Molecule

Description

Cancer

Description

SB 431542 is a potent and selective inhibitor of ALK5 with IC50 of 94 nM.

Targets

IC₅₀

In Vitro

SB 431542 inhibits the activin type I receptor ALK4 and the nodal type I receptor ALK7, which are responsible for the phosphorylation of Smad2. SB 431542 has little effect on ALK1, ALK2, ALK3, and ALK6, which show phosphorylation of Smad1. SB 431542 is a selective inhibitor of endogenous activin but has no apparent effect on BMP signaling. SB 431542 could induce both Smad2/Smad4- and Smad3/Smad4-dependent transcription. ^[2] In A498 cells, SB 431542 inhibits both TGF-β1–induced collagen Iα1 and PAI-1 mRNA with IC50 values of 60 and 50 nM, respectively. In addition, SB 431542 inhibits production of TGF-β1–induced fibronectin mRNA and protein with IC50 of 62 and 22 nM, respectively. ^[3] SB 431542 blocks the TGF-β–mediated growth factors, including PDGF-A, FGF-2 and HB-EGF, leading to an increase in proliferation of MG63 cells. SB 431542 also inhibits TGF-β–induced c-Myc and p21 ^WAF1/CIP1. ^[4] SB 431542 significantly suppresses TGF-β–induced G1 arrest, leading to accumulation of cells in the S phase of the cell cycle in FET, RIE, and Mv1Lu cells. SB 431542 also inhibits TGF-β-induced epithelial to mesenchymal transition (EMT) in NMuMG and PANC-1 cells. ^[5] SB 431542 significantly elevates the expression of CD86 in BM-DCs and that of CD83 within CD11c+ cells suppressed by TGF-β. SB 431542 is able to induce NK activity through functional maturation and IL-12 production of human DCs. ^[6]

In Vivo

SB 431542 triggers cytotoxic T lymphocyte (CTL) activities in the colon-26 carcinoma models and is most likely to produce antitumor immunological outcomes through alteration of DC function suppressed by TGF-β. ^[6]

Clinical Trials

Features

Flashplate assay for ALK5

SB 431542 is dissolved in DMSO at a concentration of 10 mM. The kinase domain of TGFβRI, from amino acid 200 to the C-terminus, and the full-length Smad3 protein are expressed as N-terminal glutathion S-transferase (GST) fusion proteins in the baculovirus expression system. Proteins are purified with glutathion Sepharose beads 4B. Basic FlashPlates are coated with 0.1 M sterile filtered sodium bicarbonate, pH 7.6, containing 700 ng of GST-Smad3 per 100 μL. Assay buffer contains 50 mM HEPES (pH 7.4), 5 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, 100 mM GTP, 3 μM ATP plus 0.5 μCi/well ?³³P-ATP, and 85 ng of GST-ALK5 with or without SB 431542. Plates are incubated at 30 °C for 3 hours. The assay buffer is removed by aspiration, and the plate is counted on a Packard TopCount 96-well scintillation plate reader.

Cell Lines

MG63 and NIH3T3

Concentrations

0.3 μM

Incubation Time

30 minutes

Methods

To explore the effects of ligands, MG63 and NIH3T3 cells are seeded at a density of 8 × 10⁴ cells/well in 6-well plates and starved (0.1% FCS for MG63 cells and 0.5% FCS for NIH3T3 cells) for 24 hours before ligand stimulation. Media containing various ligands are exchanged at 48-hours intervals. Cells are trypsinized and counted by a Coulter counter on days 2, 4, and 6 after ligand stimulation. To explore the effects of constitutively active receptors, cells are seeded at a density of 2 × 10⁵ cells/well in 6-well plates. The next day, cells are infected with adenoviruses carrying various cDNAs at a multiplicity of infection of 100. Cells are trypsinized and counted on day 3. Cell proliferation assay is performed in the presence of 0.3 μM SB 431542.

Animal Models

BALB/c mice receive intraperitoneal (i.p.) injections of colon-26 tumor cells.

Formulation

DMSO

Doses

1 μM solution, 100 μL/mouse

Administration

Directly injected into peritoneal cavity