beta 2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor

The structures of acetylcholine-binding protein ( AChBP) and nicotinic acetylcholine receptor ( nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha 3 beta 2 model to identify beta 2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha 3 beta 2 nAChR by two-electrode voltage clamp analysis. Although a beta 2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta 2-F117A, beta 2-V109A, and beta 2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta 2-F117A mutant was combined with the alpha 4 instead of the alpha 3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta 2-F117A, beta 2-V109A, and beta 2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha 3 beta 2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.

Isolation and characterization of novel cyclotides from Viola hederaceae - Solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 ( vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H-1 NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β- sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pK(a) of 3.0 for Glu(3) and ∼ 5.0 for Glu(14) to be determined. Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μ m.

Single turn peptide alpha helices with exceptional stability in water

Cyclic pentapepticles are not known to exist in a-helical conformations. CD and NMR spectra show that specific 20-membered cyclic pentapepticles, Ac-(cyclo-1,5) [KxxxD]-NH2 and Ac-(cyclo-2,6)R[KxxxD]-NH2, are highly a-helical structures in water and independent of concentration, TFE, denaturants, and proteases. These are the smallest a-helical peptides in water.

Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites ( Ile(116), Arg(175), Arg(206), Glu(199), Asp(282), and Val(286)). Agonist peptide 1 (Phe-Lys-Pro-D-cyclohexylalanine-cyclohexylalanine-D-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 ( peptide 2). Conversely, mutation of C5aR transmembrane residue Ile(116) to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394 - 3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] ( peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.

Comparison of the binding of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines with their isosteric sulfonamides to the active site of phenylethanolamine N-methyltransferase

3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (14, 16, and 18-22) are highly potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT). Molecular modeling studies with 3-fluoromethyl-7-(N-alkyl aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines, such as 16, suggested that the sulfonamide -NH-could form a hydrogen bond with the side chain of Lys57. However, SAR studies and analysis of the crystal structure of human PNMT (hPNMT) in complex with 7 indicated that the sulfonamide oxygens, and not the sulfonamide -NH-, formed favorable interactions with the enzyme. Thus, we hypothesized that replacement of the sulfonamide -NH-with a methylene group could result in compounds that would retain potency at PNMT and that would have increased lipophilicity, thus increasing the likelihood they will cross the blood brain barrier. A series of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines (23-30) were synthesized and evaluated for their PNMT inhibitory potency and affinity for the R2-adrenoceptor. A comparison of these compounds with their isosteric sulfonamides (14, 16, and 18-22) showed that the sulfones were more lipophilic but less potent than their corresponding sulfonamides. Sulfone 24 (hPNMT K-i = 1.3 mu M) is the most potent compound in this series and is quite selective for PNMT versus the R2-adrenoceptor, but 24 is less potent than the corresponding sulfonamide, 16 (hPNMT K-i = 0.13 mu M). We also report the crystal structure of hPNMT in complex with sulfonamide 15, from which a potential hydrogen bond acceptor within the hPNMT active site has been identified, the main chain carbonyl oxygen of Asn39. The interaction of this residue with the sulfonamide -NH-is likely responsible for much of the enhanced inhibitory potency of the sulfonamides versus the sulfones.

Enhancement of drug affinity for cell membranes by conjugation with lipoamino acids II. Experimental and computational evidence using biomembrane models

Lipoamino acids (LAAs) are promoieties able to enhance the amphiphilicity of drugs, facilitating their interaction with cell membranes. Experimental and computational studies were carried out on two series of lipophilic amide conjugates between a model drug (tranylcypromine, TCP) and LAA or alkanoic acids containing a short, medium or long alkyl side chain (C-4 to C-16). The effects of these compounds were evaluated by monolayer surface tension analysis and differential scanning calorimetry using dimyristoylphosphatidylcholine nnonolayers and liposomes as biomembrane models. The experimental results were related to independent calculations to determine partition coefficient and blood-brain partitioning. The comparison of TCP-LAA conjugates with the related series of TCP alkanoyl amides confirmed that the ability to interact with the biomembrane models is not due to the mere increase of lipophilicity, but mainly to the amphipatic nature and the kind of LAA residue. (C) 2005 Elsevier B.V. All rights reserved.

Modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus

A 13-residue peptide sequence from a respiratory syncitial virus fusion protein was constrained in an alpha-helical conformation by fusing two back-to-back cyclic alpha-turn mimetics. The resulting peptide, Ac-(3 -> 7; 8 -> 12)-bicyclo-FP[KDEFD][KSIRD]V-NH2, was highly alpha-helical in water by CD and NMR spectroscopy, correctly positioning crucial binding residues (F488, I491, V493) on one face of the helix and side chain-side chain linkers on a noninteracting face of the helix. This compound displayed potent activity in both a recombinant fusion assay and an RSV antiviral assay (IC50 = 36 nM) and demonstrates for the first time that back-to-back modular alpha-helix mimetics can produce functional antagonists of important protein-protein interactions.

