Our understanding of G protein-coupled receptors (GPCRs) has been greatly aided by their relative tractability in terms of pharmacological targeting. These receptors are fairly easy to express in cells and their signaling pathways are amenable to high throughput screening (HTS) technologies. GPCRs couple to a trimeric G-protein structure composed of an alpha subunit and a beta/gamma subunit. The alpha subunit dissociates from beta/gamma upon stimulation of the GPCR and the duration of the alpha subunit signaling is determined by its intrinsic GTPase activity. This GTPase activity can be modulated by regulator of G-protein signaling (RGS) proteins. In terms of GPCR signaling the vast majority of attention has been paid to alpha subunits and part of the reason for this is the availability of molecules (e.g. pertussis and cholera toxins) that target those subunits. Despite this, it is well known that beta/gamma subunits are also capable of generating signaling as these little proteins are known to activate phospholipase C (PLC), PI3Kinase (PI3K) and G-protein receptor kinases (GRKs). Additionally, beta/gamma subunits activate G-protein coupled inwardly rectifying potassium channels (GiRKs) and inhibit certain types of voltage-gated calcium channels (VGCaC). While these signaling mechanisms for beta/gamma are well known, we know relatively little about the physiology of these processes in vivo. This is because we do not have tools to probe the function of beta/gamma pharmacologically. At least not until 2006.
Bonacci et al., Differential targeting of Gbeta/gamma-subunit signaling with small molecules, Science (2006) [free at Science], was the first paper to describe small molecules that target beta/gamma subunits. The first step in discovering these small molecules involved describing a modulatory binding site for beta/gamma function. The authors used phage display of beta/gamma subunits to screen for peptides that bound to these subunits. They discovered a small peptide, SIGK, that bound to a “hotspot” in beta/gamma. They then used this hotspot to screen several thousand molecules computationally for potential binding within this region. They came out of this “virtual screen” with a list of 85 compounds that could then be tested for interference with SIGK binding to beta/gamma. Of these 85 compounds, they found 9 with apparent binding affinity between 0.1 and 60 uM. They then focused on these compounds as potential modulators of beta/gamma signaling.
They focused on two compounds from their screen for the majority of experiments in the paper. These compounds, M119 and M201, showed relatively high affinity for beta/gamma binding so they wanted to know if they would influence beta/gamma activity in vitro and in cells. They looked at four main targets: PLCbeta2, PLCbeta3, PI3Kgamma and GRK2. They chose these targets, as I mentioned above, because they are known to be modulated specifically by beta/gamma signaling. Here is what they found (also shown in the figure below) from their in vitro assays:
1) Compound M119 inhibits PLCbeta2 and PLCbeta3 activity in vitro with an apparent IC50 of ~5uM. M119 also inhibits PI3Kgamma activity and prevents GRK2 association with beta/gamma. Hence, M119 is an apparent inhibitor of beta/gamma function.
2) Compound M201 had no effect on PLCbeta2 but augmented PLCbeta3 activity. It also increased PI3Kgamma activity. In contrast, M201 inhibited GRK2 association with beta/gamma. Hence, compound 201 has differential effects on beta/gamma depending on the downstream target of the complex.
They then turned to a cell based assay. M119 inhibited calcium liberation from intracellular stores (consistent with PLCbeta activity) in cells and also inhibited GRK2 mobilization to the membrane. M201 had no effect on calcium mobilization (consistent with the in vitro assay for PLCbeta2) but also inhibited GRK2 mobilization to the membrane. Therefore, the authors conclude that these novel small molecules are capable of modulating beta/gamma function in cellular assays.
The authors then assessed whether compound M119 had an effect in whole animals (what this scientist would call in vivo but let’s not nitpick). They took advantage of the well known effects of morphine to do this. When morphine is administered directly into the brain (or systemically for that matter) it produces profound analgesia. This analgesia is modulated by PLCbeta3 because the dose response curve for morphine is right shifted (becomes more potent) in PLCbeta3 knockout mice. Hence, they predicted that M119 would have a similar effect because it disrupts beta/gamma signaling to PLCbeta3 in their assays. When M119 was given directly into the brain in wildtype mice it made the effect of morphine analgesia more potent, consistent with findings in PLCbeta3 knockout mice. When M119 was given directly into the brain of PLCbeta3 knockout mice, it had no effect on the dose-response curve for morphine analgesia, consistent with a specific effect of this compound on beta/gamma in vivo.
The authors conclude with the following statements:
Protein interaction interfaces present difficult drug targets because of the generally large interaction surface area and flat topology of the interaction surfaces. Hotspots at protein interfaces comprise a small fraction of the overall interaction surface, yet are responsible for most of the energetics of binding, and it has been proposed that targeting such surfaces could successfully disrupt protein-protein binding. As a result of extensive screening, some protein interaction interfaces have been targeted with small molecules. Our screen of only 1990 molecules identified multiple compounds with apparent affinities in the high nM to low µM range. These molecules demonstrate that multiple small molecule binders of Gbeta/gamma could be developed that differentially modulate functions downstream of GPCRs. Numerous studies in animal models have implicated Gbeta/gamma-subunit targeting as a therapeutic strategy in diseases such as heart failure, prostate cancer, vascular disease, and inflammatory disease. Thus, more extensive screening for molecules that bind to the Gbeta/gamma hotspot will yield a repertoire of potentially therapeutically useful small molecules.
In my view, this was a landmark moment in furthering our understanding of the function of GPCRs because, for the first time, tools became available to study beta/gamma effects with some level of specificity without modulating overall gene function with either knockout or knockdown. The potential for the utilization of compounds such as M119 in heart failure was recently described by Casey et. al.. Time will tell if this will be a fruitful approach for therapeutics; however, it is clear that these tools can be used to probe the function of beta/gamma in model systems therefore enhancing our understanding of the role of these previously enigmatic yet ubiquitous proteins.
Bonacci, T. (2006). Differential Targeting of G -Subunit Signaling with Small Molecules Science, 312 (5772), 443-446 DOI: 10.1126/science.1120378