NMR2-Based Drug Discovery Pipeline Presented on the Oncogenic Protein KRAS

• DOI: 10.1021/jacs.4c16762
• Publication Date: 2025-04-14T18:48:26Z
• Authors: Matthias Bütikofer; Felix Torres; Harindranath Kada...; Nina Gämperli; Marie Jose Abi Saad; Daniel Zindel; Nicolas Coudevylle; Roland Riek; Julien Orts

Fragment-based drug discovery has emerged as a powerful approach for developing therapeutics against challenging targets, including the GTPase KRAS. Here, we report an NMR-based screening campaign employing state-of-the-art techniques to evaluate library of 890 fragments oncogenic KRAS G12V mutant bound GMP-PNP. Further HSQC titration experiments identified hits with low millimolar affinities binding within SI/SII switch region, which forms interface effector proteins. To elucidate modes, applied NMR molecular replacement (NMR2) structure calculations, bypassing need conventional protein resonance assignment. Traditionally, NMR2 relies on isotope-filtered nuclear Overhauser effect spectroscopy requiring double-labeled [13C,15N]-protein. We introduce cost-efficient alternative using relaxation-based filter that eliminates isotope labeling while preserving structural accuracy. Validation standard isotopically labeled workflows confirmed equivalence derived protein-ligand structures. This enabled determination 12 KRAS-fragment complex structures, providing critical insights into structure-activity relationships guide ligand optimization. These results demonstrate streamlined integration fragment-based pipeline composed screening, characterization, and rapid elucidation or without isotopic labeling.