MEK1 31 nM (DC50, in HT29 cells) MEK2 17 nM (DC50, in HT29 cells) MEK1 31 nM (DC50, in SK-MEL-28 cells) MEK2 9.3 nM (DC50, in SK-MEL-28 cells)

Compound 23, or MS432, has the ability to significantly lower the levels of MEK1/2 protein in COLO 205 cells (DC50 (MEK1) = 18±7 nM, DC50 (MEK2) = 11±2 nM) and UACC257 cells (DC50 (MEK1) = 56±25 nM, DC50 (MEK2) = 27±19 nM).

MS432 (Compound 23) was bioavailable in mice and can be used for in vivo efficacy studies. [1]

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In evaluated cancer cell lines, MS432 (Compound 23) demonstrated good plasma exposure, with GI50 values that were roughly 3 to 20 times greater than Compound 23[1].

Kinase Inhibition Assay[1]
The inhibition potencies of compounds against MEK1 and MEK2 kinases were determined using the HotSpot kinase assay. This assay measures MEK kinase activity on ERK phosphorylation. Briefly, after the compounds were incubated with the kinase reaction mixture of MEK and ERK proteins for 20 min at rt, 33P-ATP (specific activity 10 μCi/μL) was delivered into the reaction mixture to initiate the reaction for incubation for 2 h at rt. Radioactivity was then detected by filter-binding method. Kinase activity data were expressed as the percent remaining kinase activity in samples as compared to DMSO reactions. Purified kinase proteins, MEK1 at 100 nM, MEK2 (PV3615, Thermo Fisher Scientific) at 150 nM, and ERK kinase-dead mutant K52R at 5 μM, were used in the reactions. IC50 values were determined using 10-concentration 3-fold serial dilution (top concentrations for PD0325901, compound 23 (MS432), and compound 24 were 3, 30, and 30 μM, respectively) with DMSO as control point in two independent experiments.

Cell Viability Assay[1]
Cell Types: (HT-29, SK-MEL-28, COLO 205 and UACC 257 cell lines. Tested
Tested Concentrations: 0-1 μM.
Incubation Duration: 72 hrs (hours).
Experimental Results: Effectively inhibited proliferation of these CRC and melanoma cells in a concentration-dependent manner, with GI50 values ranging from 30 to 200 nM.

Animal/Disease Models: Male Swiss Albino mice[1].
Doses: 50 mg/kg (pharmacokinetic/PK Analysis).
Route of Administration: IP
Experimental Results: Displays good plasma exposure with the maximum plasma concentration of 1,400 nM detected at 0.5 hour post dosing and plasma concentration of 710 nM at 8 hrs (hours) post dosing.

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Mouse PK Study[1]
A standard in vivo PK study was conducted for compound 23 (MS432) using three male Swiss Albino mice. The mice were administered intraperitoneally with solution formulation of compound 23 (MS432) at a 50 mg/kg dose. Sixty microliters of blood samples was collected from each mouse at 0.5, 2, and 8 h. Plasma was harvested by centrifugation of blood and stored at −70 ± 10 °C until analysis. Pharmacokinetic analysis was performed using the NCA module of Phoenix WinNonlin (Version 7.0). Plasma samples were quantified by fit-for-purpose LC–MS/MS method (LLOQ: 5.02 ng/mL for plasma). The formulation of compound 23 (MS432) was 5% NMP, 5% Solutol HS-15, and 90% normal saline.

[1]. Discovery of a First-in-Class Mitogen-Activated Protein Kinase Kinase 1/2 Degrader. J Med Chem. 2019 Dec 12;62(23):10897-10911.

