TRP channels as targets for therapeutic intervention in obesity: focus on TRPV1 and TRPM5.

The disease of obesity is one of the greatest healthcare challenges of our time. The increasing urgency for effective treatment is driving an intensive search for new targets for anti-obesity drug discovery. The TRP channel super family represents a class of proteins now recognized to serve many functions in physiology related to maintenance of health and the development of diseases. A few of these might offer new potential for therapeutic intervention in obesity. Among the TRP channels, TRPV1 appears most closely associated with body weight homeostasis through its influence on energy expenditure. TRPM5 has been thoroughly characterized as a critical component of taste signaling and recently has been implicated in insulin release. Because of its role in taste signaling, we argue that drugs designed to modulate TRPM5 could be useful in controlling energy consumption by impacting taste sensory signals. As drug targets for obesity, both TRPV1 and TRPM5 offer the advantage of operating in compartments that could limit drug distribution to the site of action. The potential for other TRP channels as anti-obesity drug targets also is discussed.

Expansion of SAR studies on triaryl bis sulfone cannabinoid CB2 receptor ligands.

We report further expansion of the structure activity relationship (SAR) on the triaryl bis sulfone class of compounds (I), which are potent CB(2) receptor ligands with excellent selectivity over the CB(1) receptor. This study was extended to B ring changes, followed by simultaneous optimization of the A-, B-, and C-rings. Compound 42 has excellent CB(2) potency, selectivity and rat exposure.

Updating the chemistry and biology of cannabinoid CB2 receptor-specific inverse agonists.

The cannabinoid CB(2) receptor continues to be an intriguing target for the potential therapeutic benefit of cannabinoids. Because this receptor is significantly found outside the brain, compounds specific for the CB(2) receptor may be free of the side effects that have plagued cannabinoid CB(1) receptor-based therapeutics. In this review, we will discuss a class of compounds which modulate the constitutive activity of the cannabinoid CB(2) receptor, the inverse agonists. We will discuss recent chemical advances that provide new compounds to investigate the biology based on this pharmacology. We will then discuss new biology associated with the cannabinoid CB(2) receptor for hints of how these compounds can best be utilized in vivo.

Label-free screening assays: a strategy for finding better drug candidates.

The last 10 years have seen advances in automation and high-throughput biochemistry in the drug-discovery arena. However, these advances have not led to improvements in drug-discovery success. Drug programs must find new ways to identify superior compounds. Advances in label-free assay technologies may provide advantages needed for improved drug discovery. In this article, we will discuss high-throughput MS, a technology that allows screening with native substrates and with targets inaccessible to standard assay formats. We will then discuss cell-based label-free biosensors, focusing on the increased information content available when using these platforms. We will conclude with speculation on the future and ways to obtain relevant biological information early in development to ensure the best compounds are promoted to medicinal chemistry campaigns.

Non-aromatic A-ring replacement in the triaryl bis-sulfone CB2 receptor inhibitors.

The triaryl bis-sulfone 1 was modified by converting the aryl A-ring to a piperidine ring. The piperidine ring was further elaborated to a spirocyclopropyl piperidine moiety. The effect on CB2 binding potency, rat calcium channel affinity, and CYP 2C9 inhibition is described.

Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay.

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 microg/mL). The results show that mass spectrometry-based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.

Characterizing cannabinoid CB2 receptor ligands using DiscoveRx PathHunter beta-arrestin assay.

The authors have characterized a set of cannabinoid CB(2) receptor ligands, including triaryl bis sulfone inverse agonists, in a cell-based receptor/beta-arrestin interaction assay (DiscoveRx PathHunter). The results were compared with results using a competitive ligand binding assay, and with effects on forskolin-stimulated cAMP levels (PerkinElmer LANCE). The authors show good correlation between the 3 assay systems tested, with the beta-arrestin protein complementation assay exhibiting a more robust signal than the cAMP assay for cannabinoid CB(2) agonists. Further assay validation shows that DiscoveRx PathHunter HEK293 CB(2) beta-arrestin assay can be carried out from cryopreserved cell suspensions, eliminating variations caused by the need for multiple cell pools during live cell screening campaigns. These results, and the authors' results evaluating a test set of random library compounds, validate the use of ligand-induced interaction between the human cannabinoid CB(2) receptor and beta-arrestin as an appropriate and valuable screening platform for compounds specific for the cannabinoid CB(2) receptor.

Screening for antiviral inhibitors of the HIV integrase-LEDGF/p75 interaction using the AlphaScreen luminescent proximity assay.

