Biomedical subjects

J R Barbier

Publications and source records attributed to J R Barbier.

At least 19 recordsLinked to original sources

PubMed2001

Structural requirements for conserved arginine of parathyroid hormone.

Arg-20 is one of two residues conserved in all peptides known to activate the parathyroid hormone (PTH) receptor. Previous studies have failed to find any naturally encoded analogues of residue 20 that had any adenylyl cyclase (AC) stimulating activity. In this work we have studied substitutions of Arg-20 with nonencoded amino acids and conformationally constrained analogues with side chains mimicking that of Arg. No analogue had more than 20% of the AC-stimulating ability of the natural Arg-20-bearing peptide. In descending order of activity, the most active analogues had (S)-4-piperidyl-(N-amidino)glycine (PipGly), norleucine (Nle), citrulline (Cit), or ornithine (Orn) at residue 20. Analogues with Arg-20 substituted with L-4-piperidyl-(N-amidino)alanine, Lys, Glu, Ala, Gln, (S)-2-amino-4-[(2-amino)pyrimidinyl]butanoic acid, or L-(4-guanidino)phenylalanine had very low or negligible activity. Low or negligible activities of Lys or Orn analogues suggested ionic interactions play a minor role in the Arg interaction with the receptor. The conformational constraints imposed by the PipGly ring had a negative effect on its ability to substitute for Arg. The side-chain H-bonding potential of the Cit ureimido group was likely an important factor in its mimicry of Arg. The increase in amphiphilicity, as demonstrated by its greater high-performance liquid chromatographic retention, and increased alpha-helix, as shown by circular dichroic spectroscopy, likely contributed to the activity of the Nle-20 analogue. The data demonstrated that specific H-bonding, hydrophobicity of the side chain, stabilization of alpha-helix, and possibly specific cation positioning were all important in the interaction of Arg-20 with receptor groups.

Adenylyl Cyclases↗

PubMed2001

The effect of monocyclic and bicyclic analogs of human parathyroid hormone (hPTH)-(1-31)NH2 on bone formation and mechanical strength in ovariectomized rats.

The [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 lactam is a stronger stimulator of adenylyl cyclase activity and a better stimulator of trabecular bone in the ovariectomized (OVX) rat model of osteopenia than hPTH-(1-31)NH2. This enhanced activity is due in large part to the stabilization of the amphiphilic receptor-binding alpha-helix in the Ser17-Gln29 region. The goal of the present study was to determine whether further cyclization could produce a more active hPTH analog. To this end, we compared the relative bioactivities of the bicyclic hPTH analog [Glu17,Leu27]cyclo(Lys13-Glu17,Glu22-Lys26)-hPTH-(1-31)NH2, made by replacing Ser17 with Glu17 and introducing a second lactam linkage between Lys13 and Glu17. The relative EC50 for adenylyl cyclase stimulation by the bicyclic hPTH analog was similar to the EC50 of the monocyclic [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, but the bicyclic analog was still more active than hPTH-(1-31)NH2. As expected from adenylyl cyclase stimulation being responsible for PTH's anabolic action, the bicyclic hPTH analog [Glu17, Leu27]cyclo(Lys13-Glu17, Glu22-Lys26)-hPTH-(1-31)NH2 was able to increase femoral trabecular volume and thickness and mechanical strength in OVX rats, but it was no more effective than [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 when injected once daily in a dose of 0.8 nmol/100 g body weight. Thus, further constraint of the conformation of hPTH-(1-31)NH2 by introducing two lactam link-ages between Lys13-Glu17 and Glu22-Lys26 did not raise the osteogenicity above that of the monocyclic analog.

Adenylyl Cyclases↗

PubMed2000

Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones.

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.

Adenylyl Cyclases↗

PubMed2000

Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone.

The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.

Amino Acid Sequence↗

PubMed2000

The stimulation of vertebral and tibial bone growth by the parathyroid hormone fragments, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2.

