An overview of the binding models of FGFR tyrosine kinases in complex with small molecule inhibitors

Accepted Manuscript An overview of the binding models of FGFR tyrosine kinases in complex with small molecule inhibitors Weiyan Cheng, Mixiang Wang, Xin Tian, Xiaojian Zhang PII:

S0223-5234(16)30998-9

DOI:

10.1016/j.ejmech.2016.11.052

Reference:

EJMECH 9085

To appear in:

European Journal of Medicinal Chemistry

Received Date: 12 September 2016 Revised Date:

19 October 2016

Accepted Date: 7 November 2016

Please cite this article as: W. Cheng, M. Wang, X. Tian, X. Zhang, An overview of the binding models of FGFR tyrosine kinases in complex with small molecule inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.052. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

ACCEPTED MANUSCRIPT An Overview of the Binding Models of FGFR Tyrosine Kinases in Complex with Small Molecule Inhibitors Weiyan Cheng1, Mixiang Wang2, Xin Tian1* and Xiaojian Zhang1* 1

Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China

Department of Pharmacy, The First Affiliated Hospital of Nanyang Medical College, Nanyang 473000,

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2

China Abstract

The fibroblast growth factor receptor (FGFR) family receptor tyrosine kinase (RTK) includes four

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structurally related members, termed as FGFR1, FGFR2, FGFR3, and FGFR4. Given its intimate role in the progression of several solid tumors, excessive FGFR signaling provides an opportunity for

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anticancer therapy. Along with extensive pharmacological studies validating the therapeutic potential of targeting the FGFRs for cancer treatment, co-crystal structures of FGFRs/inhibitors are continuously coming up to study the mechanism of actions and explore new inhibitors. Herein, we review the reported co-crystals of FGFRs in complex with the corresponding inhibitors, main focusing our attention on the binding models and the pharmacological activities of the inhibitors. FGFR;

small

pharmacological activity

molecule

inhibitor;

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Keywords:

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*Corresponding author: Xin Tian, Xiaojian Zhang Department of Pharmacy,

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The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.

E-mail: [email protected]; [email protected]

crystal

structure;

DFG-in/out;

irreversible;

ACCEPTED MANUSCRIPT 1. Introduction The fibroblast growth factor receptors (FGFRs) are a family members of receptor tyrosine kinase (RTK) that represent attractive therapeutic targets for anti-cancer therapy gaining more and more attention in recent years [1, 2]. This family members are comprised by FGFR1, FGFR2, FGFR3, and FGFR4, which share significant sequence homology [3]. Like other RTKs [4-6], each of the receptors consists

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of an extracellular ligand binding domain, a single transmembrane domain, and a cytosolic region with a split tyrosine kinase binding domain [7].

For signal transduction (Figure 1), the extracellular fibroblast growth factors (FGFs) bind to a

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cell-surface FGFR in a ternary complex consisting of FGF, FGFR, and heparan sulfate proteoglycans (HPSG) [8]. Dimerization of the ternary FGF/FGFR/HPSG complex leads to a conformational shift in

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the FGFR structure, resulting in intermolecular transphosphorylation of the intracellular tyrosine kinase domain and carboxy-terminal tail [9]. Subsequent downstream signaling occurs through two main pathways via the intracellular receptor substrates FGFR substrate 2 (FRS2) and phospholipase Cγ (PLCγ), leading ultimately to upregulation of the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)-Akt, diacylglycerol-Protein kinase C (DAG-PKC), and inositol

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trisphosphate (IP3)-Ca2+-releasing signaling pathways [10, 11].

The FGFR family tyrosine kinases serve as high affinity receptors for the FGFs that control cell proliferation, migration, apoptosis, and differentiation and are involved in both developmental and adult tissue homeostasis [12]. Besides, many cancer cell types have been recognized to overexpress

and so on.

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FGFRs, which include breast cancer [13], lung cancer [14], gastric cancer [15], endometrial cancer [16],

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As for FGFRs playing such an important role in tumors progression, the crystal structures of these kinases have been confirmed and the binding models of ATP in complex with these kinases have been determined in order to understand their mechanism of actions [17-20]. Besides, according to the binding features, a large number of FGFR inhibitors have been explored and meaningful clinical outcomes of these inhibitors have been acquired [21-25]. Many published articles have reviewed the development of FGFR inhibitors [26-30], however, the reports that specially highlight the co-crystal structures of FGFRs in complex with inhibitors have not been found. In consideration of their vital roles in the exploration of molecules’ mechanism of actions and development of new drugs, we conclude the reported co-crystals of FGFRs complexed with the corresponding inhibitors herein, main

ACCEPTED MANUSCRIPT focusing our attention on the binding models and the pharmacological activities of the inhibitors.



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2. Categorization of the binding models of FGFR inhibitors in complex with receptors The binding models of FGFR inhibitors in complex with receptors can be grouped as Asp-Phe-Gly (DFG)-in bindings, DFG-out bindings, and irreversible bindings. DFG is a conserved activation loop playing an important role in regulating kinase activity. Inhibitors binding as the DFG-in conformation

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are named as Type I inhibitors, they are ATP-competitive inhibitors that bind to the active forms of kinases with the aspartate residue of the DFG motif facing into the active site of the kinase (Figure

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2a). Correspondingly, Type II inhibitors exhibit the DFG-out binding conformation, they bind to the inactive forms of kinase with the aspartate residue of the DFG motif protruding outward from the ATP-binding site of the kinase. Importantly, the DFG-out state opens a new allosteric pocket directly adjacent to the ATP binding pocket which facilitates inhibitor binding (Figure 2b). Inhibitors with irreversible bindings tend to covalently bind with a reactive nucleophilic cysteine residue proximal to

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the ATP-binding site, resulting in the blockage of the ATP site and irreversible inhibition (Figure 2c) [31-33].