MEK1 and MEK2 (also known as MAP2K1 and MAP2K2) are the "gatekeepers" of the ERK signaling output with redundant roles in controlling ERK activity. Numerous inhibitors targeting MEK1/2 have been developed including three FDA-approved drugs. However, acquired resistance to MEK1/2 inhibitors has been observed in patients, and new therapeutic strategies are needed to overcome the resistance. Here, we report a first-in-class degrader of MEK1/2, MS432 (23), which potently and selectively degraded MEK1 and MEK2 in a VHL E3 ligase- and proteasome-dependent manner and suppressed ERK phosphorylation in cells. It inhibited colorectal cancer and melanoma cell proliferation much more effectively than its negative control MS432N (24), and its effect was phenocopied by MEK1/2 knockdown. Compound 23 was highly selective for MEK1/2 in global proteomic profiling studies. It was also bioavailable in mice and can be used for in vivo efficacy studies. We provide two well-characterized chemical tools to the biomedical community.[1]

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In this study, researchers discovered compound 23 (MS432) as a first-in-class degrader of MEK1 and MEK2, the gatekeepers of ERK signaling. Compound 23 is a PD0325901-based VHL-recruiting small-molecule degrader of MEK1 and MEK2 with high potency and selectivity. We also developed compound 24 as a degrader negative control for compound 23. Compound 24 is a diastereoisomer of compound 23, thus sharing very high structural similarity with compound 23. We show compounds 23 and 24 inhibited the catalytic activity of MEK1 and MEK2 in vitro with similar potencies. In cellular experiments, compound 23 potently induced the degradation of MEK1 and MEK2 in a concentration- and time-dependent manner with durable effect. In contrast, the negative control compound 24 did not decrease MEK1/2 protein levels in cells. Our rescue experiment results indicate that MEK1/2 degradation induced by compound 23 is mediated through recruiting VHL E3 ligase for MEK1/2 polyubiquitination and proteasome-dependent proteolysis. Our quantitative global proteomic profiling analyses revealed that compound 23 is a highly selective degrader of MEK1 and MEK2. Furthermore, we found that compound 23, but not compound 24, effectively inhibited CRC and melanoma cell proliferation, and knockdown of MEK1/2 via shRNAs phenocopied the antiproliferative effect of compound 23 in BRAFV600E cancer cells. Moreover, compound 23 exhibited good plasma exposure in mice; thus it is suitable for in vivo efficacy studies. Overall, compounds 23 and 24 are a pair of well-characterized chemical tools for the research community to investigate the therapeutic potential of targeting MEK1 and MEK2. Further optimization of compound 23 into drug candidates may lead to novel, effective therapeutics for the treatment of CRC, melanoma, and other cancers.[1]

C50H65F3IN7O6S

1076.06

1075.371

2672512-44-4

PD0325901-O-C2-dioxolane;2581116-22-3

145712394

White to off-white solid powder

10.4

6

13

26

68

1570

4

C(N1C[C@@H](C[C@H]1C(=O)N[C@H](C1C=CC(C2=C(N=CS2)C)=CC=1)C)O)(=O)[C@H](C(C)(C)C)NC(=O)CCCCCCCCCCNCCCONC(C1C=CC(=C(C=1NC1C=CC(=CC=1F)I)F)F)=O

KCBAMQOKOLXLOX-BSZYMOERSA-N

InChI=1S/C50H65F3IN7O6S/c1-31(33-16-18-34(19-17-33)45-32(2)56-30-68-45)57-48(65)41-28-36(62)29-61(41)49(66)46(50(3,4)5)59-42(63)15-12-10-8-6-7-9-11-13-24-55-25-14-26-67-60-47(64)37-21-22-38(51)43(53)44(37)58-40-23-20-35(54)27-39(40)52/h16-23,27,30-31,36,41,46,55,58,62H,6-15,24-26,28-29H2,1-5H3,(H,57,65)(H,59,63)(H,60,64)/t31-,36+,41-,46+/m0/s1

(2S,4R)-1-[(2S)-2-[11-[3-[[3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzoyl]amino]oxypropylamino]undecanoylamino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO: 100 mg/mL (92.93 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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