Structures of neuropeptide γ from goldfish and mammalian neuropeptide γ, as determined by 1H NMR spectroscopy

Abstract: Neuropeptide γ belongs to tachykinin families which have a common C‐terminal amino acid sequence (Phe‐X‐Leu‐Met‐NH₂) and which induce various biological responses including salivation, hypotension, and contraction of gastrointestinal, respiratory, and urinary smooth muscle. In the present study, we present the solution structures of neuropeptide γ (NPγ) from gold fish (G‐NPγ) and mammalian NPγ (M‐NPγ), as determined by nuclear magnetic resonance (NMR) spectroscopy in 50% trifluoroethanol (TFE)/water (1 : 1, v/v) solution and 200 mm sodium dodecyl sulfate (SDS) micelles. In aqueous TFE solution, G‐NPγ has a α‐helical conformation in the region of His¹²–Met²¹ and a short helix in the N‐terminal region, and has a β‐turn from Arg⁹ to Arg¹¹ in between. In aqueous TFE solution, M‐NPγ also has α‐helical conformations both in the C‐terminal region and the N‐terminal region and a β‐turn from His⁹ to Arg¹¹ in between. In SDS micelle, the structure of G‐NPγ contains a stable α‐helix from His¹² to Met²¹ and a β‐turn from Arg⁹ to Arg¹¹, while M‐NPγ has a short helix from Ser¹⁶ to Met²¹. The region from His¹² to Met²¹ corresponds to the amino acid sequence of neurokinin A. Neuropeptide γ may act as a precursor of neurokinin A and the post‐translational processing of this peptide involves the enzymatic attack of the basic β‐turn region from residue 9 to residue 11 in the middle. From our relaxation study, it could be suggested that in fish system G‐NPγ induces the biological actions corresponding to those of substance P in mammalian system. The structures of G‐NPγ and M‐NPγ contain α‐helical structures at the C‐terminus and this helix seems to promote the affinity for NK₁ and/or NK₂ receptor.     

Conformational properties of hybrid peptides containing α‐ and ω‐amino acids*

Abstract: This review briefly surveys the conformational properties of guest ω‐amino acid residues when incorporated into host α‐peptide sequences. The results presented focus primarily on the use of β‐ and γ‐residues in αω sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between α‐peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and β‐hairpin conformations permits the characterization of backbone conformational parameters for β‐ and γ‐residues inserted into regular α‐polypeptide structures. Substituted β‐ and γ‐residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral β,β‐disubstituted γ‐amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C^β–C^γ (θ₁) and C^α–C^β (θ₂) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.     

Functional activation of Gαq/11 protein via α1‐adrenoceptor in rat cerebral cortical membranes

Although it is recognized that α₁‐adrenoceptors are coupled to diverse intracellular signalling pathways, its primary transduction mechanisms are evoked by activating phospholipase C in the cell membrane through Gα_q/11, resulting in production of inositol 1,4,5‐trisphosphate and diacylglycerol. However, there have been few studies that indicate directly the involvement of Gα_q/11 proteins in this signalling pathway in the central nervous system. In the current study, we tried to pharmacologically characterize (?)‐adrenaline‐stimulated [³⁵S]GTPγS binding to Gα_q/11 in rat brain membranes. Functional activation of Gα_q/11 coupled to α₁‐adrenoceptor was investigated by using [³⁵S]GTPγS binding/immunoprecipitation assay in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. The specific [³⁵S]GTPγS binding to Gα_q/11 was stimulated by (?)‐adrenaline in a concentration‐dependent and saturable manner in rat cerebral cortical membranes. In hippocampal or striatal membranes, the stimulatory effects of (?)‐adrenaline were scarce. The effect of (?)‐adrenaline was potently inhibited by prazosin, a potent and selective α₁‐adrenoceptor antagonist, but not by yohimbine, a selective α₂‐adrenoceptor antagonist. The response was mimicked by cirazoline, but not by R(?)‐phenylephrine. Although oxymetazoline also stimulated the specific [³⁵S]GTPγS binding to Gα_q/11 as an apparent “super‐agonist”, detailed pharmacological characterization revealed that its agonistic properties in this experimental system were derived from off‐target effects on 5‐HT_2A receptors, but not via α₁‐adrenoceptors. In conclusion, functional coupling of α₁‐adrenoceptors to Gα_q/11 proteins are detectable in rat brain membranes by means of [³⁵S]GTPγS binding/immunoprecipitation assay. It is necessary to interpret the experimental data with caution when oxymetazoline is included as an agonist at α₁‐adrenoceptors.     

