ERK1 (IC50 = 5 nM); ERK2 (IC50 = 5 nM)

Temuterkib has an IC50 of 5 nM for both ERK1 and ERK2 in biochemical assays, making it a highly selective inhibitor of these two enzymes. Temuterkib effectively inhibits cellular phospho-RSK1 in cancer cell lines containing the BRAF and RAS mutations. Tumor cells with MAPK pathway alterations, such as BRAF, NRAS, or KRAS mutations, are typically sensitive to Temuterkib in an unbiased tumor cell panel sensitivity profiling for inhibition of cell proliferation[1].

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LY3214996 is a highly selective inhibitor of ERK1 and ERK2, with IC50 of 5 nM for both enzymes in biochemical assays. It potently inhibits cellular phospho-RSK1 in BRAF and RAS mutant cancer cell lines. In an unbiased tumor cell panel sensitivity profiling for inhibition of cell proliferation, tumor cells with MAPK pathway alterations including BRAF, NRAS or KRAS mutation are generally sensitivity to LY3214996. [1]

Temuterkib inhibits the phospho-p90RSK1 PD biomarker in tumors in tumor xenograft models, and the PD effects are correlated with compound exposures and anti-tumor activities. Comparing Temuterkib to other ERK inhibitors that have been published, it exhibits either comparable or superior anti-tumor activity in BRAF or RAS mutant cell lines and xenograft models. In BRAF or NRAS mutant melanoma, BRAF or KRAS mutant colorectal, lung, and pancreatic cancer xenografts or PDX models, oral administration of single-agent Temuterkib significantly inhibits tumor growth in vivo and is well tolerated. Temuterkib can therefore be modified for the treatment of cancers with altered MAPK pathways. Temuterkib also exhibits anti-tumor activity in a PLX4032-resistant A375 melanoma xenograft model, suggesting that it may be useful in treating melanoma patients who have received ineffective BRAF therapies. More significantly, Temuterkib can be used in preclinical models, particularly KRAS mutant models, in combination with investigational and approved agents. Temuterkib and the CDK4/6 inhibitor abemaciclib, when used in combination, are well tolerated and effectively inhibit tumor growth or cause it to shrink in a variety of in vivo cancer models, including KRAS mutant colorectal and non-small cell lung cancers[1].

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LY3214996 demonstrates potent in vivo antitumor activity in BRAF-, KRAS-, NRAS-, and MEK-mutant models as a single agent [2]
The in vivo efficacy of LY3214996 and MEK inhibitors was assessed in subcutaneous xenograft models derived from several colorectal cancer (KRAS-mutant HCT116, BRAF-mutant Colo205, and MEK1-mutant SW48), melanoma (NRAS-mutant SK-MEL-30), pancreatic cancer (KRAS-mutant MiaPaCa-2), and NSCLC (KRAS-mutant Calu6) models as representative examples of RAS/ERK pathway alterations. MEK inhibitors were dosed at predicted clinical efficacious doses in mice. LY3214996 treatment resulted in significant tumor regression of HCT116 (31%), Colo205 (76%), MiaPaCa-2 (66%), and Calu-6 (54%) xenograft tumors (Fig. 5A, B, E, and F; Supplementary Table S3). LY3214996 treatment also resulted in significant growth inhibition in SW48 colorectal cancer (%dT/C  =  11) and SK-MEL-30 melanoma (%dT/C  =  1) xenograft models (Fig. 5C and D; Supplementary Table S3). All single-agent treatments were well tolerated as represented in the HCT116 study (Supplementary Figs. S4 and S5). Taken together, our data suggest that LY3214996 has potent efficacy in xenograft models with various ERK pathway alterations including mutations of BRAF, MEK1, NRAS, or KRAS (Supplementary Table S3). Notably, the efficacy is similar compared with MEK inhibitor which reinforces the point that constant pRSK1 suppression (>50%) may not be required, especially in the most responsive tumor types. These genetic alterations are key biomarkers for patient selection and precision medicine of LY3214996 in clinical development.

