Inhibitors of the Fibroblast Growth Factor Receptor
Signaling through the fibroblast growth factor receptor (FGFR) tyrosine kinase is crucial to a number of key pharmacological processes; however, dysregulation of this signaling is observed with a number of different cancers suggesting that inhibition of FGFR may provide an important therapeutic agent in the treatment of cancers. This chapter provides an overview of the development of FGFR inhibitors beginning with the identification of nonselective FGFR inhibitors, then describing the medicinal chemistry optimization resulting in the delivery of a number of highly selective FGFR inhibitors, some of which are currently being assessed in clinical trials. The development of isoform selective FGFR inhibitors as well as covalent inhibitors and inhibitors of the inactive form of FGFR are also described.
KeywordsFibroblast growth factor receptor Receptor tyrosine kinase Selective kinase inhibitor
The development of human cancers, in which normal cells are transformed into highly malignant cells, is usually a multistage process that may extend over decades . The transformation process involves cells accumulating a number of genetic changes which result in a specific set of properties commonly referred to as the hallmarks of cancer (including sustained proliferative signaling, evasion of growth suppressors, active invasion and metastasis, replicative immortality, induction of angiogenesis, and resistance to cell death) [2, 3]. The genetic alterations acquired by the cell can result in overactivation of growth-promoting oncogenes or the inactivation of growth-inhibitory suppressor genes. Receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR, “Inhibitors of Vascular Endothelial Growth Factor Receptor”), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR, “Inhibitors of Vascular Endothelial Growth Factor Receptor”), represent an important family of genes which have been found to be altered in human cancer.
1.1 Fibroblast Growth Factor Receptor (FGFR)
1.2 The Role of FGFR in Cancer
FGFRs are known to be overexpressed in many types of human cancer cells, and our understanding of the mechanisms by which FGFR signaling is dysregulated and drives cancer has increased significantly in recent years. FGFRs are among the most commonly mutated kinase genes observed in human cancers. Mutations in FGFR that confer constitutive activation have been seen in many cancers including non-muscle invasive and high-grade bladder cancer (FGFR3 activation), endometrial cancer (FGFR2 activation), and rhabdomyosarcoma (FGFR4 activation). Amplification of FGFR genes will often result in FGFR overexpression, which can also allow ligand-independent signaling to occur. FGFR amplification has been observed in cancers including estrogen receptor-positive breast cancer (FGFR1 amplification), squamous non-small cell lung cancer (FGFR1 amplification), and gastric cancer (FGFR2 amplification). FGFR translocations have been identified in hematologic malignancies, which result in a protein fusing to the intracellular tyrosine kinase domain of an FGFR. This fusion results in the permanent dimerization, and therefore activation, of the protein as well as the removal of mechanisms of feedback inhibition. Such translocations have been observed to result in FGFR3 overexpression and continuous signaling. In addition to aberrations within the receptor proteins, overexpression of the FGF ligands has also been shown to play a role in cancer development; FGF5 overexpression has been observed in a number of lung, esophagus, melanoma, and colon and prostate tumor cell lines, and overexpression/upregulation of FGF2, 8, 17, 18, and 19 has been observed in hepatocellular carcinomas (HCC).
2 The Development of Nonselective FGFR Inhibitors
3 The Development of Selective FGFR Inhibitors
3.1 Pyrido[2,3-d]pyrimidine-Based Inhibitors of FGFR
PD173074 (7) was shown to inhibit the autophosphorylation of FGFR1 in NIH 3T3 cells with an IC50 of 0.001–0.005 μM, whereas the autophosphorylation of VEGFR2 in the same cell line was inhibited with significantly reduced potency (IC50 = 0.1–0.2 μM). PD173074 (7) inhibited the bFGF-stimulated HUVEC growth (IC50 = 0.007 μM) but was less active against VEGF-stimulated HUVEC growth (IC50 = 0.194 μM). The bioavailable nature of 7 allowed for the in vivo characterization of the compound, and it was shown to inhibit bFGF-induced angiogenesis in an in vivo mouse cornea model but was significantly less effective at blocking VEGF-induced angiogenesis in the same model .
In parallel with the optimization of hit 1, the researchers at Parke–Davis also optimized a related pyrido[2,3-d]pyrimidin-7-one hit 10 with the expectation of identifying kinase inhibitors with differing profiles with respect to inhibitory activity, metabolism, and pharmacokinetic properties . Optimization of this scaffold resulted in the discovery of PD166285 (11), a potent inhibitor of a range of tyrosine kinases (including FGFR, PDGFR, EGFR, and c-Src) which displays excellent physicochemical properties .