The native human parathyroid hormone, hPTH-(1-84), and certain carboxyl truncated analogs such as hPTH-(1-34) and even smaller fragments such as hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2 stimulate femoral trabecular and cortical bone growth in ovariectomized (OVX) rats. Here we show that when injected once daily for 6 weeks starting 2 weeks after OVX in doses of 1 or 2 nmol/100 g of body weight, hPTH-(1-31)NH2, [Leu27] cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 prevented the loss of trabecular volume in the L5 vertebrae induced by OVX. In fact, by the end of the sixth week of injections (i.e., the eighth week after OVX) the fragments had increased the volume and trabecular thickness significantly above the values in vehicle-injected sham-operated rats. hPTH-(1-30)NH2 can stimulate vertebral bone growth as much as the larger fragments, but 10-25 times more of it was needed to do so. The same daily doses of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 also raised the trabecular volume and thickness in the L5 vertebrae of rats well above the values in vehicle-treated animals when the injections were started 9 weeks after OVX. This restoration of trabecular bone in the L5 vertebrae in estrogen-deprived animals was accompanied by a significant increase in the bone mineral density (BMD) of the L1-L4 vertebrae and tibias. However, there was no significant drop in the pelvic BMD in the estrogen-deprived animals and the effects of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-(Lys) hPTH-(1-31)NH2, and hPTH-(1-34)NH2 on the pelvic BMD were equivocal.

Animals↗

PubMed2000

High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli.

A fully active analog of human parathyroid hormone (hPTH) has been produced by recombinant expression in Escherichia coli. Initially, a nucleotide sequence encoding hPTH(1-34)-Asp-Pro was ligated to a proinsulin gene in the plasmid pUC8, for the eventual expression of a fusion protein of 137 amino acids. Unexpectedly, the proinsulin gene and 340 bp downstream were deleted by an unknown mechanism during transformation of the E. coli. This resulted in a new plasmid encoding a small (72-amino acid) fusion product of hPTH(1-34)-Asp35-Pro36-X, where X is a 36-residue "arbitrary" downstream sequence of pUC8. The fusion product was efficiently expressed and the hPTH analog, [Asp35]hPTH-(1-35), was readily released by acid cleavage, with a yield of 100 mg/L. This analog had an effective concentration for half-maximal adenylyl cyclase stimulation (EC50) in rat osteosarcoma cells of 14 nM, which was identical to that for hPTH-(1-34). In the ovariectomized rat model of osteoporosis, [Asp35]hPTH-(1-35) was fully active as a bone anabolic agent.

Adenylyl Cyclases↗

PubMed2000

Lactam formation increases receptor binding, adenylyl cyclase stimulation and bone growth stimulation by human parathyroid hormone (hPTH)(1-28)NH2.

Human parathyroid hormone (1-28)NH2 [hPTH(1-28)NH2] is the smallest of the PTH fragments that can fully stimulate adenylyl cyclase in ROS 17/2 rat osteoblast-like osteosarcoma cells. This fragment has an IC50 of 110 nM for displacing 125I-[Nle8,18,Tyr34]bovine PTH(1-34)NH2 from HKRK B7 porcine kidney cells, which stably express 950,000 human type 1 PTH/PTH-related protein (PTHrP) receptors (PTH1Rs) per cell. It also has an EC50 of 23.9 nM for stimulating adenylyl cyclase in ROS 17/2 cells. Increasing the amphiphilicity of the alpha-helix in the residue 17-28 region by replacing Lys27 with Leu and stabilizing the helix by forming a lactam between Glu22 and Lys26 to produce the [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 analog dramatically reduced the IC50 for displacing 125I-[Nle8,18,Tyr34]bPTH(1-34)NH2 from hPTH1Rs from 110 to 6 nM and dropped the EC50 for adenylyl cyclase stimulation in ROS 17/2 cells from 23.9 to 9.6 nM. These modifications also increased the osteogenic potency of hPTH(1-28)NH2. Thus, hPTH(1-28)NH2 did not significantly stimulate either femoral or vertebral trabecular bone growth in rats when injected daily at a dose of 5 nmol/100 g body weight for 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stimulated the growth of trabeculae in the cancellous bone of the distal femurs and L5 vertebrae when injected at 25 nmol/100 g body weight. By contrast [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 significantly stimulated trabecular bone growth when injected at 5 nmol/100 g of body weight. Thus, these modifications have brought the bone anabolic potency of hPTH(1-28)NH2 considerably closer to the potencies of the larger PTH peptides and analogs.

