A novel agonist with homobivalent single-domain antibodies that bind the FGF receptor 1 domain III functions as an FGF2 ligand.

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC50) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling.

The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23-FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling.

Two FGF Receptor Kinase Molecules Act in Concert to Recruit and Transphosphorylate Phospholipase Cγ.

The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation.

Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases.

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.

Number and brightness analysis reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1 in live cells.

Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling; however, they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interaction remain elusive. Number of molecules and brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 in response to low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Similar instability was observed upon altering the endocytic pathway. Thus, FGF2 and NCAM induce differential FGFR1 clustering at the cell surface, which might account for the distinct intracellular fate of the receptor and, hence, for the different signaling cascades and cellular responses.

Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers.

Fibroblast growth factor receptor 1 (FGFR1) is an integral membrane protein that transmits prolife signals through the plasma membrane. Overexpression of FGFR1 has been reported in various tumor types, and therefore, this receptor constitutes an attractive molecular target for selective anticancer therapies. Here, we present a novel system for generation of intrinsically fluorescent, self-assembling, oligomeric cytotoxic conjugates with high affinity and efficient internalization targeting FGFR1. In our approach, we employed FGF1 as an FGFR1 recognizing molecule and genetically fused it to green fluorescent protein polygons (GFPp), a fluorescent oligomerization scaffold, resulting in a set of GFPp_FGF1 oligomers with largely improved receptor binding. To validate the applicability of using GFPp_FGF1 oligomers as cancer probes and drug carriers in targeted therapy of cancers with aberrant FGFR1, we selected a trimeric variant from generated GFPp_FGF1 oligomers and further engineered it by introducing FGF1-stabilizing mutations and by incorporating the cytotoxic drug monomethyl auristatin E (MMAE) in a site-specific manner. The resulting intrinsically fluorescent, trimeric cytotoxic conjugate 3xGFPp_FGF1E_LPET_MMAE exhibits nanomolar affinity for the receptor and very high stability. Notably, the intrinsic fluorescence of 3xGFPp_FGF1E_LPET_MMAE allows for tracking the cellular transport of the conjugate, demonstrating that 3xGFPp_FGF1E_LPET_MMAE is efficiently and selectively internalized into cells expressing FGFR1. Importantly, we show that 3xGFPp_FGF1E_LPET_MMAE displays very high cytotoxicity against a panel of different cancer cells overproducing FGFR1 while remaining neutral toward cells devoid of FGFR1 expression. Our data implicate that the engineered fluorescent conjugates can be used for imaging and targeted therapy of FGFR1-overproducing cancers.

Fibroblast growth factor receptor 5 (FGFR5) is a co-receptor for FGFR1 that is up-regulated in beta-cells by cytokine-induced inflammation.

Fibroblast growth factor receptor-1 (FGFR1) activity at the plasma membrane is tightly controlled by the availability of co-receptors and competing receptor isoforms. We have previously shown that FGFR1 activity in pancreatic beta-cells modulates a wide range of processes, including lipid metabolism, insulin processing, and cell survival. More recently, we have revealed that co-expression of FGFR5, a receptor isoform that lacks a tyrosine-kinase domain, influences FGFR1 responses. We therefore hypothesized that FGFR5 is a co-receptor to FGFR1 that modulates responses to ligands by forming a receptor heterocomplex with FGFR1. We first show here increased FGFR5 expression in the pancreatic islets of nonobese diabetic (NOD) mice and also in mouse and human islets treated with proinflammatory cytokines. Using siRNA knockdown, we further report that FGFR5 and FGFR1 expression improves beta-cell survival. Co-immunoprecipitation and quantitative live-cell imaging to measure the molecular interaction between FGFR5 and FGFR1 revealed that FGFR5 forms a mixture of ligand-independent homodimers (∼25%) and homotrimers (∼75%) at the plasma membrane. Interestingly, co-expressed FGFR5 and FGFR1 formed heterocomplexes with a 2:1 ratio and subsequently responded to FGF2 by forming FGFR5/FGFR1 signaling complexes with a 4:2 ratio. Taken together, our findings identify FGFR5 as a co-receptor that is up-regulated by inflammation and promotes FGFR1-induced survival, insights that reveal a potential target for intervention during beta-cell pathogenesis.

