We are currently expanding the repertoire of bioorthoganal ligation reactions, which can be used in chemical tools to modify or to interact with biological systems. We have focused on the development of highly efficient ligation reactions, which will enable us to develop innovative chemical proteomics, MS proteome analysis, or chemical biology technology tools.
The laboratory applied an innovative approach using the 96 well plate format to evaluate the kinetic profile, as well as, the characteristics of two types of chemical reactions. Initially, we investigated the selectivity of various electrophilic functions for the labelling the N-terminus of specific amino acids. The first chemo-specific reactive groups identified are currently being evaluated for their ability to specifically tag distinctive proteins. The second type of reactions that we have explored concern bio-orthogonal reactions, which can proceed in the presence of complex biological constituents without modulating them. To identify such reactions, we used the test described above and combined various highly energetic reactive groups. In particular, we assessed the reaction kinetics and reaction sensitivity to poisoning by biological constituents. Initial new “click like” candidates have been identified in vitro.
To advance our studies further, a system to evaluate intracellular reaction efficiency and bio-compatibility was developed. This system is based on the actine-phalloïdine interaction. Reference molecules have been tested have validated the system.
The laboratory applied an innovative approach using the 96 well plate format to evaluate the kinetic profile, as well as, the characteristics of two types of chemical reactions. Initially, we investigated the selectivity of various electrophilic functions for the labelling the N-terminus of specific amino acids. The first chemo-specific reactive groups identified are currently being evaluated for their ability to specifically tag distinctive proteins. The second type of reactions that we have explored concern bio-orthogonal reactions, which can proceed in the presence of complex biological constituents without modulating them. To identify such reactions, we used the test described above and combined various highly energetic reactive groups. In particular, we assessed the reaction kinetics and reaction sensitivity to poisoning by biological constituents. Initial new “click like” candidates have been identified in vitro.
To advance our studies further, a system to evaluate intracellular reaction efficiency and bio-compatibility was developed. This system is based on the actine-phalloïdine interaction. Reference molecules have been tested have validated the system.
Research in this area is part of the BioChemLig Marie Curie initial training network
The chemical proteomics domain revolves around developing the necessary molecular functions: to capture of the bio-active molecule’s targets, to ligate the captured targets using functional probes, extraction, purification, identification and profiling of capture targets.
The advent of this holistic drug discovery approach highlights the need for new chemical and molecular bio-compatible tools to identify a drug’s target (target deconvolution). Currently, the identification of active compounds by phenotypic screening in drug discovery lacks an effective method to determine compounds’ targets.
Activity-based protein profiling (ABPP) is a strategy used for target deconvolution. We have developed a mild approach exploiting the characteristics of a low-dissociation-rate inhibitor and advantageously the system does not require covalent bonds to stabilise the complex. We used bacterial type II topoisomerase, DNA gyrase, as the target protein and its inhibitor, the aminocoumarin novobiocin. This drug has a high affinity with a low dissociation constant (Kd<10−7 M) with DNA gyrase. The probe was also labelled very specificity using fluorescence. The target-probe-label complex was be stabilised by using a mild Staudinger ligation and was compatible with non-denaturing protein analysis.
We then developed a cleavable linker that could be used in conjunction with this mild approach for ABPP. We optimised the structure of azo–arenes for a dithionite reduction and developed an efficient HAZA (2-(4′-hydroxy-2′-alkoxy phenylazo) benzoic acid) linker. This new orthogonally deprotected cleavable linker can be derivatized with a range of tags and can be cleaved in significantly milder conditions than previous generations of the linker.
This cleavable linker enabled us to capture and release native functional proteins complexes in extremely mild conditions. We chose novobiocin as the molecular hook for the linker to determine its protein targets. The antibiotic is known to target the DNA gyrase B subunit ATP binding site. Using our system, we were able to purify the DNA gyrase complex A2B2 in its native form and to preserve its enzymatic activity. In addition, we isolated about fifty proteins (interacting or secondary targets) and they were characterized by mass spectrometry. This ABPP of novobiocine is currently being biochemical validated to assess novobiocine’s mode of action. (Collaboration: Dr Valérie Lamour, IGBMC)
Having developed the molecular tools and the "target deconvolution" techniques required, we will focus on other biological systems. We are therefore creating probes for profiling PARP, HDAC and PRMT inhibitors (Collaboration Dr Valérie Schreiber, ESBS; Dr Laurent Brino IGBMC and Prof. Jean Cavarelli, IGBMC).