Adenylyl Cyclases↗

PubMed1999

Stimulation of membrane-associated protein kinase-C activity in spleen lymphocytes by hPTH-(1-31)NH2, its lactam derivative, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, and hPTH-(1-30)NH2.

Human parathyroid hormone, hPTH-(1-34), stimulates adenylyl cyclase and phosphatidylinositol-bisphosphate-specific phospholipase-C (PIP2-PLC), as indicated by increased membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. The C-terminally truncated hPTH-(1-31)NH2 stimulates adenylyl cyclase as strongly as hPTH-(1-34) in these cells, but it does not stimulate PKC activity. Even [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, a 6-fold stronger adenylyl cyclase stimulator than hPTH-(1-34), cannot stimulate PKC activity in ROS cells. Therefore PTH required its 32-34 region to stimulate PIP2-PLC/PKCs in this osteosarcoma line. In contrast, hPTH-(1-31)NH2 [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 and even hPTH-(1-30)NH2 can stimulate PKC activity in freshly isolated rat spleen lymphocytes as strongly as hPTH-(1-34)NH2. The difference in the ability of membrane-associated PKC activity in spleen lymphocytes, but not in ROS cells, to be stimulated by C-terminally truncated PTH fragments might be due to different receptor densities or to the lymphocyte's atypical PTH/PTHrP receptor.

Animals↗

PubMed1999

Prolonged low-dose infusion of human parathyroid hormone does not increase femoral cancellous bone volume in ovariectomized rats.

OBJECTIVE: Daily injections of human parathyroid hormone (hPTH) increase bone volume in various animal species and in osteoporotic women. For hPTH to be widely accepted as an anabolic therapy for treating postmenopausal osteoporosis alternative delivery options need to be explored to replace the need for daily patient subcutaneous self-injection. Among these are inhalation, oral delivery and the use of programmable implanted minipumps to deliver the peptide. While infusion of high doses of PTH causes bone loss and hypercalcemia, no studies have assessed the effects of prolonged infusion of low doses of PTH on bone growth. DESIGN AND METHODS: [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2) was delivered by Alzet minipumps to ovariectomized rats for 6 weeks after which histomorphometric indices (cancellous bone volume, trabecular thickness, mean trabecular number) of bone formation were measured in distal femurs. RESULTS: Infusing low doses (0.05 and 0.1 nmole/100g body weight/day) of the hPTH analog, [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2), for 6 weeks does not prevent the ovariectomy-induced loss of rat femoral cancellous bone volume, trabecular thickness or trabecular number. CONCLUSION: These results support the absolute requirement of daily injections for the osteogenic action of hPTH on bone.

Animals↗

PubMed1998

Comparison of the abilities of human parathyroid hormone (hPTH)-(1-34) and [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 to stimulate femoral trabecular bone growth in ovariectomized rats.

hPTH-(1-31)NH2, so far the smallest of the potently anabolic N-terminal fragments of the human parathyroid hormone, stimulates trabecular growth in the distal femurs of ovariectomized (OVX) rats as strongly as hPTH-(1-34) when injected at a high daily dose such as 1 nmol/100 g of body weight, but it is only about 70% as effective as hPTH-(1-34) when injected at the suboptimal 0.6 nmol/100 g of body weight. A lactam derivative of hPTH-(1-31)-NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, is a much more effective stimulator of adenylyl cyclase in ROS 17/2 rat osteoblast-like cells and a significantly more effective stimulator of femoral trabecular growth in OVX rats than hPTH-(1-31)NH2. We have now shown that [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 prevents the OVX-induced loss of femoral trabeculae significantly more effectively than hPTH-(1-34) and stimulates the thickening of the trabeculae remaining in severely depleted femoral trabecular bone of OVX rats as effectively as hPTH-(1-34) when injected at 0.6 nmol/100 g of body weight.

Adenylyl Cyclases↗

PubMed1997

Bioactivities and secondary structures of constrained analogues of human parathyroid hormone: cyclic lactams of the receptor binding region.

