Determinants of potency on alpha-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors

Alpha-conotoxin MII, a peptide toxin isolated from Conus magus, antagonizes a subset of neuronal nicotinic receptors. Rat alpha3beta2 receptors, expressed in Xenopus oocytes, are blocked with an IC(50) of 3.7 +/- 0.3 nM. To identify structural features that determine toxin potency, a series of alanine-substituted toxins were synthesized and tested for the ability to block the function of alpha3beta2 receptors. Circular dichroism and protein modeling were used to assess the structural integrity of the mutant toxins. Three residues were identified as major determinants of toxin potency. Replacement of asparagine 5, proline 6, or histidine 12 with alanine resulted in >2700-fold, 700-fold, and approximately 2700-fold losses in toxin potency, respectively. A decrease in pH improved toxin potency, while an increase in pH eliminated toxin blockade, suggesting that, in the active form of the toxin, histidine 12 is charged. The imidazole ring of histidine 12 protrudes from one side, while asparagine 5 and proline 6 are located at the opposite end of the toxin structure. The side chains of these three residues are exposed on the surface of the toxin, suggesting that they directly interact with the alpha3beta2 receptor.     

Synthesis and properties of NBD-n-acylcholines, fluorescent analogs of acetylcholine

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N-7-(4-nitrobenzo-2-oxa-1,3-diazolyl)-omega-amino-n-alkanoic acid beta (N,N,N-trialkylammonium) ethylesters (NBD-n-acylcholines) and report here on their physiological and biochemical properties. All NBD-n-acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length. The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n = 4 and n = 3. The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 10(8) M-1 S-1 and do not vary significantly with the chain length of the NBD-n-acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length. The quenching of fluorescence of NBD-n-acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the omega-amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein. With their advantageous fluorescence properties, their simple pharmacology, and their clear structure-function relationships, these compounds are useful tools for the study of cholinergic mechanisms.     

Synthesis of the chromophores of fluorescent proteins and their analogs

Members of the green fluorescent protein (GFP) family are widely used in experimental biology as genetically encoded fluorescent tags. Chromophores of GFP-like proteins share a common structural core: 3,5-dihydro-4H-imidazol-4-one. This review covers synthetic approaches to 3,5-dihydro-4H-imida-zol-4-ones, substituted at different positions. General, as well as specific methods, represented by single examples are considered. The most popular synthetic route to substituted 3,5-dihydro-4H-imidazol-4-ones includes synthesis of azlactones, followed by transformation into N-acyldehydroamino acids and, finally, cyclization into target heterocycles. Accordingly, the review is divided into three parts: the first part covers syntheses of azlactones, the second part covers main approaches to N-acyldehydroamino acids, and in the third part we summarize cyclizations of N-acyldehydroamino acids, as well as all other approaches to 3,5-dihydro-4H-imidazol-4-ones.     

Demonstration of direct binding of cIAP1 degradation-promoting bestatin analogs to BIR3 domain: Synthesis and application of fluorescent bestatin ester analogs

Overexpression of cIAP1 correlates with resistance to radiotherapy and chemotherapy in various cancers. Recently, we reported that a class of bestatin ester analogs represented by MeBS (2) destabilized and promoted the degradation of cIAP1 through auto-ubiquitination, and thereby sensitized cancer cells to apoptosis. Herein, we present chemical evidence that bestatin ester analogs directly interact with the cIAP1-BIR3 domain by means of fluorescence polarization assay and photoaffinity labeling assay using fluorescent probes.     

Synthesis, receptor binding activity and fluorescence property of fluorescent enkephalin analogs containing L-1-pyrenylalanine

The novel fluorescent amino acid, L-1-pyrenylalanine (L-Pya), was prepared by the asymmetric hydrogenation of cyclic dehydrodipeptide. Fluorescent enkephalins containing one or two Pya residues at position 1,4 or 5 of [D-Ala2, Leu5]enkephalin were synthesized by the solution method. Mono-Pya-enkephalins showed strong fluorescence intensities and potent binding affinities with specificity and selectivity for opiate receptors. However, di-Pya-enkephalins showed markedly decreased receptor binding affinities. These results indicate that the incorporation of two Pya residues into enkephalin makes the peptide unable to interact with the opiate receptors, although introduction of one Pya residue is effective to elicit a specific receptor interaction. Di-Pya-enkephalins showed intramolecular excimer spectra, indicating that the peptides are able to take possible folded conformations.     

