Ras Assays for Drug Discovery
The small GTPase, K-Ras, is a known oncogene that is frequently mutated in a large percentage of cancers and is associated with poor disease prognosis. Mutated K-Ras 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 K-Ras 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 K-Ras with exchange factors and effector proteins.
Reaction Biology is committed to quality service in every aspect of our relationships with clients: before, during, and after the study, the assigned business development manager will be just one phone call away to answer any questions. Robust and reproducible assay formats for our ras assay suite ensure high-quality data.
Reaction Biology provides a variety of services to discover new inhibitors targeting the K-Ras pathway including:
- K-Ras Nucleotide Exchange Assay (also known as Ras Activation Assay) to monitor SOS mediated exchange of GDP to GTP
- Direct K-Ras binding assays via thermal shift or surface plasmon resonance techniques
- HTRF assays for testing the interactions of K-Ras::SOS, or K-Ras::Raf
- Activity assays for testing of kinase inhibitors
- Recombinant proteins such as K-Ras, GEFs, GAP, and others
The K-Ras Pathway
The K-Ras pathway can be inhibited by numerous compounds at different stages. The initial signaling can be inhibited with EGRF family inhibitors. K-Ras activation can be intercepted by inhibiting guanidin exchange factors such as SOS. K-Ras processing as well as K-Ras itself can be inhibited by siRNA or allele-specific RAS inhibitors. Finally, the two K-Ras downstream signaling pathways MAPK and PI3K signaling cascades can be inhibited at each kinase step.
Assays Formats Targeting the K-Ras Pathway
K-Ras::SOS1 Protein:Protein Interaction (PPI) Assay
Disruption of SOS1 binding to KRas can be used as an orthogonal method for studying SOS1 specific compounds. The assay uses an HTRF-based detection of interaction.
K-Ras::cRAF Protein:Protein Interaction (PPI) Assay
cRAF recognizes the GTP-bound form of K-Ras. cRAF binding assay can be used for the identification of disruptors of interaction between K-Ras and cRAF, as well as quantification of the nucleotide exchange reaction. This assay can be used as an alternative to the regular NEA with an optional examination of SOS1 independent GTP binding. The assay uses an HTRF-based detection of interaction.
The Protein-Protein Interaction assay is available for wild-type KRas and various K-Ras G12 mutants. Please inquire about custom-tailored GTPase assay development.
The K-Ras Nucleotide Exchange Assay (NEA) allows the monitoring of SOS1/2 mediated exchange of fluorescently labeled GDP to GTP.
The main application of the assay is to identify compounds that lock K-Ras in the inactive “OFF” state by preventing GTP binding.
An alternative nucleotide exchange assay format utilizes GTP labelled with DY-647P1 and monitors the increase in HTRF signal observed upon GTP* binding to K-Ras.The assay is performed at lower GTP concentrations compared to the standard K-Ras Nucleotide Exchange Assay and can evaluate various modes of nucleotide exchange inhibition.
The K-Ras Nucleotide Exchange Assay is available for K-Ras and various G12 mutants thereof. Please inquire for custom-tailored assay development.
Thermal shift assays are used to assess the effects of compounds on protein stability. Selectivity of compounds ARS-1620 and AMG-510 for K-Ras mutant G12C is clearly shown among K-Ras wt and mutants. The melting temperature of K-Ras 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 K-Ras G12C incubated with BI-2852, however, did not result in a significant shift showing that BI-2852 does not bind to K-Ras G12C.
The Thermal Shift Assay is available for K-Ras and various G12 mutants thereoff as well as SOS1 and SOS2. Please inquire for custom-tailored assay development.
Surface Plasmon Resonance (SPR) is used to quantify the binding affinity of the molecule as well as binding kinetics. A comparison between K-Ras WT and mutant proteins can be performed to determine selectivity.
K-Ras and various G12 mutants thereof as well as SOS1 are established for SPR analysis. Please inquire for custom-tailored assay development.
Example study: The K-Ras G12D mutant selective peptide KRpep-2d was used to show the difference in the binding of KRpep-2d to mutant G12D versus wild type K-Ras 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 K-Ras recombinant proteins, with 6xHis tag, GST tag, and 8xHis plus Biotin.
In addition, we offer K-Ras mutants G12C, G12D, and G12V. G13 mutant variants are in production.
Our K-Ras-related recombinant proteins are available for purchase, please inquire for more information.
A variety of K-Ras pathway related recombinant proteins were produced in house and are available for screening.