Isolation and Characterization of a Chinese Hamster Ovary Heparan Sulfate Cell Mutant Defective in Both Met Receptor Binding and Hepatocyte Growth Factor NK1/Met Signaling.

BACKGROUND/AIMS: The up-regulation of hepatocyte growth factor/receptor, HGF/Met, signal transduction is observed in most of human cancers. Specific heparan sulfate structures enhance the HGF/Met signaling at both cell and animal-based model systems. Biochemical studies indicate that heparan sulfate interacts with HGF and a natural occurring splicing variant NK1 of HGF with similar affinity. However, it is currently unknown if cell surface heparan sulfate binds to Met at physiological conditions and if specific cell surface heparan sulfate structures are required for effective HGF/Met or NK1/Met signaling. METHODS: An established flow sorting strategy was used to isolate a soluble Met recombinant protein-binding positive or negative CHO cell clones different only in specific heparan sulfate structures. The cell surface bindings were imaged by confocal microscopy and flow cytometry analysis. Glucosamine vs. galactosamine contents from media-, cell surface-, and cell association glycosaminoglycans were quantified by HPLC. 35S-sulfate labeled glycosaminoglycans were characterized by anion exchange and size-exclusion HPLC. Heparan sulfate disaccharide compositions were determined by HPLC-MS analysis. Western blot analyses of MAPK-p42/44 were used to monitor HGF- and NK1-facillated Met signaling. RESULTS: CHO-Positive but not CHO-Negative cell surface heparan sulfate bound to Met recombinant protein and HGF/NK1 further promoted the binding. Overall glycosaminoglycan analysis results indicated that the CHO-Negative cells had reduced amount of heparan sulfate, shorter chain length, and less 6-O-sulfated disaccharides compared to that of CHO-Positive cells. Moreover, CHO-Negative cells were defective in NK1/Met but not HGF/Met signaling. CONCLUSIONS: This study demonstrated that soluble Met recombinant protein bound to cell surface HS at physiological conditions and a Met /HGF or NK1/HS ternary signaling complex might be involved in Met signaling. Shorter HS chains and reduced 6-O-sulfation might be responsible for reduced Met binding and the diminished NK1-initiated signaling in the CHO-Negative cells. The unique CHO-Positive and CHO-Negative cell clones established in current study should be effective tools for studying the role of specific glycosaminoglycan structures in regulating Met signaling. Such knowledge should be useful in developing glycosaminoglycan-based compounds that target HGF/Met signaling.

α-Klotho's effects on mineral homeostasis are fibroblast growth factor-23 dependent.

PURPOSE OF REVIEW: α-Klotho (Klotho) occurs in three isoforms, a membrane-bound form acting as a coreceptor for fibroblast growth factor-23 (FGF23) signalling, a shed soluble form consisting of Klotho's large ectodomain thought to act as an enzyme or a hormone, and a secreted truncated form generated by alternative splicing of the Klotho mRNA with unknown function. The purpose of this review is to highlight the recent advances in our understanding of Klotho's function in mineral homeostasis. RECENT FINDINGS: A number of seminal discoveries have recently been made in this area, shifting existing paradigms. The crystal structure of the ternary FGF receptor (FGFR)-1c/Klotho/FGF23 complex has been uncovered, revealing how the ligand FGF23 interacts with FGFR1c and the coreceptor Klotho at atomic resolution. Furthermore, it was shown that soluble Klotho lacks any glycosidase activity and serves as a bona fide coreceptor for FGF23 signalling. Experiments with a combination of Klotho and Fgf23-deficient mouse models demonstrated that all isoforms of Klotho lack any physiologically relevant, FGF23-independent functions in mineral homeostasis or ageing. Finally, it was demonstrated that the alternatively spliced Klotho mRNA is degraded and is not translated into a secreted Klotho protein isoform in humans. SUMMARY: Taken together, there is now overwhelming evidence that the main physiological function of transmembrane and soluble Klotho for mineral homeostasis is their role as coreceptors mediating FGF23 actions. In light of these findings, the main pathophysiological consequence of the downregulation of Klotho observed in acute and chronic renal failure may be the induction of renal FGF23 resistance.

