Cell-based screen identifies porphyrins as FGFR3 activity inhibitors with therapeutic potential for achondroplasia and cancer

Overactive fibroblast growth factor receptor 3 (FGFR3) signaling drives pathogenesis in a variety of cancers and a spectrum of short-limbed bone dysplasias, including the most common form of human dwarfism, achondroplasia (ACH). Targeting FGFR3 activity holds great promise as a therapeutic approach for treatment of these diseases. Here, we established a receptor/adaptor translocation assay system that can specifically monitor FGFR3 activation, and we applied it to identify FGFR3 modulators from complex natural mixtures. An FGFR3-suppressing plant extract of Amaranthus viridis was identified from the screen, and 2 bioactive porphyrins, pheophorbide a (Pa) and pyropheophorbide a, were sequentially isolated from the extract and functionally characterized. Further analysis showed that Pa reduced excessive FGFR3 signaling by decreasing its half-life in FGFR3-overactivated multiple myeloma cells and chondrocytes. In an ex vivo culture system, Pa alleviated defective long bone growth in humanized ACH mice (FGFR3ACH mice). Overall, our study presents an approach to discovery and validation of plant extracts or drug candidates that target FGFR3 activation. The compounds identified by this approach may have applications as therapeutics for FGFR3-associated cancers and skeletal dysplasias.

The full-length cDNA for Renilla reniformis green fluorescent protein (GFP) was amplified by polymerase chain reaction (PCR) and subcloned into the pcDNA3.1/Hygroexpression vector (Invitrogen) using NotI and XbaI sites.SH2 proteins fused to the N-terminus of GFP were generated by amplifying the Src homology 2 (SH2) domain of human SH2Bb and the SH2 domains of human PLCg from genomic DNA and subcloning them into BamHI and NotI sites in the GFP expression vector.Full-length cDNA for human FGFR3 (OriGene, Rockville, MD, USA) was subcloned into the BamHI and XbaI sites of the pcDNA3.1 expression vector (Invitrogen).Mutations were introduced into FGFR3 by oligonucleotide-directed mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA).The mutagenic oligonucleotides used are as follows: Y373C (TDI), 5′-cga ggc ggg cag tgt gtg tgc agg cat-3′ (forward) and 5′-atg cct gca cac aca ctg ccc gcc tcg-3′ (reverse); G380R (ACH), 5′gca tcc tca gct aca ggg tgg gct tct tc-3′ (forward) and 5′-gaa gaa gcc cac cct gta gct gag gat gc-3′ (reverse).All plasmids were verified by DNA sequencing.

Immunoprecipitation and immunoblotting
Cells were lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany).For immunoprecipitation experiments, cell lysates were pre-cleared with protein A/G PLUS-Agarose (sc-2003; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with 2 µg of a polyclonal anti-FGFR3 antibody (C-15; Santa Cruz Biotechnology) for 1 h at 4°C.Protein A/B beads were then added and incubated overnight at 4°C.The immunoprecipitated samples were washed four times with lysis buffer and analyzed by immunoblotting.For experiments not requiring immunoprecipitation, lysates were immunoblotted with antibodies.The signals were detected using enhanced chemiluminescence (ECL; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).
For high-content screening, all steps of sample-plate preparation, including compound treatment, fixation, and plate washing, were fully automated and were performed using a Bio-Tek EL405uv system (Bio-Tek Instruments, Inc., Winooski, VT, USA).U2OS FGFR3-TD1/SH2-GFP were seeded at a density of 4 × 10 3 cells/well in black 96-well Packard Viewplates, incubated overnight in complete medium at 37°C in a CO2 incubator, and then transferred to 100 μl serum-free medium containing PKC412 or plant extract.The final DMSO concentration was 1%.After incubating for 1 h at 37°C in a CO2 incubator, cells were fixed with 4.5% formaldehyde and cell nuclei were labeled with a 2 μg/ml Hoechst stain solution (Sigma-Aldrich).Cells treated with either control (DMSO) or 4 μM PKC412 were used for protocol optimization.