Small-molecule inhibitors of HIV integrase (HIV IN) have emerged as a promising new class of antivirals for the treatment of HIV/AIDS. The compounds currently approved or in clinical development specifically target HIV DNA integration and were identified using strand-transfer assays targeting the HIV IN/viral DNA complex. The authors have developed a second biochemical assay for identification of HIV integrase inhibitors, targeting the interaction between HIV IN and the cellular cofactor LEDGF/p75. They developed a luminescent proximity assay (AlphaScreen) designed to measure the association of the 80-amino-acid integrase binding domain of LEDGF/p75 with the 163-amino-acid catalytic core domain of HIV IN. This assay proved to be quite robust (with a Z' factor of 0.84 in screening libraries arrayed as orthogonal mixtures) and successfully identified several compounds specific for this protein-protein interaction.

KCa3.1: target and marker for cancer, autoimmune disorder and vascular inflammation?

KCa3.1 is a calcium-activated intermediate-conductance potassium ion channel. In humans the channel is expressed in several secretory organs and subtypes of hematopoietic cells, but not detected in excitable tissues. The mRNA level for KCa3.1 is upregulated in activated leukocytes, mitogen-induced endothelial cells and vascular smooth muscle cells, and several types of human cancers, suggesting a possible role for the channel in inflammatory and oncology diseases. Several potent and selective KCa3.1 blockers, including clotrimazole and its analogs TRAM-34 and ICA-17043, have been used to investigate the involvement of the channel in human disease. The compounds have been shown to suppress the proliferation of several cancer cells in vitro and the growth of the corresponding cancers in vivo, consistent with an oncologic indication. TRAM-34 also ameliorates symptoms in experimental autoimmune encephalomyelitis and several models of cardiovascular diseases, arguing for a role of the channel in inflammatory diseases. These results suggest several important opportunities for therapeutics based on KCa3.1. Further efforts will establish the optimal indication for these ion channel inhibitors.

Cannabinoid CB(2)-selective inverse agonist protects against antigen-induced bone loss.

Work to improve the therapeutic properties of cannabinoid CB(2) receptor-selective inverse agonists has led to the development of Sch.036, an aryl substituted triaryl bis-sulfone with improved oral pharmacokinetic parameters. In this report, we show that this compound blocks in vivo trafficking of various leukocyte populations, a property consistent with other members of this chemical series. This CB(2)-selective compound also shows efficacy in leukocyte recruitment models when added in concert with suboptimal doses of selected anti-inflammatory agents, consistent with its unique function and indicative of its potential therapeutic utility. Finally, studies with Sch.036 show that this cannabinoid CB(2)-specific inverse agonist can ameliorate bone damage in a rat model of relapsing-remitting arthritis. This result suggests that a cannabinoid CB(2)-selective inverse agonist may help ameliorate a particularly harmful property of this inflammatory joint disease.

Himbacine derived thrombin receptor (PAR-1) antagonists: SAR of the pyridine ring.

The structure-activity relationship (SAR) of the vinyl pyridine region of himbacine derived thrombin receptor (PAR-1) antagonists is described. A 2-vinylpyridyl ring substituted with an aryl or a heteroaryl group at the 5-position showed the best overall PAR-1 affinity and pharmacokinetic properties. One of the newly discovered analogs bearing a 5-(3-pyridyl) substituent showed excellent PAR-1 affinity (Ki = 22 nM) and oral activity with reduced ClogP and improved off-target selectivity compared to an earlier development candidate.

Optimization of triaryl bis-sulfones as cannabinoid-2 receptor ligands.

Structure-activity relationship on our recently reported triaryl bis-sulfone class of cannabinoid-2 (CB2) receptor selective inverse agonists was explored. Modifications to the methane sulfonamide, substitutions to B and C phenyl rings, and replacements of the C-ring were investigated. A compound with excellent CB2 activity, selectivity for CB2 over CB1, and in vivo plasma levels was identified.

Targeting the CB2 receptor for immune modulation.

Early work on the biology of the components of Cannabis sativa showed evidence for a potential influence on immune regulation. With the discovery of a peripheral cannabinoid receptor associated with immune cells, many laboratories have sought to link the immunoregulatory activities of cannabinoid compounds with this receptor, hoping that such compounds would lack the psychoactive effects of marijuana and other nonspecific cannabinoid agonists. In this report, the authors investigate the role of the cannabinoid CB2 receptor in immune regulation, with particular emphasis on compounds shown to regulate immune cell recruitment. The authors conclude by using the immune cell recruitment model to rationalise cannabinoid CB2 receptor-specific effects in modulating immune disease, particularly the increasing evidence for its role in experimental autoimmune encephalomyelitis and in influencing bone density.

