A synthetic approach to 'click' neoglycoprotein analogues of EPO employing one-pot native chemical ligation and CuAAC chemistry.

The clinical significance of batch-wise variability on the pharmacokinetics and potency of commercial erythropoietin (EPO), prepared recombinantly as a heterogeneous mixture of glycoforms, necessitates the development of synthetic strategies to afford homogenous EPO formulations. Herein we present a previously unexplored and divergent route towards 'click' neoglycoprotein analogues of EPO, employing one-pot native chemical ligation (NCL) of alkynylated peptides and copper-catalysed azide-alkyne cycloaddition (CuAAC) with azido monosaccharides. By design, our synthetic platform permits glycosylation at virtually any stage, providing flexibility for the synthesis of various glycoforms for biological analysis. Insights obtained from attempted folding of our 'click' neoglycoprotein EPO analogue, bearing four different neutral sugar moieties, highlight the important role played by the charged oligosaccharides present in native EPO glycoproteins.

The role for cyclic Glycine-Proline, a biological regulator of insulin-like growth factor-1 in pregnancy-related obesity and weight changes.

Cyclic Glycine-Proline (cGP) regulates the homeostasis of insulin-like growth factor (IGF)-1 function and cGP/IGF-1 ratio determines IGF-1 bioactivity in vitro and in vivo. Plasma IGF-1 represents largely inactive IGF-1 and weakly associated with human obesity and hypertension. We evaluated the regulatory role for cGP in pregnancy-related obesity and hypertension, and in obesity status between pregnancy and postpartum. Women were recruited in their first pregnancy. A cross-sectional study compared plasma concentration of cGP, IGF-1 and IGF binding protein (IGFBP)-3 in women with obesity and/or hypertension to normal controls 6-year postpartum using UPLC-MS and ELISA. A longitudinal study compared the changes of these peptides from 15-week gestation to 6-year post-partum in the women who remained normal weight, remained obese or changed to obese or to normal respectively. Study 1 is a cross-sectional study. The obese group had lower IGF-1(p = 0.001), higher cGP/IGF-1 ratio (p = 0.0055) and the hypertensive group had lower IGFBP-3 (p = 0.046) and cGP (p = 0.043) than the controls. Study 2 is a longitudinal study. Women with weight loss had increased cGP/IGF-1 ratio (p = 0.0026) and decreased IGFBP-3 (p = 0.0001) compared with women whose weight remained normal. Women with weight gain had lower IGFBP-3 (p less than 0.0001) only. Women who remained obese had increased cGP/IGF-1 ratio (p = 0.006) only. Increase in cGP/IGF-1 ratio is associated with obesity, but not hypertension. Changes of IGFBP-3 and/or cGP/IGF-1 ratio are associated with weight changes. The data suggest the role for cGP in obesity through autocrine regulation of IGF-1.

A double-click approach to the protecting group free synthesis of glycoconjugates.

The use of a bi-functional linker, containing an alkyne and an alkene, allows the protecting group free conjugation of reducing sugars to thiols via a double click process. Firstly the linker is attached to the sugar via one-pot glycosyl azide formation and Cu-catalysed azide-alkyne cycloaddition. Photochemical thiol-ene click reaction then allows conjugation to a range of thiols, including cysteine residues of peptides.

Protecting group free synthesis of glycosyl thiols from reducing sugars in water; application to the production of N-glycan glycoconjugates.

Glycosyl thiols may be accessed from the corresponding reducing sugars in water without recourse to any sugar projecting groups by way of a DMC mediated reaction with thioacetic acid in the presence of base, and hydrolysis of the anomeric thioacetate. Glycosyl thiols produced by this method may be used to access glycoconjugates, such as glycopeptides by use of the thiol-ene click reaction.

Fluorescent IGF-II analogues for FRET-based investigations into the binding of IGF-II to the IGF-1R.

The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two non-native IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners.

Site-specific cross-linking of collagen peptides by lysyl advanced glycation endproducts.

Cross-linking of proteins by advanced glycation endproducts (AGEs) causes a host of pathological conditions but their exact roles are unknown. Cross-linking lysyl AGEs were synthesized and incorporated into two types of collagen peptides. The utility of these cross-linked peptides for biochemical investigations was demonstrated by proteolysis studies and circular dichroism.

