KRas Pathway Assay Services
The small GTPase, KRas, is a known oncogene that is frequently mutated in a large percentage of cancers and is associated with poor disease prognosis. Mutated KRas is locked in the activated GTP bound state and facilitates enhanced Ras signaling in cancer cells. While being a desirable target, the absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful.
The recent identification of a unique binding pocket and successful inhibition of the KRas G12C mutant by covalent chemical modifiers have led to the resurgence of interest in the design of inhibitors targeting KRas directly. Alternative efforts are undertaken at inhibition of interactions of KRas with exchange factors and effector proteins.
Reaction Biology provides a variety of services to discover new inhibitors targeting the KRAS pathway including:
- Nucleotide exchange assay to monitor SOS mediated exchange of GDP to GTP
- Direct binding assays via thermal shift or surface plasmon resonance techniques
- HTRF assays for testing the disruption of protein:protein interactions of KRas with SOS or Raf proteins
- Activity assays for testing of kinase inhibitors
- Recombinant proteins such as KRas, GEFs, GAP and others
The KRas Pathway
The KRas pathway can be inhibited by numerous compounds at different stages. The initial signaling can be inhibited with EGRF family inhibitors. KRas activation can be intercepted by inhibiting guanidin exchange factors such as SOS. KRas processing as well as KRas itself can be inhibited by siRNA or allele-specific RAS inhibitors. Finally, the two KRas downstream signaling pathways MAPK and PI3K signaling cascades can be inhibited at each kinase step.
List of KRas-related targets
| Target | HGNC Symbol | Additional Synonyms | Target Class | Mutants Available | Available Assay Formats |
|---|---|---|---|---|---|
| Caspase3 | CASP3 | Apopain, Cysteine protease CPP32, Protein Yama, SREBP cleavage activity 1, CPP32 | Apoptosis | no | Fluorescence-based Assay |
| Caspase7 | CASP7 | Apoptotic protease Mch-3, CMH-1, ICE-like apoptotic protease 3, MCH3 | Apoptosis | no | Fluorescence-based Assay |
| Caspase8 | CASP8 | Apoptotic cysteine protease, Apoptotic protease Mch-5, CAP4, FADD-homologous ICE/ced-3-like protease, FADD-like ICE, ICE-like apoptotic protease 5, MORT1-associated ced-3 homolog, MCH5 | Apoptosis | no | Fluorescence-based Assay |
| SOS1 | SOS1 | Son of sevenless homolog 1, GF1, GGF1, GINGF, HGF, NS4 | GEF | no | Nucleotide Exchange Assay; Thermal Shift; Kras Binding Assay |
| SOS2 | SOS2 | Son of sevenless homolog 2 | GEF | no | Nucleotide Exchange Assay; Thermal Shift; Kras Binding Assay |
| KRas | KRAS | C-K-RAS, CFC2, K-RAS2A, K-RAS2B, K-RAS4A, K-RAS4B, KI-RAS, KRAS1, KRAS2, NS, NS3, RASK2 | GTPase | n0 | Nucleotide Exchange Assay |
| AKT1 | AKT1 | PKB, RAC, PRKBA, RAc-ALPHA | Kinase | yes | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay, NanoBRET, Western blot |
| AKT2 | AKT2 | PRKBB, PKBBETA, RAc-BETA | Kinase | yes | HotSpot, ³³PanQinase, NanoBRET |
| AKT3 | AKT3 | PKBG, PKB gamma, PRKBG, RAC-gamma, RAC-PK-gamma, STK-2 | Kinase | yes | HotSpot, ³³PanQinase |