3.2 Indolin-2-One-Based FGFR Inhibitors
Interestingly, throughout the reported SAR investigation, the authors note an apparent disconnect between biochemical potency and cell potency. They postulate that poor cell permeability, related to the physicochemical properties of some of the molecules described, contributes to this observed disconnect.
Computational modeling was also used to prioritize the SAR investigations into substitution of the 5-position of the indolin-2-one core. As a result of this in silico approach, NP506 (19) was synthesized and shown to possess good activity against FGFR1, VEGFR2, and PDGFRβ in biochemical assays (IC50’s = 0.10 μM, 0.07 μM, and 0.04 μM, respectively) but was less active against EGFR (IC50 = 3.48 μM) . The proposed binding mode for 19 locates the phenylhydrazine moiety more deeply in to the hydrophobic back pocket compared to the 3,5-dimethoxyphenyl moiety of 18 and forms a π–π interaction with Phe642. However, despite the improved biochemical potency of 19, little difference is observed between the potency of this compound in a cellular antiproliferation assay compared with 18 (IC50 = 18.2 and 27.4 μM, respectively). The authors postulate that the physicochemical properties of 19 may result in poor cellular permeability. Compound 19 also appears to show activity against the PI3K/AKT-signaling pathway perhaps indicating that the compound is less selective than 18.
3.3 Benzimidazol-2-Ylhydroquinolin-2-One-Based FGFR Inhibitors
In addition to being a potent inhibitor of FGFR1, PDGFRβ, and VEGFR2 in biochemical assays, dovitinib (27) also inhibits VEGFR1 and 3, FGFR3, c-Kit, CSF1R, and Flt3 with IC50’s ranging between 0.001 and 0.036 μM. It potently inhibits the phosphorylation of FGFR1, VEGFR2, and PDGFRβ in cells (EC50’s = 0.166 μM, 0.046 μM, and 0.051 μM, respectively) . Dovitinib (27) shows increased aqueous solubility and attractive pharmacokinetic properties with good oral bioavailability (>70% in mice and rat), moderate to high clearance, and a large volume of distribution resulting in a terminal half-life of approximately 3 h. Dovitinib (27) shows potent inhibition of bFGF-driven angiogenesis in a mouse Matrigel model of neovascularization and tumor regression in both KM12L4a and HCT116 xenograft models at doses >60 mg/kg. The observed efficacy is associated with a significant reduction of phosphorylated PDGFRβ and a reduction of phosphorylation of the downstream protein ERK in the tumor cells. Based on the preclinical data, dovitinib (27) has been described as a multi-targeted growth factor receptor kinase inhibitor and has been studied in a number of clinical studies, including a phase I/II study in patients with advanced melanoma; a phase II study in patients with previously treated, metastatic, HER2-negative breast cancer; and a phase II study in combination with fulvestrant in postmenopausal, HER2-negative/HR-positive advanced breast cancer patients.
3.4 Pyrimidinyl Ureas as FGFR Inhibitors
Compound 30 was a moderately potent inhibitor of FGFR3 in both a biochemical assay using constitutively active FGFR3-K650E kinase (IC50 = 0.505 μM) and a cell assay using an FGFR3-dependent BaF3 cell line (IC50 = 1.5 μM). A homology model of FGFR3 was also constructed to aid compound design. The authors appreciated the requirement for the 3,5-dimethoxyphenyl ring to adopt an orthogonal orientation with respect to the pyrimidine ring to best compliment the hydrophobic back pocket and so focused on molecular conformational. Ab initio calculations predict that 7 is likely to have a torsional angle of 127° between the 3,5-dimethoxyphenyl ring and the pyrido[2,3-d]pyrimidine ring when in a relaxed conformation in water and that an energy penalty of 0.5 kcal/mol would be required to access the perpendicular conformation observed in the binding mode (tortional angle of ~90°). In contrast, the 3,5-dimethoxyphenyl ring in compound 30 is predicted to be coplanar with the pyrimidinyl urea motif in the relaxed conformation (torsional angle of 180°) with an energy penalty of 1.5 kcal/mol required to access the postulated binding conformation. This energy difference arises from the higher deconjugation energy penalty for compound 30 compared to 7, and the hypothesis is consistent with the observed reduction in potency. The introduction of ortho-substituents to the 3,5-dimethoxyphenyl ring was anticipated to reduce the deconjugation energy, and compound 31, bearing diortho-substitution, was predicted to have a torsional angle of 109° in the relaxed conformation with an energy penalty of only 0.1 kcal/mol to access the postulated binding conformation. Compound 31 does indeed show significantly increased potency in both biochemical and cell assays. Modeling of 31 in the FGFR3 homology model suggests a tridentate interaction with the kinase hinge between the anilino NH, the adjacent pyrimidine nitrogen, and the pyrimidine C(2)-H as well as the potential for a water-mediated interaction between the urea carbonyl and the side chain of Lys508 (Lys514 in FGFR1). The optimized fit of the tetra-substituted phenyl ring was predicted to result in productive interactions between the methoxy substituent and Asp635 (Asp641 in FGFR1) as well as favorable hydrophobic interactions between the two chlorine atoms and the Val555 and Ala634 residues (Val561 and Ala640 in FGFR1). Interestingly, the latter residue is replaced by a larger cysteine in VEGFR2, and the authors speculate that as a result of increased steric congestion an energy penalty might be incurred when the compound binds to VEGFR2.
