Daunorubicin induces GLI1­dependent apoptosis in colorectal cancer cell lines.

Daunorubicin, also known as daunomycin, is a DNA­targeting anticancer drug that is used as chemotherapy, mainly for patients with leukemia. It has also been shown to have anticancer effects in monotherapy or combination therapy in solid tumors, but at present it has not been adequately studied in colorectal cancer (CRC). In the present study, from a screening using an FDA­approved drug library, it was found that daunorubicin suppresses GLI­dependent luciferase reporter activity. Daunorubicin also increased p53 levels, which contributed to both GLI1 suppression and apoptosis. The current detailed investigation showed that daunorubicin promoted the ß­TrCP­mediated ubiquitination and proteasomal degradation of GLI1. Moreover, a competition experiment using BODIPY­cyclopamine, a well­known Smo inhibitor, suggested that daunorubicin does not bind to Smo in HCT116 cells. Administration of daunorubicin (2 mg/kg, ip, qod, 15 days) into HCT116 xenograft mice profoundly suppressed tumor progress and the GLI1 level in tumor tissues. Taken together, the present results revealed that daunorubicin suppresses canonical Hedgehog pathways in CRC. Ultimately, the present study discloses a new mechanism of daunorubicin's anticancer effect and might provide a rationale for expanding the clinical application of daunorubicin.

Discovery of N-benzylbenzamide-based allosteric inhibitors of Aurora kinase A.

Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6h) of N-benzylbenzamide backbone. Compound 6h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6h against AurkA (IC50 = 6.50 µM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure-activity relationship. In addition, 6h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors.

Identification of a novel potent CDK inhibitor degrading cyclinK with a superb activity to reverse trastuzumab-resistance in HER2-positive breast cancer in vivo.

CDK12 is overexpressed in HER2-positive breast cancers and promotes tumorigenesis and trastuzumab resistance. Thus CDK12 is a good therapeutic target for the HER2-positive breast tumors resistant to trastuzumab. We previously reported a novel purine-based CDK inhibitor with an ability to degrade cyclinK. Herein, we further explored and synthesized new derivatives, and identified a new potent pan-CDK inhibitor degrading cyclinK (32e). Compound 32e potently inhibited CDK12/cyclinK with IC50 = 3 nM, and suppressed the growth of the both trastuzumab-sensitive and trastuzumab-resistant HER2-positive breast cancer cell lines (GI50's = 9-21 nM), which is superior to a potent, clinical pan-CDK inhibitor dinaciclib. Moreover, 32e (10, 20 mg/kg, ip, twice a week) showed a dose-dependent inhibition of tumor growth and a more dramatic anti-cancer effect than dinaciclib in mouse in vivo orthotopic breast cancer model of trastuzumab-resistant HCC1954 cells. Kinome-wide inhibition profiling revealed that 32e at 1 µM exhibits a decent selectivity toward CDK-family kinases including CDK12 over other wildtype protein kinases. Quantitative global proteomic analysis of 32e-treated HCC1954 cells demonstrated that 32e also showed a decent selectivity in degrading cyclinK over other cyclins. Compound 32e could be developed as a drug for intractable trastuzumab-resistant HER2-positive breast cancers. Our current study would provide a useful insight in designing potent cyclinK degraders.

Discovery of 3-((3-amino-1H-indazol-4-yl)ethynyl)-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide (AKE-72), a potent Pan-BCR-ABL inhibitor including the T315I gatekeeper resistant mutant.

BCR-ABL inhibition is an effective therapeutic approach for the treatment of chronic myeloid leukaemia (CML). Herein, we report the discovery of AKE-72 (5), a diarylamide 3-aminoindazole, as a potent pan-BCR-ABL inhibitor, including the imatinib-resistant mutant T315I. A focussed array of compounds 4a, 4b, and 5 has been designed based on our previously reported indazole I to improve its BCR-ABLT315I inhibitory activity. Replacing the morpholine moiety of I with the privileged tail (4-ethylpiperazin-1-yl)methyl afforded 5 (AKE-72) with IC50 values of < 0.5 nM, and 9 nM against BCR-ABLWT and BCR-ABLT315I, respectively. Moreover, AKE-72 potently inhibited a panel of other clinically important mutants in single-digit nanomolar IC50 values. AKE-72 elicited remarkable anti-leukemic activity against K-562 cell line (GI50 < 10 nM, TGI = 154 nM). In addition, AKE-72 strongly inhibited the proliferation of Ba/F3 cells expressing native BCR-ABL or its T315I mutant. Overall, AKE-72 may serve as a promising candidate for the treatment of CML, including those harbouring T315I mutation.

Protective effects of esomeprazole against cisplatin-induced ototoxicity: an in vitro and in vivo study.

