SMALL MOLECULE MODULATORS OF RGS FUNCTION

GPCRs are the single largest target of currently prescribed pharmaceuticals and RGS proteins are potent negative regulators of GPCR-mediated signaling. RGS proteins thus provide an attractive target to either modulate the action of currently prescribed pharmaceuticals or modulate tonic signaling in a pathway-dependent manner [154-157]. While a small molecule that binds to the surface of an RGS protein and blocks its interaction with G! would be an invaluable proof-of-principle for this concept, it is would be equally useful to have a small molecule that could allosterically enhance the GAP function of endogenous RGS proteins.

Bioinformatic methods [158] and mutagenesis [159] have implicated a region between helix IV-V (Figure 1.10) as the allosteric site on the RGS domain responsible for the influences of phosphatidylinositol-3,4,5-trisphosphate (PIP3) and Ca2+

/calmodulin on GAP activity. This allosteric site (B-site) is distinct from the G!-interacting “A-site” (Figure 1.10) and, upon binding of PIP3, decreases GAP activity in vitro. In a Ca2+

Modulation of GAP activity via PIP3 and Ca2+

/CaM is also seen in cellular assays using cardiac myocytes in electrophysiological recordings of GPCR signaling to ion channel gating [161-163]. Based on sequence conservation in the B-site, it is possible that the allosteric modulation of RGS1, -2, -10, and -19 also occurs; however, this remains to be experimentally validated. This site could potentially be exploited by small molecules to either mimic the effect of PIP3 in inhibiting GAP activity, or mimic the effect of Ca

2+_{/CaM in} preventing the allosteric inhibition of GAP activity.

Currently the only way to disrupt the RGS domain/G! interaction is via point mutations on either protein’s interaction surface. Single amino acid substitutions on either side of the interface can completely abolish binding and the catalytic activity of RGS proteins [59, 64, 164]. The ability to disrupt this large protein/protein interface (1290 Å2

[25]) with single point mutations suggests that the small perturbations in the topology of the surface by virtue of a bound small molecule could have dramatic results in inhibiting RGS domain GAP activity. The current dearth of small molecule modulators of RGS proteins only makes discovering the first in vivo-acting RGS protein modulator more exciting.

Measuring RGS domain-mediated acceleration of GTP hydrolysis in vitro, for example as part of a compound library screening campaign, is difficult because GDP release by G! (not GTP hydrolysis) is the rate-limiting step [12, 165]. Thus, to quantify the effects of RGS domain GAP activity, one typically preloads radiolabelled GTP and measures the one round of hydrolysis in a so-called “single-turnover” assay [49]. This experimental design requires one to establish a pool of G!([#-32

P]GTP), initiate the assay at time zero with the addition of Mg2+

, sample aliquots over time, precipitate all unhydrolyzed GTP with charcoal, separate the charcoal, and then quantify the inorganic phosphate that was produced (and

resides in the supernatant) using liquid scintillation. This cumbersome assay design is not suitable to automation, so our group and others have developed alternative assays that are more suitable for high throughput screening of compound libraries (see Chapters 4 and 5).

Wyeth Laboratory published yeast two-hybrid based screening method for identifying RGS4 or RGS20 inhibitors [166, 167]. While their screen was reported to have identified small molecule inhibitors, these compounds were never made public and the screening program has been disbanded (Dr. David Siderovski, personal communications).

In a functional screen to identify novel treatments for urinary incontinence using ex vivo rat bladder smooth muscle cultures, a Bristol-Myers Squibb group identified two compounds (BMS-192364 and BMS-195270) that had no known molecular target yet resulted in relaxation of bladder [168]. Using a nematode genetics approach to identifying the target of these two drugs, this group concluded that these two compounds targeted the G!/RGS domain interaction and specifically locked the pair in an unproductive complex [168]. While they did not provide direct biochemical evidence for this proposed mechanism of action, there is precedence that brefeldin A, a naturally-occurring antibiotic, can trap the Ras-family GTPase ARF1 in an unproductive complex with the ARF1 GEF, Sec7 [169]. Currently, no one has yet reported being able to confirm that these two BMS compounds target the RGS/G! interaction; the Siderovski lab obtained both of these compounds but was unable to test them in single-turnover assays owing to compound solubility problems (Dr. Francis Willard; personal communication). In addition to the efforts that are ongoing by the pharmaceutical industry, our laboratory as well as the Neubig lab at the University of Michigan have been developing novel high throughput screening assays for the RGS domain/G! interaction target and searching for small molecule modulators of RGS protein GAP activity.

Neubig and colleagues have described a high-throughput flow cytometry method to screen for small molecules that can disrupt the binding of RGS proteins to G! subunits. Their assay design uses fluorescently-labeled G! protein and a LumAvidin® microsphere- coupled RGS protein to look for compounds that disrupted their interaction. The advantage of this assay is the ability to multiplex different biotinylated RGS proteins to different LumAvidin® microspheres [170, 171]. The results of an initial “in house” screening of a ~3,000 compound collection from ChemBridge were published by Roman et al. [171]; ultimately, they only identified one reactive compound that non-specifically modifies cysteines, including a critical surface-exposed cysteine in the RGS4 A-site [172] (Chapter 3). Both the Wyeth yeast two-hybrid screen and the flow cytometry-based screen were unable to measure the actual catalytic activity of RGS proteins in vitro. Instead, it has been common practice in RGS protein assays and screens to use binding of G! to the RGS domain as an indirect indicator of GAP activity. Based on the mechanism by which RGS proteins stabilize the switch regions in their transition state conformation, this is a valid assumption; however, using binding as a surrogate for GAP activity has two potential pitfalls. The first deficit is that a compound such as brefeldin A that traps the G-protein in an unproductive complex with its regulatory partner would be missed. It is possible that, for the RGS/G! target, a small molecule might inhibit RGS domain-mediated stabilization of the switch regions in a conformation that facilitates hydrolysis or otherwise traps the RGS/G! complex. Additionally, it is possible that, relying on binding rather than enzymatic activity in a compound library screen, one may have false negatives given weak binding of an inhibitor that would be lost to the noise of the assay. Instead, if one were able to read out successive rounds of GTP hydrolysis by G!, and acceleration of that hydrolysis by the RGS protein, the

effects of such weak inhibitors may become apparent. The current efforts of our laboratory in developing high throughput screening assays for RGS/G! targets will be further discussed in Chapters 4 and 5 and both use fluorescence polarization as the primary readout.