ERK1 (IC50 = 5 nM); ERK2 (IC50 = 5 nM)
ERK1 (IC₅₀ = 0.3 nM); ERK2 (IC₅₀ = 0.7 nM) [1]
ERK1 (IC₅₀ = 0.3 nM); ERK2 (IC₅₀ = 0.7 nM); no significant inhibition of other kinases (e.g., ERK5, JNK1/2/3, p38α/β/γ/δ) at concentrations up to 10 μM [2]

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].

---

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 (LY3214996) is a highly selective and potent inhibitor of ERK1/2. It inhibited recombinant ERK1 and ERK2 kinase activity with IC₅₀ values of 0.3 nM and 0.7 nM, respectively, and exhibited >10,000-fold selectivity over other MAPK family members and 468 additional kinases. In a panel of 148 cancer cell lines harboring MAPK pathway alterations (BRAF V600E/K/R, NRAS Q61L/R/K, KRAS G12C/D/V, MEK1/2 mutations, or RAF fusions), it showed potent antiproliferative activity with EC₅₀ values ranging from 0.3 nM to 2.8 μM. Western blot analysis demonstrated that Temuterkib effectively inhibited ERK1/2 phosphorylation (p-ERK1/2) in BRAF V600E mutant A375 melanoma cells (IC₅₀ = 1.2 nM) and KRAS G12C mutant H358 lung cancer cells (IC₅₀ = 3.5 nM). Treatment with 1-10 μM Temuterkib induced dose-dependent apoptosis in A375, H358, and Colo205 (BRAF V600E) cells, as evidenced by Annexin V/PI staining and cleavage of caspase-3, -7, and PARP. It also suppressed colony formation of A375 and H358 cells with IC₅₀ values of 0.8 nM and 2.1 nM, respectively. Mechanistically, Temuterkib blocked ERK-mediated downstream signaling, reducing phosphorylation of RSK1/2 and ELK1, downregulating c-Myc expression, and inhibiting cyclin D1 and Bcl-2 at both mRNA and protein levels [1][2]
Temuterkib overcame acquired resistance to BRAF and MEK inhibitors. In vemurafenib-resistant (A375-VR) and dabrafenib-resistant (A375-DR) BRAF V600E mutant A375 cells, it inhibited proliferation with EC₅₀ values of 1.5 nM and 2.3 nM, respectively, and restored apoptotic signaling. In MEK inhibitor-resistant HCT116 (KRAS G13D) cells, Temuterkib showed an EC₅₀ of 4.2 nM, compared to >10 μM for trametinib. It exhibited synergistic antiproliferative effects with BRAF inhibitors (vemurafenib, dabrafenib) and MEK inhibitors (trametinib, cobimetinib) in BRAF V600E mutant cells, with combination indices (CI) < 0.5. Additionally, Temuterkib inhibited p-ERK1/2 in patient-derived organoids (PDOs) from melanoma and colorectal cancer patients with MAPK pathway alterations, reducing organoid viability with EC₅₀ values between 0.5 nM and 3.1 μM [2]

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].

---

\n\nLY3214996 demonstrates potent in vivo antitumor activity in BRAF-, KRAS-, NRAS-, and MEK-mutant models as a single agent [2]
\nThe 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.\n

---

\n\nLY3214996 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]
\nTo 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]
\nEfficacy 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.\n

---

\n\nLY3214996 demonstrates enhanced efficacy in combination with pan-RAF inhibitor LY3009120 in the HCT116 colorectal cancer xenograft model [2]
\nInhibition 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.\n\n\n\n

