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Presentation

G-Protein Coupled Receptors (GPCRs) are known as 7 transmembrane domain (7TMD) receptors and represent the largest superfamily of all receptor types (approximately 3 to 4% of the human genome). They have been classified into three families (I, II and III) according to sequences. They are involved in all physiological processes and are highly tractable drug targets for the pharmaceutical industry with more than one-third of currently approved drugs acting through this target family. At Domain Therapeutics, we have developed DTect-All™, a unique and proprietary technology that enables us to identify novel drug candidates acting on GPCRs of the three classes including those that are considered intractable such as orphan and peptidic receptors.

What is the principle of DTect-All™

The general principle of DTect-All™ is to setup a FRET binding assay between the targeted receptor fused to the GFP and a fluorescent ligand selected from Domain’s unique and proprietary library of GPCR frequent hitter probes. The assay is then used to screen a library of druggable compounds with the aim of identifying every molecule able to displace the fluorescent ligand or to modify the receptor conformation. In both cases, this will result in a measurable change of the FRET signal. The combination of high specificity of the assay, due to double labelling, and of low potency of the probe allows the detection of more chemical scaffolds than the classical binding and functional assays.

DTect-All™ advantages

DTect-All™ has the capacity to selectively identify allosteric modulators. Indeed, by removing the orthosteric binding sites of classes Ib, Ic, II and III GPCRs, our technology will focus only on finding binders of the 7TMD, the region known to host allosteric modulator binding sites.

DTect-All™ is also perfectly adapted to orphan GPCRs as the technology does not require a known ligand of the receptor. The initial ligand used to setup the FRET assay can be selected amongst our collection of fluorescent GPCR frequent hitter probes. This collection was designed from pharmacophores of non-selective GPCR frequent-hitters identified in the course of several screening campaigns.

DTect-All™ sensitivity is higher than that of assays developed with standard technologies. This is illustrated with its capacity to identify Silent Allosteric Modulators (SAMs), a new class of ligands that binds to the receptor but is devoid of functional activity in a specific functional assay. SAMs enlarge the chemical space available for allosteric modulator identification as they represent a novel source of positive or negative allosteric modulators.

Technological aspects of DTect-All™

The technology breaks down into three steps (see figure below):

  • Firstly, the Nter domain of the GPCR of interest is truncated and fused to GFP (Green Fluorescent Protein). This step will induce “orphanisation” of GPCRs that need the Nter domain for the binding of the endogenous ligand (families Ib, Ic, II and III) and will allow the selective identification of ligands interacting only with the 7TMD.
  • Secondly, a tagged probe interacting with the 7TMD is identified. For that, we use our proprietary library of fluorescent GPCR frequent hitters that allow us to work with every GPCRs including those lacking known ligand such as the orphan GPCRs
  • Once a stable cell line expressing the GFP-truncated GPCR fusion protein at the cell surface and an appropriate tagged probe are obtained, the assay can be used for screening purposes. As FRET signal is distance-dependant, this screening step will enable identification of molecules that change distance between GFP and the tag either by competition with the probe of by interaction with any site resulting in a receptor conformation modification.