Plant extract preparation
To generate ethanol plant extracts, the aerial parts of plants were collected and washed with water.Then whole plants were dried in a 65°C oven for 13 h and ground into powder.The ground powders were extracted in a 10-fold volume of 95% ethanol with continuous stirring at room temperature for 16 h.The ethanol extracts were dried by evaporation under reduced pressure in a rotary evaporator and were dissolved in DMSO to a final concentration of 37 mg/ml.For preliminary screening, ethanol extracts from whole plants of 101 different species from 75 families collected from Taiwan were tested at a concentration of 10 μg/ml in 1% DMSO.The ethanol extracts of the second batch of Hit 4 (A.spinosus) and two closely related species, A. viridis (Hit 4-1) and A. tricolor (Hit 4-2) were prepared using the same method.For further fractionation, the ethanol extracts were condensed and stored at 4°C for further fractionation.Large-scale preparations of A. viridis extracts were prepared by the Industrial Technology Research Institute, Hsinchu, Taiwan.Briefly, fresh A. viridis plants (500 Kg) were collected, washed with water, dried in an oven at 65°C for 13 h, and chopped into small strips.The dried strips (47.3 kg) were extracted by 95% ethanol reflux extraction for 24 h with intermittent stirring at 2 h intervals, repeated once.The extract was concentrated via an evaporator to a final solid content of 6.43% in 25.7 L of ethanol.The extract was stored at 4°C before use.

Analytical thin layer chromatography (TLC) and pooling of fractions
The content of each eluted fraction was spotted on pre-coated (silica gel F254) aluminium plates in a small chromatographic tank.Fractions were characterized based on their relative mobilities in solvent systems and color reactions with UV light.The fractions were pooled into 12 fractions based on the similarity of the TLC patterns (F4-IS1 to F4-IS12).

Identification of bioactive compounds from A. viridis that inhibit FGFR3 activation
The most potent active fractions to inhibit FGFR3 activity in the cell-based assay system were further fractionated and the main chemical constituents of active fractions were identified.Each fraction was tested for cytotoxicity and its ability to inhibit FGFR3 activity using the cell-based assay system.The most potent fraction without cytotoxicity was further fractionated, and the main chemical constituents were identified by AnlytiCon, Potsdam, Germany.F4-IS11 and F4-IS-12 fractions were further fractionated into 227 fractions (Supplemental Figure 3), and finally 8 fractions (Supplemental Figure 4).The final 2 most potent fractions (N100-N3 and N100-N5) showed high purity based on HPLC and MS analysis (Supplemental Figure 4) were analyzed for their main chemical constituents using high-resolution MA, MS/MS, IR and NMR.

Primary chondrocytes isolation
Murine articular chondrocytes were harvested as previously described (1).Briefly, the articular cartilage from homozygous FGFR3 ACH mice was isolated on postnatal day 5, followed by digestion with 3 mg/ml collagenase D (Roche) in DMEM culture medium for 45 minutes at 37℃ and continued digestion with diluted digestion solution (0.5 mg/ml collagenase D) at 37℃ overnight.The suspension of isolated cells was filtered through a 48 μm cell strainer for subsequent counting and seeding.

RNA isolation and quantitative real-time RT-PCR analysis
Total RNA from various cell lines was isolated using TRIzol reagent (Invitrogen).Next, total RNA was further purified with the QIAGEN RNeasy Mini Kit, treated with DNase (DNase I, 30 U/μg total RNA; QIAGEN), and reverse transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen).The levels of FGFR1, 2, 3 and 4 mRNA were quantitatively detected by real-time RT-PCR using SYBR Green PCR Master Mix and an ABI Prism 7900HT Sequence Detection System (Applied Biosystems).The primers used for RT-PCR are as follows: