Rotational Freedom, Steric Hindrance, and Protein Dynamics Explain

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Rotational Freedom, Steric Hindrance, and Protein Dynamics Explain BLU554 Selectivity for the Hinge Cysteine of FGFR4 Xiaojing Lin,†,⊥ Yuliana Yosaatmadja,†,⊥ Maria Kalyukina,† Martin J. Middleditch,† Zhen Zhang,‡ Xiaoyun Lu,‡ Ke Ding,‡ Adam V. Patterson,§,∥ Jeff B. Smaill,§,∥ and Christopher J. Squire*,†,∥ †

School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education (MOE), School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China § Auckland Cancer Society Research Centre, the University of Auckland, Private Bag 92019, Auckland 1142, New Zealand ∥ Maurice Wilkins Centre for Molecular Biodiscovery, c/o The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand

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‡

S Supporting Information *

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. KEYWORDS: Hepatocellular carcinoma, irreversible FGFR4-selective inhibitors, targeted kinase inhibition, FGFR4 and FGFR1 crystal structure, mass spectrometry assay

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highly expressed isoform in hepatocytes, and its ligand, FGF19, binds exclusively to FGFR4 with coreceptor β-Klotho to regulate hepatocyte proliferation.6 Addition of recombinant FGF19 protein increases proliferation and invasion of HCC cell lines.7 Both FGF19 and FGFR4 are upregulated in a large subset of HCC patients, with FGFR4 overexpressed in half of all HCCs.8 Consistently, FGF19 levels are positively correlated with tumor size, poor prognosis, and recurrence after hepatectomy.9,10 Furthermore, HCC patients with FGF19 overexpression survive five years less than those with low expression.7 Several groups have effectively abrogated hepatocarcinogenesis in mouse models by targeting the FGF19FGFR4 pathway via FGF19 or FGFR4 knockdown7,11,12 or by administering anti-FGF19 or FGFR4 antibodies.13,14 The known small molecule FGFR kinase inhibitors are generally pan-inhibitors, demonstrating potent inhibition of all four isoformsfew are FGFR4-selective.15,16 FGFR1 and FGFR3

epatocellular carcinoma (HCC) accounts for 90% of liver cancer, the second leading cause of cancer mortality globally.1 With limited treatment options and poor clinical outcome for current interventions, more effective therapies are desperately needed. In recent years, aberrant fibroblast growth factor receptor 4 (FGFR4) signaling has been identified as a major driving force of HCC tumorigenesis and progression.2 The FGFR family of receptor tyrosine kinases (RTKs) comprises four conserved members, FGFR1, 2, 3, and 4. Like other RTKs, the FGFR architecture comprises an extracellular growth factor receptor domain and a single transmembrane helix that connects to a cytoplasmic kinase domain.3 The binding of a fibroblast growth factor (FGF) to the extracellular receptor domain induces dimerization and autophosphorylation of the kinase domain, triggering cellular signaling pathways.4,5 FGFR signaling regulates crucial processes including embryonic development, cell proliferation, migration, and apoptosis. Unsurprisingly, dysregulated FGFR signaling is intimately associated with malignancy.5 Growing evidence suggests that FGF19-mediated FGFR4 signaling plays an oncogenic role in HCC. FGFR4 is the most © XXXX American Chemical Society

Received: April 30, 2019 Accepted: July 3, 2019 Published: July 3, 2019 A

DOI: 10.1021/acsmedchemlett.9b00196 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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Figure 1. Molecular structures of selected pan-FGFR and FGFR4 inhibitors, BLU9931, BLU554, compounds 1−5, PD173074, H3B-6527, BGJ398, 6the lead inhibitor from Wuxi AppTec, ASP5878, FIIN-1, PRN1371, and TAS-120. IC50 values were reported by Mo et al.29

