TGF-β receptor type 1/ALK5 (IC50 = 38.2 nM)

LY3200882 potently suppresses TGFβ induced SMAD phosphorylation in vitro in tumor and immunological cells in a dosage dependent fashion[1]. LY3200882 exhibits potent anti-tumor action in the orthotopic 4T1-LP model of triple negative breast cancer and this activity correlates with augmented tumor infiltrating lymphocytes in the tumor microenvironment[1]. In in vitro immune suppression experiments, LY3200882 has showed the ability to rescue TGFβ1 suppressed or T regulatory cell suppressed naive T cell activity and restore proliferation[1]. LY3200882 decreases NIH3T3 cell viability with an IC50 of 82.9 nM[2].

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Mechanism of action studies reveal revealed that LY3200882 inhibits various pro-tumorigenic activities. LY3200882 potently inhibits TGFβ mediated SMAD phosphorylation in vitro in tumor and immune cells. In in vitro immune suppression assays, LY3200882 has shown the ability to rescue TGFβ1 suppressed or T regulatory cell suppressed naïve T cell activity and restore proliferation. Therefore, LY3200882 shows promising activity as an immune modulatory agent. In addition, LY3200882 has shown anti-metastatic activity in vitro in migration assays as well as in vivo in an experimental metastasis tumor model (intravenous EMT6-LM2 model of triple negative breast cancer).[1]

Treatment with LY3200882 (60 mg/kg; oral gavage; twice daily; 21 days; female BALB/C mice) dramatically slows the formation of tumors in the CT26 model[2]. In vivo, LY3200882 significantly and dose-dependently suppresses TGFβ-mediated SMAD phosphorylation in subcutaneous tumors[1]. In an experimental metastasis tumor model (intravenous EMT6-LM2 model of triple negative breast cancer), LY3200882 has demonstrated anti-metastatic efficacy in vivo[1].

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In preclinical tumor models, LY3200882 showed potent anti-tumor activity in the orthotopic 4T1-LP model of triple negative breast cancer and this activity correlated with enhanced tumor infiltrating lymphocytes in the tumor microenvironment. Durable tumor regressions in the orthotopic 4T1-LP model were observed and rechallenge of congenic tumors resulted in complete rejection in all mice.Finally, LY3200882 shows combinatorial anti-tumor benefits with checkpoint inhibition (anti-PD-L1) in the syngeneic CT26 model.[1]

ALK5 inhibitory activity[2]
A luminescent ADP detection assay was used to assess the ALK5 binding capacity of compounds. Serially dilute the stock solution of 10 mM 3-fold in DMSO to obtain a ten-point dilution curve with final compound concentrations ranging from 3.333 μM to 0.5 nM. Assay measurements were performed in 1X kinase reaction buffer containing 40 mM Tris pH 7.5, 20 mM MgCl2, 0.1% BSA, 1 mM DTT in a final assay volume of 5 μL. Briefly 2.5 μL of ALK5 protein, final concentration of 3 μg/mL, was added to each well of a 384 well assay plate containing 100 nL of each concentration of test compound dissolved in DMSO. 2.5 μL of TGF-βR1 peptide, final concentration 3 μg/mL, and ATP, final concentration 1 mM. Following incubation for 120 min at 28 °C, add 5 μL ADP-Glo™ Reagent to terminate the kinase reaction and deplete the remaining ATP. After incubation for 120 min at 28 °C, add 10 μL of Kinase Detection Reagent to convert ADP to ATP and record luminescence.

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In vitro p38α enzymatic activity assay[2]
All of the enzymatic reactions were conducted at 28 °C for 40 min. The 25 μL reaction mixture contains 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 25 ng kinase, 10 μM ATP, and the FAM-labled peptide. The compounds were tested from 100 μM, 3-fold dilution, 10 concentration. The assay was performed by ChemPartner. It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The IC50 values were calculated in XLFit excel add-in version 5.4.0.8 to obtain IC50 values using the equation: Y=Bottom + (Top-Bottom)/(1+(IC50/X) ^Hill Slope) where X is Compound Concentration and Y is Inhibition Rate(%).

Cell-based luciferase reporter assay for TGF-β type 1 receptor activity[2]
The aim of this experiment is to identify compounds which interfere with SMAD 2,3-dependent gene expression selectively in cell-based assays demonstrating that they inhibit ALK5 at cellular level. Plate the Luc-Smad 2/3-NIH3T3 cells from assay-ready frozen stocks at 4000 cells per well in 96-well plates in DMEM medium. After overnight attachment of the cells, media was changed to 2% FBS. Prepare test compounds in DMSO to make 4 mM stock solutions. Serially dilute the stock solutions 4-fold in DMSO to obtain an eight-point dilution curve with final compound concentrations ranging from 20 μM to 1.22 nM and test compounds are added. After 24 h, add Glo Lysis Buffer and Bright-Glo Luciferase assay system to each well to double the well volume. Transfer aliquots (180 μL) to white solid bottom plates for reading luminescence on a plate reader (1 s read).

Animal/Disease Models: BALB/C female mice (5-8 weeks old) injected with CT26 cells[2]
Doses: 60 mg/kg
Route of Administration: po (oral gavage); twice a day; for 21 days
Experimental Results: A statistically significant tumor growth delay in CT26 model was observed.

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Pharmacokinetics procedures[2]
This study was conducted in BALB/C male subjects to investigate the pharmacokinetic profiles of test compounds. The mice were randomized and divided into two groups consisting of 3 mice/group. Prepare test compounds [15r (a LY3200882 analog)] in PEG200-EtOH-solutol-physiological saline (4:1:1:14) to make 0.5 mg/mL solutions for oral gavage and to make 0.1 mg/mL solutions for intravenous injection. Sample collection was performed as follows: 1) single oral administration (PO) group: 5 mg/kg, 0.2 mL/10 g; 2) single tail vein injection (IV) group: 1 mg/kg, 0.1 mL/10 g. Blood was collected from orbital venous plexus in heparinized EP tube at 5, 15, 30 min, 1, 2, 6, 10, 24 h after intravenous or oral administration, and the contents of the blood were analyzed by LC-MS/MS(API 4500).