Total Chemical Synthesis of an Orf Virus Protein, ORFV002, an Inhibitor of the Master Gene Regulator NF-κB.

ORFV002 is a novel orf viral protein (117 Aa) that inhibits nuclear events through the regulation of the transcriptional activity of NF-κB, a master regulator of human gene expression (Diel et al., J Virol 2011, 85, 264-275). It is identified as the first nuclear inhibitor of NF-κB produced by orf virus (ORFV) and no homologues in other genera of the Chordopoxvirinae subfamily have been reported to date (Diel et al., J Virol 2011, 85, 264-275). Our molecular structure predictions suggest that ORFV002 may mimic part of IκB, an inhibitor and natural human partner of NF-κB. Recent advances in total chemical synthesis of proteins have provided solutions in overcoming challenges of current recombinant methods of protein isolation for structure elucidation. Aided by Boc solid phase peptide synthesis and native chemical ligation, ORFV002 was successfully synthesized in multimilligram amounts in good yield and high purity.

Identification of key residues involved in adrenomedullin binding to the AM1 receptor.

BACKGROUND AND PURPOSE: Adrenomedullin (AM) is a peptide hormone whose receptors are members of the class B GPCR family. They comprise a heteromer between the GPCR, the calcitonin receptor-like receptor and one of the receptor activity-modifying proteins 1-3. AM plays a significant role in angiogenesis and its antagonist fragment AM22-52 can inhibit blood vessel and tumour growth. The mechanism by which AM interacts with its receptors is unknown. EXPERIMENTAL APPROACH: We determined the AM22-52 binding epitope for the AM1 receptor extracellular domain using biophysical techniques, heteronuclear magnetic resonance spectroscopy and alanine scanning. KEY RESULTS: Chemical shift perturbation experiments located the main binding epitope for AM22-52 at the AM1 receptor to the C-terminal 8 amino acids. Isothermal titration calorimetry of AM22-52 alanine-substituted peptides indicated that Y52, G51 and I47 are essential for AM1 receptor binding and that K46 and P49 and R44 have a smaller role to play. Characterization of these peptides at the full-length AM receptors was assessed in Cos7 cells by cAMP assay. This confirmed the essential role of Y52, G51 and I47 in binding to the AM1 receptor, with their substitution resulting in ≥100-fold reduction in antagonist potency compared with AM22-52 . R44A, K46A, S48A and P49A AM22-52 decreased antagonist potency by approximately 10-fold. CONCLUSIONS AND IMPLICATIONS: This study localizes the main binding epitope of AM22-52 to its C-terminal amino acids and distinguishes essential residues involved in this binding. This will inform the development of improved AM receptor antagonists.

Peripheral administration of a novel diketopiperazine, NNZ 2591, prevents brain injury and improves somatosensory-motor function following hypoxia-ischemia in adult rats.

The current study describes the neuroprotective effects of an endogenous diketopiperazine, cyclo-glycyl-proline (cyclic GP), in rats with hypoxic-ischemic brain injury and the pre-clinical development of an analogue, cyclo-L-glycyl-L-2-allylproline (NNZ 2591), modified for improved bioavailability. The compounds were given either intracerebroventricularly or subcutaneously 2h after hypoxia-ischemia. Histology, immunohistochemistry and behavior were used to evaluate treatment effects. The central uptake of NNZ 2591 was also examined in normal and hypoxic-ischemic injured rats by HPLC-mass spectrometry. Central administration of cyclic GP or NNZ 2591 reduced the extent of brain damage in the lateral cortex, the hippocampus and the striatum (p<0.001), with NNZ 2591 being more potent. NNZ 2591 was stable in the plasma and crossed the blood-brain barrier independent of hypoxic-ischemic injury. The level of NNZ 2591 in the CSF was maintained for 2 h after a single subcutaneous dose, and modest neuroprotection was seen after a bolus subcutaneous administration (overall p<0.001). Treatment with NNZ 2591 for 5 d subcutaneously improved somatosensory-motor function (p<0.05) and long-term histological outcome (overall p<0.0001). NNZ 2591 treatment not only reduced both caspase-3 mediated apoptosis and microglial activation but also enhanced astrocytic reactivity, which may mediate its protective effect. The pharmacokinetic profile and potent long-term protective effects of NNZ 2591 suggests its utility for the treatment of ischemic brain injury and other neurological conditions requiring chronic intervention.