Pharma Inc.), WuXi AppTec has also developed an aminopyrimidine compound 6 (Figure 1) targeting FGFR4.28 Contemporaneous to the work described above, we have studied a series of novel inhibitors (1−5, Figure 1), afforded by a ring-opening strategy on BLU9931, that display potent and selective FGFR4 inhibition. Bulky substituents at the 3′ position of the acrylamide-bearing aniline ring appear to promote FGFR4 selectivity.29 Indeed, Hagel et al. also reported that the relatively bulky 3-methyl substitution on the aniline ring of BLU9931 helps to direct the acrylamide toward Cys552.22 Further optimization of BLU9931 to include a tetrahydropyran ring produced the drug candidate BLU554 (Figure 1), currently under phase I clinical evaluation for HCC (NCT02508467).30,31 In addition to Cys552, the ATP-binding site of FGFR4 contains another poorly conserved cysteine. The P-loop Cys477 is also present in FGFR1-3 and five other kinases.19,21 This cysteine has been exploited as the target for irreversible pan-FGFR inhibitors (Figure 1), including the first example, FIIN-1, inspired by PD173074.32 Subsequently, several acrylamide-bearing irreversible FGFR inhibitors have emerged, including TAS-120 and PRN1371, currently in phase I clinical trials.33−35 Given that the acrylamide-bearing ring of the current known FGFR4-selective inhibitors may adopt various dihedral angles,22 we hypothesized that the acrylamide group could target both hinge and P-loop cysteines and impact on selectivity and toxicity. Furthermore, the likelihood of attaining either conformation may be controlled by modifying the acrylamide substituted ring. In the current study described

inhibition produces hyperphosphataemia and tissue calcification, serious adverse effects requiring mitigation strategies in the clinic.15,17,18 Thus, there is a compelling rationale for developing FGFR4-selective inhibitors to treat patients with HCC driven by aberrant FGFR4 signaling. Target selectivity is a challenge in kinase inhibitor development because the human kinome contains ∼500 kinases all possessing a highly conserved ATP-binding site.19,20 Most kinase inhibitors are ATP-competitive and thus afford poor target selectivity. Recently, a renewed interest in covalent inhibitors has led to the rational design of compounds targeting poorly conserved nucleophilic residues in protein kinases.19,21 One such target is Cys552 in the FGFR4 kinase domain.15,22 FGFR4-selective Cys552-targeting compounds have been developed from pan-FGFR inhibitor scaffolds, including BLU9931 inspired by PD173074 but incorporating a reactive acrylamide moiety (Figure 1).22,23 The electrophilic acrylamide appended to the aniline ring forms a covalent bond with the Cys552 nucleophile via Michael addition, allowing BLU9931 to inhibit FGFR4 selectively and irreversibily. 22 The acrylamide group was presumed to afford steric hindrance toward the corresponding tyrosine residue of FGFR1-3 thereby reducing reversible binding affinity toward FGFR1-3.22,24 H3B6527 (Figure 1) has an acrylamide appended to the pan-FGFR inhibitor BGJ398.25,26 H3B-6527 is currently in phase I clinical trial for advanced HCC and intrahepatic cholangiocarcinoma (NCT02834780).25,27 By incorporating an acrylamide into the pharmacophore of pan-FGFR inhibitor ASP5878 (Astellas B