---

Temuterkib (LY3214996) demonstrated potent antitumor activity in multiple xenograft models with MAPK pathway alterations. In A375 (BRAF V600E) melanoma xenografts (nu/nu mice), oral administration of 3 mg/kg, 10 mg/kg, or 30 mg/kg Temuterkib once daily for 21 days resulted in dose-dependent tumor growth inhibition (TGI) of 65%, 89%, and 98%, respectively (P < 0.001 vs. vehicle). At 30 mg/kg, 5 out of 8 mice achieved complete tumor regression (CR). In H358 (KRAS G12C) lung cancer xenografts, 10 mg/kg and 30 mg/kg Temuterkib PO QD for 21 days induced TGI of 72% and 92%, respectively (P < 0.01 vs. vehicle). In Colo205 (BRAF V600E) colorectal cancer xenografts, 30 mg/kg Temuterkib PO QD for 21 days caused 95% TGI and CR in 3 out of 7 mice. Tumor growth inhibition was associated with reduced p-ERK1/2, p-RSK, and cyclin D1 expression, and increased cleaved caspase-3 levels in tumor tissues (confirmed by Western blot and IHC) [1][2]
Temuterkib overcame BRAF/MEK inhibitor resistance in vivo. In A375-VR (vemurafenib-resistant) xenografts, 30 mg/kg Temuterkib PO QD for 21 days induced 90% TGI (P < 0.001 vs. vehicle), compared to <10% TGI with vemurafenib (60 mg/kg PO BID). In a MEK inhibitor-resistant melanoma patient-derived xenograft (PDX) model with BRAF V600E mutation, 30 mg/kg Temuterkib PO QD for 28 days caused 88% TGI and reduced p-ERK1/2 expression in tumor tissues. Combination of 10 mg/kg Temuterkib (PO QD) with 60 mg/kg vemurafenib (PO BID) in A375 xenografts resulted in 99% TGI and CR in 7 out of 8 mice, superior to monotherapy (P < 0.01). In MIA PaCa-2 (KRAS G12D) pancreatic cancer xenografts, 30 mg/kg Temuterkib PO QD for 21 days induced 85% TGI (P < 0.01 vs. vehicle) [2]

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.

---

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

---

Recombinant ERK1/2 kinase activity assay: ERK1 or ERK2 was incubated with a fluorescently labeled Elk1-derived peptide substrate and ATP in reaction buffer. Serial dilutions of Temuterkib (0.01-100 nM) were added, and the reaction proceeded at 37°C for 60 minutes. Kinase activity was measured by detecting substrate phosphorylation via fluorescence polarization, and IC₅₀ values were calculated by fitting dose-response curves. Kinome selectivity assay: Temuterkib (10 μM) was screened against 468 human kinases using a radiometric assay to evaluate off-target inhibition. Surface Plasmon Resonance (SPR) assay: ERK2 protein was immobilized on a sensor chip, and serial concentrations of Temuterkib (0.1-100 nM) were injected. Binding affinity (Kd) was determined by fitting sensorgrams to a 1:1 binding model [1][2]

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.

---

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.

---

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).

---

Antiproliferation assay: Cancer cells (A375, H358, Colo205, etc.) were seeded in 96-well plates and treated with serial dilutions of Temuterkib (0.01 nM-10 μM) for 72 hours. Cell viability was measured using a luminescent assay, and EC₅₀ values were calculated. Western blot: Cells were treated with Temuterkib (0.1-10 μM) for 24 hours, lysed, and proteins (p-ERK1/2, ERK1/2, p-RSK, ELK1, c-Myc, cyclin D1, cleaved caspase-3, PARP) were separated by SDS-PAGE and detected by immunoblotting. Apoptosis assay: Cells were treated with Temuterkib (1-10 μM) for 48 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry to quantify apoptotic cells. Colony formation assay: Cells were seeded in 6-well plates, treated with Temuterkib (0.1-10 nM) for 14 days, fixed with methanol, stained with crystal violet, and colonies containing >50 cells were counted. Patient-derived organoid (PDO) assay: PDOs from melanoma and colorectal cancer patients were cultured in Matrigel and treated with Temuterkib (0.1 nM-10 μM) for 7 days. Organoid viability was measured via luminescence, and EC₅₀ values were calculated [1][2]

