Thèse Chromatographie d'Affinité Miniaturisée Wac Couplée à la Spectrométrie de Masse dans la Découverte de Médicaments Décryptage des Bibliothèques de Microsynthèse Combinatoire H/F - 69

Établissement : Université Claude Bernard Lyon 1
École doctorale : Chimie de Lyon
Laboratoire de recherche : ISA - Institut des sciences analytiques
Direction de la thèse : Claire DEMESMAY-GUILHIN ORCID 0000000286137870
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-05-04T23:59:59

Context: The design of candidate drugs starting from the so-called fragment-like molecules (Fragment-Based Drug Discovery) has strongly modified the generation of therapeutic compounds1. The method consists in identifying small organic compounds (fragment hits) that bind proteins associated with human diseases. Based on structural information, hits are growth to generate complex molecules with high activity and specificity. Traditionally, this growing process driven by medicinal chemistry approach require iteratives' synthesis steps coupled to protein-binding characterization. Biophysical characterization methods (NMR, ITC, SPR) at this stage generally involve purification steps before use. Therefore, new improvements are welcome, in order to shorten the process and limit its environmental impact. In this context, the Techsep team has developed a breakthrough approach that relies on the extreme miniaturization of Weak Affinity Chromatography (nanoWAC) coupled to MS detector2 allowing characterization of protein-ligand interactions from ligands mixture from a small sample volume. This method avoids tedious purification steps and uses very small amounts of protein and sample.
This development therefore makes it possible to consider scaling up ligands using combinatorial chemistry approaches, since it is suitable for mixture analysis and mass spectrometry detection allows for the identification of molecules of interest. Development of new assays that would be adapted for screening combinatorial libraries from microsyntheses would be highly valuable for lead discovery optimization and should revitalize drug discovery programs.
Thesis project: In this context, we propose to develop a powerful analytical workflow for the characterization and identification of the best hits while reducing reagent consumption. Miniaturized affinity chromatography in-line coupled with mass spectrometry (mAC-MS) will be at the heart of the analytical strategy.
Ligand's libraries will be generated by combinatorial chemistry at the microscale (microsynthesis). The combinatorial library mixture will be deciphered on an affinity column on which an therapeutic protein is immobilized. The specific interaction of ligands for the protein induces retention times proportional to the affinity (Ka). Ligands with no and/or low affinity for the protein are not retained and eliminated (simplification of the mixture). The high binders are then eluted in ascending order of affinity and detected and identified by mass spectrometry coupled on-line to the column.
We recently reported a proof-of-concept of the application of mAC-MS for the identification of high binderd (using Concanavalin A as an protein model) in a combinatorial library.1
The objective of this work is to evaluate the potential of low affinity chromatography in the evolution stages of fragments and more particularly for the analysis of combinatorial micro-synthesis mixtures (Figure ).

The approach will first be evaluated on a simple mixture resulting from combinatorial chemistry microsynthesis. Complex mixtures will then be studied.

Therapeutic target: the CK2 protein kinase. This enzyme regulates numerous cellular processes essential for development and differentiation. Its dysregulation is observed in many pathologies, including cancer; this protein is therefore an attractive target for the development of pharmacological inhibitors.

This work will be carried out in collaboration with other team specialized in the chemical synthesis (I. Krimm).

Bilbiography:
(1)Jeanroy, F.; Demontrond, F.; Vidal, F.-X.; Gueyrard, D.; Vidal, S.; Demesmay, C.; Dugas, V. Deciphering Dynamic Combinatorial Libraries of Glycoclusters with Miniaturized Weak Affinity Chromatography Coupled with Mass Spectrometry (Nano-FAC-MS). *****. Chim. Acta 2023, 1261, 341227. https://doi.org/10.1016/j.aca.2023.341227.

The design of candidate drugs starting from the so-called fragment-like molecules (Fragment-Based Drug Discovery) has strongly modified the generation of therapeutic compounds1. The method consists in identifying small organic compounds (fragment hits) that bind proteins associated with human diseases. Based on structural information, hits are growth to generate complex molecules with high activity and specificity. Traditionally, this growing process driven by medicinal chemistry approach require iteratives' synthesis steps coupled to protein-binding characterization. Biophysical characterization methods (NMR, ITC, SPR) at this stage generally involve purification steps before use. Therefore, new improvements are welcome, in order to shorten the process and limit its environmental impact. In this context, the Techsep team has developed a breakthrough approach that relies on the extreme miniaturization of Weak Affinity Chromatography (nanoWAC) coupled to MS detector2 allowing characterization of protein-ligand interactions from ligands mixture from a small sample volume. This method avoids tedious purification steps and uses very small amounts of protein and sample.
This development therefore makes it possible to consider scaling up ligands using combinatorial chemistry approaches, since it is suitable for mixture analysis and mass spectrometry detection allows for the identification of molecules of interest. Development of new assays that would be adapted for screening combinatorial libraries from microsyntheses would be highly valuable for lead discovery optimization and should revitalize drug discovery programs.

dèdévelopper un couplage Chromatographie d'affinité miniaturisée (WAC) - spectrométrie de masse miniaturisé pour le décryptage des bibliothèques de microsynthèse combinatoire

: In this context, we propose to develop a powerful analytical workflow for the characterization and identification of the best hits while reducing reagent consumption. Miniaturized affinity chromatography in-line coupled with mass spectrometry (mAC-MS) will be at the heart of the analytical strategy.
Ligand's libraries will be generated by combinatorial chemistry at the microscale (microsynthesis). The combinatorial library mixture will be deciphered on an affinity column on which an therapeutic protein is immobilized. The specific interaction of ligands for the protein induces retention times proportional to the affinity (Ka). Ligands with no and/or low affinity for the protein are not retained and eliminated (simplification of the mixture). The high binders are then eluted in ascending order of affinity and detected and identified by mass spectrometry coupled on-line to the column.