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2.1. FGFR/inhibitor complexes with DFG-in binding models



2.1.1. FGFR1 in complex with SU4984 (1, Figure 3) and SU5402 (2, Figure 3) SU4984

and

SU5402

are

two

structure

similar

compounds

bearing

the

same

3-benzylideneindolin-2-one core. Both compounds are confirmed to inhibit FGFR1 with the IC50 values of 10 to 20 µM in the presence of 1 mM ATP. In addition, auto-phosphorylation of FGFR1 induced by FGF can also be inhibited by the two compounds with the IC50 values of 20 to 40 µM for SU4984 and 10 to 20 µM for SU5402 [34].

ACCEPTED MANUSCRIPT The co-crystal structures of FGFR1 in complex with SU4984 and SU5402 are firstly reported. In these complexes (Figure 4, PDB code: 1AGW for SU4984; 1FGI for SU5402), the two inhibitors bind to FGFR1 in the same general region as ATP in DFG-in model. The oxindole of the inhibitors occupies the same site as the ATP adenine. The chemical groups attached to C-3 of the oxindole emerge from

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the cleft at approximately right angles to the direction taken by the rest of the ATP molecule. The oxindole makes two hydrogen bonds to the protein backbone of FGFR1: between N-1 of the oxindole and the carbonyl oxygen of Glu562, and between O-2 of the oxindole and the amide nitrogen of Ala564. The cavity in which the oxindole binds is lined with numerous hydrophobic residues including

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Val492, Ala512, Ile545, Val561, Ala564, and Leu630. In addition, Leu484 and Tyr563 provide a hydrophobic environment for the ring proximal to the oxindole: a phenyl in SU4984 and a pyrrole in

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SU5402. In terms of the binding difference between the two complexes, in the FGFR1/SU4984 complex, the phenyl ring of SU4984 makes an oxygen-aromatic contact with the carbonyl oxygen of Ala564, the piperazine ring of SU4984 is in van der Waals contact with Gly567, and the terminal formyl group of SU4984 is disordered. While in the FGFR1/SU5402 structure, the NH of the pyrrole ring makes an intramolecular hydrogen bond with the O of the oxindole, the methyl group of the

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pyrrole ring is in van der Waals contact with Gly567, and the carboxyl-ethyl group attached to the pyrrole ring is hydrogen bonded to the side chain of Asn568 [34].

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2.1.2. FGFR1 in complex with CH5183284 (3, Figure 3)

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CH5183284 or Debio1347, a 1-(1H-benzimidazol-5-yl)-5-aminopyrazole derivative that currently advanced in phase I clinical trials, is a potent, and orally available FGFR1, FGFR2, and FGFR3 inhibitor with the IC50 values of 9.3, 7.6, and 22 nM, respectively, while less potently inhibits FGFR4 (IC50: 290 nM), KDR (IC50: 2100 nM), and other kinases [25, 35]. To understand the mechanism of selectivity of CH5183284 for FGFR1, FGFR2, and FGFR3 over other kinases, the three-dimensional structure of a complex of CH5183284 and the protein kinase domain of FGFR1 is solved (Figure 5, PDB code: 5B7V). Analysis of the co-crystals reveals that CH5183284 binds to the ATP-binding site of FGFR1 in a DFG-in mode, and five hydrogen bonds are found. Two of these hydrogen bonds occur between the benzimidazole moiety of CH5183284 and a backbone nitrogen atom of Asp641 and a side

ACCEPTED MANUSCRIPT chain oxygen atom of Glu531 of FGFR1. The remaining three hydrogen bonds are identified between the hinge binder of CH5183284 and the hinge region of FGFR1 at Glu562, Tyr563, and Ala564. The benzimidazole moiety interacts with FGFR1 at the back pocket, and three unique interactions are suggested. One of them is an interaction between Phe642 in FGFR1 and the methyl group at the

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benzimidazole moiety of CH5183284, which enhances molecule’s selectivity and potency against FGFR. Another unique interaction is between sulfur atom of Met535 in FGFR1 and the nitrogen atom and the methyl group of the benzimidazole moiety of CH5183284, which explains the compound’s selectivity towards KDR. The third unique interaction is between Val561, a gatekeeper residue of

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FGFR1, and aromatic ring of the benzimidazole moiety of CH5183284. Although this interaction is conserved among FGFRs, CH5183284 has wider space between FGFR1 and the aromatic ring because

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CH5183284 does not have bulky methoxy moiety common to other FGFR selective inhibitors [36-41], so CH5183284 might be able to accept the mutation at a gatekeeper residue which is insensitive to other FGFR inhibitors. By interacting with these three unique residues, CH5183284 obtains FGFR selective kinase inhibitory activity, and this interaction mode is different from other FGFR inhibitors

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[42].



2.1.3. FGFR1 in complex with PD173074 (4, Figure 3)

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PD173074 is a pyrido[2,3-d]pyrimidine derivative that effectively and selectively inhibits FGFR1 and FGFR2 with the IC50 values of 21.5 and 5 nM, respectively [43-45]. For crystallographic studies, the

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co-crystals of PD173074/FGFR1 are obtained (Figure 6, PDB code: 2FGI and 5A4C). In this complex, PD173074 binds in the ATP-binding cleft of FGFR1 in a DFG-in conformation, which lies between the two lobes of the kinase. The pyrido[2,3-d]pyrimidine ring system occupies the same position as the ATP adenine. Besides, N-3 of the pyrimidine ring is hydrogen-bonded to the amide nitrogen of Ala564, and the nitrogen of the butylamino group is hydrogen-bonded to the carbonyl oxygen of Ala564. A third hydrogen bond is observed between one of the methoxyl groups of PD173074 and the amide nitrogen of Asp641, a residue which is part of the protein kinase-conserved DFG located at the beginning of the activation loop. The urea group attached to the pyrido[2,3-d]pyrimidine at the C-7 position makes an internal hydrogen bond with N-8 of the ring system and is hydrogen-bonded to a

ACCEPTED MANUSCRIPT water molecule, this water molecule in turn is hydrogen-bonded to the side chains of protein kinase-conserved Lys514 and Asp641 [44, 46].



