Maurotoxin and the Kv1.1 channel: voltage‐dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31–Cys34

Abstract: Maurotoxin (MTX) is a 34‐amino acid polypeptide cross‐linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half‐cystine pairings at Cys³–Cys²⁴, Cys⁹–Cys²⁹, Cys¹³–Cys¹⁹ and Cys³¹–Cys^34. This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C‐terminal ‘14‐membered disulfide ring’ (i.e. cyclic domain 31–34), We therefore studied structure–activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C‐terminus {i.e. MTX (1–29), [Abu_31,34]‐MTX and [Cys_31,34, Tyr₃₂]_d ‐MTX} or mimicking the cyclic C–terminal domain [i.e. MTX (31–34)]. Unexpectedly, the absence of a disulfide bridge Cys³¹–Cys³⁴ in [Abu _31,34]‐MTX and MTX (1–29) resulted in MTX‐unrelated half‐cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX (31–34) was also biologically inactive. [Cys_31,34, Tyr₃₂]_d ‐MTX, which had a ‘native’, MTX‐related, disulfide bridge organization, but a d ‐residue‐induced reorientation of the C–terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide‐induced Kv1.1 blockage was voltage‐dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on‐rate changes in ligand binding. Thus, the cyclic C–terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.     

Expedient synthesis of [18F]‐labeled α‐trifluoromethyl ketones

Several [¹⁸F]‐labeled α‐trifluoromethyl ketones have been synthesized. Reactions of 2,2‐difluoro‐1‐aryl‐1‐trimethylsiloxyethenes ( 1a–d ) with [¹⁸F]‐F₂ at low temperature produced [¹⁸F]‐labeled α‐trifluoromethyl ketones ( 2a–d ). Radio‐labeled products were isolated by purification with column chromatography in 22–28% yields, decay corrected (d.c.) in three runs per compound. Radiochemical purity was >99% with specific activities 15–20 GBq/mmol at the end of synthesis (EOS). The synthesis time was 35–40 min from the end of bombardment (EOB). This one‐step simple method is highly useful for the radiochemical synthesis of potential biologically active [¹⁸F]‐labeled α‐trifluoromethyl ketones for PET imaging. Copyright © 2003 John Wiley & Sons, Ltd.     

Relationship between temporary inhibition and structure of disulfide‐linkage analogs of marinostatin,a natural ester‐linked protein protease inhibitor

Abstract: A 12‐residue marinostatin [MST(1–12): ¹FATMRYPSDSDE¹²] which contains two ester linkages of Thr³–Asp⁹ and Ser⁸–Asp¹¹ strongly inhibits subtilisin. In order to study the relationship between the inhibitory activity, structure, and stability of MST, MST analogs were prepared by changing ester linkages to a disulfide linkages. The analogs without the disulfide linkage between 3 and 9 positions lost their inhibitory activity. The K_i value of 1SS(C³–C⁹) (¹FACMRYPSCSDE¹²), which has a single disulfide linkage of Cys³–Cys⁹ was comparable with those of MST(1–12) and MST‐2SS (¹FACMRYPCCSCE¹²), a doubly linked analog of Cys³–Cys⁹ and Cys⁸–Cys¹¹. However, 1SS(C³–C⁹) and MST‐2SS showed temporary inhibition, but not MST(1–12): These analogs were inactivated after incubation with subtilisin for 30 min, and were specifically hydrolyzed at the reactive site. ¹H NMR study showed that 1SS(C³–C⁹) has two conformations, which contain a cis‐ (70%) or trans‐ (30%) Pro residue, while MST‐2SS as well as MST(1–12) takes a single conformation containing only a cis‐Pro residue. Hydrogen–deuterium exchange rate of the Arg⁵ (P1′) NH proton of the MST analogs was about 100 times faster than that of MST(1–12). These results indicate that the linkage between the positions 8 and 11 plays a role for fixing the cis‐conformation of the Pro⁷ residue, and that the linkage between 3 and 9 is indispensable for the inhibition, but not enough for stable protease‐inhibitor complex.     