More thorough testing revealed that 31 was an exceptionally potent inhibitor of FGFR1–3 (IC50 ~0.001 μM) but was a significantly less potent inhibitor of FGFR4 (IC50 = 0.060 μM) and VEGFR2 (IC50 = 0.18 μM). Compound 31 was largely inactive when tested against a panel of 76 kinases with only KIT and LYN showing IC50 values below 1 μM. When tested against a panel of 31, BaF3 cell line 31 was highly potent in the FGFR1–3 lines (IC50 ~0.001 μM) but was approximately 400 times less active against VEGFR2 lines and inactive against most other lines. The compound displays high clearance and high volume of distribution in mouse and rat with moderate to good bioavailability and some evidence of increased exposure in tumor tissue compared to plasma. Efficacy was observed in orthotopic mouse xenograft models and subcutaneous rat xenograft models, and this was associated with in vivo knockdown of pFRS2 and pMAPK. It also inhibited bFGF-stimulated angiogenesis in a murine agar chamber model but had no effect on VEGF-stimulated angiogenesis, further demonstrating the selectivity for FGFR over VEGFR. Compound 31 was evaluated in a phase I study in patients with advanced solid tumors and is currently undergoing evaluation phase II trials in patients with solid tumors, glioblastoma multiforme, and melanoma.
3.5 Pyrazolylaminopyrimidines and Pyrazolylbenzamides as FGFR Inhibitors
Exploration of different regions of the molecule quickly established that the bromine could be removed with little impact on potency, 36. This change is associated with a reduction in logP of approximately one unit, thereby demonstrating improved binding efficiency as measured by lipophilicity ligand efficiency (LLE). Comparison of the structures of both 34 and PD173074 (7) bound to FGFR1 suggested an opportunity to target the hydrophobic back pocket of the ATP-binding site by building from the 5-position of the pyrazole ring. The incorporation of a 3,5-dimethyoxyphenyl ring, known to have high complementarity to the back pocket in other scaffolds, linked to the pyrazole with a two-carbon spacer, resulted in compound 37 which shows a dramatic improvement in FGFR1 potency while retaining good selectivity over VEGFR2. However, despite the promising profile of 37, the lipophilic nature of the compound resulted in poor aqueous solubility (0.6 μM) and was felt to preclude the in vivo characterization of the compound.
A crystal structure of compound 39 bound in to FGFR1 showed the N-methylpiperazinyl unit to be situated in the solvent channel of the kinase and thus might provide an opportunity to modulate physicochemical and pharmacokinetic properties. More detailed profiling of 39 revealed high clearance of the compound in rat hepatocyte incubations (Clint = 60 μL/min/106 cells) and in rat in vivo pharmacokinetic studies (Cl = 46 mL/min/kg). Metabolite identification studies highlighted significant N-demethylation and N-oxidation of the N-methylpiperazinyl functionality. The high clearance of the compound, driven by metabolism of the N-methylpiperazinyl motif, limited the oral bioavailability of the compound. The strategies adopted to lower clearance included the removal of the N-methyl on the piperazine ring as well as reducing the potential of the piperazine to undergo both N-oxidation and N-dealkylation by increasing the pKa, thereby increasing the fraction of the basic center existing in a protonated form. Removal of the N-methyl group, 40, was well tolerated and did result in a reduction of clearance in both in vitro rat hepatocyte incubations (Clint = 12 μL/min/106 cells) and in rat in vivo pharmacokinetic studies (Cl = 23 mL/min/kg); however, oral bioavailability in rat was low (3%) presumably due to compromized permeability. The optimization goal was therefore to balance reduced