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LY3214996 shows durable response in an A375 melanoma parent model and potent antitumor activity in an A375 model resistant to vemurafenib or colorectal cancer PDX model with intrinsic resistance to vemurafenib [2]
To further test if LY3214996 can overcome BRAF inhibitor resistance, we generated an in vivo acquired resistance model to vemurafenib using A375 melanoma cells. In the parental A375 xenograft model, LY3214996 (100 mpk qd) showed significant tumor regression resulting in four of six complete responses and complete cure as those animals were tumor free for 115 days after 21 days of treatment (Fig. 6A). We used the same model to generate an in vivo acquired resistance to vemurafenib model by administering vemurafenib (15 mpk b.i.d.) over time. Acquired resistance was first demonstrated after 45 days (Supplementary Fig. S6). Tumor fragments from those resistant tumors were implanted for the LY3214996 efficacy study shown in Fig. 6B. LY3214996 dosed at 50 mpk b.i.d. showed 95% tumor growth inhibition (%dT/C  =  5), whereas the vehicle control grew in the presence of vemurafenib (15 mpk b.i.d.; Fig. 6B). These results suggest that LY3214996 can overcome acquired resistance to vemurafenib in BRAF V600E–mutant melanoma.[2]
Efficacy of LY3214996 was also tested in PDX models that maintain morphologic similarities and recapitulate molecular profiling of the original tumors. In a BRAF V600E–mutant colorectal cancer PDX model CTG-0652, which is intrinsically resistant to vemurafenib, LY3214996 treatment showed 83% tumor growth inhibition (%dT/C  =  17; Fig. 6C). Overall, our data suggest that LY3214996 has single-agent activity in BRAF inhibitor–resistant melanoma and colorectal cancer models.

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LY3214996 demonstrates enhanced efficacy in combination with pan-RAF inhibitor LY3009120 in the HCT116 colorectal cancer xenograft model [2]
Inhibition of multiple targets in the ERK pathway has been used to enhance therapeutic response in melanoma. Using this paradigm, we have explored combination of LY3214996 with pan-RAF inhibitor LY3009120 in a KRAS-mutant HCT116 colorectal cancer xenograft model. LY3214996 alone, LY3009120 alone, and the combination of both resulted in 52%, 29%, and 94% tumor growth inhibition, respectively, suggesting synergistic effect for the combination (P < 0.001; Fig. 6D and E). All tested doses were well tolerated as indicated by body weight measurements in the study.

Biochemical assay and Ki determination [2]
Human ERK1 and EKR2 kinase assays were performed in vitro using LanthaScreen® TR-FRET assay reagents. All reactions were imitated by adding ERK1 or ERK2 enzyme (at a final concentration of 3.6 nM or 1.7 nM respectively), prepared in kinase buffer (50 mM HEPES pH 7.4, 10 µM ATP, 5 mM MgCl2, 200 nM GFP-ATF2 (19-96), 0.1 mM EGTA, 0.01% TritonTM X-100, and 1 mM DTT) and increasing concentrations of LY3214996 in DMSO solution (final 4%, v/v) in a 384-well Proxi plate. The reactions were incubated at room temperature for 60 minutes. Then, reactions were stopped by the addition of stop buffer containing 10 mM EDTA and 2 nM Tb-(Terbium)-anti-pATF2 (pThr71) antibody, in TR-FRET dilution buffer. The plates were then incubated at room temperature for an additional 60 minutes and read on an Envision plate reader at an excitation wavelength of 340 nm. A TR-FRET ratio was calculated by dividing the GFP acceptor emission signal (at 520 nm) by the Tb donor emission signal (at 495 nM). IC50 values were determined from a concentration-response curve, which was used to calculate apparent Ki as described by Cheng and Prusoff. For kinase selectivity, LY3214996 was tested at three concentrations (20, 2, and 0.2 µM) in each of 456 kinase targets in the KINOMEscan® platform. For each target, a three-point IC50 was calculated using the LY3214996 dose concentrations and assay-derived percent control data.

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LY3214996 demonstrated an optimal balance of potency (hERK1 IC50 5 nM, hERK2 IC50 5nM, pRSK IC50 0.43 µM) and solubility.

Tumor cells with MAPK pathway alterations, including BRAF, NRAS, or KRAS mutations, are generally sensitive to LY3214996 in an unbiased tumor cell panel sensitivity profiling for inhibition of cell proliferation.

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Western blotting [2]
Cells were treated with indicated concentrations of LY3214996 for indicated time points in 10 cm dishes, and whole-cell lysates were prepared in RIPA Lysis Buffer (Millipore) supplemented with PMSF and Halt Phosphatase and Protease Inhibitor Cocktail at a final concentration of 5% for each reagent in buffer. Protein concentrations were determined via BCA assay following the manufacturer's guidance and subjected to SDS-PAGE and Western blotting with primary antibodies pCRAF, CRAF, pMEK1/2, MEK1/2, pERK1/2, ERK1/2, EGR1, c-MYC, DUSP4, pRSK1, and SPRY4 and Beta-Actin. Secondary antibodies used were Alexa Fluor 680 goat anti-rabbit, and goat anti-mouse and donkey anti-rabbit (LI-COR). Blots were read using the LI-COR Odyssey Classic Infrared Imaging System at 700 and 800 nm. Images were processed and analyzed using Image Studio version 3.1.

