34) could increase the biological activity by up to 13.89% and 6.34%. After daily subcutaneous injection (for 13 weeks) of 5, 10 and 20 microg of Gly1-Gln26-rhPTH(1-34)/1000g body weight, the mean Bone Material Density (BMD) of ovariectomized (OVXed) rats increased to 7.95-30.54% and 1.98-23.32%, compared to control-vehicle group (OVX, P<0.001) and sham- operated group (SHAM, P<0.01), respectively.     

Solution structures of human parathyroid hormone fragments hPTH(1-34) and hPTH(1-39) and bovine parathyroid hormone fragment bPTH(1-37)

Parathyroid hormone (PTH) is involved in regulation of the calcium level in blood and has an influence on bone metabolism, thus playing a role in osteoporosis therapy. In this study, the structures of the human PTH fragments (1-34) and (1-39) as well as bovine PTH(1-37) in aqueous buffer solution under near physiological conditions were determined using two-dimensional nuclear magnetic resonance spectroscopy. The overall structure of the first 34 amino acids of these three peptides is virtually identical, exhibiting a short NH(2)-terminal and a longer COOH-terminal helix as well as a defined loop region from His14 to Ser17, stabilized by hydrophobic interactions. bPTH(1-37), which has a higher biological activity, shows a better-defined NH(2)-terminal part. In contrast to NH(2)-terminal truncations, which cause destabilization of helical structure, neither COOH-terminal truncation nor elongation significantly influences the secondary structure. Furthermore, we investigated the structure of hPTH(1-34) in 20% trifluoroethanol solution. In addition to its helix-stabilizing effect, trifluorethanol causes the loss of tertiary hydrophobic interactions.     

Chemical and biological properties of synthetic, sulfur-free analogues of parathyroid hormone.

Several analogues of the biologically active fragment of bovine parathyroid hormone (bPTH), based on the sequence of the NH2-terminal 34 amino acids, were prepared by solid phase synthesis and bioassayed in the in vitro adenylyl cyclase assay to provide further information concerning structure-activity relations in parathyroid hormone. In two analogues both methionines of the natural hormone were replaced with the sulfur-free and closely isosteric amino acid norleucine (Nle). The synthetic analogue [Nle-8, Nle-18]bPTH-(1-34) was highly active in the in vitro rat adenylyl cyclase bioassay, thus demonstrating that neither of the methionines, found in the native sequence, is indispensable for biological activity. Tyrosine was substituted for phenylalanine at position 34 in the synthesis of two other hormone analogues, [Try-34]bPTH-(1-34) and [Nle-8,Nle-18,Tyr-34]bPTH-(1-34). Both derivatives were exposed to conventional iodination procedures involving use of the oxidant chloramine T. Although iodination of [Try-34]bPTH-(1-34) resulted in virtually complete loss of biological activity, [Nle-8,Nle-18,Tyr-34]-bPTH-(1-34), which lacks methionine, could be exposed to oxidants and labeled efficiently with iodine with retention of nearly complete biological activity. These findings confirm that the loss of biological activity after oxidation of bPTH, as previously observed with the native hormone, is indeed attributable to the oxidation lability of methionine rather than to any other modifications. This sulfur-free, radioiodinated, biologically active analogue of parathyroid hormone may prove useful in studies of interaction of the hormone with the membrane receptors of target tissues and in studies of the metabolism of parathyroid hormone.     

Potentiation of antigen-induced mast cell activation by 1-34 bovine parathyroid hormone

Peptides such as parathyroid hormone (PTH), somatostatin, and gastrin have been reported to stimulate mast cell mediator release. Preincubation of rat serosal mast cells with synthetic 1-34 bovine parathyroid hormone (1-34bPTH) significantly enhanced antigen-induced 5-hydroxytryptamine (5-HT) release. Enhancement of 5-HT release by 1-34bPTH was dose dependent between 5 and 2000 nM. In the absence of antigen, mean net 5-HT release was less than 1% when naive or passively sensitized mast cells were incubated with 1000 nM 1-34bPTH for time intervals up to 90 min. These findings indicate that 1-34bPTH, at relatively low concentration, potentiates antigen-induced 5-HT release from mast cells.