G protein betagamma subunits as targets for small molecule therapeutic development.

Department of Pharmacology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Combinatorial chemistry & high throughput screening (Impact Factor: 1.22). 07/2008; 11(5):382-95.
Source: PubMed


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.

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    • "Additionally, evidence has accumulated indicating that Gβγ subunits also transduce cellular signals. In fact, Gβγ subunits have been reported to directly regulate a diverse array of effector molecules including ion channels, enzymes, and intracellular regulators [27]. These interactions provide a role for Gβγ in important physiological functions such as cardiac membrane potential, heart rate, inflammation, pain modulation, and neurotransmitter release [17], [28]–[29]. "
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    ABSTRACT: Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson's disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.
    Full-text · Article · Mar 2013 · PLoS ONE
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    • "Mutagenesis studies (Ford et al., 1998) and structural analyses (Yokogawa et al., 2011) have shown that Kir3 association with Gbg subunits takes place at a surface that normally interacts with Ga subunits, known as the " hot spot. " For the hot spot to become available for effector interaction, Ga subunits must exchange GDP for GTP (Smrcka et al., 2008). In keeping with this mechanism, ligand-induced BRET changes at the pair monitoring Gbg/Kir3.1 interaction were sensitive to pertussis toxin (DPDPE-induced BRET changes in CTL cells: 0,022 6 0.005 and in cells pre-exposed to PTX: 20,002 6 0,005; P , 0.01; n 5 6, unpaired t test). "
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    ABSTRACT: This study assessed how conformational information encoded by ligand binding to δ-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. HEK293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to independently evaluate reciprocal interactions among signaling partners. These and co-immunoprecipitation studies indicated that DORs, Gβγ and Kir3 subunits constitutively interacted with one another. GαoA associated with DORs and Gβγ but, despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified, but modulated BRET between DOR-GαoA, DOR-Gβγ, GαoA-Gβγ and Gβγ-Kir3 interfaces. Ligand-induced BRET changes assessing Gβγ-Kir3.1 subunit interaction: i) followed similar kinetics as those monitoring the GαoA-Gβγ interface, ii) displayed same order of efficacy as those observed at the DOR-Gβγ interface, iii) were sensitive to pertussis toxin and iv) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gβγ/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gβγ-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gαβγ with respect to DORs, GαoA and channel subunits. Further, the fact that BRET changes at the Gβγ-Kir3 interface are predictive of ligand ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation.
    Full-text · Article · Nov 2012 · Molecular pharmacology
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    • "More recently, direct signaling of βγ dimers toward PI3K, Src etc. has been documented in the regulation of cell motility, growth and differentiation [87,88]. A specific βγ inhibitor (M119K) has been identified, but, so far, very limited efforts have been made to act pharmacologically on any of the 5 β- and 12 γ- subunits expressed by the human genome [89]. "
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    ABSTRACT: Hundreds of G protein coupled receptor (GPCR) isotypes integrate and coordinate the function of individual cells mediating signaling between different organs in our bodies. As an aberration of the normal relationships that organize cells’ coexistence, cancer has to deceive cell-cell communication in order to grow and spread. GPCRs play a critical role in this process. Despite the fact that GPCRs represent one of the most common drug targets, current medical practice includes only a few anticancer compounds directly acting on their signaling. Many approaches can be envisaged to target GPCRs involved in oncology. Beyond interfering with GPCRs signaling by using agonists or antagonists to prevent cell proliferation, favor apoptosis, induce maturation, prevent migration, etc., the high specificity of the interaction between the receptors and their ligands can be exploited to deliver toxins, antineoplastic drugs or isotopes to transformed cells. In this review we describe the strategies that are in use, or appear promising, to act directly on GPCRs in the fight against neoplastic transformation and tumor progression.
    Full-text · Article · Dec 2011 · Pharmaceuticals
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