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta 2 and gamma 1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha 1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha 1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha 1, laminins beta 2 and gamma 1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.

Structural and functional characterization of the conserved salt bridge in mammalian Paneth cell alpha-defensins - Solution structures of mouse cryptdin-4 AND (E15D)-cryptdin-4

Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell α-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.

Combinatorial solid phase synthesis of multiply substituted 1,4-benzodiazepines and affinity studies on the CCK2 receptor (Part 1)

One hundred sixty-eight multiply substituted 1,4-benzodiazepines have been prepared by a five-step solid-phase combinatorial approach using syn-phase crowns as a solid support and a hydroxymethyl-phenoxy-acetamido linkage (Wang linker). The substituents of the 1,4-benzodiazepine scaffold have been varied in the -3, -5, -7, and 8-positions and the combinatorial library was evaluated in a cholecystokinin (CCK) radioligand binding assay. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCK-B (CCK2) receptor have been optimized on the lipophilic side chain, the ketone moiety, and the stereochemistry at the 3-position. Various novel 3-alkylated compounds were synthesized and [S]3-propyl-5-phenyl-1,4-benzodiazepin-2-one, [S]NV-A, has shown a CCK-B selective binding at about 180 nM. Fifty-eight compounds of this combinatorial library were purified by preparative TLC and 25 compounds were isolated and fully characterized by TLC, IR, APCI-MS, and 1H/13C-NMR spectroscopy.

Synthesis and evaluation of asperlicin analogues as non-peptidal cholecystokinin-antagonists

The SAR of Asperlicin analogues is reported, leading to bioactive 1,4-benzodiazepine-2-ones, which were prepared in a 3 step reaction sequence. The Asperlicin substructure was built up using Tryptophan and readily available 2-amino-acetophenones. This template, containing a 1,4-benzodiazepin-2-one moiety with a 3-indolmethyl side chain, was transformed into mono- and di-substituted 3-indol-3 '-yl-methyl-1,4-benzodi-azepine-2-ones by selective alkylation and acylation reactions. The SAR optimization of the 1,4-benzodiazepine scaffold has included variations at the 5-, 7-, 8-position, at the N1, N-indole nitrogen and the configuration of the C3-position. The most active Asperlicin analogue, having an IC50 of 1.6 microM on the CCKA receptor subtype, was obtained from Tryptophan in only 3 steps in an overall yield of 48%.

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells:Studies with chimeric and other peptides

1. Structure-activity relationships for the binding of human α-calcitonin gene-related peptide 8-37 (hαCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (hαCGRP stimulation of adenylate cyclase). 2. On SK-N-MC cells the potency order was hαCGRP8-37 > hαCGRP19-37 = AC187 > rat amylin8-37 > hα[Tyr0]-CGRP28-37 (apparent pKBS of 7.49 ± 0.25, 5.89 ± 0.20, 6.18 ± 0.19, 5.85 ± 0.19 and 5.25 ± 0.07). The SK-N-MC receptor appeared CGRP1-like. 3. On Col 29 cells, only hαCGRP8-37 of the above compounds was able to antagonize the actions of hαCGRP (apparent pKB = 6.48 ± 0.28). Its receptor appeared CGRP2-like. 4. hα[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal α-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than hαCGRP8-37. 5. On SK-N-MC cells, hαCGRP(8-18, 28-37) (M433) and mastoparan-hαCGRP28-37 (M432) had apparent pKBS of 6.64 ± 0.16 and 6.42 ± 0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. 6. M433 was almost as potent as hαCGRP8-37 on Col 29 cells (apparent pKB = 6.17 ± 0.20). Other antagonists were inactive.

