The deteriorating effectiveness of antibiotics is propelling researchers worldwide towards alternative techniques such as phage therapy: curing infectious diseases using viruses of bacteria called bacteriophages. In a previous paper, we isolated phage EFDG1, highly effective against both planktonic and biofilm cultures of one of the most challenging pathogenic species, the vancomycin-resistant Enterococcus (VRE). Thus, it is a promising phage to be used in phage therapy. Further experimentation revealed the emergence of a mutant resistant to EFDG1 phage: EFDG1r. This kind of spontaneous resistance to antibiotics would be disastrous occurrence, however for phage-therapy it is only a minor hindrance. We quickly and successfully isolated a new phage, EFLK1, which proved effective against both the resistant mutant EFDG1r and its parental VRE, Enterococcus faecalis V583. Furthermore, combining both phages in a cocktail produced an additive effect against E. faecalis V583 strains regardless of their antibiotic or phage-resistance profile. An analysis of the differences in genome sequence, genes, mutations, and tRNA content of both phages is presented. This work is a proof-of-concept of one of the most significant advantages of phage therapy, namely the ability to easily overcome emerging resistant bacteria.

G proteins mediate the action of G protein coupled receptors (GPCRs), a major target of current pharmaceuticals and a major target of interest in future drug development. Most pharmaceutical interest has been in the development of selective GPCR agonists and antagonists that activate or inhibit specific GPCRs. Some recent thinking has focused on the idea that some pathologies are the result of the actions of an array of GPCRs suggesting that targeting single receptors may have limited efficacy. Thus, targeting pathways common to multiple GPCRs that control critical pathways involved in disease has potential therapeutic relevance. G protein betagamma subunits released from some GPCRs upon receptor activation regulate a variety of downstream pathways to control various aspects of mammalian physiology. There is evidence from cell- based and animal models that excess Gbetagamma signaling can be detrimental and blocking Gbetagamma signaling has salutary effects in a number of pathological models. Gbetagamma regulates downstream pathways through modulation of enzymes that produce cellular second messengers or through regulation of ion channels by direct protein-protein interactions. Thus, blocking Gbetagamma functions requires development of small molecule agents that disrupt Gbetagamma protein interactions with downstream partners. Here we discuss evidence that small molecule targeting Gbetagamma could be of therapeutic value. The concept of disruption of protein-protein interactions by targeting a "hot spot" on Gbetagamma is delineated and the biochemical and virtual screening strategies for identification of small molecules that selectively target Gbetagamma functions are outlined. Evaluation of the effectiveness of virtual screening indicates that computational screening enhanced identification of true Gbetagamma binding molecules. However, further refinement of the approach could significantly improve the yield of Gbetagamma binding molecules from this screen that could result in multiple candidate leads for future drug development.

G protein-coupled receptors transduce signals through heterotrimeric G protein G␣ and G␤␥ subunits, both of which interact with downstream effectors to regulate cell function. G␤␥ signaling has been implicated in the pathophysiology of several diseases, suggesting that G␤␥ could be an important pharmaceutical target. Previously, we used a combination of virtual and manual screening to find small molecules that bind to a proteinprotein interaction "hot spot" on G␤␥ and block regulation of physiological effectors. One of the most potent and effective compounds from this screen was selenocystamine. In this study, we investigated the mechanism of action of selenocystamine and found that selenocysteamine forms a covalent complex with G␤␥ by a reversible redox mechanism. Mass spectrometry and site-directed mutagenesis suggest that selenocysteamine preferentially modifies G␤Cys204, but also a second undefined site. The high potency of selenocystamine in G␤␥ inhibition seems to arise from both high reactivity of the diselenide group and binding to a specific site on G␤. Using structural information about the "hot spot," we developed a strategy to selectively target redox reversible compounds to a specific site on G␤␥ using peptide carriers such as SIGCAFKILGY (-cysteamine) [SIGC(-cysteamine)]. Mass spectrometry and site-directed mutagenesis indicate that SIGC(-cysteamine) specifically and efficiently leads to cysteamine (half-cystamine) modification of a single site on G␤, likely G␤Cys204, and inhibits G␤␥ more than a hundred times more potently than cystamine. These data support the concept that covalent modifiers can be specifically targeted to the G␤␥ "hot spot" through rational incorporation into molecules that noncovalently bind to G␤␥.

Several studies have suggested that disrupting interactions of the G protein βγ subunits with downstream binding partners might be a valuable study for pharmaceutical development. Recently, small molecules have been found which bind to Gβγ with high apparent affinity in an enzyme-linked immunosorbent assay (ELISA), have demonstrated selective inhibition of interactions of Gβγ with downstream signaling partners, and have been shown to increase antinociceptive effects of morphine and inhibit inflammation in vivo. In this paper we examine several docking and scoring protocols for estimating binding affinities for a set of 830 ligands from the NCI diversity set to the Gβ1γ2 subunit and compared these with IC50s measured in a competition ELISA with a high-affinity peptidic ligand. The best-performing docking protocol used a consensus score and ensemble docking and resulted in a 6-fold enrichment of high-affinity compounds in the top-ranked 5% of the ligand data set.

The βγ‐subunit of G protein (Gβγ) dissociates from the α‐subunit upon activation of G protein‐coupled receptors. Once free, Gβγ is able to regulate many target proteins, including phospholipase C β2 (PLCβ2), phosphoinositide 3‐kinase γ (PI3Kγ) and G protein‐coupled receptor kinase 2 (GRK2). Our group has used a combination of virtual and manual screening to find small molecules that inhibit the interaction between Gβγ and the aforementioned proteins with affinities in the high nM range. Recently, we discovered another set of compounds (some with heavy metals, Hg2+, Cu2+ and Ag+ in their structure) that binding of a Gβγ‐binding peptide (SIGK) in an enzyme‐linked immunosorbent assay (ELISA) at concentrations in the low nM range. The effect of these small molecules persists after washing them away, and it is both prevented and reversed by dithiothreitol (DTT). This suggests that these compounds act directly at Gβγ by a reversible redox mechanism. Investigation of the mechanism of action of these compounds may lead to uncovering mechanisms for Gβγ regulation by both natural and synthetic redox active molecules.This work was funded by National Institutes of Health GM060286