Chemical syntheses and biological activities of lactam analogues of alpha-conotoxin SI.

Bicyclization represents an effective method for the introduction of conformational constraints into small, biologically important peptides. Several strategies have been developed for the preparation of bicyclic lactam analogues of alpha-conotoxin SI, a 13-residue peptide neurotoxin found in cone snail venom. Four analogues of the natural regioisomer of alpha-conotoxin SI were designed and synthesized, each with one of the two paired cysteines of the parent peptide being replaced by a side-chain lactam bridged glutamic acid/lysine pair. Solid-phase lactamization was studied to determine rates of formation of the two possible loops and to document the extent of dimerization and higher oligomerization. Radioligand binding assays were carried out on all synthesized peptides, including the naturally occurring two-disulfide form, in order to determine their affinities for nicotinic acetylcholine receptors (nAChRs). Replacement of the Cys(2)-Cys(7) loop of alpha-conotoxin SI with a lactam bridge resulted in complete loss of activity, whereas replacement of the Cys(3)-Cys(13) disulfide loop resulted in a approximately 60-fold reduction in affinity for one orientation and a approximately 70-fold increase in affinity for the other. The two active lactam analogues retain the selectivity exhibited by the naturally occurring peptide for the alpha/delta subunit of nAChRs, as judged by competition experiments with the curariform antagonist metocurine.

Evaluation of electrochemiluminescent technology for inhibitors of granulocyte colony-stimulating factor receptor binding.

An electrochemiluminescent (ECL) assay was developed to identify compounds that inhibit the interaction of granulocyte colony-stimulating factor (GCSF) with its recombinant human receptor. The ECL technology uses a tris-(bipyridine) chelate of ruthenium, which, in the presence of excess tripropylamine, undergoes a redox reaction cycle to produce light. Paramagnetic beads with primary antibody were coated with secondary anti-GCSF receptor antibody, which were then bound with GCSF receptor. These samples were incubated with ruthenylated GCSF in the presence and absence of test compounds. The bead density, receptor and ligand concentrations, and incubation time were optimized in the assay. A set of mixed compound plates was screened to examine the feasibility of using this technology in high throughput screening. The results from this format were found to be comparable to the assay performed using a time-resolved fluorescence format.

Validation of FLIPR membrane potential dye for high throughput screening of potassium channel modulators.

A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive K+ channel; K(ATP)) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference K(ATP) channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.

Screening for inhibitors of histone deacetylase by incorporating a spraying method to micro-arrayed compound screening.

We have developed a method of spraying assay reagents onto a target gel in the Micro-Arrayed Compound Screening ( micro ARCS) format. After application of target gels to compound sheets, subsequent reagents can be applied by spraying onto the target gel. The spraying method conserves on assay reagents by up to 10-fold, eliminates the need for casting additional agarose gels, and increases the throughput of a screen by 3-fold. To demonstrate the efficacy of applying the spraying method to micro ARCS, we screened over 600,000 compounds for inhibitors of histone deacetylase (HDAC). Commercially available HDAC substrate and reaction developer were sprayed directly onto the gel to initiate the reaction and to amplify the signal, respectively. Picks in the primary screen were retested at a density of 384 compounds per sheet in the micro ARCS format. IC(50) values for active compounds were confirmed in a 96-well plate assay. The screen identified several small molecule inhibitors of the enzyme, including members of several classes of known HDAC inhibitors. The combination of the high-density format of micro ARCS, the efficiency of the spraying method, and a timed sequence of adding assay reagents permitted a screening throughput of 200,000 tests an hour. We present the details of the screening format and the analysis of the hits from the screen.

Characterization of RNA hairpin loop stability.

Fifteen RNA hairpins that share the same stem sequence and have homopolymer loops of A, C and U residues which vary in length from three to nine nucleotides were synthesized and their thermal stabilities determined. Tm varies as a function of loop size but is almost independent of loop composition. Loops of four or five nucleotides are found to be the most stable loop size. This is consistent with the observation that four-membered loops are the most prevalent loop size in 16S-like RNAs. The contribution of each loop to hairpin stability was calculated by subtracting the known contribution of the helical stem. These data should be useful for predicting the stability of other hairpins.

Thermal stability of RNA hairpins containing a four-membered loop and a bulge nucleotide.

Fourteen RNA hairpins containing a four-membered loop and a bulge nucleotide were synthesized and their thermal stabilities determined. The combined contribution of a four-membered loop and bulge A to the free energy of a hairpin is calculated to be 9.3 kcal/mol at 37 degrees C and successfully predicts the stability of an independent RNA hairpin. The introduction of a bulge nucleotide to the helical stem of an RNA hairpin destabilizes the molecule in a sequence-dependent manner. The individual thermodynamic contributions of a four-membered loop and bulge A, G, and U residues to the stability of an RNA hairpin loop are presented.

Lophotoxin is a slow binding irreversible inhibitor of nicotinic acetylcholine receptors.

