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...

Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits α, β, γ mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to Gβγ and interfere with or enhance binding of select effectors with Gβγ. To understand the molecular mechanisms of selectivity and assess binding of compounds to Gβγ, we used biophysical and biochemical approaches to directly monitor small molecule binding to Gβγ. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to Gβγ with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the koff was slow for most of the compounds in physiological buffers, they could be removed from Gβγ with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-Gβγ interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of Gβγ-effector interactions is by specific direct non-covalent and reversible binding of small molecules to Gβγ. This is highly relevant to development of Gβγ targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity.

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six ␣ helices [␣1-␣6] out of which [␣2-␣5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the ␣1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[␣2-␣6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the ␣2 and ␣3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the ␣1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of ␣1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.Mitochondria are dynamic organelles that undergo constant shape changes through processes including fission and fusion of the organelle tubules (1). Mitochondrial fission and fusion are evolutionarily conserved processes, suggesting their importance in basic cell function. It is presumed that fission and fusion of mitochondria allow the mixing of the mitochondrial genome as well as other mitochondrial components such as the respiratory chain complexes to maintain adequate organelle function (2-5). Mitochondrial dynamics is also required for the proper distribution of this organelle within cells especially in neurons (6 -8) and to ensure correct inheritance of this organelle to daughter cells during cell division (9). Defects in the fission or fusion machinery result in human disease conditions, such as subtypes of Charcot-Marie-Tooth disease and autosomal dominant optic atrophy, indicating their functional importance (10 -13). In addition, abnormal phenotypes of mitochondrial morphology have been reported in several neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington diseases, and recent studies indicate the direct involvement of mitochondrial fission/fusion in these diseases (14 -18).The two major processes that govern mitochondrial morphology are fission and fusion of the organelle. Studies on mitochondrial fusion in mammals have shown that dynamin-related large GTPases, mitofusin (Mfn) and OPA1, participate in the fusion of outer and inner mitochondrial membranes, respectively (19 -24). Recent ...