New Cell Signaling Pathways Mapped

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June 15, 2018 (Vol. 38, No. 12)

Vivienne Raper Ph.D. Freelance Journalist

Focus on Visualizing Signal Molecules and Reducing Drug Side Effects

Over the last 30 years, cell signaling has come to play a major role in cell and molecular biology research, as well as in drug discovery. The process by which cells communicate is—at its most fundamental—a description of life itself, and is implicated in areas of healthcare as diverse as treating heart disease, Alzheimer’s, and drug addiction.

The process of cell signaling begins when a ligand, a signaling molecule such as a hormone or neurotransmitter, released by one cell interacts with a compatible receptor on another cell’s surface. One of the largest and most varied group of membrane-protein receptors on eukaryotic cells are G-protein-coupled receptors (GPCRs).

GPCRs mediate a huge range of functions within the human body, which makes them a popular target for drug development, and the focus of the 7th Focused Meeting on Cell Signalling, which took place last month at the East Midlands Conference Centre in Nottingham, U.K. Among the topics covered at the meeting were microscopy techniques for visualizing individual molecules, and research targeting individual signaling pathways to reduce drug side effects.


Aiding Alzheimer’s Patients

Today, an estimated one third to one half of all marketed drugs bind to GPCRs. According to a 2016 review co-authored by Sophie Bradley, Ph.D., a research fellow at the Institute of Molecular Cell and Systems Biology, University of Glasgow, their popularity as a drug target is due to their versatility – they respond to a myriad of substances, including hormones, neurotransmitters, odorants and even light. Moreover, they selectively interact with small-molecule ligands in a way that can be replicated with synthetic molecule drugs (Figure 1).

One potential GPCR target for drug discovery is the M1 muscarinic acetylcholine receptor (M1 mAChR). “We’re interested in M1 mAChR as a therapeutic target for Alzheimer’s disease,” says Dr. Bradley, who co-authored a paper published in 2017 on targeting M1 mAChR in mice engineered to experience prion disease. She presented new published and unpublished research at the 7th Focused Meeting.

According to Dr. Bradley, the current frontline treatments for Alzheimer’s are acetylcholinesterase inhibitors, which increase transmission of a signaling molecule, acetylcholine, in the parasympathetic nervous system (PSNS). Acetylcholine signaling to key brain regions is reduced in Alzheimer’s.

Unfortunately, the non-specificity of acetylcholinesterase inhibitors means they have unpleasant side effects at higher doses. “Targeting M1 mAChR could provide a more selective approach,” says Dr. Bradley. “By targeting M1 receptors in mice with degenerative disease, we reversed cognitive decline and increased the lifespan of the mice,” she says.

Dr. Bradley also explained how targeting the M1 mAChR receptor may have additional benefits in treating Alzheimer’s. “Alzheimer’s patients don’t just suffer memory decline, but also experience a variety of other symptoms, such as listlessness and agitation,” she says. Dr. Bradley and her team have discovered that targeting M1 mAChR could decrease hyper-anxiety and incessant movement, an indicator of agitation, in genetically modified mice. “This shows that, perhaps, by pharmacologically targeting M1 mAChR in human beings, we can also reduce agitation.”


Figure 1. A hypothetical example of stimulus bias shows a GPCR that is able to couple to two distinct pathways, pathway A and pathway B. On the left, agonist A is able to activate pathway A preferentially compared with pathway B; whereas agonist B, on the right, has the opposite effect. The design of ligands that display selectivity for one pathway over another may offer a means of driving signaling down a therapeutically beneficial arm while minimizing signaling down pathways that lead to toxic/adverse outcomes. [Figure supplied by Sophie Bradley, Ph.D. Text from Bradley and Tobin. Annu. Rev. Pharmacol. Toxicol. 2016.56.535-559. Doi 10.1146/annurev-pharmtox-011613-140012]

Choosing the Right Pathway

Adverse side effects aren’t just a problem for acetylcholinesterase inhibitors. Non-specific interactions between pharmaceuticals and GPCRs explain—in part—why, according to a review co-authored by Dr. Bradley in 2016, only 15% of the around 390 non-odorant GPCRs in the human genome have been successfully targeted by drugs.

