Cell-based screen identifies porphyrins as FGFR3 activity inhibitors with therapeutic potential for achondroplasia and cancer
Yun-Wen Lin, Hsiao-Jung Kao, Wei-Ting Chen, Cheng-Fu Kao, Jer-Yuarn Wu, Yuan-Tsong Chen, Yi-Ching Lee
Yun-Wen Lin, Hsiao-Jung Kao, Wei-Ting Chen, Cheng-Fu Kao, Jer-Yuarn Wu, Yuan-Tsong Chen, Yi-Ching Lee
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Research Article Bone biology Genetics

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

Abstract

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.

Authors

Yun-Wen Lin, Hsiao-Jung Kao, Wei-Ting Chen, Cheng-Fu Kao, Jer-Yuarn Wu, Yuan-Tsong Chen, Yi-Ching Lee

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Figure 2

High-throughput imaging system (receptor/adaptor translocation assay) to quantify FGFR3 activation and identify hits that inhibit FGFR3 activation.

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High-throughput imaging system (receptor/adaptor translocation assay) to...
(A and B) U2OS-TDI/SH2BGFP cells were treated with 4 μM PKC412 or vehicle control (DMSO) for 1 hour. Scale bars, 20 μm. (A) Confocal images show SH2(SH2B)/GFP signals (green). (B) Representative raw images acquired on the ArrayScan VTI HCS Reader (left panels) and automated identification of spots (yellow dots) within cytoplasmic area (green ring) (right panels). (C and D) Dose-response curves of PKC412 plotted by ring spot count (C) or ring spot intensity (D) relative to vehicle control. (E) FGFR3 activity was quantified based on ring spot counts in U2OS-SH2(SH2B)/GFP cells expressing TDI FGFR3, Y724F/760F TDI FGFR3, or vector control (Mock). (F) U2OS-TDI/SH2BGFP cells were treated with different plant extracts. Ring spot count per cell was plotted relative to vehicle control. Data from 3 independent treatments are shown. Hits: red circles; PKC412: black circles. (G) Linear regression analysis of experiments 1 and 2 showing PKC412 treatments and hits (lower-left corner) and active responder (upper-right corner). (H–J) Dose-response curves of (H) 4 identified hits (Hits 1–4), (I) 2 different batches of Hit 4 (Hit 4 B1 and Hit 4 B2), and (J) 2 additional plant extracts from species closely related to Hit 4: Hit 4-1 (A. viridis) and Hit 4-2 (A. tricolor). Data in C–E and H–J represent the mean ± SEM of triplicates. (K) KMS-11 cells were treated with vehicle control or different doses of plant extracts for 48 hours. Cell viability was analyzed using WST-1 assay and normalized to the vehicle control. Data represent the mean ± SEM of 3 independent experiments. Student’s 2-tailed t tests were performed. *P < 0.05, ***P < 0.001. (L) KMS-11 cells were treated with indicated concentrations of plant extracts, PKC412, or vehicle control (Vehicle) for 1 hour. Total and phosphorylated protein levels of FGFR3 and its downstream effectors were detected by immunoblotting.