(1021-B) Combination of Affinity Selection Mass Spectrometry with Biophysical approaches to identify and characterize biomolecules binders

Abstract: Small molecule drug discovery is benefiting since many years from the continual development of novel scientific methodologies to occasion therapeutic advances. Among those, Affinity Selection Mass Spectrometry (AS-MS) is now a well-known approach in the HTS world as valuable complement or alternative to traditional drug discovery technologies.
This in solution and label-free approach, implemented for more than 7 years at Evotec, shown great achievements with different kind of targets in hit identification (proteins, but also RNAs which are emerging now as new targets of interest). Many improvements were done during those years of success, mainly with the aim to enhance the throughput of the global process. This is allowing Evotec to be in line with the growing interest for this approach from partners and customers.
In brief, principle of the AS-MS approach used at Evotec is based on three main steps: 1- Incubation of the target with mixture of compounds; 2- separation of the protein-ligands complexes from unbound molecules by fast Size Exclusion Chromatography (SEC); 3- Online reverse phase HPLC to dissociate target-small molecules complexes and release binders to allow their MS identification.
Standard Evotec workflow is using dedicated pooled chemical libraries, with up to 640 compounds per well, allowing us to assess at the primary screen level more than 60k compounds per day. Over the past two years, we also made huge improvements of the data analysis workflow, leading to the implementation of a fully dedicated software solution, developed in association with Virscidian.
Resulting set of target-selective chemical features then need to be confirmed as binders with some orthogonal assays/techniques, that are also used to deeper characterize their binding on the targets. In that way, Evotec is able to perform Affinity ranking by using same AS-MS technique. This approach allows to get an affinity-based comparison between the binders, even without providing classical parameters used to characterize binding, such as Kd.
Evotec is also very well equipped to perform widely used biophysical approaches for binding evidence, such as Surface Plasmon Resonance (SPR), Dynamic Scanning Fluorimetry (DSF), IsoThermal Calorimetry (ITC), Nuclear Magnetic Resonance (NMR). Within Evotec, those different technologies have shown great achievements in the target druggability investigation with small molecules. X-ray crystallography or cryoElectroMicroscopy (cryoEM) were also used to get direct visualization of the target-small molecule complexes, and more recently, Evotec has also Native MS might be considered as an approach worth to develop at Evotec to get similar information.
Finally, Evotec has been able to successfully apply a combination of AS-MS technique as primary HTS technique with classical biophysical approaches within several full drug discovery programs. Evotec is still keen to strengthen those integrated programs of drug discovery by doing improvements on existing technologies, but also developing new approaches.