Thiol-reactive analogues of galanthamine, codeine and morphine as potential probes to interrogate allosteric binding within nAChRs

Alkaloids including galanthamine (1) and codeine (2) are reported to be positive allosteric modulators of nicotinic acetylcholine receptors (nAChRs) but the binding sites responsible for this activity are not known with certainty. Analogues of galanthamine (1), codeine (2) and morphine (3) with reactivity towards cysteine thiols were synthesised including conjugated enone derivatives of the three alkaloids 4-6 and two chloro-alkane derivatives of codeine 7 and 8. The stability of the enones was deemed sufficient for use in buffered aqueous solutions and their reactivity towards thiols was assessed by determining the kinetics of reaction with a cysteine derivative. All three enone derivatives were of sufficient reactivity and stability to be used in covalent trapping, an extension of the substituted cysteine accessibility method (SCAM), to elucidate the allosteric binding sites of galanthamine and codeine at nAChRs

Alpha9 nicotinic acetylcholine receptors and the treatment of pain

Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. α-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as α-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of α9α10 nAChRs. A recent study also reported that these α9α10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on α9* nAChRs.11* indicates the possible presence of additional subunits.Fil: McIntosh, J. Michael. University of Utah; Estados UnidosFil: Absalom, Nathan. The University of Sydney; AustraliaFil: Chebib, Mary. The University of Sydney; AustraliaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Farmacología; ArgentinaFil: Vincler, Michelle. Wake Forest University Health Sciences; Estados Unido

Identification of GABA receptors in chick cornea

Purpose: The cornea has an important role in vision, is highly innervated and many neurotransmitter receptors are present, e.g., muscarine, melatonin, and dopamine receptors. γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and central nervous system, but it is unknown whether GABA receptors are present in cornea. The aim of this study was to determine if GABA receptors are located in chick cornea. Methods: Corneal tissues were collected from 25, 12-day-old chicks. Real time PCR, western blot, and immunohistochemistry were used to determine whether alpha1 GABAA, GABAB, and rho1 GABAC receptors were expressed and located in chick cornea. Results: Corneal tissue was positive for alpha1 GABAA and rho1 GABAC receptor mRNA (PCR) and protein (western blot) expression but was negative for GABAB receptor mRNA and protein. Alpha1 GABAA and rho1 GABAC receptor protein labeling was observed in the corneal epithelium using immunohistochemistry. Conclusions: These investigations clearly show that chick cornea possesses alpha1 GABAA, and rho1 GABAC receptors, but not GABAB receptors. The purpose of the alpha1 GABAA and rho1 GABAC receptors in cornea is a fascinating unexplored question

Efficiency considerations in the construction of interpolated potential energy surfaces for the calculation of quantum observables by diffusion Monte Carlo

A modified Shepard interpolation scheme is used to construct global potential energy surfaces (PES) in order to calculate quantum observables--vibrationally averaged internal coordinates, fully anharmonic zero-point energies and nuclear radial distribution functions--for a prototypical loosely bound molecular system, the water dimer. The efficiency of PES construction is examined with respect to (a) the method used to sample configurational space, (b) the method used to choose which points to add to the PES data set, and (c) the use of either a one- or two-part weight function. The most efficient method for constructing the PES is found to require a quantum sampling regime, a combination of both h-weight and rms methods for choosing data points and use of the two-part weight function in the interpolation. Using this regime, the quantum diffusion Monte Carlo zero-point energy converges to the exact result within addition of 50 data points. The vibrationally averaged O-O distance and O-O radial distribution function, however, converge more slowly and require addition of over 500 data points. The methods presented here are expected to be applicable to both other loosely bound complexes as well as tightly bound molecular species. When combined with high quality ab initio calculations, these methods should be able to accurately characterize the PES of such species.D.L.C. would like to acknowledge the financial support of an Australian Postgraduate Research Award. This work has also been supported by Large Grant No. A00104447 from the Australian Research Council and by grants of computer time from the Australian Partnership in Advanced Computing (APAC) National Merit Allocation Scheme

