Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

Non-native disulfide isomers of ␣-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of ␣-conotoxin AuIB have been characterized on ␣3␤4 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to ϳ50%. The maximal inhibition was stoichiometry-dependent because altering ␣3:␤4 RNA injection ratios either increased AuIB(ribbon) efficacy (10␣:1␤) or completely abolished blockade (1␣:10␤). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity ␣3␤4 nAChRs with corresponding EC 50 values of 22.6 and 176.9 M, respectively. Increasing the agonist concentration antagonized the inhibition of LS ␣3␤4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS ␣3␤4 nAChRs by AuIB-(globular) was unaffected. Inhibition of LS and HS ␣3␤4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both ␣3␤4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS ␣3␤4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS ␣3␤4 nAChRs competitively, whereas AuIB-(globular) inhibits ␣3␤4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.Conotoxins are short disulfide-rich bioactive peptides that have been originally isolated from venoms of carnivorous mollusk cone snails, belonging to the genus Conus. ␣-Conotoxins are among the largest class of conotoxins found in the venom of most cone snail species (1). This class of conotoxins targets various subtypes of nicotinic acetylcholine receptors (nAChRs) 2 and is distinguished by four cysteines arranged in a CC-C-C pattern.␣-Conotoxins have attracted considerable attention as some of them, such as Vc1.1 and RgIA, have been shown to possess analgesic activity in rodent behavioral models of neuropathic pain (2, 3). Interestingly, AuIB has recently been shown to be analgesic in vivo despite the fact that it acts on the ␣3␤4 nAChR subtype different from the ␣9␣10 nAChR targeted by Vc1.1 and RgIA.3 Vc1.1 and RgIA have been shown to suppress N-type Ca 2ϩ channel currents in dorsal root ganglion (DRG) neurons of neonatal and adult rats and wild type and ␣9 knock-out mice via activation of GABA B G protein-coupled receptors (2). Similarly, AuIB inhibits N-type Ca 2ϩ channels in rat DRG neurons analogous to Vc1.1 and RgIA and its effect can be blocked with selective GABA B receptor antagonists.4 GABA B -mediated inhibition of N-type Ca 2ϩ channels...

Background: ␣-Conotoxin AuIB interacts with ␣3␤4 nAChRs and GABA B receptors, but structural determinants of these interactions are unknown. Results: Using alanine scanning mutagenesis and molecular dynamics, we identified residues crucial for AuIB⅐␣3␤4 nAChR interaction. Conclusion:We identified the key residues that mediate AuIB⅐␣3␤4 nAChR interaction. Significance: Ability to direct ␣-conotoxin binding to nAChRs or GABA B receptors will improve analgesic conopeptides.

Neuronal Ca2ϩ influx via NMDA receptors (NMDARs) is essential for the development and plasticity of synapses but also triggers excitotoxic cell death when critical intracellular levels are exceeded. Therefore, finely equilibrated mechanisms are necessary to ensure that NMDAR function is maintained within a homeostatic range. Here we describe a pronounced difference in the modulation of NMDA currents in two closely related hippocampal cell types, the CA1 and the CA3 pyramidal cells (PCs). Manipulations that increase intracellular Ca 2ϩ levels strongly depressed NMDA currents in CA3 with only minor effects in CA1 PCs. Furthermore, activation of G q -coupled metabotropic receptors potentiated NMDA currents in CA1 PCs but depressed them in CA3 PCs. Interestingly, the CA3 type modulation of NMDARs could be converted into CA1-like behavior, and vice versa, by increasing Ca 2ϩ buffering in CA3 cells or decreasing Ca 2ϩ buffering in CA1 cells, respectively. Our data suggest that a differential Ca 2ϩ sensitivity of the regulatory cascades targeting NMDARs plays a key role in determining the direction and magnitude of NMDA responses in various types of neurons. These findings may have important implications for NMDA receptor-dependent synaptic plasticity and the differential sensitivity of CA1 and CA3 PCs to NMDARdependent ischemic cell death.

α-Conotoxins are peptides isolated from the venom ducts of cone snails that target nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential applications for treating a range of conditions in humans, including pain. However, like all peptides, conotoxins are susceptible to degradation, and to enhance their therapeutic potential it is important to elucidate the factors contributing to instability and to develop approaches for improving stability. AuIB is a unique member of the α-conotoxin family because the nonnative "ribbon" disulfide isomer exhibits enhanced activity at the nAChR in rat parasympathetic neurons compared with the native "globular" isomer. Here we show that the ribbon isomer of AuIB is also more resistant to disulfide scrambling, despite having a nonnative connectivity and flexible structure. This resistance to disulfide scrambling does not correlate with overall stability in serum because the ribbon isomer is degraded in human serum more rapidly than the globular isomer. Cyclization via the joining of the N- and C-termini with peptide linkers of four to seven amino acids prevented degradation of the ribbon isomer in serum and stabilized the globular isomers to disulfide scrambling. The linker length used for cyclization strongly affected the relative proportions of the disulfide isomers produced by oxidative folding. Overall, the results of this study provide important insights into factors influencing the stability and oxidative folding of α-conotoxin AuIB and might be valuable in the design of more stable antagonists of nAChRs.

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Positive allosteric modulators (PAMs) of α7 nAChRs can have different properties with respect to their effects on channel kinetics. Type I PAMs amplify peak channel response to acetylcholine but do not appear to influence channel desensitization kinetics, whereas Type II PAMs both increase channel response and delay receptor desensitization. Both Type I and Type II PAMs are reported in literature, but there are limited reports describing their structure−kinetic profile relationships. Here, we report a novel class of compounds with either Type I or Type II behavior that can be tuned by the relative stereochemistry around the central cyclopropyl ring: for example, (R,R)-13 (BNC375) and its analogues with RR stereochemistry around the central cyclopropyl ring are Type I PAMs, whereas compounds in the same series with SS stereochemistry (e.g., (S,S)-13) are Type II PAMs as measured using patch-clamp electrophysiology. Further fine control over the kinetics has been achieved by changing the substitutions on the aniline ring: generally the substitution of aniline with strong electron withdrawing groups reduces the Type II character of these compounds. Our structure−activity optimization efforts have led to the discovery of BNC375, a small molecule with good CNS-drug like properties and clinical candidate potential.

Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The a7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with a7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective a7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked a7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13 C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that a7 nAChR PAMs have multiple advantages over orthosteric a7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. SIGNIFICANCE STATEMENTBNC375 is a novel and selective a7 nicotinic acetylcholine receptor (nAChR) positive allosteric modulator (PAM) that potentiates acetylcholine-evoked a7 currents in in vitro assays with little to no effect on the desensitization kinetics. In vivo, BNC375 demonstrated robust procognitive effects in multiple preclinical models across a wide exposure range. These results suggest that a7 nAChR PAMs have therapeutic potential in central nervous system diseases with cognitive impairments.This work was funded by Merck & Co., Inc. and Bionomics Limited.