Context-Dependent Pharmacology Exhibited by Negative Allosteric Modulators of Metabotropic Glutamate Receptor 7

Phenotypic studies of mice lacking metabotropic glutamate receptor subtype 7 (mGluR7) suggest that antagonists of this receptor may be promising for the treatment of central nervous system disorders such as anxiety and depression. Suzuki et al. (J Pharmacol Exp Ther 323: 147-156, 2007) recently reported the in vitro characterization of a novel mGluR7 antagonist called 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), which noncompetitively inhibited the activity of orthosteric and allosteric agonists at mGluR7. We describe that MMPIP acts as a noncompetitive antagonist in calcium mobilization assays in cells coexpressing mGluR7 and the promiscuous G protein G alpha(15). Assessment of the activity of a small library of MMPIP-derived compounds using this assay reveals that, despite similar potencies, compounds exhibit differences in negative co-operativity for agonist-mediated calcium mobilization. Examination of the inhibitory activity of MMPIP and analogs using endogenous G(i/o)-coupled assay readouts indicates that the pharmacology of these ligands seems to be context-dependent, and MMPIP exhibits differences in negative cooperativity in certain cellular backgrounds. Electrophysiological studies reveal that, in contrast to the orthosteric antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxyclycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), MMPIP is unable to block agonist-mediated responses at the Schaffer collateral-CA1 synapse, a location at which neurotransmission has been shown to be modulated by mGluR7 activity. Thus, MMPIP and related compounds differentially inhibit coupling of mGluR7 in different cellular backgrounds and may not antagonize the coupling of this receptor to native G(i/o) signaling pathways in all cellular contexts. The pharmacology of this compound represents a striking example of the potential for context-dependent blockade of receptor responses by negative allosteric modulators.

Toenail trace element status and risk of Barrett's oesophagus and oesophageal adenocarcinoma. Results from the FINBAR study

Trace elements have been cited as both inhibitory and causative agents of cancer but importantly exposure to them is potentially modifiable. Our study aimed to examine toenail trace element status and risk of Barrett's oesophagus (BO) and oesophageal adenocarcinoma (OAC). Toenail clippings from each hallux were obtained from 638 participants of the FINBAR (Factors Influencing the Barrett's Adenocarcinoma Relationship) study comprising 221 healthy controls, 98 reflux oesophagitis, 182 BO and 137 OAC cases. The concentrations of eight toenail trace elements were determined using instrumental neutron activation analysis. Using multivariable adjusted logistic regression analysis, odds ratios (OR) and 95% confidence intervals (CIs) were calculated within tertiles of trace element concentrations. A twofold increased risk of BO was observed, but not OAC, among individuals in the highest tertile of toenail zinc status OR 2.21 (95% CI, 1.11-4.40). A higher toenail selenium status was not associated with risk of OAC OR 0.94 (95% CI, 0.44-2.04) or BO OR 0.89 (95% CI, 0.37-2.12). A borderline significant increased risk of BO was detected with a higher toenail cobalt concentration, OR 1.97 (95% CI, 1.01-3.85). No association was found between toenail levels of chromium, cerium, mercury and OAC or BO risk. This is the first case-control study to investigate a variety of trace elements in relation to OAC and BO risk. Despite antioxidant and proapoptotic properties, no associations were found with selenium. Higher concentrations of toenail zinc and cobalt were associated with an increased BO risk, but not OAC. These findings need confirmation in prospective analysis.

Nematode neuropeptide modulation of the vagina vera of Ascaris suum: in vitro effects of PF1, PF2, PF4, AF3 and AF4

Ascaris suum possesses a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8/PF3) have been shown to modulate the intrinsic, rhythmic activity of the vagina vera of A. suum in vitro. In the present study, the effects of the nematode FaRPs, SDPNFLRFamide (PF1), SADPNFLREamide (PF2) and KPNFIRFamide (PF4) (from Panagrellus redivivus) and AVPGVLRFamide (AF3) and GDVPGVLRFamide (AF4) (from A. suum) on the in vitro activity of the vagina vera were examined. The effects of each of the peptides were qualitatively and quantitatively distinct. All 3 FaRPs from P. redivivus were inhibitory, causing a cessation of contractions. PF2 was 3 times more potent than PF1, with a threshold of 1 nM. Although PF4 was the least potent (threshold, 10 nM), its effects at greater than or equal to 10 nM were quantitatively the greatest. Both AF3 and AF4 (1 mu M) induced complex, multiphasic responses consisting of an initial contraction and spastic paralysis followed by a return of contractile activity of increased amplitude. AF3 was 3 times more potent than AF4. The effects of these peptides had some similarities to those observed on A. suum somatic body wall muscle in vitro, with PF1, PF2 and PF4 being inhibitory and AF3 and AF4 being excitatory.

