k ras inhibitors  (MedChemExpress)

Journal: Neurotherapeutics

Article Title: PET in neurotherapeutic discovery and development

doi: 10.1016/j.neurot.2024.e00498

Figure Lengend Snippet: (A) Structure of radiolabeled [ 11 C]AZD4747. (B) [ 11 C]AZD4747 PET images (average SUV 5–123 ​min) in two cynomolgus monkeys. Adapted with permission from Kettle, J.G. et al. Discovery of AZD4747, a Potent and Selective Inhibitor of Mutant GTPase KRASG12C with Demonstrable CNS Penetration. J Med Chem 2023; 66(13):9147–9160. Copyright 2023 American Chemical Society.

Article Snippet: Recently, researchers at AstraZeneca discovered a potent inhibitor selective for the glycine to cysteine mutation at codon 12 of the K-Ras GTPase (KRAS G12C ) [ ].

Techniques: Mutagenesis

Journal: Cancer cell

Article Title: A covalent inhibitor of K-Ras(G12C) induces MHC-I presentation of haptenated peptide neoepitopes targetable by immunotherapy

doi: 10.1016/j.ccell.2022.07.005

Figure Lengend Snippet: K-Ras(G12C)-derived peptides covalently modified by the investigational inhibitor ARS1620 form functional complexes with MHC Class I heavy chain and β2-microglobulin. A. Conjugate addition from the acquired cysteine (Cys12) on K-Ras(G12C) to the acrylamide group in ARS1620 yields a covalent ARS1620-K-Ras(G12C) adduct. B. ARS1620-modified peptides form functional complexes with MHC Class I heavy chain and β2-microglobulin. Recombinant MHC-I complexes were prepared by refolding of the indicated heavy chain in the presence of β2-microglobulin and the indicated peptide. For sandwich ELISA detection, the complexes were captured by the conformation-specific MHC Class I heavy chain antibody W6/32 and detected by an β2-microglobulin-specific antibody (BBM.1) (One-way ANOVA with Dunnett’s correction for multiple comparisons, ns, not significant, ****, p<0.0001). Individual data points are shown with mean ±SD indicated. C. ARS1620-modified peptides stabilize MHC Class I on the surface of the TAP-deficient cell line T2 (One-way ANOVA with Dunnett’s correction for multiple comparisons, ns, not significant, ****, p<0.0001). Individual data points are shown with mean ±SD indicated. D. Thermal stability of A*02:01 MHC-I complexes loaded with various K5, K-Ras-derived peptides as determined by differential scanning fluorimetry. Data is represented as mean ± SD of four replicates.

Article Snippet: KMS is an inventor on patents covering covalent inhibitors of K-Ras (G12C) owned by UCSF and licensed to Wellspring Biosciences.

Techniques: Derivative Assay, Modification, Functional Assay, Recombinant, Sandwich ELISA

Journal: Cancer cell

Article Title: A covalent inhibitor of K-Ras(G12C) induces MHC-I presentation of haptenated peptide neoepitopes targetable by immunotherapy

doi: 10.1016/j.ccell.2022.07.005

Figure Lengend Snippet: P1A4 is a recombinant antibody that specifically recognizes the K-Ras(G12C) inhibitor ARS1620. A. Amino acid sequences of the CDRs of five unique Fabs identified in the phage display selection. B. Biolayer interferometry sensograms of P1A4 Fab binding to the peptide Biotin-KLVVVGAC*GV, where the cysteine residue is modified by ARS1620. Dissociation constant ( K d ) was determined by fitting the steady-state response to a 1:1 equilibrium binding model. C. Biolayer interferometry sensograms of P1A4 Fab binding to the K5-ARS A*02:01 MHC-I complex. Fit as described in A. D. Biolayer interferometry sensograms of P1A4 Fab binding to the V7-ARS A*03:01 MHC-I complex. Fit as described in A. E. X-ray crystal structure of ARS1620 bound to Fab P1A4 (PDB: 7KKH). The heavy chain CDRs are shown in blue and the light chain CDRs in purple. Ordered water molecules in the pocket are shown as red spheres. Fo-Fc omit map for ARS1620 is shown in mesh, contoured at 1.0 σ. F. P1A4 IgG detects ARS1620-modified K-Ras(G12C) as a recombinant protein or from ARS1620-treated cell lysates. G. Sandwich ELISA of recombinant MHC-I complexes prepared by refolding of the indicated heavy chain in the presence of β2-microglobulin and the indicated peptide. The complexes were captured by the conformation-specific antibody W6/32 and detected by an β2-microglobulin-specific antibody (BBM.1) or an ARS1620-specific antibody (P1A4) (One-way ANOVA with Dunnett’s correction for multiple comparisons, ns, not significant, ****, p<0.0001). Individual data points are shown with mean ±SD indicated. H. ARS1620-modified peptide-stabilized MHC complexes on the surface of the TAP-deficient cell line T2 are detectable by the conformational specific antibody W6/32 (left y axis) as well as by P1A4 (right y axis) (One-way ANOVA with Dunnett’s correction for multiple comparisons, ns, not significant, **, p<0.01, ***, p<0.001, ****, p<0.0001). Individual data points are shown with mean ±SD indicated.