Computational Simulation Studies on the Binding Selectivity of 1-(1H-Benzimidazol-5-yl)-5-aminopyrazoles in Complexes with FGFR1 and FGFR4.

Fibroblast growth factor receptor 1 (FGFR1) has become a potential target for the treatment of cancer. Designing FGFR1-selective inhibitors remains fundamental to the development of anti-cancer drugs because of highly sequential homology among FGFR subtypes. In present work, four inhibitors were examined with intermolecular interaction patterns with FGFR1 and FGFR4, respectively, for the exploration of binding mechanisms by applying a combined approach of computational techniques, including flexible docking, binding site analyses, electronic structure computations, molecular dynamic simulations, and binding free energy predictions. Molecular simulation-predicted binding conformations and pharmacophoric features of these molecules in the active pocket of either FGFR1 or FGFR4. MMPB(GB)SA-calculated binding free energies were accordant with the ordering of their tested potency values. Furthermore, in silico mutations of two residues (FGFR1: Tyr563 and Ser565) were also performed to check their impact on ligand binding by applying MD simulations and binding free energy calculations. The present studies may provide a structural understanding of the FGFR1-selective mechanism. The viewpoints from computational simulations would be valuable guidelines for the development of novel FGFR1-selective inhibitors.

Nuclear magnetic resonance analysis of the conformational state of cancer mutant of fibroblast growth factor receptor 1 tyrosine kinase domain.

Tyrosine kinases are key enzymes that play critical roles in growth signaling, the abnormal activation of which is associated with various human cancers. Activation of tyrosine kinases is mediated by tyrosine phosphorylation in the activation-loop, which transforms the catalytic domain to the active state conformation. Cancer mutations are supposed to transform the conformation of the catalytic domain into the active-form independent of the phosphorylation state of the activation-loop. Here, we report structural and biophysical analyses of cancer mutations of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1). Based on the nuclear magnetic resonance analyses, phosphorylation of the activation-loop exhibited cooperative structural transition in the activation-loop, C-helix and P-loop regions, whereas cancer mutations induced structural transformation at either one or two of these regions.

Biophysical characterization of drug-resistant mutants of fibroblast growth factor receptor 1.

Over-expression and aberrant activation of tyrosine kinases occur frequently in human cancers. Various tyrosine kinase inhibitors (TKIs) are under clinical use, but acquisition of resistance to these drugs is a major problem. Here, we studied the interaction between two drug-resistant mutants of fibroblast growth factor receptor 1 (FGFR1), N546K and V561M, and four ATP-competitive inhibitors, ponatinib, dovitinib, PD173074 and BGJ-398. Among these protein-drug systems, the only marked reduction in affinity was that of PD173074 for the V561M mutant. We also examined the interaction of these FGFR1 variants to AMP-PNP, a nonhydrolyzable analogue of ATP, and showed that N546K showed increased affinity for the ATP analogue as compared with the wild type. These findings will help to clarify the mechanism of drug resistance in mutant tyrosine kinases.

Theoretical studies on FGFR isoform selectivity of FGFR1/FGFR4 inhibitors by molecular dynamics simulations and free energy calculations.

The activation and overexpression of fibroblast growth factor receptors (FGFRs) are highly correlated with a variety of cancers. Most small molecule inhibitors of FGFRs selectively target FGFR1-3, but not FGFR4. Hence, designing highly selective inhibitors towards FGFR4 remains a great challenge because FGFR4 and FGFR1 have a high sequence identity. Recently, two small molecule inhibitors of FGFRs, ponatinib and AZD4547, have attracted huge attention. Ponatinib, a type II inhibitor, has high affinity towards FGFR1/4 isoforms, but AZD4547, a type I inhibitor of FGFR1, displays much reduced inhibition toward FGFR4. In this study, conventional molecular dynamics (MD) simulations, molecular mechanics/generalized Born surface area (MM/GBSA) free energy calculations and umbrella sampling (US) simulations were carried out to reveal the principle of the binding preference of ponatinib and AZD4547 towards FGFR4/FGFR1. The results provided by MM/GBSA illustrate that ponatinib has similar binding affinities to FGFR4 and FGFR1, while AZD4547 has much stronger binding affinity to FGFR1 than to FGFR4. A comparison of the individual energy terms suggests that the selectivity of AZD4547 towards FGFR1 versus FGFR4 is primarily controlled by the variation of the van der Waals interactions. The US simulations reveal that the PMF profile of FGFR1/AZD4547 has more peaks and valleys compared with that of FGFR4/AZD4547, suggesting that the dissociation process of AZD4547 from FGFR1 are easily trapped into local minima. Moreover, it is observed that FGFR1/AZD4547 has much higher PMF depth than FGFR4/AZD4547, implying that it is more difficult for AZD4547 to escape from FGFR1 than from FGFR4. The physical principles provided by this study extend our understanding of the binding mechanisms and provide valuable guidance for the rational design of FGFR isoform selective inhibitors.

