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  2. DEL for GPCRs


G protein coupled receptors (GPCRs) are a family of multi-transmembrane proteins with typical 7 transmembrane regions. Many marketed drugs are known to target GPCRs. Given the difficulty in preparing the purified protein with right conformation, traditional GPCR ligand discovery utilizes cell based high throughput screening (HTS), virtual screening and in fewer cases, protein based screening. DNA encoded library (DEL) selection for GPCRs has also been intensively explored at HitGen and a variety of selection approaches are utilized to enable GPCR ligand discovery.


Cell based DEL selection

Cell based DEL selection has been applied for GPCR targets, especially for targets that are more difficult to be purified. DEL selection is a binding process with binding equilibrium driven mainly by target concentration, therefore, target density on the surface is the key for a successful screen. A direct comparison of expression level and enrichment ratio in cell based DEL selection was reported (ACS Comb Sci. 2015 Dec 14;17(12):722-31), when below certain expression level, the nM binding ligand could not be enriched. Another confounding factor is the complexity of other cell surface membrane proteins, for a regular protein based selection, the target purity can be regarded as 100%, however, for cell based selection, target purity may be less than 1%. Though with difficulty, cell based DEL selection for GPCRs has been validated at HitGen. Target was prepared by overexpression on HEK293 cells, and a positive ligand on-DNA conjugate was made to validate selection condition and served as a positive control for DEL selection (Figure 1). DEL selection revealed relatively weak features and the corresponding compound was validated as an antagonist with uM potency.



Figure 1, A) FACs to confirm the overexpression of target GPCR. B) The positive conjugate was successfully enriched in DEL selection.


Membrane prep based DEL selection

Preparing cells with high level of GPCR overexpression is labor intensive and difficult to achieve, as high exogenous proteins sometimes impedes cell proliferation. As an alternative, membrane prep based selection provides a new approach to expand DEL applications. It maintains the conformation of GPCR in the natural membrane environment, offers higher target density by various strategies and saves the effort of purifying protein from membranes. HitGen has validated membrane prep based DEL selection for a GPCR, with the purified detergent stabilized protein based selection performed in parallel for comparison, similar features were enriched for both selections, and the corresponding compounds were validated as nM binders (Figure 2).


Figure 2, A) Both membrane and protein based selection revealed similar features. B) SPR validated nM binding compound from DEL selection.


Purified protein based DEL selection

Due to the hydrophobicity of the transmembrane region, GPCRs tend to precipitate in aqueous buffer. Therefore, different methods including detergent, nanodisc and other particles are utilized to stabilize the protein. Detergents provide hydrophobic protection for GPCRs and maintain the protein in lipid-free system, however, detergents must be kept in the downstream process. Nanodisc offers a more membrane like structure for GPCRs and sometimes is capable of stabilizing the more difficult proteins, but it is more laborious to make and the scaffold binders need to be excluded. HitGen has performed several detergent/nanodisc stabilized GPCR selections, and different agonists or antagonists were identified with targets complexed with or without G proteins (Figure 3).


Figure 3, A) Different forms of GPCRs used in DEL selection. B) Chemical properties and potency of representative hits from DEL selection. C) Structure novelty analysis of DEL hits according to reported method (ACS Med Chem Lett. 2020 Nov 12; 11(11): 2114–2119).


DEL for GPCR at HitGen

The aforementioned approaches provide versatile selection strategies using DEL for ligand discovery against GPCR targets. Protein based selection gives a higher target concentration and the binding process is maintained in a simpler context. If the stabilized protein can be obtained, protein based selection affords a quick option for GPCR ligand discovery. If purified protein is difficult to be prepared, membrane prep and cell based DEL selection offer alternative approaches. In cell based selection, target is maintained in the natural context with the presence of interaction proteins and cofactors, but high target density is the key and sometimes very difficult to achieve. As an alternative, membrane prep based selection also preserves the target in the cell membrane, saves the efforts to solubilize the protein, and enables target enrichment by immobilization strategies. HitGen has built up good experience in GPCR biology, offers various strategies for GPCR DEL selection and provides customized selection solutions for your GPCR:

● Cell/membrane prep/protein based selection offers different selection strategies.

●Different protein forms/constructs allow for ligand identification of different binding sites and agonist/antagonist mechanisms.

● Large diversity of DEL molecules maximizes hit rate, especially for orphan receptors.

● Quick turnaround time and demonstrated successful cases.

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