Isolation, structure, and activity of GID, a novel alpha 4/7-conotoxin with an extended N-terminal sequence

Using assay-directed fractionation of Conus geographus crude venom, we isolated a-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective alpha-conotoxins, alpha-GID has a four amino acid N-terminal tail, gamma-carboxyglutamate (Gla), and hydroxyproline (0) residues, and lacks an amidated C terminus. GID inhibits alpha7 and alpha3beta2 nAChRs with IC50 values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the alpha1beta1gammadelta, alpha3beta4, and alpha4beta4 combinations. GID also potently inhibits the alpha4beta2 subtype (IC50 of 150 nm). Deletion of the N-terminal sequence (GIDDelta1-4) significantly decreased activity at the alpha4beta2 nAChR but hardly affected potency at alpha3beta2 and alpha7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg(12) contributed to alpha4beta2 and alpha7 activity but not to alpha3beta2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other a-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other alpha4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater alpha7 versus alpha3beta2 selectivity

Identification of a novel class of nicotinic receptor antagonists - Dimeric conotoxins VxXIIA, VxXIIB, and VxXIIC from Conus vexillum

The venoms of predatory marine snails ( Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, alpha D-VxXIIA, alpha D-VxXIIB, and alpha D-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an similar to 11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunitsexhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that alpha D-VxXIIA, alpha D-VxXIIB, and alpha D-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for alpha 7 and alpha 2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs

Isolation, structure, and activity of GID, a novel α4/7-conotoxin with an extended N-terminal sequence

Using assay-directed fractionation of Conus geographus crude venom, we isolated α-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective α-conotoxins, α-GID has a four amino acid N-terminal tail, γ-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits α7 and α3β2 nAChRs with IC50 values of 5 and 3 nM, respectively and is at least 1000-fold less potent at the α1β1γδ, α3β4, and α4β4 combinations. GID also potently inhibits the α4β2 subtype (IC50 of 150 nM). Deletion of the N-terminal sequence (GIDΔ1-4) significantly decreased activity at the α4β2 nAChR but hardly affected potency at α3β2 and α7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg12 contributed to α4β2 and α7 activity but not to α3β2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other α-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other α4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater α7 versus α3β2 selectivity

alpha-conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors

The Xenopus laevis oocyte expression system was used to determine the activities of alpha-conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, alpha-EpI showed no significant activity on oocyte-expressed alpha3beta4 and alpha3beta2 nAChRs but blocked the alpha7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the alpha7/5HT(3) chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed alpha3beta4 receptors but unlike in native cells, where it was 10-fold more potent than native alpha-AuIB, had 25-fold lower activity. These results indicate that as yet unidentified factors influence alpha-conotoxin pharmacology at native versus oocyte-expressed nAChRs. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies

Inhibition of neuronal nicotinic acetylcholine receptor subtypes by alpha-conotoxin GID and analogues

alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha 4 beta 2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha 4 beta 2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alaninescan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha 7, alpha 3 beta 2, and alpha 4 beta 2 subtypes. Although the proline residue was the most critical residue for activity at the alpha 3 beta 2 subtype, Asp3, Arg12, and Asn14 are also critical at the alpha 7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha 4 beta 2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha 4 beta 2 subtype to date. Overall, our findings contribute to the understanding of the structureactivity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents

Inhibition of neuronal nicotinic acetylcholine receptor subtypes by α-conotoxin GID and analogues

α-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. α-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the α4β2 subtype of the nAChR. To date, only four conotoxins that inhibit the α4β2 subtype have been characterized but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in α-conotoxins is important for activity at the α7, α3β2, and α4β2 subtypes. Although the proline residue was the Most critical residue for activity at the α3β2 subtype, Asp3, Arg12 and Asn14 are also critical at the α7 subtype. Interestingly, very few of the mutations tested retained activity at the α4β2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the α4β2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of α-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents

Chromosomal instability determines taxane sensitivity - supplementary materials

Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these ‘‘CIN-survival’’ genes is associated with poor outcome in estrogen receptor–positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents

New mutations in the ATM gene and clinical data of 25 AT patients

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, oculocutaneous telangiectasias, chromosomal instability, radiosensitivity, and cancer predisposition. The gene mutated in the patients, ATM, encodes a member of the phosphatidylinositol 3-kinase family proteins. The ATM protein has a key role in the cellular response to DNA damage. Truncating and splice site mutations in ATM have been found in most patients with the classical AT phenotype. Here we report of our extensive ATM mutation screening on 25 AT patients from 19 families of different ethnic origin. Previously unknown mutations were identified in six patients including a new homozygous missense mutation, c.8110T > C (p.Cys2704Arg), in a severely affected patient. Comprehensive clinical data are presented for all patients described here along with data on ATM function generated by analysis of cell lines established from a subset of the patients