Gregory S. Welmaker

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.

Combinatorial Libraries As a Tool for the Discovery of Novel, Broad-Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens.

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.

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 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.

Highly Selective and Potent α4β2 nAChR Antagonist Inhibits Nicotine Self-Administration and Reinstatement in Rats

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.

Differential regulation of alcohol taking and seeking by antagonism at α4β2 and α3β4 nAChRs

RationaleAlcoholism 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.ObjectiveHere, it was hypothesized that α4β2 nAChR antagonism is a viable approach for treatment of alcohol use disorders.ResultsWhen 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.ConclusionsThese 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.

Identification of a Novel Polyamine Scaffold With Potent Efflux Pump Inhibition Activity Toward Multi-Drug Resistant Bacterial Pathogens

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.