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2.1.4. FGFR1 in complex with JNJ-42756493 (5, Figure 3) JNJ-42756493 is a potent, oral pan-FGFR inhibitor with the IC50 values in the low nanomolar range for all FGFR family members, while with minimal activity on VEGFRs (approximately 20-fold potency difference) [47]. According to the binding model of FGFR1 in complex with JNJ42756493 (Figure 7,

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PDB code: 5EW8), JNJ42756493 occupies the ATP-binding cleft of FGFR1, and the activation loop clearly exhibits a DFG-in conformation. The quinoxaline core of JNJ42756493 is observed to form a

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single hydrogen bond to the hinge region via the main chain amide of Ala564, while the dimethoxyphenyl ring is orientated perpendicular to the quinoxaline core and occupies the hydrophobic pocket located behind the gatekeeper residue (Val561). One of the methoxyl oxygen atoms is involved in a hydrogen bond with the backbone nitrogen atom of the DFG aspartate (Asp641). The methyl pyrazole solubilizing group extends away from the hinge region towards the solvent channel and does

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not make any specific interactions with the protein. A unique feature of JNJ42756493 is the amide side chain which extends into the region of the binding site normally occupied by the α-phosphate of ATP where it forms a hydrogen bond to the side chain of Asp641. In addition, the terminal isopropyl group

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of this side chain also makes good van der Waals interactions with the protein in a shallow pocket formed by the side chains of Asn628, Leu630, Ala640, and Asp641 that has been referred to as the “pit”

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region [48].



2.1.5. FGFR1 in complex with BGJ398 (6, Figure 3) BGJ398, a N-aryl-N’-pyrimidin-4-yl urea derivative, potently inhibits FGFR1, FGFR2, and FGFR3 with the respective IC50 values of 1 nM, and inhibits FGFR4 with an IC50 value of 60 nM, while exhibiting promising selectivity against other kinases including FYN, LCK, YES, and ABL [40, 49]. According to the co-crystal structure of FGFR1 in complex with BGJ398 at 2.8 Å resolution (Figure 8, PDB code: 3TT0), the 4-(4-ethyl-piperazin-1-yl)-phenylamine NH and the adjacent pyrimidine

ACCEPTED MANUSCRIPT nitrogen of BGJ398 are involved in critical hydrogen bonds with the carbonyl and the amino groups of Ala564, respectively. An additional hydrogen bond occurs between the backbone carbonyl group of Glu562 and the pyrimidine C-2 hydrogen, which behaves as a hydrogen bond donor by virtue of the polarization induced by the two adjacent nitrogen atoms. It is noteworthy that the urea carbonyl group

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also engages in a water-mediated hydrogen bond with the side chain amino group of Lys514, whereas the aryl ring of the 4-(4-ethyl-piperazin-1-yl)-phenylamine is in contact with the hydrophobic side chains of two amino acid residues Gly567 and Leu484. The optimized substitution pattern of the urea aniline is responsible for productive interactions such as the hydrogen bond between the methoxyl O

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and the NH of Asp641 in addition to further hydrophobic contacts. The optimal fitting of the tetra-substituted phenyl ring in the complementary hydrophobic cavity results from the perpendicular

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orientation of this ring with respect to the plane of the pseudobicyclic system. Such a rigid orthogonal arrangement is enforced by the two chlorine atoms which prevent aromatic conjugation. These atoms are involved in favorable contacts with the gate keeper Val561 and with Ala640, respectively [49].



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2.1.6. FGFR1C488A/C584C in complex with lucitanib (7, Figure 3) Lucitanib or E-3810, currently advanced in phase II clinical trials [50], is a dual inhibitor of FGFR and VEGFR, it potently inhibits FGFR1 and FGFR2 with the IC50 values of 17.5 and 82.5 nM, respectively,

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and inhibits VEGFR1, VEGFR2, and VEGFR3 with the IC50 values of 7, 25 and 10 nM, respectively [51, 52]. Lucitanib binds FGFR1C488A/C584C as a Type I inhibitor (Figure 9, PDB code: 4RWL). In this

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binding model, one hydrogen bond is formed between lucitanib’s quinolin N and the backbone NH of hinge residue Ala564. Besides, the N-methyl-1-naphthamide moiety of lucitanib extends towards the deep pocket comprised by Val561, Ile545, Asp641, Glu531, and Lys514, and two hydrogen bonds are found between this moiety and Asp641 and Glu531. Moreover, an indirect hydrogen bond is formed between the NH2 of the 1-aminocyclopropylmethyl group and the carbonyl O of Glu571 through a water molecule [53].



2.1.7. Wild-type and mutant FGFR1 in complex with AZD4547 (8, Figure 3)

ACCEPTED MANUSCRIPT AZD4547, advanced in phase II clinical studies [54], is a highly potent inhibitor of FGFR1, FGFR2, and FGFR3 in vitro (IC50 values of 0.2, 2.5, and 1.8 nM, respectively) and displays weaker activity against FGFR4 (IC50 value of 165 nM) [41]. Tucker and coworkers report the complex of AZD4547 bound to the kinase domain of wild-type (WT)

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FGFR1 at 1.65 Å resolution (Figure 10a, PDB code: 4V05) [55]. In the complex, the AZD4547 molecule is located deeply at the ATP-binding site, which is consistent with the active kinase conformation of the protein. Three hydrogen bonds are formed in the hinge region of the protein, specifically between N2 and Ala564 O, between N4 and Ala564 N and between N3 and Glu562 O. A

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hydrogen bond is also formed from the dimethoxyphenyl ring O1 atom to the C-terminal lobe atom Asp641 N. In another co-crystal structure of FGFR1WT/AZD4547 (Figure 10b, PDB code: 4WUN)

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[56], it is found that the carbonyl O of AZD4547 is hydrogen-interacted with a solvent network which is comprised by three water molecules and related residues (Leu484, Glu486, Ala488, Phe489, and Gly490).

