Kinase experiment:

Multiwell plates are precoated with a synthetic polymer substrate poly-Glu-Tyr (polyGT 4:1). The reaction is carried out in the presence of kinase buffer (10×: 50 mM HEPES buffer, pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2, and 0.2 mM Na3VO4) supplemented with ATP and dimethyl sulfoxide (DMSO) for the positive control (C+) or SAR131675 (ranging from 3-1,000 nM). ATP is used at 30 μM for VEGFR-1 and VEGFR-3 and at 15 μM for VEGFR-2. The phosphorylated poly-GT is probed with a phosphotyrosine specific monoclonal antibody (mAb) conjugated to horseradish peroxidase and developed in the dark with the HRP chromogenic substrate (OPD). The reaction is then stopped by the addition of 100 μL 1.25 mol/L H2SO4, and absorbance is determined using an Envision spectrophotometer at 492 nm[1].

Cell experiment:

HLMVECs are seeded in 96-well plates coated with 0.3% gelatin (5000 cells per well). Cells are incubated in RPMI 0.1% FCS with VEGFA (10 ng/mL) VEGFC (300 ng/mL), VEGFD (300 ng/mL), or FGF2 (10 ng/mL) in the absence or presence of SAR131675. Five days later, viable cells are quantified with the cell Titer-glo luminescent cell viability assay[1].

Animal experiment:

Mouse: Sterile sponge disks impregnated with 200 μg of FGF2 or PBS are subcutaneously introduced on the back of anaesthetized mice. FGF2 is reinjected into the sponges the first 2 days. Daily oral treatment with SAR131675 (30, 100, and 300 mg/kg/d) started the day of sponge implantation. Seven days later, the animals are euthanatized and the sponges are removed, harvested, and lysed in RIPA buffer at 4℃. After a centrifugation at 6,000 × g, the supernatants are collected for further analysis[1].

SAR131675 is a selective inhibitor of VEGFR-3 with IC50 value of 20nM [1].

SAR131675 is an ATP-competitive inhibitor. It inhibits recombinant human VEGFR-3 with IC50 value of 23nM and inhibits the kinase activity of VEGFR-3 with Ki value of 12nM. SAR131675 is found to be cell permeable and inhibits VEGFR-3 autophosphorylation in HEK cells with IC50 values ranging from 30nM to 50nM. SAR131675 shows IC50 values of >3μM and 235nM against VEGFR-1 and VEGFR-2, respectively. It also havs no inhibitory effect on a panel of 65 kinases, 107 nonkinase enzymes and receptors and on 21 ion channels. Moreover, SAR131675 inhibits lymphatic cell survival induced by VEGFC and VEGFD with IC50 values of 14nM and 17nM. On another hand, SAR131675 exerts inhibition of the migration induced by VEGFA or VEGFC with IC50 values of 100nM and

In vivo, SAR131675 abrogates lymphangiogenesis and angiogenesis induced by FGF2. It also shows antitumor activity in the mice models. Treatment with SAR131675 significantly reduces the tumor volume of 4T1 mammary carcinoma tumors [1]

References:
[1] Alam A, Blanc I, Gueguen-Dorbes G, et al. SAR131675, a Potent and Selective VEGFR-3–TK Inhibitor with Antilymphangiogenic, Antitumoral, and Antimetastatic Activities. Molecular cancer therapeutics, 2012, 11(8): 1637-1649.

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