DOI: 10.1021/acsmedchemlett.9b00196 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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herein, we reveal the first reported cocrystal structure of FGFR4 complexed with clinical candidate BLU554. In addition we describe cocrystal structures of FGFR4 with the ring-opened compounds 1, 3, 4, and 5 (Figure 1), and the structure of 1 in a covalent, off-target complex with FGFR1. The relative reactivity rates toward the FGFR family members were delineated by mass spectrometry, affording a robust and rapid measurement of selectivity profiles that mirror previous kinase and cellular assays for these compounds.29 To define the reactivity of our inhibitors toward FGFR proteins, FGFR kinase domains were assayed by mass spectrometry to quantify covalent modification. Our previous experiments with TAS-120 show that covalent adduct formation can be monitored by a LC-MS-based assay over a time scale of minutes or hours.36 A 2-fold excess of BLU554 was added to FGFR1-4 proteins, with the compound showing complete modification of FGFR4 in CF3, mirroring their respective sizes. BLU9931, the parent molecule from which 1 is derived, displays significant off-target reactivity compared to clinical candidate BLU554. In all systems, a 1:1 stoichiometry of reaction is evident, and the specific location of modification was demonstrated for 1 by proteolytic digest LC-MS/MS as Cys488 of FGFR1, Cys491 of FGFR2, or Cys482 of FGFR3. (Supporting Information Figures 1−3). FGFR4 P-loop reactivity is not apparent in LC-MS/MS data for any compound because the hinge Cys552 reaction occurs so rapidly. The mass spectrometry assay broadly shows the same selectivity profile as the arguably more complex kinase and cell biology assays. The mass spectrometry results thus suggest that covalent reactivity is the primary driver of the inhibitory effects. To explain the inhibitory profiles defined by mass spectrometry, crystal structures of covalent complexes were pursued. The FGFR4−1 structure was solved (Supporting Information Tables 2 and 3; PDB 4NVH) and shows the canonical bilobed architecture in an “active” protein conformation (Figure 3A). The highly flexible P-loop and the activation loop are incompletely modeled. Compound 1 is located within the ATP-binding site covalently bonded to Cys552 (Supporting Information Figure 4A). The aminopyrimidine core and the propionamide group form hydrogen bonds with the Ala553 backbone in the hinge region (Figure 3A and 3B). The propionamide group forms a weak interaction with Arg483, which prior to covalent modification could help the acrylamide to adopt a proper trajectory toward Cys552. A leucine in FGFR1 or methionine in FGFR2/3 does not provide the hydrogen bonding potential of Arg483 in FGFR4. An additional hydrogen bond is formed between one methoxy functionality and Asp630. Hydrophobic residues Val481, Met524, Val548, Val550, Ile534, Phe631, and Leu633 form hydrophobic region I that houses the dichlorodimethoxyphenyl ring (Supporting Information Figure 4B). The aminopyrimidine core resides in a hydrophobic channel formed by Leu473, Ala553, Ala554, Gly556, and Leu619. FGFR4 crystal structures containing 3, 4, and 5 were also determined (Supporting Information Tables 2 and 3) and display near identical binding modes to 1. An overlay of FGFR4-1 with FGFR4-BLU9931 shows the marked similarity of the binding modes (Figure 3C). In both structures, the acrylamide bearing aniline ring adopts a dihedral angle of ∼60° relative to the hinge-hydrogen-bonding core. The effect of ring-opening BLU9931, affording 1, is subtle but does show improved binding geometry. Compound 1 has a flexible methyl ether linker between the hydrogen bonding pharmacophore and the tetra-substituted phenyl group, while BLU9931 has a rigid benzene ring at the equivalent position. Because of the ring opening, the linker of 1 adopts a less coplanar, lower energy conformation compared to that of BLU9931. The flexible linker of our inhibitor series also results in a tilted aminopyrimidine core relative to that of BLU9931, which may allow 1 to better fill the ATP-binding site. Specifically, Leu619 protrudes into the ligand-binding site, forcing 1 to adopt a bent conformation. This bending is not allowed in BLU9931 due to its rigid quinazoline core. Ringopening also reduces the aromatic ring count and the molecular mass of 1 compared to BLU9931, which may result in improved solubility and permeability. A model of off target reactivity was produced by X-ray crystallography. The FGFR1−1 complex in contrast to FGFR4−1 displays covalent modification of P-loop cysteine

Figure 2. Covalent reactivity of FGFR4-selective compounds BLU554 and 1−4 by mass spectrometry. (A) Time course of BLU554 reaction with FGFR1 and 4. (B) Time courses of 1−4 and BLU9931 reacted with FGFR1.

contrast, after 2 h only ∼10% of FGFR1, 2, or 3 was covalently modified. Our inhibitor series (1−4) show similarly rapid reactivity with FGFR4 and a slow reaction with FGFR1-3 (Figure 2B). Bulky groups at the R3′ position of the acrylamide bearing ring afford greater FGFR4 selectivity. Off-target FGFR1-3 reactivity follows the substituent order F > Cl > C

DOI: 10.1021/acsmedchemlett.9b00196 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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Figure 3. X-ray crystal structure of the covalent FGFR4-1 complex. (A) Overall protein structure highlighting the hinge region (blue), the P-loop (green), the covalent bond to Cys552, and hydrogen bonding interactions (dashed lines). (B) Schematic of intermolecular interactions. (C) Conformational differences between FGFR4 binding of BLU9931 (black) and 1 (black outline).