Article Snippet: KMS is an inventor on patents covering covalent inhibitors of K-Ras (G12C) owned by UCSF and licensed to Wellspring Biosciences.

Techniques: Recombinant, Selection, Binding Assay, Residue, Modification, Sandwich ELISA

Journal: Cancer cell

Article Title: A covalent inhibitor of K-Ras(G12C) induces MHC-I presentation of haptenated peptide neoepitopes targetable by immunotherapy

doi: 10.1016/j.ccell.2022.07.005

Figure Lengend Snippet: ARS1620-modified peptides are presented by MHC Class I on K-Ras(G12C)-mutant cells. A. H358 cells treated with 10 μM ARS1620 show increased surface staining by P1A4. B. ARS1620 treatment leads to increase surface staining by P1A4 for three K-Ras(G12C) mutant cell lines (unpaired two-tailed t -test). Individual data points are shown with mean ±SD indicated. C. MHC Class I heavy chain and β2-microglobulin coimmunoprecipitate with ARS1620 in drug-treated cells. D. Proximity ligation assay reveals colocalization of ARS1620 and MHC Class I on the surface of K-Ras(G12C) mutant cells lines.

Article Snippet: KMS is an inventor on patents covering covalent inhibitors of K-Ras (G12C) owned by UCSF and licensed to Wellspring Biosciences.

Techniques: Modification, Mutagenesis, Staining, Two Tailed Test, Proximity Ligation Assay

Journal: Cancer cell

Article Title: A covalent inhibitor of K-Ras(G12C) induces MHC-I presentation of haptenated peptide neoepitopes targetable by immunotherapy

doi: 10.1016/j.ccell.2022.07.005

Figure Lengend Snippet: Bispecific antibodies induce ARS1620 dependent killing of K-Ras(G12C)-mutant cells. A. K-Ras(G12C) mutant cell lines were treated with ARS1620 and cell viability was assessed after 72 h. Data is represented as mean ± SD of three replicates. B. SW1573 cells stably expressing nucleus-restricted mKate fluorescent protein were pulse-treated with ARS1620 and co-incubated with unstimulated PBMCs at 10:1 effector:target ratio in the presence or absence of P1A4xCD3, and cell viability was monitored by live fluorescence imaging for 72 h (One-way ANOVA with Dunnett’s correction for multiple comparisons, ns, not significant, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001). Data is presented as viable cell count relative to time 0. Individual data points are shown with mean ±SD indicated. C. At the end of the experiment in panel B, PBMCs were analyzed by flow cytometry. D. P1A4xCD3 induces ARS1620-dependent killing of K-Ras(G12C) mutant cell lines in a dose-dependent fashion (unpaired two-tailed t -test with Holm-Šídák correction for multiple comparisons, ns, not significant, *, p<0.05, **, p<0.01, ***, p<0.001). Individual data points are shown with mean ±SD indicated. E. SW1573 cells stably expressing nucleus-restricted mKate fluorescent protein were grown in media containing DMSO or 10 μM ARS1620 for 14 days, co-incubated with unstimulated PBMCs at 10:1 effector:target ratio in the presence or absence of P1A4xCD3, and cell viability was monitored by live fluorescence imaging for 72 h (unpaired two-tailed t -test, ns, not significant, *, p<0.05, **, p<0.01, ***, p<0.001). Data is presented as viable cell count relative to time 0. Individual data points are shown with mean ±SD indicated. F. H358 cells (H358 Parent) or H358 cells stably expressing K-Ras(G12V) (H358-G12V), each stably expressing nucleus-restricted mKate fluorescent protein were pulse-treated with ARS1620 and co-incubated with unstimulated PBMCs at 10:1 effector:target ratio in the presence or absence of P1A4xCD3, and cell viability was monitored by live fluorescence imaging for 72 h (One-way ANOVA with Dunnett’s correction for multiple comparisons, ns, not significant, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001). Data is presented as viable cell count relative to time 0. Individual data points are shown with mean ±SD indicated. G. mice bearing H358 xenografts were treated with covalent K-Ras(G12C) inhibitors, and tumors were dissected and analyzed by flow cytometry.