A review of neuroprotective agents.

The brain remains an area where little corrective surgery can be performed and the reversal of damage is almost impossible. Recently, reports of agents offering neuroprotection have begun to appear in the literature. The concept of neuroprotection is the administration of some agent, which should reverse some of the damage or prevent further damage. Some agents offer protection against cell degeneration to the neuronal cells. Still other agents specifically protect the dopamine neurons and the retina. The majority of neuroprotective agents are antioxidants. An immunosuppressive calcineurin inhibitor, NOS inhibitor, sigma-1 modulator, AMPA antagonist and Ca2+ channel blocker have all shown neuroprotective activity. An estrogen agonist and two glycoprotein IIb/IIIa antagonists also exhibit neuroprotective activity. Most of the synthetic compounds presented were not originally designed as neuroprotective agents but were found to possess neuroprotective activity in later studies. Many of these compounds are biologically active natural products, either plant extracts or endogenous peptides/proteins. This review will present the most recent reports on these agents.

Influence of oxytocin on sodium excretion in the anaesthetized Brattleboro rat.

The contribution of oxytocin to the maintenance of renal Na+ excretion in the Brattleboro rat has been examined in animals infused with hypotonic saline. Brattleboro rats exhibited hypernatraemia and hyperosmolality associated with greatly increased plasma concentrations of oxytocin by comparison with Long-Evans control rats. Neurohypophysectomy to remove the secretion of the remaining posterior pituitary peptide, oxytocin, led to greatly diminished rates of Na+ excretion in the Brattleboro rat. Oxytocin replacement to achieve plasma levels equivalent to those in intact Brattleboro rats produced a substantial and sustained natriuresis in the neurohypophysectomized animal. Oxytocin secretion evoked in response to saline infusion would thus appear to be effective in promoting renal Na+ excretion in the absence of vasopressin in the Brattleboro rat.

Influence of neurohypophysectomy on the renal actions of aldosterone in the adrenalectomized rat.

The influence of aldosterone administration on urine flow, Na+ and K+ excretion was examined in hypotonic saline infused, Inactin anesthetised rats following removal of the adrenals or adrenals and posterior pituitary. Plasma adrenal steroid levels were considerably depressed but still detectable 10-14 days after adrenalectomy. Removal of the posterior pituitary markedly reduced Na+ excretion in adrenalectomized animals implying that Na+ retention following neurohypophysectomy is not dependent on adrenal gland function. In adrenalectomized rats aldosterone administration at 42 pmol/min reduced Na+ excretion and urine flow without significantly changing K+ excretion, though plasma K+ was reduced. In adrenalectomized/neurohypophysectomized rats aldosterone further reduced the already low rate of Na+ excretion and increased K+ excretion, though there was no observable effect on urine flow. The results obtained indicate that the Na(+)-retaining actions of aldosterone are largely independent of posterior pituitary influence. The K(+)-losing action of aldosterone was, however, only observed in animals in which the posterior pituitary was absent.

Effect of fungal metabolite peramine and analogs on feeding and development of argentine stem weevil (Listronotus bonariensis).

Peramine, a pyrrolopyrazine alkaloid produced by the fungal endophyte of perennial ryegrassAcremonium lolii, deterred the feeding of both adults and larvae of the graminacious herbivore, the Argentine stem weevil (Listronotus bonariensis), at 0.1 µg/g and 10 µg/g, respectively. In a no-choice test fewer stem weevil larvae fed and developed on diet containing as little as 2 µg/g peramine. The proportion of larvae which did not develop beyond the first instar was higher on diet containing peramine and appeared to be due to a higher proportion of larvae which did not feed. For larvae which fed on the peramine-containing diet, feeding scores and times to pupation were not significantly different from those of controls. A number of simple peramine analogues showed feeding-deterrent activity against adult weevils, indicating the importance of the pyrrolopyrazine ring system of peramine in determining feeding-deterrent activity.

Natriuretic action of arginine vasopressin in the conscious unrestrained rat.

