Evidence has emerged suggesting a role for the cannabinoid CB 2 receptor in immune cell motility. This provides a rationale for a novel and generalized immunoregulatory role for cannabinoid CB 2 receptor-specific compounds. In support of this possibility, we will review the biology of a class of cannabinoid CB 2 receptor-specific inverse agonist, the triaryl bis-sulfones. We will show that one candidate, Sch.414319, is potent and selective for the cannabinoid CB 2 receptor, based on profiling studies using biochemical assays for 45 enzymes and 80 G-protein coupled receptors and ion channels. We will describe initial mechanistic studies using this optimized triaryl bis-sulfone, showing that the compound exerts a broad effect on cellular protein phosphorylations in human monocytes. This profile includes the down regulation of a required phosphorylation of the monocyte-specific actin bundling protein L-plastin. We suggest that this observation may provide a mechanism for the observed activity of Sch.414319 in vivo. Our continued analysis of the in vivo efficacy of this compound in diverse disease models shows that Sch.414319 is a potent modulator of immune cell mobility in vivo, can modulate bone damage in antigeninduced mono-articular arthritis in the rat, and is uniquely potent at blocking experimental autoimmune encephalomyelitis in the rat.

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, 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 5Ј-3-O-(thio)triphosphate (GTP␥S) binding to membranes containing hCB 2 , by the ability of GTP␥S 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 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.The identification of a second cannabinoid receptor present primarily in peripheral immune tissues and cells (Munro et al., 1993) suggested an immunomodulatory role for endocannabinoids independent of the effects mediated by their interaction with the "brain" cannabinoid CB 1 receptor. Libraries of selective compounds now exist, based either on the structure of known ligands for the cannabinoid receptors or on results of random compound library screening (Huffman, 2000). With the help of CB 2 -specific compounds and by characterization of a CB 2 Ϫ/Ϫ mouse strain (Buckley et al., 2000), Article, publication date, and citation information can be found at

This report describes a joint hybridoma and combinatorial antibody library approach to elicit catalysts for primary amide bond hydrolysis. By immunization with a trigonal boronic acid hapten 3a and construction of a Fab (antigen-binding fragment) library, a diastereoselective catalyst for hydrolysis of the tripeptide primary amide substrate 1a was selected. In contrast, no antibody catalyst was isolated by standard hybridoma methods of monoclonal antibody production following immunizations with hapten 3a. The active Fab, BL25, obeys Michaelis−Menten kinetic behavior (k cat/k uncat ca. 4 × 104, K m = 150 μM) and is competitively inhibited by a boronic acid hapten analog 3b (K i = 9 μM). Kinetic and binding studies both point to Fab selection of a hydrated tetrahedral anionic form of the boronic acid hapten 3a which serves to mimic the putative transition state 4 for catalysis of water addition to the primary amide bond. Fab-BL25 exhibits exquisite substrate selectivity, as a methyl ester analog of 1a is not accepted as a substrate. This work emphasises the power of the direct selection strategy when linked to screening of antibody combinatorial libraries and discloses the utility of boronic acids as haptens in acyl transfer processes.

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