Direct-reversible binding of small molecules to G protein ßγ subunits.

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

Identification and characterization of unique proline-rich peptides binding to the mitochondrial fission protein hFis1.

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 alpha helices [alpha1-alpha6] out of which [alpha2-alpha5] 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 alpha1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[alpha2-alpha6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the alpha2 and alpha3 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 alpha1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of alpha1-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.

Small molecule disruption of G protein beta gamma subunit signaling inhibits neutrophil chemotaxis and inflammation.

G protein betagamma subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) gamma catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gbetagamma-dependent signaling, including Gbetagamma-dependent activation of PI3-kinase gamma and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gbetagamma 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 Gbetagamma-regulation may be an effective anti-inflammation strategy.