Molecule

Description

GW788388 is a potent and selective inhibitor of ALK5 with IC50 of 18 nM.

Targets

IC₅₀

In Vitro

GW788388 shows anti-TGF-β activity with IC50 of 93 nM in cellular assay. ^[1] GW788388 shows some inhibitory to activin type II receptor (ActRII) but no inhibitory to bone morphogenic protein (BMP) type II receptor. GW788388 shows no toxicity in Namru murine mammary gland (NMuMG), MDA-MB-231, renal cell carcinoma (RCC)4, and U2OS cells at 4 nM to 15 μM. GW788388 blocks TGF-β-induced Smad activation and target gene expression, while decreasing epithelial–mesenchymal transitions and fibrogenesis. GW788388 inhibits ALK5, ALK4, ALK7 and TGF-β-mediated growth arrest. ^[2]

In Vivo

GW788388 exhibits an adequate pharmacokinetic profile in rats (plasma clearance less than 40 mL/min/kg and half-life more than 2 hours). GW788388 significantly reduces the expression of collagen IA1 mRNA by 80% in a model of puromycin aminonucleoside-induced renal fibrosis at 10 mg/kg. ^[1] GW788388 attenuates TGF-β signalling and effectively reduces hallmarks of fibrogenesis in mice suffering from late-stage diabetic nephropathy at 2 mg/kg. ^[2] Treatment with GW788388 significantly attenuates systolic dysfunction in the myocardial infarction (MI) animals, together with the attenuation of the activated (phosphorylated) Smad2, α-smooth muscle actin, and collagen I in the noninfarct zone of MI rats. Cardiomyocyte hypertrophy in MI hearts is also attenuated by GW788388 inhibition. ^[3] GW788388 reduces the fibrotic response in bleomycin-injected animals at 2 mg/kg. ^[4]

Clinical Trials

Features

ALK5 Fluorescence Polarization Binding Assay

GW788388 binding to ALK5 is tested on purified recombinant GST?ALK5 (residues 198-503). Displacement of rhodamine green fluorescently labeled ATP competitive inhibitor by different concentrations of GW788388 is used to calculate a binding pIC50. GST?ALK5 is added to a buffer containing 62.5 mM N-(2-hydroxyethyl)piperazine-N‘-2-ethanesulfonic acid (Hepes), pH 7.5, 1 mM dithiothreitol (DTT), 12.5 mM MgCl₂, 1.25 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and 1 nM rhodamine green-labeled ligand so that the final ALK5 concentration is 10 nM based on active-site titration of the enzyme. The enzyme/ligand reagent (40 μL) is added to 384-well assay plates containing 1 μL of different concentrations of GW788388. The plates are read immediately on a LJL Acquest fluorescence reader with excitation, emission, and dichroic filters of 485, 530, and 505 nm, respectively. The fluorescence polarization for each well is calculated by the Acquest and is then imported into curve-fitting software for construction of concentration?response curves.

Cellular Assays To Measure Anti-TGF-β Activity of ALK5 Inhibitors

Activity of GW788388 is tested in a transcriptional assay in HepG2 cells. Cells are stably transfected with a PAI-1 promoter driving a luciferase (firefly) reporter gene. The stably transfected cells responded to TGF-β stimulation by a 10-20-fold increase in luciferase activity compares to control conditions. To test anti-TGF-β activity of GW788388, cells are seeded in 96-well microplates at a concentration of 3.5 × 10⁴ cells/well in 200 μL of serum-containing medium. Microplates are then placed for 24 hours in a cell incubator at 37 °C in a 5% CO₂ atmosphere. GW788388 dissolved in dimethyl sulfoxide (DMSO) is then added at concentrations of 50 nM?10 μM (final concentration of DMSO 1%) for 30 minutes prior to addition of recombinant TGF-β (1 ng/mL). After an overnight incubation, cells are washed with phosphate-buffered saline (PBS) and lysed by addition of 10 μL of passive lysis buffer. Inhibition of luciferase activity relative to control groups is used as a measure of GW788388 activity. A concentration?response curve is constructed, from which IC50 is determined graphically.

Cell Lines

Namru murine mammary gland (NMuMG), MDA-MB-231, renal cell carcinoma (RCC)4, and U2OS cells

Concentrations

4 nM - 15 μM

Incubation Time

72 hours

Methods

Cell viability/proliferation assays are done according to the manufactures instructions (CellTiter 96 Aqueous One Solution Cell Proliferation Assay). Viability and proliferation are measured after 72 hours GW788388 treatment in the presence or absence of TGF-β.

Animal Models

Sprague–Dawley rats with dimethylnitrosamine- (DMN-) induced liver disease or puromycin aminonucleoside-induced renal fibrosis

Formulation

4% DMSO and 96% [0.5% HPMC/5% Tween/20%HCl (1 M) in NaH₂PO₄ (0.1 M)

Doses

3 or 10 mg/kg

Administration

Oral gavage