Investigating the role of loop c hydrophilic residue 'T244' in the binding site of ρ1 GABAC receptors via site mutation and partial agonism

The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand

Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors : evidence of a novel benzodiazepine site in the α1-α1 interface

Zolpidem is not a typical GABAA receptor hypnotic. Unlike benzodiazepines, zolpidem modulates tonic GABA currents in the rat dorsal motor nucleus of the vagus, exhibits residual effects in mice lacking the benzodiazepine binding site, and improves speech, cognitive and motor function in human patients with severe brain injury. The receptor by which zolpidem mediates these effects is not known. In this study we evaluated binary α1β3 GABAA receptors in either the 3α1:2β3 or 2α1:3β3 subunit stoichiometry, which differ by the existence of either an α1-α1 interface, or a β3-β3 interface, respectively. Both receptor stoichiometries are readily expressed in Xenopus oocytes, distinguished from each other by using GABA, zolpidem, diazepam and Zn2+. At the 3α1:2β3 receptor, clinically relevant concentrations of zolpidem enhanced GABA in a flumazenil-sensitive manner. The efficacy of diazepam was significantly lower compared to zolpidem. No modulation by either zolpidem or diazepam was detected at the 2α1:3β3 receptor, indicating that the binding site for zolpidem is at the α1-α1 interface, a site mimicking the classical α1-γ2 benzodiazepine site. Activating α1β3 (3α1:2β3) receptors may, in part, mediate the physiological effects of zolpidem observed under distinct physiological and clinical conditions, constituting a potentially attractive drug target

Novel Approach for the Search for Chemical Scaffolds with Activity at Both Acetylcholinesterase and the α7 Nicotinic Acetylcholine Receptor: A Perspective on Scaffolds with Dual Activity for the Treatment of Neurodegenerative Disorders

Publisher's version (útgefin grein)Neurodegenerative disorders, including Alzheimer’s disease, belong to the group of the most difficult and challenging conditions with very limited treatment options. Attempts to find new drugs in most cases fail at the clinical stage. New tactics to develop better drug candidates to manage these diseases are urgently needed. It is evident that better understanding of the neurodegeneration process is required and targeting multiple receptors may be essential. Herein, we present a novel approach, searching for dual active compounds interacting with acetylcholinesterase (AChE) and the α7 nicotinic acetylcholine receptor (nAChR) using computational chemistry methods including homology modelling and high throughput virtual screening. Activities of identified hits were evaluated at the two targets using the colorimetric method of Ellman and two-electrode voltage-clamp electrophysiology, respectively. Out of 87,250 compounds from a ZINC database of natural products and their derivatives, we identified two compounds, 8 and 9, with dual activity and balanced IC50 values of 10 and 5 µM at AChE, and 34 and 14 µM at α7 nAChR, respectively. This is the first report presenting successful use of virtual screening in finding compounds with dual mode of action inhibiting both the AChE enzyme and the α7 nAChR and shows that computational methods can be a valuable tool in the early lead discovery processThis research was supported by the Australian National Health and Medical Research Council [NHMRC grant number APP1069417], The Australian Research Council [ARC grant number LP140100781], a doctoral grant and financial support from the University of Iceland, a grant from the Bergthóru and Thorsteins Scheving Thorsteinsson Fund, and the Icelandic Research Fund [grant number: 152604051].Peer Reviewe

Concatenated γ-aminobutyric acid type A receptors revisited: Finding order in chaos

