Opioids are powerful painkillers that act on the brain, but they have a range of harmful side effects including addiction. Researchers have developed a tool that gives deeper insights into the brain's response to opioids. Using mass spectrometry, they determined changes of proteins' phosphorylation patterns - the molecular switches of proteins - in five different regions of the brain and assigned them to the desired and the undesired effects of opioid treatment. Their results, which are published in the journal Science, will lead the way for identification of novel drug targets and design of a new class of painkillers with fewer side effects.
Researchers use mass spectrometry - a method that determines the identity and quantity of proteins in a sample - to describe phosphorylation patterns of thousands of proteins in many organ specimens, a term coined as phosphoproteomics. In the recent study, they analyzed the activation of signaling pathways in different regions of the brain, responding to opioid-like drugs. To achieve this goal, the researchers used a recently developed method named EasyPhos.
To understand how drugs like opioids work, researchers must know their influence on the brain. "With phosphoproteomics, we can analyze more than 50,000 phosphorylation sites at once and get a snapshot of all pathways that are active in the brain samples during that time. We found more than 1,000 changes after exposure to an opioid-like drug, showing a global effect of these drugs on signaling in the brain," says the lead author of the study. Previous methods could not capture protein phosphorylations at a comparable scale and missed many important signaling pathways that were switched on or off.
Authors observed strong regional specificity of KOR signaling attributable to differences in protein-protein interaction networks, neuronal contacts, and the different tissues in neuronal circuitries. Agonists with distinct signaling profiles elicited differential dynamic phosphorylation of synaptic proteins, thereby linking GPCR signaling to the modulation of brain functions.
The most prominent changes occurred on synaptic proteins associated with dopaminergic, glutamatergic, and γ-aminobutyric acid–mediated (GABAergic) signaling and synaptic vesicle release. The large-scale dephosphorylation of synaptic proteins in the striatum after 5 min of agonist stimulation was partially blocked by protein phosphatase 2A (PP2A) inhibitors, underscoring the involvement of PP2A in KOR-mediated synaptic functions.
Pathway analysis revealed enrichment of mTOR (mechanistic target of rapamycin) signaling by agonists associated with aversion. Strikingly, mTOR inhibition during KOR activation abolished aversion while preserving therapeutic antinociceptive and anticonvulsant effects.
The group performed behavior experiments using two drugs and found that they have similar analgesic effects, but very different levels of side effects. Brains of animals treated with the two drugs were analyzed by MPIB for phosphoproteomic differences, which were found to belong to a few signaling pathways. Inhibition of one of the identified pathways greatly reduced some of the side effects.
Phosphoproteomic analysis of brain's response to opioids
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