Parasitic peptides! The structure and function of neuropeptides in parasitic worms

Parasitic worms come from two very different phyla-Platyhelminthes (flatworms) and Nematoda (roundworms). Although both phyla possess nervous systems with highly developed peptidergic components. there are key differences in the structure and action of native neuropeptides in the two groups. For example, the most abundant neuropeptide known in platyhelminths is the pancreatic polypeptide-like neuropeptide F, whereas the most prevalent neuropeptides in nematodes an FMRFamide-related peptides (FaRPs), which are also present in platyhelminths. With respect to neuropeptide diversity, platyhelminth species possess only one or two distinct FaRPs, whereas nematodes have upwards of 50 unique FaRPs. FaRP bioactivity in platyhelminths appears to be restricted to myoexcitation, whereas both excitatory and inhibitory effects have been reported in nematodes. Recently interest has focused on the peptidergic signaling systems of both phyla because elucidation of these systems will do much to clarify the basic biology of the worms and because the peptidergic systems hold the promise of yielding novel targets for a new generation of antiparasitic drugs. (C) 1999 Elsevier Science Inc. All rights reserved.

Structural characterisation and pharmacology of KHEYLRFamide (AF2) and KSAYMRFamide (PF3/AF8) from Haemonchus contortus

The FMRFamide-related peptides (FaRPs), KHEYLRFamide (AF2) and KSAYMRFamide (PF3) were structurally characterised from the parasitic nematode of sheep, Haemonchus contortus (MH isolate). Both peptides were sequenced in a single gas-phase sequencing run and their structure confirmed by mass spectrometry which identified peptides of 920 Da (C-terminally amidated AF2) and 902/918 Da (C-terminally amidated non-oxidised/oxidised PF3, respectively). AF2 had inhibitory effects on H. contortus muscle and inhibited acetylcholine (ACh, 10 mu M)-induced contractions, with a threshold for activity of I mu M. PF3 induced concentration-dependent contractions of H. contortus (activity threshold, 10 nM) and enhanced ACh contractions. Compared with the MH isolate, an isolate of H. contortus which has reduced sensitivity to cholinergic drugs (Lawes isolate) was less sensitive to the effects of PF3. The concentration-response curves for the cholinergic compounds ACh and levamisole (LEV), and PF3, but not a control, KPNFIRFamide (PF4), showed a statistically similar shift. This study implicates PF3 in the modulation of cholinergic function in H. contortus. (C) 1999 Elsevier Science B.V. All rights reserved.

Isolation, pharmacology and gene organization of KPSFVRFamide: A neuropeptide from Caenorhabditis elegans

To date, 53 peptides with C-terminal RFamides have been identified by the genome sequencing project in the nematode, Caenorhabditis elegans. In this study the FMRFamide-related peptide (FaRP) KPSFVRFamide (879.90 Da [MH](+)) was structurally characterized from extracts of the nematode, Caenorhabditis elegans. Two copies of KPSFVRFamide are encoded by a gene designated flp-9. RT-PCR identified a single cDNA product which was confirmed as flp-9 by sequence determination. Flp-9 cDNA was isolated from larval stages of C. elegans but was not detected-in adult worms, indicating that its expression is may be developmentally regulated. KPSFVRFamide displays sequence homology to the nematode peptide, KPNFIRFamide (PF4). The physiological effects of KPSFVRFamide, PF4 and the chimeras, KPNFVRFamide and KPSFIRFamide, were measured on body wall muscle and the vagina vera of the parasitic nematode, Ascaris suum. KPNFVRFamide and KPNFIRFamide had Cl--dependent inhibitory activity on innervated and denervated muscle-preparations, whereas KPSFVRFamide and KPSFIRFamide did not elicit a detectable physiological effect. Although all 4 peptides had inhibitory effects on the vagina vera, KPSFVRFamide and KPSFIRFamide (threshold, greater than or equal to 0.1 mu M) were less potent than KPNFVRFamide and KPNFIRFamide (threshold, greater than or equal to 10 nM). (C) 1999 Academic Press.

APEASPFIRFamide, a novel FMRFamide-related decapeptide from Caenorhabditis elegans: Structure and myoactivity

To date, 9 FMRF amide-related peptides (FaRPs) have been identified in Caenorhabditis elegans. Eight of these peptides are encoded on the flp-1 gene. However, AF2 (KHEYLRF amide) which was not co-encoded was the most abundant FaRP identified in ethanolic extracts. Further radioimmunometrical screening of acidified ethanol extracts of C. elegans has revealed the presence of other novel FaRPs, which are not encoded on the flp-l gene. One of these peptides has been isolated by sequential rpHPLC and subjected to Edman degradation analysis and gas-phase sequencing and the unequivocal primary structure of the decapeptide Ala-Pro-Glu-Ala-Ser-Pro-Phe-Ile-Arg-Phe-NH2 was determined following a single gas-phase sequencing run. The molecular mass of the peptide was found to be 1133.7 Ha, determined using a time-of-flight mass spectrometer. Synthetic replicates of this peptide were found to induce a profound relaxation of both dorsal and ventral somatic muscle-strip preparations of Ascaris suum with a threshold for activity of 10 nM. The inhibitory response was not dependent on the presence of nerve cords, indicating a post-synaptic site-of-action. The relaxation was Ca++- and Cl--independent but was abolished in high-KI medium and could be distinguished from those of other inhibitory nematode FaRPs, including PF1 (SDPNFLRFamide)and PF1 (KPNFIRF amide). (C) 1997 Academic Press.