Synthesis of an M + 7 stable isotope of γ‐cyhalothrin

γ‐Cyhalothrin is a single isomer, synthetic pyrethroid insecticide. This material was originally developed and marketed by Pytech Chemicals, a joint venture between Dow AgroSciences and Cheminova A/S. Cheminova A/S now wholly owns Pytech Chemical. As a part of registration studies, there was a need for a stable isotope of γ‐cyhalothrin to serve as an internal standard. This paper will discuss the 11‐step synthesis that was used to prepare an M + 7 stable isotope of γ‐cyhalothrin by utilizing triethyl phosphonoacetate‐d₂ and acetone‐d₆ to incorporate deuterium into the molecule. In the end, the diastereomers were separated by preparative reverse‐phase HPLC to give γ‐cyhalothrin‐d₇ in an overall yield of 6%. Copyright © 2012 John Wiley & Sons, Ltd.     

The effect of a side chain‐backbone swap on protein stability

Abstract: To assess the relative importance of backbone hydrogen bonding (H‐bonding) vs. side chain hydrophobicity in protein structural formation, a method called side chain‐backbone swap is proposed. Such a method swaps the side chain and backbone portions of certain amino acid residues, such as Asp, Glu, Asn, Gln, Lys, and Arg. Such a swap retains the sequence of a polypeptide and preserves the identity of the backbone linkage. On the other hand, the swap disrupts backbone H‐bonding geometry because of the introduction of extra methylene groups into the peptide backbone. In this project, we chose the two‐stranded α‐helical coiled‐coil to implement side chain‐backbone swap. A pair of 36‐residue peptides was designed. The two peptides have identical sequence with four residues in each heptad repeat occupied by glutamyl residues. Each glutamic acid was incorporated either as α‐glutamyl residue (the peptide is denoted as α‐Glu‐36) or as γ‐glutamyl residue (the peptide is denoted as γ‐Glu‐36). The inter‐conversion between the two peptides constitutes a side chain‐backbone swap. Residues constituting the hydrophobic core of the coiled‐coil, however, are left unchanged. The peptide pair was characterized by circular dichroism spectroscopy, reversed‐phase liquid chromatography (RPLC), and two‐dimensional nuclear magnetic resonance (NMR). The results indicate that α‐Glu‐36 is a two‐stranded α‐helical coiled‐coil while γ‐Glu‐36 lacks stable structural elements. It is concluded that, at least for coiled‐coils where hydrophobic interactions are predominantly long‐range, local backbone H‐bonding is a required for structural formation, consistent with a hierarchic folding mechanism. The methodological implication of side chain‐backbone swap is also discussed.     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

Suppression of tumour necrosis factor‐α by Schizonepeta tenuifolia water extract via inhibition of IκBα degradation and Jun N‐terminal kinase/stress‐activated protein kinase activation

Objectives The anti‐inflammatory effects of an aqueous extract of Schizonepeta tenuifolia on lipopolysaccharide (LPS)‐induced tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) in vivo and in vitro have been investigated. Methods C57BL/6 mice were orally administered phosphate‐buffered saline (control) or S. tenuifolia water extract (50, 200, 500 or 1000 mg/kg) for 10 days before intraperitoneal administration of LPS (1.3 mg/kg). Blood samples were obtained 1 h after LPS challenge, followed by determination of TNF‐α and IL‐6 levels. Peritoneal macrophages from thioglycollate‐injected mice were obtained and stimulated with LPS and S. tenuifolia water extract for viability assay, cytokine analysis, real‐time RT PCR and Western blotting. Key findings Oral administration of S. tenuifolia water extract to mice significantly reduced LPS‐induced serum levels of TNF‐α, but not IL‐6. When peritoneal macrophages were treated in vitro with S. tenuifolia water extract, the inhibition of LPS‐induced TNF‐α was more pronounced than that of IL‐6 at the level of secreted protein and mRNA. S. tenuifolia water extract reduced the degradation of IκBα and the nuclear relocation of p65 NF‐κB, but the phosphorylation of IκBα was not affected. Inhibition of c‐Jun N‐terminal kinase/stress‐activated protein kinase (JNK/SAPK) by S. tenuifolia water extract led secondarily to the inhibition of phospho‐c‐Jun and phospho‐ATF‐2. Conclusions These results indicated that the downregulation of TNF‐α by S. tenuifolia water extract may have involved the inhibition of both IκBα degradation and activation of c‐Jun and ATF‐2 involving suppression of JNK/SAPK.     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