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Anderson’s group determines two co-crystal structures of FGFR1 mutations in complex with AZD4547: the

FGFR1C488A/C584S/AZD4547

complex

and

the

FGFR1

gatekeeper

mutation

FGFR1C488A/C584S/V561M/AZD4547 complex [53]. The binding model of FGFR1C488A/C584S/AZD4547

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(Figure 10c, PDB code: 4RWJ) is similar to one of the WT bindings (Figure 10a), except that an additional hydrogen bond is observed between the AZD4547 carbonyl O and the backbone NH of

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Gly485. Surprisingly, in the FGFR1C488A/C584S/V561M/AZD4547 complex, AZD4547 binds to each of the molecules of the asymmetric unit of FGFR1C488A/C584S/V561M structure in two different conformations: WT-like inhibitor conformation and bent inhibitor conformation (PDB code: 4RWK). In the WT-like inhibitor conformation (Figure 11a), AZD4547 binds to FGFR1C488A/C584S/V561M in a very similar fashion to FGFR1C488A/C584S, however, the hydrogen bond is lost between the AZD4547 and the backbone of Gly485 that is observed in the FGFR1C488A/C584S/AZD4547 complex. In addition, minor adjustments are observed for AZD4547 in the P-loop and hinge region in order to accommodate the increase in length that occurs upon replacing a valine residue with a methionine. In the bent inhibitor conformation (Figure 11b), significant changes are observed compared with the WT-like model.

ACCEPTED MANUSCRIPT Severe bending in the ethyl linker connecting the dimethoxyphenyl and the pyrazole moieties of AZD4547 occurs in order to better accommodate the methionine mutation. These findings suggest that regions of flexibility in the inhibitor and inhibitor binding pocket are critical for developing future



2.1.8. Optimization of compound 9 (Figure 3)

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FGFR inhibitors with delayed resistance profiles.

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Compound 9 is a FGFR1 inhibitor that explored utilizing the scaffold hopping strategy based on the structure of AZD4547. It shows good enzymatic inhibitory (IC50 value of 15.0 nM against FGFR1) and

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modest cellular inhibitory (IC50 value of 642.1 nM against SNU-16 cells) activities. To characterize the mechanism of FGFR1 inhibition by 9, the co-crystal structure of FGFR1 bound to compound 9 is obtained (Figure 12, PDB code: 4ZSA). Analysis of the bindings indicates that compound 9 fits well into the ATP binding site of FGFR1 as a Type I inhibitor, and the 3-aminoindazole ring system participates in hydrogen-bonding interactions with the backbone amides in the hinge region of the

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protein. In detail, the 2-N and the 3-NH of the 3-aminoindazole form two hydrogen bonds with Ala564, and the indazole intro-NH forms another hydrogen bond with Glu562. Besides, the 3-methoxyphenyl substituted at 3-aminoindazole forms strong hydrophobic interactions with the hydrophobic pocket residues Phe489, Lys514, Leu547, and Val561. Co-crystal structure analysis of compound 9 reveals

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that an additional halogen substituents in the methoxylphenyl moiety may be helpful to improve the membrane permeability and enhance cellular potencies. Therefore, a series of compounds bearing a

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fluorine or chlorine atom at the ortho-, meta- or para-position of the phenyl ring are obtained. Fortunately, compound 10 containing a fluorine atom at the ortho-position is found to be the most potent one both in enzymatic (IC50 value of 2.9 nM against FGFR1) and cellular (IC50 value of 40.5 nM against NU-16 cells) inhibitory activities [57].



2.1.9. Structure based design of compound 15 (Figure 3)



ACCEPTED MANUSCRIPT Norman and coworkers use protein−ligand crystal structure information to guide the design of selective FGFR inhibitors [58]. Firstly, compound 11 (Figure 3) is chosen as a template. 11 is a 2, 4-diaminopyrimdine derivative that potently inhibits FGFR1, KDR, and IGF1R with the pIC50 values of 6.3, 6.2 and 5.2, respectively. The binding model of FGFR1/11 (PDB code: 4F63) is shown in

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Figure 13a, in this model, three hydrogen bonds are found between the 3-aminopyrazol moiety of compound 11 and the hinge residues of Glu562 and Ala564, the 5-Me on the pyrazole is directed toward the gatekeeper Val561. Meanwhile, two conformations are observed in the two FGFR1/11

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complexes in the crystal asymmetric unit due to the flexibility of compound 11. The NHCH2CH2 linker is sufficiently flexible that it can presumably adapt to different steric environment (FGFR1 and KDR)

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in the ATP pocket but be adverse for selectivity.

Compound 12 (Figure 3) is more conformationally rigid than 11, it shows the pIC50 values of 7.2, 5.9 and 4.7 against FGFR1, KDR, and IGF1R, respectively. In the co-crystal structure of 12 in complex with FGFR1 (Figure 13b, PDB code: 4F64), only one conformation is observed in the two molecules of the crystallographic dimer. Besides the same hydrogen bonds presented in FGFR1/11 complex, the

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structure also shows that the methyl group on the isoxazole is directed toward the “pit” region which is caused due to the small side chain of Ala640, suggesting a beneficial hydrophobic interaction that would also favor this conformation [58].

In the binding models of compound 11 in complex with FGFR1, it can be seen that the 5-substituent on

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the pyrazole is directed toward the gatekeeper Val561. The author hypothesizes that an aryl group suitably linked at this position can be tolerated. Basis on this hypothesis, compound 13 (Figure 3) is

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designed and selected for screen. As a result, 13 exhibits increased potency against FGFR1 and KDR (pIC50 vales of 8.6 and 7.6, respectively). Meanwhile, the author’s hypothesis is confirmed by the co-crystal structures of compound 13 bound to FGFR1 (Figure 13c, PDB code: 4F65). In this complex, the phenyl group does indeed occupy the hydrophobic pocket, fitting tightly against the gatekeeper Val561, while the methyl isoxazole protrudes into the “pit” near Ala640. This indicates that 13 utilizes the various specificity-enhancing features of FGFR1. However, the lipophilicity of compound 13 is higher and is implicated as the cause of a number of problems including poor solubility. Meanwhile, the bromine substituent on the pyrimidine ring is not considered an essential element for FGFR inhibition.