A method for studying the renal action of AVP in the saline-infused conscious unrestrained rat is described. Conscious rats infused iv with 0.077 mol/l NaCl at 150 microliter/min had a plasma AVP concentration of 0.70 +/- 0.15 mol/l. AVP administration at 6 and 24 microU/min resulted in plasma AVP levels of 0.97 +/- 0.14 and 2.27 +/- 0.30 mU/l, respectively. AVP administration at 6 microU/min increased Na+ excretion from 11.9 +/- 0.80 to 14.0 +/- 0.9 mumol/min and at 24 microU/min AVP raised Na+ excretion from 10.3 +/- 0.7 to 14.0 +/- 1.1 mumol/min. The increases were accompanied by the expected reductions in urine flow. As the changes in plasma AVP are within the range occurring in response to moderate dehydration, the observed increases in Na+ excretion provide support for the view that AVP is involved in the physiological regulation of Na+ excretion.

The influence of neurohypophysial hormones on renal function in the acutely hypophysectomized rat.

1. Renal function and the effect of neurohypophysial hormone replacement was investigated in anaesthetized, acutely hypophysectomized, male rats. 2. Although urine production was only slightly lower over the 8 h post-operative study period in hypophysectomized rats, sodium excretion was greatly depressed reaching only 3.5 +/- 1.4 mumol/min compared with a peak of 13.2 +/- 1.0 mumol/min in intact animals. 3. In association with a decline in mean arterial blood pressure, glomerular filtration rate in hypophysectomized rats fell to 2.1 +/- 0.2 ml/min 8 h after operation by comparison with a mean rate in intact rats of 3.2 +/- 0.2 ml/min. 4. Plasma corticosterone levels were much lower in hypophysectomized (4 +/- 2 ng/ml) than in intact (36 +/- 4 ng/ml) rats, plasma aldosterone was reduced to a lesser extent (0.41 +/- 0.08 compared with 0.76 +/- 0.04 ng/ml). While oxytocin was not detectable in hypophysectomized rat plasma, trace levels of vasopressin (0.16 +/- 0.04 mu u./ml) were found. In intact unanaesthetized rats basal plasma levels of oxytocin were 0.32 +/- 0.13 mu u./ml and vasopressin were 0.85 +/- 0.19 mu u./ml. 5. Administration of oxytocin at 150 mu u./min, which produced plasma hormone levels (24.0 +/- 2.5 mu u./ml) greatly in excess of basal concentrations, increased renal sodium excretion but did not alter urine flow. Oxytocin administration at the lower rate of 15 mu u./min producing plasma hormone levels of 2.60 +/- 0.1 mu u./ml, did not alter renal sodium excretion. 6. Arginine vasopressin administered at 12 mu u./min induced plasma hormone levels of 1.54 +/- 0.09 mu u./ml and produced a large antidiuresis and small increase in the rate of sodium excretion. 7. The natriuretic response to vasopressin was potentiated by concurrent administration of oxytocin at 15 mu u./min. The peak sodium excretion of 5.8 +/- 1.0 mumol/min, however, remained well below that seen in intact rats. 8. It is concluded that, as restoration of posterior pituitary hormones at or above the physiological range only partially restored sodium excretion, the absence of anterior pituitary factors may also contribute directly or indirectly to the renal sodium retention of the hypophysectomized rat.

A synergistic effect of oxytocin and vasopressin on sodium excretion in the neurohypophysectomized rat.

1. Renal function and the effect of oxytocin and vasopressin replacement have been examined in anaesthetized male neurohypophysectomized rats. 2. Rates of urine flow were higher but sodium excretion markedly lower in neurohypophysectomized rats than in intact animals receiving hypotonic saline infusion (33.8 +/- 2.3 vs. 27.0 +/- 0.7 ml and 472 +/- 84 vs. 1946 +/- 124 mumol respectively for the third to sixth hour of study). 3. In intact animals, mean arterial blood pressure stabilized at 106 mmHg. Haematocrit (46%) remained stable but glomerular filtration rates declined slightly over the 8 h of study to 2.5 +/- 0.2 ml/h. These values in neurohypophysectomized rats did not differ significantly from those in intact rats. 4. Although plasma corticosterone levels (54 +/- 13 ng/ml) did not differ significantly from those in intact rats, neurohypophysectomy was associated with greatly reduced aldosterone concentration (0.12 +/- 0.03 vs. 0.76 +/- 0.04 ng/ml). Trace levels of vasopressin (0.17 +/- 0.03 microunit/ml) were found in neurohypophysectomized rat plasma. 5. Oxytocin administration at 15 microunits/min, which produced plasma hormone levels of 1.62 +/- 0.19 microunit/ml, had no detectable effect on sodium excretion but increased urine flow. Arginine vasopressin administration (12 microunits/min) inducing plasma levels of 1.24 +/- 0.08 microunit/ml, reduced urine flow by 80% and produced a small increase in sodium excretion. 6. Concurrent administration of oxytocin (15 microunits/min) potentiated the natriuretic response to vasopressin (12 microunits/min). Total sodium excretion during the 3 h combined hormone infusion (1256 +/- 149 mumol) greatly exceeded that in animals receiving vasopressin alone (549 +/- 132 mumol) and approached that observed in intact animals (1946 +/- 124 mumol). Combined hormone administration at the lower rate of 5 microunits/min oxytocin and 4 microunits/min vasopressin produced a similar large increment in sodium excretion. 7. It is concluded that replacement of both neurohypophysial hormones, at plasma levels within the physiological range, largely reverses the renal sodium retention of neurohypophysectomized rats, oxytocin considerably potentiating the natriuretic action of vasopressin. This synergism between the two neurohypophysial peptides to promote salt excretion may be an important component of the non-steroidal management of sodium.

Renal sodium retention and vasopressin induced kaliuresis in ethanol anaesthetised rats.

Renal electrolyte excretion has been examined in water loaded ethanol anaesthetised rats receiving continuous iv saline (0.077 M NaCl) infusion. These animals exhibited very low rates of Na+, K+ and Cl- excretion by comparison with Inactin anaesthetised rats. Water loaded Inactin anaesthetised rats also showed a degree of Na retention but both Na+ and K+ excretory rates were higher than in ethanol anaesthetised animals. Plasma aldosterone levels did not differ between ethanol and Inactin anaesthetised groups. Vasopressin administration did not effect Na+ but potentiated K+ excretion in ethanol anaesthetised animals. This contrasted with the potent natriuretic and weak kaliuretic action of vasopressin in water loaded Inactin anaesthetised rats. The significance of abnormal renal electrolyte handling and the marked kaliuretic effect of vasopressin to the use of ethanol anaesthetised animals for vasopressin bioassay is discussed.

Natriuretic response of the rat to plasma concentrations of arginine vasopressin within the physiological range.

The relationship of plasma vasopressin concentrations in the physiological range to renal electrolyte excretion was investigated. Unanaesthetized rats, when normally hydrated, were found to have a plasma vasopressin concentration of 1.13 +/- 0.15 mu u./ml. 16 h water deprivation raised this to 1.98 +/- 0.21 mu u./ml. Inactin-anaesthetized rats infused with 0.45% NaCl had a plasma vasopressin concentration of 1.19 +/- 0.18 mu u./ml. Administration of synthetic arginine vasopressin at 6 and 24 mu u./min raised plasma vasopressin levels to 1.88 +/- 0.17 and 4.26 +/- 0.43 microunits./ml respectively. In addition to the expected antidiuresis, vasopressin at a rate of 6 microunits./min also produced a highly significant increase in Na+ excretion from 8.9 +/- 0.6 to 10.5 +/- 0.6 mumol/min and Cl- excretion from 9.1 +/- 0.7 to 10.5 +/- 0.7 mumol/min. At 24 microunits./min it produced larger increases in Na+ and Cl- excretion. Inactin-anaesthetized hypophysectomized rats infused with 0.45% NaCl had a plasma vasopressin concentration of only 0.17 +/- 0.04 microunits./ml. Administration of vasopressin at 6 and 24 microunits./ml raised plasma vasopressin levels in these animals to 0.63 +/- 0.17 and 2.20 +/- 0.11 microunits./ml respectively. Hypophysectomized rats failed to exhibit a natriuresis in response to the lower dose of vasopressin, despite exhibiting an undiminished antidiuresis. The failure of the natriuresis may be related to the lower plasma vasopressin concentration achieved. It is concluded that in the rat plasma vasopressin concentrations within the physiological range do influence Na+ and Cl- excretion by the kidney as well as controlling urine flow rate.