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Membrane Protein Workflow at Evotec: From Protein Production to Fragment Screening
Transmembrane proteins (TMPs) are proteins that span across cell membranes. Due to the crucial role of TMPs in different physiological processes, they have become prominent targets for drug discovery. Many classes of TMPs exist, each class containing many TMPs that are of pharmacological interest including: G protein-coupled receptors (GPCRs), solute carriers (SLCs), ion channels and tetraspanins to name a few1.
Due to their attractiveness as pharmacological targets, biophysical characterization of TMP: drug interactions is extremely valuable in the identification, validation and characterization of new therapeutics. Unfortunately, the study of TMPs remains highly challenging. This is due to the large mass of the TMP embedded in a detergent micelle as well as their intrinsic instability once removed from their native membrane environment - a crucial step for structure-based drug discovery and biophysical characterization of TMPs.
To address these challenges, Evotec has developed a set of capabilities to conduct high-quality biophysical characterization of TMPs. Starting with construct design and expression/purification optimization2, the highest quality protein is obtained for downstream applications. Once purified, successful implementation of Grating-Coupled Interferometry (GCI) technology, has opened the door to investigate direct target engagement with wild-type TMPs. Taking advantage of the enhanced sensitivity of GCI, as well as the waveRAPID injection method, we have successfully established binding platforms for multiple challenging TMP targets. These efforts have led to the successful prioritization of bona fide ligands that were cross-validated in functional assays and subsequent cryo-EM structures of protein-ligand complexes. A recent highlight in this area is a fragment screening campaign against multiple wild-type GPCRs.
In the present work, we show a representative workflow for biophysical characterization of TMP ligand interactions, using the adenosine A2a GPCR as a model system. A fragment screening campaign, evaluating 704 fragments, successfully identified 16 fragment binders. Hit selection criteria were devised to provide a robust method to identify fragments directly from the primary screen. Dissociation constants of the validated fragments ranged from low to high µM, with reasonable kinetic profiles. The identified fragments were retested in a multi-cycle kinetic approach as a follow up to the primary screen. The hit identification campaign took less than a week to complete and required less than 80 µg of protein.
Sara Crespillo-Torreno3, Alicia Churchill-Angus3, Tamas Yelland3, Edoardo Fabini3, Byron Carpenter3 and Cedric Fiez-Vandal3
1. Yin H, Flynn AD. Drugging Membrane Protein Interactions. Annu Rev Biomed Eng. 2016 Jul 11;18:51-76
2. Errey JC, Fiez-Vandal C. Production of membrane proteins in industry: The example of GPCRs. Protein Expr Purif. 2020 May;169:105569
3. Evotec Ltd, Milton Park, Abingdon, OX14 4RY, UK
Date: 10 November 2024
Time: 7:00 - 9:30pm CET
Presented by Thomas Yelland, Structural Biologist at Evotec