clearance with acceptable permeability. The introduction of methyl groups to sterically “mask” the hydrogen bond donor, while concurrently increasing lipophilicity, resulted in the discovery of compound 41, subsequently given the corporate identifier AZD4547, which retains excellent potency against FGFR1 in biochemical and cell assays while displaying low clearance in in vitro rat hepatocyte incubations (Clint = 11 μL/min/106 cells) and in rat in vivo pharmacokinetic studies (Cl = 16 mL/min/kg) and good oral bioavailability in rat (54%). Gavine et al. report on the detailed pharmacological profiling of AZD4547 (41) and show the compound to be an extremely potent inhibitor of FGFR1–3 in biochemical assays (IC50’s = 0.0002 μM, 0.0025 μM and 0.0018 μM, respectively) but less active against FGFR4 (IC50 = 0.165 μM). A similar profile was observed when looking at the inhibition of phosphorylation of FGFR in Cos-1 cells transfected with FGFR1–4 (IC50’s = 0.013 μM, 0.002 μM, 0.040 μM and 0.142 μM, respectively). AZD4547 (41) showed reduced activity for the inhibition of phosphorylation of VEGFR2 in HUVEC cells (IC50 = 0.258 μM) and showed excellent selectivity when tested against a broad panel of kinases. AZD4547 (41) has potent in vitro antiproliferative effects on tumor cell lines with dysregulated FGFR expression, such as the FGFR3 driven KMS11 cell line, but was inactive in over 100 additional tumor cell lines. This result is believed to indicate that the antiproliferative effects observed are driven by FGFR inhibition rather than any nonspecific cytotoxicity. AZD4547 (41) showed dose proportional antitumor efficacy in KMS11 xenograft models with concurrent pharmacodynamic modulation of FGFR phosphorylation. AZD4547 (41) did not induce any significant changes in blood pressure in conscious telemetered rats at in vivo exposure levels equivalent to those observed to give efficacy in mouse xenograft models. This observation supports the suggestion that at these efficacious doses there is no significant in vivo activity against VEGFR2 . At the time of writing, AZD4547 (41) was being evaluated in phase II clinical trials in patients with solid tumors.
3.6 Indazoles as FGFR Inhibitors
LY2874455 (42) is reported to inhibit all four isoforms of FGFR to a similar degree in a biochemical assay (IC50’s = 0.0028 μM, 0.0026 μM, 0.0064 μM, and 0.006 μM, for FGFR 1–4, respectively), in contrast to many of the selective FGFR inhibitors reported that show a preference for FGFR1–3. However, 42 is also a potent inhibitor of VEGFR2 in biochemical assays (IC50 = 0.007 μM). The authors state that the lack of high quality antibodies against pFGFRs has hampered their attempts to measure the impact of 42 on the phosphorylation of the individual FGFR isoforms in cellular systems. However, the compound was shown to inhibit the FGF2- and FGF9-stimulated phosphorylation of ERK in HUVEC and RT-112 cells (average IC50 between 0.3 and 0.8 nM) as well as showing potent antiproliferative effects in the FGFR3 driven KMS11 and OPM-2 cell lines (IC50’s = 0.57 and 1.0 nM, respectively). LY2874455 (42) inhibits both FGF2- and VEGF-induced tube-forming activities in an assay (IC50’s = 0.0006 and 0.0036 μM, respectively). A similar preference (six- to ninefold) for 42 to inhibit FGF-induced signaling in vivo over VEGF-induced signaling was observed in an IVTI assay to measure VEGFR-2 phosphorylation in the heart tissues of mice, suggesting a potential margin between FGFR-mediated effects and VEGFR-mediated effects exists in vivo. Compound 42 was reported to cause a significant regression of tumor growth in RT-112, SNU-16, and OPM-2 tumor xenograft models at doses of 3 mg/kg bid. At the time of writing, 42 had now completed a phase I clinical trial in patients with advanced cancer.