Lophotoxin and the bipinnatins are members of the lophotoxin family of marine neurotoxins, which covalently react with Tyr190 in the alpha-subunits of the nicotinic acetylcholine receptor. Bipinnatin-A, -B, and -C are protoxins that have been shown to spontaneously convert from inactive to active toxins during preincubation in buffer. However, in this report, we show that preincubation of lophotoxin did not result in an increase in the subsequent rate of irreversible inhibition of nicotinic receptors. Thus, unlike the bipinnatins, lophotoxin does not appear to be an inactive protoxin. Lophotoxin preferentially inhibited one of the two acetylcholine-binding sites on the receptor, and this preference resulted from both a higher reversible affinity and a faster rate of irreversible inhibition at this site. Association of 125I-alpha-bungarotoxin in the presence of lophotoxin was analyzed to obtain the apparent reversible association and dissociation rate constants for lophotoxin. The apparent association rate constant of lophotoxin was approximately 10(6)-fold slower than expected for a diffusion-limited interaction, indicating that lophotoxin is a slow binding irreversible inhibitor. The kinetic constants that describe the interaction of lophotoxin with the receptor did not change in the presence of dibucaine, suggesting that, unlike agonists, the slow apparent association of lophotoxin does not result from a slow transition of the receptor to a desensitized conformation.

Determinants involved in the affinity of alpha-conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors from muscle contain two functionally active and pharmacologically distinct acetylcholine-binding sites located at the alpha/gamma and alpha/delta subunit interfaces. The alpha-conotoxins are competitive antagonists of nicotinic receptors and can be highly site-selective, displaying greater than 10,000-fold differences in affinities for the two acetylcholine-binding sites on a single nicotinic receptor. The higher affinity site for alpha-conotoxins GI, MI, and SI is the alpha/delta site on mouse muscle-derived BC3H-1 receptors. However, alpha-conotoxins GI and MI exhibit higher affinity for the other site (alpha/gamma site) on nicotinic receptors from Torpedo californica electric organ. alpha-Conotoxin SI does not distinguish between the two acetylcholine-binding sites on Torpedo receptors. In this study, alpha-conotoxins [K10H]SI and [K10N]SI displayed wild-type affinity for the two acetylcholine-binding sites on BC3H-1 receptors but a 10-20-fold decrease in apparent affinity at one of the two acetylcholine-binding sites on Torpedo receptors. alpha-Conotoxin [P9K]SI displayed a selective and dramatic increase in the apparent affinity for the alpha/delta site of BC3H-1 receptors and for the alpha/gamma site of Torpedo receptors. alpha-Conotoxin [R9A]GI displayed a reduction in affinity for both acetylcholine-binding sites on BC3H-1 receptors, although the extent of its selectivity for the alpha/delta site was retained. alpha-Conotoxin [R9A]GI also displayed a loss of affinity for the two acetylcholine-binding sites on Torpedo receptors, but its site-selectivity was apparently abolished. These results indicate that positions 9 and 10 in alpha-conotoxins GI and SI are involved in complex species- and subunit-dependent interactions with nicotinic receptors.

Irreversible inhibition of nicotinic acetylcholine receptors by the bipinnatins. Toxin activation and kinetics of receptor inhibition.

Bipinnatin-A, -B, and -C belong to a family of naturally occurring marine neurotoxins known as the lophotoxins. The lophotoxins are unique in that they irreversibly inhibit nicotinic acetylcholine receptors by forming a covalent bond with a tyrosine residue at position 190 in the alpha-subunit of the receptor. In this study, we show that the inhibitory activity of the bipinnatins against the nicotinic receptor increased with preincubation of the toxins in aqueous buffer prior to incubation with the receptor. The parent species of the bipinnatins displayed little, if any, affinity for the nicotinic receptor. Preincubation of the toxins appeared to produce a single, relatively stable, active toxin species that irreversibly inhibited the two acetylcholine-binding sites on the nicotinic receptor with two distinguishable apparent pseudo first-order rates. The difference in the rates of irreversible inhibition of the two binding sites on the receptor was exploited to selectively inhibit one site for the pharmacological investigation of the other. The bipinnatins preferentially inhibited the binding site near the alpha/delta-subunit interface that displays low affinity for metocurine and high affinity for acetylcholine. The bimolecular reaction constants for the interaction of the bipinnatins with the nicotinic receptor decreased in the order bipinnatin-B > bipinnatin-A > bipinnatin-C for both acetylcholine-binding sites. The ratio of the bimolecular reaction constants for the two binding sites on the receptor was not the same for the three bipinnatins. This indicates that the reaction of the bipinnatins with the nicotinic receptor is sensitive to differences in the structure of the two acetylcholine-binding sites. The bipinnatins may be useful in the design of novel drugs for the nicotinic receptor that exclusively inhibit one of the two binding sites and for the investigation of structural differences between the two acetylcholine-binding sites of the receptor.

Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.

A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.

alpha-Conotoxins selectively inhibit one of the two acetylcholine binding sites of nicotinic receptors.