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode, Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu(5)] PF4, [Ala(2)]PF4, [Gly(2)]PF4, [Ala(2),Leu(5)]PF4, and [Gly(2),Leu(5)]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu(5)] PF4 >> [Ala(2)]PF4 = [Ala(2),Leu(5)] PF4 >> [Gly(2)] PF4 = [Gly(2),Leu(5)] PF4. Leu(5) for Ile(5) substitutions in PF4 did not alter the activity of this peptide; however, Gly(2)/Ala(2) for Pro(2) substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly(2)]PF4(2-7) and -(3-7) and [Ala(2)]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro(2) in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases. Copyright (C) 1996 Elsevier Science Inc.

EFFECT OF IN-VIVO AND IN-VITRO LOVASTATIN TREATMENT ON MAST-CELL ACTIVATION

The hydroxymethylglutaryl coenzmye A (HMG CoA) reductase inhibitor lovastatin is used to treat hyperlipidaemia. This agent prevents the isoprenylation of some proteins involved in signal transduction processes and inhibits IgE-receptor-linked mediator release from RBL-2H3 cells. In this study the effect of in vivo and in vitro administration of lovastatin on histamine release from rat peritoneal mast cells was examined. Lovastatin (4 mg/kg/day for 2 weeks) inhibited histamine release induced by concanavalin A (con A) from rat peritoneal mast cells of Hooded-Lister rats and both homozygous lean and obese Zucker rats. In contrast, release induced by antirat IgE (anti-IgE) was only significantly inhibited in cells derived from Hooded-Lister rats and that induced by compound 48/ 80 was not altered. Lovastatin (20 mu M, 24 h, in vitro) caused a significant inhibition of the subsequent histamine release to con A, anti-IgE and compound 48/80 but not to the calcium ionophore A 23187. It is important to determine whether such inhibitory effects are also observed after the chronic, clinical administration of lovastatin and other HMG CoA reductase inhibitors.

MODULATORY ACTION OF HELICOBACTER-PYLORI ON HISTAMINE-RELEASE FROM MAST-CELLS AND BASOPHILS IN-VITRO

Helicobacter pylori is important in the aetiology of peptic ulceration. Despite inducing an inflammatory response in the mucosa, the organism persists, suggesting that it has efficient protective mechanisms. Some bacterial and viral products modulate histamine secretion from inflammatory cells. Therefore, this study examined the modulatory effects of H. pylori preparations on histamine release from rat peritoneal mast cells and human basophils. Eleven clinical isolates of H. pylori were prepared in different ways: as whole washed bacteria, washed sonicated bacteria, and formalin-killed bacteria, and as outer-membrane and lipopolysaccharide (LPS) extracts. Histamine release from mast cells or basophils was not elicited by any of these bacterial preparations alone. However, when mixed with various secretory stimulants, the bacterial preparations caused inhibition of histamine release from rat mast cells (calcium ionophore A23187, compound 48/80, concanavalin A, anti-rat IgE) and human basophils (A23187, N-formyl Met-Leu-Phe). The degree of inhibition ranged from 48 % to 97 %. These results indicate that H. pylori exerts an inhibitory effect on cells of the immune system that contributes to its persistence within the gastric mucosa.

A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu?Pro at position 2 and Phe?Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20µM and 150µM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His?Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5µM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His?Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40µm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.

Evaluation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy

Purpose: To characterize the importance of cellular Fas-associated death domain (FADD)–like interleukin 1ß-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)–promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)–targeted therapy.

Experimental Design: c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions.

Results: c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P < 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P < 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7–mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance.

Conclusion: c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC. Clin Cancer Res; 18(14); 3822–33.

Pharmacological Activity of Ibuprofen Release from Measoporous Silica

Novel drug delivery systems (DDS) to improve the pharmacokinetic profile of hydrophobic drugs following oral administration are an area of keen interest in drug research. An ideal DDS should not adversely affect drug activity, be capable of delivering a therapeutic dose of drug, and allow homogenous drug loading and drug release. Mesoporous silica has been proposed for this application, with ibuprofen employed as the model drug. It was hypothesised that mesoporous silica MCM-41 is capable of delivering a pharmacologically therapeutic dose of ibuprofen. Ibuprofen-loaded MCM-41 can be prepared reproducibly at a drug to carrier ratio of 30% (wt/wt). The release profile was seen to be 90% within 2 h. Initial assessment of COX-1 inhibitory activity suggests the absence of adverse effects attributable to drug-carrier interaction. The results of this study provide further evidence in support of the proposed use of mesoporous silica in drug delivery.

Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.