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Cell proliferation assay[2]
Sixty human lung, colorectal, pancreatic, and skin cancer cell lines were obtained from the ATCC. The cells were maintained in RPMI 1640 or DMEM supplemented with 10% FBS, sodium pyruvate, nonessential amino acids, l-glutamine, and penicillin–streptomycin (Invitrogen). All cultures were maintained in a humidified incubator at 37°C under 5% CO2/95% air free of mycoplasma and pathogenic murine viruses. The cells were used for experiments at passages < 7 after recovery from frozen stocks. Cells (3,000/well) were plated in 96-well black plates and cultured in the RPMI 1640 or DMEM with 10% FBS for 24 hours. The cells were treated with DMSO or LY3214996 at nine final dilutions from a 10 mmol/L stock solution (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol/L) in medium with 0.1% DMSO and 5% FBS or 10% FBS (melanoma cells) for 120 hours. Cell viability was measured by CellTiter-Glo luminescent cell viability assay. Data were presented as absolute IC50 (Abs IC50) and analyzed using XLfit (IDBS).

Briefly, 5 × 106 (HCT116, Colo205, SK-MEL-30, Calu6, MiaPaCa-2) or 10 × 10~6 (SW48) tumor cells in a 1:1 matrigel mix (0.2 mL total volume) were injected s.c. in to the right hind flank of 8- to 12-week-old (22–25 g) female athymic nude mice. For CTG-0652 patient-derived xenograft (PDX) studies, tumor fragments were used for implantation. After tumors reached 200 to 300 mm3, animals were randomized into groups of 5 or 6. All drugs were administered orally (gavage) in 0.2  mL volume of vehicle. Tumor growth and body weight were monitored over time to evaluate efficacy and signs of toxicity (24). The combination study of LY3214996 and LY3009120 in the HCT116 colorectal cancer xenograft model was performed in female NIH nude rats. Statistical analyses are described in the Supplementary Methods.[2]
For pharmacodynamic (PD) studies in HCT116 colorectal cancer xenograft model, animals were randomized into groups of 5 and dosed orally with LY3214996 in hydroxyethylcellulose 1% (w/v)/P80 0.25% (v/v)/antifoam 1510-US 0.05% (v/v), trametinib in 20% captisol in 25 mmol/L phosphate buffer, pH 2.0, or cobimetinib in 1% HEC/0.25% tween-80/0.05% antifoam. Tumors and blood samples were collected after dose at indicated time points. Tumors were flash frozen and stored at -80°C until tumor lysates were processed for pRSK1 levels by ELISA method as described in the Supplementary Methods. TEC50 and TED50 were calculated using Excel and XLfit. Blood was collected in EDTA-coated tubes, spun down to isolate plasma, and frozen at −80°C in a 96-well plate. Drug concentrations were measured by LC/MS-MS.[2]

For pharmacodynamic (PD) studies in HCT116 colorectal cancer xenograft model, animals were randomized into groups of 5 and dosed orally with LY3214996 in hydroxyethylcellulose 1% (w/v)/P80 0.25% (v/v)/antifoam 1510-US 0.05% (v/v) [2]

Vemurafenib-resistant A375 melanoma xenograft model and multiple in vivo cancer models, including KRAS mutant colorectal and non-small cell lung cancers

LY3214996 demonstrates good PK/PD correlation in tumors that correspond to potent tumor growth inhibition [2]
The kinetics of drug–target interactions can be quantified to predict in vivo PD and antitumor activity. A detailed PK/PD (pRSK1 inhibition) relationship of LY3214996 was generated in a KRAS-mutant HCT116 colorectal cancer xenograft model. After administration of a single dose of LY3214996 (6.25, 12.5, 25, 50, and 100 mpk) for dose–response study in nude mice bearing HCT116 xenografts, tumors were harvested at 4 hours after dosing, and pRSK1 was measured by sandwich ELISA (Fig. 4A).The PD effects correlated well with drug levels in the plasma (Fig. 4A). LY3214996 treatment showed dose-dependent increase in plasma drug exposure and inhibition of pRSK1 in tumors. LY3214996 was also evaluated at two different efficacious dose levels (50 and 100  mpk qd) for time-dependent plasma drug exposure and pRSK1 inhibition in the HCT116 colorectal cancer xenograft model. PD effects (pRSK1 inhibition) correlated well with PK (drug levels) in the plasma (Fig. 4B and C). After fitting to a four-parameter sigmoidal logistical model using XL fit, estimated TEC50 and TED50 (4 hours) values were 1,107 nmol/L and 16 mpk respectively. Our data suggest good PK and PD correlation of LY3214996 in a KRAS-mutant HCT116 colorectal cancer xenograft model.

[1]. Abstract 4973: Discovery of LY3214996, a selective and novel ERK1/2 inhibitor with potent antitumor activities in cancer models with MAPK pathway alterations. Cancer Res (2017) 77 (13_Supplement): 4973. https://doi.org/10.1158/1538-7445.AM2017-4973.