List of 82 K-Ras-related targets available for compound testing at Reaction Biology
| Target | HGNC Symbol | Additional Synonyms | Target Class | Mutants Available | Available Assay Formats |
|---|---|---|---|---|---|
| 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 |
| 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 |
| AMPKalpha1 | PRKAA1 | AMPK A1 | Kinase | yes | ³³PanQinase |
| 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 |
| c-MET | MET | c-MET, HGFR, RCCP2 | Kinase | yes | HotSpot; ³³PanQinase; Cellular Phosphorylation Assay |
| 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 |
| CDK2/cyclin A | CDK2-CCNA2 | P33 | Kinase | no | HotSpot; ³³PanQinase |
| CDK2/cyclin A1 | CDK2-CCNA1 | P33 | Kinase | no | HotSpot |
| CDK2/cyclin D1 | CDK2-CCND1 | p33 | Kinase | no | ³³PanQinase |
| 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 |
| CDK4/cyclin D1 | CDK4-CCND1 | CMM3, PSK-J3 | Kinase | no | HotSpot; NanoBRET; ³³PanQinase |
| CDK4/cyclin D2 | CDK4-CCND2 | CMM3, PSK-J3 | Kinase | no | ³³PanQinase |
| CDK4/cyclin D3 | CDK4-CCND3 | CMM3, PSK-J3 | Kinase | no | HotSpot; 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 |
| ERK1 | MAPK3 | ERT2, HS44KDAP, HUMKER1A, P44ERK1, P44MAPK, PRKM3 | Kinase | no | HotSpot; ³³PanQinase; Western blot |
| ERK2 | MAPK1 | ERT1, P42MAPK, PRKM1 | Kinase | no | HotSpot; ³³PanQinase; NanoBRET; Western blot |
| 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 |
| KRas | KRAS | C-K-RAS, CFC2, K-RAS2A, K-RAS2B, K-RAS4A, K-RAS4B, KI-RAS, KRAS1, KRAS2, NS, NS3, RASK2 | GTPase | yes | Nucleotide Exchange Assay; SOS1 PPI Assay; RAF1 PPI Assay; Thermal Shift Assay; SPR Assay |
| KSR1 | KSR1 | Kinase suppressor of Ras 1 | Kinase | yes | HotSpot |
| KSR2 | KSR2 | Kinase suppressor of Ras 2 | Kinase | yes | HotSpot |
| LKB1 | STK11 | LKB1 | Kinase | no | HotSpot; NanoBRET |
| MEK1 | MAP2K1 | MAPKK1, MKK1, PRKMK1 | Kinase | yes | HotSpot; ³³PanQinase |
| MEK2 | MAP2K2 | MAPKK2, MKK2, PRKMK2 | Kinase | no | HotSpot; ³³PanQinase |
| MST1 | STK4 | MST1 | Kinase | no | HotSpot; ³³PanQinase |
| MST2 | STK3 | MST2, KRS1 | Kinase | no | HotSpot; ³³PanQinase |
| mTOR | MTOR | FRAP1, FRAP, FRAP2, RAFT1, RAPT1 | Kinase | no | HotSpot; ³³PanQinase |
| 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 |
| 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 |
| 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) |
| 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 |
| RAF1 | RAF1 | c-Raf | Kinase | no | HotSpot; ³³PanQinase; Kras Protein-Protein Interaction 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 |
| RSK2 | RPS6KA3 | RSK2, HU-3, MRX19, CLS | Kinase | yes | HotSpot; ³³PanQinase; NanoBRET |
| RSK3 | RPS6KA2 | RSK3, HU-2 | Kinase | no | HotSpot; ³³PanQinase; NanoBRET |
| RSK4 | RPS6KA6 | RSK4 | Kinase | no | HotSpot; ³³PanQinase; NanoBRET |
| SOS1 | SOS1 | Son of sevenless homolog 1, GF1, GGF1, GINGF, HGF, NS4 | GEF | no | Nucleotide Exchange Assay; SPR Assay; Thermal Shift; Kras Protein-Protein-Interaction Assay |
| SOS2 | SOS2 | Son of sevenless homolog 2 | GEF | no | Nucleotide Exchange Assay; Thermal Shift |
| TBK1 | TBK1 | CDC37, HSPCA, HSPCB, IKBKG, NAK, T2K, TANK, TRAF2 | Kinase | no | HotSpot; ³³PanQinase; NanoBRET |
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
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