Biology subjects

Watts, V. J.

Publications and source records attributed to Watts, V. J..

5 recordsLinked to original sources

bioRxiv2025

Inhibition of adenylyl cyclase 1 (AC1) and exchange protein directly activated by cAMP (EPAC) restores ATP-sensitive potassium (KATP) channel activity after chronic opioid exposure

Prolonged exposure to Gi/o receptor agonists such as opioids can lead to a sensitization of adenylyl cyclases (ACs), resulting in heterologous sensitization or cyclic AMP (cAMP) overshoot. The molecular consequences of cAMP overshoot are not well understood, but this adaptive response is suggested to play a critical role in the development of opioid tolerance and withdrawal. We found that genetic reduction of AC1 and simultaneous upregulation of ATP-sensitive potassium (KATP) channel subunits, SUR1 or Kir6.2, significantly attenuated morphine tolerance and reduced naloxone-precipitated withdrawal. In vitro models utilized an EPAC2-GFP-cAMP biosensor to investigate sensitization of adenylyl cyclase in SH-SY5Y neuroblastoma cells and HEK{Delta}AC3/6 knockout cells. Acute application of DAMGO significantly decreased the cAMP signal from the EPAC2-GFP-cAMP biosensor, while chronic DAMGO administration resulted in enhanced cAMP production following AC stimulation. Inhibition of cAMP overshoot was observed with naloxone (NAL), pertussis toxin (PTX), and the neddylation inhibitor, MLN4924 (Pevonedistat), as well as co-expression of {beta}-adrenergic receptor kinase C-terminus ({beta}ARK-CT). After establishment of the AC1-EPAC sensitization in the in vitro models, we found that inhibition of AC1 or EPAC enhanced potassium channel activity after chronic morphine treatment, using a thallium-based assay in SH-SY5Y cells. Similar data were obtained in mouse dorsal root ganglia (DRG) after chronic morphine treatment. This study presents evidence for investigating further AC1 signaling as a target for opioid tolerance and withdrawal, by increasing EPAC activity and affecting potassium channels downstream of opioid receptors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/636278v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@185bb82org.highwire.dtl.DTLVardef@d97ea4org.highwire.dtl.DTLVardef@1097addorg.highwire.dtl.DTLVardef@1616fcf_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

neuroscience↗

bioRxiv2024

Discovery of Novel and Selective GPR17 Antagonists as Pharmacological Tools for Developing New Therapeutic Strategies in Diabetes and Obesity

G protein coupled receptors (GPCRs) are promising targets for diabetes and obesity therapy due to their roles in metabolism and excellent potential for pharmacological manipulation. We previously reported that Gpr17 ablation in the brain-gut axis leads to improved metabolic homeostasis, suggesting GPR17 antagonism could be developed for diabetes and obesity treatment. Here, we performed high throughput screening (HTS) and identified two new GPR17 antagonists (compound 978 and 527). Both compounds antagonized downstream Gi/o, Gq and {beta}-arrestin signaling with high selectivity for GPR17, but not the closely related purinergic and cysteinyl leukotriene receptors. The molecular mechanisms of antagonism were revealed through Schild analysis, structure-activity relationship (SAR) studies and homology modelling. Compound 978 and its analog (793) attenuated GPR17 signaling and promoted glucagon-like peptide-1 (GLP-1) secretion in enteroendocrine cells. In summary, we identified selective GPR17 antagonists through HTS, which represent promising pharmacological tools for developing new therapeutic strategies in diabetes and obesity. SignificanceOur work highlights the therapeutic potential of GPR17 antagonism in the treatment of diabetes and obesity by leveraging its role in metabolic regulation. In previous studies, we have shown that Gpr17 ablation improves metabolic homeostasis, and here we expanded our research by identifying two novel small molecule antagonists of GPR17 through high-throughput screening. The compounds inhibited multiple downstream signaling pathways of GPR17 with high selectivity over other closely related receptors. Of particular significance, compound 978 and its analogs not only attenuated GPR17 signaling but also increased glucagon-like peptide-1 (GLP-1) secretion, a critical hormone for glucose homeostasis and appetite regulation. These findings shed new light into the molecular mechanisms of GPR17 antagonism and introduce valuable pharmacological tools for further exploration of therapeutic strategies in diabetes and obesity.