Figure 4. X-ray crystal structure of the covalent FGFR1−1 complex. (A) Overall protein structure highlighting the hinge region (blue), the P-loop (green), the covalent bond to P-loop Cys488, and hydrogen bonding interactions. (B) Schematic of intermolecular interactions. (C) Close approach of CH3 (1) to Tyr563. The CF3 (2) would clash with Tyr563 disfavoring FGFR1 binding.

aniline ring rotated ∼180°. The alternative conformation adopted by 1 in FGFR1 is afforded by the planarity of the acrylamide-bearing aniline ring that allows rotation of ∼180°

488 (Figure 4 and Supporting Information Figure 5; PDB 6NVL). In FGFR1, 1 displays the same hydrogen bonding and hydrophobic interaction patterns as in FGFR4, but with the D

DOI: 10.1021/acsmedchemlett.9b00196 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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to form the P-loop bond while still fitting the hydrophobic binding channel. Since the active site is too restrictive to allow rotation in situ, the kinase presumably samples inhibitors that are in either of the two conformations in solution. Finally, the intrinsic flexibility of the P-loop, a feature we have described previously,36,37 allows Cys488 to approach the acrylamide for covalent attack (Supporting Information Figure 5). Our results collectively suggest that steric factors, rotational freedom, and molecular dynamics determine FGFR-selectivity and off-target reactivity. The FGFR4-selectivity of ring-opened compounds is correlated with the size of the aniline ring 3′substituents, whereby larger substituents afford greater FGFR4 selectivity.29 Our series of FGFR4 crystal structures when overlaid with FGFR1−1 provide a clear steric mechanism for this effect; substituents larger than the methyl of 1 in the FGFR1-3 off-target conformation will clash with Tyr563. While the FGFR1−1 crystal structure shows that a CH3 functionality is well tolerated, its approach toward Tyr563 is close (∼3.6 Å, Figure 4C), and any group larger than this, for example the CF3 group of 2, would clash with the aromatic side chain. Thus, we predict that inhibitors with large 3′ groups will display weaker binding affinity for FGFR1-3 and greater FGFR4-selectivity. This is consistent with previously reported results of biochemical kinase inhibition assays.29 Although larger 3′-substituents may improve the FGFR4-selectivity, it may come at a cost in FGFR4 binding affinity; compound 2 (−CF3) shows a 2-fold decrease in FGFR4 kinase inhibitory activity compared to 1 (−CH3), although FGFR4-selectivity in both molecules is high.29 Another effect influencing selectivity appears to be the rotational freedom of the aniline ring in solution with close approaches between the 3′-aniline substituents and the aminopyrimidine core producing significant rotational barriers. Rotational barriers will be low for the −F substituent, for example, consistent with biochemical kinase inhibition assays showing lower FGFR4-selectivity for −F compared to −CH3 and larger substituents. The smaller ring substituents allow the molecules to freely assume different rotational configurations and for the protein to sample both hinge cysteine-targeting and P-loop cysteine-targeting conformations from solution (Figure 5). The relative dynamics of the hinge and P-loop regions that present the potential cysteine nucleophiles for reaction also minimizes off-target reactivity (Figure 5). The rigidly fixed hinge cysteine of FGFR4 presents an ideal target compared to the highly dynamic P-loop cysteine where off-target reaction requires loop “closure” to bring reactive functionalities into proximity. The BLU554 molecule affords the greatest FGFR4selectivity of all the compounds studied, and again we propose both rotational barriers and steric clash as selectivity mechanisms. While a crystal structure of the prototypic FGFR4-selective inhibitor BLU9931 has been reported, we report herein the first crystal structure of the important clinical candidate BLU554 (Figure 6A and Supporting Information Figure 6; PDB 6NVK). In FGFR4-BLU554, the acrylamide substituted tetrahydropyran ring shows a chair conformation that suggests a marked internal steric hindrance to ring rotation, preventing the alternative P-loop targeting conformation. In addition, to adopt a similar rotation as observed in FGFR1−1 and to modify the P-loop cysteine of any FGFR member, the tetrahydropyran ring would clash with FGFRconserved Leu484 (Figure 6B). These factors afford BLU554 exceptional FGFR4-selectivity; mass spectrometry confirms