In a search for analogues of human parathyroid hormone (hPTH) with improved activities and bioavailabilities, we have prepared the following three lactam analogues of hPTH-(1-31)-NH2 (1) or [Leu27]hPTH-(1-31)-NH2 (2): [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 (3), [Leu27]cyclo(Lys26-Asp30)-hPTH-(1-31)-NH2 (4), and cyclo(Lys27-Asp30)-hPTH-(1-31)-NH2 (5). Analogues 1, 2, and 5 had seven or eight residues of alpha-helix, as estimated from their circular dichroism (CD) spectra, in contrast to 12 residues in cyclic analogues 3 and 4. Thus, lactams 3 and 4 stabilized a helix previously shown to exist within residues 17-29. The adenylyl cyclase activity (EC50), measured in rat osteosarcoma 17/2 cells, of 5 (40.3 +/- 2.3 nM) was half that of its linear form 1 (19.9 +/- 3.9 nM). The linear Leu27 mutant 2 was twice as active (11.5 +/- 5.2) as analogue 1, and lactam analogue 3 was 6-fold more active (3.3 +/- 0.3 nM). Lactam analogue 4 had less activity (16.9 +/- 3.3 nM) than 2, its linear form. Peptides hPTH-(1-30)-NH2 (6), [Leu27]hPTH-(1-30)-NH2 (7), and [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-30)-NH2 (8) all had AC-stimulating activities similar to that of 1. When injected intravenously, with a dose of 0.8 nmol/100 g of analogue in acid saline, hypotensive effects paralleled their adenylyl cyclase activities. They behaved quite differently when applied subcutaneously. Analogues 1, 5, and 6, the weakest, showed about half the drop in blood pressure observed with 3 and 4, the most active. In contrast, the time required to reach a maximum drop in blood pressure of 4-8, after subcutaneous administration, was 2-4 times that of the other analogues. Thus, the bioavailabilities of the lactam analogues, unlike their adenylyl cyclase-stimulating activities, were highly dependent on the presence or conformation of Val31.

Adenylyl Cyclases↗

PubMed1997

Comparison of the ability of recombinant human parathyroid hormone, rhPTH-(1-84), and hPTH-(1-31)NH2 to stimulate femoral trabecular bone growth in ovariectomized rats.

A recombinant human parathyroid hormone, rhPTH-(1-84), which is currently in Phase II clinical trial, and hPTH-(1-31)NH2 (Ostabolin) are promising anabolic agents for treating osteoporosis because they can stimulate cortical and trabecular bone growth in osteopenic, ovariectomized (OVX) rats and in osteoporotic, postmenopausal women when injected subcutaneously and intermittently at low doses. We have now found that, despite their different sizes and signaling properties (rhPTH-(1-84) stimulates adenylyl cyclase and phospholipase C; hPTH-(1-31)NH2 only stimulates adenylyl cyclase), they are equally osteogenic in OVX rats. Thus daily subcutaneous injections of 0.6 nmol/100 g of body weight of rhPTH-(1-84) or hPTH-(1-31)NH2 into 3-month-old OVX rats for 6 weeks starting 2 weeks after OVX equally reduced the otherwise large OVX-triggered loss of femoral trabecular bone. Daily subcutaneous injections of 0. 4 or 0.8 nmol/100 g of body weight of the two agents for 6 weeks also equally increased the mean thickness of the remaining femoral trabeculae in 3-month-old and 1-year-old OVX rats to 20 to 80% above the value in normal animals when started 9 weeks after ovariectomy.

Animals↗

PubMed1997

The hypotensive actions of osteogenic and nonosteogenic parathyroid hormone fragments.