Hydrogels containing linear and cyclic polyethers

Hydrogels are a unique class of polymer which swell, but do not dissolve in, water. A range of 2-hydroxyethyl methacrylate based copolymer hydrogels containing both cyclic and linear polyethers have been synthesised and are described in this thesis. Initially, cyclic polyethers were occluded within the polymer matrix and the transport properties investigated. The results indicated that the presence of an ionophore can be used to modulate ion transport and that ion transport is described by a dual-sorption mechanism. However, these studies were limited due to ionophore loss during hydration. Hence, the synthesis of a range of acrylate based crown ether monomers was considered. A pure sample of 4-acryolylaminobenzo-15-crown-5 was obtained and a terpolymer containing this monomer was prepared. Transport studies illustrated that the presence of a `bound' ionophore modulates ion transport in a similar way to the occluded systems. The transport properties of a series of terpolymers containing linear polyethers were then investigated. The results indicated that the dual-sorption mechanism is observed for these systems with group II metal cations while the transport of group I metal cations, with the exception of sodium, is enhanced. Finally, the equilibrium water contents (EWC) surface and mechanical properties of these terpolymers containing linear polyethers were examined. Although subtle variations in EWC are observed as the structure of the polyether side chain varies, generally EWC is enhanced due to the hydrophilicity of the polyether side chain. The macroscopic surface properties were investigated using a sessile drop technique and FTIR spectroscopy. At a molecular level surface properties were probed using an in vitro ocular spoilation model and preliminary cell adhesion studies. The results indicate that the polyethylene oxide side chains are expressed at the polymer surface thus reducing the adhesion of biological species.

Synthesis and cytotoxity of oxysterols:studies on the A,B ring polyoxygenated sterols

Oxysterols (OS), the polyoxygenated sterols, represent a class of potent regulatory molecules for important biological actions. Cytotoxicity of OS is one of the most important aspects in studies of OS bioactivities. However, studies, the structure-activity relationship (SAR) study in particular, have been hampered by the limited availability of structurally diverse OS in numbers and amounts. The aim of this project was to develop robust synthetic methods for the preparation of polyhydroxyl sterols, thereof, evaluate their cytotoxicity and establish structure-activity relationship. First, we found hydrophobicity of the side chain is essential for 7-HC's cytotoxicity, and a limited number of hydroxyl groups and a desired configuration on the A, B ring are required for a potent cytotoxicity of an OS, after syntheses and tests of a number of 7-HC's analogues against cancer cell lines. Then polyoxygenation of cholesterol A, B rings was explored. A preparative method for the synthesis of four diastereomerically pure cholest-4-en-3,6-diols was developed. Epoxidation on these cholest-4-en-3,6-diols showed that an allyl group exerts an auxiliary role in producing products with desired configuration in syntheses of the eight diastereomerically pure 45-epoxycholestane-3,6-diols. Reduction of the eight 45-epoxycholestane-3,6-diols produced all eight isomers of the cytotoxic 5α-acholestane 3β,5,6β-triol (CT) for the first time. Epoxide ring opening with protic or Lewis acids on the eight 45-epoxycholestane-3,6-diols are carefully studied. The results demonstrated a combination of an acid and a solvent affected the outcomes of a reaction dramatically. Acyl group participation and migration play an important role with numbers of substrates under certain conditions. All the eight 4,5-trans cholestane- 3,4,5,6-tetrols were synthesised through manipulation of acyl participation. Furthermore these reaction conditions were tested when a number of cholestane-3,4, 5,6,7-pentols and other C3-C7 oxygenated sterols were synthesised for the first time. Introduction of an oxygenated functional group through cholest-2-ene derivatives was studied. The elimination of 3-(4-toluenesulfonate) esters showed the interaction between the existing hydroxyls or acyls with the reaction centre often resulted in different products. The allyl oxidation, epoxidation and Epoxide ring opening reactions are investigated with these cholest-2-enes.

Combinatorial approach to multi-substituted 1,4-Benzodiazepines as novel non-peptide CCK-antagonists

For the drug discovery process, a library of 168 multisubstituted 1,4-benzodiazepines were prepared by a 5-step solid phase combinatorial approach. Substituents were varied in the 3,5, 7 and 8-position on the benzodiazepine scaffold. The combinatorial library was evaluated in a CCK radiolabelled binding assay and CCKA (alimentary) and CCKB (brain) selective lead structures were discovered. The template of CCKA selective 1,4-benzodiazepin-2-ones bearing the tryptophan moiety was chemically modified by selective alkylation and acylation reactions. These studies provided a series of Asperlicin naturally analogues. The fully optimised Asperlicin related compound possessed a similar CCKA activity as the natural occuring compound. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCKB receptor subtype were optimised on A) the lipophilic side chain and B) the 2-aminophenyl-ketone moiety, together with some stereochemical changes. A C3 unit in the 3-position of 1,4-benzodiazepines possessed a CCKB activity within the nanomolar range. Further SAR optimisation on the N1-position by selective alkylation resulted in an improved CCKB binding with potentially decreased activity on the GABAA/benzodiazepine receptor complex. The in vivo studies revealed two N1-alkylated compounds containing unsaturated alkyl groups with anxiolytic properties. Alternative chemical approaches have been developed, including a route that is suitable for scale up of the desired target molecule in order to provide sufficient quantities for further in vivo evaluation.