KRAS G12C

Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRASG12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRASG12C is described[1].

KRASG12C Target Engagement[1]
Tumor fragments were homogenized in 6 M guanidine–HCl, 50 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) (pH 7.5), and 5 mM TCEP. Following centrifugation, the protein concentration of the supernatant was determined using a Bradford assay. An internal standard (13C15N recombinant KRASG12C) and 20 mM iodoacetamide were added to 200 μg of tumor protein in 200 μL of lysis buffer, and samples were incubated at 37 °C for 30 min in the dark. Following alkylation, 100 μL of the reaction was exchanged into 1 M guanidine–HCl, 50 mM HEPES (pH 7.5), using a 96-well Zeba spin plate. Proteins were digested with 1 μg of trypsin/Lys-C mix at 37 °C for 18 h. Peptides were desalted using a C18 spin plate, and the solvent was removed by evaporation. Peptides were solubilized in 0.1% formic acid, 5% acetonitrile, 95% water, for LCMS analysis. A targeted method on a Sciex TripleTOF instrument was used to monitor the Cys-12-containing KRASG12C peptide, an internal reference peptide, as well as the corresponding isotope-labeled peptides. KRASG12C engagement was calculated as previously reported.

Cell-Based Phospho-ERK Assay[1]
All experiments were performed under standard conditions (37 °C and 5% CO2). IC50 values were calculated by dose response curve fitting using a four-parameter method. NCI-H358 cells harboring the KRASG12C mutation were seeded in 96-well plates in RPMI supplemented with 10% fetal bovine serum. Plates were incubated overnight. After incubation of cells with an inhibitor for 3 h, cells were washed once with phosphate-buffered saline (PBS), fixed with 3.8% formaldehyde, and permeabilized with ice cold methanol. The plates were then incubated with Li-Cor blocking buffer. Subsequently, phosphorylation of ERK was assessed by an in-cell western method by incubating with primary antibodies against GAPDH (mouse) and phospho-ERK (rabbit). The plates were then incubated with fluorescent secondary antibodies specific for mouse or rabbit. The plates were imaged on a Li-Cor fluorescent plate reader at 680 and 800 nm wavelengths. The phospho-ERK signal was normalized to the GAPDH signal, and POC values were generated[1].

In Vivo Studies[1]
Mice were maintained under pathogen-free conditions, and food and water were provided ad libitum. 6–8 week-old, female, athymic nude-Foxn1nu mice were injected subcutaneously with either NCI-H358 or MIA PaCa-2 cells in 100 μL of PBS and Matrigel matrix in the right hind flank with 5.0 × 106 cells 50:50 cells/Matrigel. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 × L × W2 in which L refers to length and W refers to the width of each tumor. When tumors reached an average tumor volume of ∼350 mm3, mice were randomized into treatment groups. Mice were treated by oral gavage with either vehicle consisting of 10% research grade Captisol in 50 mM citrate buffer pH 5.0 or study compound in vehicle at indicated doses. For efficacy studies, animals were orally administered study compound or vehicle and monitored daily, tumors were measured 3 times per week and body weights were measured 2 times per week. Study day on efficacy plots indicates the day after which compound treatment was initiated. For PK/PD studies, tumors and plasma were collected after a single dose at the time points and concentration range indicated.

[1]. Jay B Fell, et al. Identification of the Clinical Development Candidate MRTX849, a Covalent KRAS G12C Inhibitor for the Treatment of Cancer. J Med Chem. 2020 Jul 9;63(13):6679-6693.

Stability—Liver Cytosol Supplemented with GSH[1]
Compounds (1 μM) were incubated at 37 °C for 30 min in a 1 mg/mL liver cytosol, supplemented with 5 mM GSH in KPB buffer. At the end of the designated time point, samples were quenched with acetonitrile spiked with 40 mM NEM and 0.2 μM concentration of labetalol (internal standard). Samples were centrifuged, and supernatants were analyzed by LC–MS/MS.

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Whole Blood Stability[1]
Compounds (5 μM) were incubated at 37 °C for 30, 60, and 240 min in a 1:1 (v/v) blood and PBS, pH 7.4. Diluted blood was preincubated for 15 min at 37 °C and 100% humidity before the reactions were initiated with the dosing of the compound. At the end of each designated time point, the red blood cells were lysed 1:1 with water, mixed at 600 rpm for 1 min, and stopped with acetonitrile containing 0.625 μM labetalol (internal standard). Samples were centrifuged, and supernatants were analyzed by LC–MS/MS.

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Measurement of kinact/KI[1]
Recombinant KRASG12C “Lite” (C51S/C80L/C118S) was reacted with a range of MRTX849 concentrations in 25 mM HEPES (pH 7.0), 150 mM NaCl, 5 mM MgCl2, 10 mM octyl β-glucopyranoside, and 0.5 mM TCEP, for 0–45 s, at room temperature. At each time-point, the reaction was quenched with 50 mM HCl, and 0.25 μg of pepsin was added. KRASG12C was digested for 4 h at 37 °C, and the resulting Cys-12-containing peptide was analyzed by LCMS. The percent of modified KRASG12C at each time-point was calculated from the 0 s control sample for each concentration of MRTX849, and kobs was subsequently calculated from the slope of the ln(POC) versus time data. Rate versus concentration data fit the Michaelis–Menten equation.