Bioactive N‐terminal undecapeptides derived from parathyroid hormone: the role of α‐helicity*

Abstract: The N‐terminal 1–34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N‐terminal fragments presenting the principal activating domain such as PTH(1–11) and PTH(1–14) with helicity‐enhancing substitutions yield potent analogues with PTH(1–34)‐like activity. To further investigate the role of α‐helicity on biological potency, we designed and synthesized by solid‐phase methodology the following hPTH(1–11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix‐promoting C^α‐tetrasubstituted α‐amino acids α‐amino isobutyric acid (Aib), 1‐aminocyclopentane‐1‐carboxylic acid (Ac₅c) and 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c): Ac₅c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( I ); Aib‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( II ); Ac₆c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( III ); Aib‐V‐Ac₆c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( IV ); Aib‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( V ); S‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VI ), S‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VII ); Ac₅c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VIII ); Ac₆c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( IX ); Ac₅c‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( X ); Ac₆c‐V‐Ac₆c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( XI ). All analogues were biologically evaluated and conformationally characterized in 2,2,2‐trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I – V , which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand‐stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III , VI and VII are very weakly active and analogues IV , V , VIII–XI are inactive. The most potent analogue, I exhibits biological activity 3500‐fold higher than that of the native PTH(1–11) and only 15‐fold weaker than that of the native sequence hPTH(1–34). Remarkably, the two most potent analogues, I and II , and the very weakly active analogues, VI and VII , exhibit similar helix contents. These results indicate that the presence of a stable N‐terminal helical sequence is an important but not sufficient condition for biological activity.     

A Meccano set approach of joining trpzip a water soluble β‐hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide

Abstract: A 16 residues long, water soluble, monomeric β‐hairpin peptide ‘trpzip’ (Cochran et al. Proc. Natl. Acad. Sci. U.S.A., 98 (2001), 5578), stabilized by tryptophan zipper has been linked via a tetraglycyl linker to a hydrophobic didehydrophenylalnine (ΔF) containing helical octapeptide. Circular dichroism studies of this 28 residues long peptide, ‘trpzipalpha’ (Ac‐GEWTWDDATKTWTWTE‐GGGG‐ΔFALΔFALΔFA‐NH₂) in water have revealed the presence of both the β‐hairpin and the helical conformations. This is the first instance where a ΔF containing peptide has been found to display a helical fold in water. The fluorescence emission wavelengths of tryptophan in Ac‐G‐W‐G‐NH₂, trpzip and trpzipalpha were 341.5, 332.8 and 332.6 nm, respectively. The fluorescence quantum yield of trpzip was 2.6‐fold higher than trpzipalpha suggesting that proximal interactions between the β‐hairpin and the helix caused the quenching of tryptophan fluorescence in the former by the ΔFs in the latter. The molar ellipticity of the far UV couplet characteristic of trpzip was reduced in trpzipalpha and the CD based thermal melting temperatures at 228 nm were 62 °C (trpzip) and 57 °C (trpzipalpha). A concentration‐dependent variable temperature CD study in water showed that in trpzipalpha, increasing temperature is detrimental to the β‐hairpin, but it augments the helical motif, perhaps by intermolecular oligomerization. Our results show that in water, trpzipalpha exhibits long‐range interactions between two different secondary structures. In contrast to trpzip, trpzipalpha has shown a greater tendency to oligomerize in water.     

Synthesis,conformation, receptor binding and biological activities of monobiotinylated human insulin‐like peptide 3*

Abstract: Biotin‐avidin immobilization has been routinely used as a tool to study peptide–receptor and peptide–antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides’ receptor, and to analyse ligand‐receptor binding. Insulin‐like peptide 3 (INSL3) is a peptide hormone which contains A‐ and B‐chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein‐coupled receptor, we chemically synthesized N^α‐mono‐biotinylated human INSL3 (B‐hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with ³³P‐labelled relaxin H2 (B₃₃). The modified B‐hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B‐hINSL3 contains a higher α‐helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N‐terminal region of the A‐chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N‐terminus of the A‐chain promoted conformational stability which, in turn, permitted better receptor activation.     

