Dr. Geneviève Seabrook - Cancer culprits: KRAS inhibitor mechanisms of action and direct site-specific HIF1α prolyl hydroxylation identification

17 December 2024 11 h 00 min - 12 h 00 min
Dr. Geneviève Seabrook - Cancer culprits: KRAS inhibitor mechanisms of action and direct site-specific HIF1α prolyl hydroxylation identification

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Location: IPBS-Toulouse, Seminar room
205 Route de Narbonne,Toulouse

Geneviève Seabrook

The OCI/UHN High Field NMR Facility, Toronto, Canada

Cancer culprits: KRAS inhibitor mechanisms of action and direct site-specific HIF1α prolyl hydroxylation identification

Nuclear Magnetic Resonance (NMR) Spectroscopy is a very powerful tool for structural and functional studies. It allows in-depth analysis of protein-drug interactions. Ultimately, it increases the success in discovering new therapeutic drug molecules, along with understanding potential drug resistance mechanisms. Here, NMR is used as the main tool to decipher two cases (1) how MRTX1133 inhibits KRasG12D and (2) the implications of HIF1α site-specific P402 and P564 hydroxylation. In both cases, we used real-time 1H and 13C direct detection NMR to monitor enzymatic processes.
(1) Proto oncogene KRas, as the most frequently mutated oncogene, deserves special scrutiny. While labelled as a challenging ‘undruggable’ therapeutic target for decades, due to a lack of pockets, more recently, high affinity inhibitors have been discovered. Several drugs have made their way to clinical trials with some modest success. However, mechanisms underlying drugs clinical efficacy and acquired resistance still need to be uncovered. We have used a real-time NMR assay and H/D exchange experiment to elucidate the nebulous MRTX1133 molecular mechanism of action when bound to oncogenic KRas-G12D, in its GDP/GTP nucleotide bound states. Our NMR results suggest that binding of MRTX1133 to an allosteric pocket in the vicinity of switch II leads to the rigidification of the GTPase P-loop and switch I/II regions, resulting in complete arrest of the GTPase cycle and reduced binding to the effector protein RAF kinase.
(2) Intrinsically disordered proteins or regions (IDP/IDR) make up more than a third of the eukaryotic proteome. They have been found to be involved in a multitude of biological cellular processes such as recognition, signaling and regulation amongst others. Their lack of a 3D structure makes them quite difficult to study. Therefore, new NMR methodologies must be developed to study this class of proteins. Here, we present for the first time, a real-time 13C-detection NMR-based assay allowing the thorough analysis of IDR hypoxia-inducible factor HIF1α hydroxylation at two proton-less sites, P402 and P564, by the enzyme prolyl hydroxylase PHD2. NMR technological advancement allowed us to directly monitor proline hydroxylation with the use of multiple receivers, leading to a much shorter acquisition time.


Registration / Contact :

Location: IPBS-Toulouse, Seminar room
205 Route de Narbonne,Toulouse