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Pharmacogenomic Analyses Using Microplate Reader Based BRET Assays

We read with great interest the recent Cell paper entitled ‘Pharmacogenomics of GPCR Drug Targets’. The authors used a PHERAstar FS to monitor a BRET assay that looked at activation of GPCR. To see activation a downstream effector molecule was labeled with nano-luciferase while the beta/gamma subunits of the G-protein were labeled with the Venus fluorophore.  What makes this interesting is that this real-time assessment of receptor activation was done in the context of known mutants of the receptor. Thus this approach can serve as an assessment of the effect that these mutations have on the ability of the receptor to respond to the normal ligand as well as the drugs that have been designed to modify the response.

pharmacogenomic analyses
Figure 1 Effects of Natural Mutations on Drug Activity and G-Protein Coupling – A portion of Figure 5 in ‘Pharmacogenomics of GPCR Drug Targets’ A) the location of the mutations in the receptor that were analyzed for the opioid receptor. B) BRET assay scheme D) results comparing the efficacy of response of the ligand and 3 drugs on activation of normal and mutant receptors

The results shown in the figure above are quite striking in clearly pointing out how differently the receptors respond to different treatments. The wild-type receptor exhibits the expected responses: full response to ligand and agonist, a distinct response to partial agonist and no response to antagonist. The variant in red exhibits a partial loss of function with reduced responses to full and partial agonists. The other 2 variants have enhanced responses, especially to the partial agonist and antagonist. Since these drugs are usually used to treat opioid overdose or abuse their application to an individual carrying these variants could have deleterious effects.

This new article sheds additional light onto our burgeoning understanding of the world of pharmacogenomics. The goal of pharmacogenomics is to use our understanding of an individuals’ genome to inform decisions on what drug and how much of a drug to prescribe. Since drugs that target GPCR’s are more than 1/3 of all FDA approved drugs they are certainly relevant for analysis. The small portion of the data shown above fully supports the impact that this kind of research can have.

What we are realizing is that there are very important differences that can appear in these druggable targets. In some cases these differences will go unnoticed but in the right set of circumstances the results can be vastly different from what is expected. The lock and key model for substrate / enzyme interaction is still a useful visualization tool and pharmacologists often extend it to help visualize the ligand (or drug) interaction with a receptor. If you are like me you have one door in your house for which you have made a lot of copies. These are useful if your family is visiting and you are going to be at work when they arrive or if your neighbor needs to feed the plants and water the cat while you are on vacation. Do they all work the same? In my experience no.  In the lock-key metaphor the ligand is usually considered to be the key and the receptor the lock. Yes that key can still be made to work but the effort needed to make it turn might be much more than another key. If we flip this thinking a little we can start to appreciate how small differences in the receptor can alter effectiveness to certain drugs. This is what is shown in the data above and a part of what we can hope to learn by more extensive pharmacogenomics research.

enzyme substrate complex
Figure 2 By Stephjc – Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=61795681

Drug discovery has by necessity proceeded with the mindset that one treatment will be suitable for all. This approach is still mostly useful and it might not be the place for attempting to anticipate every eventuality. But what they can do is perform more complete characterization of a drug based on the known most common variations, similar to the approach shown with the BRET assays in figure 1. This could begin to alleviate some of the economic burden that arises from prescription of ineffective treatments or the need to employ trial and error to discover the dose suitable for an individual. This type of testing could also be useful in efficacy trials as knowledge of the individuals’ potential to respond to a drug could be understood before a treatment is given.

Characterization of receptor variation is really only one aspect that is scrutinized within the field of pharmacogenomics. Drug metabolism is another aspect that has received much attention. Since many drugs are actually pro-drugs they require a certain amount of metabolism within the patient before they can elicit their beneficial effects. The proteins that perform this metabolism can be expressed to different levels or can have alternate forms that have different efficacy in performing the metabolism. So in this case characterization of the variant enzymes involved in drug metabolism is essential to understanding how an individual will respond.

So even though two individual humans will only differ by about 0.5% when their genomes are compared the location of these differences will be vital in individualized medicine that is envisioned by the practitioners of pharmacogenomics. Even the difference of a few base pairs in the right location could have a dramatic effect on how an individual will respond to a particular treatment. As the tools improve to test the effects of these variations their use will become more common. It is possible that in the foreseeable future you will have the opportunity to determine treatments that are almost completely to tailored to you and you alone.