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Metabolite Identification in Hepatocytes[1]
Cryopreserved hepatocytes from mouse, rat, dog, and human were thawed and diluted to a viable cell density of 1 × 106 cells/mL using Dulbecco’s modified Eagle medium. MRTX849 (10 μM) was added to 1 mL of each hepatocyte suspension, and samples were incubated at 37 °C for 2 h. Metabolism was quenched with the addition of 1% formic acid, samples were centrifuged, and the supernatant was loaded onto a 3cc Oasis HLB cartridge. MRTX849 and metabolites were washed with 5% methanol in water and eluted with 100% methanol. The solvent was evaporated under a stream of nitrogen gas, and metabolites were reconstituted with 150 μL of 30:70 acetonitrile/water (v/v). Metabolites were separated on a Gemini NX-C18 column and detected by absorbance at 290 nm and mass spectrometry. Relative amounts of metabolites were determined by A290nm peak intensity, and the biotransformation was characterized by the associated m/z and MSMS fragmentation patterns.

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NCI-H358 Proteome Cysteine Selectivity Assay[1]
NCI-H358 cells were treated with 3 μM MRTX849 for 3 h and then lysed with 1% NP-40-containing buffer and sonication. Cell extracts were treated with 5 mM iodoacetamide desthiobiotin for 1 h at room temperature. Proteins were precipitated with acetone and resuspended in a buffer containing 6 M guanidine–HCl, 50 mM HEPES, pH 7.5, and 5 mM TCEP and incubated for 15 min at 65 °C. Following treatment with iodoacetamide and buffer exchange into 1 M guanidine-HCl, 50 mM HEPES (pH 7.5), proteins were digested with trypsin/Lys-C overnight. Desthiobiotinylated peptides were enriched using streptavidin agarose and eluted with 50% acetonitrile and 0.1% TFA. The solvent was removed by evaporation and peptides were resuspended in 0.1% formic acid, 5% acetonitrile, and in water. Samples were analyzed on a Sciex 6600 TripleTOF instrument. Protein Pilot 5 was used to identify peptides labeled with desthiobiotin from data-dependent MS/MS scans and relative quantitation (MTRX849-treated vs control) was conducted using SWATH analysis.

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Click Chemistry Target Identification[1]
Two flasks of NCI-H358 cells (60 million cells/T225 flask) in the RPMI 1640 SILAC light media were treated with DMSO as the control, and two flasks of cells in the RPMI 1640 SILAC heavy media (13C6-lysine, 13C6,15N4-arginine) were treated with 1 μM MRTX849 for 3 h at 37 °C. All cells were then treated with 2 μM compound 24 for 3 h at 37 °C. Cells were then lysed with 50 mM HEPES, 150 NaCl, 0.5% Triton-X 100, 1 mM EDTA, 1 mM EGTA, and HALT protease inhibitor cocktail, for 5 min, probe sonicated, and centrifuged, and the supernatant was filtered with a 45 μm syringe filter. For each replicate, 4 mg of protein in “light”’ lysate was combined with 4 mg of protein in “heavy” lysate for sample processing. Proteins were isolated using chloroform/methanol precipitation, resuspended in 0.18% sodium dodecyl sulfate (SDS) in 50 mM HEPES pH 7.5, and ‘clicked’ to azide agarose by incubating with 5 mM ascorbate, 1 mM CuSO4, and 2 mM BTTAA, for 2.5 h at room temperature. Resin-bound proteins were treated with 10 mM DTT for 40 min and then with 20 mM iodoacetamide for 30 min and washed with (1) 100 mM Tris, pH 8.0, 250 mM NaCl, 1% SDS, 5 mM EDTA, (2) 8 M urea, 100 mM Tris, pH 8.0, and (3) 20% acetonitrile in water. Trypsin/Lys-C (1 μg) was added to the protein-bound resin, suspended in 20 mM Tris pH 8.0, 2 mM CaCl2, 10% acetonitrile, and incubated for 18 h at 37 °C. Eluted peptides were desalted on C18 spin columns, solvent was removed by evaporation, and peptides were resuspended in 0.1% formic acid, 5% acetonitrile, and in water for LCMS analysis. Samples were analyzed on a Sciex 6600 TripleTOF instrument. Protein Pilot 5 was used to identify peptides from data-dependent MS/MS scans of an unlabeled sample, and relative quantitation (MTRX849-treated vs control) was conducted using SWATH analysis of the SILAC samples.

C36H39CLFN7O2

656.19

655.283

2490716-96-4

Adagrasib;2326521-71-3

162642619

Typically exists as White to off-white solid at room temperature

6

0

9

10

47

1200

2

C=C(C(=O)N1CCN(C[C@@H]1CC#N)C2=NC(=NC3=C2CCN(C3)C4=CC=CC5=C4C(=CC=C5)Cl)OC[C@@H]6CCCN6CCCC#C)F

YUPCFANWZUHYAJ-NSOVKSMOSA-N

InChI=1S/C36H39ClFN7O2/c1-3-4-5-17-42-18-8-11-28(42)24-47-36-40-31-23-43(32-13-7-10-26-9-6-12-30(37)33(26)32)19-15-29(31)34(41-36)44-20-21-45(35(46)25(2)38)27(22-44)14-16-39/h1,6-7,9-10,12-13,27-28H,2,4-5,8,11,14-15,17-24H2/t27-,28-/m0/s1

2-[(2S)-4-[7-(8-chloronaphthalen-1-yl)-2-[[(2S)-1-pent-4-ynylpyrrolidin-2-yl]methoxy]-6,8-dihydro-5H-pyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroprop-2-enoyl)piperazin-2-yl]acetonitrile

MRTX-849 analog 24; MRTX849 analog 24

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~76.20 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)