Discovery of Futibatinib: The First Covalent FGFR Kinase Inhibitor in Clinical Use.

Deregulating fibroblast growth factor receptor (FGFR) signaling is a promising strategy for cancer therapy. Herein, we report the discovery of compound 5 (TAS-120, futibatinib), a potent and selective covalent inhibitor of FGFR1-4, starting from a unique dual inhibitor of mutant epidermal growth factor receptor and FGFR (compound 1). Compound 5 inhibited all four families of FGFRs in the single-digit nanomolar range and showed high selectivity for over 387 kinases. Binding site analysis revealed that compound 5 covalently bound to the cysteine 491 highly flexible glycine-rich loop region of the FGFR2 adenosine triphosphate pocket. Futibatinib is currently in Phase I-III trials for patients with oncogenically driven FGFR genomic aberrations. In September 2022, the U.S. Food & Drug Administration granted accelerated approval for futibatinib in the treatment of previously treated, unresectable, locally advanced, or metastatic intrahepatic cholangiocarcinoma harboring an FGFR2 gene fusion or other rearrangement.

TAS-119, a novel selective Aurora A and TRK inhibitor, exhibits antitumor efficacy in preclinical models with deregulated activation of the Myc, ß-Catenin, and TRK pathways.

Aurora kinase A, a mitotic kinase that is overexpressed in various cancers, is a promising cancer drug target. Here, we performed preclinical characterization of TAS-119, a novel, orally active, and highly selective inhibitor of Aurora A. TAS-119 showed strong inhibitory effect against Aurora A, with an IC50 value of 1.04 nmol/L. The compound was highly selective for Aurora A compared with 301 other protein kinases, including Aurora kinase B. TAS-119 induced the inhibition of Aurora A and accumulation of mitotic cells in vitro and in vivo. It suppressed the growth of various cancer cell lines harboring MYC family amplification and CTNNB1 mutation in vitro. In a xenograft model of human lung cancer cells harboring MYC amplification and CTNNB1 mutation, TAS-119 showed a strong antitumor activity at well-tolerated doses. TAS-119 induced N-Myc degradation and inhibited downstream transcriptional targets in MYCN-amplified neuroblastoma cell lines. It also demonstrated inhibitory effect against tropomyosin receptor kinase (TRK)A, TRKB, and TRKC, with an IC50 value of 1.46, 1.53, and 1.47 nmol/L, respectively. TAS-119 inhibited TRK-fusion protein activity and exhibited robust growth inhibition of tumor cells via a deregulated TRK pathway in vitro and in vivo. Our study indicates the potential of TAS-119 as an anticancer drug, especially for patients harboring MYC amplification, CTNNB1 mutation, and NTRK fusion.

Futibatinib Is a Novel Irreversible FGFR 1-4 Inhibitor That Shows Selective Antitumor Activity against FGFR-Deregulated Tumors.