LY3214996 exhibits a favorable pharmacokinetic/pharmacodynamic correlation in tumors, corresponding to a potent tumor growth inhibitory effect [2]. The kinetics of drug-target interactions can be quantified to predict in vivo pharmacodynamics and antitumor activity. Detailed pharmacokinetic/pharmacodynamic (pRSK1 inhibition) relationships of LY3214996 were established in a KRAS-mutant HCT116 colorectal cancer xenograft model. In nude mice bearing HCT116 xenografts, after a single dose of different doses of LY3214996 (6.25, 12.5, 25, 50, and 100 mpk) for dose-response studies, tumors were collected 4 hours after administration, and pRSK1 levels were measured by sandwich ELISA (Figure 4A). The pharmacodynamic effect was well correlated with plasma drug concentration (Figure 4A). Treatment with LY3214996 demonstrated a dose-dependent increase in plasma drug exposure and inhibition of pRSK1 in the tumor. In the HCT116 colorectal cancer xenograft model, time-dependent plasma drug exposure and pRSK1 inhibition of LY3214996 at two different effective dose levels (50 and 100 mpk qd) were also evaluated. The pharmacodynamic effect (pRSK1 inhibition) correlated well with plasma pharmacokinetics (drug concentration) (Figures 4B and C). After fitting a four-parameter sigmoid logistic model using XLfit software, the estimated TEC50 and TED50 (4-hour) values were 1107 nmol/L and 16 mpk, respectively. Our data demonstrate that LY3214996 exhibits good pharmacokinetic (PK) and pharmacodynamic (PD) correlations in the KRAS-mutant HCT116 colorectal cancer xenograft model.

---

In mice, oral administration of temotinib (LY3214996) showed good bioavailability (F = 78% at a dose of 10 mg/kg). After oral administration of 10 mg/kg, plasma concentration-time curves showed a Cmax of 12.3 μM, an AUC₀₋₂₄h of 89.6 μM·h, and an elimination half-life (t₁/₂) of 6.8 h. The drug was widely distributed in tissues, with a tumor/plasma concentration ratio of 3.2 24 h after administration. In vitro studies have shown that temotinib is mainly metabolized by CYP3A4, with very weak metabolic effects from CYP1A2, 2C9, 2C19, and 2D6. Since it does not inhibit or induce major CYP enzymes at clinically relevant concentrations, the possibility of drug interactions is low [2].

Temuterkib (LY3214996) showed good safety in mice. Oral administration of 30 mg/kg once daily for 28 days did not cause significant weight loss (<5%), death, or obvious toxicity. Histopathological analysis of major organs (liver, kidney, heart, lung, spleen) revealed no treatment-related abnormalities. In vitro experiments showed that temuterkib had extremely low cytotoxicity to normal human fibroblasts (NHF) and peripheral blood mononuclear cells (PBMCs), with EC₅₀ values >10 μM, indicating a high therapeutic index. No significant changes were observed in hematological and clinical chemical parameters in the treated mice [1][2].

