Alyssa Calloway
Research Mentor(s): John Traynor
Mentor Department: Pharmacology
Authors: Alyssa Calloway, Benjamin Clements, John Traynor
Session: Session 7 (4:00pm – 4: 50pm)
Presentation Type: Oral
Abstract
It is estimated that around 21% of adults in the United States experience chronic pain. To treat this chronic pain, opioids are the most powerful medications available, but use is limited due to risks of constipation, abuse, and overdose. Positive allosteric modulators (PAMs) are compounds that aid in opioids binding to its receptor as well as increasing the efficacy of the agonist. PAMs also effectively increase the pain-relieving effects of opioids in rodent models when administered together without increasing the negative side effects. These preliminary experiments suggest that this combination could decrease the dose of opioid administered to treat pain. However, knowledge of the mechanism of PAMs at cellular levels is limited. For example, two structurally-distinct compounds, BMS-122 (a thiazolidine) and BMS-187 (a xanthene-dione), both potentiate opioid signalling at the µ-opioid receptor. This project aims to better understand the interactions of these two PAMs at the µ-opioid receptor through in vitro work. While we know that these PAMs bind to an allosteric site on the receptor, it is unknown if both BMS-122 and BMS-187 bind at the same allosteric site. In this project I measured activation of the µ-opioid receptor using the GTP?[35S] assay. In brief, GTP?[35S] will bind to the G protein upon activation of the opioid receptor. Then, using scintillation counting, we are able to determine how much GTP?[35S] binds to the G protein with increasing concentrations of PAMs. Using this assay, I completed both antagonistic and synergistic experiments. In the antagonistic assay, I used one active PAM, either BMS-122 or BMS-187, a specific orthosteric agonist to the µ-opioid receptor, and an inactive PAM, BMS-124. Since BMS-122 and BMS-187 have very different chemical structures, it was hypothesized that they would have unique binding sites. Alternatively, my data demonstrates that the activity of both BMS-122 and BMS-187 are antagonized by BMS-124 suggesting a possible shared binding site. In the synergistic protocol, both of the active PAMs, BMS-122 or BMS-187, were added at the same time. There does not seem to be a strong synergistic relationship between the two active PAMs which suggests that both of the active PAMs have a similar binding site and compete for the site instead of synergizing. This project has shown that all three of the PAMs likely have binding sites in close proximity to each other on the µ-opioid receptor. These facts aim to better understand the mechanisms and gain background knowledge on the PAMs to develop more effective and receptor-specific PAMs in the future.
