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 pathway output, playing a redundant role in controlling ERK activity. Several inhibitors targeting MEK1/2 have been developed, including three FDA-approved drugs. However, acquired resistance to MEK1/2 inhibitors has been observed in patients, necessitating novel therapeutic strategies to overcome this resistance. This article reports a first-in-class MEK1/2 degrader, MS432 (23), which efficiently and selectively degrades MEK1 and MEK2 in a VHL E3 ligase- and proteasome-dependent manner, and inhibits ERK phosphorylation in cells. Compared to the negative control MS432N (24), MS432 more effectively inhibited the proliferation of colorectal cancer and melanoma cells, and MEK1/2 knockdown mimicked its inhibitory effect. Compound 23 exhibited high selectivity for MEK1/2 in a full proteomics analysis. The compound also has bioavailability in mice and can be used for in vivo efficacy studies. We have provided the biomedical community with two well-defined chemical tools. [1] In this study, researchers discovered compound 23 (MS432), which is the first degrader of MEK1 and MEK2 (key regulators of the ERK signaling pathway). Compound 23 is a VHL-recruiting small molecule MEK1 and MEK2 degrader based on PD0325901, with high efficiency and selectivity. We also developed compound 24 as a negative control for degrading compound 23. Compound 24 is a diastereomer of compound 23 and therefore has a very high structural similarity to compound 23. We demonstrated that compounds 23 and 24 inhibited the catalytic activity of MEK1 and MEK2 in vitro with similar potency. In cell experiments, compound 23 potently induced the degradation of MEK1 and MEK2 in a concentration- and time-dependent manner with a long-lasting effect. In contrast, the negative control compound 24 did not reduce the level of MEK1/2 protein in cells. Our rescue experiments showed that the MEK1/2 degradation induced by compound 23 was achieved through the recruitment of VHL E3 ligase to mediate the polyubiquitination and proteasome-dependent proteolysis of MEK1/2. Our quantitative proteomics analysis showed that compound 23 is a highly selective degrader of MEK1 and MEK2. In addition, we found that compound 23 (but not compound 24) effectively inhibited the proliferation of CRC and melanoma cells, and that the antiproliferative effect of compound 23 could be mimicked by knocking down MEK1/2 with shRNA in BRAFV600E cancer cells. Furthermore, compound 23 had good plasma exposure in mice, making it suitable for in vivo efficacy studies. In summary, compounds 23 and 24 are a pair of well-defined chemical tools that researchers can use to investigate the therapeutic potential of targeting MEK1 and MEK2. Further optimization of compound 23 to make it a drug candidate may lead to novel and effective therapies for 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.)