3.7 Imidazopyridine-Based Inhibitors of FGFR
Although detailed SAR investigations for this scaffold have yet to be published, the structure-based design resulted in the identification of urea-containing compounds, such as 44, which showed good potency against FGFR1–4 (IC50’s = 0.013 μM, 0.033 μM, 0.003 μM, and 0.034 μM, respectively) and moderate selectivity over VEGFR1-3 (IC50’s = 0.013 μM, 0.100 μM, and 0.068 μM, respectively). A crystal structure of 44 bound into FGFR1 highlighted good surface complementarity of the 7-aryl substituent with the protein and also indicated that the urea group forms an interaction between the two urea NHs and the carboxylate of Asp641. The urea carbonyl also appears to be involved in a water-mediated interaction with the side chain of Arg627. Interestingly, the authors note that the positioning of Arg627 is shifted with respect to its position in other FGFR1 structures. The importance of the urea group on both FGFR potency and selectivity is stated to be apparent from the detailed SAR (data not disclosed), and the authors speculate that the unusual positioning of Arg627 may contribute to the selectivity observed. Further optimization of the scaffold, playing particular attention to the reduction of lipophilicity, resulted in the identification of compound 45. Compound 45 shows good potency against FGFR1–4 (IC50’s = 0.078 μM, 0.066 μM, 0.015 μM, and 0.094 μM, respectively) and selectivity over VEGFR1-3 (IC50’s = 0.44 μM, 0.38 μM, and 0.32 μM, respectively). Compounds 44 and 45 both show antiproliferative effects in BaF3 cell lines engineered to express constitutively active forms of FGFR1, 3, and 4 and were also shown to inhibit the phosphorylation of FGFR3 in KMS11 cells. Compounds 44 and 45 both exhibit high bioavailability in mice (79% and 100%, respectively) and are efficacious in FGFR-dependent tumor xenograft models but show no efficacy in FGFR-independent models.
3.8 Quinoxaline- and Naphthyridine-Based Inhibitors of FGFR
Later disclosures from the group highlight the discovery of potent inhibitors of FGFR based on similar compounds with a pyrazolyl naphthyridine scaffold, such as 47 [29, 30]. In contrast to 46, 47 does not appear to be retained in the lysosomes, and there is no evidence of cellular accumulation. Compound 47 is reported to show a more even distribution of compound between plasma and lung tissue when compared to 46. Patent literature from this group also highlights the potential to cyclize the aminoalkyl chain onto the 3,5-dimethoxyphenyl moiety to give compounds such as 48, although potency against FGFR appears reduced .
3.9 Aminopyrazolyl Inhibitors of FGFR
Western blot analysis shows that CH518284 (51) can potently inhibit the phosphorylation of FGFR1–3 in cells but that it has significantly reduced activity against FGFR4, VEGFR2, and PDGFRβ. Compound 51 shows strong antiproliferative activity against a range of cancer cell lines harboring genetic alterations in FGFR and activity in a range of xenograft models which also harbor genetic alterations in FGFR. At doses similar to those shown to be efficacious in certain xenograft models, 51 shows little impact on the in vivo diastolic blood pressure in rats and has no effect in an in vitro VEGF-induced tube-forming assay at concentrations of 1 μM. The authors also report that 51 shows activity in both in vitro and in vivo models of a relevant FGFR2 gatekeeper mutant (V564F), and they speculate that this may differentiate the compound from other FGFR inhibitors reported. At the time of writing, CH518284 (51) was undergoing phase I clinical evaluation in selected patients harboring genetic alterations in FGFR.
3.10 Selected Other FGFR Kinase Inhibitors
A wide variety of additional ATP-competitive inhibitors of FGFR have been reported but with only limited details on the structure and medicinal chemistry discovery disclosed. A brief description of the available data for a selection of compounds which have recently entered clinical investigation is reported below.
4 Irreversible Inhibitors of FGFR
A different approach to the identification of selective FGFR inhibitors has been described by Gray et al. from the Dana-Farber Cancer Institute, Harvard Medical School, working in collaboration with researchers at the Scripps Research Institute and the Massachusetts General Hospital Cancer Center. The approach described involved the design of compounds to inhibit FGFR in an irreversible manner . Irreversible inhibitors developed to date usually possess electrophilic functionality, often α,β-unsaturated carbonyls such as acrylamides, to react with nucleophilic thiol groups on cysteine residues in a binding site. Irreversible inhibitors can exploit both the inherent selectivity resulting from non-covalent binding interactions, in addition to the requirement for a protein to possess a suitably located cysteine residue, to achieve high levels of selectivity. The authors identified a suitable cysteine present at the rim of the P-loop in FGFR1 (Cys486), which is conserved across all four FGFR isoforms and thus looked to develop irreversible inhibitors targeting this residue. In addition to increased selectivity, it was also appreciated that the kinetics of irreversible binding may, in part, overcome issues with suboptimal pharmacokinetics, such as rapid clearance, which may have limited the efficacy of other FGFR inhibitors described.