Characterization of peripheral human cannabinoid receptor (hCB2) expression and pharmacology using a novel radioligand, [35S]Sch225336.

Studies to characterize the endogenous expression and pharmacology of peripheral human cannabinoid receptor (hCB2) have been hampered by the dearth of authentic anti-hCB2 antibodies and the lack of radioligands with CB2 selectivity. We recently described a novel CB2 inverse agonist, N-[1(S)-[4-[[4-methoxy-2-[(4methoxyphenyl)sulfonyl] phenyl]sulfonyl] phenyl]ethyl]methane-sulfonamide (Sch225336), that binds hCB2 with high affinity and excellent selectivity versus hCB1. The precursor primary amine of Sch225336 was prepared and reacted directly with [(35)S]mesyl chloride (synthesized from commercially obtained [(35)S]methane sulfonic acid) to generate [(35)S]Sch225336. [(35)S]Sch225336 has high specific activity (>1,400 Ci/mmol) and affinity for hCB2 (65 pm). Using [(35)S]Sch225336, we assayed hemopoietic cells and cell lines to quantitate the expression and pharmacology of hCB2. Lastly, we used [(35)S]Sch225336 for detailed autoradiographic analysis of CB2 in lymphoid tissues. Based on these data, we conclude that [(35)S]Sch225336 represents a unique radioligand for the study of CB2 endogenously expressed in blood cells and tissues.

A novel cannabinoid peripheral cannabinoid receptor-selective inverse agonist blocks leukocyte recruitment in vivo.

The expression of the cannabinoid peripheral cannabinoid receptor (CB(2)) receptor on peripheral immune cells suggests that compounds specific for CB(2) might be effective anti-inflammatory agents. In this report, we present the initial biological profiling of a novel triaryl bis-sulfone, Sch.336 (N-[1(S)-[4-[[4-methoxy-2-[(4-methoxyphenyl)sulfonyl]phenyl]-sulfonyl]phenyl]ethyl]methanesulfonamide), which is selective for the human cannabinoid CB(2) receptor (hCB(2)). Sch.336 is an inverse agonist at hCB(2), as shown by its ability to decrease guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to membranes containing hCB(2), by the ability of GTPgammaS to left-shift Sch.336 binding to hCB(2) in these membranes, and by the compound's ability to increase forskolin-stimulated cAMP levels in CHO cells expressing hCB(2). In these systems, Sch.336 displays a greater potency than that reported for the CB(2)-selective dihydropyrazole, SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo [2.2.1]heptan2-yl]-5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-1H-pyrazole-3-carboxamide). In vitro, Sch.336 impairs the migration of CB(2)-expressing recombinant cell lines to the cannabinoid agonist 2-arachidonylglycerol. In vivo, the compound impairs migration of cells to cannabinoid agonist HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo [b,d] pyran-9-methanol]. Oral administration of the Sch.336 significantly inhibited leukocyte trafficking in several rodent in vivo models, induced either by specific chemokines or by antigen challenge. Finally, oral administration of Sch.336 blocked ovalbumin-induced lung eosinophilia in mice, a disease model for allergic asthma. We conclude that selective cannabinoid CB(2) inverse agonists may serve as novel immunomodulatory agents in the treatment of a broad range of acute and chronic inflammatory disorders in which leukocyte recruitment is a hallmark of disease pathology.

Triaryl bis-sulfones as cannabinoid-2 receptor ligands: SAR studies.

We recently reported that compound 1 is a potent inhibitor of the CB2 receptor with high selectivity over CB1. This paper describes the SAR development for this class of compounds. Variation of the substitution pattern on the aromatic rings, as well as the groups linking them together, led to sub-nanomolar inhibitors of the CB2 receptor, with high selectivity over CB1.

Triaryl bis-sulfones as a new class of cannabinoid CB2 receptor inhibitors: identification of a lead and initial SAR studies.

A novel class of cannabinoid CB2 receptor ligands is described. These triaryl bis-sulfones are nanomolar inhibitors of the CB2 receptor and show high selectivity over the cannabinoid CB1 receptor. One example of this new class decreases ligand-induced GTPgammaS binding to recombinant CB2 cell membranes, identifying the compound as a CB2-selective inverse agonist.