Chronic itch associated diseases such as eczema and psoriasis will affect over 20% of human beings during their lifetime (Adikaram et al., 2019). With a statistic as immense as this, there are surprisingly few anti-chronic itch drugs available. Those that do exist usually come with unwanted side effects such as somnolence, weight gain and gastrointestinal symptoms (Yosipovitch, Rosen, and Hashimoto, 2019). Fortunately, Poorni Adikaram and the rest of William F. Simonds’ research group are working on a solution to this crisis. With the previous discovery that R7BP is a master itch regulator, Simonds’ group found that targeting this protein’s interaction with the R7-RGS/Gβ5 duplex could pave the way for anti-itch drugs that have fewer unwanted side effects.
At Nicoya, our mission is to improve human life by helping scientists succeed. We are ecstatic to share the amazing work done by Simonds and his team on his latest publication in Communications Biology: “Development of R7BP inhibitors through crosslinking coupled mass spectrometry and integrated modelling.” Simonds’ group made significant findings that were supported by quantitative binding kinetics data measured using the OpenSPR™. Let’s take a look at the novel approach to treating chronic itch Simonds’ group discovered and the techniques they used to get there.
Using XL-MS guided I-TASSER models to predict protein structure
Ultimately, the Simonds group aimed to find a way to inhibit the protein-protein interactions (PPIs) between R7BP (a master itch regulator) and the R7-RGS/Gβ5 duplex. Typically, designing PPI inhibitors requires detailed structural information of the proteins involved. The techniques normally used to produce this structural information require a crystallized protein, and R7BP is, unfortunately, a challenging protein to crystallize. Additionally, R7BP shares no protein domains with homologs that already have structural information.
To combat this, the authors chose instead to use computational modelling assisted by protein cross-linking coupled with mass spectrometry (XL-MS). XL-MS is a fast and cost-effective method that aids in determining protein interaction sites by measuring the spatial proximity of amino acid pairs after treatment with chemical crosslinkers. The deduced distance restraints from this technique can then be used to calibrate molecular models of the proteins produced by I-TASSER, a powerful protein structure prediction server designed by Yang Zhang’s lab.
To summarize, XL-MS was used to obtain experimental structural data that could then be applied to I-TASSER to predict more accurate protein models. This unique approach was used several times throughout this publication, but the Simonds group first used this method to predict the structure of R7BP accurately. Note that this approach did NOT require protein crystallization.
Using surface plasmon resonance to quantify binding interactions
Next, the Simonds group used the OpenSPR™ to run surface plasmon resonance (SPR) binding experiments to measure quantitative binding kinetics of the PPIs between R7BP and several R7-RGS/Gβ5 duplexes (the R7-RGS subfamily consists of RGS 6, 7, 9, and 11). Purified, His-tagged R7BP was immobilized onto a Streptavidin Sensor Chip, followed by injection of the four purified duplexes as separate analytes. Each experiment was performed in duplicate with 3-5 analyte concentrations to ensure the accuracy and reliability of the data. Interestingly, R7BP had a 10-fold lower binding affinity to the RGS7 duplex compared to the other analytes. This potential avenue for PPI inhibition was explored further using the XL-MS calibrated I-TASSER method described above.
Using the structural data for R7BP that Simonds’ team initially produced, they were able to design antibodies against the N-terminal 38 amino acids of R7BP (ab38). By running SPR experiments, these antibodies were found to bind R7BP with high affinity. These experiments and the following SPR experiments were performed in triplicate using R7BP as the ligand.
Finally, the antibody’s ability to inhibit interactions between R7BP and the RGS7/Gβ5 duplex was then tested using SPR. R7BP was first immobilized and saturated with either excess ab38 or a negative control antibody as the first analyte. The duplex was then injected as a second analyte. It was found that, relative to the control antibody, ab38 significantly blocked the interaction between the PPI and the RGS7/Gβ5 duplex. The Antibodies were quantified relative to the R7BP/RGS7/Gβ5 triplex, and they specifically interacted with only the R7BP in the presence of all three proteins.
To recap: By identifying binding regions from the XL-MS calibrated I-TASSER model of R7BP, Simonds’ group was able to develop first-generation R7BP antibodies. Through extensive SPR analysis, it was found that these antibodies inhibited R7BP’s binding to the RGS7/Gβ5 duplex by more than 50%. This unique method eliminated the need for protein crystallization and identified a novel approach to treating chronic-itch diseases with potentially fewer side effects to options currently available.
OpenSPR™ gets you the data you need to publish
All of us at Nicoya are ecstatic about this great accomplishment by the Simonds group. With more and more reviewers demanding quantitative binding kinetics over traditional yes/no binding data, Simonds was able to publish this work in a prestigious journal like Communications Biology using SPR. Simonds writes:
“…SPR is a reliable method to probe and test the PPIs understudy and quantify the binding affinities of various protein partners. Such an approach can shed light on potential interaction mechanisms and reveal selectivity among different partners, as demonstrated here between full-length and mutant R7BP and duplexes containing the different R7-RGS proteins.”
The OpenSPR™ provided Simonds with the data he needed to support his claims. The reliability of the OpenSPR™ allowed his team to triplicate their binding experiments with consistent results. With the OpenSPR™ being an affordable and easy-to-use SPR instrument, it has never been easier to get your next big idea published in a high impact journal.
Get your research published with reliable and affordable SPR instrumentation.BOOK WEB DEMO
- Adikaram, P. R., Zhang, J.-H., Kittock, C. M., Pandey, M., Hassan, S. A., Lue, N. G., … Simonds, W. F. (2019). Development of R7BP inhibitors through cross-linking coupled mass spectrometry and integrated modeling. Communications Biology, 2(338). doi: 10.1038/s42003-019-0585-1
- Yosipovitch, G., Rosen, J. D., Hashimoto, T. (2018). Itch: From mechanism to (novel) therapeutic approaches. J Allergy Clin Immunol., 142(5). doi: 10.1016/j.jaci.2018.09.005.