The selectivity of FIIN-1 (61) was investigated against a panel of 402 different kinase-binding assays using the Ambit KinomeScan technology at a concentration of 10 μM, and dissociation constants (KD’s) were determined for those kinases which were displaced to greater than 90% of the DMSO control. Compound 61 was seen to bind strongly to FGFR1–3 (KD’s = 0.0028 μM, 0.0069 μM and 0.0054 μM, respectively) but was significantly less potent against FGFR4 (KD = 0.12 μM). Only two other kinases were found to have KD values below 0.1 μM; these were Blk (0.065 μM) and Flt1 (0.032 μM). FIIN-1 (61) is reported to exhibit good selectivity over kinases such as c-Src, TNK1, and YES which have cysteine residues located in the same region of the binding site. Together these results indicate that 61 is a potent and selective inhibitor of FGFR1–3. Interestingly, similar biochemical potency was observed for FRIN-1 (62) against FGFR1–4 (KD = 0.0031 μM, 0.0056 μM, 0.0054 μM, and 0.28 μM, respectively) suggesting that the majority of the binding energy arises from non-covalent interactions within the binding site. Washout experiments in MCF10A cells demonstrate that 61 gives sustained inhibition of phosphorylation of both FGFR1 and ERK1/2 following washout of the drug, consistent with the proposed mechanism of irreversible inhibition. In contrast, similar experiments with either 7 or 62 demonstrate that inhibitory activity is almost completely eradicated by the washout procedure suggesting these agents act in a reversible manner. Further evidence supporting the covalent binding of 61 was developed by synthesizing biotinylated versions of both FIIN-1 (61) and FRIN-1 (62). These biotinylated analogues maintained cellular potency and FIIN-1-biotin, but not FRIN-1-biotin was found to covalently label FGFR1. Furthermore, FIIN-1-biotin was shown to covalently label wild-type FGFR1 but not an FGFR1C486S construct supporting the initial hypothesis that Cys486 is the site of covalent modification.
5 Isoform-Selective FGFR Inhibitors
Although significant progress has been made in identifying FGFR inhibitors devoid of broad spectrum kinase activity and with promising selectivity over closely related growth factor receptor tyrosine kinases, such as VEGFR2, identifying compounds which display selectivity between the individual isoforms of FGFR remains a challenge. Given the high degree of homology between the ATP-binding sites of the four FGFR isoforms, it is perhaps not surprising that the inhibitors discussed so far have shown a pan-FGFR profile; although it is worth noting that a number of the more selective pan-FGFR inhibitors developed to date actually show a preference for FGFR1–3 over FGFR4. It has been widely postulated that the ability to selectivity inhibit a single isoform of FGFR may result in compounds with improved therapeutic margins. In particular, there is evidence that inhibition of FGFR1 is associated with mineralization in preclinical models and may cause dose-limiting effects in the clinic .
5.1 FGFR4-Selective Inhibitors
The preferential inhibition of FGFR1–3, displayed by many compounds including BGJ-398 (31), AZD4547 (41), and FIIN-1 (61), has been appreciated by a number of groups who suggest such compounds may be unsuited to treat FGFR4-mediated disease as a result of the concomitant FGFR1–3-mediated pharmacology. FGFR4 is overexpressed in several cancers including colon, liver, breast, and prostate, although the role of FGFR4 in cancer is yet to be fully elucidated . FGFR4 has also been implicated in rhabdomyosarcoma (RMS) in which mutations have been identified in 7–8% of RMS tumors, and high expression of FGFR4 is associated with poor prognosis . More recently evidence has emerged demonstrating a key role for FGFR4 in hepatocellular cancer (HCC). FGF19 has unique specificity for FGFR4, and hence the FGFR4 receptor is considered the principal controller of FGF19 signaling. For FGF19 to bind to FGFR4, an additional transmembrane protein β-Klotho is required. High expression levels of both FGFR4 and β-Klotho are seen in the liver thereby strengthening the evidence that the FGFR4–FGF19 signaling axis is a key driver in HCC. Recent results have shown that in preclinical models of HCC, an FGFR4-neutralizing antibody inhibits tumor formation and development in FGF19 transgenic mice, thus suggesting the potential for FGFR4 inhibition to have therapeutic utility .
