Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Nov 11;111(45):E4869-77.
doi: 10.1073/pnas.1403438111. Epub 2014 Oct 27.

Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

Li Tan  1 , Jun Wang  2 , Junko Tanizaki  3 , Zhifeng Huang  4 , Amir R Aref  5 , Maria Rusan  6 , Su-Jie Zhu  7 , Yiyun Zhang  8 , Dalia Ercan  3 , Rachel G Liao  9 , Marzia Capelletti  3 , Wenjun Zhou  1 , Wooyoung Hur  10 , NamDoo Kim  11 , Taebo Sim  12 , Suzanne Gaudet  13 , David A Barbie  2 , Jing-Ruey Joanna Yeh  8 , Cai-Hong Yun  7 , Peter S Hammerman  14 , Moosa Mohammadi  15 , Pasi A Jänne  16 , Nathanael S Gray  17
Affiliations

Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

Li Tan et al. Proc Natl Acad Sci U S A. .

Abstract

The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket.

Keywords: cancer drug resistance; drug discovery; kinase inhibitor; structure-based drug design.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) Chemical structures of clinical-stage FGFR inhibitors. (B) Evolution of FIIN-2 and FIIN-3 from FIIN-1. Structures of the reversible counterparts FRIN-2 and FRIN-3 are shown also.
Fig. 2.
Fig. 2.
Inhibition of FGFR-dependent signaling by BGJ398, FIIN-2, and FIIN-3 in Tel/FGFR2 V564M Ba/F3 cells. Cells were treated with a dose escalation of inhibitors for 6 h and then were lysed and subjected to Western blot for the indicated proteins or phosphoproteins.
Fig. 3.
Fig. 3.
(A and B) FIIN-2 (purple stick) covalently binds to Cys477 in the P-loop of FGFR4 (green ribbons) and results in the DFG-out conformation of FGFR4. (C) FIIN-3 (pink stick) binds to Cys477 of FGFR4 V550L (green ribbons) with a similar binding mode. (D) FIIN-3 binds to Cys797 of EGFR L858R (blue ribbons) covalently and in a DFG-in conformation.
Fig. 4.
Fig. 4.
Inhibition of FGFR-dependent signaling by BGJ398, FIIN-2, and FIIN-3 in H1581 (FGFR1 WT or V561M) cells. Cells were treated with indicated inhibitors at 1.0 µM for 12 h and then were lysed and subjected to Western blot for the indicated proteins or phosphoproteins.
Fig. 5.
Fig. 5.
Effects of FIIN-2, FIIN-3, and FRIN-3 on FGF- and EGF-induced dispersion of SKOV-3 cells in a 3D microfluidic device. Images show representative spheroids of SKOV-3 cells after indicated treatment with FGF (A) or EGF (B), in the presence or absence of the FIIN-2 and FIIN-3. Objective magnification power is indicated in the lower right corner. (A) Phase-contrast images of SKOV-3 spheroids at the indicated times after the addition of FGF. In contrast to the cell dispersal observed in control-treated devices, treatment with 1.0 μM of FIIN-2, FIIN-3, or FRIN-2 inhibited FGF1-induced dispersion of SKOV-3 cells. (B) Phase-contrast images of the SKOV-3 spheroids induced to disperse with EGF and subjected to control, FIIN-2, or FIIN-3 treatment. At 1.0 µM FIIN-3, but not FIIN-2, fully inhibited the EGF-induced dispersion of SKOV-3 cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Breccia M, Alimena G. Nilotinib: A second-generation tyrosine kinase inhibitor for chronic myeloid leukemia. Leuk Res. 2010;34(2):129–134. - PubMed
    1. O’Hare T, et al. Inhibition of wild-type and mutant Bcr-Abl by AP23464, a potent ATP-based oncogenic protein kinase inhibitor: Implications for CML. Blood. 2004;104(8):2532–2539. - PubMed
    1. Solca F, et al. Target binding properties and cellular activity of afatinib (BIBW 2992), an irreversible ErbB family blocker. J Pharmacol Exp Ther. 2012;343(2):342–350. - PubMed
    1. Zhou W, et al. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature. 2009;462(7276):1070–1074. - PMC - PubMed
    1. Tiong KH, Mah LY, Leong CO. Functional roles of fibroblast growth factor receptors (FGFRs) signaling in human cancers. Apoptosis. 2013;18(12):1447–1468. - PMC - PubMed

Publication types

MeSH terms

Substances