| ALK | ALK | CD246, NBLST3 | Kinase | yes | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay |
| ARAF | ARAF | A-RA, ARAF1, PKS2, RAFA1 | Kinase | no | HotSpot, ³³PanQinase |
| BRAF | BRAF | B-Raf, B-raf-1, BRAF1, RAFB1 | Kinase | yes | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay, NanoBRET |
| CDK2/cyclin A | CDK2/CCNA2 | P33 | Kinase | no | HotSpot, ³³PanQinase |
| CDK2/cyclin A1 | CDK2/CCNA1 | P33 | Kinase | no | HotSpot |
| CDK2/cyclin E | CDK2/CCNE1 | p33 | Kinase | no | HotSpot, ³³PanQinase, NanoBRET |
| CDK2/cyclin E2 | CDK2/CCNE2 | p33 | Kinase | no | HotSpot |
| CDK2/cyclin O | CDK2/CCNO | P33 | Kinase | no | HotSpot |
| CDK2/cyclin D1 | CDK2/CCND1 | p33 | Kinase | no | ³³PanQinase |
| CDK4/cyclin D1 | CDK4/CCND1 | CMM3, PSK-J3 | Kinase | no | HotSpot, NanoBRET, ³³PanQinase |
| CDK4/cyclin D3 | CDK4/CCND3 | CMM3, PSK-J3 | Kinase | no | HotSpot, ³³PanQinase |
| CDK4/cyclin D2 | CDK4/CCND2 | CMM3, PSK-J3 | Kinase | no | ³³PanQinase |
| CDK6/cyclin D1 | CDK6/CCND1 | PLSTIRE | Kinase | no | ³³PanQinase, HotSpot |
| CDK6/cyclin D2 | CDK6/CCND2 | PLSTIRE | Kinase | no | ³³PanQinase |
| CDK6/cyclin D3 | CDK6/CCND3 | PLSTIRE | Kinase | no | ³³PanQinase, HotSpot, NanoBRET |
| EGF-R | EGFR | ERBB, mENA, ERBB1 | Kinase | yes | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay |
| ERBB2 | ERBB2 | NEU, NGL, HER2, TKR1 | Kinase | yes | HotSpot, ³³PanQinase, In Vivo Kinase Activity Model, Cellular Phosphorylation Assay |
| FGFR1 | FGFR1 | H2, H3, H4, H5, CEK, FLG, FLT2, KAL2, BFGFR, C-FGR, N-SAM | Kinase | yes | HotSpot, ³³PanQinase, NanoBRET |
| FGFR2 | FGFR2 | BEK, BFR-1, CD332, CEK3, CFD1, ECT1, JWS, KGFR, KSAM, K-SAM, TK14, TK25 | Kinase | yes | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay, NanoBRET |
| FGFR3 | FGFR3 | CH, CD333, CEK2, HSFGFR3EX, JTK4 | Kinase | yes | HotSpot, ³³PanQinase, NanoBRET |
| FGFR4 | FGFR4 | CD334, JTK2, TKF | Kinase | yes | HotSpot, ³³PanQinase, NanoBRET |
| FLT3 | FLT3 | CD135, FLK-2, FLK2, STK1 | Kinase | yes | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay, NanoBRET |
| IR | INSR | IR | Kinase | no | HotSpot, ³³PanQinase |
| IRR | INSRR | IRR | Kinase | no | HotSpot, ³³PanQinase |
| KSR1 | KSR1 | Kinase suppressor of Ras 1 | Kinase | yes | HotSpot |
| KSR2 | KSR2 | Kinase suppressor of Ras 2 | Kinase | yes | HotSpot |
| MEK1 | MAP2K1 | MAPKK1, MKK1, PRKMK1 | Kinase | yes | HotSpot, ³³PanQinase |
| MEK2 | MAP2K2 | MAPKK2, MKK2, PRKMK2 | Kinase | no | HotSpot, ³³PanQinase |
| ERK2 | MAPK1 | ERT1, P42MAPK, PRKM1 | Kinase | no | HotSpot, ³³PanQinase, NanoBRET, Western blot |
| ERK1 | MAPK3 | ERT2, HS44KDAP, HUMKER1A, P44ERK1, P44MAPK, PRKM3 | Kinase | no | HotSpot, ³³PanQinase, Western blot |
| c-MET | MET | c-MET, HGFR, RCCP2 | Kinase | yes | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay, NanoBRET |
| mTOR | MTOR | FRAP1, FRAP, FRAP2, RAFT1, RAPT1 | Kinase | no | HotSpot, ³³PanQinase |
| PAK1 | PAK1 | PAKalpha | Kinase | no | HotSpot, ³³PanQinase |
| PAK2 | PAK2 | PAK65, PAKgamma | Kinase | no | HotSpot, ³³PanQinase |
| PAK3 | PAK3 | MRX30, OPHN3, PAK3beta, bPAK | Kinase | no | HotSpot, ³³PanQinase |
| PAK4 | PAK4 | PAK4 | Kinase | no | HotSpot, ³³PanQinase, NanoBRET |
