Su5416

SU4312, SU5416, Sorafenib, PTK787 and valproic acid (VPA) were purchased from Sigma-Aldrich with a purity range of > 98% (Table 1).Table 1

Chemical properties of tyrosine kinase inhibitors

Inhibitor name	Targets	CAS RN	Purity (%)	Log Kow	Log Dlipw at pH 7.4	Molecular weight [Da]
SU4312	VEGFR2, PDGFR, EGFR, HER-2, IGF (Sun et al. 1998 (link))	5812-07-7	98	3.26	3.73	264.32
SU5416	VEGFR2, PDGFR, FIt-1, FIt-4, c-kit (Haddad 2012 (link))	204005-46-9	98	3.06	2.09	238.28
PTK787	VEGFR2, VEGFR-1, PDGF, FIt-4, and c-Kit (Gotink and Verheul 2010)	212141-51-0	98	4.98	0.78	419.73
Sorafenib	VEGFR, PDGFR, FGFR1, KIT, RAF (Gotink and Verheul 2010)	284461-73-0	98	5.30	5.53	464.82
Valproic acid	HDAC1 (Phiel et al. 2001 (link))	99-66-1	98	2.75	1.9	144.21

References for the observation of inhibition and mechanism of action are provided within the “Target” column. D_lipw values were obtained as described in Klüver et al. (2019 (link))

Stock solutions were prepared in 100% dimethyl-sulfoxide (DMSO) and diluted in exposure medium as specified in ISO 7346-3 (1996) [80 mM CaCl₂·2H₂O, 20 mM MgSO₄·7H₂O, 31 mM NaHCO₃, 3.1 mM KCl]. The final concentration of DMSO was 0.01% (v/v). The pH of the highest test concentration was adjusted to pH 7.4 and all other test concentrations were established by dilution of the highest concentration.

Anti-β-actin (A5316) and –PGC1α (AB3242) monoclonal antibodies were from Sigma (St. Louis, MO). Anti-VEGFR2 (2479) and –ERRα (13826) antibodies were from Cell Signaling (Danvers, MA). Anti-CD31 (102409) and –CD45 (103113) antibodies were from BioLegend (San Diego, CA). Anti-VE-cadherin (562243) and -CD31 (553370) antibodies were from BD Pharmingen (San Diego, CA). Anti-Twist1 antibody (sc-15393) was from Santa Cruze Biotechnology (Dallas, TX). SU5416 was purchased from Millipore Sigma (Burlington, MA).

All animal protocols were approved by the Laval University and the Institut Universitaire de Cardiologie et de Pneumologie de Québec (IUCPQ) Biosafety and Ethics Committees (#2019-311). All experiments were in accordance with recent recommendations on optimal preclinical studies in PAH, including animal randomization, sequence allocation concealment and blinded assessments [43 (link),44 (link)]. Sugen-hypoxia (Su/Hx) and monocrotaline (MCT) PAH rat models were used in the present study. For the Su/Hx model, both adult male and female Sprague Dawley rats (Charles River Laboratories, Montreal, QC, Canada) were injected with 20 mg/kg of SU5416 (Sugen, MilliporeSigma, Oakville, ON, Canada) and put in hypoxia (10% O₂) for 3 weeks. For the MCT model, rats were injected subcutaneously with 60 mg/kg of monocrotaline (MilliporeSigma, Oakville, ON, Canada). As females are traditionally considered as more resistant than males to developing MCT-induced pulmonary hypertension, only males were used. Once PAH was established (at days 14 and 21 post-MCT or -SU5416 injection, respectively), rats were randomly divided into two groups and received trifluoperazine (5 mg/kg delivered intraperitoneally, three times a week) or its vehicle for 2 additional weeks.

Three VEGF inhibitors, SU5416 (Merck KGaA, Darmstadt, Germany), bevacizumab (Avastin, Roche, Basel, Switzerland), and ranibizumab (Lucentis, Novatris, Stein Switzerland) were used as candidate drugs to reverse abnormal retinal neovascularization. SU5416 targets the VEGF receptor 2 tyrosine kinase and is a potent, selective competitive inhibitor of Flk-1/KDR [1 (link)]. bevacizumab is a humanized monoclonal antibody that is derived from antibody A4.6.1 [32 (link)] and binds to membrane-bound receptors (VEGF receptors-1 and -2) [33 (link), 34 (link)]. bevacizumab blocks VEGF receptor binding and signaling; its epitope overlaps the epitopes of VEGF receptors-1 and -2. Similarly, ranibizumab [35 (link)] is a recombinant humanized IgG1 kappa isotype monoclonal antibody fragment with a high binding affinity for all forms of VEGF. All of these inhibitors have been shown to inhibit angiogenesis [36 (link), 37 (link)]. Zebrafish were treated with 0.5 μM SU5416 [38 (link)], 2.5 μg/mL of bevacizumab [39 (link)], or 2.5 μg/mL of ranibizumab as previously described to examine the reverse efficiency of neovascularization in the current model. Furthermore, 2.5 μg/mL of normal human control IgG (R&D system, Minneapolis, MN) was used as a control.

Zebrafish embryos were then treated at 4 hpf with the snail derivatives LH, LM, LH3, and LM2, and then at 15 hpf with the VEGF inhibitor SU5416 (Sunitinib, Merck KGaA, Darmstadt, Germany) at a final concentration of 5 µg/mL in fish water and incubated in 3 cm diameter well-plates. Treated and untreated embryos were incubated at 28 °C and staged at 36 hpf for WISH.

Dechorionated embryos were treated with 1 μM SU5416 (Merck KGaA; 676487), 20 μM GSK2656157 (MedChemExpress; HY-13820), 10 μM GCN2-IN-1 (MedChemExpress; HY-100877), 10 μM GCN2iB (MedChemExpress; HY-112654), or 50 μM 4-Phenylbutyric acid (4-PBA) (Sigma Aldrich; P21005) from 24 to 54 hpf. To inhibit global protein translation, the embryos were treated with 360 μM CHX (Sigma-Aldrich; C7698). While 4-PBA was dissolved in water, the other chemicals were dissolved in Dimethyl sulfoxide (DMSO) (Sigma-Aldrich; D8418). The solvents, water or DMSO, were used as controls, respectively, and the final concentration of DMSO is 0.1%.