[2]. ERK Inhibitor LY3214996 Targets ERK Pathway–Driven Cancers: A Therapeutic Approach Toward Precision Medicine. Mol Cancer Ther. 2020 Feb;19(2):325-336

Temuterkib is an orally available inhibitor of extracellular signal-regulated kinase (ERK) 1 and 2, with potential antineoplastic activity. Upon oral administration, temuterkib inhibits both ERK 1 and 2, thereby preventing the activation of mitogen-activated protein kinase (MAPK)/ERK-mediated signal transduction pathways. This results in the inhibition of ERK-dependent tumor cell proliferation and survival. The MAPK/ERK pathway is often upregulated in a variety of tumor cell types and plays a key role in tumor cell proliferation, differentiation and survival.

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The RAS/MAPK pathway is dysregulated in approximately 30% of human cancers, and the extracellular-signal-regulated kinases (ERK1 and ERK2) serves as key central nodes within this pathway. The feasibility and clinical impact of targeting the RAS/MAPK pathway has been demonstrated by the therapeutic success of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma. However, resistance develops frequently through reactivation of the pathway. Therefore, simultaneous targeting of multiple effectors such as RAF, MEK and ERK in this pathway, offers a potential for enhanced efficacy while delaying and overcoming resistance. LY3214996 is a highly selective inhibitor of ERK1 and ERK2, with IC50 of 5 nM for both enzymes in biochemical assays. It potently inhibits cellular phospho-RSK1 in BRAF and RAS mutant cancer cell lines. In an unbiased tumor cell panel sensitivity profiling for inhibition of cell proliferation, tumor cells with MAPK pathway alterations including BRAF, NRAS or KRAS mutation are generally sensitivity to LY3214996. In tumor xenograft models, LY3214996 inhibits PD biomarker phospho-p90RSK1 in tumors and the PD effects are correlated with compound exposures and anti-tumor activities. LY3214996 shows either similar or superior anti-tumor activity as compared to other published ERK inhibitors in BRAF or RAS mutant cell lines and xenograft models. Oral administration of single-agent LY3214996 significantly inhibits tumor growth in vivo and is well tolerated in BRAF or NRAS mutant melanoma, BRAF or KRAS mutant colorectal, lung and pancreatic cancer xenografts or PDX models. Therefore, LY3214996 can be tailored for treatment of cancers with MAPK pathway alteration. In addition, LY3214996 has anti-tumor activity in a vemurafenib-resistant A375 melanoma xenograft model due to MAPK reactivation, may have potential for treatment of melanoma patients who have failed BRAF therapies. More importantly, LY3214996 can be combined with investigational and approved agents in preclinical models, particularly KRAS mutant models. Combination treatment of LY3214996 and CDK4/6 inhibitor abemaciclib was well tolerated and results in potent tumor growth inhibition or regression in multiple in vivo cancer models, including KRAS mutant colorectal and non-small cell lung cancers. Here, we first report the preclinical characterization of LY3214996, a novel small molecule ERK1/2 inhibitor currently in Phase I clinical trials in patients with advanced and metastatic cancers (NCT02857270).[1]

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The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).[2]

C22H27N7O2S

453.56

453.194

C, 58.26; H, 6.00; N, 21.62; O, 7.05; S, 7.07

1951483-29-6

1951483-29-6;2365171-00-0 (mesylate);

121408882

White to yellow solid powder

1.4±0.1 g/cm3

711.5±70.0 °C at 760 mmHg

384.1±35.7 °C

0.0±2.3 mmHg at 25°C

1.723

1.36

1

8

6

32

677

0

S1C(C2C([H])=C([H])N=C(N([H])C3=C([H])C([H])=NN3C([H])([H])[H])N=2)=C([H])C2C(N(C([H])([H])C([H])([H])N3C([H])([H])C([H])([H])OC([H])([H])C3([H])[H])C(C([H])([H])[H])(C([H])([H])[H])C1=2)=O

JNPRPMBJODOFEC-UHFFFAOYSA-N

InChI=1S/C22H27N7O2S/c1-22(2)19-15(20(30)29(22)9-8-28-10-12-31-13-11-28)14-17(32-19)16-4-6-23-21(25-16)26-18-5-7-24-27(18)3/h4-7,14H,8-13H2,1-3H3,(H,23,25,26)

6,6-dimethyl-2-[2-[(2-methylpyrazol-3-yl)amino]pyrimidin-4-yl]-5-(2-morpholin-4-ylethyl)thieno[2,3-c]pyrrol-4-one

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)

Solubility in Formulation 1: ≥ 2 mg/mL (4.41 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 20.0 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.

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Solubility in Formulation 2: ≥ 2 mg/mL (4.41 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 20.0 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 3: ≥ 2 mg/mL (4.41 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 20.0 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.)