Human α6ß4 Nicotinic Acetylcholine Receptor: Heterologous Expression and Agonist Behavior Provide Insights into the Immediate Binding Site.

Study of α6ß4 nicotinic acetylcholine receptors (nAChRs) as a pharmacological target has recently gained interest because of their involvement in analgesia, control of catecholamine secretion, dopaminergic pathways, and aversive pathways. However, an extensive characterization of the human α6ß4 nAChRs has been vitiated by technical difficulties resulting in poor receptor expression. In 2020, Knowland and collaborators identified BARP (ß-anchoring and regulatory protein), a previously known voltage-gated calcium channel suppressor, as a novel human α6ß4 chaperone. Here, we establish that co-expression of human BARP with human α6ß4 in Xenopus oocytes, resulted in the functional expression of human α6ß4 receptors with acetylcholine-elicited currents that allow an in-depth characterization of the receptor using two electrode voltage-clamp electrophysiology together with diverse agonists and receptor mutations. We report: 1) an extended pharmacological characterization of the receptor, and 2) key residues for agonist-activity located in or near the first shell of the binding pocket. SIGNIFICANCE STATEMENT: The human α6ß4 nicotinic acetylcholine receptor has attained increased interest because of its involvement in diverse physiological processes and diseases. Although recognized as a pharmacological target, development of specific agonists has been hampered by limited knowledge of its structural characteristics and by challenges in expressing the receptor. By including the chaperone ß-anchoring and regulatory protein for enhanced expression and employing different ligands, we have studied the pharmacology of α6ß4, providing insight into receptor residues and structural requirements for ligands important to consider for agonist-induced activation.

The Perceived Impact of the COVID-19 Pandemic on the Social Needs of Adult Emergency Department Patients.

Objectives: We aim to assess the influence of COVID-19 on the social needs of emergency department (ED) patients, and assess patients' access to social services. Methods: We conducted a cross-sectional survey of 175 purposively sampled adult ED patients. Results: Approximately half of participants stated that COVID-19 negatively impacted their social needs with statistically significant differences observed for race, ethnicity, and insurance status. Many participants did not know of available social services, and a majority welcomed assistance from the ED. Conclusion: This study suggests that unmet social needs have risen because of COVID-19, and EDs may be positioned to identify and assist affected patients.

Characterization of Binding Site Interactions and Selectivity Principles in the α3ß4 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine receptors (nAChRs) play an important role in neurotransmission and are also involved in addiction and several disease states. There is significant interest in therapeutic targeting of nAChRs; however, achieving selectivity for one subtype over others has been a longstanding challenge, given the close structural similarities across the family. Here, we characterize binding interactions in the α3ß4 nAChR subtype via structure-function studies involving noncanonical amino acid mutagenesis and two-electrode voltage clamp electrophysiology. We establish comprehensive binding models for both the endogenous neurotransmitter ACh and the smoking cessation drug cytisine. We also use a panel of C(10)-substituted cytisine derivatives to probe the effects of subtle changes in the ligand structure on binding. By comparing our results to those obtained for the well-studied α4ß2 subtype, we identify several features of both the receptor and agonist structure that can be utilized to enhance selectivity for either α3ß4 or α4ß2. Finally, we characterize binding interactions of the α3ß4-selective partial agonist AT-1001 to determine factors that contribute to its selectivity. These results shed new light on the design of selective nAChR-targeted ligands and can be used to inform the design of improved therapies with minimized off-target effects.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

Total Synthesis of Kibdelomycin.

A modular total synthesis of kibdelomycin is disclosed that should enable structure-activity relationship (SAR) studies of this interesting class of antibiotics. The route uses simple building blocks and addresses lingering questions about its structural assignment and relationship to amycolamicin, a recently described natural product reported to have a similar structure. Initial antibacterial assays reveal that both C-22 epimers (the N-glycosidic linkage) of the natural product have similar activity while structurally truncated analogs lose activity.

Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by >30-fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Enantio- and Diastereoselective, Complete Hydrogenation of Benzofurans by Cascade Catalysis.

We report an enantio- and diastereoselective, complete hydrogenation of multiply substituted benzofurans in a one-pot cascade catalysis. The developed protocol facilitates the controlled installation of up to six new defined stereocenters and produces architecturally complex octahydrobenzofurans, prevalent in many bioactive molecules. A unique match of a chiral homogeneous ruthenium-N-heterocyclic carbene complex and an in situ activated rhodium catalyst from a complex precursor act in sequence to enable the presented process.

A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors.

Changeux et al. (Changeux et al. C. R. Biol. 343:33-39.) recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs) and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs and of the spike to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behavior of the bound Y674-R685 is highly dependent on the receptor subtype; it adopts extended conformations in the α4ß2 and α7 complexes but is more compact when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket in which it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1, and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a nonparallel arrangement to one another.

A conserved arginine with non-conserved function is a key determinant of agonist selectivity in α7 nicotinic ACh receptors.

BACKGROUND AND PURPOSE: The α7 and α4ß2* ("*" denotes possibly assembly with another subunit) nicotinic acetylcholine receptors (nAChRs) are the most abundant nAChRs in the mammalian brain. These receptors are the most targeted nAChRs in drug discovery programmes for brain disorders. However, the development of subtype-specific agonists remains challenging due to the high degree of sequence homology and conservation of function in nAChRs. We have developed C(10) variants of cytisine, a partial agonist of α4ß2 nAChR that has been used for smoking cessation. The C(10) methyl analogue used in this study displays negligible affinity for α7 nAChR, while retaining high affinity for α4ß2 nAChR. EXPERIMENTAL APPROACH: The structural underpinning of the selectivity of 10-methylcytisine for α7 and α4ß2 nAChRs was investigated using molecular dynamic simulations, mutagenesis and whole-cell and single-channel current recordings. KEY RESULTS: We identified a conserved arginine in the ß3 strand that exhibits a non-conserved function in nAChRs. In α4ß2 nAChR, the arginine forms a salt bridge with an aspartate residue in loop B that is necessary for receptor expression, whereas in α7 nAChR, this residue is not stabilised by electrostatic interactions, making its side chain highly mobile. This lack of constrain produces steric clashes with agonists and affects the dynamics of residues involved in agonist binding and the coupling network. CONCLUSION AND IMPLICATIONS: We conclude that the high mobility of the ß3-strand arginine in the α7 nAChR influences agonist binding and possibly gating network and desensitisation. The findings have implications for rational design of subtype-selective nAChR agents.

Simulations support the interaction of the SARS-CoV-2 spike protein with nicotinic acetylcholine receptors.

Changeux et al. recently suggested that the SARS-CoV-2 spike (S) protein may interact with nicotinic acetylcholine receptors (nAChRs). Such interactions may be involved in pathology and infectivity. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the S protein, to investigate this 'nicotinic hypothesis'. We examine the binding of the Y674-R685 loop of the S protein to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results indicate that Y674-R685 has affinity for nAChRs and the region responsible for binding contains the PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. In particular, R682 has a key role in the stabilisation of the complexes as it forms interactions with loops A, B and C in the receptor's binding pocket. The conformational behaviour of the bound Y674-R685 region is highly dependent on the receptor subtype, adopting extended conformations in the α4ß2 and α7 complexes and more compact ones when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket where it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1 and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of the simulations of the full-length S protein show that the Y674-R685 region is accessible for binding, and suggest a potential binding orientation of the S protein with nAChRs.

The thermodynamic profile and molecular interactions of a C(9)-cytisine derivative-binding acetylcholine-binding protein from Aplysia californica.

Cytisine, a natural product with high affinity for clinically relevant nicotinic acetylcholine receptors (nAChRs), is used as a smoking-cessation agent. The compound displays an excellent clinical profile and hence there is an interest in derivatives that may be further improved or find use in the treatment of other conditions. Here, the binding of a cytisine derivative modified by the addition of a 3-(hydroxypropyl) moiety (ligand 4) to Aplysia californica acetylcholine-binding protein (AcAChBP), a surrogate for nAChR orthosteric binding sites, was investigated. Isothermal titration calorimetry revealed that the favorable binding of cytisine and its derivative to AcAChBP is driven by the enthalpic contribution, which dominates an unfavorable entropic component. Although ligand 4 had a less unfavorable entropic contribution compared with cytisine, the affinity for AcAChBP was significantly diminished owing to the magnitude of the reduction in the enthalpic component. The high-resolution crystal structure of the AcAChBP-4 complex indicated close similarities in the protein-ligand interactions involving the parts of 4 common to cytisine. The point of difference, the 3-(hydroxypropyl) substituent, appears to influence the conformation of the Met133 side chain and helps to form an ordered solvent structure at the edge of the orthosteric binding site.

Integrating Allyl Electrophiles into Nickel-Catalyzed Conjunctive Cross-Coupling.

Allylation and conjunctive cross-coupling represent two useful, yet largely distinct, reactivity paradigms in catalysis. The union of these two processes would offer exciting possibilities in organic synthesis but remains largely unknown. Herein, we report the use of allyl electrophiles in nickel-catalyzed conjunctive cross-coupling with a non-conjugated alkene and dimethylzinc. The transformation is enabled by weakly coordinating, monodentate aza-heterocycle directing groups that are useful building blocks in synthesis, including saccharin, pyridones, pyrazoles, and triazoles. The reaction occurs under mild conditions and is compatible with a wide range of allyl electrophiles. High chemoselectivity through substrate directivity is demonstrated by the facile reactivity of the ß-γ alkene of the starting material, whereas the ϵ-ζ alkene of the product is preserved. The generality of this approach is further illustrated through the development of an analogous method with alkyne substrates. Mechanistic studies reveal the importance of the dissociation of the weakly coordinating directing group to allow the allyl moiety to bind and facilitate C(sp3 )-C(sp3 ) reductive elimination.

