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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.
| Targets |
KRAS G12C
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|---|---|
| ln Vitro |
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].
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| 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 |
| References |
[1]. De Vita E, et al. The Missing Link between (Un)druggable and Degradable KRAS. ACS Cent Sci. 2020;6(8):1281-1284.
[2]. Bond MJ, et al. Targeted Degradation of Oncogenic KRASG12C by VHL-Recruiting PROTACs. ACS Cent Sci. 2020;6(8):1367-1375. |
| Molecular Formula |
C59H71CLFN11O7S
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|---|---|
| Molecular Weight |
1132.78095459938
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| Exact Mass |
1131.4931
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| Elemental Analysis |
C, 62.56; H, 6.32; Cl, 3.13; F, 1.68; N, 13.60; O, 9.89; S, 2.83
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| CAS # |
2502156-03-6
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| Related CAS # |
Adagrasib;2326521-71-3
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| Appearance |
Off-white to brown solid
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| LogP |
7.3
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| tPSA |
238Ų
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| 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
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| InChi Key |
ZCGQZLKPUVGCBQ-HLMPTVQRSA-N
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| 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
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| 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
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| Synonyms |
LC2; LC 2; LC-2
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~44.14 mM)
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|---|---|
| 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 |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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