ACCEPTED MANUSCRIPT 14 (Figure 3) is a desbromine analogue of compound 13 (pIC50 value of 8.1 against FGFR1) [59], the FGFR1-bound conformation of 14 (Figure 13d, PDB code: 4NKS) is similar to that of compound 13. In this structure the plane of the phenyl ring is perpendicular to the plane of the molecule core. An alternative conformation of the ethyl linker between the pyrazole and the phenyl rings of 14, however,

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positions the phenyl group of 14 close to the hydrophobic side chains of Val491, Val561, and Val559. Compound 15 (Figure 3), in which the bromine atom is removed and the phenyl ring is substituted by 3, 5-dimethoxyphenyl compared with compound 13, is targeted for further study. The biological evaluation results indicate that 15 maintains the same level of inhibition of FGFR1 (pIC50: 8.7) as 13

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while having the same or improved selectivity compared to KDR, IGF1R, and InsR [58]. In the crystal structure of FGFR1 in complex with 15 (Figure 13e, PDB code: 4NK9) [59], the

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pyrazolylaminopyrimidine core forms three hydrogen bonds to the hinge region of the kinase domain (Glu562 and Ala564). The methyl isoxazole protrudes into a constricted indentation at the base of the ATP pocket (the “pit” region), and the nitrogen of this heterocycle makes a further hydrogen bond to a water molecule. This water molecule in turn is hydrogen-bonded to the side chains of protein kinase-conserved Lys514 and Asp641 and the backbone nitrogen of Ala488. Moreover, the dimethoxy

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phenyl group of 15 binds to the hydrophobic pocket, where it packs against the gatekeeper residue Val561. One of the methoxy groups makes a hydrogen bond with the amide nitrogen of Asp641, part of the protein kinase-conserved DFG triad at the beginning of the activation loop.

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Compound 16 (Figure 3), changing the substitution pattern of the phenyl ring as 3, 4-dimethoxy, exhibits a dramatic loss of inhibitory potency against FGFR1 (pIC50 value of 7.0) compared with the 3,

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5-dimethoxy-bearing compound 15 (pIC50 value of 8.7). The crystal structure of the FGFR1/16 complex (Figure 13f, PDB code: 4NKA) provides insights in this regard. It shows that replacement of the 3, 5-dimethoxyphenyl group in 15 by a 3, 4-dimethoxyphenyl moiety significantly changes the FGFR1-bound inhibitor conformation. In contrast to 15, which binds to FGFR1 with the planes of the phenyl ring approximately perpendicular to the plane of the pyrazolyl-amino-pyrimidine, the 3, 4-dimethoxy substitution in 16 forces the phenyl and the pyrazolyl-amino-pyrimidine rings into a parallel geometry, most likely for steric reasons [59]. 2.1.10. Dovitinib (17, Figure 3) in complex with FGFR1 and FGFR4 Dovitinib is a multi-target kinase inhibitor that shows activity against FGFRs (IC50 values of 8 and 9 nM against FGFR1 and FGFR3, respectively), VEGFRs (IC50 values of 10, 13 and 8 nM against

ACCEPTED MANUSCRIPT VEGFR1 VEGFR2, and VEGFR3, respectively) and other RTKs (FLT3, c-KIT, CSF-1R/c-fms, and PDGFR, IC50 values of 1-36 nM) [60]. Three co-crystal structures of dovitinib in complex with FGFRs (FGFR1WT, FGFR1V561M, and FGFR4) have been reported.

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According to the co-crystal structure of dovitinib/FGFR1WT (Figure 14a, PDB code: 5AM6, 5A46), dovitinib binds as a Type I inhibitor, it is stabilized by hydrogen bonds, hydrophobic and van der

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Waals contacts. Five hydrogen bonds (involving residues Glu562, Ala564 and Ser565) and ten amino acid residues involved in hydrophobic interactions provide a direct contact between FGFR1WT and

with atom N29 of dovitinib [46, 61]. Structural

insights

highlight

the

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dovitinib and stabilizes the drug-protein complex, meanwhile, one water molecule is found to interact

underpinning

differences

between

the

complexes

of

dovitinib/FGFR1WT and dovitinib/FGFR1V561M (Figure 14b, PDB code: 5AM7). In the mutant binding complex, dovitinib binding is strengthened by the gatekeeper residue Met561, at its binding site by increasing the number of hydrophobic contacts. Met561 pushes two water molecules and Lys514 out of

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the binding cleft, which affect the bindings in the dovitinib/FGFR1WT structure. Thus in the gatekeeper mutant, the polar charge contributed by the two water molecules and Lys514 are void at the binding cleft, leading to a stronger affinity of dovitinib to the kinase [61].

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The DFG-in conformation of FGFR4 is seen in complex with dovitinib (Figure 14c, PDB code: 4TYI). The quinolone group of dovitinib binds in the ATP-binding pocket of FGFR4 and is stabilized by the

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hydrophobic contacts with the gatekeeper residue Val550, Val481 and Leu619. In addition, the backbone atoms of Ala553 form three hydrogen bounds with the carbonyl oxygen of the quinolone group and the nitrogen of the benzimidazole group [20]. 2.2. FGFR/inhibitor complexes with DFG-out binding models



2.2.1. ARQ069 (18, Figure 15) in complex with FGFR1 and FGFR2 ARQ069 is a potent inhibitor of FGFR1 and FGFR2 with the IC50 values of 0.84 and 1.23 µM, respectively. ARQ069 binds both FGFR1 and FGFR2 in their inactive forms. In the co-crystal structure

ACCEPTED MANUSCRIPT of ARQ069 in complex with FGFR1 (Figure 16a, PDB code: 3RHX), the aminopyrimidine group makes two hydrogen bond interactions with the hinge region residue of Ala564. The 5, 6-dihydrobenzo[h]quinazolin-2-amine core of ARQ069 is sandwiched in a hydrophobic cleft between residues Val492, Leu484, Ala512, Tyr563, and Leu630. The plane of the phenyl ring of ARQ069 is

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perpendicular to the 5, 6-dihydrobenzo-[h]quinazolin-2-amine core and occupies the main pocket. This phenyl ring makes hydrophobic contacts with the gatekeeper residue Val561 and the methionine residue of the α-helix (Met535). One major distinguishing feature between apo-FGFR1 and the FGFR1/ARQ069 complex is the significant difference found within the glycine-rich loop. When the

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inhibitor binds, the phenylalanine (Phe489) of the glycine-rich loop makes a downward movement and establishes van der Waals interactions with the fused phenyl ring of ARQ069. The binding mode of

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ARQ069/FGFR2 (Figure 16b, PDB code: 3RI1) is very similar with that of FGFR1, with only the distinct glycine-rich loop conformations [62].



