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LC-2 (LC2) is a novel KRAS G12C degrader based on PROTAC technology with DC50s between 0.25 and 0.76 μM. It is composed of a ligand for the von Hippel Lindau E3 ligase joined to the KRAS inhibitor MRTX849. KRAS is mutated in ∼20% of human cancers and is one of the most sought-after targets for pharmacological modulation, despite having historically been considered "undruggable." The discovery of potent covalent inhibitors of the KRASG12C mutant in recent years has sparked a new wave of interest in small molecules targeting KRAS. While these inhibitors have shown promise in the clinic, we wanted to explore PROTAC-mediated degradation as a complementary strategy to modulate mutant KRAS. Herein, we report the development of LC-2, the first PROTAC capable of degrading endogenous KRASG12C. LC-2 covalently binds KRASG12C with a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained KRASG12C degradation leading to suppression of MAPK signaling in both homozygous and heterozygous KRASG12C cell lines. LC-2 demonstrates that PROTAC-mediated degradation is a viable option for attenuating oncogenic KRAS levels and downstream signaling in cancer cells.
Physicochemical Properties
| Molecular Formula | C59H71CLFN11O7S |
| Molecular Weight | 1132.78095459938 |
| Exact Mass | 1131.493 |
| Elemental Analysis | C, 62.56; H, 6.32; Cl, 3.13; F, 1.68; N, 13.60; O, 9.89; S, 2.83 |
| CAS # | 2502156-03-6 |
| Related CAS # | Adagrasib;2326521-71-3 |
| PubChem CID | 154727765 |
| Appearance | Off-white to brown solid |
| Density | 1.3±0.1 g/cm3 |
| Index of Refraction | 1.614 |
| LogP | 3.07 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 16 |
| Rotatable Bond Count | 21 |
| Heavy Atom Count | 80 |
| Complexity | 2170 |
| Defined Atom Stereocenter Count | 5 |
| SMILES | ClC1=CC=CC2C=CC=C(C=21)N1CC2=C(CC1)C(=NC(=N2)OC[C@@H]1CCCN1CCCOCCC(N[C@H](C(N1C[C@@H](C[C@H]1C(NCC1C=CC(C2=C(C)N=CS2)=CC=1)=O)O)=O)C(C)(C)C)=O)N1CCN(C(C(=C)F)=O)[C@@H](CC#N)C1 |
| InChi Key | ZCGQZLKPUVGCBQ-HLMPTVQRSA-N |
| InChi Code | InChI=1S/C59H71ClFN11O7S/c1-37(61)56(76)71-27-26-70(32-42(71)19-22-62)54-45-20-25-69(48-14-7-11-40-10-6-13-46(60)51(40)48)34-47(45)65-58(67-54)79-35-43-12-8-23-68(43)24-9-28-78-29-21-50(74)66-53(59(3,4)5)57(77)72-33-44(73)30-49(72)55(75)63-31-39-15-17-41(18-16-39)52-38(2)64-36-80-52/h6-7,10-11,13-18,36,42-44,49,53,73H,1,8-9,12,19-21,23-35H2,2-5H3,(H,63,75)(H,66,74)/t42-,43-,44+,49-,53+/m0/s1 |
| Chemical Name | (2S,4R)-1-((S)-2-(3-(3-((S)-2-(((7-(8-chloronaphthalen-1-yl)-4-((S)-3-(cyanomethyl)-4-(2-fluoroacryloyl)piperazin-1-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)oxy)methyl)pyrrolidin-1-yl)propoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide |
| Synonyms | LC2; LC 2; LC-2; (2S,4R)-1-((S)-2-(3-(3-((S)-2-(((7-(8-Chloronaphthalen-1-yl)-4-((S)-3-(cyanomethyl)-4-(2-fluoroacryloyl)piperazin-1-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-2-yl)oxy)methyl)pyrrolidin-1-yl)propoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; PROTAC KRASG12C Degrader-LC-2; CHEMBL5174597; LC-2 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
KRAS G12C (DC50 = 0.25~0.76 μM); VHL The target of LC-2 is KRASG12C, and it recruits the E3 ligase VHL. |
| ln Vitro |
LC-2 covalently binds to KRASG12C via a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained degradation of endogenous KRASG12C in both homozygous and heterozygous KRASG12C cell lines. This degradation leads to the suppression of MAPK signaling, as evidenced by reduced levels of phosphorylated ERK (pERK) [2] In several KRAS mutant cancer cells (NCI-H2030, MIA PaCa-2, SW1573, NCI-H23, and NCI-H358 cells), LC-2 causes endogenous KRASG12C to degrade, with a DC50 range from 0.25 to 0.76 μM. KRASG12C is degraded by LC-2 through a genuine PROTAC mechanism. 2.5 μM LC-2 was applied to MIA PaCa-2, NCI-H23, and SW1573 cells during 6, 24, 48, and 72 hours. Maximum KRAS degradation starts in 24 hours and lasts for up to 72 hours in all three cell lines [1]. In both heterozygous and homozygous KRAS mutant cell lines, Erk signaling is influenced by LC-2-induced (2.5 μM; 6-24 hours) KRAS G12C degradation [1]. |