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.     

New fluorescent cholesterol analogs as membrane probes.

New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.     

Novel fluo-4 analogs for fluorescent calcium measurements

We report new fluorescent calcium indicators based on fluo-4. Attachment of a carboxamide or methylenecarboxamide moiety to the BAPTA chelator portion of fluo-4 allowed for the attachment of dextrans, protein-reactive moieties, and biotin. In particular, a high affinity fluo-4 dextran conjugate was prepared and shown to be functional in brain slices. All new probes were characterized spectroscopically and exhibited large fluorescence increases upon calcium-binding. The biotinylated version of fluo-4 formed a persistent streptavidin complex which still responded to increasing calcium concentrations with a large fluorescence increase.     

Solution structure of alpha-conotoxin SI

The nuclear magnetic resonance solution structure of alpha-conotoxin SI has been determined at pH 4.2. The 36 lowest energy structures show that alpha-conotoxin SI exists in a single major solution conformation and is stabilized by six hydrogen bonds. Comparisons are made between the SI solution structure and the solution and crystal structures of alpha-conotoxin GI. Surprisingly, a high degree of similarity between the backbone conformations of the GI crystal and the SI solution structures is seen in the region of lowest sequence homology, namely residues Gly-8 to Ser-12. This similarity is more surprising when considering that in SI a proline replaces the Arg-9 found in GI. The correspondence in conformation in this region provides the definitive evidence that it is the loss of the arginine basic charge at residue 9 which determines the differences in toxicity between GI and SI, rather than any changes in conformation induced by the cyclic proline residue.     

Novel fluorescent prothrombin analogs as probes of staphylocoagulase-prothrombin interactions

Staphylocoagulase (SC) is a potent nonproteolytic prothrombin (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase fragment containing residues 1-325 (SC-(1-325)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain 1 of SC (D1, residues 1-146) interacts with the 148 loop of thrombin and prethrombin 2 and the south rim of the catalytic site, whereas domain 2 of SC (D2, residues 147-325) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(1-325) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening 10 such derivatives were used to characterize quantitatively equilibrium binding of SC-(1-325) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(1-325) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of 17-72 pM. The results obtained for isolated SC domains indicate that D2 binds ProT with approximately 130-fold greater affinity than D1, yet D1 binding accounts for the majority of the fluorescence enhancement that accompanies SC-(1-325) binding. The SC-(1-325).(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tripeptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(1-325) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(1-325).(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.     

Synthesis of macrolide prostaglandin analogs

Prostaglandin analogs of the E- and F₂α-functional type, which are constrined to conformations in which the side-chains are close in space and specifically aligned in the terminal portions by covalent bonding, have been synthesized. These analogs are 1, (ω-1)-macrolides. The syntheses proceed from aldehyde intermediate I $\text{via}$ the Emmon's condensation with dimethyl n-(dimethyl-t-butyl-silyloxy) 2-oxoalkylphosphonate anions (IIa or b). The macrolide closures were performed using 2,2′-dipyridyl disulfide. For the synthesis of 9-ketoprostaglandin macrolides, a free 9-hydroxy is available for oxidation after macrolide closure, so long as the 9-position is protected as the acetate rather than benzoate. Chiroptical data revealed that the conformations of the macrolide prostaglandins are unchanged (relative to the natural uncosntrained prostaglandins) in the vicinity of the five-membered ring and the allyl alcohol unit by the formation of the macrolide linkage.     