To avoid this problem, researchers are beginning to study how drug candidates can trigger a cascade of undesirable processes within a cell by targeting multiple similar receptors or signaling pathways. “A large number of drug trials focused on the M1 receptor have failed,” she says. “We wanted to work out which pathways generate these adverse responses.”

According to Dr. Bradley, her team is the first to target the M1 receptor by using chemogenetic techniques in mice. Through selectively knocking out a phosphorylation-dependent signaling pathway, they discovered that certain M1 receptor ligands triggered seizures. “Drugs should potentially be designed to promote phosphorylation and hence reduce the likelihood of adverse responses,” she says.


Tackling Cardiovascular Disease

Anthony Davenport, Ph.D., reader in cardiovascular pharmacology at the University of Cambridge, U.K., is also working to improve the selectivity of pharmaceuticals, focused on cardiovascular drugs that target the apelin receptor (APJ), a GPCR activated by a peptide called apelin (Figure 2).

“We’re particularly into apelin because it has two beneficial effects when infused into humans: it causes the blood vessels to relax or dilate, and it increases the output of the heart,” Dr. Davenport says. In cardiovascular disease, the heart often loses its ability to pump, and the blood vessels narrow, forcing the heart to pump harder to force blood through. Drugs targeting the APJ pathway are “potentially valuable in treating cardiovascular disease,” he explains, because they address both problems at the same time. This is a new target and apelin drugs have not yet entered the clinic.

Unfortunately, many of the drugs targeting the family of receptors that include apelin don’t just produce the beneficial effects of activating the G-protein signaling pathway inside the cell. They also activate a second group of intercellular proteins, called beta-arrestins, which reduce the sensitivity of APJ to further signaling and mean higher doses of the drug are needed to generate the same effect.

Dr. Davenport and his team have found a small molecule, potentially suitable as an oral once-a-day drug that selectively activates APJ without activating the beta-arrestin pathway. “What we’ve shown, for the first time, is that we can produce the benefit of increasing cardiac output, but without desensitizing the receptor,” he says. Dr. Davenport hopes this will provide an avenue for the development of new drugs.


Figure 2. Computer model of the small molecule apelin agonist binding to two key residues in the apelin receptor (green). [Read et al. Biochem Pharmacol 2016 116:63-72. doi:10.1016/ j.bcp.2016.07.018. Image and caption supplied by Anthony Davenport, Ph.D.]

Understanding Overdose

“Our overall topic is trying to understand why people die from heroin overdose when they haven’t taken much,” says Graeme Henderson, Ph.D., professor of pharmacology at the University of Bristol, U.K. His talk at the 7th Focused Meeting on Cell Signalling also covered how GPCRs can be desensitized to drugs—in this case, to morphine and methadone.

“Our basic premise is that when you take heroin regularly you become tolerant to it, and the tolerance mechanisms involve switching off the opioid receptors by enzymes or kinases,” says Dr. Henderson. He explains that the kinase that switches off the receptor is different for morphine and methadone so, although an addict can become tolerant to both, the cell signaling mechanism is different.

For morphine, the enzyme involved is protein kinase C (PKC) whereas, for methadone, it’s an enzyme family called G-protein-coupled receptor kinase (GRK). What’s novel about his research, he explains, is that his team has moved from studying the basic cell signaling mechanisms to understanding, in an animal model, which enzymes are important in overdose deaths.

When animals were chronically treated with methadone or morphine, they experienced a greater amount of respiratory depression if they were given ethanol or pregabalin, a prescription drug that is also abused.

“What we’ve shown is that ethanol (alcohol) and pregabalin reverse tolerance to morphine, but not tolerance to methadone,” Dr. Henderson says. “The obvious interpretation is that ethanol and pregabalin inhibit PKC, but not GRK.” Since drug addicts often take many drugs at the same time, “if you have a drink before shooting up, you’ll get more effect of the heroin, more respiratory depression, and then you can die.”