Conformational studies of highly potent 1‐aminocyclohexane‐1‐carboxylic acid substituted V2 vasopressin agonists

Abstract: Conformational studies of three agonists of V₂ receptor modified with 1‐aminocyclohexane‐1‐carboxylic acid (Acc), [Acc²,DArg⁸]VP, [Acc³]AVP and [Cpa¹,Acc³]AVP, using 2D NMR and theoretical calculations are presented in this paper. It is believed that α,α‐disubstituted amino acids, such as Acc, affect the formation of either 3₁₀ or α‐helical conformation. Moreover, a peptide with Acc may adopt either the γ‐ or an inverse γ‐turn over it. Thus, incorporation of Acc into the arginine‐vasopressin sequence induced C₇‐membered ring conformation with Acc at the top of it, and additional formation of β‐bend involving residues in the 2–5 fragment of the peptides. Furthermore, the analogues are also characterized by type I of β‐turn involving residues Acc³‐Cys⁶ in [Acc³]AVP and [Cpa¹,Acc³]AVP, and by type IV or II′ in [Acc²,DArg⁸]VP. Replacement of Tyr at position 2 of [Acc²,DArg⁸]VP with Acc afforded a hydrogen bond between the guanidine moiety of DArg⁸ and the side chain of either Asn⁵ or Gln⁴. In the remaining analogues, the β‐turn comprising the Cys⁶‐Gly⁹ residues allows the positively charged side chain of Arg⁸ to be directed toward Tyr². The substitution of Cys¹ with Cpa¹ enhances hydrophobic properties of N‐terminal part of the molecule strengthening thereby the affinity to the binding pocket of receptors.     

The synthetic cathinone psychostimulant α‐PPP antagonizes serotonin 5‐HT2A receptors: In vitro and in vivo evidence

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT_2A receptors (5‐HT_2AR) and muscarinic M₁ receptors (M₁R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M₁R (minimal displacement of [~K_d] [³H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K_i values at 5‐HT_2AR. In 5‐HT_2AR–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT_2AR functional activity of α‐PPP, and observed it competitively antagonized 5‐HT_2AR signaling stimulated by the 5‐HT₂R agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; K_b = 851 nM). To assess in vivo 5‐HT_2AR activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT_2AR mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT_2AR K_is > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT_2AR in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT_2AR antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.     

Paralytic activity of (des-Glu1)conotoxin GI analogs in the mouse diaphragm

A series of 20 peptide analogs of (des-Glu¹) conotoxin GI were prepared by solid phase synthesis. The peptides were tested for their abilities to inhibit contractions in the mouse-diaphragm-with-phrenic-nerve assay. (Des-Glu¹) conotoxin has an IC₅₀, of 2.7 × 10^?7 M in this assay. Results from this assay show that total loss of paralytic activity occurs when Pro is replaced by Gly, Tyr by D-Tyr, or Gly by D-Phe. In most cases loss or change in length of one of the disulfide rings eliminates paralytic activity except with compound 17, which is weakly active, IC₅₀= 7.0 × 10^?5 M. Replacement of the Cys¹-Cys⁶ disulfide bond with an amide bond (compound 9) greatly lowers paralytic activity, IC₅, = 3.7 × 10^?5 M.     

Differential contribution of the conserved tyrosine residues to activity and structural stability of Ophiophagus hannahα‐neurotoxins

Abstract: Two α‐neurotoxins, Oh‐4 and Oh‐7, from king cobra (Ophiophagus hannah) venom were subjected to Tyr modification with tetranitromethane. Selective nitration of Tyr⁴ in Oh‐4 caused a slight decrease in lethal toxicity of 11% and a decrease in nicotinic acetylcholine receptor (nAchR)‐binding activity of 28%, whereas nitration of Tyr⁴ in Oh‐7 resulted in an ≈ 60% decrease in lethality and nAchR‐binding activity. When the Tyr²³ in Oh‐4 or Tyr²² in Oh‐7 appears to be ‘buried’ in the toxin following further modification, the toxins lost their biological activity and conformational change concurrently. Nevertheless, the dinitrated Oh‐4 retained a β‐sheet structure as revealed by CD spectra and exhibited a precipitin reaction with anti‐Oh‐4 sera. These results indicate that both Tyr⁴ and Tyr²² play a crucial role in the neurotoxicity of Oh‐7, whereas intact Tyr²³ is involved in the manifestation of the toxicity of Oh‐4 to a greater extent. In contrast to Oh‐4, the conformational stability of Oh‐7 depends heavily upon the integrity of Tyr²².     

