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In an effort to improve the activities and bioavailabilities of stromal cell-derived factor-1 (SDF-1, CXCL12) sdf-(1−67)-OH (1), we have prepared a linear peptide analogue [sdf-(1−31)-NH2 (2)] and two lactam analogues [cyclo(Lys²⁰-Glu²⁴)-sdf-(1−31)-NH2 (3) and cyclo(Glu²⁴-Lys²⁸)-sdf-(1−31)-NH2 (4)], consisting of the N-terminal region (amino acids 1−14) joined by a four-glycine linker to the C-terminal region (amino acids 56−67) of 1. Analogues 2 and 4 had eight residues of α-helix, as estimated from its circular dichroism (CD) spectra, in contrast to 10 residues in analogue 3. Cyclization of analogue 2 at residues 20 and 24 to give analogue 3 resulted in only a slight change to the θ222/θ209 ratio (0.81 to 0.86, where 1.09 is considered a perfect α-helix), although an increase in the α-helix length of analogue 3 was observed. In contrast, cyclization between residues 24 and 28 by lactamization to give analogue 4 only slightly affected the helical content but clearly resulted in a more classical α-helical structure (θ222/θ209 = 0.98). Cyclization of the linear analogue 2 enhanced the SDF-1 receptor CXCR4 binding approximately 114-fold, where the IC50 values derived from ¹²⁵I-SDF-1 competitive binding assays with CEM cells were found to be 39.5 ± 5.9 nM, 28.9 ± 6.3 μM, 225.8 ± 11.8 nM, and 254.1 ± 5.4 nM for analogues 1−4, respectively. Intracellular calcium mobilization ([Ca²⁺]i) induced after interaction with CXCR4, as measured by EC50, was significantly reduced in analogue 4 compared to 3, and approached the EC50 of native SDF-1, indicating a correlation between the degree of α-helix and biological activity. Therefore, the biological activity of small peptide SDF-1 analogues is highly dependent on the conformation of its C-terminal region.

Abstract.: 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.

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 α-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.

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)NH2prevents 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.

N-terminal fragments of PTH and PTHrP, such as hPTH-(1-34) and hPTHrP-(1-34), are sufficiently similar with respect to amino acid sequence, location of functional domains, and higher order configuration to activate the same PTH/PTHrP receptor and the same two signal enzymes, adenylyl cyclase and phospholipase-Cβ. Therefore, it was expected that hPTHrP-(1-31)NH2would stimulate bone growth in ovariectomized rats as strongly as hPTH-(1-31)NH2. Like hPTH-(1-31)NH2, hPTHrP-(1-31)NH2stimulated adenyly cyclase in ROS 17/2 osteosarcoma cells as strongly as the standard hPTH-(1-34) and like hPTH-(1-31)NH2, triggered a large drop in mean blood pressure when injected intravenously. Unlike hPTH-(1-31)NH2, however, hPTHrP-(1-31)NH2could not stimulate trabecular growth in the distal femurs of young, sexually mature, 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‐Val31amphiphilic α‐helix in an attempt to develop improved analogs for the treatment of osteoporosis. Replacing the polar Lys27with an apolar Leu on the hydrophobic face of this α‐helix and stabilizing the helix with a lactam between Glu22and Lys26produced a fragment, [Leu27]‐cyclo(Glu22‐Lys26)‐hPTH(1–31)NH2, which had six times the AC‐stimulating ability of hPTH(1–31)NH2in ROS 17/2 rat osteosarcoma cells, but the other helix‐stabilizing lactam derivative [Leu27]‐cyclo(Lys26‐Arg30)‐hPTH(1–31)NH2did 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)NH2was, 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)NH2or [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.

The secretion of 1,4-beta-D-glucanases by the basidiomycete Schizophyllum commune in response to cellulose or cellobiose has been studied. The proteins were labeled with 35S, and the secretion of enzymes was measured by beta-glucosidase and carboxymethyl cellulase activities and by immunoprecipitation with specific antibodies. The antigen proteins used were a beta-glucosidase (Mr, 93,000), an avicelase (avicelase II; Mr, 64,000), and a carboxymethyl cellulose (carboxymethyl cellulase I; Mr 41,000). The beta-glucosidase was initially secreted as an Mr 110,000 form, which was followed later by lower-molecular-weight (88,000 to 93,000) forms. The avicelase II, which accounted for about 50% of the secreted labeled protein, had an Mr of 64,000. Secretion of the related avicelase I (Mr 61,000) followed later. The carboxymethyl cellulose I was secreted in two molecular weight forms, Mr 44,000 and 41,000. The evidence is consistent with the idea that three genes account for the secreted glucanase activities. Other species result from different glycosylation or proteolytic cleavage processing, which may occur during or after secretion. The beta-glucosidase secretion appears to be regulated differently than that of avicelase II or carboxymethyl cellulase I; the latter two were regulated coordinately under the conditions used in this work. No common immune determinants between the three antigens were observed.

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, ovariec-tomized (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)NH2only 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)NH2into 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.