Pike et al. have described the work undertaken within the laboratories of AstraZeneca to identify selective small molecule inhibitors of FGFR4 . When screening approaches failed to identify FGFR4-selective chemical equity within the existing AstraZeneca compound collection, the researchers looked to adopt a strategy of structure-guided drug design. Detailed inspection of the ATP-binding sites in the different FGFR isoforms highlighted a number of subtle differences in the residues lining the solvent channel of FGFR4 compared with FGFR1–3. A more significant difference was observed in the hinge region of the kinase where a bulky tyrosine residue in FGFR1–3 (Tyr563 in FGFR1) is replaced with a smaller cysteine residue in FGFR4 (Cys552 in FGFR4). The authors hypothesized that compounds that protrude into these regions may display differential binding to the individual isoforms thereby delivering compounds with selectivity for FGFR4. Interestingly, only five other kinases in the kinome contain a cysteine in this position suggesting that an FGFR4-selective inhibitor may also show excellent selectivity against a broad spectrum of kinases. For the optimization of such compounds, the authors selected an imidazopyridine scaffold which was known to bind to FGFR from in-house kinase selectivity screening. A crystal structure of compound 63 bound into FGFR1 highlights a key interaction between the imidazopyridine N1 and Ala564 in the kinase hinge region. In addition, hydrogen-bonding interactions are seen between Asp641 and both the terminal amide group and aminopyrazine NH. The aminoamide aryl substituent is positioned in a hydrophobic pocket under the P-loop, and the orientation is controlled by the chirality of the terminal substituents. Importantly, while compound 63 shows no inherent selectivity for FGFR4, in fact it shows a preferential inhibition of FGFR1 in biochemical assays and preferential inhibition of phosphorylation of FGFR in Cos-1 cells transfected with either FGFR1 or FGFR4; the imidazopyridine scaffold was appreciated to contain suitable vectors for substitution to target the identified differences in both the solvent channel and the hinge region of the protein.
The presence of an FGFR4-specific cysteine residue in the ATP-binding site also allows the possibility of gaining FGFR4 selectivity through the use of an irreversible inhibitor. Pike et al. also describe the synthesis of compound 69 which contains electrophilic functionality directed toward the kinase hinge. Compound 69 shows selectivity for FGFR4 over FGFR1, and the irreversible nature of the inhibition is supported by the observation of covalent adducts between FGFR4 and 69 upon incubation of compound with protein.
Testing of BLU9931 (70) against a broad panel of 456 wild type and disease-relevant mutant kinases at 3 μM resulted in only two kinases showing inhibition of greater than 90% of DMSO control (FGFR4 = 99.7%, CSF1R = 90.1%) thereby highlighting the exceptional kinase selectivity of the molecule. Compound 70 showed potent antiproliferative effects in HCC cell lines known to overexpress FGF19 and contain functional FGFR4 (such as Hep3B EC50 = 0.07 μM, Huh-7 EC50 = 0.11 μM, JHH-7 EC50 = 0.02 μM). Compound 70 displays a moderate bioavailability of 18% and a half-life of 2.3 h in mouse and showed tumor regression in mice bearing Hep3B tumors following administration of 100 mg/kg twice a day for 21 days. Furthermore, two of the animals from this treatment group showed no regrowth of tumor 30 days after cessation of treatment suggesting that selective inhibition of FGFR4 can give durable efficacy in FGFR4–FGF19-driven HCC tumors.
Information issued by Novartis regarding the current equity within its pipeline reports the development of a compound, FGF401, which binds to and inhibits the activity of FGFR4. They report that FGF401 is at least 1,000-fold more potent against FGFR4 than it is against other kinases, including FGFR1–3. They report that the compound inhibits the proliferation of HCC cell lines and shows blockage of both FGFR4 signaling and tumor growth in animal models of human HCC. At the time of writing, a phase I clinical trial to evaluate FGF401 in patients with HCC or solid malignancies characterized by positive FGFR4 and β-Klotho was recruiting, but no specific biological data or structural data concerning the compound or close analogues had been disclosed.
5.2 FGFR3-Selective Inhibitors
More recently Winski et al. have reported the identification of novel inhibitors developed in the laboratories of Array BioPharma, in collaboration with researchers from LOXO Oncology, Stamford, which display nanomolar inhibition of FGFR3 but relatively spare FGFR1 . The authors report that these inhibitors have minimal activity against a panel of over 200 kinases. Although no structures have yet been disclosed for these inhibitors, they are reported to give high oral exposure in rodents and demonstrate tumor regression in the FGFR3-driven RT-122/84 subcutaneous xenograft models, when dosed for 14 days at either 30 or 45 mg/kg/day. The authors report only minimal hyperphosphatemia at these doses suggesting in vivo selectivity for FGFR3 over FGFR1 and supporting the hypothesis that isoform-selective FGFR inhibitors may provide treatments with improved efficacy and tolerability when compared to the pan-FGFR inhibitors currently undergoing clinical trials.
