VemuRafenib 是一种B-Raf选择性抑制剂，抑制 RafV600E和 c-Raf-1 的IC50分别为 31 nM 和 48 nM。它可诱导细胞自噬。

Vemurafenib (PLX4032) is a novel and potent B-Raf (V600E) inhibitor (IC50: 31 nM).

Vemurafenib (PLX4032, RG7204) displays similar potency for B-RAFV600E (31nM) and c-RAF-1 (48nM) and selectivity against many other kinases, including wild type B-RAF (100nM) [1]. In 17 melanoma cell lines, RG7204 was a potent inhibitor of proliferation in those expressing BRAFV600E but not BRAFWT. RG7204 also potently inhibited proliferation of melanoma cell lines expressing other codons 600 BRAF mutations (V600D, V600K, and V600R) [2]. Melanoma cells were more sensitive to PLX4032 than CRC cells. The inhibition of EGFR does not significantly affect the proliferation of EGFR in WiDr cells. In contrast, suppression of EGFR in combination with PLX4032 caused a marked inhibition of proliferation in WiDr cells [3].

In BRAFV600E-mutant xenograft, PLX4032 (6 or 8 mg/kg) demonstrated dose-dependent inhibition of tumor growth, with higher exposures resulting in tumor regression [1]. In mice bearing Colo829 tumor xenografts, RG7204 at 100 mg/kg bid for 21 days showed greatly improved antitumor activity at the end of the study on day 38 after the tumor cell implant. There was complete tumor regression in all 10 mice treated with RG7204 by the end of the study [2]. In the dose-escalation cohort, among the 16 patients with melanoma whose tumors carried the V600E BRAF mutation and who were receiving 240 mg or more of PLX4032 twice daily, 10 had a partial response and 1 had a complete response [4].

Expression and purification of B-RAF, structure determination, and protein kinase activity measurements were carried out as previously described. To obtain co-crystals of B-RAFV600E with PLX4032, the protein solution was initially mixed with the compound dissolved in DMSO at a final compound concentration of 1 mM. This complex was co-crystallized by a sitting drop vapor diffusion experiment in which equal volumes of complex (at 10 mg/ml concentration) and reservoir solution (100mM BisTris at pH 6.0, 12.5% 2,5-hexanediol, and 12% PEG3350) were mixed and allowed to equilibrate against the reservoir at 4°C. The crystal was soaked in cryosolvent, followed by flash-freezing in liquid nitrogen. The data were collected at Beamline ALS831 with the wavelength of 1.11?. The Ramachandran plot from the refined structure shows that 94%, 5.6% and 0.4% residues are in the most favored, additional allowed and generously allowed regions, respectively. A summary of the crystallography statistics is included in Supplementary Table 3. COLO205 tumor xenograft studies (Molecular Imaging Research, Ann Arbor, MI) were carried out as previously described either using a conventional formulation (5%DMSO, 1% methylcellulose) or using the MBP formulation [1].

Cellular proliferation was evaluated by MTT assay. Briefly, cells were plated in 96-well microtiter plates at a density of 1,000 to 5,000 cells per well in a volume of 180 μL. For the assay, RG7204 was prepared at 10 times the final assay concentration in media containing 1% DMSO. Twenty-four hours after cell plating, 20 μL of the appropriate dilution were added to plates in duplicate. The plates were assayed for proliferation 6 days after the cells were plated according to the procedure originally described by Mosmann [2].

All animal procedures were approved by the Ethical Commission of the Institute for Cancer Research and Treatment and by the Italian Ministry of Health. WiDr cells were injected subcutaneously into the right posterior flanks of 7-week-old immunodeficient NODSCID female mice (6 mice per group). Tumour formation was monitored twice a week, and tumour volume based on caliper measurements was calculated by the modified ellipsoidal formula: tumour volume = 1/2 length × width. When tumours reached a volume of approximately 200–250 mm^3, mice were randomly assigned to treatment with vehicle or drug(s) [3].

918504-65-1

C23H18ClF2N3O3S

489.92

PLX4032;维罗非尼;RG7204;RO5185426

DMSO:90 mg/mL (183.7 mM)
Ethanol:<1 mgml
H2O:<1 mgml
Powder: -20°C for 3 years
In solvent: -80°C for 2 years