FGFR signaling is deregulated in many human cancers, and FGFR is considered a valid target in FGFR-deregulated tumors. Here, we examine the preclinical profile of futibatinib (TAS-120; 1-[(3S)-[4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3, 4-d] pyrimidin-1-yl]-1-pyrrolidinyl]-2-propen-1-one), a structurally novel, irreversible FGFR1-4 inhibitor. Among a panel of 296 human kinases, futibatinib selectively inhibited FGFR1-4 with IC50 values of 1.4 to 3.7 nmol/L. Futibatinib covalently bound the FGFR kinase domain, inhibiting FGFR phosphorylation and, in turn, downstream signaling in FGFR-deregulated tumor cell lines. Futibatinib exhibited potent, selective growth inhibition of several tumor cell lines (gastric, lung, multiple myeloma, bladder, endometrial, and breast) harboring various FGFR genomic aberrations. Oral administration of futibatinib led to significant dose-dependent tumor reduction in various FGFR-driven human tumor xenograft models, and tumor reduction was associated with sustained FGFR inhibition, which was proportional to the administered dose. The frequency of appearance of drug-resistant clones was lower with futibatinib than a reversible ATP-competitive FGFR inhibitor, and futibatinib inhibited several drug-resistant FGFR2 mutants, including the FGFR2 V565I/L gatekeeper mutants, with greater potency than any reversible FGFR inhibitors tested (IC50, 1.3-50.6 nmol/L). These results indicate that futibatinib is a novel orally available, potent, selective, and irreversible inhibitor of FGFR1-4 with a broad spectrum of antitumor activity in cell lines and xenograft models. These findings provide a strong rationale for testing futibatinib in patients with tumors oncogenically driven by FGFR genomic aberrations, with phase I to III trials ongoing. SIGNIFICANCE: Preclinical characterization of futibatinib, an irreversible FGFR1-4 inhibitor, demonstrates selective and potent antitumor activity against FGFR-deregulated cancer cell lines and xenograft models, supporting clinical evaluation in patients with FGFR-driven tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/22/4986/F1.large.jpg.

Aurora A Inhibitor TAS-119 Enhances Antitumor Efficacy of Taxanes In Vitro and In Vivo: Preclinical Studies as Guidance for Clinical Development and Trial Design.

TAS-119 is a novel orally active, selective inhibitor of Aurora kinase A identified as a clinical candidate for efficacy testing in combination with taxanes. In vitro, TAS-119 enhanced cell growth inhibition of paclitaxel in multiple human cancer cell lines derived from various tissues, including paclitaxel-resistant cell lines. Interestingly, TAS-119 did not enhance paclitaxel antitumor activity in normal lung diploid fibroblast cell lines WI-38 and MRC5. In vivo, TAS-119 enhanced the antitumor efficacy of paclitaxel and docetaxel in multiple models at doses inhibitory to Aurora A in tumors. Moreover, the drug combination was well tolerated, and TAS-119 did not exaggerate clinically documented side effects of taxanes, neutropenia and neurotoxicity, in rats. The same TAS-119 concentration enhanced the cell growth inhibitory activity of three clinically approved taxanes, paclitaxel, docetaxel, and cabazitaxel. The degree of enhancement calculated as fold of change of the IC50 value for each taxane was almost the same among the three taxanes. We conducted in vitro and in vivo experiments to develop an optimized combination therapy regimen for TAS-119 with paclitaxel/docetaxel. Using in vitro and in vivo models, we tested the drug administration order for TAS-119 combined with paclitaxel and the TAS-119 treatment duration. The best regimen in preclinical models was combining paclitaxel or docetaxel treatment with 4 days of TAS-119 dosing, which was initiated on the same day as the paclitaxel or docetaxel administration or one day later. This information provided guidance for the design of a clinical trial of TAS-119 and paclitaxel or docetaxel combination.

MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo.

The serine/threonine kinase Akt lies at a critical signaling node downstream of phosphatidylinositol-3-kinase and is important in promoting cell survival and inhibiting apoptosis. An Akt inhibitor may be particularly useful for cancers in which increased Akt signaling is associated with reduced sensitivity to cytotoxic agents or receptor tyrosine kinase inhibitors. We evaluated the effect of a novel allosteric Akt inhibitor, MK-2206, in combination with several anticancer agents. In vitro, MK-2206 synergistically inhibited cell proliferation of human cancer cell lines in combination with molecular targeted agents such as erlotinib (an epidermal growth factor receptor inhibitor) or lapatinib (a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 inhibitor). Complementary inhibition of erlotinib-insensitive Akt phosphorylation by MK-2206 was one mechanism of synergism, and a synergistic effect was found even in erlotinib-insensitive cell lines. MK-2206 also showed synergistic responses in combination with cytotoxic agents such as topoisomerase inhibitors (doxorubicin, camptothecin), antimetabolites (gemcitabine, 5-fluorouracil), anti-microtubule agents (docetaxel), and DNA cross-linkers (carboplatin) in lung NCI-H460 or ovarian A2780 tumor cells. The synergy with docetaxel depended on the treatment sequence; a schedule of MK-2206 dosed before docetaxel was not effective. MK-2206 suppressed the Akt phosphorylation that is induced by carboplatin and gemcitabine. In vivo, MK-2206 in combination with these agents exerted significantly more potent tumor inhibitory activities than each agent in the monotherapy setting. These findings suggest that Akt inhibition may augment the efficacy of existing cancer therapeutics; thus, MK-2206 is a promising agent to treat cancer patients who receive these cytotoxic and/or molecular targeted agents.