2.2.2. Ponatinib (19, Figure 15) in complex with FGFR1 and FGFR4

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Ponatinib, approved by the USFDA for the treatment of chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, is a multi-targeted tyrosine kinase inhibitor that potently inhibits ABL, Lyn, VEGFR2, and FGFR1 with the IC50 values of 0.37, 0.24, 1.5, and 2.2 nM,

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respectively [63].

Ponatinib in complex with FGFR1 adopts a DFG-out binding conformation (Figure 17a, PDB codes:

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4V01 and 4V04). In this binding model, the N-1 of the imidazopyridazine moiety is hydrogen bonded with the hinge residue of Ala564, the ethynyl-linked benzamide group is positioned in the hydrophobic pocket which is comprised by Val561, Lys514, Glu531, and Asp641, two additional hydrogen bonds are generated between the carbonyl O and the backbone NH of Ala641, and between the carbonylamino NH and the carbonyl O of Glu531 in this region. The trifluoromethylphenyl moiety inserts deeply into the pocket, while the methylpiperazine group extends in the solvent portion, one of the piperazin N makes two hydrogen bonds with the carbonyl O atoms of Ile620 and His621 [55].



ACCEPTED MANUSCRIPT The co-crystal structure of FGFR4/ponatinib (Figure 17b, PDB codes: 4TYJ, 4QRC, and 4UXQ) likes that of FGFR1. In the complex, ponatinib stretches along the ATP-binding cleft into the allosteric binding pocket generated by a flip out of Phe631 located in the DFG-motif. The imidazo-pyridazine group makes a polar contacts with the hinge region (Ala553). The FGFR4 gatekeeper Val550 is

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stabilized by the benzamide group of ponatinib. Besides, a typical double hydrogen bonds are established in the active site: between the ponatinib’s benzamide nitrogen and Glu520, and between the carbonyl oxygen and Asp630 from the DFG-motif. The trifluoromethyl group is involved in a polar interactions within the deep pocket of FGFR4 and the terminal piperazine moiety of ponatinib is

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exposed to solvent [20, 55, 64].

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2.3. FGFR/inhibitor complexes with irreversible binding models



2.3.1. FGFR4 in complex with FIIN-2 (20, Figure 18) and FIIN-3 (21, Figure 18) FIIN-2 is a PD173074-based compound in which the cyclic urea N of the pyrido-pyrimidine scaffold

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has been derivatized with an acryl-amido-benzyl substituent possessing a reactive acrylamide in the para position. It potently inhibits FGFR1, FGFR2, FGFR3, and FGFR4 with the EC50 values of 1, 4, 93, and 32 nM, respectively, and inhibits the gatekeeper mutant of FGFR2 (FGFR2V564M) with an EC50

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value of 58 nM. FIIN-3 is a substituted anilino-pyrimidine derivative, it not only effectively inhibits FGFRs (EC50 values of 1, <1, 41, 22, and 64 nM against FGFR1, FGFR2, FGFR3, FGFR4, and

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FGFR2V564M, respectively), but also shows potent efficiency towards EGFRL858R (EC50 value of 16.8 nM), a vital mutations for drug resistance [65]. Besides, the kinase selectivity of FIIN-2 and FIIN-3 are assessed against a diverse panel of 456 kinases using an in vitro ATP-site competition binding assay at a concentration of 1.0 µM, both compounds display strong binding to FGFRs and exhibit good overall kinase selectivity [66].

The co-crystal structure of FGFR4WT kinase domain bound to FIIN-2 (Figure 19a, PDB code: 4QQC) is solved for studying the binding modes. In the structure, the two nitrogen atoms from the pyrimidine moiety of FIIN-2 form two hydrogen bonds with Ala553 in the hinge binding region, while the oxygen atom from one of the methoxyl groups forms a hydrogen bond with Asp630. A covalent bond is formed between the reactive acrylamide group of FIIN-2 and Cys477 in the kinase P-loop. This

ACCEPTED MANUSCRIPT covalent bonding pulls down the adjoining Phe478 from the P-loop, allowing it to engage in aromatic contacts with the acrylamidobenzyl group of the compound. Importantly, this conformational change creates favorable intramolecular π–π stacking contacts between Phe478 from the P-loop and Phe631 from the DFG motif, permitting Phe631 to interact with the 3, 5-dimethoxylphenyl group and the

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4-acrylamidobenzyl group in a “π-stacking sandwich” fashion [31, 66]. To understand how FIIN-2 is capable of overcoming gate-keeper mutations, the crystal structure of the FGFR4V550L mutant in complex with FIIN-2 (Figure 19b, PDB code: 4QQ5) is also solved, which shows similar binding with the WT complex [64].

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The FGFR4V550L/FIIN-3 co-crystal structure (Figure 19c, PDB code: 4R6V) is solved as well, which exhibits a conformation and binding mode very similar to that of FGFR4WT/FIIN-2. The pseudo

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six-membered ring next to the 4, 6-pyrimidine core in FIIN-3 adopts a conformation almost identical to the bicyclic core of FIIN-2. In addition, the covalent binding of FIIN-3 with EGFRL858R (Figure 19d, PDB code: 4R5S) explains the promising potency of FIIN-3 against this kinase [66].

