G-Protein-Coupled Receptor (GPCR)
GPCRs as the Biggest Drug Target
Subgroups and development statuses of GPCRs
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.
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.
Dozens of tractable GPCRs (low-hanging fruits) are well exploited, whereas other GPCRs (high-hanging fruits) are underexploited.
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
Breakthrough in GPCR Drug Discovery
To overcome the barriers of GPCR drug discovery and promote drug development, we have innovated a high-performance functional assay. Our assay
- works for all receptors coupled with any of the major Gα proteins including Gq, Gs, Gi/o, and G12/13
- is higher in sensitivity and S/B ratio than any existing methods,
- is low in cost and high in throughput, and
- makes orphan receptors practical targets.
Our GPCR Functional Assay Platform
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.
- 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
- Screens for agonists, antagonists, reverse agonists, PAM, and NAM
- Low in cost and high in throughput
- Single cell line for all assays; no need to maintain hundreds of receptor stably expressing cell lines
- Receptors transiently transfected on microplates
- No special equipment or reagent required
Core Technology for GPCR functional assay
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.
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 99.1% (216 out of 218) of non-orphan GPCRs
- Also works with other membrane receptors such as receptor tyrosine kinases
- International patent filed and exclusively licensed from the University of Tokyo to Tanso Biosciences
Gα protein cocktail
This one-fits-all 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 human GPCR library
Industry-leading collection of 313 human GPCRs including 218 non-sensory receptors and 95 orphan receptors. Use of native sequences minimizes signal artifacts.
List and figures
Validation of Our Assay Method
Our assay has yielded consistent readouts with results from existing methods and at high sensitivity and S/B ratio.
AGTR1, a peptide-ligand receptor
In our assay, AGTR1 responded to its agonist at high sensitivity and S/B ratio and consistently with the results from existing methods.
Binding assayNature. 1991;351:233-6.
|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|
Inositol phosphate formationJ Biol Chem. 1995;270:12846-50.
Inositol phosphate formation by transfected wild-type AT1 receptor and the D281A mutant. Inositol phosphate formed in response to [Sar1] Ang II, [ Sar1,Gln2] Ang II and the non-peptide agonist, L-162,313, in COS 1 cells transfected with wild-type and the D281A mutant. The affinity constant (Ki) of wild-type and mutant D281A receptors, respectively, for [Sar1] Ang II is 0.32 nM and 83.2 nM, for [ Sar1,Gln2] Ang II is 52.7 nM and 304 nM, and for L-162,313 is 56.2 nM and 50.4 nM.
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.
Ca2+ assayNature. 2004;429:188-93.
Flt3: a receptor tyrosine kinase (RTK)
Our method proved to also work for other membrane receptors than GPCRs.
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.
Cell proliferationRetrieved from R&D Systems' website
Frequently Asked Questions
- 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.
- Can your test cells be sub-licensed?
- No, we do not sub-license our cells for now.
- 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.