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In vivo tumor xenograft model[2]
A well-established tumorigenesis assay was used to evaluate the antitumor effect of compound 15r (a LY3200882 analog) in BALB/C female mice model. All mice were housed under standard specific-pathogen-free (SPF) conditions and the animal experiments strictly complied with protocols approved by the Animal Welfare and Ethics Committee (AWEC). 1 × 106 cells/mouse of CT26 cells were injected subcutaneously into the 5-to-8-week-old BALB/C female mice. All compounds were administrated by oral gavage. Mice were examined thrice a week for the development of tumors by palpation, and tumor volumes calculated using formula V = 0.5 × length × width2. The investigators were not blinded to allocation during experiments and outcome assessment. Mice were randomly allocated to three groups consisting of 6 mice/group by an independent person in the laboratory. No statistical method was used to predetermine sample size. The antitumor effect of the compound was assessed by tumor growth inhibition (TGI) or relative tumor proliferation rate (T/C): TGI(%) = [1-(Vt1-Vt0)/(Vc1-Vc0)] × 100%, where Vc1 and Vt1 are the mean volumes of control and treated groups at time of tumor extraction, while Vc0 and Vt0 are the same groups at the start of dosages; T/C (%) = TRTV/CRTV × 100%, where TRTV is the relative tumor volume (RTV) of treated groups, while CRTV is the RTV of control groups. (RTV = Vt/V0, Vt is the mean volumes of treated groups at time of tumor extraction, V0 is the mean volumes of the same groups at the start of dosages).

[1]. Abstract 955: LY3200882, a novel, highly selective TGFβRI small molecule inhibitor. AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 955.

[2]. Synthesis and biological evaluation of 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole derivatives as novel potent transforming growth factor-β type 1 receptor inhibitors. Eur J Med Chem. 2020 Apr 29;198:112354.

LY-3200882 is under investigation in clinical trial NCT04158700 (A Study of LY3200882 and Pembrolizumab in Participants With Advanced Cancer).
TGFbeta Inhibitor LY3200882 is an orally bioavailable agent that targets transforming growth factor-beta (TGFb), with potential antineoplastic activity. Upon administration, LY3200882 specifically targets and binds to TGFb, which prevents both the binding of TGFb to its receptor TGFbR and TGFb-mediated signal transduction. This may lead to a reduction in TGFb-dependent proliferation of cancer cells. The TGFb signaling pathway is often deregulated in tumors, and plays a key role in the regulation of cell growth, differentiation, apoptosis, motility, invasion, angiogenesis, and various immune responses.

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The transforming growth factor β (TGFβ) signaling pathway is a pleiotropic cellular pathway that plays a critical role in cancer. In fact, aggressive tumors are typically associated with high ligand levels and thus associated with poor prognosis in various tumor types. Cancer cells use autocrine and paracrine TGFβ signaling to modulate tumor cells and the tumor microenvironment leading to a highly invasive and metastatic phenotype, inducing and increasing tumor vascularization, modulating the extracellular matrix in the stroma, and inhibiting immune surveillance and antitumor immunity. Clinical studies with galunisertib (aka LY2157299 monohydrate), a small molecule inhibitor targeting the TGFβ pathway, have provided proof of concept data supporting the role of TGFβ in cancer and the utility of targeting the TGFβ pathway. Here we describe the identification of LY3200882, a next generation small molecule inhibitor of TGF-β receptor type 1 (TGFβRI). The molecule is a potent, highly selective inhibitor of TGFβRI embodied in a structural platform with a synthetically scalable route. It is an ATP competitive inhibitor of the serine-threonine kinase domain of TGFβRI. In conclusion, we have developed a novel potent and highly selective small molecule inhibitor of TGFβRI for the treatment of cancer.[1]

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Inhibition of transforming growth factor β (TGF-β) type 1 receptor (ALK5) provides a feasible approach for the treatment of fibrotic diseases and malignant tumors. In this study, we designed and synthesized a new series of 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole derivatives, and evaluated biologically as TGF-β type 1 receptor inhibitors. The most potent compound 15r inhibited the ALK5 enzyme and NIH3T3 cell viability with IC50 values of 44 and 42.5 nM, respectively. Compound 15r also displayed better oral plasma exposure and excellent bioavailability than LY-3200882, and in vivo inhibited 65.7% of the tumor growth in a CT26 xenograft mouse model.[2]

C24H29N5O3

435.518765211105

435.227

C, 66.19; H, 6.71; N, 16.08; O, 11.02

1898283-02-7

121249291

Off-white to light yellow solid powder

2.1

2

7

7

32

612

0

O1CCC(CC1)C1C(=CN(C2CC2)N=1)OC1C=CN=C(C=1)NC1C=CN=C(C=1)C(C)(C)O

PNPFMWIDAKQFPY-UHFFFAOYSA-N

InChI=1S/C24H29N5O3/c1-24(2,30)21-13-17(5-9-25-21)27-22-14-19(6-10-26-22)32-20-15-29(18-3-4-18)28-23(20)16-7-11-31-12-8-16/h5-6,9-10,13-16,18,30H,3-4,7-8,11-12H2,1-2H3,(H,25,26,27)

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: ≥ 2.5 mg/mL (5.74 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 25.0 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: ≥ 2.5 mg/mL (5.74 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 3: ≥ 2.5 mg/mL (5.74 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.)