Dr. Henderson is currently researching how alcohol and pregabalin might operate to inhibit PKC inside the cell. He explains that PKC and GRK sit a little way from the receptor inside the cell but move to the receptor when it’s activated by a drug—a process called translocation. Although the team has no definitive evidence, Dr. Henderson believes that alcohol and pregabalin stop the translocation process of the enzyme, reducing its activity at the receptor.


Studying Individual Receptors at Work

“We don’t fully understand how G-protein-coupled receptors decode signals from the outside,” says Davide Calebiro, Ph.D., professor of molecular endocrinology at the Institute of Metabolism and Systems Research, University of Birmingham, U.K., and Centre of Membrane Proteins and Receptors (COMPARE) at both Birmingham and Nottingham Universities, U.K. (Figure 3).

He explains that, until recently, scientists believed these receptors were simple on-off switches. They now understand these systems are far more complex—for example, with different drugs producing different effects by acting on the same receptor. Understanding these processes, however, remains difficult, he says, because standard microscopy and biochemical techniques don’t have the spatial and temporal resolution required to study receptors on the scale at which crucial signaling events take place.

Dr. Calebiro’s talk was about single-molecule microscopy as a tool for studying individual signaling proteins as they move and interact on the surface of living cells. “We can now address fundamental questions about how individual receptors work,” he says. His team is among the first to use the technique for GPCR studies.

“Our recent study (Sungkaworn et al., Nature 2017) was the first where someone could visualize individual receptors and G proteins, which transmit their signals inside cells, as they interact to produce specific effects—this is very new.”

Crucial improvements in technology making this work feasible include the development of new ultrasensitive cameras, he explains, with new models able to capture the few photons emitted by a single molecule. “Until recently, only a few labs in the world could do this kind of work. Now, with current technology, we can look for a longer time, at different types of molecules with different colors simultaneously and go for real biological applications,” he says.


Figure 3. Individual receptors (green) and G proteins (magenta) imaged on the surface of a living cell. [Modified from Sungkaworn et al. Nature 2017 Oct 26;550(7677):543-547. doi: 10.1038/nature24264 (image and caption provided by Davide Calebiro, Ph.D.)]

Role of Redox Signaling in Wound Healing

Tissue oxygenation is a critical determinant of the overall wound healing process. Apart from fueling the high energy demands of tissue repair, tissue oxygen is expended to generate reactive oxygen species (ROS) at the injury site.

In an open skin wound, infection is a common threat. It causes immune cells of the body to mount a “respiratory burst” where phagocytic oxidases utilize oxygen to generate ROS and its derivatives to disinfect the wound. Thus, the wound fluid is observed to be one of the compartments of the body with highest reported concentrations of hydrogen peroxide, an ROS.

Reversible oxidation-reduction processes play a key role in multiple aspects of the wound healing process, says Chandan Sen, Ph.D., from the Ohio State University Wexner Medical Center, where he serves as executive director of the, Ohio State Comprehensive Wound Center. He is also editor-in-chief of Antioxidants & Redox Signaling, published by Mary Ann Liebert, Inc.

“Almost all non-phagocytic cells at the wound site are equipped with enzymatic systems to deliberately generate ROS for redox signaling.  Redox signaling is directly implicated in epithelial migration that helps the epidermis cover the wound,” says Dr. Sen. “Hydrogen peroxide and nitric oxide work in tandem to draw vasculature to the site of tissue repair. The wound bed provides the extracellular matrix support for tissue repair.”

“Redox signaling is central to numerous aspects of collagen formation and maturation—processes that are responsible for healthy tensile strength of the repaired skin,“ continues Dr. Sen. “A defective extracellular matrix compromises skin biomechanics, thus causing wound recurrence, a major public health burden.”

“Of major concern are emergent findings reporting that the biofilm form of infection, often not detected by standard clinical tests, is able to arrest the ability of mammalian cells to enzymatically generate ROS,” says Dr. Sen. “Such forms of infection, known to be associated with over two-thirds of all clinically presented chronic wounds, is likely to derail redox signaling cascades required for healthy repair of skin wounds. “

























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