Structure-guided drug design: Conferring selectivity among neuronal nicotinic receptor and acetylcholine-binding protein subtypes

Abstract

Neuronal nicotinic receptors, encoded by nine genes of the alpha and three of the beta type of subunits, and whose gene products assemble in distinct permutations as pentameric molecules, constitute a fertile area for structure-guided drug design. Design strategies are augmented by a wide variety of peptide, alkaloid and terpenoid toxins from various marine and terrestrial species that interact with nicotinic receptors. Also, acetylcholinebinding proteins from mollusks, as structural surrogates of the receptor that mimic its extracellular domain, provide atomic resolution templates for analysis of structure and response. Herein, we describe a structure-guided approach to nicotinic ligand design that employs crystallography of this protein as the basic template, but also takes into consideration the dynamic properties of the receptor molecules in their biological media. We present the crystallographic structures of several complexes of various agonists and antagonists that associate with the agonist site and can competitively block the action of acetylcholine. In so far as the extracellular domain is involved, we identify additional noncompetitive sites at those subunit interfaces where agonists do not preferentially bind. Ligand association at these interface sites may modulate receptor function. Ligand binding is also shown by solution-based spectroscopic and spectrometric methods to affect the dynamics of discrete domains of the receptor molecule. The surrogate receptor molecules can then be employed to design ligands selective for receptor subtype through the novel methods of freeze-frame, click chemistry that uses the very structure of the target molecule as a template for synthesis of the inhibitor.

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

“…Hence, an alternative methodology must be considered. In Cys-loop receptors, the neurotransmitter-binding pockets lie at the interface between adjacent subunits (1)(2)(3)(4)(5)(6)(7)(8)(9). One could therefore use site-specific mutagenesis and biophysical characterization of activation mechanisms in recombinant receptors to find the types of subunits that line the agonist-binding pockets.…”

“…They belong to the Cys-loop receptor superfamily of transmembrane oligomers that open an intrinsic cationic or anionic channel pore upon binding of neurotransmitters, such as ACh, serotonin, GABA, Gly, histamine, or Glu (1)(2)(3)(4)(5)(6)(7)(8)(9). GluClRs are specific targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat filarial diseases like onchocerciasis (river blindness) and elephantiasis (lymphatic filariasis) that afflict hundreds of millions of people worldwide (10,11).…”

Creating an α7 Nicotinic Acetylcholine Recognition Domain from the Acetylcholine-binding Protein

“…1 and supplemental Table S3), as well as a third mutant (mutant IV) that added Q162E, V163A, L165I, and S167G to mutant III were chosen as further intermediates to test for h␣7-nAChR selectivity. Because previous reports suggest that the ␤9 -10 linker (C-loop) plays a defined role in ligand binding and gating (21,38,39), we looked at mutant II, which lacked mutation sets 5 and 1 of the ␤9 -10 linker from mutant III. Mutant IV proved unstable in yielding consistent binding characteristics and FPLC profiles (data not shown); therefore, mutant III was chosen as a final mutant of the mutated ligand-binding domain.…”

“…Several studies have shown expression of ␣7 nAChR extracellular domains (9 -11) or variants (12)(13)(14), yet none have resulted in crystal structures. Studies involving the Lymnaea stagnalis (Ls) and Aplysia californica (Ac) acetylcholine-binding proteins (AChBP) have yielded many structural analyses of the binding site for these mollusk homologues (15)(16)(17)(18)(19)(20)(21). Recently determined structures of multiple ligand complexes with the AcAChBP (18,19), along with binding and mutagenesis studies, have begun to define the structural determinants of ligand specificity (4,8).…”

Basic and modern concepts on cholinergic receptor: A review

“…In situ and in vitro, their behavior resembles that expected for a high-affinity antagonist. Since cholinergic neurotransmission mediates motor activity in marine vertebrates and mammals, a large number of peptide, terpinoid, and alkaloid toxins that block the nicotinic receptors have evolved to enhance predation or protect plant and animal species from predation [23] . Purification of the receptor from Torpedo ultimately led to isolation of complementary DNAs (cDNAs) that encode each of the subunits.…”