A novel synthesis of 6′′‐[18F]‐fluoromaltotriose as a PET tracer for imaging bacterial infection

The aim of this study was to develop a positron emission tomography (PET) tracer to visualize and monitor therapeutic response to bacterial infections. In our continued efforts to find maltose based PET tracers that can image bacterial infections, we have designed and prepared 6′′‐[¹⁸F]fluoromaltotriose as a second generation PET imaging tracer targeting the maltodextrin transporter of bacteria. We have developed methods to synthesize 6′′‐deoxy‐6′′‐[¹⁸F]fluoro‐α‐D‐glucopyranosyl‐(1‐4)‐O‐α‐D‐glucopyranosyl‐(1‐4)‐O‐D‐glucopyranose (6′′‐[¹⁸F]‐fluoromaltotriose) as a bacterial infection PET imaging agent. 6′′‐[¹⁸F]fluoromaltotriose was prepared from precursor, 2′′,3′′,4′′‐tri‐O‐acetyl‐6′′‐O‐nosyl‐α‐D‐glucopyranosyl‐(1‐4)‐O‐2′,3′,6′‐tri‐O‐acetyl‐α‐D‐glucopyranosyl‐(1‐4)‐1,2,3,6‐tetra‐O‐acetyl‐D‐glucopyranose (per‐O‐acetyl‐6′′‐O‐nosyl‐maltotriose 4 ). This method utilizes the reaction between precursor 4 and anhydrous [¹⁸F]KF/Kryptofix 2.2.2 in dimethylformamide (DMF) at 85°C for 10 minutes to yield per‐O‐acetyl‐6′′‐deoxy‐6‐′′ [¹⁸F]‐fluoromaltotriose ( 7) . Successive acidic and basic hydrolysis of the acetyl protecting groups in 7 produced 6′′‐[¹⁸F]fluoromaltotriose ( 8 ). Also, cold 6′′‐ [¹⁹F]fluoromaltotriose was prepared from per‐O‐acetyl‐6′′‐hydroxymaltotriose via a diethylaminosulfur trifluoride reaction followed by a basic hydrolysis. A successful synthesis of 6′′‐[¹⁸F]‐fluoromaltotriose has been accomplished in 8 ± 1.2% radiochemical yield (decay corrected). Total synthesis time was 120 minutes. Serum stability of 6′′‐[¹⁸F]fluoromaltotriose at 37°C indicated that 6′′‐[¹⁸F]‐fluoromaltotriose remained intact up to 2 hours. In conclusion, we have successfully synthesized 6′′‐[¹⁸F]‐fluoromaltotriose via direct fluorination of an appropriate precursor of a protected maltotriose.     

From pro defensins to defensins: synthesis and characterization of human neutrophil pro α‐defensin‐1 and its mature domain

Abstract: Human neutrophil α‐defensins (HNPs) are small, cationic, Cys‐rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre‐proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro‐peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45‐residue pro‐peptide and the C‐terminal functional domain. Here we described, total chemical synthesis of the 75‐residue human neutrophil pro α‐defensin‐1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met⁴⁵–Ala⁴⁶ peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys¹–Cys⁶, Cys²–Cys⁴ and Cys³–Cys⁵, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro‐peptide binds to HNP1 intermolecularly with an apparent K_d value of 6.2 μm at pH 7.4, confirming the mode of intramolecular inactivation of human α‐defensin precursors.     

