Periodic Reporting for period 1 - TEDCIP (Targeting epigenetic demethylases: development of covalent inhibitors and PROTACs (Proteolysis Targeting Chimeras).)
Période du rapport: 2017-08-01 au 2019-07-31
Since covalent inhibitors possess time-dependent inhibition due to the kinetics of covalent binding to the protein, their activity was better assessed through determination of the kinetic parameter kinact/Ki, rather than a simple IC50 .These parameters were calculated by using an established method to derive Ki and kinact directly from time-dependant IC50 values. The IC50 values were determined after optimization of the biochemical assay, and we showed that compounds possessed time-dependent inhibition through covalent binding to KDM5B leading to potent nanomolar IC50 values. As well as improving their potency, I performed selectivity and 2-OG competition assays, which showed that the compounds were selective towards the KDM5 members and to our delight, the addition of a covalent warhead to the inhibitors also reduce competition with 2-OG. The covalent binding of the inhibitors with KDM5B was confirmed through MS-labelling experiments. I have also developed a NanoBRET tracer that was used as a tool to test cellular target engagement of the compounds, which was also confirmed. I have finally performed ChIP-seq on the most promising covalent compounds, which showed an increase in H3K4me3 and confirming the efficiency of the compounds (Vazquez-Rodriguez et al. Angew. Chem. Int. Ed., 2019).
After the successful development of the covalent KDM5 inhibitors, I moved into the next main objective, which was the development of KDM5 PROTACs. PROTACs are synthetic chimeric molecules composed of at least two distinct molecular moieties that have separate functional activities. For our purpose, one molecular component is a KDM5 inhibitor scaffold, and the second component, which binds to a protein involved in endogenous protein degradation, is either cereblon or VHL. By bringing the target protein into contact with protein degradation machinery, these chimeric molecules catalyze the degradation of the target protein using the natural machinery of the cells.
I synthesized a variety of PROTACs based on a non-covalent version of the most promising covalent inhibitors previously developed. A variety of linkers taking into account the hydrophobicity and hydrophilicity ratio, the length and the nature of the attachment to the KDM5 and E3 ligase ligands was obtain. A toolbox of E3 ligand-linkers allowed combinatorial synthesis for the final steps of the synthesis of the PROTACs. Biochemical assays showed that the compounds were in the nanomolar range of potency, as their covalent counterparts. Cellular target engagement with the NanoBRET technology confirmed the engagement with both targets, KDM5 and the corresponding E3 ligase. Cellular timecouse also showed that the PROTAC strategy was also effective, degrading the KDM5 in a time and dose-dependent manner. There results are currently under preparat
On the PROTACs, the project was also successful. To our knowledge, at the time this report was written,the KDM5 PROTACs were the first degrader compounds on eraser epigenetic enzymes. The synthesis of the compounds was in most cases successful but yields of the final step reaction were significantly low. Despite confirming degradation of the KDM5B, PROTAC dosing was higher than expected, which could also be due to cell permeability issues. Further studies would add significant value to the understanding of KDM5 PROTAC mechanism of action and KDM5 role in regulation and structural features. Currently, a manuscript is under preparation for publication of this results.