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2.3.2. FGFR4 in complex with BLU9931 (22, Figure 18) BLU9931, a 2-anilinoquinazoline derivative bearing an acrylamide at the ortho -position of the aniline

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and a substituted phenyl group at the 6-quinazoline position, is a potent and irreversible FGFR4 inhibitor. The crystal structure of BLU9931 and FGFR4 kinase complex is determined (Figure 20, PDB

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code: 4XCU): BLU9931 binds within the ATP-binding pocket of FGFR4, forming a covalent bond with Cys552. The anilinoquinazoline core of BLU9931 makes a bidentate hydrogen bonding interaction with the hinge residue (Ala553) of FGFR4, whereas the dichloro-dimethoxy-phenyl group occupies the hydrophobic pocket, providing kinase selectivity, a hydrogen bond is formed between one of the methoxyl group and Asp630. Distinctly, the aniline phenyl ring adopts a dihedral rotation of approximately 60°, stabilized in part by the methyl substituent at the C-3 position. This rotation directs the ortho-substituted acrylamide toward the Cys552 sulfur. To achieve covalency, the reactive acrylamide moiety adopts a trans-amide conformation, which positions the terminal carbon proximal to the Cys552 sulfur.

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Consistent with structural predictions, the covalent bond formation with Cys552 in FGFR4 affords BLU9931 exquisite potency and paralog selectivity. BLU9931 shows potent inhibition for FGFR4

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(IC50: 3 nM), but weak inhibition for FGFR1 (IC50: 591 nM), FGFR2 (IC50: 493 nM), and FGFR3 (IC50: 150 nM). Meanwhile, the ability of BLU9931 to form a covalent bond with FGFR4 affords significant potency, as a noncovalent analogue with a reduced acrylamide moiety exhibits much less potent inhibition for FGFR4 (IC50: 938 nM). In addition, kinome-wide selectivity is demonstrated by

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BLU9931, at a 3 µM concentration, BLU9931 displays significant binding to only two of the 398 wild-type kinases (FGFR4 and CSF1R). Moreover, BLU9931 exhibits remarkable antitumor activity in

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FGF19-expressing HCC xenograft models with an intact FGFR4 signaling pathway at doses that are well tolerated [67].

3. General features of FGFRs and inhibitors

3.1. An overview of the crystal binding models and comparison of the inhibitory activities An overview of the crystal binding models and inhibitors’ inhibitory activities are listed in Table 1. It

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can be seen that most of the inhibitors bind with FGFR1 in the DFG-in conformation, except few multi-target inhibitors exhibiting the DFG-out conformation (ARQ069 and ponatinib). Different to FGFR1, FGFR4 displays all of the three conformations when binding with inhibitors, while the only

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FGFR2/inhibitor complex exhibits DFG-out conformation. The additional allosteric pocket of DFG-out conformation provides Type II inhibitors more binding sites, thus Type II inhibitors are likely to

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present higher selectivity towards targets along with lower dissociation rate constant in biochemical activity compared to Type I inhibitors [68]. Similarly, the covalent binding bestows irreversible inhibitors with infinite affinity for the ATP binding site [32]. However, although the inhibitory activities of these inhibitors show some extent of diversity, we cannot see the activity change regulation among the three binding models. Maybe because the number of the inhibitors and the data of the reported inhibitory activities are not enough for comparison, or maybe because the inhibitory activities of these inhibitors are not merely dependent by the binding models, they are also influenced by other factors such as the inhibitor’s physicochemical property, the affinities of inhibitor binding with receptor in other regions.

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3.2. Detailed characteristics of the receptors and inhibitors. In terms of the receptors, exampled as FGFR1, two regions are summarized as the main binding site for

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inhibitors: the hinge region and the hydrophobic region. In the hinge region, two hinge residues, Ala564 and Glu562, are crucial for inhibitors, which commonly interact with inhibitors by forming hydrogen bonds. The hydrophobic pocket is mainly comprised by the residues of Val492, Ala512, Lys514, Ile545, Val561, Ala564, and Leu630. Especially, the “pit” region composed by the side chains

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of Asn628, Leu630, Ala640, and Asp641 is among the hydrophobic pocket, which contribute to the selectivity of inhibitors. The inhibitors binding regions for FGFR2 and FGFR4 are similar to FGFR1,

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the main difference is that two cysteines (Cys477 and Cys552) are around the binding pocket of FGFR4, which facilitate to develop irreversible inhibitors (Figure 21).



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As for the structure characteristics of inhibitors, all of them bear an aromatic heterocycle core, for example, the indolin-2-one in SU4984, the pyridopyrimidine in PD173074, and the quinoxaline in JNJ-42756493, so as to form hydrogen bonds with the kinase hinge region. Besides, a substituted

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lipophilic aromatic ring (benzimidazole in CH5183284, 3, 5-dimethoxyphenyl in AZD4547, et al) is essential for inhibitors, which occupies the hydrophobic region of kinase. The lipophilic aromatic ring

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is in van der Waals contact with the hydrophobic residues. Meanwhile, a hydrogen-bond is often observed between the inhibitor and the hydrophobic residue, for example, between one of the methoxyl groups in JNJ-42756493 and Asp641 in FGFR1, and between the methoxyl group in BLU9931 and Asp630 in FGFR4. These interactions not only enhance the binding affinity but also improve inhibitors’ selectivity. In addition, small lipophilic groups (isopropyl in JNJ-42756493, methyl in compound 15, et al) are preferred for inhibitors to interact with the “pit” region of kinase. With regard to the irreversible FGFR4 inhibitors, an acrylamide group and a proper linker are necessary so that the reaction group is approximate enough to the cysteines of the kinase to carry on Michael Addition [69] (Figure 21). 5. Conclusion Given the critical role of FGFRs in the progression of tumors, the co-crystal structures of these kinases

ACCEPTED MANUSCRIPT in complex with inhibitors are determined in order to clarify the mechanism of actions and explore new efficient inhibitors. This manuscript highlights the co-crystal structures of FGFRs in complex with inhibitors and the pharmacological activities of these inhibitors. As for the crystallization method is firstly well established [34], most of the reported co-crystal structures are FGFR1/inhibitor complexes.

