Molecular Mechanisms for Inhibition of Regulators of G-protein Signaling by Small Molecules.

Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric Gα proteins. RGS proteins have become attractive targets for the purpose of manipulating GPCR-mediated cellular responses. The RGS family comprises thirty-seven proteins with differential expression patterns throughout the body. RGS4 is enriched in the CNS and has been proposed as a therapeutic target for treatment of neurological disorders including epilepsy and Parkinson's disease. Therefore, our lab has focused on the identification of small molecule inhibitors of RGS4. To date, all small molecule inhibitors of RGS proteins function through covalent modification of cysteine residues, yet substantial specificity has been observed for RGS4 over other closely related homologs. The work in this thesis details the molecular mechanism of inhibition by a potent RGS4 inhibitor (CCG-50014; IC50 = 30 nM), and reveals the importance of RGS4 dynamics in the exposure of key cysteine residues that upon binding the inhibitor prevent the protein from reaching native conformations. Elucidating this mechanism has allowed us to propose a novel cryptic site (C-site) that is formed around the buried cysteine residues, and may be more druggable than previously proposed sites on RGS4. In addition, new chemical scaffolds have been identified using a cell-based high-throughput screen that also inhibit RGS4 through a cysteine-dependent mechanism, but are significantly reversible in contrast to the first cell-active RGS4 inhibitors. Furthermore, I employed IP6 as a derivative of an endogenous negative regulator, PIP3, to map the binding site on RGS4 and the corresponding effects on protein stability and function. This study shows the direct interactions of a non-covalent small molecule with an allosteric site on the RGS4 structure, and provides insight into the mechanism of endogenous regulation of RGS4. In conclusion, the studies described within this thesis provide new pharmacological tools for the study of RGS function in a cellular context, and describe in detail the molecular interactions of both endogenous and pharmacological modulators of RGS4. These results provide the theoretical framework to pursue drug discovery strategies that are expected to significantly advance the field of RGS drug discovery.PHDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107061/1/storaska_1.pd

Selectivity and anti-Parkinson's potential of thiadiazolidinone RGS4 inhibitors

Many current therapies target G protein coupled receptors (GPCR), transporters, or ion channels. In addition to directly targeting these proteins, disrupting the protein−protein interactions that localize or regulate their function could enhance selectivity and provide unique pharmacologic actions. Regulators of G protein signaling (RGS) proteins, especially RGS4, play significant roles in epilepsy and Parkinson’s disease. Thiadiazolidinone (TDZD) inhibitors of RGS4 are nanomolar potency blockers of the biochemical actions of RGS4 in vitro. Here, we demonstrate the substantial selectivity (8- to >5000-fold) of CCG-203769 for RGS4 over other RGS proteins. It is also 300-fold selective for RGS4 over GSK-3β, another target of this class of chemical scaffolds. It does not inhibit the cysteine protease papain at 100 μM. CCG-203769 enhances Gαq-dependent cellular Ca2+ signaling in an RGS4-dependent manner. TDZD inhibitors also enhance Gαi-dependent δ-OR inhibition of cAMP production in SH-SY-5Y cells, which express endogenous receptors and RGS4. Importantly, CCG-203769 potentiates the known RGS4 mechanism of Gαi-dependent muscarinic bradycardia in vivo. Furthermore, it reverses raclopride-induced akinesia and bradykinesia in mice, a model of some aspects of the movement disorder in Parkinson’s disease. A broad assessment of compound effects revealed minimal off-target effects at concentrations necessary for cellular RGS4 inhibition. These results expand our understanding of the mechanism and specificity of TDZD RGS inhibitors and support the potential for therapeutic targeting of RGS proteins in Parkinson’s disease and other neural disorders

Perturbing the Ubiquitin Pathway Reveals How Mitosis Is Hijacked to Denucleate and Regulate Cell Proliferation and Differentiation In Vivo

The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip), a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed

Conformational Dynamics of a Regulator of G‑Protein Signaling Protein Reveals a Mechanism of Allosteric Inhibition by a Small Molecule

Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the G_α-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations and NMR spectroscopy, we have studied in molecular detail the mechanism of action of CCG-50014, a potent small molecule inhibitor of RGS4 that covalently binds to cysteine residues on RGS4. We apply temperature-accelerated molecular dynamics (TAMD) to carry out enhanced conformational sampling of <i>apo</i> RGS4 structures, and consistently find that the α₅-α₆ helix pair of RGS4 can spontaneously span open-like conformations, allowing binding of CCG-50014 to the buried side-chain of Cys95. Both NMR experiments and MD simulations reveal chemical shift perturbations in residues in the vicinity of inhibitor binding site as well as in the RGS4-G_α binding interface. Consistent with a loss of G-protein binding, GAP activity, and allosteric mechanism of action of CCG-50014, our simulations of the RGS4-G_α complex in the presence of inhibitor suggest a relatively unstable protein–protein interaction. These results have potential implications for understanding how the conformational dynamics among RGS proteins may play a key role in the sensitivity of inhibitors