Rotational Freedom, Steric Hindrance, and Protein Dynamics Explain BLU554 Selectivity for the Hinge Cysteine of FGFR4

Abstract

Aberration in FGFR4 signaling drives carcinogenesis and progression in a subset of hepatocellular carcinoma (HCC) patients, thereby making FGFR4 an attractive molecular target for this disease. Selective FGFR4 inhibition can be achieved through covalently targeting a poorly conserved cysteine residue in the FGFR4 kinase domain. We report mass spectrometry assays and cocrystal structures of FGFR4 in covalent complex with the clinical candidate BLU554 and with a series of four structurally related inhibitors that define the inherent reactivity and selectivity profile of these molecules. We further reveal the structure of FGFR1 with one of our inhibitors and show that offtarget covalent binding can occur through an alternative conformation that supports targeting of a cysteine conserved in all members of the FGFR family. Collectively, we propose that rotational freedom, steric hindrance, and protein dynamics explain the exceptional selectivity profile of BLU554 for targeting FGFR4.

Advances of Fibroblast Growth Factor/Receptor Signaling Pathway in Hepatocellular Carcinoma and its Pharmacotherapeutic Targets

“…Considering the established roles of aberrant FGF/FGFR signaling in liver cancer oncogenesis, inhibitors of the FGF/FGFR signaling axis may be promising for HCC treatment, which slow or halt HCC tumor growth, target angiogenesis and metastasis, and reverse acquired resistance to anticancer agents. The development of FGFR inhibitors started from the earliest multi-target inhibitors to pan-FGFR inhibitors and then to selective FGFR4 inhibitors and irreversible FGFR4 inhibitors (French et al, 2012;Shen et al, 2013;Katoh and Nakagama, 2014;Mellor, 2014;Choi et al, 2015;Joshi et al, 2017;Ettrich and Seufferlein, 2018;Spallanzani et al, 2018;Kim et al, 2019a;Lin et al, 2019a;Doycheva and Thuluvath, 2019;Hatlen et al, 2019;Weiss et al, 2019).…”

Discovery of a Potent Degrader for Fibroblast Growth Factor Receptor 1/2

“…To improve selectivity,astrategy of covalently targeting the conserved cysteine on the activation loop of FGFR paralogs has been achieved by FIIN1-3, [9] PRN1371, [10] BLU554, [11] and TAS-120. [12] Separately,s econd generation reversible FGFR inhibitors such as AZD4547, [13] BGJ398, [14] JNJ-42756493 [15] and INCB054828 [16] have improved selectivity for the FGFR family and have demonstrated efficacy against FGFR-dependent cancers in clinical trials,leading to the recent approval of JNJ-42756493 and INCB054828 for the treatment of advanced urothelial and biliary tract cancers, respectively (Figure 1).…”

“…However,given the high structural similarity in the ATP-binding pocket between FGFR1-3, developing selective inhibitors is challenging and none have been reported to date.C urrently,t he only small molecule with selectivity within the FGFR family is BLU554, which targets au nique cysteine (Cys 554) located in the hinge region of FGFR4. [11] Recently,degradation strategies that utilize heterobifunctional molecules to recruit an E3 ubiquitin ligase complex to at arget protein for ubiquitination and subsequent proteasome-mediated degradation have been employed to increase target selectivity when compared to their parental inhibitors. [21,22] Fore xample,adegrader that was constructed with ap romiscuous kinase inhibitor TAE684 and CRBN ligand caused selective degradation of only asubset of the TAE684 binding kinase targets.…”

“…However, given the high structural similarity in the ATP‐binding pocket between FGFR1‐3, developing selective inhibitors is challenging and none have been reported to date. Currently, the only small molecule with selectivity within the FGFR family is BLU554, which targets a unique cysteine (Cys 554) located in the hinge region of FGFR4 [11] …”

Discovery of a Potent Degrader for Fibroblast Growth Factor Receptor 1/2

“…To improve selectivity, a strategy of covalently targeting the conserved cysteine on the activation loop of FGFR paralogs has been achieved by FIIN1‐3, [9] PRN1371, [10] BLU554, [11] and TAS‐120 [12] . Separately, second generation reversible FGFR inhibitors such as AZD4547, [13] BGJ398, [14] JNJ‐42756493 [15] and INCB054828 [16] have improved selectivity for the FGFR family and have demonstrated efficacy against FGFR‐dependent cancers in clinical trials, leading to the recent approval of JNJ‐42756493 and INCB054828 for the treatment of advanced urothelial and biliary tract cancers, respectively (Figure 1).…”