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Meanwhile, in order to overcome drug resistance, irreversible FGFR4 inhibitors are successfully acquired utilizing the cysteines around the binding site. In addition, co-crystal structures of one inhibitor in complex with two or more receptors are got to compare the binding characteristics and the mechanism of actions, which provide novel insights for drug design. However, although the crystal

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structures of FGFR2 and FGFR3 have been reported, the co-crystals of these kinases in complex with inhibitors are few or none, which suggest a direction for further development.

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Conflict of interest

The authors confirm that this article content has no conflict of interest. Acknowledgements

Dr Weiyan Cheng thanks the support of Young Scholar Fund from The First Affiliated Hospital of Zhengzhou University. The authors also thank Prof. Yongzhou Hu (Zhejiang Province Key Laboratory

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of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, China) for providing software assistance for computer-aided drug design. The authors are indebted to one of the anonymous reviewers for his or her detailed and constructive comments, which have been very helpful

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Schnell,

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3-(2,

6-dichloro-3,

5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-ur

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Figure captions:

Figure 1 The FGFRs signaling pathways.

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Figure 2 Binding models of receptor/inhibitor complex (a, DFG-in bindings; b, DFG-out bindings; c, irreversible bindings).

Figure 3 Chemical structures of FGFR inhibitors exhibiting DFG-in binding conformations.

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Figure 4 Co-crystal structures of FGFR1/SU4984 (a, PDB code: 1AGW) and FGFR1/SU5402 (b, PDB code: 1FGI).

Figure 5 Co-crystal structures of CH5183284 bound to FGFR1 (PDB code: 5B7V). Figure 6 Co-crystal structures of PD173074 bound to FGFR1 (PDB code: 2FGI). Figure 7 Co-crystal structures of JNJ-42756493 bound to FGFR1 (PDB code: 5EW8). Figure 8 Co-crystal structures of BGJ398 bound to FGFR1 (PDB code: 3TT0). Figure 9 Co-crystal structures of lucitanib bound to FGFR1C488A/C584C (PDB code: 4RWL). Figure 10 Co-crystal structures of AZD4547 bound to FGFR1WT (a, PDB code: 4V05; b, PDB code: 4WUN) and FGFR1C488A/C584S (c, PDB code: 4RWJ). Figure 11 Co-crystal structures of AZD4547 bound to FGFR1C488A/C584S/V561M (a, WT-like inhibitor conformation; b, bent inhibitor conformation. PDB code: 4RWK).

ACCEPTED MANUSCRIPT Figure 12 Co-crystal structures of compound 9 bound to FGFR1 (PDB code: 4ZSA). Figure 13 Co-crystal structures of compounds 11-16 bound to FGFR1. Panel a: 11/FGFR1 co-crystal structure (yellow: one conformation of 11; white: another conformation of 11, PDB code: 4F63); panel b: 12/FGFR1 co-crystal structure (PDB code: 4F64); panel c: 13/FGFR1 co-crystal structure (PDB code: 4F65); panel d: 14/FGFR1 co-crystal structure (PDB code:

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4NKS); panel e: 15/FGFR1 co-crystal structure (PDB code: 4NK9); Panel f: 16/FGFR1 co-crystal structure (PDB code: 4NKA).

Figure 14 Co-crystal structures of dovitinib bound to FGFR1WT (a, PDB code: 5AM6), FGFR1V561M (b,

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PDB code: 5AM7), and FGFR4 (c, PDB code: 4TYI).

Figure 15 Chemical structures of FGFR inhibitors exhibiting DFG-out binding conformations.

PDB code: 3RI1).

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Figure 16 Co-crystal structures of FGFR1/ARQ069 (a, PDB code: 3RHX) and FGFR2/ARQ069 (b,

Figure 17 Co-crystal structures of ponatinib bound to FGFR1 (a, PDB code: 4V01) and FGFR4 (b, PDB code: 4TYJ).

Figure 18 Chemical structures of FGFR inhibitors exhibiting irreversible binding models. Figure 19 Co-crystal structures of FGFR4WT/FIIN-2 (a, PDB code: 4QQC), FGFR4V550L/FIIN-2 (b,

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PDB code: 4QQ5), FGFR4V550L/FIIN-3 (c, PDB code: 4R6V), and EGFRL858R/FIIN-3 (d, PDB code: 4R5S).

Figure 20 Co-crystal structures of FGFR4/BLU9931 (PDB code: 4XCU).

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Figure 21 Main binding regions of FGFRs and structure characteristic of the inhibitors.

ACCEPTED MANUSCRIPT Table 1 An overview of the crystal binding models and inhibitory activities of FGFR inhibitors. Inhibitor SU4984 (1)

Activity (IC50, nM)

Crystal Binding

Binding

FGFR1

FGFR2

FGFR3

FGFR4

Receptor

Model

103-203

-

-

-

FGFR1

DFG-in

3

3

10 -20

-

-

-

FGFR1

9.3

7.6

22

290

FGFR1

PD173074 (4)

21.5

5

-

-

FGFR1

JNJ-42756493 (5)

Low nM

Low nM

Low nM

Low nM

FGFR1

BGJ398 (6)

1

1

1

60

FGFR1

Lucitanib (7)

17.5

82.5

-

-

FGFR1

AZD4547 (8)

0.2

2.5

1.8

165

FGFR1

Compound 9

15

-

-

-

FGFR1

Compound 15

8.7 (pIC50)

-

-

-

FGFR1

Dovitinib (17)

8

-

9

-

ARQ069 (18)

840

1230

-

-

Ponatinib (19)

2.2

-

-

-

FIIN-2 (20)

1

4

FIIN-3 (21)

1

<1

BLU9931 (22)

591

493

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FGFR1, FGFR2

DFG-out

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93

32

FGFR4

41

22

FGFR4

150

3

FGFR4

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SU5402 (2) CH5183284 (3)

Irreversible

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ACCEPTED MANUSCRIPT Highlights: Summarized the binding models of FGFRs/inhibitors. Pharmacological activities of the inhibitors are concluded.

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General binding features of FGFRs/inhibitors are highlighted.