Muscle subtypes of the nicotinic acetylcholine receptor contain two acetylcholine binding sites that can be distinguished pharmacologically. The affinities of several alpha-conotoxins for the two acetylcholine binding sites on nicotinic receptors from BC3H1 cells and Torpedo electric organ were investigated. alpha-Conotoxins MI, GI, and SIA each inhibited the binding of 125I-alpha-bungarotoxin to nicotinic acetylcholine receptors on BC3H1 cells with two distinct and independent affinities, which differed by > 10,000-fold. The affinities of alpha-conotoxins SI and SII were significantly lower and the differences in the affinities of each of these toxins for the two sites were < 400-fold. alpha-Conotoxins MI, GI, SIA, and SI had higher affinity for the acetylcholine binding site near the alpha/delta subunit interface of nicotinic receptors from BC3H1 cells. However, when assessed using nicotinic receptors from Torpedo electric organ, alpha-conotoxin MI displayed higher affinity for the acetylcholine binding site near the alpha/gamma subunit interface. These observations suggest that species variations in the sequences of the gamma and delta subunits resulted in a dramatic reversal of the relative affinities of the alpha-conotoxins for each acetylcholine binding site. Some of the practical implications of these observations are discussed.

CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes.

The mRNA of bacteriophage T4 contains a strikingly abundant intercistronic hairpin. Within the 55 kilobases of known T4 sequence, the hexanucleotide sequence CTTCGG is found 13 times in the DNA strand equivalent to mRNA sequences. In 12 of those occurrences, the sequence is flanked by inverted repeats predictive of RNA hairpins with UUCG in the loop. Avian myeloblastosis virus reverse transcriptase, which can traverse hairpins of larger calculated stability, terminates efficiently at these CUUCGG hairpins. Thermal denaturation studies of model hairpins show that the loop sequence UUCG dramatically stabilizes RNA hairpins when compared to a control sequence. These data, when combined with previously described parameters of helix stability, suggest that T4 has utilized this loop sequence to optimize the stability of intercistronic hairpins. The stability of CUUCGG hairpins is also utilized in the RNAs of many organisms besides T4.

High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.

alpha-Conotoxin EI, a new nicotinic acetylcholine receptor antagonist with novel selectivity.

We report the isolation and characterization of a novel nicotinic acetylcholine receptor (nAChR) ligand. The toxin is an 18 amino acid peptide and is the first reported alpha-conotoxin from an Atlantic fish-hunting Conus. The peptide was purified from the venom of Conus ermineus and is called alpha-conotoxin EI. The sequence diverges from that of previously isolated alpha-conotoxins. We demonstrate that this structural divergence has functional consequences. In Torpedo nAChRs, alpha-conotoxin EI selectively binds the agonist site near the alpha/delta subunit interface in contrast to alpha-conotoxin MI which selectively targets the alpha/gamma agonist binding site. In mammalian nAChRs alpha-conotoxin EI shows high affinity for both the alpha/delta and alpha/gamma subunit interfaces (with some preference for the alpha/delta site), whereas alpha-conotoxin MI is highly selective for the alpha/delta ligand binding site. The sequence of the peptide is: Arg-Asp-Hyp-Cys-Cys-Tyr-His-Pro-Thr-Cys-Asn-Met-Ser-Asn-Pro-Gln-Ile-Cys- NH2, with disulfide bridging between Cys4-Cys10 and Cys5-Cys18, analogous to those of previously described alpha-conotoxins. This sequence has been verified by total chemical synthesis. Thus, alpha-conotoxin EI is a newly-available tool with unique structure and function for characterization of nAChRs.

Differential targeting of nicotinic acetylcholine receptors by novel alphaA-conotoxins.

We describe the isolation and characterization of two peptide toxins from Conus ermineus venom targeted to nicotinic acetylcholine receptors (nAChRs). The peptide structures have been confirmed by mass spectrometry and chemical synthesis. In contrast to the 12-18 residue, 4 Cys-containing alpha-conotoxins, the new toxins have 30 residues and 6 Cys residues. The toxins, named alphaA-conotoxins EIVA and EIVB, block both Torpedo and mouse alpha1-containing muscle subtype nAChRs expressed in Xenopus oocytes at low nanomolar concentrations. In contrast to alpha-bungarotoxin, alphaA-EIVA is inactive at alpha7-containing nAChRs even at micromolar concentrations. In this regard, alphaA-EIVA is similar to the previously described alpha-conotoxins (e.g. alpha-MI and alpha-GI) which also selectively target alpha1- versus alpha7-containing nAChRs. However, alpha-MI and alpha-GI discriminate between the alpha/delta versus alpha/gamma subunit interfaces of the mouse muscle nAChR with 10,000-fold selectivity. In contrast, alphaA-conotoxin EIVA blocks both the alpha/gamma site and alpha/delta site with equally high affinity but with distinct kinetics. The alphaA-conotoxins thus represent novel probes for the alpha/gamma as well as the alpha/delta binding sites of the nAChR.