Cheryl Dowell

α-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between α6β4 and α6β2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release

α6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of α6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. α-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of α-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC(50) by 2800-fold at α6/α3β2β3 but only by 6-fold at α6/α3β4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in α6-null mutant mice. α-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in β4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain α6β2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related α6* nAChRs; α6β4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.

α-Conotoxin OmIA Is a Potent Ligand for the Acetylcholine-binding Protein as Well as α3β2 and α7 Nicotinic Acetylcholine Receptors

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the α-subunit of nicotinic acetylcholine receptors and in particular the homomeric α7 nicotinic receptor. We report the isolation and characterization of an α-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the α7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the α3β2 nAChR indicating that α-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of α3β2 nAChRs. α-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first α-conotoxin with higher affinity for the closely related receptor subtypes, α3β2 versus α6β2, and selectively blocks these two subtypes when compared with α2β2, α4β2, and α1β1δϵ nAChRs.

Atypical α-Conotoxin LtIA from Conus litteratus Targets a Novel Microsite of the α3β2 Nicotinic Receptor

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. α-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of α-conotoxins may serve as a platform for development of new therapies. Previously characterized α-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel α-conotoxin LtIA, which lacks this highly conserved motif but potently blocked α3β2 nAChRs with a 9.8 nm IC50 value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing α-conotoxin MII. Nevertheless, pre-block of α3β2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR β subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for α3β2 versus α3β4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC50 value on α3β2(F119Q) versus wild type nAChRs, whereas T59K and V111I β2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the α3β2 nAChR that includes β2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

The atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. α-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of α-conotoxins may serve as a platform for development of new therapies. Previously characterized α-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel α-conotoxin LtIA, which lacks this highly conserved motif but potently blocked α3β2 nAChRs with a 9.8 nm IC50 value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing α-conotoxin MII. Nevertheless, pre-block of α3β2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR β subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for α3β2 versus α3β4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC50 value on α3β2(F119Q) versus wild type nAChRs, whereas T59K and V111I β2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the α3β2 nAChR that includes β2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

Synthesis and Characterization of 125I-α-Conotoxin ArIB[V11L;V16A], a Selective α7 Nicotinic Acetylcholine Receptor Antagonist

The α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-α-bungarotoxin is commonly used to identify α7 nAChRs specifically. However, α-bungarotoxin also interacts potently with α1* and α9α10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the α7 subtype. [3H]Methyllycaconitine is also frequently used as an α7-selective antagonist, but it has significant affinity for α6* and α9α10 nAChR subtypes. In this study, we have developed a highly α7-selective α-conotoxin radioligand by iodination of a naturally occurring histidine. Both mono- and diiodo derivatives were generated and purified (specific activities were 2200 and 4400 Ci mmol-1, respectively). The properties of the mono- and diiodo derivatives were very similar to each other, but the diiodo was less stable. For monoidodo peptide, saturation binding to mouse hippocampal membranes demonstrated a Kd value of 1.15 ± 0.13 nM, similar to that of 125I-α-bungarotoxin in the same preparations (0.52 ± 0.16 nM). Association and dissociation kinetics were relatively rapid (kobs for association at 1 nM was 0.027 ± 0.007 min-1; koff = 0.020 ± 0.001 min-1). Selectivity was confirmed with autoradiography using α7-null mutant tissue: specific binding was abolished in all regions of α7-/- brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. 125I-α-conotoxin ArIB[V11L; V16A] should prove useful where α7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on α7 nAChRs, something that is impossible with 125I-α-bungarotoxin.

Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist

Significance The α9α10 nicotinic AChR (nAChR) subtype is a recently identified target for the development of breast cancer chemotherapeutics and analgesics, particularly to treat neuropathic pain. Structure/function analyses of antagonists of this subtype are therefore essential for the development of specific therapeutic compounds. The Conus genus is a rich source of pharmacologically active peptides, and we report here that the αO-conotoxin GeXIVA is a potent and selective antagonist of the α9α10 nAChR subtype. GeXIVA displays unique structural properties among other Conus peptides and represents a previously unidentified template for molecules active against neuropathic pain. We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.

Discovery of non-peptide, small molecule antagonists of α9α10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain

A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of α9α10 nAChRs and greater selectivity over α7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.