Radiosynthesis and in vitro evaluation of 1‐(tetrahydro‐5‐hydroxy‐6‐(hydroxymethyl)‐2H‐pyran‐3‐yl)‐5‐[125I]iodouracil: A new potential agent for HSV1‐tk reporter gene monitoring

Synthesis, radiolabelling, and in vitro evaluation of a new ¹²⁵I‐labelled iodouracil hexitol nucleoside analogue are reported. The target compound was successfully synthesized by an iodination–destannylation method and then purified by reverse phase HPLC. The radiochemical purity of the product was >99% with decay‐corrected yields of 48±3%. In vitro cellular uptake testing was carried out using MCA and MCA‐tk cell lines for comparison of compound 1 with [¹⁸F]FHBG. The newly synthesized compound 1 showed higher accumulation in herpex simplex virus type 1 thymidine kinase (HSV1‐tk) gene expression cell line (MCA‐tk cell line) than in the wild type MCA cell line compared with [¹⁸F]FHBG. The MCA‐tk to MCA cellular uptake ratio for compound 1 was higher than that of [¹⁸F]FHBG from 2 h after incubation. The radioiodine‐labelled compound 1 (I‐125, t_1/2=59.37 days) has a longer physical half‐life than F‐18‐(t_1/2=110 min) labelled FHBG. Radioiodine‐labelled compound 1 could be used for monitoring gene expression for a long time. The selectivity for MCA‐tk cell line makes compound 1 a promising imaging agent for HSV1‐tk expression. Copyright © 2010 John Wiley & Sons, Ltd.     

Controlled syntheses of natural and disulfide-mispaired regioisomers ofα-conotoxin SI*

Abstract: Methods are reported for the unambiguous syntheses of all three possible disulfide regioisomers with the sequence of α-conotoxin SI, a tridecapeptide amide from marine cone snail venom that binds selectively to the muscle subtype of nicotinic acetylcholine receptors. The naturally occurring peptide has two ‘interlocking’ disulfide bridges connecting Cys²–Cys⁷ and Cys³–Cys¹³ (2/7&3/13), while in the two mispaired isomers the disulfide bridges connect Cys²–Cys¹³ and Cys³–Cys⁷ (2/13 & 3/7, ‘nested’) and Cys²–Cys³ and Cys⁷–Cys¹³ (2/3 & 7/13, ‘discrete’), respectively. Alignment of disulfide bridges was controlled at the level of orthogonal protection schemes for the linear precursors, assembled by Fmoc solid-phase peptide synthesis on acidolyzable tris(alkoxy)benzylamide (PAL) supports. Side-chain protection of cysteine was provided by suitable pairwise combination of the S-9H-xanthen-9-yl (Xan) and S-acetamidomethyl (Acm) protecting groups. The first disulfide bridge was formed from the corresponding bis(thiol) precursor obtained by selective deprotection of S-Xan, and the second disulfide bridge was formed by orthogonal co-oxidation ofS-Acm groups on the remaining two Cys residues. It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice versa). The highest overall yields were obtained when both disulfides were formed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overall yields and poorer selectivities towards the desired isomers.     

The Biological Consequences of Replacing d‐Ala in Biphalin with Amphiphilic α‐Alkylserines

Biphalin, a synthetic opioid peptide with a broad affinity for all opioid receptors (δ, μ, and κ) and high antinociceptive activity, has been under extensive study as a potential analgesic drug. This study presents the synthesis and biological properties of four new analogues of biphalin containing amphiphilic α‐alkylserines in position 2 and 2′. The incorporation of bulky α,α‐disubstituted amino acids in the peptide chain using standard peptide chemistry is often unsuccessful. We synthesized depsipeptides, and then, the desired peptides were obtained by internal O,N‐migration of the acyl residue from the hydroxyl to the amino group under mild basic conditions. The potency and selectivity of the new analogues were evaluated by a competitive receptor‐binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). Their binding affinity is strongly dependent on the chirality of α‐alkylserine, as analogues containing (R)‐α‐alkylserines displayed higher μ receptor affinity and selectivity than those incorporating the (S)‐isomers.     

Immunological Response to (1,4)‐α‐d‐Glucan in the Lung and Spleen of Endotoxin‐Stimulated Juvenile Rats

Abstract: We investigated the effects of (1,4)‐α‐D‐glucan (α‐DG), a novel immune stimulatory drug from Tinospora cordifolia, on the concentration of pro‐ and anti‐inflammatory cytokines (interleukin [IL]‐1β, IL‐6, tumour necrosis factor‐α [TNF‐α], γ‐interferon [IFN‐γ] and IL‐10) in the lung and spleen of endotoxin‐stimulated juvenile rats. Experimental groups (n = 16/group) included controls with an intraperitoneal injection of saline, endotoxaemic rats with a non‐lethal dose of 10 mg/kg Escherichia coli endotoxin, and endotoxaemic rats treated with two doses of 10 mg/kg α‐DG, intraperitoneally, 2 and 4 hr after endotoxin injection. At 24 hr of treatment, rats were euthanized and lungs and spleen were removed for cytokines determination and lung injury. Endotoxaemia increased IL‐1β concentration by fivefold in both organs, while creating a moderate pulmonary hypercellularity (demonstrated by about 11% increase in the alveolar‐septal thickening and 11% decrease in the alveolar‐interstitial space ratio). In the lung, α‐DG treatment reduced concentrations of IL‐1β by 30% (p > 0.05), IL‐6 by 43% (p < 0.01), IFN‐γ by 46% (p < 0.01) and the anti‐inflammatory cytokine, IL‐10, by 31% (p > 0.05) compared to endotoxaemia. In the spleen, α‐DG treatment decreased the ratio of IL‐1β to IL‐10 by 55% (p < 0.05), demonstrating an anti‐inflammatory trend. These data suggest that α‐DG differentially modulates cytokine response in the lung and spleen and modifies the pro‐ and anti‐inflammatory balance during an early period of endotoxaemia in juvenile rats.     

Methylation of α‐Amino Acids and Derivatives Using Trimethylsilyldiazomethane

A study of the methylation of N‐nosyl‐α‐amino acids and derivatives with trimethylsilyldiazomethane is here reported. Trimethylsilyldiazomethane allows the chemo‐specific methylation of the carboxyl function of N‐nosyl‐α‐amino acids in high yields and purity. This method provides a practical route to N‐methyl‐α‐amino acids avoiding the use of the more toxic and explosive diazomethane. This simple and safe methylation methodology of α‐amino acids and derivatives is not limited to organic synthesis and involves the use of a commercially available reagent as well.     

Synthesis,biological activity,and solution structures of a cyclic dodecapeptide from the EGF‐2 domain of blood coagulation factor VII

Abstract: The cyclic dodecapeptide, disulfide‐cyclo‐[H‐Cys‐Val‐Asn‐Glu‐Asn‐Gly‐Gly‐Cys(Acm)‐Glu‐Gln‐Tyr‐Cys‐OH], which corresponds to the 91–102 sequence of the second epidermal growth factor domain of human blood coagulation factor VII, was synthesized using solid‐phase procedures. It was shown to be an inhibitor at the key step in the induction of coagulation by the extrinsic pathway, i.e. the factor VII/tissue factor‐catalyzed activation of coagulation factor X. The solution structure of this peptide was investigated by NMR spectroscopy and was computer‐modeled via molecular mechanics. Structures were calculated based on 112 distance and nine dihedral angle constraints. The resulting backbone structures were classified into two structural subsets: one which exhibited a twisted ‘8’‐shaped folding and another describing an open, circular ‘O’ outline. The local backbone structures of segments Asn³‐Glu⁴‐Asn⁵, Gly⁷‐Cys⁸ and Gln¹⁰‐Tyr¹¹ were well preserved among the two subsets. Apart from the unrestrained N‐ and C‐termini, Gly⁶ and Glu⁹ sites exhibited marked local disorder between the two subsets, suggesting localized flexible hinges likely to govern tertiary structure interconversion between the two subsets. Two transient hydrogen bonds were identified from pH chemical shift titrations by matching the pK_a values of NH and carboxylate groups, which supported the occurrence of the ‘8’ structure, and agreed with temperature coefficients of peptidyl NH resonances. Structure?function relationships of the peptide were discussed in terms of the likely physiological function of the disulfide‐bonded loop in factor VII which the peptide represents.     

Synthesis of [13C2, 15N]‐1,3‐2H‐1‐benzyl‐(Z)‐3‐(benzylidene)indolin‐2‐one

Parkinson disease (PD) is a neurodegenerative disorder characterized by the accumulation of α‐synuclein into Lewy bodies. 3‐Benzylidine‐indolin‐2‐one represents a class of compounds, which are known to inhibit the accumulation of α‐synuclein. In this paper, we report the synthesis of [¹³C] and [¹⁵N] labelled 1‐benzyl‐(Z)‐3‐(benzylidene)indolin‐2‐one from commercially available [¹³C₂]‐chloroacetic acid and [¹⁵N]‐aniline in five steps. The product will be used to study its metabolites in human liver microsomes by liquid chromatography‐tandem mass spectrometry.     

Unravelling the cardiovascular effects induced by α‐terpineol: A role for the nitric oxide–cGMP pathway

1. α‐Terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by α‐terpineol in rats. 2. In normotensive rats, administration of α‐terpineol (1, 5, 10, 20 and 30 mg/kg, i.v.) produced a dose‐dependent hypotension (?10 ± 3, ?20 ± 8, ?39 ± 16, ?52 ± 21 and ?57 ± 23 mmHg, respectively; n = 5) followed by tachycardia. The hypotensive responses to 1, 5, 10, 20 and 30 mg/kg, i.v., α‐terpineol were significantly attenuated following the administration of N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME; 20 mg/kg, i.v.; ?2 ± 1, ?5 ± 2, ?7 ± 3, ?22 ± 9 and ?22 ± 10 mmHg, respectively; P < 0.05; n = 5). 3. In 10 μmol/L phenylephrine (PE)‐precontracted mesenteric artery rings, α‐terpineol (10^?12 to 10^?5 mol/L) caused a concentration‐dependent relaxation (maximum relaxation 61 ± 6%; n = 7). After removal of the endothelium, the vasorelaxation elicited by α‐terpineol was attenuated (maximum relaxation 20 ± 1%; P < 0.05; n = 7). In addition, vasorelaxation induced by α‐terpineol in rings pretreated with 100 or 300 μmol/L l ‐NAME, 30 μmol/L hydroxocobalamin or 10 μmol/L 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one was attenuated (maximum relaxation 18 ± 3, 23 ± 3, 24 ± 7 and 21 ± 1%, respectively; n = 6; P < 0.05). 4. Furthermore, in a rabbit aortic endothelial cell line, 10^?6, 10^?5 and 10^?4 mol/L α‐terpineol induced concentration‐dependent increases in nitric oxide (NO) levels (12 ± 6, 18 ± 9 and 34 ± 12%Δ fluorescence, respectively; n = 3). 5. In conclusion, using combined functional and biochemical approaches in the present study, we were able to demonstrate that α‐terpineol‐induced hypotension and vasorelaxation are mediated, at least in part, by the endothelium, most likely via NO release and activation of the NO–cGMP pathway.     

Chemical synthesis and receptor binding of catfish somatostatin: a disulfide‐bridged β‐d‐Galp‐(1→3)‐α‐d‐GalpNAc O‐glycopeptide*

Abstract: The glycopeptide hormone catfish somatostatin (somatostatin‐22) has the amino acid sequence H‐Asp‐Asn‐Thr‐Val‐Thr‐Ser‐Lys‐Pro‐Leu‐Asn‐Cys‐Met‐Asn‐Tyr‐Phe‐Trp‐Lys‐Ser‐Arg‐Thr‐Ala‐Cys‐OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains d ‐GalNAc and d ‐Gal O‐glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc‐Cys(Trt)‐PAC‐PEG‐PS support, using stepwise Fmoc solid‐phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin‐22 were accessed by incorporating as building blocks, respectively, N^α‐Fmoc‐Thr(Ac₃‐α‐D‐GalNAc)‐OH and N^α‐Fmoc‐Thr(Ac₄‐β‐D‐Gal‐(1→3)‐Ac₂‐α‐D‐GalNAc)‐OH. Acidolytic deprotection/cleavage of these peptidyl‐resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl‐protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N^α‐dithiasuccinoyl (Dts)‐glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2‐fold tighter binding.     