| Enzyme Assay |
Competition, Proteasome Inhibition, and Neddylation Inhibition Experiments [2] Between 2.5 x 105 and 5.0 x 105 cells were seeded into 6-well plates. The next day cells were pretreated with DMSO, 500 μM or 1 mM VHL ligand, 1 μM epoxomicin, 1 μM MLN4924, or 100 nM M bafilomycin A1 for 1 h. Media was then removed and cells were treated with DMSO, 2.5 μM LC-2 plus DMSO, 2.5 μM LC-2 Epimer plus DMSO, or cotreated with 2.5 μM LC-2 and the corresponding competitor/inhibitor. NCI-H2030 cells were treated for 4 h and NCI-H23 cells were treated for 24 h, after which cells were lysed by scraping in RIPA buffer supplemented as described previously. For an individual experiment conducted on a given day, two separate wells of cells were treated identically for every condition and harvested side-by-side. |
| Cell Assay |
Western blot analysis[1] Cell Types: MIA PaCa-2 cells and NCI-H23 cells Tested Concentrations: 2.5 μM Incubation Duration: 6-24 hrs (hours) Experimental Results: Inhibition and degradation of KRAS G12C reduces homozygous MIA PaCa at 6 and 24 hrs (hours) pErk Signaling-2 Cells Cells (homozygous and heterozygous KRASG12C lines) were treated with LC-2 at specified concentrations. Following treatment, cell lysates were prepared, and Western blot analysis was performed to detect levels of KRASG12C and phosphorylated ERK (pERK), allowing assessment of KRASG12C degradation and suppression of MAPK signaling [2] |
| References |
[1]. The Missing Link between (Un)druggable and Degradable KRAS. ACS Cent Sci. 2020;6(8):1281-1284. [2]. Targeted Degradation of Oncogenic KRASG12C by VHL-Recruiting PROTACs. ACS Cent Sci. 2020;6(8):1367-1375. |
| Additional Infomation |
LC-2 is the first PROTAC capable of degrading endogenous KRASG12C. Its design leverages a MRTX849 warhead for covalent binding to KRASG12C and a moiety that recruits the E3 ligase VHL, enabling targeted degradation of the oncogenic mutant KRASG12C as a complementary strategy to existing KRASG12C inhibitors [2] KRAS is mutated in ∼20% of human cancers and is one of the most sought-after targets for pharmacological modulation, despite having historically been considered "undruggable." The discovery of potent covalent inhibitors of the KRASG12C mutant in recent years has sparked a new wave of interest in small molecules targeting KRAS. While these inhibitors have shown promise in the clinic, we wanted to explore PROTAC-mediated degradation as a complementary strategy to modulate mutant KRAS. Herein, we report the development of LC-2, the first PROTAC capable of degrading endogenous KRASG12C. LC-2 covalently binds KRASG12C with a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained KRASG12C degradation leading to suppression of MAPK signaling in both homozygous and heterozygous KRASG12C cell lines. LC-2 demonstrates that PROTAC-mediated degradation is a viable option for attenuating oncogenic KRAS levels and downstream signaling in cancer cells.[2] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~50 mg/mL (~44.14 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (1.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.08 mg/mL (1.84 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.8828 mL | 4.4139 mL | 8.8278 mL | |
| 5 mM | 0.1766 mL | 0.8828 mL | 1.7656 mL | |
| 10 mM | 0.0883 mL | 0.4414 mL | 0.8828 mL |