ELTD1 (ADGRL4) in glioblastoma: structure and therapeutic targeting

Background: Glioblastoma is the most common and aggressive malignant brain tumour and is generally incurable. Improving outcomes is a serious unmet clinical need.

ELTD1 (also known as ADGRL4), is a promising new therapeutic target in glioblastoma and other tumour types (breast, head & neck, renal, colorectal, liver and ovarian cancers).

ELTD1, an adhesion GPCR (AGPCR) is upregulated within glioblastoma tumour cells and tumour-associated endothelial cells and plays a role in tumour proliferation, migration and invasion.

Aims: My project seeks to use cryo-electron microscopy (cryo-EM) to determine ELTD1’s structure in both active and inactive states at high resolution, to be followed by in silico small molecule screening and in vitro candidate testing. This grant application is specifically to cover the initial structuring work leading to cryo-EM over 24-months.

To date, only 1 AGPCR has been partially structured.

Method: Various engineered ELTD1 constructs will be designed and synthesised (incorporating various mutations/ truncations to improve expression, stability, or to trigger self-activation).

Structuring will be approached through three different approaches: 1) Inactive state receptor, stabilised using the KOR-ICL3-nanobody6 graft approach. 2) Active state receptor, stabilised with its specific mini-G protein.

This will be preceded by signalling experiments with all known G proteins done in collaboration with Dr Jayesh Patel (Sosei Heptares, Cambridge) using a proximity-based split nano-luciferase approach (SmBiT/LgBiT). 3) Activate state receptor, stabilised by interacting partner.

This will be done in collaboration with Prof Lynn Butler (Karolinska Institute, Sweden) using tagged BioID2 to identify protein-protein interactions assessed using mass-spectrometry).

All ELTD1 constructs will be overexpressed in insect lines, followed by purification and cryo-EM recording and data processing (the final milestone of this grant).

How the results of this research will be used: Results from this work (the high-resolution resolved active/inactive ELTD1 structures) will be submitted for publication in reputable high-impact journals and used for the next phase of this project: in silico small molecule screening and in vitro candidate testing.

Results will also be used to apply for further funding.

This research project is expected to generate a large amount of cryo-EM data which will be of significant interest to adhesion GPCR community and to the wider GPCR and structural biology communities.

After publication, all cryo-EM data will be deposited at the "The Electron Microscopy Public Image Archive" (EMPIAR) at the EMBL-EBI whilst the high resolution protein structure will be deposited at the “RCSB Protein Data Bank” (PDB).