Third‐generation dihydropyridine‐type calcium channel blocker labedipinedilol‐B displays α/β‐adrenoceptor blocking activities

The pharmacological properties of labedipinedilol‐B {N‐[4‐[2‐hydroxy‐3‐(2‐methoxy‐1‐oxyethylaminobenzene) propoxy]‐benzyl]‐2,6‐dimethyl‐3,5‐dicarbomethoxy‐1,4‐dihydropyridine} were investigated in vivo and in vitro in comparison with labedipinedilol‐A. Intravenous labedipinedilol‐B (0.5, 1.0, and 3.0 mg kg^–1), produced dose‐dependent hypotensive and bradycardia responses in pentobarbital‐anesthetized Wistar rats. Pretreatment with labedipinedilol‐B (1.0 mg kg^–1, iv) also inhibited phenylephrine (10 μg kg^–1)‐induced hypertensive and (–)isoproterenol (0.5 μg kg^–1)‐induced tachycardia effects. In the isolated Wistar rat right and left atria and guinea pigs tracheal strips experiments, labedipinedilol‐B (10^–7, 10^–6, and 10^–5 M) competitively antagonized the (–)isoproterenol‐induced positive chronotropic and inotropic effects and tracheal relaxation responses in a concentration‐dependent manner. The parallel shift to the right of the concentration–response curve of (–)isoproterenol suggested that labedipinedilol‐B was a β₁/β₂‐adrenoceptor competitive antagonist. Labedipinedilol‐B (10^–7, 10^–6, and 10^–5 M) also prevented the rate‐increasing effects of increased extracellular Ca²⁺ (3.0–9.0 mM) in a concentration‐dependent manner. In the isolated rat aorta, labedipinedilol‐B (10^–7, 10^–6, and 10^–5 M) competitively antagonized the CaCl₂ and norepinephrine‐induced contractions with pKCa^–1 and pA₂ values of 8.02 ± 0.04 and 7.55 ± 0.05 in a concentration‐dependent manner. The parallel shift to the right of the concentration–response curves of norepinephrine suggested that labedipinedilol‐B was an α‐adrenoceptor competitive antagonist. Furthermore, labedipinedilol‐B, in an equal antagonist activity, inhibited norepinephrine‐induced phasic and tonic contraction. In the isolated rat aorta, labedipinedilol‐B also competitively antagonized CaCl₂‐induced contractions and made the parallel shift to the right of the concentration–response curve of CaCl₂. In cultured blood vessel smooth muscle cells (A7r5 cell lines), Bay K 8644‐induced intracellular calcium changes were decreased after application of labedipinedilol‐B, suggesting that the compound was a calcium channel blocker. The binding characteristics of labedipinedilol‐B were evaluated in [³H]CGP‐12177 binding to ventricle and lung and [³H]prazosin binding to brain membranes in rats. Labedipinedilol‐B also was evaluated in [³H]nitrendipine binding to brain membranes in rats. These results indicated that labedipinedilol‐B, similar to labedipinedilol‐A, has α‐adrenoceptor blocking, β‐adrenoceptor blocking, and calcium entry blocking activities in a single compound. We suggest that these two compounds represent a new generation of 1,4‐dihydropyridine‐type calcium channel blockers. Drug Dev. Res. 52:462–474, 2001. © 2001 Wiley‐Liss, Inc.     

Preparation and a simple one‐step purification of [His1‐mono‐125I‐Tyr10,Nle27]‐hGHRH(1‐32)‐NH2

A one‐step purification of [His¹‐mono‐¹²⁵I‐Tyr¹⁰,Nle²⁷]‐hGHRH(1‐32)‐NH₂, prepared using chloramine‐T, by HPLC with isocratic elution is described. The labeled GHRH analog was suitable for GHRH receptor binding assays. Copyright © 2002 John Wiley & Sons, Ltd.