Parathyroid hormone (PTH), hPTH-(1-84), and its hPTH-(1-34), hPTH-(1-31)NH2, and hPTH-(1-30)NH2 fragments reduced the tail artery pressure in anesthetized female Sprague-Dawley rats by 42.4-67.1% within about 1 minute after injection into a femoral vein, but reduced the pressure by only 8.5-36.2% 2-19 minutes after subcutaneous injection. hPTH-(1-84) and hPTH-(1-34) stimulate both adenylyl cyclase and phospholipase-C in their target cells, but the hypotensive action must have been stimulated specifically by adenylyl cyclase activation, because hPTH-(1-30)NH2 and hPTH-(1-31)NH2, which can only stimulate adenylyl cyclase, were potently hypotensive when injected intravenously whereas hPTH-(7-84), which can only stimulate phospholipase-C, was not significantly hypotensive when injected intravenously. Since PTH's osteogenic action is also mediated by adenylyl cyclase stimulation, it was expected that the hypotensive response might be used to screen new PTH constructs for possible osteogenicity. Indeed, the osteogenic activities of subcutaneously injected hPTH-(1-31)NH2, hPTH-(1-34), and hPTH-(1-84) correlated closely to their hypotensive activities, with hPTH-(1-34) being much more hypotensive and significantly more osteogenic than the other two molecules. hPTH-(1-31)NH2 and hPTH-(1-84) were equally osteogenic and hypotensive. However, this correlation broke down with hPTH-(1-30)NH2 which does not stimulate bone formation, but in the present study it stimulated adenylyl cyclase and reduced tail artery pressure almost as much as hPTH-(1-31)NH2 and hPTH-(1-34). Nevertheless, the ability to significantly reduce arterial pressure is a common property of osteogenic PTH and PTH fragments and is thus a rapidly determinable preliminary indicator of in vivo bioactivity of PTH fragments.

Adenylyl Cyclases↗

PubMed1997

Cyclization by a specific lactam increases the ability of human parathyroid hormone (hPTH)-(1-31)NH2 to stimulate bone growth in ovariectomized rats.

Human parathyroid hormone (hPTH)-(1-31)NH2 (Ostabolin), which only stimulates adenylyl cyclase (AC) instead of AC and phospholipase-C as do hPTH(1-84) and hPTH(1-34), strongly stimulates femoral cortical and trabecular bone growth in ovariectomized (OVX) rats. Two side-chain lactams have been introduced in the hydrophilic face of the receptor-binding region of the fragment's Ser17-Val31 amphiphilic alpha-helix in an attempt to develop improved analogs for the treatment of osteoporosis. Replacing the polar Lys27 with an apolar Leu on the hydrophobic face of this alpha-helix and stabilizing the helix with a lactam between Glu22 and Lys26 produced a fragment, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2, which had six times the AC-stimulating ability of hPTH(1-31)NH2 in ROS 17/2 rat osteosarcoma cells, but the other helix-stabilizing lactam derivative [Leu27]-cyclo(Lys26-Arg30)-hPTH(1-31)NH2 did not have a greater AC-stimulating ability than hPTH(1-31)NH2, to stimulate AC in ROS 17/2 rat osteosarcoma cells. As expected from AC stimulation being responsible for PTH's anabolic action, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2 was, depending on the experimental design, a 1.4 to 2 times better stimulator of trabecular bone growth in the OVX rat model than either hPTH(1-31)NH2 or [Leu27]-cyclo(Lys26-Arg30)-hPTH(1-31)NH2. Thus, there is now a more potently anabolic derivative of hPTH(1-31)NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH(1-31)NH2, which might ultimately prove to be one of the more effective therapeutics for osteoporosis.

Adenylyl Cyclases↗

PubMed1996

Stimulation of the growth of femoral trabecular bone in ovariectomized rats by the novel parathyroid hormone fragment, hPTH-(1-31)NH2 (Ostabolin).

The human parathyroid hormone, hPTH-(1-84), and its hPTH-(1-34) fragment are promising anabolic agents for treating osteoporosis because they can strongly stimulate the production of biomechanically effective cortical and trabecular bone in osteopenic ovariectomized (OVX) rats and trabecular bone in osteoporotic postmenopausal humans. The ideal PTH fragment for treating osteoporosis would be the smallest and functionally simplest fragment that activates only one signal mechanism and still strongly stimulates trabecular bone growth. A new PTH fragment, hPTH-(1-31)NH2, which only stimulates adenylyl cyclase instead of stimulating both adenylyl cyclase and phospholipase-C as do hPTH-(1-84) and hPTH-(1-34), is this minimum, high-potency anabolic fragment. hPTH-(1-31)NH2 (which we have named Ostabolin) can greatly thicken trabeculae and increase the dry weight and calcium content of trabecular bone in the distal femurs of osteopenic, young, sexually mature OVX Sprague-Dawley rats when injected subcutaneously each day for 6 weeks at doses between 0.4 and 1.6 nmole/100 g of body weight.