Publisher's version (útgefin grein)γ-aminobutyric acid type A receptors (GABAARs), the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are arguably the most challenging member of the pentameric Cys-loop receptors to study due to their heteromeric structure. When two or more subunits are expressed together in heterologous systems, receptors of variable subunit type, ratio, and orientation can form, precluding accurate interpretation of data from functional studies. Subunit concatenation is a technique that involves the linking of individual subunits and in theory allows the precise control of the uniformity of expressed receptors. In reality, the resulting concatemers from widely used constructs are flexible in their orientation and may therefore assemble with themselves or free GABAAR subunits in unexpected ways. In this study, we examine functional responses of receptors from existing concatenated constructs and describe refinements necessary to allow expression of uniform receptor populations. We find that dimers from two commonly used concatenated constructs, β-23-α and α-10-β, assemble readily in both the clockwise and the counterclockwise orientations when coexpressed with free subunits. Furthermore, we show that concatemers formed from new tetrameric α-10-β-α-β and α-10-β-α-γ constructs also assemble in both orientations with free subunits to give canonical αβγ receptors. To restrict linker flexibility, we systematically shorten linker lengths of dimeric and pentameric constructs and find optimized constructs that direct the assembly of GABAARs only in one orientation, thus eliminating the ambiguity associated with previously described concatemers. Based on our data, we revisit some noncanonical GABAAR configurations proposed in recent years and explain how the use of some concatenated constructs may have led to wrong conclusions. Our results help clarify current contradictions in the literature regarding GABAAR subunit stoichiometry and arrangement. The lessons learned from this study may guide future efforts in understanding other related heteromeric receptors.This work was supported by the Australian Research Council (LP160100560) and the Australian National Health and Medical Research Council (APP1124567 and APP1081733 to M. Chebib, P.K. Ahring, and T. Balle). N.M. Kowal was supported by the Icelandic Research Fund (grant number 152604). The authors declare no competing financial interests. Author contributions: P.K. Ahring designed the research; V.W.Y. Liao, P.K. Ahring, H.C. Chua, N.M. Kowal, and T. Balle performed the research; P.K. Ahring analyzed the data and wrote the manuscript. P.K. Ahring, T. Balle, and M. Chebib acquired the necessary funding. All authors approved the final version of the manuscript. Richard W. Aldrich served as editor.Peer Reviewe

The synthesis and evaluation of fluoro-, trifluoromethyl, and iodomuscimols as GABA agonists

MAFAM acknowledges the Ministry of Education of Malaysia and Universiti Teknologi MARA for a scholarship. MC acknowledges the funding support of the Australian National Health and Medical Research Council (APP1081733).Halogenated analogues of the neurotoxic alkaloid muscimol were prepared with fluorine, iodine or trifluoromethyl at the 4 position of the isoxazole ring system. These compounds were investigated as agonists for GABAA receptors. Only the C-4 fluorine containing analogue proved to be an active compound in these assays. The fluoro analogue was less active than muscimol, however it showed differential activity between synaptic (α1β2γ2) and extrasynaptic (α4β2γ) GABAA receptors, having a similar potency to the neurotransmitter GABA for the extrasynaptic (α4β2γ) receptor.PostprintPostprintPeer reviewe

Cannabigerolic acid, a major biosynthetic precursor molecule in cannabis, exhibits divergent effects on seizures in mouse models of epilepsy

Background and Purpose: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties. Experimental Approach: We used the Scn1a+/− mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a+/− mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets. Key Results: The initial screen identified three phytocannabinoids with novel anticonvulsant properties: CBGA, cannabidivarinic acid (CBDVA) and cannabigerovarinic acid (CBGVA). CBGA was most potent and potentiated the anticonvulsant effects of clobazam against hyperthermia-induced and spontaneous seizures, and was anticonvulsant in the MES threshold test. However, CBGA was proconvulsant in the 6-Hz threshold test and a high dose increased spontaneous seizure frequency in Scn1a+/− mice. CBGA was found to interact with numerous epilepsy-relevant targets including GPR55, TRPV1 channels and GABAA receptors. Conclusion and Implications: These results suggest that CBGA, CBDVA and CBGVA may contribute to the effects of cannabis-based products in childhood epilepsy. Although these phytocannabinoids have anticonvulsant potential and could be lead compounds for drug development programmes, several liabilities would need to be overcome before CBD is superseded by another in this class