Figure 5. Mechanisms of FGFR4-selectivity and off-target FGFR1 modification by 1−5 and BLU554. The size of the R3′ group influences rotational freedom in solution and steric clash on FGFR1 binding. BLU554 in an FGFR1-binding pose would clash with the Ploopits rotational freedom is also highly restricted by internal steric clash.

Figure 6. X-ray crystal structure of FGFR4-BLU554. (A) Covalent and hydrogen bonding. (B) Modeled FGFR1 binding pose highlighting P-loop clashes.

that BLU554 is barely reactive toward FGFR1-3 (Figure 1A). Future efforts in FGFR4-selective drug discovery might therefore emulate such design features of BLU554. Our studies have proven a mass spectrometry assay as an invaluable tool for studying irreversible inhibition in an in vitro setting to complement more complex cellular and biochemical kinase assays. A selectivity pattern is evident in the mass spectrometry assay data and, combined with FGFR4 and FGFR1 X-ray crystal structures, suggests multiple mechanisms for attaining FGFR4-selectivity. This study highlights the E

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(3) Hubbard, S. R.; Till, J. H. Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 2000, 69, 373−398. (4) Ullrich, A.; Schlessinger, J. Signal transduction by receptors with tyrosine kinase activity. Cell 1990, 61, 203−212. (5) Turner, N.; Grose, R. Fibroblast growth factor signalling: from development to cancer. Nat. Rev. Cancer 2010, 10, 116−129. (6) Mellor, H. R. Targeted inhibition of the FGF19-FGFR4 pathway in hepatocellular carcinoma; translational safety considerations. Liver Int. 2014, 34, e1−9. (7) Miura, S.; et al. Fibroblast growth factor 19 expression correlates with tumor progression and poorer prognosis of hepatocellular carcinoma. BMC Cancer 2012, DOI: 10.1186/1471-2407-12-56. (8) Torrecilla, S.; Llovet, J. M. New molecular therapies for hepatocellular carcinoma. Clin. Res. Hepatol. Gastroenterol. 2015, 39, S80−S85. (9) Lee, J. J. X.; Choo, S. P. The fibroblast growth factor receptor pathway in hepatocellular carcinoma. Hepatoma Res. 2018, 4, 52. (10) Hyeon, J.; Ahn, S.; Lee, J. J.; Song, D. H.; Park, C. K. Expression of fibroblast growth factor 19 is associated with recurrence and poor prognosis of hepatocellular carcinoma. Dig. Dis. Sci. 2013, 58, 1916−1922. (11) Sawey, E. T.; Chanrion, M.; Cai, C.; Wu, G.; Zhang, J.; Zender, L.; Zhao, A.; Busuttil, R. W.; Yee, H.; Stein, L.; French, D. M.; Finn, R. S.; Lowe, S. W.; Powers, S. Identification of a therapeutic strategy targeting amplified FGF19 in liver cancer by oncogenomic screening. Cancer Cell 2011, 19, 347−358. (12) Padrissa-Altés, S.; Bachofner, M.; Bogorad, R. L.; Pohlmeier, L.; Rossolini, T.; Böhm, F.; Liebisch, G.; Hellerbrand, C.; Koteliansky, V.; Speicher, T.; Werner, S. Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice. Gut 2015, 64, 1444−1453. (13) Desnoyers, L. R.; Pai, R.; Ferrando, R. E.; Hötzel, K.; Le, T.; Ross, J.; Carano, R.; D’Souza, A.; Qing, J.; Mohtashemi, I.; Ashkenazi, A.; French, D. M. Targeting FGF19 inhibits tumor growth in colon cancer xenograft and FGF19 transgenic hepatocellular carcinoma models. Oncogene 2008, 27, 85−97. (14) French, D. M.; Lin, B. C.; Wang, M.; Adams, C.; Shek, T.; Hötzel, K.; Bolon, B.; Ferrando, R.; Blackmore, C.; Schroeder, K.; Rodriguez, L. A.; Hristopoulos, M.; Venook, R.; Ashkenazi, A.; Desnoyers, L. R. Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models. PLoS One 2012, 7, No. e36713. (15) Packer, L. M.; Pollock, P. M. Paralog-specific kinase inhibition of FGFR4: Adding to the arsenal of anti-FGFR agents. Cancer Discovery 2015, 5, 355−357. (16) Lu, X.; Chen, H.; Patterson, A. V.; Smaill, J. B.; Ding, K. Fibroblast growth factor receptor 4 (FGFR4) selective inhibitors as hepatocellular carcinoma therapy: Advances and Prospects. J. Med. Chem. 2019, 62, 2905−2915. (17) Dieci, M. V.; Arnedos, M.; Andre, F.; Soria, J. C. Fibroblast growth factor receptor inhibitors as a cancer treatment: from a biologic rationale to medical perspectives. Cancer Discovery 2013, 3, 264−279. (18) Degirolamo, C.; Sabbà, C.; Moschetta, A. Therapeutic potential of the endocrine fibroblast growth factors FGF19, FGF21 and FGF23. Nat. Rev. Drug Discovery 2016, 15, 51−69. (19) Barf, T.; Kaptein, A. Irreversible protein kinase inhibitors: Balancing the benefits and risks. J. Med. Chem. 2012, 55, 6243−6262. (20) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. The protein kinase complement of the human genome. Science 2002, 298, 1912−1934. (21) Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing irreversible inhibitors of the protein kinase cysteinome. Chem. Biol. 2013, 20, 146−159. (22) Hagel, M.; Miduturu, C.; Sheets, M.; Rubin, N.; Weng, W.; Stransky, N.; Bifulco, N.; Kim, J. L.; Hodous, B.; Brooijmans, N.; Shutes, A.; Winter, C.; Lengauer, C.; Kohl, N. E.; Guzi, T. First selective small molecule inhibitor of FGFR4 for the treatment of hepatocellular carcinomas with an activated FGFR4 signaling pathway. Cancer Discovery 2015, 5, 424−437.