Animals↗

PubMed1996

Peptide synthesis on chitin.

The use of chitin as a support for solid-phase peptide synthesis is described and illustrated by synthesis of four peptides, varying in length from 10 to 29 residues. Syntheses were performed in a continuous-flow peptide synthesizer, using Fmoc chemistry. A cleavable linker, p-[(R,S)-alpha-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-di methoxybenzyl]- phenoxyacetic acid, was attached to chitosan at the desired substitution level, and the complex acetylated to yield a linker substituted chitin. The effects of temperature, solvents and degree of linker substitution on the syntheses were studied. Acyl carrier peptide (ACP) synthesis studies indicated that temperature was the single most important parameter. Increasing the temperature of the synthesis from 20 to 55 degrees C resulted in an enormous improvement of this synthesis, with about 90% of the crude product being the correct peptide. Denaturing solvents, such as DMSO, could be used without significant effect on the flow properties of the support. The synthesis of one peptide was mainly improved by lowering the degree of substitution from 0.3 to 0.1 mmol/g, suggesting peptide aggregation was a problem in this case. The results of three syntheses on chitin were comparable with those obtained with a commonly used commercial support. This work shows that, under appropriate conditions, chitin can be utilized directly as a support for peptide synthesis.

Amino Acid Sequence↗

PubMed1995

Solution structure and adenylyl cyclase stimulating activities of C-terminal truncated human parathyroid hormone analogues.

Analogues of human parathyroid hormone (hPTH) truncated at the C-terminal end have been studied for adenylyl cyclase (AC) activity and for solution conformation by circular dichroism (CD) spectroscopy. Analogues of hPTH-(1-34)-NH2, containing the first 28-31 residues, had only a slightly diminished ability to stimulate AC in rat osteosarcoma (ROS) cells as compared to that of the parent analogue. CD data on hPTH-(16-34)-NH2 and C-terminal deletion mutants of hPTH-(1-34)-NH2 supported the presence of a partially stable alpha-helix over residues 17-28. A carboxyl-terminal mutant, hPTH-(1-30)-OH, showed both reduced helix and greatly reduced AC-stimulating activity as compared to the corresponding amide analogue. In contrast, both of these analogues, in the presence of palmitoyloleoylphosphatidylserine (POPS) vesicles, showed an equal stabilization of alpha-helix. All other analogues showed at least some enhancement of alpha-helix in the presence of POPS. However, both in neutral, aqueous buffer and in POPS, the relative amount of alpha-helix decreased greatly as the peptide was shortened below the 1-28 sequence. These data provide additional support for an amphiphilic alpha-helix over residues 21-28 being the conformation for receptor binding of hPTH for stimulation of AC activity. Modeling human parathyroid hormone-related peptide as an alpha-helix over this same region, and comparison to hPTH, suggests that both may bind via the hydrophobic face to the receptor.

Adenylyl Cyclases↗

PubMed1991

Internal ribosome-binding site directs expression of parathyroid hormone analogue (8-84) in Escherichia coli.

Expression of the human parathyroid hormone (PTH) gene in E. coli yielded intact PTH and PTH-(8-84). To determine if PTH-(8-84) is the result of a competing translation initiated from methionine codon-8 or degradation of the intact PTH, twelve new gene constructs with or without an internal ribosome-binding site (iRBS) in the PTH-(1-5) region were prepared via substitution with degenerate codons. Expression of constructs without iRBS produced only intact PTH. Constructs with weak iRBS, including one that resembles the cDNA sequence, yielded PTH-(8-84) as a minor product. In contrast, constructs with strong iRBS produced predominantly or exclusively this shorter analogue.

Binding Sites↗