The PhD researcher Maria Ricart from Medicinal Chemistry & Synthesis (MCS) group is defending her thesis next  4th December 2020 at 10:00h.


Innovative tool to decipher the mode of action of light-regulated ligands for GPCRs and enzymes.

Directors: Amadeu Llebaria (IQAC-CSIC) and Cyril Goudet  (IGF-UM-CNRS, France)

The thesis will be streamed in this link


G Protein-Coupled Receptors (GPCRs), including the metabotropic glutamate 5 receptor (mGlu5), are important therapeutic targets and the development of allosteric ligands targeting GPCRs has become a desirable approach toward modulating receptor activity. Traditional pharmacological approaches toward modulating GPCR activity are still limited since precise spatiotemporal control of a ligand is lost as soon as it is administered. Photopharmacology proposes the use of photoswitchable ligands to overcome this limitation, since their activity can be reversibly controlled by light with high precision. As this is still a growing field, the molecular mechanisms underlying the light-induced changes of existing photoswitchable ligands are not well-understood and new light-regulated approaches can be useful to expand the toolbox of optical switches.

We studied the mechanisms of action of alloswitch-1 and MCS0331, two freely diffusible mGlu5 photoswitchable negative allosteric modulators (NAMs), through an innovative mass spectrometric (MS) binding methodology. We developed and validated a highly sensitive, rapid and robust MS binding assay to quantify the binding of mGlu5 allosteric ligands. This MS-based direct binding assay in combination with photochemical, cell-based, and in vivo photopharmacological approaches allowed then to investigate the effects of trans-to-cis azobenzene photoisomerisation on the functional activity and binding ability of these ligands to the mGlu5 allosteric pocket. From these results, we conclude that photoisomerisation can take place inside and outside the ligand binding pocket, and this leads to a reversible loss in affinity, in part, due to changes in dissociation rates from the receptor. Ligand activity for both photoswitchable ligands deviates from high-affinity mGlu5 negative allosteric modulation (in the trans configuration) to reduced affinity for the mGlu5 in their cis configuration. Importantly, this mechanism translates to dynamic and reversible control over pain following local injection and illumination of negative allosteric modulators into a brain region implicated in pain control.

A selective covalent labelling directed by the ligand affinity in a native protein was studied as an alternative approach to expand the toolbox of optical switches. The approach is based on obtaining an affinity label (AL) through a bioorthogonal reaction which contains a short-lived highly reactive anchoring group and the ligand. We designed non-photoswitchable and photoswitchable ALs to react with lysine residues exposed on the protein surface near the binding pocket. As a proof of concept, we selected Imiglucerase as target protein to be covalently modified. Among the obtained ALs, AL 3 was formed using a cooper-free click reaction and successfully labelled Imiglucerase. A proteomic assay following a bottom-up approach was then set up to verify the selectivity of the covalent modification. The results obtained from non-labelled and labelled samples demonstrated the power of this approach to detect the desired peptide fragments. Nevertheless, the preliminary data obtained indicated that the Imiglucerase was non-selectively labelled by AL 3 since non-desired lysine residues were targeted. Future experiments should be performed to verify the AL 3 labelling selectivity and to develop new photoswitchable AL compounds.