6 Inhibitors of Inactive Forms of FGFR
Targeting the inactive form of a kinase has long been considered an attractive concept due to the hypothesis that the inactive form would be more likely to adopt a distinct conformation, thereby presenting an opportunity to identify inhibitors of greater selectivity. The most widely reported approach to targeting an inactive form of a kinase focuses on the identification of so-called type II inhibitors. Such inhibitors characteristically induce a DFG-out conformation in which the Phe side chain of the activation loop (the F of the DFG) is flipped out to leave a hydrophobic pocket which is subsequently occupied by the inhibitor. Norman et al. at AstraZeneca have reported detailed crystallographic studies into the binding of the nonselective inhibitor ponatinib (“Inhibitors of Vascular Endothelial Growth Factor Receptor”) in both FGFR1 and FGFR4 . These studies reveal that ponatinib acts as a type II inhibitor of FGFR and induces a DFG-out conformation. Previous to this disclosure, all reported crystal structures of FGFR-inhibitor complexes have shown the FGFR protein in the DFG-in conformation. The authors speculate that the ability to induce a DFG-out conformation of FGFR might open new avenues for the design of novel type II inhibitors of FGFR with differing selectivity profiles and the potential for different inhibition kinetics.
ARQ069 (73) was found to inhibit the inactive (unphosphorylated) forms of both FGFR1 and FGFR2 in biochemical assays (IC50’s = 0.84 μM and 1.23 μM, respectively), whereas 72 did not. Neither compound showed significant activity against the phosphorylated (active) forms of FGFR1 or FGFR2. The inhibition of FGFR1 and FGFR2 by 73 was found to be independent of ATP concentration. In KATO-III cells, which are known to overexpress FGFR2, 73 inhibited the phosphorylation of FGFR with an IC50 value of 9.7 μM. Consistent with the observed lack of biochemical activity, 72 was inactive in this assay. A crystal structure of 73 in the autoinhibited form of FGFR1 shows that the aminopyrimidine group forms a bidentate interaction with the hinge region, while the 5,6-dihydrobenzo[h]quinazolin-2-amine core is sandwiched in a hydrophobic cleft. The phenyl substituent of 73 is positioned orthogonal to the 5,6-dihydrobenzo[h]quinazolin-2-amine core occupying the main pocket and making hydrophobic interactions with the gatekeeper residue (Val561). The authors suggest that the ability to target kinases in the autoinhibited conformation may provide a new generation of kinase inhibitors that exhibit a high degree of selectivity across the kinome.
7 Future Perspectives
The increasing evidence for dysregulated FGF/FGFR signaling to play a key role in the development of human cancers means the appetite for efficacious and well-tolerated FGFR inhibitors remains strong. The nonselective FGFR inhibitors represent the most advanced clinical agents and have shown activity in patients; however, the multi-kinase inhibition profile of these compounds (in particular VEGFR inhibition) does result in a toxicity profile which will limit their utility. The more recent emergence of selective FGFR inhibitors has allowed for a more targeted approach to FGFR inhibition but has also revealed a different toxicology profile, namely, hyperphosphatemia and tissue mineralization, which are thought to represent on-target class effects. While it may be possible to manage these effects in the clinic through the modification of diet or with additional drugs, it is not surprising that recent attention has turned toward making isoform-selective FGFR inhibitors. While it is too early to say with any certainty whether such compounds will show clinical utility, it is anticipated that such compounds would possess a more favorable toxicity profile. As with many targeted therapies, the most likely role for FGFR inhibitors is likely to be in combination with chemotherapy, radiotherapy, or other molecularly targeted agents. This means that to achieve a “best in class” profile for an FGFR inhibitor, the right balance between clinical effectiveness and patient toxicity will need to be attained. Given the important role FGF/FGFR signaling plays in normal human biology and the multitude of different aberrations identified in cancer cells, the successful treatment of patients will require a personalized medicine approach based on the genetic aberrations presented, to select those patients who will gain the maximum clinical benefit from FGFR inhibition. In addition, technology designed to give tissue-specific exposure of drugs may also help increase the therapeutic margin for FGFR inhibitors.
As the use of FGFR inhibitor in the clinic increases, it would seem likely that resistance mechanisms will start to emerge as has been observed following the inhibition of other RTKs, such as EGFR. Mutations in the ATP-binding site, such as gatekeeper mutations similar to those observed to cause resistance to first generation EGFR inhibitors, may decrease the effectiveness of current FGFR inhibitors therefore requiring the development of new inhibitors. Irreversible inhibition of FGFR, or the targeting of inactive forms of the protein, may provide opportunities to overcome resistance mutations. In addition to the small molecule inhibitors of FGFR discussed above, research is also underway to develop antibody approaches to target both FGF ligands and the ligand-binding domains of FGFR. Such approaches might also provide effective inhibition of single FGFR isoforms although it is currently unclear whether the long duration of action often associated with antibody therapies would be a benefit or concern in the context of FGFR inhibition.
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