Effect of alpha-conotoxin MII and its N-terminal derivatives on Ca2+ and Na+ signals induced by nicotine in neuroblastoma cell line SH-SY5Y

Nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation ofintracellular Ca2+-dependent processes in cells both in normal and pathological states, alpha-Conotoxins isolated from Conus snails venom are a valuable tool for the study of pharmacological properties and functional role of nAChRs. In the present study the alpha-conotoxin MII analogue with the additional tyrosine attached to the N terminus (Y0-MII) was prepared. Also we synthesized analogs with the N-terminal glycine residue labeled with the Bolton- Hunter reagent (BH-MII) or fluorestsein isothiocyanate (FITC-MII). Fluorescence microscopy studies of the neuroblastoma SH-SY5Y cells loaded with Ca2+ indicator Fura-2 or with Ca2+ and Na+ indicators Fluo-4 and SBFI were performed to examine effect of MII modification on its ability to inhibit nicotin-induced increases in intracellular free Ca2+ and Na+ concentrations ([Ca2+] and [Na+]i respectively). Monitoring of individual cell [Ca2+]i and [Na+]i signals revealed different kinetics of [Ca2+]i and [Na+]i rise and decay in responses to brief nicotine (Nic) applications (10-30 microM, 3-5 min), which indicates to different mechanisms of Ca2+ and Na+ homeostasis control in SH-SY5Y cells. MII inhibited in concentration-dependent manner the both [Ca2+]i and [Na+]i increase induced by Nic. Additional tyrosine in the Y0-MII or, especially, more sizeable label in FITC-MII significantly reduced the inhibitory effect of MII. Whereas the efficiency of the Ca2+ response inhibition by BH-MII was found to be close to the efficiency of its inhibition by natural alpha-conotoxin MII, radioiodinated derivatives BH-MII can be used in radioligand assay.     

Synthesis of acyclic analogs of ribavirin

A number of ribavirin analogues were prepared in which the ribose moiety was replaced with acyclic substituents imitating some fragments of the ribose ring: 2,3-dihydroxy-prop-1-yl, 3-hydroxymethyl-4-hydroxy-2-oxabut-1-yl, 4,5-dihydroxy-2-oxapent-1-yl and 1,5-dihydroxy-3-oxapent-2-yl. These analogues were synthesized by direct alkylation of ethyl 1,2,4-triazole-3-carboxylate with suitable agents followed by ammonolysis. New convenient methods for preparing the alkylating agents were developed.     

Synthesis of some analogs of estradiol

As part of a search for estradiol derivatives designed for conjugation to carboxyl or amine functions of anti-cancer agents or suitable derivatives thereof, estradiol analogs with side chains at the C-16 or -17 position were prepared for biological assay. These analogs include several which have a substituted nitrogenous function at C-17. The avidity of some of these analogs for binding to estrogen receptor was found to be of a low order.     

Synthesis of fluorescent lactosylceramide stereoisomers

The intracellular distribution of synthetic glycosphingolipids (GSLs) bearing a fluorophore can be monitored in living cells by fluorescence microscopy. We reported previously that variation in the length of the long-chain base and in the structure of the carbohydrate-containing polar head group of (2S,3R) (or D-erythro-)-beta-lactosylceramide (LacCer) did not alter the mechanism of endocytic uptake from the plasma membrane of various mammalian cell types [Singh, R.D., Puri, V., Valiyaveettil, J.T., Marks, D.L., Bittman, R., Pagano, R.E., 2003. Selective caveolin-1-dependent endocytosis of glycosphingolipids. Mol. Biol. Cell 14, 3254-3265]. To extend our examination of the molecular features in LacCer that are responsible for its uptake by the caveolar-requiring endocytic pathway, we have synthesized the three unnatural stereoisomers [(2R,3R)-, (2S,3S)-, and (2R,3S)] of dipyrromethene difluoride (BODIPY)-LacCer. These analogues will be used to probe the role of stereochemistry in the long-chain base of LacCer in the mechanism of endocytic uptake.     

Synthesis of some aromatic prostaglandin analogs

Some aromatic prostaglandin analogs, having a benzene (2a, 2b) and a dimethoxybenzene (1) ring in place of the cyclopentane moiety, have been synthesized. The key intermediates in the syntheses were lactols 9 and 5, which were elaborated to the final products via two olefination reactions. Compound 2b was twice as potent as phenylbutazone and nine times as potent as aspirin in inhibiting prostaglandin synthetase activity.     

Synthesis and cytotoxicity of aurilide analogs

The artificial analogs of aurilide (1), a potent cytotoxic cyclodepsipeptide of marine origin, were synthesized, and the structure–activity relationships were investigated.