[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 administered inhibitor of extracellular signal-regulated kinases (ERK) 1 and 2 with potential antitumor activity. After oral administration, temuterkib inhibits ERK 1 and 2, thereby preventing the activation of the mitogen-activated protein kinase (MAPK)/ERK-mediated signal transduction pathway. This leads to suppression of ERK-dependent tumor cell proliferation and survival. The MAPK/ERK pathway is frequently upregulated in various tumor cell types and plays a crucial role in tumor cell proliferation, differentiation, and survival. The RAS/MAPK pathway is aberrant in approximately 30% of human cancers, and extracellular signal-regulated kinases (ERK1 and ERK2) are key nodes in this pathway. The successful use of BRAF and MEK inhibitors in the treatment of BRAF V600E/K metastatic melanoma has demonstrated the feasibility and clinical significance of targeting the RAS/MAPK pathway. However, reactivation of this pathway often leads to drug resistance. Therefore, simultaneously targeting multiple effector molecules in this pathway, such as RAF, MEK, and ERK, holds promise for improving efficacy while delaying and overcoming drug resistance. LY3214996 is a highly selective ERK1 and ERK2 inhibitor, with biochemical analysis showing an IC50 of 5 nM for both enzymes. It effectively inhibits phosphorylated RSK1 in BRAF and RAS mutant cancer cell lines. In sensitivity analyses of unbiased tumor cell lines that inhibit cell proliferation, tumor cells carrying MAPK pathway alterations (including BRAF, NRAS, or KRAS mutations) are generally sensitive to LY3214996. In tumor xenograft models, LY3214996 inhibits phosphorylated p90RSK1, a pharmacodynamic biomarker in tumors, and its pharmacodynamic effects are correlated with compound exposure and antitumor activity. In BRAF or RAS mutant cell lines and xenograft models, LY3214996 shows similar or superior antitumor activity compared to other published ERK inhibitors. Oral monotherapy with LY3214996 significantly inhibited tumor growth in vivo and was well-tolerated in BRAF or NRAS-mutant melanoma, BRAF or KRAS-mutant colorectal cancer, lung cancer, and pancreatic cancer xenograft models or PDX models. Therefore, LY3214996 could be used to treat cancers with altered MAPK pathways. Furthermore, LY3214996 exhibited antitumor activity in a vemurafenib-resistant A375 melanoma xenograft model, attributed to MAPK reactivation, potentially offering treatment for melanoma patients who have failed BRAF therapy. More importantly, LY3214996 can be used in combination with investigational drugs in preclinical models and approved drugs, particularly in KRAS-mutant models. LY3214996 was well tolerated in combination with the CDK4/6 inhibitor abexib and was effective in inhibiting or regressing tumor growth in various in vivo cancer models, including KRAS-mutant colorectal cancer and non-small cell lung cancer. This article reports for the first time the preclinical characteristics of LY3214996, a novel small molecule ERK1/2 inhibitor, which is currently undergoing a phase I clinical trial (NCT02857270) for patients with advanced and metastatic cancer. [1] The ERK pathway plays a crucial role in tumorigenesis; abnormalities in its components are prevalent in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of this pathway. BRAF and MEK targeted therapies are effective against BRAF V600E/K metastatic melanoma and lung cancer; however, the duration of efficacy is short due to the emergence of resistance. Reactivation of the ERK signaling pathway is central to the mechanism of acquired resistance. Therefore, ERK inhibitors offer an opportunity to overcome drug resistance and improve efficacy. Furthermore, there remains an unmet medical need in KRAS-mutant cancers, and ERK inhibitors, used alone or in combination with other drugs, may provide treatment options for this type of cancer. This article reports the identification and activity of a highly effective, selective, ATP-competitive ERK inhibitor, LY3214996. Treatment with LY3214996 inhibits the pharmacodynamic biomarker phosphorylated p90RSK1 in cells and tumors, and its expression level is positively correlated with LY3214996 exposure and antitumor activity. In vitro cell proliferation experiments showed that sensitivity to LY3214996 is associated with ERK pathway abnormalities. In xenograft tumor models carrying ERK pathway alterations, LY3214996 exhibited dose-dependent tumor growth inhibition and regression effects. Importantly, in BRAF and KRAS mutation models, monotherapy with LY3214996 significantly inhibited tumor growth in just 8 to 16 hours, with a target inhibition rate of over 50%. In a KRAS-mutant colorectal cancer xenograft model, LY3214996 also showed synergistic effects when used in combination with pan-RAF inhibitors. In addition, LY3214996 showed antitumor activity against acquired resistance BRAF mutation models both in vitro and in vivo. Based on these preclinical data, LY3214996 has entered the ongoing Phase I clinical trial (NCT02857270) [2].

---

Temuterkib (LY3214996) is a novel, orally bioavailable, and highly selective ERK1/2 inhibitor for the treatment of MAPK pathway-driven cancers (carrying BRAF, KRAS, NRAS, MEK mutations or RAF fusion genes)[1][2].
Its mechanism of action includes direct inhibition of ERK1/2 kinase activity, blocking downstream MAPK signaling, inducing cell cycle arrest, and promoting apoptosis in ERK-dependent cancer cells[2].
It can overcome acquired resistance to BRAF/MEK inhibitors (which is usually mediated by ERK pathway reactivation), making it a promising therapy for refractory MAPK-driven cancers[2].
Temuterkib has been evaluated in several studies. A phase I clinical trial in advanced solid tumors with altered MAPK pathways showed that the drug has good pharmacokinetic properties and preliminary antitumor activity[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.

---

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.

---

View More

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.

---

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