BPS2026 – 19F NMR to probe the cryptic pocket in “undruggable” Ras-like GTPases

Ras-like GTPases are molecular switches that govern fundamental cellular processes coupled with their regulators and effectors. The Ras superfamily of small GTPases (smG) is diverse, including Ras, Rho, Rab, Arf, and Ran GTPases. Often, the activity of single smGs is assessed using biochemical assays, and 2D 1 H- 15 N/ 13 C NMR experiments can be employed to assess structural features. Unfortunately, high protein concentrations, long acquisition times, and expensive isotope labeling adversely affect the commonly used heteronuclear NMR experiments. F-labeling of proteins or ligands and 19 F-NMR can address some of these shortcomings. 19 F is an excellent reporter and virtually absent in cells but abundantly represented in drug libraries. We have established the utility of 19 F-labeled smGs with position-specific tryptophan to distinguish between the nucleotide states of RhoA, KRas and Rac1 that permit screening for smG and nucleotide-specific ligands under true physiological conditions. While there has been recent success in developing cysteine-targeting covalent inhibitors of KRas, the development of selective inhibitors for other oncogenic Ras-family proteins is lacking behind. We tested whether the cryptic switch-II pocket, beyond KRas, present in Rho and Rab GTPases can be target and probed by 19 F-NMR. We evaluate F-Trp labeled Rho-, Rab-, and Ras-like GTPase for their ability to engage inhibitors at an equivalent cysteine residue by employing established K-Ras inhibitors and molecular glues that stabilize Ras-CypA interactions. Although 19 F-Trp probes are versatile, Ras GTPases often lack a native Trp residue. Therefore, we also exploited trifluoromethyl-bearing amino acid reporters like trifluoromethyl-methionine (tfmM) for KRas. We achieved complete incorporation of tfmM via cell-free synthesis into KRas and its oncogenic variants and evaluated the enzymatic activity of tfmM K-Ras and inhibitor binding to KRas. Our work paves the way for studying various functional aspects of smG using highly sensitive 19 F-probes.