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

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

Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

Unliganded fibroblast growth factor receptor 1 forms density-independent dimers.

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Förster resonance energy transfer combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full-length FGFR1 at the cell-surface with or without the coreceptor ßKlotho. Using this approach we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR) and was further increased by the addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and ßKlotho. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface.

Structural analysis of the mechanism of phosphorylation of a critical autoregulatory tyrosine residue in FGFR1 kinase domain.

Receptor and nonreceptor tyrosine kinases are enzymes that play important roles in regulating signal transduction pathways in a variety of normal cellular process and in many pathological conditions. Ordered phosphorylation is required for receptor tyrosine kinase (RTK) activation, a process mediated by transient dimer formation of the kinase domains. This process is triggered by the tyrosine phosphorylation in the activation loop. Here, we report structural and biochemical analyses of the tyrosine kinase domain interaction of fibroblast growth factor receptor 1 (FGFR1) required for the initial phosphorylation step. On the basis of nuclear magnetic resonance (NMR) analysis and covalent cross-linking experiments, we propose a parallel symmetric dimer model where specific contacts are formed between the N-lobes and C-lobes, respectively, in the FGFR1 kinase domains. Moreover, assignment of the contact sites between two FGFR1 kinase domains are supported by a trans-phosphorylation assay and by mutational analyses. The present report shows the molecular mechanism underlying the control of trans-phosphorylation of a critical auto-regulatory site in FGF receptors' catalytic domain.

Expanding the structural diversity of diarylureas as multi-target tyrosine kinase inhibitors.

Recently approved multi-target inhibitors of receptor tyrosine kinases (RTKs) have significantly improved the clinical treatment of cancers. A series of N,N'-diarylureas incorporated with aromatic heterocycle have been designed, synthesized and evaluated as novel multi-target RTK inhibitors. The preliminary biological evaluation indicated that several compounds exhibited comparable potency with Sorafenib. Among them, compound 6f was identified as the most potent multikinase inhibitor of EGFR, KDR and FGFR1 with IC50 values of 14.83nM, 21.57nM, and 28.23nM, respectively. These compounds expanded the structural diversity of diarylureas as RTK inhibitors. The results demonstrated that compound 6f could be served as novel lead compound for further development of multi-target RTK inhibitors.

Design, Synthesis and Biological Evaluation of 6-(2,6-Dichloro-3,5-dimethoxyphenyl)-4-substituted-1H-indazoles as Potent Fibroblast Growth Factor Receptor Inhibitors.

Tyrosine kinase fibroblast growth factor receptor (FGFR), which is aberrant in various cancer types, is a promising target for cancer therapy. Here we reported the design, synthesis, and biological evaluation of a new series of 6-(2,6-dichloro-3,5-dimethoxyphenyl)-4-substituted-1H-indazole derivatives as potent FGFR inhibitors. The compound 6-(2,6-dichloro-3,5-dimethoxyphenyl)-N-phenyl-1H-indazole-4-carboxamide (10a) was identified as a potent FGFR1 inhibitor, with good enzymatic inhibition. Further structure-based optimization revealed that 6-(2,6-dichloro-3,5-dimethoxyphenyl)-N-(3-(4-methylpiperazin-1-yl)phenyl)-1H-indazole-4-carboxamide (13a) is the most potent FGFR1 inhibitor in this series, with an enzyme inhibitory activity IC50 value of about 30.2 nM.