Overcoming the Regulatory Hurdles for the Production of Hand Sanitizer for Public Health Protection: The UK and US Academic Perspective.

The COVID-19 pandemic has led to an acute shortage of hand sanitizer, which is crucial to keeping people safe and to preventing the spread of the SARS-CoV-2 virus. However, universities across the world have used their expertise to help to meet urgent demand from public bodies and the emergency services for supplies of safe and effective sanitizer. We explore here the experience of the University of Bristol, UK, in negotiating the regulatory demands and logistical challenges facing its own sanitizer production efforts. We also reflect on the different regulatory situation for US colleagues pursuing similar activities, and we share our advice for other universities wishing to follow a similar path.

A General Mechanism for Signal Propagation in the Nicotinic Acetylcholine Receptor Family.

Nicotinic acetylcholine receptors (nAChRs) modulate synaptic activity in the central nervous system. The α7 subtype, in particular, has attracted considerable interest in drug discovery as a target for several conditions, including Alzheimer's disease and schizophrenia. Identifying agonist-induced structural changes underlying nAChR activation is fundamentally important for understanding biological function and rational drug design. Here, extensive equilibrium and nonequilibrium molecular dynamics simulations, enabled by cloud-based high-performance computing, reveal the molecular mechanism by which structural changes induced by agonist unbinding are transmitted within the human α7 nAChR. The simulations reveal the sequence of coupled structural changes involved in driving conformational change responsible for biological function. Comparison with simulations of the α4ß2 nAChR subtype identifies features of the dynamical architecture common to both receptors, suggesting a general structural mechanism for signal propagation in this important family of receptors.

Probing Binding Interactions of Cytisine Derivatives to the α4ß2 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine receptors (nAChRs) are crucial for communication between synapses in the central nervous system. As such, they are also implicated in several neuropsychiatric and addictive diseases. Cytisine is a partial agonist of some nAChRs and has been used for smoking cessation. Previous studies have established a binding model for several agonists to several nAChR subtypes. Here, we evaluate the extent to which this model applies to cytisine at the α4ß2 nAChR, which is a subtype that is known to play a prominent role in nicotine addiction. Along with the commonly seen cation-π interaction and two hydrogen bonds, we find that cytisine makes a second cation-π interaction at the agonist binding site. We also evaluated a series of C(10)-substituted cytisine derivatives, using two-electrode voltage-clamp electrophysiology and noncanonical amino acid mutagenesis. Double-mutant cycle analyses revealed that C(10) substitution generally strengthens the newly established second cation-π interaction, while it weakens the hydrogen bond typically seen to LeuE in the complementary subunit. The results suggest a model for how cytisine derivatives substituted at C(10) (as well as C(9)/C(10)) adjust their binding orientation, in response to pyridone ring substitution.

Identification of the Initial Steps in Signal Transduction in the α4ß2 Nicotinic Receptor: Insights from Equilibrium and Nonequilibrium Simulations.

Nicotinic acetylcholine receptors (nAChRs) modulate synaptic transmission in the nervous system. These receptors have emerged as therapeutic targets in drug discovery for treating several conditions, including Alzheimer's disease, pain, and nicotine addiction. In this in silico study, we use a combination of equilibrium and nonequilibrium molecular dynamics simulations to map dynamic and structural changes induced by nicotine in the human α4ß2 nAChR. They reveal a striking pattern of communication between the extracellular binding pockets and the transmembrane domains (TMDs) and show the sequence of conformational changes associated with the initial steps in this process. We propose a general mechanism for signal transduction for Cys-loop receptors: the mechanistic steps for communication proceed firstly through loop C in the principal subunit, and are subsequently transmitted, gradually and cumulatively, to loop F of the complementary subunit, and then to the TMDs through the M2-M3 linker.

(-)-Cytisine: Access to a stereochemically defined and functionally flexible piperidine scaffold.

N-Benzyl cytisine undergoes an efficient C(6)-N(7) cleavage via directed C(6) lithiation, borylation and oxidation to provide a "privileged" heterocyclic core unit comprising a highly functionalised, cis-3,5-disubstituted piperidine in enantiomerically pure form. The potential offered by this unit as a means to explore chemical space has been evaluated and methods have been defined (and illustrated) that allow for selective manipulation of N(1), C(3'), and the pyridone N. The pyridone core can also be diversified via bromination (at C(3'') and C(5'')) which is complementary to direct C-H activation based on Ir-catalyzed borylation to provide access to C(4''). The use of a boronate-based 1,2-migration as an alternative trigger to mediate C(6)-N(7) cleavage of cytisine was evaluated but failed. However, the stability of the intermediate boronate opens a new pathway for the elaboration of cytisine itself using both Matteson homologation and Zweifel olefination.