Differential regulation of alcohol taking and seeking by antagonism at α4ß2 and α3ß4 nAChRs.

RATIONALE: Alcoholism is a serious public health problem throughout the world. Current pharmacotherapies for the treatment of this disorder are poorly effective. Preclinical and clinical findings point to nicotinic acetylcholine receptors (nAChRs) as a promising target for the development of novel and effective medications. Assuage Pharmaceuticals, in collaboration with Torrey Pines Institute for Molecular Studies, has discovered a new class of potent and selective α4ß2 nAChR antagonists. OBJECTIVE: Here, it was hypothesized that α4ß2 nAChR antagonism is a viable approach for treatment of alcohol use disorders. RESULTS: When tested in rats, one lead compound, AP-202, attenuated both operant alcohol and nicotine self-administration in a paradigm in which the two reinforcers were concurrently available. The conotoxin TP2212-59, a selective α3ß4 nAChR antagonist, was only effective in reducing nicotine self-administration. AP-202 also reduced alcohol but not food responding when alcohol was presented as the only reinforcer, whereas the commercially available α4ß2 nAChR antagonist dihydro-ß-erythroidine failed to alter alcohol self-administration. AP-202 did not block relapse-like behavior induced by previously alcohol-associated stimuli or yohimbine stress. In a reinstatement paradigm, in which alcohol seeking was triggered by a nicotine challenge, a behavior successfully inhibited by the nonselective nAChR antagonist mecamylamine, AP-202 was not effective, while pretreatment with TP2212-59 abolished nicotine-induced reinstatement of alcohol seeking. CONCLUSIONS: These findings suggest differential roles for α4ß2 and α3ß4 nAChR on alcohol taking and seeking with selective blockade of α4ß2 nAChR being more implicated in modulating alcohol taking while selective blockade of α3ß4 nAChR is involved in nicotine-induced alcohol seeking.

Highly Selective and Potent α4ß2 nAChR Antagonist Inhibits Nicotine Self-Administration and Reinstatement in Rats.

The α4ß2 nAChR is the most predominant subtype in the brain and is a well-known culprit for nicotine addiction. Previously we presented a series of α4ß2 nAChR selective compounds that were discovered from a mixture-based positional-scanning combinatorial library. Here we report further optimization identified highly potent and selective α4ß2 nAChR antagonists 5 (AP-202) and 13 (AP-211). Both compounds are devoid of in vitro agonist activity and are potent inhibitors of epibatidine-induced changes in membrane potential in cells containing α4ß2 nAChR, with IC50 values of approximately 10 nM, but are weak agonists in cells containing α3ß4 nAChR. In vivo studies show that 5 can significantly reduce operant nicotine self-administration and nicotine relapse-like behavior in rats at doses of 0.3 and 1 mg/kg. The pharmacokinetic data also indicate that 5, via sc administration, is rapidly absorbed into the blood, reaching maximal concentration within 10 min with a half-life of less than 1 h.

Scaffold ranking and positional scanning utilized in the discovery of nAChR-selective compounds suitable for optimization studies.

Nicotine binds to nicotinic acetylcholine receptors (nAChR), which can exist as many different subtypes. The α4ß2 nAChR is the most prevalent subtype in the brain and possesses the most evidence linking it to nicotine seeking behavior. Herein we report the use of mixture based combinatorial libraries for the rapid discovery of a series of α4ß2 nAChR selective compounds. Further chemistry optimization provided compound 301, which was characterized as a selective α4ß2 nAChR antagonist. This compound displayed no agonist activity but blocked nicotine-induced depolarization of HEK cells with an IC50 of approximately 430 nM. 301 demonstrated nearly 500-fold selectivity for binding and 40-fold functional selectivity for α4ß2 over α3ß4 nAChR. In total over 5 million compounds were assessed through the use of just 170 samples in order to identify a series of structural analogues suitable for future optimization toward the goal of developing clinically relevant smoking cessation medications.

Shifting from the single to the multitarget paradigm in drug discovery.

Increasing evidence that several drug compounds exert their effects through interactions with multiple targets is boosting the development of research fields that challenge the data reductionism approach. In this article, we review and discuss the concepts of drug repurposing, polypharmacology, chemogenomics, phenotypic screening and high-throughput in vivo testing of mixture-based libraries in an integrated manner. These research fields offer alternatives to the current paradigm of drug discovery, from a one target-one drug model to a multiple-target approach. Furthermore, the goals of lead identification are being expanded accordingly to identify not only 'key' compounds that fit with a single-target 'lock', but also 'master key' compounds that favorably interact with multiple targets (i.e. operate a set of desired locks to gain access to the expected clinical effects).

A small molecule inhibitor of the Wnt antagonist secreted frizzled-related protein-1 stimulates bone formation.

Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a K(D) of 0.35 microM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC(50) of 3.9 microM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a K(D) of 0.08 microM and inhibited it with an EC(50) of 0.65 microM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 microM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.

Modulation of Wnt signaling through inhibition of secreted frizzled-related protein I (sFRP-1) with N-substituted piperidinyl diphenylsulfonyl sulfonamides.

The diphenylsulfonyl sulfonamide scaffold represented by 1 (WAY-316606) are small molecule inhibitors of the secreted protein sFRP-1, an endogenous antagonist of the secreted glycoprotein Wnt. Modulators of the Wnt pathway have been proposed as anabolic agents for the treatment of osteoporosis or other bone-related disorders. Details of the structure-activity relationships and biological activity from the first structural class of this scaffold will be discussed.