Structure-based design of a dual-warhead covalent inhibitor of FGFR4

Abstract

The fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR4) signaling pathways play critical roles in a variety of cancers, such as hepatocellular carcinoma (HCC). FGFR4 is recognized as a promising target to treat HCC. Currently, all FGFR covalent inhibitors target one of the two cysteines (Cys477 and Cys552). Here, we designed and synthesized a dual-warhead covalent FGFR4 inhibitor, CXF-009, targeting Cys477 and Cys552 of FGFR4. We report the cocrystal structure of FGFR4 with CXF-009, which exhibits a dual-warhead covalent binding mode. CXF-009 exhibited stronger selectivity for FGFR4 than FGFR1-3 and other kinases. CXF-009 can also potently inhibit the single cystine mutants, FGFR4(C477A) and FGFR4(C552A), of FGFR4. In summary, our study provides a dual-warhead covalent FGFR4 inhibitor that can covalently target two cysteines of FGFR4. CXF-009, to our knowledge, is the first reported inhibitor that forms dual-warhead covalent bonds with two cysteine residues in FGFR4. CXF-009 also has the potential to overcome drug induced resistant FGFR4 mutations and might serve as a lead compound for future anticancer drug discovery.

Fig. 1. Design of dual-warhead covalent inhibitor of FGFR4.

a, b Kinases with the cysteines at the equivalent position of Cys477 (a) and Cys552 (b) in FGFR4 within the human kinome. c Design the new dual-warhead covalent inhibitors CXF-009 and CXF-008 for Cys477 and Cys552 of FGFR4. 4-acrylamidepiperazin-1-propyl from PRN1371 is colored red, and the 2-acrylamidebenzamine group from BLU9931 is indicated in blue.

Fig. 2. Synthesis of compound CXF-008 and CXF-009.

CXF-008 and CXF-009 were synthesized using 5-hydroxymethyluracil as the starting material via eight steps.

Fig. 3. MALDI-TOF MS determination of protein (black) and protein/inhibitor complexes (blue) for CXF-009.

a Apo FGFR4 and FGFR4/CXF-009 mixture; b Apo FGFR4(C477A) and FGFR4(C477A)/CXF-009 mixture; c Apo FGFR4(C552A) and FGFR4(C552A)/CXF-009 mixture; d Apo FGFR4(C477A, C552A) and FGFR4(C477A, C552A)/CXF-009 mixture.

Fig. 4. Crystal structure of FGFR4/CXF-009 complex.

a The overall structure of FGFR4/CXF-009 complex. FGFR4 kinase is colored as wheat, and CXF-009 is shown as cyan. Hinge loop (warm-pink) and P-loop (green) are high-light in the structure. b The 2Fo-Fc omit map (black mesh, contoured at 0.7σ) of the FGFR4/CXF-009 co-crystal structure. c The hydrogen bonds between FGFR4 and CXF-009. Hydrogen bond distance is <3.3 Å.

Fig. 5. CXF-009 selectively inhibited FGFR4.

a Potency and selectivity of CXF-009 against wild-type FGFR1-4 and FGFR4 mutations using kinase assay. b Cellular activity determination of CXF-009 using a Ba/F3 cell model. Data are presented as dot, n = 3 independent replicates. Error bars indicate mean ± SD. c Kinase inhibition profile of CXF-009 with 185 kinases at 1000 nM.