ras proteins  (Jena Bioscience)

Journal: Nature Communications

Article Title: Identification and characterization of binders to a cryptic and functional pocket in KRAS

doi: 10.1038/s41467-025-65844-3

Figure Lengend Snippet: A Schematic representation of the display technologies applied to RAS proteins, for identification of RAS-selective macrocyclic peptides and nanobodies. Illustration created using BioRender (Maira, M., 2025): https://BioRender.com/lk2zlor . B Illustration of the binding sites of the three macrocyclic peptides MPB1, MPB2 and MPB3 identified in the mRNA display screens and obtained upon co-crystallization studies either with GDP-loaded KRAS G12D (MPB1, PDB code 9GLU) or GTP-loaded NRAS Q61R (MPB2, PDB code 9GLW; MPB3, PDB code 9GLX). C Illustration of the binding site for the KM12-AM nanobody, identified from a yeast display screen using a lama naive library, and solved upon co-crystallization studies with GMPPnP-loaded KRAS G12D (PDB code 9GLZ). The “back-pocket” induced by KM12-AM binding is further emphasized in the included inlets, when omitting the nanobody from the complex structure (inlet 1) in comparison to the same region of KRAS in the apo form when not bound to KM12-AM present in the crystal (inlet 2, PDB code 5USJ). The lower inlet (inlet 3) shows an overlay of both structures as ribbon diagrams (KM12-AM and PDB code 5USJ structures shown in gray and orange, respectively) with a semitransparent surface of inlet 1. The side chains of Tyr157 and Lys42 are shown as stick model.

Article Snippet: Nucleotide exchange was performed by incubating the RAS proteins during 1 h at room temperature with a 24-molar excess of nucleotide (GMPPnP from Jena Bioscience, GTP from Sigma or GDP from Sigma) in presence of 25 mM EDTA.

Techniques: Binding Assay, Crystallization Assay, Comparison

Journal: Nature Communications

Article Title: Identification and characterization of binders to a cryptic and functional pocket in KRAS

doi: 10.1038/s41467-025-65844-3

Figure Lengend Snippet: A Overlay and overlap of KM12-AM (PDB code 9GLZ) and cRAF1 RBD-CRD (PDB code 6QUU) binding modes on RAS (left panel) and cRAF constructs used in this study (right panel). B cRAF proteins mentioned in ( A ) were tested in the GMPPnP loaded KRAS G12D /KM12-AM TR-FRET assay and potential competition determined by quantification of IC 50 (concentration needed for 50% maximal inhibition achieved with the respective proteins tested) and A inf (maximum amplitude of the effect) values, as described in Methods. While CRD-containing constructs appear to result in (close to) complete competition with binding of KM12-AM, RBD alone results in only partial displacement (~50%). The assay is validated by titration of untagged KM12-AM or GMPPnP-loaded KRAS G12D that both fully displace with IC 50 (K i ) in range with their affinity as measured by SPR (see Tables and ). TR-FRET curves, K i and A inf values shown ( n = 2 biological independent replicates) are derived from one representative experiment. This experiment was repeated at least two times with similar results. Source Data is provided as a source Data file. C A-B-A SPR experiment studies using MRTX1133 (right panels) or not (DMSO, left panels) as a switch II pocket binder during A-step, for K D determination of either cRaf RBD (by steady-state affinity measurement, top panels) or KM12 (by kinetic affinity measurement, bottom panels) added in B-Step, on immobilized GMPPnP-loaded KRAS G12D , as described in Methods. For steady-state affinity measurements, insets representing the corresponding R eq versus concentration curves have been incorporated. For kinetic affinity measurements, sensorgrams are reported as overlay of fitted (in black for each concentration) and experimental (one color per concentration) curves. K D , R max and Chi values derived from one representative experiment are reported on the right side of each panel.

Article Snippet: Nucleotide exchange was performed by incubating the RAS proteins during 1 h at room temperature with a 24-molar excess of nucleotide (GMPPnP from Jena Bioscience, GTP from Sigma or GDP from Sigma) in presence of 25 mM EDTA.

Techniques: Binding Assay, Construct, Concentration Assay, Inhibition, Titration, Derivative Assay

Journal: Nature Communications

Article Title: Identification and characterization of binders to a cryptic and functional pocket in KRAS

doi: 10.1038/s41467-025-65844-3

Figure Lengend Snippet: A HEK293 cells stably expressing LgBit-KRAS G12D were transiently transfected with expression vectors containing either with the cDNAs of the mentioned proteins or not (mock control). Cells were then lysed, cell extracts used for anti-HA co-immuno-precipitations and bound RAS proteins revealed by western blotting (anti-HA Pull-down panels), as described in Methods. Straight western blots (Input panels) with the same cell extracts were also performed to estimate expression levels across conditions. Both GFP-NLuc and HA-Myc-GFP-NLuc constructs are Nanoluciferase fusions used as unrelated controls, that can be revealed with anti-LgBit antibodies. a and b labels refer to endogenous RAS WT and LgBit-KRAS G12D exogenous proteins, respectively. This experiment has been repeated at least three times with similar results. B LgBit-KRAS G12V and SmBit-cRAF knock-in engineered PATU8988 cells were transiently transfected or not (mock control) with expression vectors encoding either KM12-AM*, its binding deficient mutant (KM12-AM*-AA) or with the NS1 monobody. Cells were then either incubated with NanoGlo Live TM substrate, and bioluminescence determined (left panel) or lyzed and analyzed by straight western blots to estimate expression levels across conditions (right panel), as described in methods. Bioluminescence data are represented as mean values ± SD of n = 6 biological independent replicates. p -values were determined using two-tailed t -test. This experiment has been repeated three times with similar results. C HCT116 cells were transiently transfected or not (mock control) with expression vectors encoding either with GFP alone, GFP-fused KM12-AM* or its binding deficient mutant KM12-AM*-AA. Cells were then fixed, permeabilized, nuclei stained with Hoechst and then analyzed by confocal microcopy High Content Imaging using anti-pERK antibodies to reveal nuclei (blue fluorescence) and pERK levels (red fluorescence) in cells expressing GFP or GFP-fused KM12-AM*/KM12-AM*-AA express (green fluorescence) or not as described in Methods. Representative examples are shown in the left panel and quantification of multiple fields is shown in the right panel. Quantification data are presented as violin plot of the distribution of the log value of pERK intensity. Dots represent scoring for individual cells, boxplots represent median and first and third quartiles, and whiskers extend to 95 th percentile, statistical significance was calculated using welch-corrected T -test. (Control n = 3745, KM12-AM*-AA n = 5582, KM12-AM* n = 582). This experiment has been repeated two times with similar results. For ( A – C ), Source Data are provided as a source Data file.

Article Snippet: Nucleotide exchange was performed by incubating the RAS proteins during 1 h at room temperature with a 24-molar excess of nucleotide (GMPPnP from Jena Bioscience, GTP from Sigma or GDP from Sigma) in presence of 25 mM EDTA.

Techniques: Stable Transfection, Expressing, Transfection, Control, Western Blot, Construct, Knock-In, Binding Assay, Mutagenesis, Incubation, Two Tailed Test, Staining, Imaging, Fluorescence