FRAX597, a small molecule inhibitor of the p21-activated kinases, inhibits tumorigenesis of neurofibromatosis type 2 (NF2)-associated Schwannomas

Abstract

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.

Keywords: Enzyme Inhibitors; Merlin; Protein Kinases; Schwann Cells; Signal Transduction; Small Molecules; nf2; p21-activated Kinase.

FIGURE 1.

Characterization of FRAX597. A, chemical structures and IC₅₀ of FRAX019 and FRAX414. B, chemical structure of FRAX597. C, inhibition profiles for FRAX597 against group I PAKs1–3 and group II PAK4. Each of the 10 concentration points was done in duplicate. D, Western blot analysis of FRAX597 inhibition of PAK1 autophosphorylation (p-PAK1). SC4 cells were treated in culture for 2 h at the indicated FRAX597 concentrations and then extracted for protein. Actin was used as a loading control.

FIGURE 2.

Structure of the PAK1/FRAX597 complex. A, overall structure of PAK1 with FRAX597 bound in the active site. The N-terminal lobe is shown in light gray, the hinge region is shown in dark gray, and the C-terminal lobe is shown in salmon. The C-helix is labeled. B, electron density corresponding to FRAX597. A Fo−Fc electron density map is shown in green and contoured at 2 σ as calculated before building the inhibitor model.

FIGURE 3.

Detailed view of the interactions of FRAX597 within the PAK1 active site. A, view of the PAK1 active site in complex with FRAX597. Residues that form the binding pocket for the inhibitor are labeled. Numbering for important carbon atoms of FRAX597 is shown. B, surface view of the gatekeeper residue and the back cavity of the ATP binding site. Residues involved in inhibitor binding are labeled. C, Van der Waals sphere representation of the inhibitor and surface representation of the residues forming the back cavity of the ATP binding site. D, inhibition profiles for FRAX597 against wild type PAK1 and Val342 mutants. Each of the 10 concentration points was done in duplicate at 1 μm ATP and 20 nm PAK1.

FIGURE 4.

Structural basis for FRAX597 potency and selectivity toward group I PAKs. A, superimposition of the co-crystal structures of PAK1 with FL172 (PDB ID 3FXZ), Ru-phthalimide (PDB ID 4DAW), and FRAX597. The protein chain is only displayed for the PAK1/FRAX597 structure for clarity. B, superimposed co-crystal structures of PAK4/CGP74514A (PDB ID 2CDZ) and PAK1/FRAX597 illustrating the difference in size of the back pocket between two kinases. PAK4 is shown in blue, and PAK1 is shown in gray. C, superimposed cocrystal structures of RET (PDB ID 2IVU) and PAK1 kinases showing that the thiazole ligand of FRAX597 is not optimally accommodated by the back pocket of RET (shown in “green”). D, surface view and cartoon representation of the RET and PAK1 demonstrating the difference in the size of the back pockets and position of C-helices. The RET surface and C-helix are outlined in green and the PAK1 C-helix in gray.

FIGURE 5.

FRAX597 inhibits tumor cells proliferation and tumor growth in vivo. A, SC4 Nf2-null cells were plated in triplicate wells and treated with FRAX597 (1 μm) or DMSO control daily for 4 days, and cell numbers were scored daily. The experiment shown is representative of three independent experiments. B, percentage of treated SC4 cells in different phases of the cell cycle. The DNA content of treated SC4 cells was determined by staining with PI and analysis by FACS. The experiment shown is representative of three independent experiments. C, representative images from bioluminescence imaging (BLI) of mice carrying orthotopic tumors treated with FRAX597 (100 mg/kg) or vehicle control at day 14 of treatment. NOD/SCID mice were injected intraneurally with 5 × 10⁴ SC4/pLuc-mCherry cells and were enrolled into treatment after 10 days. Mice were treated daily for 14 days and imaged every 3 days to follow tumor development. D, quantitative analysis of the flux reading from treated cohorts. A mixed-effect model analysis indicated that the speed of tumor growth in treatment group is significantly slower than that in control group (p = 0.0002). E, distribution of tumor/body weight ratios in the cohorts treated with FRAX597 or vehicle control. The results of t test with equal variances show that FRAX-A group has significant lower average tumor weight ratio than that observed in control group (p = 0.0001). For the in vivo experiments the n = 6 in each cohort, experiments repeated three times.

FIGURE 6.

Inhibition of group I PAKs mediates FRAX597 inhibition of SC4 cells. A, Western blot analysis of Yes1, TEK, CSF1R and Ret expression in SC4 cells compared with relevant positive control cells. Tubulin was used as a loading control. B, SC4 Nf2-null cells were plated in triplicate wells and treated with PF3758309 (1 μm) or DMSO control daily for 4 days, and cell numbers were scored daily. The experiment shown is representative of three independent experiments.