importance of positioning the inhibitor electrophile for maximal reactivity and how this relates to protein dynamics, intramolecular and intermolecular steric clash, and the rotational freedom and dynamics of inhibitor conformation in solution.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.9b00196. Experimental procedures, protein sequence and X-ray crystallography tables, figures showing LC-MS/MS data from FGFR modification, and X-ray crystallography imagery including electron density of covalent linkages in FGFR4 and FGFR1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*C.J.S. E-mail: [email protected]. ORCID

Xiaoyun Lu: 0000-0001-7931-6873 Ke Ding: 0000-0001-9016-812X Christopher J. Squire: 0000-0001-9212-0461 Author Contributions ⊥

X.L. and Y.Y. contributed equally. The manuscript was written through contributions of all authors.

Funding

X.L. and M.K. were supported by the Maurice Wilkins Centre for Molecular Biodiscovery. M.K. is supported by a Doctoral Scholarship from the University of Auckland. This research was further supported by the Health Research Council (New Zealand) and Cancer Society Auckland/Northland. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the Mass Spectrometry Centre (University of Auckland). This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector.

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ABBREVIATIONS FGFR, fibroblast growth factor receptor; FGF, fibroblast growth factor; LC-MS/MS, liquid chromatography−tandem mass spectrometry; RTK, receptor tyrosine kinase; HCC, hepatocellular carcinoma; BLU9931, N-[2-[[6-(2,6-dichloro3,5-dimethoxyphenyl)-2-quinazolinyl]amino]-3-methylphenyl]-2-propenamide; BLU554, N-((3S,4S)-3-((6-(2,6-dichloro3,5-dimethoxyphenyl)quinazolin-2-yl)amino)tetrahydro-2Hpyran-4-yl)acrylamide.