Cerenkov luminescence imaging of αvβ6 integrin expressing tumors using 90Y‐labeled peptides

Cerenkov luminescence imaging (CLI) is an emerging preclinical molecular imaging modality that tracks the radiation emitted in the visible spectrum by fast moving charged decay products of radionuclides. The aim of this study was in vitro and in vivo evaluation of the two radiotracers, ⁹⁰Y‐DOTA‐PEG₂₈‐A20FMDV2 (⁹⁰Y‐1) and ⁹⁰Y‐DOTA‐Ahx‐A20FMDV2 (⁹⁰Y‐2) (>99% radiochemical purity, 3.7 GBq/µmol specific activity) for noninvasive assessment of tumors expressing the integrin α_vβ₆ and their future use in tumor targeted radiotherapy. Cell binding and internalization in α_vβ₆‐positive cells was ⁹⁰Y‐1: 10.1 ± 0.8%, 50.3 ± 2.1%; ⁹⁰Y‐2: 22.4 ± 1.7%, 44.7 ± 1.5% with <5% binding to α_vβ₆‐negative control cells. Biodistribution studies showed maximum α_vβ₆‐positive tumor uptake of the radiotracers at 1‐h post injection (p.i.) (⁹⁰Y‐1: 0.64 ± 0.15% ID/g; ⁹⁰Y‐2: 0.34 ± 0.11% ID/g) with high renal uptake (>25% ID/g at 24 h). Because of the lower tumor uptake and high radioactivity accumulation in kidneys (that could not be reduced by pre‐administration of either lysine or furosemide), the luminescence signal from the α_vβ₆‐positive tumor was not clearly detectable in CLI images. The studies suggest that CLI is useful for indicating major organ uptake for both radiotracers; however, it reaches its limitation when there is low signal‐to‐noise ratio.     

Synthesis of deoxyadenosine‐5′‐(35S)‐thiomonophosphate [dAMP(35S)] of high specific activity – a key intermediate for the synthesis of Sp‐deoxyadenosine‐5′‐(alpha;‐35S)‐thiotriphosphate [Sp‐dATP (α‐35S)]

Unprotected deoxyadenosine 1 was treated with an excess of phosphorus acid and stoichiometric proportions of N, N′‐di‐p‐tolylcarbodiimide in anhydrous pyridine to give deoxyadenosine‐5′‐monophosphite 2 . The latter was activated with trimethylsilyl chloride followed by sulphurisation with elemental ³⁵S (specific activity>1000 Ci/mmol) in toluene solution to give deoxyadenosine‐5′‐(³⁵S)‐thiomonophosphate [dAMP(³⁵S)] 3 . Enzymatic conversion of deoxyadenosine‐5′‐(³⁵S)‐thiomonophosphate to Sp‐deoxyadenosine‐5′‐(α‐³⁵S)‐thiotriphosphate [Sp‐dATP (α‐³⁵S)] 5 was carried out following a standard reaction protocol. Copyright © 2001 John Wiley & Sons, Ltd.     

The Anti‐tumor Effects of Androstene Steroids Exhibit a Strict Structure–Activity Relationship Dependent upon the Orientation of the Hydroxyl Group on Carbon‐17

Androstene steroids are metabolites of dehydroepiandrosterone and exist as androstene‐diols or ‐triols in α‐ and β‐epimeric forms based upon the placement of the hydroxyl groups relative to the plane of the Δ⁵cycloperhydrophenanthrene ring. 5‐Androstene‐3β,17β‐diol (3β,17β‐AED) functions to upregulate immunity and the addition of a third hydroxyl group at C‐7 in the α‐ or β‐orientation (3β,7α,17β‐AET and 3β,7β,17β‐AET, respectively) enhances the immunological activity of the molecule. In contrast, 5‐androstene‐3β,17α‐diol (3β,17α‐AED) possesses potent anti‐tumor activity. We synthesized a new androstene by adding a third hydroxyl group at C‐7 to make 5‐androstene‐3β,7α,17α‐triol (3β,7α,17α‐AET) and compared the anti‐tumor activity of this steroid to the four existing androstenes. The results showed that this modification reduced the activity of 3β,17α‐AED. The ranking of the anti‐tumor activities of these steroids and their IC50 on human glioblastoma and lymphoma cells was: 3β,17α‐AED (～10 μm ) > 3β,7α,17α‐AET (～30 μm ) >> 3β,7α,17β‐AET (～150 μm )> 3β,7β,17β‐AET (not achievable) ≥ 3β,17β‐AED (not achievable). 3β,17α‐AED and 3β,7α,17α‐AET induced autophagy in T98G glioblastoma cells and apoptosis in U937 lymphoma cells. These results indicate that the position of the hydroxyl group on C‐17 dictates the anti‐tumor activity of the androstenes and must be in the α‐configuration, demonstrating a strict structure–activity relationship.     