pharmacology and toxicology↗

bioRxiv2024

G protein-coupled receptor 17 inhibits glucagon-like peptide-1 secretion via a Gi/o-dependent mechanism in enteroendocrine cells

Gut peptides, including glucagon-like peptide-1 (GLP-1), regulate metabolic homeostasis and have emerged as the basis for multiple state-of-the-art diabetes and obesity therapies. We previously showed that G protein-coupled receptor 17 (GPR17) is expressed in intestinal enteroendocrine cells (EECs) and modulates nutrient-induced GLP-1 secretion. However, the GPR17-mediated molecular signaling pathways in EECs have yet to be fully deciphered. Here, we expressed the human GPR17 long isoform (hGPR17L) in GLUTag cells, a murine EEC line, and we used the GPR17 synthetic agonist MDL29,951 together with pharmacological probes and genetic approaches to quantitatively assess the contribution of GPR17 signaling to GLP-1 secretion. Constitutive hGPR17L activity inhibited GLP-1 secretion, and MDL29,951 treatment further inhibited this secretion, which was attenuated by treatment with the GPR17 antagonist HAMI3379. MDL29,951 promoted both Gi/o and Gq protein coupling to mediate cyclic AMP (cAMP) and calcium signaling. hGPR17L regulation of GLP-1 secretion was Gq-independent and dependent upon Gi/o signaling, but was not correlated with MDL29,951-induced whole-cell cAMP signaling. Our studies revealed key signaling mechanisms underlying the role of GPR17 in regulating GLP-1 secretion and suggest future opportunities for pharmacologically targeting GPR17 with inverse agonists to maximize GLP-1 secretion O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/623413v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@18d0e45org.highwire.dtl.DTLVardef@1a3e431org.highwire.dtl.DTLVardef@da8118org.highwire.dtl.DTLVardef@1518576_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

molecular biology↗

bioRxiv2023

Isoform Specific Regulation of Adenylyl Cyclase 5 by Gβγ

The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein Gs, but their response to G{beta}{gamma} regulation is isoform-specific. For example, AC5 is conditionally activated by G{beta}{gamma}. Here, we report cryo-EM structures of ligand-free AC5 in complex with G{beta}{gamma} and of a dimeric form of AC5 that could be involved in its regulation. G{beta}{gamma} binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the G{beta}{gamma} interaction with both purified proteins and cell-based assays. The interface with G{beta}{gamma} involves AC5 residues that are subject to gain-of-function mutations in humans with familial dyskinesia, indicating that the observed interaction is important for motor function. A molecular mechanism wherein G{beta}{gamma} either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core, is proposed. Because our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

biophysics↗

bioRxiv2020

Reduced activity of Adenylyl Cyclase 1 Attenuates Morphine Induced Hyperalgesia and Inflammatory Pain in Mice

Opioid tolerance and opioid-induced hyperalgesia during repeated opioid administration and chronic pain are associated with upregulation of adenylyl cyclase activity. The objective of this study was to test the hypothesis that a reduction in adenylyl cyclase 1 (AC1) activity or expression would attenuate morphine tolerance and hypersensitivity, and inflammatory pain using murine models. To investigate opioid tolerance and opioid-induced hyperalgesia, mice were subjected to twice daily treatments of saline or morphine using either a static (15 mg/kg, 5 days) or an escalating tolerance paradigm (10-40 mg/kg, 4 days). Systemic treatment with an AC1 inhibitor, ST03437 (5 mg/kg, ip), reduced morphine tolerance and morphine hyperalgesia in mice. Lumbar intrathecal administration of a vector incorporating adeno-associated virus and short-hairpin RNA against Adcy1 reduced morphine induced hypersensitivity compared to control vector treated mice. In contrast, morphine antinociception, along with baseline thermal paw withdrawal latencies, motor performance, exploration in an open field test, and burrowing behaviors were not affected by intrathecal Adcy1 knockdown. Knockdown of Adcy1 by intrathecal injection also attenuated inflammatory mechanical hyperalgesia after intraplantar administration of Complete Freunds Adjuvant (CFA) after one week post injection. This Adcy1 knockdown strategy also increased burrowing and nesting activity after CFA injection when compared to controls. Together, these data indicate targeting AC1 to mitigate opioid-induced adverse effects, or as a method to treat chronic pain, are appropriate as a clinical approach and further development into generating pharmaceuticals targeting these genes/proteins may prove beneficial in the future.

neuroscience↗