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 α4β2 nAChR antagonists.OBJECTIVE:Here, it was hypothesized that α4β2 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 α3β4 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 α4β2 nAChR antagonist dihydro-β-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 α4β2 and α3β4 nAChR on alcohol taking and seeking with selective blockade of α4β2 nAChR being more implicated in modulating alcohol taking while selective blockade of α3β4 nAChR is involved in nicotine-induced alcohol seeking.

We have previously reported the use of combinatorial chemistry to identify broad-spectrum antibacterial agents. Herein, we extend our analysis of this technology toward the discovery of anti-resistance molecules, focusing on efflux pump inhibitors. Using high-throughput screening against multi-drug resistant Pseudomonas aeruginosa, we identified a polyamine scaffold that demonstrated strong efflux pump inhibition without possessing antibacterial effects. We determined that these molecules were most effective with an amine functionality at R1 and benzene functionalities at R2 and R3. From a library of 188 compounds, we studied the properties of 5 lead agents in detail, observing a fivefold to eightfold decrease in the 90% effective concentration of tetracycline, chloramphenicol, and aztreonam toward P. aeruginosa isolates. Additionally, we determined that our molecules were not only active toward P. aeruginosa, but toward Acinetobacter baumannii and Staphylococcus aureus as well. The specificity of our molecules to efflux pump inhibition was confirmed using ethidium bromide accumulation assays, and in studies with strains that displayed varying abilities in their efflux potential. When assessing off target effects we observed no disruption of bacterial membrane polarity, no general toxicity toward mammalian cells, and no inhibition of calcium channel activity in human kidney cells. Finally, combination treatment with our lead agents engendered a marked increase in the bactericidal capacity of tetracycline, and significantly decreased viability within P. aeruginosa biofilms. As such, we report a unique polyamine scaffold that has strong potential for the future development of novel and broadly active efflux pump inhibitors targeting multi-drug resistant bacterial infections.

The α4β2 nAChR is the most predominant subtype in the brain and is a well-known culprit for nicotine addiction. Previously we presented a series of α4β2 nAChR selective compounds that were discovered from a mixture-based positional-scanning combinatorial library. Here we report further optimization identified highly potent and selective α4β2 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 α4β2 nAChR, with IC50 values of approximately 10 nM, but are weak agonists in cells containing α3β4 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.

Ovarian cancer (OC) is the most lethal gynecological malignancy. OC mortality is associated with a high rate of recurrence (~80%). Recurrent OC is typified by treatment resistant cells; thus, conventional therapies are often ineffective. Therefore, it is imperative to find novel drugs to eliminate recurrent OC cells. We propose that targeting mitochondrial‐cell communication in recurrent OC will increase chemotherapeutic efficacy. Preliminary data demonstrate that increasing the concentration of the mitochondrial scaffold protein Sab sensitizes OC cells to paclitaxel by inducing apoptotic priming. These studies suggest that increasing Sab levels in OC will induce a pre‐apoptotic state and sensitize the tumor cells to conventional therapies, thus rendering resistant OC cells sensitive. We developed a high throughput assay to screen a scaffold ranking and positional scanning library in order to identify novel chemicals that increase Sab expression in SK‐OV‐3 cells. An In‐Cell Western (ICW) assay was used to detect Sab levels, and this assay was optimized for screening (z‐score of 0.81). The Sab ICW was co‐stained with the TO‐PRO‐3 DNA stain for normalization and to assess viability. We screened 50 scaffolds in SK‐OV‐3 cells. Scaffolds selected for further evaluation increased Sab expression greater than 30% and demonstrated minimal toxicity. Using this method we identified six candidate scaffolds. Of these, N‐Methyl‐1,4,5‐tri‐substituted‐2,3‐diketopiperazines were considered to be the best candidates. We predict that this approach will identify novel chemotherapeutic drugs that will improve OC treatment and mortality.

Mixture based synthetic combinatorial libraries offer a tremendous enhancement for the rate of drug discovery, allowing the activity of millions of compounds to be assessed through the testing of exponentially fewer samples. In this study, we used a scaffold-ranking library to screen 37 different libraries for antibacterial activity against the ESKAPE pathogens. Each library contained between 10000 and 750000 structural analogues for a total of >6 million compounds. From this, we identified a bis-cyclic guanidine library that displayed strong antibacterial activity. A positional scanning library for these compounds was developed and used to identify the most effective functional groups at each variant position. Individual compounds were synthesized that were broadly active against all ESKAPE organisms at concentrations <2 μM. In addition, these compounds were bactericidal, had antibiofilm effects, showed limited potential for the development of resistance, and displayed almost no toxicity when tested against human lung cells and erythrocytes. Using a murine model of peritonitis, we also demonstrate that these agents are highly efficacious in vivo.

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).

Nicotine binds to nicotinic acetylcholine receptors (nAChR), which can exist as many different subtypes. The α4β2 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 α4β2 nAChR selective compounds. Further chemistry optimization provided compound 301, which was characterized as a selective α4β2 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 α4β2 over α3β4 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.

Because of their ability to rapidly build molecular complexity, reactions that construct several carbon‐carbon bonds are of special value in organic synthesis. Among these transformations, cascade reactions form several bonds by an orchestrated sequence in which the first bond‐forming step reveals functionality that allows subsequent bond forming transformations. In 1979 such a sequence for the synthesis of 3‐acylpyrrolidines and azacyclic structures that contain this unit was reported. This reaction is now commonly called the aza‐Cope/Mannich reaction, a [3,3]‐sigmatropic rearrangement of an unsaturated iminium cation generates the iminium ion and enol that are participants in a subsequent intramolecular Mannich reaction. The aza‐Cope/Mannich reaction has not been the subject of comprehensive review. This chapter covers the common version of the transformation discussed above along with the formation of 3‐acylpyrrolidines by a related base‐promoted cascade sequence.

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 Writ 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 suifonamide, bound to sFRP-1 with a K-D of 0.35 mu M in a tryptophan fluorescence quenching assay. This Compound also selectively inhibited sFRP-1 with an EC50 of 3.9 mu M 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 KD of 0.08 mu M and inhibited it with an EC50 of 0.65 mu M. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 mu M. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation. (C) 2009 Elsevier Inc. All rights reserved.

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A simplified approach to the uncatalyzed Paal-Knorr condensation using microwave irradiation in water is described.

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.