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Method “Matches” Medicines to Their Unique Protein Receptors

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A receptor (drug target) can shift from a single subunit to a multi-structure in the presence of a ligand (drug compound) – a process known as "oligomerization". Having a method to determine exactly how different drug compounds target the same receptor is key to understanding why and how some candidates are effective while others are not. 

Now, using photon excitation microscopy, researchers report that they can uncover the various protein receptor oligomers formed in the absence or presence of different drug candidates that bind to them.

We recently spoke with Michael Stoneman and Valerica Raicu to learn more about photon excitation microscopy, and the potential this technique holds for the field of drug discovery.

Molly Campbell (MC): In the
press release, you state that depending on the ligand, the same receptor can produce many different responses. Please can you expand on this and provide examples?

Michael Stoneman (MS):
Cells receive information from their surrounding environment through a class of proteins known as receptors. Signals originating from outside the cell are typically triggered by the binding of signaling molecules, known as ligands, to a particular type of receptor. This in turn triggers an event inside the cell. The binding of the ligand to the receptor either causes a change or stabilizes a particular conformation of said receptor, which then initiates a cascade of biochemical events, ultimately resulting in a physiological response from the cell.

Many receptors can initiate their signaling pathway even in the absence of a ligand; this is typically referred to as the basal signaling level of the receptor. The role of the natural binding ligand to a particular receptor is to increase its signaling activity above the basal level. These types of ligands are referred to as agonists. An inverse agonist is a ligand that binds to the same receptor as an agonist but induces a pharmacological response which is different than that of the agonist. For example, an inverse agonist could decrease the activity of the receptor below the basal level. Finally, an antagonist is a ligand which binds to a particular receptor but produces no response other than to block agonists and inverse agonists from binding to the receptor.

A well-known example of a group of receptors exhibiting ligand specific responses are the adrenergic receptors. These receptors are targets of a number of endogenous ligands, e.g. epinephrine, which are naturally produced by the body. The cascade of reactions induced by binding of epinephrine to adrenergic receptors stimulates the sympathetic nervous system (SNS), which is responsible for the "fight-or-flight" response. In turn, excessive amounts of epinephrine can lead to conditions such as heart disease and high blood pressure. A common group of medications called beta blockers are typically given to combat such adverse conditions. Beta blockers are inverse agonists which bind to and reduce the activity of the adrenergic receptors. There are a plethora of drugs in the beta-blocker class with varying levels of specificity and efficacy across the class.

Laura Lansdowne (LL): Your method tracks a chemical process called oligomerization, could you tell us more about oligomerization?

Valerica Raicu (VR):
A membrane receptor, which is a protein, consists of multiple amino acids connected along a line by strong covalent bonds. This line of amino acids, or polypeptide chain, then folds into a distinctive shape, called tertiary structure, which is suitable for its biological function. The properly folded amino acid chain is the protein of interest.

A protein oligomer is a complex which is comprised of two (dimer) or more (trimer, tetramer, pentamer, etc.) properly folded proteins bound together by relatively weak forces (compared to the covalent bonds).
The properties of protein oligomers are incredibly diverse: some protein oligomers may be homooligomers, meaning each of the protein subunits is identical, while others form heterooligomers, i.e. two or more different subunits form the complex.

he association between each of the oligomer subunits varies in strength and duration across different protein complexes. Some proteins form one specific active oligomeric state that is essentially permanent. However, some proteins are involved in weaker interactions, forming a dynamic oligomerization equilibrium between several oligomeric states. A large group of proteins forming these dynamic interactions have been shown to be involved in triggering important cell signaling cascades which occur upon environmental changes. However, these interactions are poorly investigated, mainly due to methodological limitations. Of course, in certain instances, oligomerization of proteins can also lead to adverse effects. For example, unwanted oligomerization is thought to be a key contributing factor in both Parkinson’s and Alzheimer's disease.

MC: How can photon excitation microscopy be used to help characterize protein receptor responses to drug compounds?

Proteins cannot be visualized in the traditional sense that you put the cells they are expressed in under a benchtop microscope, and watch their distances and motions relative to one another in order to determine whether they are bound to one another or separated.