A cell-based screening method for specifically detecting kinase activity.

No universal approach has been reported for specific monitoring of the catalytic activity of a wide range of kinases in cells. The present study describes an original platform for detecting the autonomous activity of serine/threonine kinases in cells through the introduction of expression vectors encoding modified substrate kinase fusion proteins. The surrogate substrate used consists of the p53 tumor suppressor protein fused with individual kinase domains (Chk1, DYRK3, and Cdk5) at its carboxy-terminal through four tandem Gly-Gly-Gly-Gly-Ser repeats. After transfection into cells, phosphorylation of the p53 moiety could be specifically induced by the catalytic activity of kinases contained in the fusion protein. Moreover, p53 phosphorylation was significantly blocked when a kinase-inactive mutant was used as the fusion partner instead of the active kinase. Using this system, the cell-based evaluation of several Cdk5 inhibitors was demonstrated. Thus, this approach provides a novel platform for the specific, cell-based screening of inhibitors of a wide prospective range of protein kinases and is of tremendous potential for drug discovery efforts.

Dual enzyme-linked immunosorbent assay system for detection of endogenous kinase activities of mitogen- and stress-activated protein kinase-1/2.

The kinase signaling cascades related to mitogen- and stress-activated protein kinase-1 and -2 (MSK1 and MSK2, respectively) are attractive targets for pharmaceutical intervention, especially for neural injury. Therefore, we have developed a high throughput and cost-effective detection platform for measuring selective activity of MSK1/MSK2 in cells. Through the serial monitoring of both the p38 mitogen-activated protein kinase (stress-activated protein kinase 2B)-MSK1/MSK2- cyclic AMP response element binding protein (CREB)/activating transcription factor 1 (ATF1) pathway and the p38-mammalian heat shock protein 27 (Hsp27) pathway in HeLa cells treated with anisomycin, two selective MSK1 inhibitors showed inhibition of CREB (Ser-133) and ATF1 (Ser-63) phosphorylation and no interference with Hsp-27 phosphorylation (Ser-82). On the other hand, the p38 inhibitor SB-220025 showed equipotent inhibition of CREB/ATF1 and Hsp27 phosphorylation. This study demonstrated that the specific inhibition of a target kinase could be subsequently monitored by a secondary assay that measures the intervention arising from the modulation of off-target kinases. Our established system is applicable to inhibitor screening and drug discovery related to MSK1/MSK2.

A robust, target-driven, cell-based assay for checkpoint kinase 1 inhibitors.

Checkpoint kinase 1 (Chk1), a serine/threonine kinase, plays an important role in DNA damage checkpoint control and is an attractive target for cancer treatment. To develop a Chk1-specific cell-based assay, stable clones were established in which Chk1 kinase domain fused at its N-terminus with p53 through 4 tandem repeats of Gly-Gly-Gly-Gly-Ser was expressed in an inducible manner. Chk1 kinase specificity of the phosphorylation of fused p53 was confirmed by the experiments with a kinase-inactive Chk1. Only in the presence of an inducer molecule was phosphorylation of p53 at Ser-15 in the stable clones induced. Furthermore, its assay performance proved acceptable for high-throughput screening applications, judging from the Z' factor values (> 0.77). Finally, the cell-based assay thus established yielded structure-activity relationship data for a small set of test inhibitors of Chk1 within cells. Collectively, these results demonstrate that the established cell-based assay provides a novel and highly sensitive cellular platform for Chk1 inhibitor discovery.