KMUP 880708: A vanyllilamide‐based β1‐adrenoceptor antagonist with α‐adrenoceptor blocking and potassium channel opening activities

A vanillylamide‐based propanolamine derivative, KMUP 880708, was first investigated both in vivo and in vitro. KMUP 880708 (0.1, 0.5, 1.0, and 2.0 mg kg^–1, iv) produced dose‐dependent hypotensive and bradycardia responses in pentobarbital‐anesthetized Wistar rats. KMUP 880708 (0.1, 0.5, and 1.0 mg kg^–1, iv) also markedly inhibited both the tachycardia effects induced by (–)isoproterenol and arterial pressor responses induced by phenylephrine. KMUP 880708 competitively antagonized (–)isoproterenol‐induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses on isolated guinea pig tissues. The apparent pA₂ values for KMUP 880708 was 7.82 ± 0.06 in the right atria, 7.51 ± 0.13 in the left atria, and 6.31 ± 0.07 in the trachea, respectively, indicating that KMUP 880708 was selective β₁‐adrenoceptor blocker. In thoracic aorta experiments, KMUP 880708 also produced a competitive antagonism of norepinephrine‐induced contraction with pA₂ value of 7.92 ± 0.52, indicating that KMUP 880708 was α‐adrenoceptor antagonist. In isolated rat thoracic aorta, KMUP 880708 more potently relaxed the contractions induced by phenylephrine (10^–5 M) than those by high K⁺ (75 mM). KMUP 880708‐induced relaxation was significantly reduced by endothelium removal and by exposure to L‐N^G‐nitro arginine methyl ester (L‐NAME, 1 and 3 × 10^–4 M), indomethacin (3 × 10^–5 M), methylene blue (10^–5 M) and 1H‐[1,2,4]oxadiazolol[4,3,‐a]quinoxalin‐1‐one (ODQ, 10^–6 M). The vasorelaxant effect of KMUP 880708 on phenylephrine‐induced contraction was attenuated by the pretreatment with tetraethylammonium (TEA), glibenclamide, charybdotoxin, and apamin, but not by 4‐aminopyridine (4‐AP). In addition, KMUP 880708 inhibited phenylephrine‐induced biphasic contraction and affected the fast‐twitch phase more significantly than the slow tonic phase. In the radioligand‐binding assay, the K_i values of [³H]CGP‐12177 binding to rat ventricle and lung membranes were 15.14 and 524.81 nM, respectively, and the value of [³H]prazosin binding to rat brain membrane was 3.89 nM. The ranking order of inhibition for [³H]CGP‐12177 binding on β‐adrenoceptor was propranolol > labetalol > KMUP 880708 > atenolol, and that for [³H]prazosin binding to α‐adrenoceptor was KMUP 880708 > labetalol. In conclusion, KMUP 880708 was found to be a new generation α/β‐adrenoceptor blocker with selective β₁‐adrenoceptor blocking and vascular smooth muscle relaxation activities. Furthermore, the vasodilator effect of KMUP 880708 is attributed to the release of NO or NO‐related substance from vascular endothelium. While the endothelium‐independent mechanism involved in the relaxation of KMUP 880708 is probably linked to K⁺ channel activation in these vessels. Drug Dev. Res. 55:104–117, 2002. © 2002 Wiley‐Liss, Inc.     

Search for New Anticonvulsant Compounds,Part 2. Structure-Activity Relationship Studies of New N-Substituted Amides of α-Piperazine-γ-Hydroxybutyric Acid as Active Anticonvulsants

In a search for new anticonvulsants, two series of compounds, viz. derivatives of N-benzylamides of α-(4-phenylpiperazine)-γ-hydroxybutyric acid ( A and derivatives of N-benzylamides of α-(4-benzylpiperazine)-γ-hydroxybutyric acid ( B ), were investigated. These amides were obtained by aminolysis of 3-(4-phenyl-, or 4-benzylpiperazine)-tetrahydrofuran-2-one with primary arylalkylamines (i.e. 2-phenylethylamine and 2,3,4-substituted derivatives of benzylamine). Preliminary pharmacological tests, a maximal electroshock (MES) and a subcutaneous metrazole (scMet), and a rotorod toxicity assay were employed. All compounds displayed anticonvulsant activity at range of doses 100–300 mg/kg in the MES screens. In order to point to some structural features correlating with the MES anticonvulsant activity crystal structure analysis followed by conformational analysis was carried out on two representative compounds of series A and B .     

Design,Synthesis, and Biological Evaluation of γ‐Aminopropyl Silatrane–Acyclovir Hybrids with Immunomodulatory Effects

Several derivatives of γ‐aminopropyl silatrane containing acyclovir in their molecular structure were synthesized and evaluated for their immunomodulatory and antiviral activities. The structures of all these derivatives were confirmed by mass spectra, IR, and ¹H NMR. Based on WST‐1 assay in vitro, these compounds could stimulate proliferation of splenic lymphocytes at certain concentrations. Furthermore, compound 3d could also potentiate the expression of IFN‐γ, IL‐2, CD4⁺, CD8⁺, and CD4⁺/CD8⁺ in vivo. Our results show that these derivatives possess antiviral activity against herpes simplex viruses with a similar potency to acyclovir without a cellular immune response.     

Agonist function of the recombinant α4β3δ GABAA receptor is dependent on the human and rat variants of the α4‐subunit

1. It is known that the α₄‐subunit is likely to occur in the brain predominantly in α₄β₃δ receptors at extrasynaptic sites. Recent studies have revealed that the α₁‐, α₄‐, γ₂‐ and δ‐subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA_A receptors containing human (H) and rat (R) α₁/α₄‐, β₂/β₃‐ and γ_2S/δ‐subunits in Xenopus oocytes using the two‐electrode voltage‐clamp technique. 2. Both Hα₁β₃δ and Hα₄β₃γ_2S receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α₄β₃δ combination was more sensitive to agonist action than the α₄β₃γ_2S receptor, we observed extremely small GABA‐ and pentobarbital‐activated currents at the wild‐type Hα₄β₃δ receptor. However, GABA and pentobarbital activated the wild‐type Rα₄β₃δ receptor with high potency (EC₅₀ = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively). 3. Substituting the Hα₄ subunit with Rα₄ conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC₅₀ and increased I_max. When the Hα₄ subunit was combined with the Rβ₃ and Rδ subunit in a heteropentameric form, the amplitude of GABA‐ and pentobarbital‐activated currents increased significantly compared with the wild‐type Hα₄β₃δ receptor. 4. Thus, the results indicate that the Rα₄β₃δ, Hα₁β₃δ and Hα₄β₃γ_2S combinations may contribute to functions of extrasynaptic GABA_A receptors. The presence of the Rα₄ subunit at recombinant GABA_A receptors containing the δ‐subunit is a strong determinant of agonist action. The recombinant Hα₄β₃δ receptor is a less sensitive subunit composition in terms of agonist activation.     

A topographically and conformationally constrained,spin‐labeled, α‐amino acid: crystallographic characterization in peptides*

Abstract: 2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) is a topographically and conformationally restricted, nitroxide containing, C^α‐tetrasubstituted α‐amino acid. Here, we describe the molecular and crystal structures, as determined by X‐ray diffraction analyses, of a TOAC terminally protected derivative, the cyclic dipeptide c(TOAC)₂·1,1,1,3,3,3‐hexafluoropropan‐2‐ol (HFIP) solvate, and five TOAC‐containing, terminally protected, linear peptides ranging in length from tetra‐ to hepta‐peptides. Incipient and fully developed, regular or distorted 3₁₀‐helical structures are formed by the linear peptides. A detailed discussion on the average geometry and preferred conformation for the TOAC piperidine ring is also reported. The X‐ray diffraction structure of an intramolecularly cyclized side product resulting from a C‐activated TOAC residue has also been determined.