The resolution of optical microscopes, such as confocal microscopes, is simply not high enough for that.

Therefore, alternative techniques have been developed in order to study protein interactions. The field of fluorescence microscopy encompasses a multitude of such techniques for tracking proteins in living cells. Fluorescence microscopy makes use of fluorescent markers that emit colored light when illuminated with a light source, such as a laser. The cells expressing the protein of interest can be instructed, via a genetic transformation, to express the receptors with a fluorescent marker, such as the green fluorescent protein (GFP), attached to it. The fluorescence signal which is emitted by the markers within a cell exposed to, e.g., laser light, can be collected and analyzed to give information about the receptors which are attached to the markers themselves.

There are a number of experimental techniques which, when combined with fluorescence microscopy, give information on not only the location, but also the interactions of the fluorescently labeled proteins. Förster resonance energy transfer (FRET) allows the measurement of distances between two molecules on the nanometer length scale, a distance sufficiently close for molecular interactions to occur. Single particle tracking (SPT) quantifies the diffusion properties and motion paths of certain receptors, allowing the measurement of the strength of intermolecular interactions.

Our most recent approach outlined in the press release is called fluorescence intensity fluctuation (FIF) spectrometry. It measures the fluorescence fluctuations which result from variations in the protein oligomer concentration from pixel to pixel. The measured variance in this fluorescence signal is related to the size of the protein oligomer. By measuring a distribution of these fluctuations over many cells expressing the proteins, information regarding the proportion of oligomers with different sizes can be obtained. Therefore, FIF offers the ability to discriminate slight shifts in the equilibrium between the proportions of different sized oligomers. These shifts can be quantified as a function of treatment with various drug compounds (or ligands).

LL: What techniques did you use to test and validate this novel method?

We first tested the technique on samples comprised of mixtures of fluorescent protein constructs which were synthesized with varying oligomer sizes. Using an FIF based approach, we were able to measure the size of the fluorescent protein constructs and confirm that they were in agreement with the known composition of the samples prepared.

We then further tested the approach by applying it to cells expressing the epidermal growth factor receptor (EGFR). The oligomerization properties of EGFR already have been reported on using a number of fluorescence-based techniques, like FRET and SPT, which allowed us to compare the results obtained using our method to these previous studies.  

MC: What key benefits are associated with using this new method and what implications could it have on the drug development process?

The key benefit associated with this new method is the combination of (i) extracting a detailed breakdown of the proportions of various oligomeric sizes in (ii) a relatively short time window. Existing technologies have typically hit one of those two benchmarks; either they obtain detailed structural information regarding the proteins of interest but are rather slow and difficult to execute, or they are fast and easy to carry out but lack the capacity to accurately discern proportions of different oligomeric sizes.

Simultaneously hitting these two benchmarks could lead to crucial improvements in the drug development process. Drugs intended to affect a particular protein receptor can be sorted according to their ability to specifically modulate the oligomer size, either increase or decrease it, based on the results of the FIF approach.

Furthermore, the speed and simplicity of the approach offers the potential for scaling the FIF method to accommodate high-throughput screening of drug candidates that target protein oligomers.

LL: Could you tell us more about next steps? Are there any specific ligand-receptor associations you plan to investigate further?

The immediate next step will be to continue to probe the effect of ligands on the oligomeric size of various GPCRs and other receptors. We plan to expose cells expressing the receptors of interest to a bevy of agonists as well as antagonists and apply the FIF approach to obtain a detailed breakdown on the partitioning between oligomeric states for each of the ligands used. On a related note, we have also developed a user-friendly software package that incorporates every aspect of the analysis needed to carry out this technique. We have been eagerly sharing software with those interested in applying the FIF method to their receptor of interest.

In addition to applying the already established method, we plan to further develop the FIF approach on both the technology side, i.e. enhance the fluorescence microscope for image acquisition, as well as analysis procedure. The overall goal is to increase the throughput of the technique by a couple more orders of magnitude, to allow for rapid screening of drugs.

Michael Stoneman and Valerica Raicu were speaking to Laura Elizabeth Lansdowne and Molly Campbell, Science Writers for Technology Networks.