A selective cellular screening assay for B-Raf and c-Raf kinases.

The Ras/Raf signaling pathway has been recognized as an important process in cancer biology. Recently, activating mutations in the BRAF gene were reported to be present in approximately 66% of malignant melanomas as well as other malignancies such as colon cancer. Here, the authors report the development of a B-Raf-specific cellular assay to profile cell-active B-Raf inhibitors. Expression of the active B-Raf mutant (V600E) and the kinase-inactive form of its substrate, MEK1, was regulated by mifepristone, and the catalytic activity of B-Raf was monitored by following MEK1 phosphorylation. Target specificity was ensured because the phosphorylation of MEK1 was significantly inhibited when kinase-inactive B-Raf was used in place of the active kinase. A cellular c-Raf assay was similarly established to monitor the selectivity between B-Raf and c-Raf. Z' factor values were consistently above 0.50 with either kinase, indicating that assay performance was sufficiently robust for use as cellular profiling assays. The authors used this system to demonstrate that the selectivity profile of compounds targeted against B-Raf and c-Raf kinases could be quantitatively determined. This platform provides a quantitative cellular readout for a spectrum of specific inhibitors of B-Raf and c-Raf kinases that is particularly suitable for use in drug discovery.

Practical evaluation of universal conditions for four-plex quantitative PCR.

Multiplexing quantitative polymerase chain reaction (qPCR) is a powerful way to substantially increase the number of genes that can be analyzed, while also reducing sample requirements, time, and cost. However, little previous work has been done to show its feasibility for multiple gene targets. Here, we determined optimal conditions for four-color multiplex qPCR. On the basis of amplification curves, we first established that the concentration of probe-primers should be about tenfold lower than that for conventional qPCR. This condition was evaluated using four sets of probe-primers labeled with FAM, CAL Fluor Orange, TAMRA, and Quasar670, respectively. To simulate the condition that different genes have different levels of transcript abundance, a series of test samples was prepared by mixing a constant amount of two kinds of vector together with different amounts of two other vectors in a four-plex qPCR format. The PCR efficiency of the constant genes was minimally affected by the presence of the spiked vectors, and the slope factors of standard curves for the two spiked genes were sufficient for the accurate quantification. We demonstrated here that qPCR in a four-plexed format is feasible for cost-effective practical use through a combination of lower concentrations of probe-primers, an appropriate reagent, and a detection instrument.

Validation of universal conditions for duplex quantitative reverse transcription polymerase chain reaction assays.

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for measuring mRNA in biological materials. Multiplex qRT-PCR provides advantages for gene expression analysis by reducing sample requirements, saving time, and lowering experimental cost. However, there are currently no universal qRT-PCR experimental conditions validated as applicable to a large set of genes. We report here on the standardized condition for two-color real-time qRT-PCR with the Quantitect Multiplex PCR kit. We first verified lack of interferential effects of gene abundance on the efficiency of PCR amplification by an 8x8 checkerboard validation method, in which combinations of the plasmids encoding either fibronectin1 or cyclophilin mixed at 64 different ratios were amplified with the Quantitect Multiplex PCR kit. Then, a duplex analysis for 69 genes was performed to verify the universality of the reaction condition. The results were consistent with corresponding data obtained from the singleplex format, and their intra- and interassay coefficients of variance were sufficient for performing reliable quantitative analysis. This duplex format was also applicable to samples from animal experiments, with a good correlation between singleplex and duplex-assay (R(2)>0.92) observed. This duplex assay system is ready for use in high-throughput gene expression analysis without any gene-pair compatibility restrictions limiting its use.