Pharmacological Characterization of the Novel and Selective α7 Nicotinic Acetylcholine Receptor-Positive Allosteric Modulator BNC375.

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 α7 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 α7 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 α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 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 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. SIGNIFICANCE STATEMENT: BNC375 is a novel and selective α7 nicotinic acetylcholine receptor (nAChR) positive allosteric modulator (PAM) that potentiates acetylcholine-evoked α7 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 α7 nAChR PAMs have therapeutic potential in central nervous system diseases with cognitive impairments.

Identifying key amino acid residues that affect α-conotoxin AuIB inhibition of α3ß4 nicotinic acetylcholine receptors.

α-Conotoxin AuIB is a selective α3ß4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3ß4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3ß4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3ß4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3ß4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the ß4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the ß4-Trp-59 and ß4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the ß4 subunit and gave further insight into how AuIB Phe-9 interacts with the ß4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively "avoid" nAChR receptors while targeting receptors involved with nociception.

Alpha-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of alpha3beta4 nicotinic acetylcholine receptors.

Non-native disulfide isomers of alpha-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of alpha-conotoxin AuIB have been characterized on alpha3beta4 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 approximately 50%. The maximal inhibition was stoichiometry-dependent because altering alpha3:beta4 RNA injection ratios either increased AuIB(ribbon) efficacy (10alpha:1beta) or completely abolished blockade (1alpha:10beta). 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 alpha3beta4 nAChRs with corresponding EC(50) values of 22.6 and 176.9 microM, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha3beta4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS alpha3beta4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS alpha3beta4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both alpha3beta4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS alpha3beta4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS alpha3beta4 nAChRs competitively, whereas AuIB(globular) inhibits alpha3beta4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.

Solving the alpha-conotoxin folding problem: efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antagonists.

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.

Discovery of BNC375, a Potent, Selective, and Orally Available Type I Positive Allosteric Modulator of α7 nAChRs.

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.

Differential calcium-dependent modulation of NMDA currents in CA1 and CA3 hippocampal pyramidal cells.

Neuronal Ca2+ 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 Ca2+ 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 Ca2+ buffering in CA3 cells or decreasing Ca2+ buffering in CA1 cells, respectively. Our data suggest that a differential Ca2+ 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 NMDAR-dependent ischemic cell death.

Muscarinic receptor stimulation reduces NMDA responses in CA3 hippocampal pyramidal cells via Ca2+-dependent activation of tyrosine phosphatase.

N-methyl-D-aspartate (NMDA)-type glutamate receptors perform critical functions during the development of the nervous system and in the initiation of synaptic plasticity. An important mechanism in setting the gain of NMDA receptors involves the stimulation of G-protein-coupled receptors (GPCRs), which through activation of protein tyrosine kinases leads to an upregulation of NMDA receptors. In contrast, little is known about how NMDA receptors are downregulated. In the present study, we characterized a signaling pathway that mediates the depression of NMDA receptor function in response to stimulation of muscarinic acetylcholine receptors. Whole-cell patch-clamp recordings obtained from CA3 pyramidal cells in organotypic slice cultures revealed that under conditions of low intracellular calcium buffering application of muscarine-depressed NMDA receptor current. The sensitivity of this response to pirenzipine indicated that the M1 acetylcholine receptor is mediating this depression. The muscarine-induced depression of NMDA current was prevented by blocking G-protein function or after depleting intracellular Ca2+ stores with cyclopiazonic acid. Inhibitors of calmodulin prevented the depression whereas blocking calcineurin enhanced the depression of NMDA currents. Blocking tyrosine phosphatase activity with pervanandate converted the muscarine-induced depression into a potentiation of NMDA currents, whereas blocking protein kinase A (H-89), Src kinase (PP2, SU6656), or PKC (GF 109203X) failed to prevent the depression of NMDA currents. As Src tyrosine kinase is known to phosphorylate and upregulate NMDA receptors, we propose that a protein tyrosine phosphatase(s) counteracting the action of Src is the final target in the mAChR-dependent inhibitory signaling cascade. Our data are consistent with a transduction cascade comprising an M1 acetylcholine receptor-->G-protein-->Ca2+ release-->calmodulin-->tyrosine phosphatase.

Stabilization of α-conotoxin AuIB: influences of disulfide connectivity and backbone cyclization.

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.