Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

Abstract

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

Molecular basis for Ras suppressor-1 binding to PINCH-1 in focal adhesion assembly

“…By contrast, a minimal fragment (CR1) comprising RBD and CRD of Raf-1 was successfully generated using bacterial expression system and utilized to examine a potential interaction with Rsu-1. Our quantitative assessment with BLItz biosensor system revealed that while Raf-1 CR1 domain significantly bound HRAS (G12V) that is consistent with recent binding study ( 46 ), it did not interact with Rsu-1 at a physiologically meaningful affinity (data not shown), suggesting that the CR1 does not involve the binding site for Rsu-1. Confirmation of a potential physical interaction between Raf-1 and Rsu-1 and the presence of additional as-yet-uncharacterized linker molecules should await further investigation until a full-length Raf-1 protein and a diverse set of interactors become available.…”

Disrupting the LC3 Interaction Region (LIR) Binding of Selective Autophagy Receptors Sensitizes AML Cell Lines to Cytarabine

“…Soft randomization means that in each codon triplet encoding the target amino acid, the wt nucleotide occurs with a probability of 70%, while the remaining 30% are evenly distributed among the three non-wt nucleotides. For a library of 19 residues, such an approach results in a mild mutational load of five to six mutations on average, which not only maintains the overall folding of the LC3 proteins but also introduces sufficient surface variations required for a subtle optimization of existing intermolecular contacts (Ernst et al, 2013;Wiechmann et al, 2017Wiechmann et al, , 2020. After mutagenesis, our final combinatorial libraries contained in average 6.5 × 10 9 unique variants of LC3B or GATE-16 displayed on filamentous phage.…”

“…In this work, we describe the protein engineering of specific inhibitors of LIRs and characterize their binding properties in vitro and their impact on the survival of THP-1 cells, a model cell line to study acute myeloid leukemia (AML). We predicated our protein engineering approach on previous work where we demonstrated that intracellular affinity reagents can be generated by introducing targeted mutations in the binding site of a naturally occurring binding partner (Ernst et al, 2013;Wiechmann et al, 2017Wiechmann et al, , 2020. As scaffold to target LIRs, we chose the proteins LC3B and GATE-16 from the LC3/GABARAP family of LIR-binding proteins.…”

Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms

“…Intracellular variants of naturally occurring binding partners developed by phage display have been widely used as effective tools for studying PPI functional epitopes, interrogation of signaling pathways, and monitoring of intracellular pathway activity (Ernst et al, 2013;Gorelik et al, 2016;Stolz et al, 2017;Wiechmann et al, 2017Wiechmann et al, , 2020Zhang et al, 2016). We anticipate that integrating protein engineering and chemical screening has the potential to expand the targetability of other therapeutically relevant but challenging targets.…”