Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity

Abstract

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.

Fig. 1.

Structures of individual compounds leading to the discovery of PLX4720 are shown. (A) The chemical structure of 3-aminophenyl-7-azaindole (compound 1) is shown beneath its costructure with Pim-1 kinase. (B) The chemical structure of 3-(3-methoxybenzyl)-7-azaindole (compound 2) is shown beneath its costructure with the kinase domain of FGFR1. (C) The chemical structure of PLX4720 is shown beneath its costructure with B-Raf kinase.

Fig. 2.

Depiction of the three-dimensional structure of PLX4720 bound to B-Raf. (A) The structure of B-Raf^V600E bound to PLX4720 (yellow) is overlayed with an ATP model based on structures of ATP analogs in complex with other tyrosine kinases (orange). This view indicates that the PLX4720 scaffold overlaps with the adenine-binding site, but the tail of PLX4720 binds to a different pocket from the ATP ribose-triphosphate tail. The positions of the hinge, activation loop (A-loop), and phosphate-binding loop (P-loop) are also shown. (B) A surface representation shows PLX4720 binding to the B-Raf-selective pocket in the active conformation. (C) A surface representation shows PLX4720 binding to the kinase general pocket in the inactive conformation. (D) A close-up view shows the overlay PLX4720 bound to both active (green) and inactive (purple) conformations of the V600 protein, and PLX3203 (yellow) bound to V600E protein in the active kinase conformation. (E) A stereoview shows the specific interactions of PLX4720 to the active kinase conformation. In this conformation, the phenylalanine of the DFG loop is pointing in toward the compound-binding site. (F) A stereoview shows the specific interactions of PLX4720 to the inactive kinase conformation. In this conformation, the phenylalanine of the DFG loop is pointing away from the compound-binding site, and binding of PLX4720 is disfavored, leading to partial occupancy of this site even at the 1 mM compound concentration used in cocrystallography.

Fig. 3.

Selectivity and antimelanoma activity of PLX4720 in vitro. (A) Panel of melanoma cell lines (V600E+, left; B-Raf wild-type, right) were treated with various dosages of PLX4720, and protein extracts were subject to immunoblotting. Activity within the MAPK pathway is represented by levels of phosphorylated ERK; β-actin serves as a loading control. (B) B-Raf V600E+ (Left) and B-Raf wild-type (Right) cells were treated PLX4720 at the indicated dosages for 72 h. Cell number was assayed by MTT analysis. (C) 1205Lu and C8161 cells were treated with 1 μM PLX4720 for the times indicated and stained with Annexin V/FITC and propidium iodide (PI) for analysis of apoptosis. (D) Graphs represent raw data from the Annexin/PI assay. (E) Spheroids from 1205Lu and C8161 cells were treated with indicated dosages of PLX4720 and stained with calcein AM and ethidium bromide to assess overall viability. Green (calcein-AM) indicates live cells; red (EtBr) depicts apoptotic cells. (F) Synthetic skin was created by using 1205Lu (V600E+) cells and subjected to vehicle control (Upper) or 1 μM PLX4720 (Lower) for 72 h. H&E staining is depicted (Left), and immunofluorescent stains for DAPI, PCNA, and S100 are also shown. (G) Same as F, except with C8161 (B-Raf wild-type) cells.

Fig. 4.

Effect of PLX4720 on xenograft tumor growth. (A) Tumor volume measurements of COLO205 xenograft tumors treated with 5 or 20 mg/kg PLX4720 by oral gavage or treated with vehicle. Dosing occurred from days 1 to 14. (B and C) Two million cells [1205Lu (B); C8161 (C)] were s.c. injected into SCID mice. After reaching sufficient size, mice were treated by oral gavage with vehicle control (Left) or 100 mg/kg PLX4720 (Right) twice daily for the indicated times. (D) 1205Lu xenograft tumors were extracted, fixed in formalin, and paraffin embedded. Vehicle- (Left) and PLX4720- (Right) treated samples were immunostained for phospho-ERK.