Abstract
We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.
Highlights
Urinary incontinence (UI) is an increasing medical problem in ageing populations
The signal transduction pathways downstream of muscarinic G-protein coupled receptor (GPCR) are responsible for bladder muscle cell contractility, and antagonists of these receptors allow for greater bladder filling
To define the pathway of action of these small molecules, we have used genetic screens in Caenorhabditis elegans coupled with biochemical assays in mammalian systems. We demonstrate that these small molecules likely act at the intersection of regulators of G-protein signaling (RGS) and G-protein alpha subunit (G-aq) proteins, resulting in the downregulation of GPCR signaling, reduced calcium fluxes, and reduced muscle contraction
Summary
Urinary incontinence (UI) is an increasing medical problem in ageing populations. Affecting more than 12 million afflicted people in the US alone, UI is a frequent cause of confinement and lifestyle modification [1]. Current treatments for UI rely on antagonism of G-protein coupled receptors (GPCRs) of the muscarinic acetylcholine receptor class [2]. The signal transduction pathways downstream of muscarinic GPCRs are responsible for bladder muscle cell contractility, and antagonists of these receptors allow for greater bladder filling. While muscarinic GPCR antagonists are generally safe, they have unwanted side effects due to the broad tissue expression of their targets [3,4,5]. GPCRs are the most successful class of targets for disease states including hypertension, diabetes, obesity, depression, osteoporosis, and inflammation. More than half of currently marketed drugs for the condition act as modulators of this protein class [6,7]. Methods to modulate other signaling nodes downstream of GPCRs may hold potential for safer and more efficacious therapies
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