The novel small molecule α9α10 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic

Chronic pain is inadequately managed with currently available classes of analgesic drugs. Recently, peptide antagonists of the α9α10 nicotinic acetylcholine receptor were shown to be analgesic. The present study was conducted to characterize a novel small molecule, non-peptide antagonist at nicotinic receptors. The tetrakis-quaternary ammonium compound ZZ-204G was evaluated for functional activity on cloned nicotinic receptors expressed in Xenopus oocytes. In-vivo efficacy was assessed in rat models of tonic inflammatory pain (formalin test), neuropathic pain (chronic constriction nerve injury), and thermal nociception (tail flick test). ZZ-204G was an antagonist at nicotinic receptors inhibiting the α9α10 subtype with an IC₅₀ of 0.51 (0.35-0.72) nM. Antagonist activity at other nicotinic subtypes (α1β1δε, α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, α6/α3β2β3, α6/α3β4 and α7) was 10-1000-fold lower than at the α9α10 subtype. In competition binding assays, the k(i) of ZZ-204G at γ-aminobutyric acid(A), serotonin(3), γ-aminobutyric acid(B), κ- and μ-opioid receptors was 1000- to >10,000-fold lower than at α9α10 nicotinic receptors. Parenteral administration of ZZ-204G dose-dependently decreased nociceptive behaviors (paw flinches) in the formalin test and mechanical hyperalgesia in the chronic constriction nerve injury model of neuropathic pain. ZZ-204G was not antinociceptive in the tail flick assay. Results from the rotarod assay indicated that lower doses of ZZ-204G that were analgesic did not alter motor function. In summary, ZZ-204G represents a prototype small molecule antagonist for α9α10 nicotinic receptors and provides a novel molecular scaffold for analgesic agents with the potential to treat chronic inflammatory or neuropathic pain.

Autoradiographic localization of N-type VGCCs in gerbil hippocampus and failure of ω-conotoxin MVIIA to attenuate neuronal injury after transient cerebral ischemia

In the mammalian central nervous system, transient global ischemia of specific duration causes selective degeneration of CA1 pyramidal neurons in hippocampus. Many of the ischemia-induced pathophysiologic cascades that destroy the neurons are triggered by pre- and postsynaptic calcium entry. Consistent with this, many calcium channel blockers have been shown to be neuroprotective in global models of ischemia. omega-Conotoxin MVIIA, a selective N-type VGCC blocker isolated from the venom of Conus magus, protects CA1 neurons in the rat model of global ischemia, albeit transiently. The mechanism by which this peptide renders neuroprotection is unknown. We performed high-resolution receptor autoradiography with the radiolabeled peptide and observed highest binding in stratum lucidum of CA3 subfield, known to contain inhibitory neurons potentially important in the pathogenesis of delayed neuronal death. This finding suggested that the survival of stratum lucidum inhibitory neurons might be the primary event, leading to CA1 neuroprotection after ischemia. Testing of this hypothesis required the reproduction of its neuroprotective effects in the gerbil model of global ischemia. Surprisingly, we found that omega-MVIIA did not attenuate CA1 hippocampal injury after 5 min of cerebral ischemia in gerbil. Possible reasons are discussed. Lastly, we show that the peptide can be used as a synaptic marker in assessing short and long-term changes that occur in hippocampus after ischemic injury.

Discovery of a potent and selective α3β4 nicotinic acetylcholine receptor antagonist from an α-conotoxin synthetic combinatorial library

α-Conotoxins are disulfide-rich peptide neurotoxins that selectively inhibit neuronal nicotinic acetylcholine receptors (nAChRs). The α3β4 nAChR subtype has been identified as a novel target for managing nicotine addiction. Using a mixture-based positional-scanning synthetic combinatorial library (PS-SCL) with the α4/4-conotoxin BuIA framework, we discovered a highly potent and selective α3β4 nAChR antagonist. The initial PS-SCL consisted of a total of 113 379 904 sequences that were screened for α3β4 nAChR inhibition, which facilitated the design and synthesis of a second generation library of 64 individual α-conotoxin derivatives. Eleven analogues were identified as α3β4 nAChR antagonists, with TP-2212-59 exhibiting the most potent antagonistic activity and selectivity over the α3β2 and α4β2 nAChR subtypes. Final electrophysiological characterization demonstrated that TP-2212-59 inhibited acetylcholine evoked currents in α3β4 nAChRs heterogeneously expressed in Xenopus laevis oocytes with a calculated IC50 of 2.3 nM and exhibited more than 1000-fold selectivity over the α3β2 and α7 nAChR subtypes. As such, TP-2212-59 is among the most potent α3β4 nAChRs antagonists identified to date and further demonstrates the utility of mixture-based combinatorial libraries in the discovery of novel α-conotoxin derivatives with refined pharmacological activity.