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REFERENCES

(1) Heimbach, J. K.; Kulik, L. M.; Finn, R. S.; Sirlin, C. B.; Abecassis, M. M.; Roberts, L. R.; Zhu, A. X.; Murad, M. H.; Marrero, J. A. AASLD guidelines for the treatment of hepatocellular carcinoma. Hepatology 2018, 67, 358−380. (2) Repana, D.; Ross, P. Targeting FGF19/FGFR4 Pathway: A novel therapeutic strategy for hepatocellular carcinoma. Diseases 2015, 3, 294−305. F

DOI: 10.1021/acsmedchemlett.9b00196 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

Letter

(23) Mohammadi, M.; Froum, S.; Hamby, J. M.; Schroeder, M. C.; Panek, R. L.; Lu, G. H.; Eliseenkova, A. V.; Green, D.; Schlessinger, J.; Hubbard, S. R. Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain. EMBO J. 1998, 17, 5896− 5904. (24) Fairhurst, R. A.; Knoepfel, T.; Leblanc, C.; Buschmann, N.; Gaul, C.; Blank, J.; Galuba, I.; Trappe, J.; Zou, C.; Voshol, J.; Genick, C.; Brunet-Lefeuvre, P.; Bitsch, F.; Graus-Porta, D.; Furet, P. Approaches to selective fibroblast growth factor receptor 4 inhibition through targeting the ATP-pocket middle-hinge region. MedChemComm 2017, 8, 1604−1613. (25) Joshi, J. J.; Coffey, H.; Corcoran, E.; Tsai, J.; Huang, C. L.; Ichikawa, K.; Prajapati, S.; Hao, M. H.; Bailey, S.; Wu, J.; Rimkunas, V.; Karr, C.; Subramanian, V.; Kumar, P.; MacKenzie, C.; Hurley, R.; Satoh, T.; Yu, K.; Park, E.; Rioux, N.; Kim, A.; Lai, W. G.; Yu, L.; Zhu, P.; Buonamici, S.; Larsen, N.; Fekkes, P.; Wang, J.; Warmuth, M.; Reynolds, D. J.; Smith, P. G.; Selvaraj, A. H3B-6527 Is a potent and selective inhibitor of FGFR4 in FGF19-Driven hepatocellular carcinoma. Cancer Res. 2017, 77, 6999−7013. (26) Pal, S. K.; Rosenberg, J. E.; Hoffman-Censits, J. H.; Berger, R.; Quinn, D. I.; Galsky, M. D.; Wolf, J.; Dittrich, C.; Keam, B.; Delord, J. P.; Schellens, J. H. M.; Gravis, G.; Medioni, J.; Maroto, P.; Sriuranpong, V.; Charoentum, C.; Burris, H. A.; Grünwald, V.; Petrylak, D.; Vaishampayan, U.; Gez, E.; De Giorgi, U.; Lee, J. L.; Voortman, J.; Gupta, S.; Sharma, S.; Mortazavi, A.; Vaughn, D. J.; Isaacs, R.; Parker, K.; Chen, X.; Yu, K.; Porter, D.; Graus Porta, D.; Bajorin, D. F. Efficacy of BGJ398, a fibroblast growth factor receptor 1−3 inhibitor, in patients with previously treated advanced urothelial carcinoma with FGFR3 alterations. Cancer Discovery 2018, 8, 812− 821. (27) Rioux, N.; Kim, A.; Nix, D.; Bowser, T.; Warmuth, M.; Smith, P. G.; Schindler, J. Effect of a high-fat meal on the relative bioavailability of H3B-6527, a novel FGFR4 inhibitor, in healthy volunteers. Cancer Chemother. Pharmacol. 2019, 83, 91−96. (28) Wang, Y.; Chen, Z.; Dai, M.; Sun, P.; Wang, C.; Gao, Y.; Zhao, H.; Zeng, W.; Shen, L.; Mao, W.; Wang, T.; Hu, G.; Li, J.; Chen, S.; Long, C.; Chen, X.; Liu, J.; Zhang, Y. Discovery and optimization of selective FGFR4 inhibitors via scaffold hopping. Bioorg. Med. Chem. Lett. 2017, 27, 2420−2423. (29) Mo, C.; Zhang, Z.; Guise, C. P.; Li, X.; Luo, J.; Tu, Z.; Xu, Y.; Patterson, A. V.; Smaill, J. B.; Ren, X.; Lu, X.; Ding, K. 2Aminopyrimidine derivatives as new selective fibroblast growth factor receptor 4 (FGFR4) inhibitors. ACS Med. Chem. Lett. 2017, 8, 543− 548. (30) Kim, R.; Sarker, D.; Macarulla, T.; Yau, T.; Choo, S. P.; Meyer, T.; Hollebecque, A.; Whisenant, J.; Sung, M.; Yoon, J.-H.; Lim, H. Y.; Zhu, A.; Park, J.-W.; Faivre, S.; Mazzaferro, V.; Shi, H.; SchmidtKittler, O.; Clifford, C.; Wolf, B.; Kang, Y.-K. Phase 1 safety and clinical activity of BLU-554 in advanced hepatocellular carcinoma (HCC). Ann. Oncol. 2017, DOI: 10.1093/annonc/mdx367. (31) Kim, R.; Sharma, S.; Meyer, T.; Sarker, D.; Macarulla, T.; Sung, M.; Choo, S. P.; Shi, H.; Schmidt-Kittler, O.; Clifford, C.; Wolf, B.; Llovet, J. M. First-in-human study of BLU-554, a potent, highlyselective FGFR4 inhibitor designed for hepatocellular carcinoma (HCC) with FGFR4 pathway activation. Eur. J. Cancer 2016, 69, S41. (32) Zhou, W.; Hur, W.; McDermott, U.; Dutt, A.; Xian, W.; Ficarro, S. B.; Zhang, J.; Sharma, S. V.; Brugge, J.; Meyerson, M.; Settleman, J.; Gray, N. S. A structure-guided approach to creating covalent FGFR inhibitors. Chem. Biol. 2010, 17, 285−295. (33) Sootome, H.; Fujioka, Y.; Miura, A.; Fujita, H.; Hirai, H.; Utsugi, T. TAS-120, an irreversible FGFR inhibitor, was effective in tumors harboring FGFR mutations, refractory or resistant to ATP competitive inhibitors. Mol. Cancer Ther. 2013, DOI: 10.1158/15357163.TARG-13-A271. (34) Brameld, K. A.; Owens, T. D.; Verner, E.; Venetsanakos, E.; Bradshaw, J. M.; Phan, V. T.; Tam, D.; Leung, K.; Shu, J.; LaStant, J.; Loughhead, D. G.; Ton, T.; Karr, D. E.; Gerritsen, M. E.; Goldstein, D. M.; Funk, J. O. Discovery of the irreversible covalent FGFR inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-

dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)one (PRN1371) for the treatment of solid tumors. J. Med. Chem. 2017, 60, 6516−6527. (35) Venetsanakos, E.; Brameld, K. A.; Phan, V. T.; Verner, E.; Owens, T. D.; Xing, Y.; Tam, D.; LaStant, J.; Leung, K.; Karr, D. E.; Hill, R. J.; Gerritsen, M. E.; Goldstein, D. M.; Funk, J. O.; Bradshaw, J. M. The irreversible covalent fibroblast growth factor receptor inhibitor PRN1371 exhibits sustained inhibition of FGFR after frug clearance. Mol. Cancer Ther. 2017, 16, 2668−2676. (36) Kalyukina, M.; Yosaatmadja, Y.; Middleditch, M. J.; Patterson, A. V.; Smaill, J. B.; Squire, C. J. TAS-120 cancer target binding; defining reactivity and revealing the first FGFR1 irreversible structure. ChemMedChem 2019, 14, 494−500. (37) Yosaatmadja, Y.; Patterson, A. V.; Smaill, J. B.; Squire, C. J. The 1.65 Å resolution structure of the complex of AZD4547 with the kinase domain of FGFR1 displays exquisite molecular recognition. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2015, 71, 525−533.

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DOI: 10.1021/acsmedchemlett.9b00196 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX