First-in-class thyrotropin-releasing hormone (TRH)-based compound binds to a pharmacologically distinct TRH receptor subtype in human brain and is effective in neurodegenerative models.

JAK4D, a first-in-class thyrotropin-releasing hormone (TRH)-based compound, is a prospective therapeutic candidate offering a multifaceted approach to treating neurodegeneration and other CNS conditions. The purpose of these studies was to determine the ability of JAK4D to bind to TRH receptors in human brain and to evaluate its neuropharmacological effects in neurodegenerative animal models. Additionally, JAK4D brain permeation was examined in mouse, and initial toxicology was assessed in vivo and in vitro. We report that JAK4D bound selectively with nanomolar affinity to native TRH receptors in human hippocampal tissue and showed for the first time that these receptors are pharmacologically distinct from TRH receptors in human pituitary, thus revealing a new TRH receptor subtype which represents a promising neurotherapeutic target in human brain. Systemic administration of JAK4D elicited statistically significant and clinically-relevant neuroprotective effects in three established neurodegenerative animal models: JAK4D reduced cognitive deficits when administered post-insult in a kainate (KA)-induced rat model of neurodegeneration; it protected against free radical release and neuronal damage evoked by intrastriatal microdialysis of KA in rat; and it reduced motor decline, weight loss, and lumbar spinal cord neuronal loss in G93A-SOD1 transgenic Amyotrophic Lateral Sclerosis mice. Ability to cross the blood-brain barrier and a clean initial toxicology profile were also shown. In light of these findings, JAK4D is an important tool for investigating the hitherto-unidentified central TRH receptor subtype reported herein and an attractive therapeutic candidate for neurodegenerative disorders.

Biological study of the angiogenesis inhibitor N-(8-(3-ethynylphenoxy)octyl-1-deoxynojirimycin.

The alpha-glucosidase inhibitors N-methyl-1-deoxynojirimycin (MDNJ) and castanospermine have been shown to inhibit angiogenesis. A hybrid of 1-deoxynojirimycin (DNJ) and an aryl-1,2,3-triazole, which inhibits both an alpha-glucosidase and methionine aminopeptidase-2 (MetAP2), displayed properties associated with inhibition of angiogenesis (Bioorg. Med. Chem., 16, 2008, 6333-7). The biological evaluation of a structural analogue N-(8-(3-ethynylphenoxy)octyl-1-deoxynojirimycin is described herein. Although this alkyne derivative did not inhibit MetAP2, it inhibited a bacterial alpha-glucosidase, altered bovine aortic endothelial cell (BAEC) surface oligosaccharide expression and inhibited BAEC proliferation by inducing G1 phase cell cycle arrest. Experiments showed G1 arrest was attributable to the alpha-glucosidase inhibitor inducing an increase in p27(Kip1) expression and high phosphorylation of ERK1/2 without a reduction in cyclin D1. The DNJ derivative (0.1 mM) prevented capillary tube formation from bovine aortic endothelial cells, whereas DNJ or other analogues were unable to inhibit tube formation at the same concentration. Stress fiber assembly in bovine aortic endothelial cells was abolished, and BAEC migration was inhibited indicating the inhibition of tube formation by this derivative is partially a result of a reduction in cell motility. The agent also caused a reduction in secretion of MMP-2 from bovine aortic endothelial cells. Therefore, the new alpha-glucosidase inhibitor has a different mechanism by which it inhibits angiogenesis in vitro when compared with deoxynojirimycin, the deoxynojirimycin -triazole hybrid, N-methyl-1-deoxynojirimycin and castanospermine.

D2 receptor-mediated inhibition of dopamine release in the rat striatum in vitro is modulated by CB1 receptors: studies using fast cyclic voltammetry.

Cannabinoid CB(1) receptors are highly expressed in the striatum where they are known to be co-localized with dopamine D(2) receptors. There is now strong evidence that cannabinoids modulate dopamine release in the brain. Using fast cyclic voltammetry, single pulse stimulation (0.1 ms; 10 V) was applied every 5 min and peak dopamine release was measured with a carbon fibre microelectrode. Application of the D(2) receptor agonist, quinpirole, inhibited single pulse dopamine overflow in a concentration-dependent manner (IC(50): 3.25 x 10(-8) M). The CB(1) receptor agonist WIN55212-2 (WIN; 1 microM) had no effect on single pulse dopamine release (93.9 +/- 6.6% at 60 min, n = 5) but attenuated the inhibitory effect of quinpirole (30 nM; quinpirole 39.0 +/- 4.2% vs. quinpirole + WIN, 48.2 +/- 3.7%, n = 5, p < 0.05). This affect was antagonized by the CB(1) receptor antagonist [N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM-251, 1 microM). Dopamine release evoked by four pulses delivered at 1 Hz (4P1Hz) and 10 pulses delivered at 5 Hz (10P5Hz) was significantly inhibited by WIN [72.3 +/- 7.9% control (peak 4 to 1 ratio measurement) and 66.9 +/- 3.8% control (area under the curve measurement), respectively, p < 0.05; n = 6 for both]. Prior perfusion of WIN significantly attenuated the effects of quinpirole on multiple pulse-evoked dopamine release (4P1Hz: quinpirole, 28.4 +/- 4.8% vs. WIN + quinpirole, 52.3 +/- 1.2%; 10P5Hz: quinpirole, 29.5 +/- 1.3% vs. WIN + quinpirole, 59.4 +/-7.1%; p < 0.05 for both; n = 6). These effects were also antagonized by AM-251 (1 microM). This is the first report demonstrating a functional, antagonistic interaction between CB(1) receptors and D(2) autoreceptors in regulating rat striatal dopamine release.

Hybrids of 1-deoxynojirimycin and aryl-1,2,3-triazoles and biological studies related to angiogenesis.

Hybrids of 1-deoxynojirimycin (DNJ) and aryl-1,2,3-triazole have been synthesized with a view to identifying an inhibitor of both alpha-glucosidase and methionine aminopeptidase 2 (MetAP2). One compound was a potent inhibitor of alpha-glucosidase at both the enzyme and cellular level, and this agent also inhibited bovine aortic endothelial cell (BAEC) growth and tube formation. The anti-proliferative activity of this hybrid is due to its ability to induce cell-cycle arrest in the G(1) phase. The novel agent caused a reduction in the expression of cyclin D1 but did not promote apoptosis or inhibit the phosphorylation of ERK1/2. These observations indicate that its mechanism of action is distinct from fumagillin and its analogues, which inhibit MetAP2. Stress-fibre assembly in BAECs was abolished by the novel agent indicating that the inhibition of BAEC tube formation observed is partially a result of a reduction in cell motility.

Hybrid angiogenesis inhibitors: synthesis and biological evaluation of bifunctional compounds based on 1-deoxynojirimycin and aryl-1,2,3-triazoles.

Synthesis of hybrids of 1-deoxynojirimycin (DNJ) and 5-aryl-1,2,3-triazole as potential bifunctional inhibitors of angiogenesis is described. The DNJ component inhibits the biosynthesis of cell surface oligosaccharides necessary for angiogenesis, whereas the aryl-1,2,3-triazole inhibits methionine aminopeptidase II, a target in angiogenesis therapy. One bifunctional compound was a more potent inhibitor of angiogenesis in vitro than DNJ alone or the 5-aryl-1,2,3-triazole alone.

A novel TRH analog, Glp-Asn-Pro-D-Tyr-D-TrpNH2, binds to [3H][3-Me-His2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2.

Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all-L counterpart (Glp-Asn-Pro-Tyr-TrpNH(2)) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [(3)H][3-Me-His(2)]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His(2)]TRH potently displaced [(3)H][3-Me-His(2)]TRH binding from all tissues/cells investigated. Glp-Asn-Pro-D-Tyr-D-TrpNH(2) in concentrations up to 10(-5)M did not displace [(3)H][3-Me-His(2)]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all-L peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.7+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2), analogous to cortical membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.8+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2). Neither peptide, however, displaced [(3)H][3-Me-His(2)]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is not displacing [(3)H][3-Me-His(2)]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.

Inhibition of endothelial cell proliferation by per-O-acetylated mannose conjugates.

BACKGROUND: The inhibition of angiogenesis, defined as the process of new blood vessel formation, represents a promising strategy for treating cancer. MATERIALS AND METHODS: The inhibitory properties of two N-(per-O-acetylated-beta-D- mannopyranosyl)thiophene-2-carboxamides derivatives (AMTCs, [1],[2]), N-(2,3,4,6-tetra-O-ethoxycarbonyl-beta-D-mannopyranosyl)- thiophene-2-carboxamide [3] and of 2,3,4,6-tetra-O-acetyl-beta-D-mannopyranosyl-acetamide [4] on the growth of bovine aortic endothelial cells (BAECs) induced by basic fibroblast growth factor (bFGF) were assessed using a [3H]thymidine incorporation assay. The cellular uptake of AMTCs and the non-acetylated homologue (MTC) into BAEC were compared using mass spectrometry analysis of cell lysates. RESULTS: AMTCs [1],[2]), at 80 microM, reversed the increase of [3H]thymidine incorporation induced by bFGF, suggesting that these compounds inhibited bFGF-induced proliferative response in BAECs. The acetamide [4] was inactive showing the importance of the thiophene carboxamide for biological activity. The results of a study of AMTC uptake into BAEC suggest that AMTC is rapidly converted to its non-acetylated counterpart. CONCLUSION: The promising results obtained with AMTCs as inhibitors of BAEC growth could lead to the development of novel angiogenesis inhibitors.

Discovery of a dual action first-in-class peptide that mimics and enhances CNS-mediated actions of thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) displays multiple CNS-mediated actions that have long been recognized to have therapeutic potential in treating a wide range of neurological disorders. Investigations of CNS functions and clinical use of TRH are hindered, however, due to its rapid degradation by TRH-degrading ectoenzyme (TRH-DE). We now report the discovery of a set of first-in-class compounds that display unique ability to both potently inhibit TRH-DE and bind to central TRH receptors with unparalleled affinity. This dual pharmacological activity within one molecular entity was found through selective manipulation of peptide stereochemistry. Notably, the lead compound of this set, L-pyroglutamyl-L-asparaginyl-L-prolyl-D-tyrosyl-D-tryptophan amide (Glp-Asn-Pro-D-Tyr-D-TrpNH(2)), is effective in vivo at producing and potentiating central actions of TRH without evoking release of thyroid-stimulating hormone (TSH). Specifically, this peptide displayed high plasma stability and combined potent inhibition of TRH-DE (K(i) 151 nM) with high affinity binding to central TRH receptors (K(i) 6.8 nM). Moreover, intraperitoneal injection of this peptide mimicked and augmented the effects of TRH on behavioural activity in rat. Analogous to TRH, it also antagonized pentobarbital-induced narcosis when administered intravenously. This discovery provides new opportunities for probing the role of TRH actions in the CNS and a basis for development of novel TRH-based neurotherapeutics.

N-Glycosyl-thiophene-2-carboxamides: synthesis, structure and effects on the growth of diverse cell types.

A range of N-glycosyl-thiophene-2-carboxamides, including a 6H-thieno[2,3-c]pyridin-7-one and a bivalent compound, have been synthesised and assayed for their effects on DNA synthesis in bovine aortic endothelial cells or on the growth of synoviocytes. Per-O-acetylated analogues of the glycoconjugates were significantly more effective inhibitors when compared to their corresponding non-acetylated analogues, indicating that the lower potency observed for hydroxylated derivatives is due to less efficient transport of these compounds across the cell membrane. Thiophene-2-carboxamide was inactive as an inhibitor of bFGF induced proliferation, confirming the requirement of the carbohydrate residue for the observed biological properties. Glucose, mannose, galactose and 2-amino-2-deoxy-glucose analogues were active as were a variety of substituted thiophene derivatives; the 6H-thieno[2,3-c]pyridin-7-one conjugate was inactive. Conformational analysis of the title compounds was investigated. X-ray crystal structural analysis of four N-glucosyl-thiophene-2-carboxamides showed that the pyranose rings adopted the expected 4C1 conformations and that Z-anti structures were predominant (H1-C1-N-H anomeric torsion angle varied from -168.2 degrees to -175.0 degrees ) and that the carbonyl oxygen and sulfur of the thiophene adopted an s-cis conformation in three of the isomers. In a crystal structure of a 3-alkynyl derivative, the hydrogen atom of the NH group was directed toward the acetylene group. The distance between the hydrogen atom and acetylene carbons and angles between nitrogen, hydrogen and carbon atoms were consistent with hydrogen bonding and this was supported by IR and NMR spectroscopic studies. The geometries of thiophene-2-carboxamides were explored by density functional theory (DFT) and Møller-Plesset (MP2) calculations and the s-cis conformer of thiophene-2-carboxamide was found to be more stable than its s-trans isomer by 0.83 kcal mol(-1). The s-cis conformer of 3-ethynyl-thiophene-2-carboxamide was 5.32 kcal mol(-1) more stable than the s-trans isomer. The larger stabilisation for the s-cis conformer in the 3-alkynyl derivatives is explained to be due to a moderate hydrogen bonding interaction between the alkyne and NH group.

N-Glycosyl-thiophene-2-carboxamides: effects on endothelial cell growth in the presence and absence of bFGF--a significant increase in potency using per-O-acetylated sugar analogues.

Inhibitors of endothelial cell proliferation are of interest in development of therapies for angiogenesis related disease. N-Glucosyl-thiophene-2-carboxamides have been synthesized and evaluated for their effects on proliferation in bovine aortic endothelial cells. Per-O-acetylated-N-glucosyl-thiophene-2-carboxamides showed improved inhibition of both serum and bFGF stimulated uptake of [(3)H]thymidine, when compared to non-acetylated analogues.

Synthesis of disaccharides derived from heparin and evaluation of effects on endothelial cell growth and on binding of heparin to FGF-2.

The disaccharide beta-D-GlcA-(1-->4)-alpha-D-GlcNAc-1-->OMe and other small nonsulfated oligosaccharides related to heparin/heparan sulfate have been shown to bind to FGF and activated the fibroblast growth factor (FGF) signalling pathway in (F32) cells expressing the FGF receptor. Synthetic routes to beta-D-GlcA-(1-->4)-alpha-D-GlcNAc-1-->OMe and a glucose analogue beta-D-Glc-(1-->4)-alpha-D-GlcNAc-1-->OMe are described. The effects of these disaccharides on endothelial cell growth, which is relevant to angiogenesis, were evaluated and it was found they did not mimic the inhibitory effects that were observed for heparin albumin (HA) and that have also been observed by monosaccharide conjugates. They did not alter bovine aortic endothelial cell (BAEC) proliferation, in the presence of FGF-2 in serum free medium or in absence of FGF-2 in serum free and complete medium. Disaccharides (10 microg/mL) reduced by 25-31% the inhibition caused by HA (10 microg/mL) on BAEC growth in serum-free medium but had no effect in complete medium. There was no evidence obtained for the binding of these oligosaccharides to FGF-2 in competition with HA by ELISA.

Synthesis of a glucuronic acid and glucose conjugate library and evaluation of effects on endothelial cell growth.

Compounds that alter endothelial cell growth are of interest in the development of angiogenesis modulators. A structurally diverse series of saccharide derivatives (glycosylamide conjugates) have been synthesized and evaluated for their effects on bovine aortic endothelial cell (BAEC) growth. Heparin-albumin (HA) reduced BAEC growth by 32% at 10 microg/mL and a number of the novel saccharide conjugates from the library were found to mimic the effect of HA as they also inhibit endothelial cell survival under identical conditions. Two thiophene conjugates, thioglucamide (24% inhibition at 35 microM) and a related glucuronide (26% inhibition at 33 microM) were the most potent inhibitors of BAEC growth, as determined using a methylthiazol tetrazolium (MTT) assay. The effects of thioglucamide and HA on absolute cell number were also studied using cell counting experiments; thioglucamide (47% after 24 h) was more potent than indicated by the MTT assay and initially reduced the BAEC number to a greater extent than HA (30% after 24 h); however, its actions were over more rapidly than were HA's as cell growth had returned to levels of the control after 72 h where HA still caused 25% inhibition. The binding of the monosaccharide conjugates to fibroblast growth factor (FGF-2) in competition with heparin-albumin by ELISA was investigated to establish the possible mechanism by which glycoconjugates could alter growth but there was no general correlation between reduction in viable cell population and binding to FGF-2. No glycoconjugate reduced the proliferation of mouse mammary epithelial cells, nor did any alter gross cell morphology, supporting a proposal that the reduction in BAEC survival by monosaccharide conjugates such as thioglucamide is a result of the inhibition of cell proliferation rather than being an induction of cytotoxicity. These studies indicate that cell biological studies to determine the mechanism of action of the simple monosaccharide conjugates may be worthwhile.

Identification of novel inhibitors of fibroblast growth factor (FGF-2) binding to heparin and endothelial cell survival from a structurally diverse carbohybrid library.

Inhibitors of FGF-2 binding to a heparin-albumin conjugate were identified by ELISA from a library of glucuronic acid derivatives. These compounds were also inhibitors of endothelial cell survival that is dependant on FGF-2 and heparin or heparan sulfate proteoglycans. The results indicate that these bioactive compounds may prove useful as lead structures for the further development of pharmaceutical agents capable of modulating biological activity of FGF-2.

Pharmacologically distinct binding sites in rat brain for [3H]thyrotropin-releasing hormone (TRH) and [3H][3-methyl-histidine(2)]TRH.

We have used a directed peptide library, in which the histidyl residue of thyrotropin-releasing hormone (TRH) was systematically replaced by a series of 24 natural and unnatural amino acids, to characterise TRH binding sites in rat brain cortex. This was achieved by measuring the ability of library peptides to compete with [3H][3-Me-His(2)]TRH or [3H]TRH binding to rat cortical homogenates. [3H][3-Me-His(2)]TRH was observed to bind to a single population of high-affinity, low-capacity sites (K(d): 4.54+/-0.62 nM, N=5; B(max): 4.38+/-0.21 fmol/mg wet weight tissue, N=5), consistent with them being central TRH receptors. Displacement studies showed TRH to bind to these sites with an apparent K(i) of 22 nM. K(i) values for the library peptides at [3H][3-Me-His(2)]TRH-labelled sites varied from 10(-3) to 10(-9)M; the potency order was: [3-Me-His(2)]>His>Thi>Leu,Phe,Asn>Gln, Arg, Thr, Ala, HomoPhe. All other replacements had K(i) values >10(-4)M. [3H]TRH was observed to label a single population of low-affinity, high-capacity sites (K(d): 7.55+/-1.23 microM, N=6; B(max): 3.40+/-0.63 pmol/mg wet weight tissue, N=6). The affinities of the synthetic peptides for [3H]TRH-labelled sites did not correlate with their affinities for [3H][3-Me-His(2)]TRH-labelled sites (r=0.33, N=18, P>0.1). They did, however, correlate significantly with previously reported binding affinities for TRH-degrading ectoenzyme (r=0.72, N=12, P<0.01). These results strongly indicate that the identity of the low-affinity, [3H]TRH-labelled site is the membrane-bound enzyme, TRH-degrading ectoenzyme, not a subpopulation of TRH receptors. They also provide the first comprehensive description of the influence of the histidyl residue in TRH on binding of TRH to brain receptors.