Article Snippet: KMS is an inventor on patents covering covalent inhibitors of K-Ras (G12C) owned by UCSF and licensed to Wellspring Biosciences.

Techniques: Mutagenesis, Stable Transfection, Expressing, Incubation, Fluorescence, Imaging, Cell Counting, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: Mutant KRAS Drives Immune Evasion by Sensitizing Cytotoxic T‐Cells to Activation‐Induced Cell Death in Colorectal Cancer

doi: 10.1002/advs.202203757

Figure Lengend Snippet: Clinical correlation between mutant KRAS and intratumoral cytotoxic CD8 + T‐cells. A) CD8 immunostaining within tumor tissue and invasive margin in a representative human CRC case carrying mutant KRAS compared to a KRAS wild‐type case. B) Quantification of CD8 + T‐cells within tumor tissues and invasive margin in KRAS mutant (n = 76) and KRAS wild‐type cases (n = 99). C) Statistical analysis of CD8 + T‐cells within tumor tissues in dMMR (n = 56) and pMMR cases (n = 119). D) Statistics of CD8 + T‐cells within the tumor tissues in 76 CRC patients of KRAS mutations (Left panel, Others include KRAS G12C (n = 4), G12R (n = 2), G12S (n = 6), G13C (n = 1), K117N (n = 1), A146T (n = 2), and Q61H (n = 2) mutations), and in 25 CRC patients of dMMR (Middle panel, Others include KRAS G12V (n = 1), K117N (n = 1), A146T (n = 2), and Q61H (n = 2) mutations), and in 51 CRC patients of pMMR (Right panel, Others include KRAS G12C (n = 4), G12R (n = 2), G12S (n = 6), and G13C (n = 1) mutations). E) Pearson's correlation analysis between KRAS expression levels and CD8 + T‐cell density in CRC tissues. Scare bars, 100 µm (A), *** P ≤ 0.001, and ns indicates P > 0.05, by two‐tailed Student's t ‐test (B,C), one‐way ANOVA (D), or Person's correlation analysis (E).

Article Snippet: The KRAS G12C inhibitor AMG 510 (Selleck) was given daily through oral gavage at 30 mL kg −1 .

Techniques: Mutagenesis, Immunostaining, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Mutant KRAS Drives Immune Evasion by Sensitizing Cytotoxic T‐Cells to Activation‐Induced Cell Death in Colorectal Cancer

doi: 10.1002/advs.202203757

Figure Lengend Snippet: Susceptibility of tumor‐specific CTLs to AICD in KRAS mutant CRC. A) Flow cytometry analysis of the migratory abilities of CTLs from KRAS wild type versus mutant tumors (n = 5). B–E) ELISAs for CXCL9, CXCL10, CXCL12, and CCL22 expression in KRAS wild type versus mutant tumors (n = 5). F–H) ELISAs for FasL, TNF, and TRAIL expression in KRAS wild type versus mutant tumor cells (n = 5). I) Tumor‐specific CTL apoptosis induced by autologous primary tumor cells or anti‐CD3. Numerical values denote the percentage of annexin V + cells (mean ± SD). J,K) Statistics of autologous tumor cells‐ or anti‐CD3‐induced specific apoptotic rates of tumor‐specific CTLs from KRAS wild type versus mutant tumors (n = 20). L,M) Statistics of autologous tumor cells‐ or anti‐CD3‐induced specific apoptotic rates of tumor‐specific CTLs from 20 tumors of different types of KRAS mutations (Others include KRAS G12C (n = 2), G12R (n = 1), and G13C (n = 1) mutations). N–P) ELISAs for Fas, TNFR2, and TRAILER expression in CTLs from KRAS wild type versus mutant tumors (n = 5). *** P ≤ 0.001, and ns indicates P > 0.05, by two‐tailed Student's t ‐test (A–H,J,K,N–P) or one‐way ANOVA (L,M).

Article Snippet: The KRAS G12C inhibitor AMG 510 (Selleck) was given daily through oral gavage at 30 mL kg −1 .

Techniques: Mutagenesis, Flow Cytometry, Expressing, Two Tailed Test