Physiological Roles of Mammalian Transmembrane Adenylyl Cyclase Isoforms

Adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. Mammals possess nine isoforms of transmembrane ACs, dubbed AC1-9, that serve as major effector enzymes of G protein-coupled receptors. The transmembrane ACs display varying expression patterns across tissues, giving potential for them having a wide array of physiologic roles. Cells express multiple AC isoforms, implying that ACs have redundant functions. Furthermore, all transmembrane ACs are activated by Gαs so it was long assumed that all ACs are activated by Gαs-coupled GPCRs. AC isoforms partition to different microdomains of the plasma membrane and form prearranged signaling complexes with specific GPCRs that contribute to cAMP signaling compartments. This compartmentation allows for a diversity of cellular and physiological responses by enabling unique signaling events to be triggered by different pools of cAMP. Isoform specific pharmacological activators or inhibitors are lacking for most ACs, making knockdown and overexpression the primary tools for examining the physiological roles of a given isoform. Much progress has been made in understanding the physiological effects mediated through individual transmembrane ACs. GPCR-AC-cAMP signaling pathways play significant roles in regulating functions of every cell and tissue, so understanding each AC isoform\u27s role holds potential for uncovering new approaches for treating a vast array of pathophysiological conditions

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

Exploring Opioid and Cannabis Pharmacology: Biased Signaling of Endogenous Opioid Peptides and Cannabimimetic Properties of Cannabis Sativa Terpenes

The United States has roughly 100 million people suffering from pain disorders, including chronic pain, which is becoming an ever increasing medical and economic burden. Opioids and non-steroidal anti-inflammatory drugs (NSAIDs) are first line drugs for the treatment of acute and post-operative pain, however opioids can be ineffective for long-term treatment due to negative side effects such as tolerance and addiction. Recent efforts have attempted to combat pain pharmacologically through different avenues including biased agonists at the mu opioid receptor (MOR) and non-opioid targets such as cannabinoid receptors. Biased agonists attempt to preferentially activate specific downstream pathways of MOR that are therapeutically beneficial over pathways linked to negative side effects. G protein signaling through the MOR mediates its antinociceptive properties, whereas it has been proposed that barrestin2 (barr2) signaling elicits negative side effects such as tolerance and respiratory depression. Thus, recent efforts have attempted to create G protein biased agonists at MOR that have a greater therapeutic index compared to classic opioids. Although initially promising in preclinical trials, MOR biased ligands have fallen short in clinical trials. Although there is significant preclinical data in support of the therapeutic benefit of biased agonists, most of the work uses exogenous agonists. Biased signaling in terms of endogenous ligands at GPCRs is limited and most is centered on chemokine receptors. The literature is very much lacking in whether endogenous agonists at MOR induce biased signaling, if at all, and what the physiological function of this is. It thus begs the question: do endogenous opioids participate in biased signaling? An understanding of biased signaling, or lack thereof, by endogenous ligands could help resolve the disparity between promising preclinical data and failed clinical applications. While the development of biased ligands appeared auspicious, non-opioid targets were also under investigation. Targets of particular interest in the past decade include cannabinoid receptors. Cannabinoid receptors are positioned in suitable locations for the relief of pain, albeit in many other areas, unlike to opioid receptors. In recent decades the stigma of Cannabis sativa has reduced substantially. Consequently, the plant is now legally used for its medicinal properties including analgesia among other treatments. Cannabis sativa is now also available recreationally in many states. The major pharmacologically active components in Cannabis sativa exploited for both medicinal and recreational purposes are (-trans-tetrahydrocannabinol (9-THC) and cannabidiol (CBD). These compounds are part of a large family of ~150 phytocannabinoids found in Cannabis sativa. The plant also contains hundreds of terpenes: small, lipophilic hydrocarbons causing the distinct smells and tastes of Cannabis sativa chemovars. Anecdotal reports have suggested that differing cannabis strains, differing in terpene content, can induce different subjective effects. Reports also show differing therapeutic benefit of cannabinoid/terpene combinations versus just 9-THC alone. This concept is called the entourage effect and suggests interactions of terpenes and cannabinoids at some level In these studies, we have examined whether endogenous opioids participate in biased signaling, and attempted to determine mechanisms for those compounds which do. Furthermore, we sought to characterize the cannabimimetic properties of selected terpenes both in vitro and in vivo, and how these terpenes modulate the function of a cannabinoid agonist

The endomorphin-1/2 and dynorphin-B peptides display biased agonism at the mu opioid receptor

Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR.12 month embargo; published online: 28 February 2020This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Novel Molecular Strategies and Targets for Opioid Drug Discovery for the Treatment of Chronic Pain.

Opioid drugs like morphine and fentanyl are the gold standard for treating moderate to severe acute and chronic pain. However, opioid drug use can be limited by serious side effects, including constipation, tolerance, respiratory suppression, and addiction. For more than 100 years, we have tried to develop opioids that decrease or eliminate these liabilities, with little success. Recent advances in understanding opioid receptor signal transduction have suggested new possibilities to activate the opioid receptors to cause analgesia, while reducing or eliminating unwanted side effects. These new approaches include designing functionally selective ligands, which activate desired signaling cascades while avoiding signaling cascades that are thought to provoke side effects. It may also be possible to directly modulate downstream signaling through the use of selective activators and inhibitors. Separate from downstream signal transduction, it has also been found that when the opioid system is stimulated, various negative feedback systems are upregulated to compensate, which can drive side effects. This has led to the development of multi-functional molecules that simultaneously activate the opioid receptor while blocking various negative feedback receptor systems including cholecystokinin and neurokinin-1. Other novel approaches include targeting heterodimers of the opioid and other receptor systems which may drive side effects, and making endogenous opioid peptides druggable, which may also reduce opioid mediated side effects. Taken together, these advances in our molecular understanding provide a path forward to break the barrier in producing an opioid with reduced or eliminated side effects, especially addiction, which may provide relief for millions of patients.Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Addition of a new resource in agroecosystems: Do cover crops alter the trophic positions of generalist predators?

International audienc

Optimization of a Pyrimidinone Series for Selective Inhibition of Ca2+/Calmodulin-Stimulated Adenylyl Cyclase 1 Activity for the Treatment of Chronic Pain

Adenylyl cyclase type 1 is an emerging target for the treatment of chronic pain that is downstream on the analgesic pathway from the traditional µ-opioid receptor. AC1 is expressed in the central nervous system and critical for signaling in pain sensitization. Behavioral studies have revealed AC1 knockout mice exhibit reduced behavioral pain sensitization responses similar to morphine administration. AC1, and a closely related isoform AC8, are also implicated to have a role in learning and memory signaling processes. However, reports suggest selectively targeting AC1 over AC8 may be a viable strategy to eliminate potential deleterious effects on learning and memory. Our team has carried out cellular screening for inhibitors of AC1 that yielded a pyrazolyl-pyrimidinone scaffold with potency comparable to previously published AC1 inhibitors, selectivity versus AC8, and improved drug-like physicochemical properties. Structure-activity relationship (SAR) studies produced 36 analogs that balanced improvements in potency with cellular IC50 values as low as 0.25 µM and selectivity versus AC8. Prioritized analogs were selective for AC1 compared to other AC isoforms and other common neurological targets. A representative analog was assessed for efficacy in a mouse model of inflammatory pain and displayed modest anti-allodynic effects. This series of compounds represents the most potent and selective inhibitors of Ca2+/Calmodulin-stimulated AC1 activity to date with reduced off-target liabilities and improved drug-like physicochemical properties making them promising lead compounds for the treatment of inflammatory pain