Molecule

Description

Cancer

Description

PF-04217903 is a selective ATP-competitive c-Met inhibitor with IC50 of 4.8 nM.

Targets

IC₅₀

In Vitro

Being more selective than staurosporine or PF-02341066, PF-04217903 displays >1000-fold selectivity for c-Met over a panel of 208 kinases, although more susceptible to oncogenic mutations of c-Met that attenuate potency than PF-02341066. In addition to WT c-Met, PF-04217903 displays similar potency to inhibit the activity of c-Met-H1094R, c-Met-R988C, and c-Met-T1010I with IC50 of 3.1 nM, 6.4 nM, and 6.7 nM, respectively, but has no inhibitory activity against c-Met-Y1230C with IC50 of >10 μM. ^[1] PF-04217903 in combination with sunitinib significantly inhibits endothelial cells, but not the tumor cells B16F1, Tib6, EL4, and LLC ^[2] PF-04217903 significantly inhibits the clonogenic growth of LXFA 526L and LXFA 1647L with IC50 values of 16 nM, and 13 nM, respectively, yielding an additive effect when in combination with cetuximab. ^[3] PF-04217903 potently inhibits c-Met-driven processes such as cell growth, motility, invasion, and morphology of a variety of tumor cells. PF-04217903 treatment (2 μM) increased cell death of GTL-16 cells, which involves the downregulation of phosphorylated 4E-BP1, ERK/MAPK associated proteins and PI3K/AKT pathway. ^[4]

In Vivo

Although unable to inhibit tumor growth in the sunitinib-sensitive B16F1 and Tib6 tumor models, the combination of PF-04217903 and sunitinib significantly inhibits tumor growth in sunitinib-resistant EL4, and LLC tumor models compared with sunitinib or PF-04217903 alone by significantly blocking vascular expansion, indicating a functional role for HGF/c-Met axis in the sunitinib-resistant tumors. ^[2]

Clinical Trials

A Phase I study to evaluate the effect of PF-04217903 in patients with advanced cancer has been terminated.

Features

Cellular c-Met phosphorylation ELISA

A549 cells with endogenous human WT c-Met are seeded in 96-well plates in growth medium and cultured overnight. On the second day of the assay, the growth medium is replaced with serum-free medium (with 0.04% BSA). Serial dilutions of PF-04217903 are added to each well, and cells are incubated at 37 °C for 1 hour. Then 40 ng/mL HGF is added to the cells for 20 minutes. The cells are washed once with HBSS supplemented with 1 mM Na₃VO₄, and protein lysates are generated from cells using lysis buffer. Phosphorylation of c-Met is assessed by an ELISA method utilizing capture antibodies specific for c-Met and a detection antibody specific for phosphorylated tyrosine residues. Antibody-coated plates are incubated in the presence of protein lysates at 4 °C overnight and washed with 1% Tween 20 in PBS seven times. HRP-PY20 (horseradish peroxidase-conjugated anti-phosphotyrosine) is diluted 1:500 in blocking buffer and added to each plate for 30 minutes. Plates are then washed again, and TMB peroxidase substrate is added to initiate the HRP-dependent colorimetric reaction and the reaction stopped by addition of 0.09 N H₂SO₄. ELISA end points are the absorbance measured at 450 nm using a spectrophotometer. IC50 value is calculated by concentration-response curve fitting utilizing a Microsoft Excel-based four-parameter analytical method.

Cell Lines

B16F1, Tib6, EL4, and LLC, HUVECs and C166 cells

Concentrations

Dissolved in DMSO, final concentrations ~2 μM

Incubation Time

4 days

Methods

Cells are treated with different concentrations PF-04217903 for 4 days. Cell proliferation is assessed by counting content of each well using a Coulter counter machine.

Animal Models

Immunodeficient nude mice (nu/nu) subcutaneously implanted with tumor cell lines B16F1, EL4, LLC, or Tib6

Formulation

Dissolved in DMSO, and diluted in saline

Doses

45 mg/kg

Administration

Orally