G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor–dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein–coupled receptor signaling that may represent an important therapeutic strategy.

G protein ␤␥ subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) ␥ catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of G␤␥-dependent signaling, including G␤␥-dependent activation of PI3-kinase ␥ and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule G␤␥ inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting G␤␥-regulation may be an effective anti-inflammation strategy.Chemoattractant-mediated recruitment of leukocytes is responsible for many of the deleterious effects of chronic inflammatory diseases. Many chemoattractants activate G protein-coupled receptors (GPCRs) coupled to the G i family of heterotrimeric G proteins in leukocytes. Heterotrimeric G proteins are composed of G␣, G␤, and G␥ subunits. Ligand binding to receptors catalyzes the exchange of tightly bound GDP for GTP on the G␣ subunit, liberating it from the G␤␥ subunits. Dissociation of the G␣ and G␤␥ subunits can allow each to directly bind to downstream effector proteins (Gilman, 1987;Oldham and Hamm, 2006). The free G␤␥ subunits released from G i heterotrimers upon chemoattractant receptor activation initiate critical signaling pathways to direct chemoattractant-dependent neutrophil functions including chemotaxis and superoxide production (Neptune and Bourne, 1997).Key direct targets of G␤␥ subunit binding and activation in neutrophils are phosphoinositide 3-kinase ␥ (PI3-kinase ␥) (Stephens et al., 1994(Stephens et al., , 1997Stoyanov et al., 1995), Phospholipase C ␤ (PLC␤) , and P-Rex (Welch et al., 2002). PI3-kinase ␥ has been noted to be a central mediator of chemotaxis and plays a pivotal role in leukocyte recruitment to inflamed tissues (Hirsch et al., 2000;Li et al., 2000;Camps et al., 2005). PIP 3 , produced by PI3-kinase ␥ catalytic activity, is critical to the development of cell polarity, which is necessary for chemokine-mediated cell motility and directional sensing . PI3-kinase ␥-deficient neutrophils have impaired responses to various chemoattractants, including diminished chemotaxis (Hirsch et al., 2000;Li et al., 2000) and respiratory burst (Li et al., 2000;Sasaki et al., 2000), in response to GPCR activation. Small-molecule inhibitors of PI3-kinase ␥ catalytic activity have been demonstrated to suppress joint inflammation in mouse models of inflammation (Barber et al., 2005;Camps et al., 2005). Critical to the success of a method that targets PI3-kinase ␥ activity as a therapeutic anti-inflammatory a...

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One of the top priorities of ICCVAM is the identification and evaluation of non-animal alternatives for skin sensitization testing. Although skin sensitization is a complex process, the key biological events of the process have been well characterized in an adverse outcome pathway (AOP) proposed by OECD. Accordingly, ICCVAM is working to develop integrated decision strategies based on the AOP using in vitro, in chemico, and in silico information. Data were compiled for 120 substances tested in the murine local lymph node assay (LLNA), direct peptide reactivity assay (DPRA), human cell line activation test (h-CLAT), and KeratinoSens assay. Data for six physicochemical properties that may affect skin penetration were also collected, and skin sensitization read-across predictions were performed using OECD QSAR Toolbox. All data were combined into a variety of potential integrated decision strategies to predict LLNA outcomes using a training set of 94 substances and an external test set of 26 substances. Fifty-four models were built using multiple combinations of machine learning approaches and predictor variables. The seven models with the highest accuracy (89–96% for the test set and 96–99% for the training set) for predicting LLNA outcomes used a support vector machine (SVM) approach with different combinations of predictor variables. The performance statistics of the SVM models were higher than any of the non-animal tests alone and higher than simple test battery approaches using these methods. These data suggest that computational approaches are promising tools to effectively integrate data sources to identify potential skin sensitizers without animal testing.

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