Drugging DNA Repair complexes – DrugDR

The project is aimed at developing a new framework for identifying biological target(s) of small molecules with therapeutic potential. This framework combine the use of chemoproteomics, to identify the proteins associating with the small molecule in cells, and functional genomics, to identify mutations conferring resistance to the action of the small molecule. Combining the results from these two techniques should facilitate the deconvolution of the functional target of molecule of therapeutic potential such as small molecules identified through phenotypical screens. The chemoproteomics analysis relies on the design and synthesis of bioactive clickable analogues, bearing an alkyne group, of the molecules of interest.
As such, the project consists in studying molecules with therapeutic potential that would come from collaborations or identified through dedicated phenotypical screen designed to identify new sensitizers to the DNA damaging anticancer agent camptothecin. The proposed biological target and related mechanism of action will then be validated through conventional cellular and biochemical techniques.

Several phenotypical screens were performed and led to identify 3 new sensitizers to the anticancer agent camptothecin.
The proposed framework was applied to 2 interesting small molecules provided by collaborators and led for one of them to identify an original mechanism of action that can be exploited and extended to develop new drugs against various diseases.
Functionalized bioactive analogues were generated for some of the molecules of interest. These analogues led to map the cellular distribution of these molecules, which was critical to identify for one of them its mechanism of action.
A new collaboration with a chemistry lab was established to develop new analogues for one of the most promising sensitizers.

New functionalized clickable analogues will be synthesized for two more of the identified sensitizers, which will allow analysis of their subcellular distribution and, through chemoproteomics, to identify the cellular proteins that they interact with.
Functional genomics will be used to identify mutations conferring resistance to the action of the new sensitizers identified.
A mechanism of action will be proposed for these sensitizers and will be validated by conventional cell and biochemical assays.
The complete validation of the proposed mechanism of action for the two molecules identified through collaboration is ongoing and should be completed shortly. This will provide new druggable targets in the area of cancer research.
A new phenotypical screen will be developed to identify new small molecules inhibiting the first step of DNA repair by homologous recombination.

ChemBioChem (2018). «Alkyne-Tagged Analogue of Jaspine B: New Tool for Identifying Jaspine B Mode of Action.« Rozié A, Santos C, Fabing I, Calsou P, Britton S*, Génisson Y, Ballereau S*. Selected for cover.

My project is aimed at discovering new druggable proteins among DNA double-strand break (DSB) repair complexes by implementing in the host Institute an original approach to quickly identify the biological target(s) of bioactive small molecules. I propose first to use a dedicated phenotypic screen (task 1) that I previously established to identify among small molecule libraries new sensitizers to a DSB inducing drug, the topoisomerase I poison, camptothecin (CPT). CPT induces a special class of DSB that are associated to replication forks and are mainly repaired by Homologous Recombination (HR), through mechanisms I have contributed to decipher. A secondary screen based on the analysis of each steps of HR will be used to select sensitizers affecting DSB repair and to define which step is affected. Guided by the chemical biology lab of R. Rodriguez (Institut Curie, Paris), I will select sensitizers for further studies according to their amenability to analogue synthesis. Then I plan to identify the biological target(s) of these molecules by combining two methods: a chemoproteomic approach (task 2), that I previously contributed to develop and that will allow to identify the proteins interacting with our molecules, and a functional genomics approach (task 3) aimed at identifying the mutations conferring resistance against the activity of these molecules. Our chemoproteomic approach relies on the synthesis by our collaborator of bioactive analogues with a terminal alkyne for each selected sensitizer. These analogues will be used to isolate through “click chemistry” the proteins interacting with each of the sensitizers that will then be identified by the local mass spectrometry facility. At the same time, using mutagenized human haploid cells, we will select clones that are resistant to the sensitizing activity to CPT of each sensitizers. The mutations conferring the resistance in these clones will then be identified by RNA-seq at the Get-PlaGe genomic facility and with the help of bioanalysts from the GenoToul facility. By crossing the list of the proteins interacting with each sensitizers from the MS/MS analysis to the list of mutations conferring resistance to each drug it should be possible to quickly identify their biological target and to propose a mode of action for each of them. A last part of the project will be aimed at validating the most interesting biological targets (task 4).
This project will lead to implement in the host institute (Institute of Pharmacology and Structural Biology, Toulouse), a new approach that can be applied to quickly identify biological targets of small molecules of interest. It will allow me to gain knowledge in the field of genomic, to strengthen existing collaborations and to develop new ones that will be the basis of future projects. Several molecules with interesting biological properties have been identified by chemistry labs in Toulouse but most of them remains unexploited due to the lack of information on their mechanism of action/biological targets. Thanks to this project, I will be able to establish myself as an expert in the area of drug target identification. Ultimately, the identified sensitizers can be the basis for future drug development programs aimed at converting them into therapeutic agents. While the approach itself can be the subject of a publication, the best strategy will be selected with the help of our IP specialist for each of the identified sensitizers: publication, pre-maturation program, maturation program and/or patent filing.