| PDGFRa | PDGFRA | PDGFR2, CD140a | Kinase | no | HotSpot, ³³PanQinase |
| PDGFRb | PDGFRB | JTK12, PDGFR, CD140B, PDGFR1 | Kinase | no | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay |
| PDPK1 | PDPK1 | PDPK1, PKB-like, 3-phosphoinositide dependent protein kinase-1 | Kinase | no | HotSpot, ³³PanQinase |
| PI3K (p110a/p65a) | PIK3CA-PIK3R1 | MGC142161, MGC142163, p110-alpha, PI3K, PI3K-alpha | Kinase | no | ADP-Glo (US) |
| PI3Ka (p110a/p85a) | PIK3CA-PIK3R1 | MGC142161, MGC142163, p110-alpha, PI3K, PI3K-alpha | Kinase | yes | ADP-Glo (US), ADP-Glo (Germany) |
| PI3Kd (p110d/p85a) | PIK3CD | p110D, P110DELTA, PI3K, PI3K-delta, p110-delta | Kinase | no | ADP-Glo (US), ADP-Glo (Germany) |
| PI3Kg (p110g) | PIK3CG | p120-PI3K, PI3CG, PI3K, PI3Kgamma, p110-gamma | Kinase | yes | ADP-Glo (US), ADP-Glo (Germany) |
| AMPKalpha1 | PRKAA1 | AMPK A1 | Kinase | yes | ³³PanQinase |
| AMPK (A1/B1/G1) | PRKAA1/PRKAB1/PRKAG1 | AMPK (A1/B1/G1) | Kinase | no | HotSpot |
| AMPK (A1/B1/G2) | PRKAA1/PRKAB1/PRKAG2 | AMPK (A1/B1/G2) | Kinase | no | HotSpot |
| AMPK (A1/B1/G3) | PRKAA1/PRKAB1/PRKAG3 | AMPK (A1/B1/G3) | Kinase | no | HotSpot |
| AMPK (A1/B2/G1) | PRKAA1/PRKAB2/PRKAG1 | AMPK (A1/B2/G1) | Kinase | no | HotSpot |
| AMPK (A1/B2/G2) | PRKAA1/PRKAB2/PRKAG2 | AMPK (A1/B2/G2) | Kinase | no | HotSpot |
| AMPK (A1/B2/G3) | PRKAA1/PRKAB2/PRKAG3 | AMPK (A1/B2/G3) | Kinase | no | HotSpot |
| AMPK (A2/B1/G1) | PRKAA2/PRKAB1/PRKAG1 | AMPK (A2/B1/G1) | Kinase | no | HotSpot |
| AMPK (A2/B1/G2) | PRKAA2/PRKAB1/PRKAG2 | AMPK (A2/B1/G2) | Kinase | no | HotSpot |
| AMPK (A2/B1/G3) | PRKAA2/PRKAB1/PRKAG3 | AMPK (A2/B1/G3) | Kinase | no | HotSpot |
| AMPK (A2/B2/G1) | PRKAA2/PRKAB2/PRKAG1 | AMPK (A2/B2/G1) | Kinase | no | HotSpot |
| AMPK (A2/B2/G2) | PRKAA2/PRKAB2/PRKAG2 | AMPK (A2/B2/G2) | Kinase | no | HotSpot |
| AMPK (A2/B2/G3) | PRKAA2/PRKAB2/PRKAG3 | AMPK (A2/B2/G3) | Kinase | no | HotSpot |
| RAF1 | RAF1 | c-Raf | Kinase | no | HotSpot, ³³PanQinase; Kras Binding Assay |
| ROCK1 | ROCK1 | P160ROCK | Kinase | no | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay |
| ROCK2 | ROCK2 | KIAA0619, ROC2 | Kinase | no | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay |
| ROS | ROS1 | ROS, MCF3 | Kinase | yes | HotSpot, ³³PanQinase |
| RSK1 | RPS6KA1 | RSK1, HU-1, MAPKAPK1, p90Rsk | Kinase | no | HotSpot, ³³PanQinase, NanoBRET |
| RSK3 | RPS6KA2 | RSK3, HU-2 | Kinase | no | HotSpot, ³³PanQinase, NanoBRET |
| RSK2 | RPS6KA3 | RSK2, HU-3, MRX19, CLS | Kinase | yes | HotSpot, ³³PanQinase, NanoBRET |
| RSK4 | RPS6KA6 | RSK4 | Kinase | no | HotSpot, ³³PanQinase, NanoBRET |
| P70S6K | RPS6KB1 | S6K, STK14A, P70S6K | Kinase | no | HotSpot, ³³PanQinase, Cellular Phosphorylation Assay |
| P70S6Kb | RPS6KB2 | KLS, P70-beta-1, P70-beta-2, S6K-beta2, S6K2, SRK, p70S6Kb, S6K-beta | Kinase | no | HotSpot, ³³PanQinase |
| LKB1 | STK11 | ;LKB1 | Kinase | no | HotSpot, NanoBRET |
| MST2 | STK3 | MST2, KRS1 | Kinase | no | HotSpot, ³³PanQinase |
| MST1 | STK4 | MST1 | Kinase | no | HotSpot, ³³PanQinase |
| TBK1 | TBK1 | CDC37, HSPCA, HSPCB, IKBKG, NAK, T2K, TANK, TRAF2 | Kinase | no | HotSpot, ³³PanQinase, NanoBRET |
| PP1A | PPP1CA | Serine/threonine-protein phosphatase PP1-alpha catalytic subunit, PPP1A | Phosphatase | no | Fluorescence-based Assay |
| PTPN11/SHP2-FL | PTPN11 | Tyrosine-protein phosphatase non-receptor type 11, Protein-tyrosine phosphatase 1D, Protein-tyrosine phosphatase 2C, SH-PTP2, SH-PTP3, PTP2C, SHPTP2 | Phosphatase | no | Fluorescence-based Assay |
| NFKB1 | NFKB1 | EBP-1, KBF1, NF-kB1, NF-kappa-B, NF-kappaB, NFKB-p105, NFKB-p50, NFkappaB, p105, p50 | Transcription factor | no | Cell-based Reporter Assay |
The list of targets was derived from "The RAS Initiative" of the NIH.
Source: https://www.cancer.gov/research/key-initiatives/ras/ras-central/blog/2015/ras-pathway-v2
Assays for the discovery of KRas pathway inhibitors
KRAS::SOS1
Disruptionof SOS1 binding to KRas can be used as an orthogonal method for studying SOS1 specific compounds. Assay uses HTRF-based detection of interaction.
RAS::cRAF
cRAF recognizes the GTP-bound form of KRas. cRAF binding assay can be used for the identification of disruptors of interaction between KRas and cRAF, as well as quantification of nucleotide exchange reaction. This assay can be used as an alternative to the regular NEA with optional examination of SOS1 independent GTP binding. Assay uses HTRF-based detection of interaction.
The nucleotide exchange assay (NEA) allows the monitoring of SOS1/2 mediated exchange of fluorescently labeled GDP (GDP*) to GTP. The main application of the assay is to identify compounds that lock KRas in the inactive “OFF” state by preventing GTP binding.
Many of the KRas downstream kinases are available for testing. Check out our kinase assay portfolio.
Thermal shift assays are used to assess the effects of compounds on protein stability. Selectivity of compounds ARS-1620 and AMG-510 for KRas mutant G12C is clearly shown among KRas wt and mutants. The melting temperature of KRas G12C incubated with ARS-1620 for example shifts from 53.8 degree celsius to 58.5 degree Celsius with a second peak appearing at 65.6 degree Celsius. The melting temperature of KRas G12C incubated with BI-2852, however, did not result in a significant shift showing that BI-2852 does not bind to KRas G12C.
Surface Plasmon Resonance (SPR) is used to quantify the binding affinity of the molecule as well as binding kinetics. A comparison between KRas WT and mutant proteins can be performed to determine selectivity.
SOS1 is also established for SPR analysis.
Example study: The KRas G12D mutant selective peptide KRpep-2d was used to show the difference in the binding of KRpep-2d to mutant G12D versus wild type KRas and other mutants. The peptide binds to all targets, however, the binding affinity (KD) of the peptide is 15 x higher when interacting with the G12D mutant.
At Reaction Biology, we have created three versions of wild-type KRas recombinant proteins, with 6xHis tag, GST tag, and 8xHis plus Biotin.
In addition, we offer KRas mutants G12C, G12D, and G12V. G13 mutant variants are in production.
Our KRas-related recombinant proteins are available for purchase, please inquire for more information.
A variety of KRas pathway related recombinant proteins were produced in house and are available for screening.
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