Ligand gated ion channels (LGICs) are currently very important targets for drug discovery in the pharmaceutical industry. The superfamily is separated into the nicotinic receptor superfamily (muscular and neuronal nicotinic, GABA-A and-C, glycine and 5-HT3 receptors), the excitatory amino acid superfamily (glutamate, aspartate and kainate receptors) and the ATP purinergic ligand gated ion channels. These families only differ in the number of transmembrane domains found in each subunit (nicotinic—4 transmembrane domains, excitatory amino acid receptors—3 transmembrane domains, ATP purinergic LGICs—2 transmembrane domains).
Nicotinic acetylcholine receptors (nAChRs) are a family of ligand gated ion channels that control the fast permeation of cations through the postsynaptic cell membrane when stimulated by acetylcholine. Physiologically, nAChRs are key targets in drug discovery for a number of diseases, including Alzheimer's and Parkinson's disease, and have been widely discussed and investigated.
Structural and functional studies of nAChRs have led to the elucidation of three physiological states of the receptor: 1) resting (channel closed); 2) acetylcholine stimulated (channel open); and 3) a desensitized state where the ion flux is inhibited even in the presence of neurotransmitter. The overall structure of nicotinic acetylcholine receptor of Torpedo marmorata has been examined by Unwin and coworkers using cryo-electron microscopy and revealed the conical shape of the channel portion of the receptor and the relationship of the membrane-spanning helices to each other (see FIG. 1). In spite of these unprecedented advances in resolving the structures of transmembrane proteins, the detailed, atomic resolution, structure of the entire nAChR family remains unresolved.
Muscular nAChRs are located at the nerve-muscle junctions and are responsible for triggering motor motion, and neuronal nAChRs, widely distributed in the nervous system, are involved in the fast synaptic transmission of inter-neuronal communication. It is known that these receptors are structurally similar in their overall composition but differ in the exact make-up of the protein subunits forming the receptors.
The nicotinic acetylcholine receptor (nAChR) is presently the best characterized member of the ligand-gated ion channel superfamily. The nicotinic receptors are of great therapeutic importance. The subunits assemble combinatorily to form a variety of pentameric transmembrane protein subtypes.
Each receptor is formed by bringing together five separate trans-membrane proteins, each containing a large extra-cellular N-terminal domain, four membrane spanning alpha helices (M1, M2, M3, and M4) and a small C-terminal domain (see FIG. 1). Two, homologous, neurotransmitter binding sites are formed by the N-terminal domains where cholinergic agonist and competitive antagonists bind, and are the usual targets for drug design. The ion channel is formed by a pentameric arrangement of the M2 helical segments contributed by the five proteins (see FIG. 2). The channel specificity, characteristic of each receptor subtype, is controlled by the identity of each of the M2 helices.
Neuronal nicotinic acetylcholine receptors (nAChRs) are the class of ligand-gated ion channels of the central and peripheral nervous system that regulate synaptic activity. The basic structure of the nAChR is shown in FIGS. 1 and 2. Referring to FIG. 1, nAChR consists of five transmembrane subunits 1, 2, 3, 4, 5 oriented around a central pore 6 permeable to cations. Cations flow through the pore is regulated by ligand binding. The subunits in nAChR are typically α subunits and β subunits.
At present, 12 different homologous subunits have been identified in neuronal nAChRs, 9 α subunits (α2-α10) and 3 β subunits (β2-β4). The major difference between α and β subunits is the presence and location of the disulfide bond formed by two adjacent cysteines in the α systems, the absence of this feature distinguishes non-α subunits. This disulfide bond located on the extracellular domain plays an important role in neurotransmitter binding as well as the mechanism of channel opening. These subunits combine to form multiple nAChR subtypes and predominant stoichiometry is (α)2(β)3, however pentamers containing only α subunit are also known e.g., (α7)5. In case of muscular nAChR the stoichiometry is more complicated, the muscular nAChR receptor is predominantly described as (α)2βδγ.
The nAChRs are very complex systems with dozens of potential different binding domains for different classes of compounds of both endo- and exogenous origin (Arias H. R., (1997) Topology of ligand binding sites on the nicotinic acetylcholine receptor. Brain Res. Rev. 25: 133-91). Two primary cholinergic binding sites are located on the extracellular side 7 (refer to FIG. 1, approximately 30-35 Å above the membrane) in the pocket at the interface between the α and β subunits. The nAChR contains several other classes of binding sites at which non-competitive inhibitors (NCIs) bind (Arias H. R. (1998) Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor. Biochim. Biophys. Act. 1376: 173-220). One, so-called “luminal high affinity” NCI binding domain is located on the surface of the internal lumen forming the ion channel. This site is a highly polar and negatively charged domain, which primarily plays the role as a cation selector. In general, an NCI compound does not compete with the neurotransmitter ligand of the receptor for binding to the neurotransmitter ligand binding site of the receptor located on the external surface both α subunits in a pocket approximately 30-35 Å from the transmembrane portion of the subunit (that is, above the surface membrane when the receptor is expressed on in a cell), as described by Arias [Arias, H. R. (2000) Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors Neurochem. Int 36, 595-645].
Such drugs as mecamylamine, ketamine, bupropion or barbiturates bind in the narrowest region of the channel on the cell membrane level. Inhibitors acting there are mainly amines. It is believed that the ligands bind into this region and sterically plug the channel, blocking the flux of ions.
