A variety of GPCR functional assay methods are available, such as Ca2+ assay, cAMP assay, β-arrestin assay, and reporter gene assay. However, some are clumsy, costly, and time-consuming, and others work with only some GPCRs. There has been no silver bullet … until now. Our proprietary assay platform has overcome limitations of existing methods and made all GPCRs accessible for drug discovery.
GPCRs as the Biggest Drug Target
Nat Rev Drug Discov 2017;16:829-842
G-protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors and amount to approximately 800 in the human genome (left pie chart). Many of them are crucial in biology and medicine. ~200 of GPCRs respond to endogenous mediators and regulate a wide range of physiological parameters from blood pressure and blood glucose levels to cognitive functions. ~500 are sensory receptors sensing smells, tastes, and other modalities of stimulation. The remaining 100 are orphan, i.e., their ligands are yet to be identified.
Drug Browser. GPCRdb.
Although receptors for endogenous mediators are the most promising group of GPCRs in drug development, only a fraction of them have drugs to target them (right pie chart). Furthermore, recent studies have shown quite a few orphan receptors and sensory receptors to play vital roles in a variety of pathophysiology. Altogether, there remain enormous untapped opportunities with the vast majority of GPCRs.
Drugs have been developed for only a small part of GPCRs. The chart above ranks GPCRs in the ascending order according to the number of targeting drugs (the more drugs a GPCR has, the lower it sits). For example, DRD2 at the bottom has more than 80 approved drugs and compounds under R&D, but as you go up, the number of drugs per receptor drops quickly. The top 100 GPCRs keep almost all GPCR drugs to themselves, and the remaining hundreds are left out with no drugs.
Barriers to GPCR Drug Discovery
One of the barriers to GPCR drug discovery is lack of satisfactory assays.
- Binding assays are unreliable with many false-positives and false-negatives when it comes to GPCRs
- No functional assay is adequate.
- Only a small number of GPCRs are readily amenable to reporter gene assays, and G12/13- and Gi/o-coupled receptors are notoriously intractable.
- Other functional assays cost a lot of time, work, and/or money. e.g., Western blotting
To overcome the barriers of GPCR drug discovery and promote drug development, we have innovated a high-performance functional assay.
- Remarkable sensitivity and S/B ratio
- An exhaustive library of 313 human GPCRs
- Works for all receptors coupled with any major Gα proteins incliding Gq, Gs, Gi/o, and G12/13
- Makes orphan receptors practical targets.
- Screens for agonists, antagonists, reverse agonists, PAM, and NAM
The core technology for our cell-based GPCR functional assay consists of sensitive and versatile test cells, a Gα protein cocktail, and an exhaustive human GPCR library.
Proprietary Assay Cells
Outstandingly sensitive, specific, and versatile
- Equipped with an artificial gene circuit to specifically amplify GPCR-induced intracellular signals
- Works for all major Gα proteins including Gq, Gs, Gi/o, and G12/13
- Verified with 99% (208 out of 210) of non-orphan GPCRs
- Also works with other membrane receptors such as receptor tyrosine kinases
- International patent filed by the University of Tokyo and exclusively licensed to Tanso Biosciences (publication number WO/2020/026979)
Gα protein cocktail
This allround Gα protein cocktail complements our test cells and enables functional assay for any GPCR under a single protocol even without knowledege of the Gα protein the target receptor is coupled with.
Comprehensive GPCR library
Industry-leading collection of non-sensory GPCRs. The use of native sequences minimizes signal artifacts.
- Human: 218 non-orphan GPCRs, 95 orphan GPCRs, 313 receptors in total
- Mouse: 212 non-orphan GPCRs, 64 orphan GPCRs, 276 receptors in total
Our assay has yielded consistent readouts with results from existing methods and at high sensitivity and S/B ratio.
AGTR1, a peptide-ligand receptor
Inositol phosphate formation
|Ligand||Ki (nM) ± s.e.m.||n|
|Sar1-angiotensin II||0.38 ± 0.07||3|
|Sar1, Ala8-angiotensin II||0.43 ± 0.08||3|
|Sar1, Ile8-angiotensin II||0.46 ± 0.03||3|
|Ile7-angiotensin III||1.1 ± 0.3||3|
|Angiotensin II||1.6 ± 0.2||3|
|Angiotensin III||5.4 ± 0.5||3|
|Dup753||6.3 ± 0.9||3|
|Angiotensin I||74 ± 12||3|
GPR91, a low molecular ligand receptor
In our assay, GPR91 responded to its agonist at high sensitivity and S/B ratio and consistently with the results from existing methods.
Flt3, a receptor tyrosine kinase (RTK)
Cells from the same line of test cells were used in this assay as in assays for GPCRs, and Flt3 was transiently transfected into cells in the same way as GPCRs.
Recombinant Human Flt-3 Ligand Protein. R&D System
Do your test cells amplify noise as well as signals?
The artificial gene circuit in our cells is designed to amplify signals quadratically while boosting noise only by ~two-fold. The cells have responded to stimuli with >100-fold signal/background ratios and small standard errors in many GPCRs（see Validation).
Can your test cells examine biased agonism?
No, you need in vivo experiments, not cell-based assay, to test if compounds act in a biased way since biased agonism is defined by the expression pattern of Gα proteins in the target tissue.
Are your test cells capable of expressing orphan GPCRs in the plasma membrane when they are transfected?
It is essential to make sure that introduced orphan GPCRs are present in the plasma membrane all the more because they do not have agonists to prove that pharmacologically. We are testing each orphan receptor now.
Do you have animal GPCR panels?
We are expanding our library to mouse and rat GPCRs. If you are interested in a particular receptor, we can prioritize cloning it and will be ready to assay it in a few weeks.
Send us a message via the form below or write to us at [email protected].
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