From High-Throughput Screening to Target Validation: Benzo[d]isothiazoles as Potent and Selective Agonists of Human Transient Receptor Potential Cation Channel Subfamily M Member 5 Possessing In Vivo Gastrointestinal Prokinetic Activity in Rodents.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.

Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6ß2 properties: discovery of selective antagonists.

BACKGROUND AND PURPOSE: Despite growing evidence that inhibition of α6ß2-containing (α6ß2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of α6ß2*-nAChRs has delayed the discovery of α6ß2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with α6ß2 properties, to enable pharmacological characterization and the identification of novel α6ß2-selective antagonists. EXPERIMENTAL APPROACH: Different combinations of the α6, ß2, ß3, chimeric α6/3 and mutant ß3(V273S) subunits were transfected in human embryonic kidney cells and tested for activity in a fluorescent imaging plate reader assay. The pharmacology of rat immune-immobilized α6ß2*-nAChRs was determined with ¹²5I-epibatidine binding. KEY RESULTS: Functional channels were detected after co-transfection of α6/3, ß2 and ß3(V273S) subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed α6/3ß2ß3(V273S)-nAChR pharmacology was unique, and clearly distinct from α4ß2-, α3ß4-, α7- and α1ß1δε-nAChRs. Antagonist potencies in inhibiting α6/3ß2ß3(V273S) -nAChRs was similar to their binding affinity for rat native α6ß2*-nAChRs. Agonist affinities for α6ß2*-nAChRs was higher than their potency in activating α6/3ß2ß3(V273S)-nAChRs, but their relative activities were equivalent. Focussed set screening at α6/3ß2ß3(V273S)-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel α6ß2-selective antagonists. CONCLUSIONS AND IMPLICATIONS: We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native α6ß2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, α6ß2-selective antagonists. We also propose a pharmacophore model of α6ß2 antagonists, which offers a starting point for the development of new smoking cessation agents.

Identification of novel alpha7 nAChR positive allosteric modulators with the use of pharmacophore in silico screening methods.

The pharmacophore model of in house potent and selective alpha7 nAChR positive allosteric modulators is reported. The model was used to fish out commercially-available compounds from corporate 3D databases. As a result, novel alpha7 positive modulator chemotypes were identified. A rat full PK profile of a representative compound is also described.

Discovery and optimization of highly ligand-efficient oxytocin receptor antagonists using structure-based drug design.

A novel oxytocin antagonist was identified by 'scaffold-hopping' using Cresset FieldScreen molecular field similarity searching. A single cycle of optimization driven by an understanding of the key pharmacophoric elements required for activity led to the discovery of a potent, selective and highly ligand-efficient oxytocin receptor antagonist. Selectivity over vasopressin receptors was rationalized based on differences in the structure of the natural ligands.

Discovery and optimisation of a potent and selective tertiary sulfonamide oxytocin antagonist.

The optimisation of a tertiary sulfonamide high-throughput screening hit is described. A combination of high-throughput chemistry, pharmacophore analysis and in silico PK profiling resulted in the discovery of potent sulfonamide oxytocin receptor antagonists with oral exposure and good selectivity over vasopressin receptors.

Nitrogen substitution modifies the activity of cytisine on neuronal nicotinic receptor subtypes.

Cytisine very potently binds and activates the alpha 3 beta 4 and alpha 7 nicotinic subtypes, but only partially agonises the alpha 4 beta 2 subtype. Although with a lower affinity than cytisine, new cytisine derivatives with different substituents on the basic nitrogen (CC1-CC8) bind to both the heteromeric and homomeric subtypes, with higher affinity for brain [3H]epibatidine receptors. The cytisine derivatives were tested on the Ca(2+) flux of native or transfected cell lines expressing the rat alpha 7, or human alpha 3 beta 4 or alpha 4 beta 2 subtypes using Ca(2+) dynamics in conjunction with a fluorescent image plate reader. None elicited any response at doses of up to 30-100 microM, but all inhibited agonist-induced responses. Compounds CC5 and CC7 were also electrophysiologically tested on oocyte-expressed rat alpha 4 beta 2, alpha 3 beta 4 and alpha 7 subtypes. CC5 competitively antagonised the alpha 4 beta 2 and alpha 3 beta 4 subtypes with similar potency, whereas CC7 only partially agonised them with maximum responses of respectively 3% and 11% of those of 1 mM acetylcholine. Neither compound induced any current in the oocyte-expressed alpha 7 subtype, and both weakly inhibited acetylcholine-induced currents. Adding chemical groups of a different class or size to the basic nitrogen of cytisine leads to compounds that lose full agonist activity on the alpha 3 beta 4 and alpha 7 subtypes.