“Non-luminal” sites are the population of 10-30 binding sites located mostly at the lipid-protein interface for which an allosteric mechanism of non-competitive inhibition was proposed. Agents of different origin (steroids, fatty acids, alcohols, local anesthetics etc.) can bind to those sites and modulate nAChR activity.
Other classes of ligand-gated ion channels include GABA (Johnston G. A. (2002) Medicinal chemistry and molecular pharmacology of GABA(C) receptors. Curr Top Med Chem 2, 903-13), 5HT3 (D. C. Reeves, S. C. Lummis, (2002) The molecular basis of the structure and function of the 5-HT3 receptor: a model ligand-gated ion channel (review). Mol. Membr. Biol. 19, 11-26), AMPA (T. B. Stensbol, U Madsen, P. Krogsgaard-Larsen, (2002) The AMPA receptor binding site: focus on agonists and competitive antagonists. Curr. Pharm. Des. 8, 857-72) and NMDA (K. A. Macritchie, A. H. Young, (2001) Emerging targets for the treatment of depressive disorder. Expert Opin. Ther. Targets 5, 601-612) receptors, etc. Although the molecular structure of these receptors differ significantly, it is believed that the luminal domains are homologous to the luminal domain of nAChRs. There are five (or occasionally four) transmembrane helices forming the wall of the channel with “rings” of polar amino-acids exposed on the pre-forming surface and the same non-competitive inhibition phenomenon can be observed.
In summary, the luminal high affinity NCI binding domain is located on the surface of the internal lumen forming the ion channel. Drugs of different origin bind in this region and sterically plug the channel blocking the flux of ions.
Non-competitive inhibition of the nAChR can be responsible for severe adverse drug effects. On the other hand, designing ligands that specifically interact with this site can be part of the development of new treatments of Alzheimer's and Parkinson's diseases, for example by identifying compounds likely to exhibit side effects through non-competitive inhibition of a LGIC. Furthermore, the compounds identified as NCIs by the present method are likely to find use in treating Tourette's syndrome and cognitive disorders, schizophrenia, pain [see, Lloyd, G. K. and Williams, M. (2000) J. Pharmacol. Exper. Ther. 292, 461-467.], anxiety, depression, neurodegeneration and addictions caused by an overactive LGIC receptor, especially diseases in which nicotine agonist activity against a neuronal nAChR is part of the etiology (e.g. smoking addiction). The invention can also be used to evaluate cardiovascular toxicity of a compound mediated by non-competitive inhibition of a LGIC receptor, e.g. arrhythmia and GI spasming or diarrheal side effects of a compound caused by inhibition of a muscular nAChR.
Classical methods of NCI identification are time consuming and not effective in rapid screening of chemical libraries of drug candidates.
Several different molecular models of the nAChR transmembrane domain have been reported (Capener C E, Kim H J, Arinaminpathy Y, Sansom M S (2002) Ion channels: structural bioinformatics and modelling. Hum Mol Genet 11:2425-33). However, none of those models were used to investigate interaction with channel blockers. A computer based model for in silico simulations of NCI interactions with the luminal domain of LGICs is needed to better understand the phenomenon of the receptor's inhibition by NCIs.
Furthermore, in drug discovery, the potential adverse effects of drug candidates are of great importance. In-depth understanding of mechanistic interaction of luminal NCIs with different subtypes of LGICs, especially of nAChRs, is required to remove potential unwanted side effects at this site. In this respect, a rapid screening technology that would identify NCIs of LGICs, and especially of nAChRs would be greatly desired.
The functional determination and characterization of a NCI of a LGIC is very complex and time consuming. One approach is affinity chromatography based on immobilized receptor protein. This is a versatile tool for investigation of intermolecular interactions of a receptor with its ligands. The chemometric approach of affinity chromatography can be employed for determination of reliable relative affinities of ligands as well as kinetic characterization, which otherwise would be inaccessible, for a large set of compounds (Kaliszan R., Wainer I. W. (1997) Combination of Biochromatography and Chemometrics: A Potential New Research Strategy in Molecular Pharmacology and Drug Design. In Chromatographic Separations Based on Molecular Recognition. K. Jinno, editors Wiley-VCH).
Methods using nAChR and other receptors immobilized on a chromatographic support have been elaborated (U.S. Pat. Nos. 6,387,268, 6,139,735, provisional application No. 60/337,172). It was shown that the obtained stationary phases worked as selective binding materials for competitive cholinergic ligands and can be used for high throughput screening of various competitive agonists and antagonists (R. Moaddel, I. W. Wainer, (2003) Immobilized nicotinic receptor stationary phases: going with the flow in high-throughput screening and pharmacological studies J Pharm Biomed Anal. 30, 1715-24). The usefulness of such columns based on immobilized nAChR for investigations and modeling of NCI affinity has also been demonstrated. Using a novel non-linear chromatography approach off and on kinetics of ligand interaction with the receptor can be determined. (K. Jozwiak, J. Haginaka et al., (2002) Displacement and nonlinear chromatographic techniques in the investigation of interaction of noncompetitive inhibitors with an immobilized α3β4 nicotinic acetylcholine receptor liquid chromatographic stationary phase. Anal Chem 74: 4618-4624).