The advent of this holistic drug discovery approach highlights the need for new chemical and molecular bio-compatible tools to identify a drug’s target (target deconvolution). Currently, the identification of active compounds by phenotypic screening in drug discovery lacks an effective method to determine compounds’ targets.
Activity-based protein profiling (ABPP) is a strategy used for target deconvolution. We have developed a mild approach exploiting the characteristics of a low-dissociation-rate inhibitor and advantageously the system does not require covalent bonds to stabilise the complex. We used bacterial type II topoisomerase, DNA gyrase, as the target protein and its inhibitor, the aminocoumarin novobiocin. This drug has a high affinity with a low dissociation constant (Kd<10−7 M) with DNA gyrase. The probe was also labelled very specificity using fluorescence. The target-probe-label complex was be stabilised by using a mild Staudinger ligation and was compatible with non-denaturing protein analysis.
We then developed a cleavable linker that could be used in conjunction with this mild approach for ABPP. We optimised the structure of azo–arenes for a dithionite reduction and developed an efficient HAZA (2-(4′-hydroxy-2′-alkoxy phenylazo) benzoic acid) linker. This new orthogonally deprotected cleavable linker can be derivatized with a range of tags and can be cleaved in significantly milder conditions than previous generations of the linker.
This cleavable linker enabled us to capture and release native functional proteins complexes in extremely mild conditions. We chose novobiocin as the molecular hook for the linker to determine its protein targets. The antibiotic is known to target the DNA gyrase B subunit ATP binding site. Using our system, we were able to purify the DNA gyrase complex A2B2 in its native form and to preserve its enzymatic activity. In addition, we isolated about fifty proteins (interacting or secondary targets) and they were characterized by mass spectrometry. This ABPP of novobiocine is currently being biochemical validated to assess novobiocine’s mode of action. (Collaboration: Dr Valérie Lamour, IGBMC)
Having developed the molecular tools and the "target deconvolution" techniques required, we will focus on other biological systems. We are therefore creating probes for profiling PARP, HDAC and PRMT inhibitors (Collaboration Dr Valérie Schreiber, ESBS; Dr Laurent Brino IGBMC and Prof. Jean Cavarelli, IGBMC).
To be able to probe, modify or interact with intracellular biological processes chemical reagents and tools/inhibitors must be able to enter the cell.
We have developed a traceable fluorescence probe system which can be turned-on through a reaction with an exogenous biocompatible chemical stimulus. This approach was used in the discovery of novel biocompatible exogenous chemical reagents, and to develop novel traceable turn-on fluorescent probes for tumor cells.
In this system, a fluorophore and a quencher are connected by a chemical bond that is sensitive to a specific chemical stimulus. The probe is incubated with cells, then after washing, the chemical reagent is added. If the probe and the fluorophore have penetrated the cell, the fluorescence turns-on. The appearance of fluorescence enables the assessment of the probe and reagent’s penetration into the cell. A control using fixed cells allows us to confirm the reactivity of combinations, if no reaction was observed.
Based on this principle, we have initiated the development of imaging probes to visualize the cell’s physico-chemical state. Including, diagnostic probes responsive to the fluctuations of reducing conditions and oxidizing stress present hypoxic cancer cells (collaboration Dr Erwan Pencreach, CHU Strasbourg). Other sets of probes are also being developed to diagnose gastric abnormalities.
We have developed a traceable fluorescence probe system which can be turned-on through a reaction with an exogenous biocompatible chemical stimulus. This approach was used in the discovery of novel biocompatible exogenous chemical reagents, and to develop novel traceable turn-on fluorescent probes for tumor cells.
In this system, a fluorophore and a quencher are connected by a chemical bond that is sensitive to a specific chemical stimulus. The probe is incubated with cells, then after washing, the chemical reagent is added. If the probe and the fluorophore have penetrated the cell, the fluorescence turns-on. The appearance of fluorescence enables the assessment of the probe and reagent’s penetration into the cell. A control using fixed cells allows us to confirm the reactivity of combinations, if no reaction was observed.
Based on this principle, we have initiated the development of imaging probes to visualize the cell’s physico-chemical state. Including, diagnostic probes responsive to the fluctuations of reducing conditions and oxidizing stress present hypoxic cancer cells (collaboration Dr Erwan Pencreach, CHU Strasbourg). Other sets of probes are also being developed to diagnose gastric abnormalities.