In this study, we aimed to identify novel compounds that could afford protection against cisplatin-induced ototoxicity by employing both cell- and zebrafish (Danio rerio)-based screening platforms. We screened 923 US Food and Drug Administration-approved drugs to identify potential compounds exhibiting protective effects against cisplatin-induced ototoxicity in HEI-OC1 cells (auditory hair cell line). The screening strategy identified esomeprazole and dexlansoprazole as the primary hit compounds. Subsequently, we examined the effects of these compounds on cell viability and apoptosis. Our results revealed that esomeprazole and dexlansoprazole inhibited organic cation transporter 2 (OCT2), thus providing in vitro evidence that these compounds could ameliorate cisplatin-induced ototoxicity by directly inhibiting OCT2-mediated cisplatin transport. In vivo, the protective effects were validated using zebrafish; esomeprazole was found to decrease cisplatin-induced hair cell damage in neuromasts. Furthermore, the esomeprazole-treated group showed a significantly lower number of TUNEL-positive cells than the cisplatin-treated group. Collectively, our findings revealed that esomeprazole exerts a protective effect against cisplatin-induced hair cell damage in both HEI-OC1 cells and a zebrafish model.

Identification of novel class inhibitors of NSD3 methyltransferase showing a unique, bivalent binding mode in the SET domain.

NSD3/WHSC1L1 lysine methyltransferase promotes the transcription of target genes through di- or tri-methylation at histone H3K36 using SAM as a cofactor. Genetic alterations such as amplification and gain-of-function mutation of NSD3 act as oncogenic drivers in several cancers including squamous cell lung cancer and breast cancer. NSD3 is an important therapeutic target for cancers, but the reported NSD3 inhibitors targeting the catalytic SET domain are very rare and show a poor activity. Herein, from a virtual library screening and the subsequent medicinal chemistry optimization, we identified a novel class of NSD3 inhibitors. Our docking analysis and pulldown result suggested that the most potent analogue 13i shows a unique, bivalent binding mode interacting with both SAM-binding site and BT3-bindig site within the SET domain. We found 13i inhibits NSD3 activity with IC50 = 287 µM in vitro and suppresses the proliferation of JIMT1 breast cancer cells with GI50 = 36.5 µM, which express a high level of NSD3. Also, 13i downregulated the levels of H3K36me2/3 in a dose-dependent manner. Our study could provide an insight in designing high-affinity NSD3 inhibitors. Also, as the acrylamide group of 13i was predicted to position near Cys1265 in the BT3-binding site, further optimization would lead to a discovery of novel irreversible NSD3 inhibitors.

Novel 2,6,9-Trisubstituted Purines as Potent CDK Inhibitors Alleviating Trastuzumab-Resistance of HER2-Positive Breast Cancers.

HER2-positive (HER2+) breast cancer is defined by HER2 oncogene amplification on chromosome 17q12 and accounts for 15−20% population of breast-cancer patients. Therapeutic anti-HER2 antibody such as trastuzumab is used as the first-line therapy for HER2-positive breast cancers. However, more than 50% of the patients respond poorly to trastuzumab, illustrating that novel therapy is warranted to overcome the resistance. We previously reported that in the majority of HER2+ breast-cancer patients, CDK12 is co-amplified on 17q12 and involved in developing tumors and trastuzumab resistance, proposing CDK12 as a potential drug target for HER2+ breast cancers. Here, we designed and synthesized novel 2,6,9-trisubstituted purines as potent CDK12 inhibitors showing strong, equipotent antiproliferative activity against trastuzumab-sensitive HER2+ SK-Br3 cells and trastuzumab-resistant HER2+ HCC1954 cells (GI50 values < 50 nM) both of which express a high level of CDK12. Two potent analogue 30d and 30e at 40, 200 nM greatly downregulated the levels of cyclinK and Pol II p-CTD (Ser2), as well as the expression of CDK12 downstream genes (IRS1 and WNT1) in a dose-dependent manner. We also observed structure-property relationship for a subset of potent analogues, and found that 30e is highly stable in liver microsomes with lack of CYP inhibition. In addition, 30d exhibited a synergy with trastuzumab in the both cells, suggesting that our inhibitors could be applied to alleviate trastuzumab-resistance of HER2+ breast cancers and escalate the efficacy of trastuzumab as well. Our study may provide insight into developing a novel therapy for HER2+ breast cancers.

Design, synthesis, and biological evaluation of novel aminopyrimidinylisoindolines as AXL kinase inhibitors.

A novel series of aminopyrimidinylisoindoline derivatives 1a-w having an aminopyrimidine scaffold as a hinge region binding motif were designed and synthesized. Among them, six compounds showed potent inhibitory activities against AXL kinase with IC50 values of submicromolar range. Especially, compound 1u possessing (4-acetylpiperazin-1-yl)phenyl moiety exhibited extremely excellent efficacy (IC50 = <0.00050 µM). Their in vitro antiproliferative activities were tested over five cancer cell lines. Most compounds showed good antiproliferative activities against HeLa cell line. The kinase panel profiling of 50 different kinases and the selected inhibitory activities for the representative compound 1u were carried out. The compound 1u exhibited excellent inhibitory activities (IC50 = <0.00050, 0.025, and 0.050 µM for AXL, MER, and TYRO3, respectively) against TAM family, together with potent antiproliferative activity against MV4-11 cell line (GI50 = 0.10 µM) related to acute myeloid leukemia (AML).

Design, synthesis and biological evaluation of novel thiazolidinedione derivatives as irreversible allosteric IKK-ß modulators.

The kinase known as IKK-ß activates NF-κB signaling pathway leading to expression of several genes contributing to inflammation, immune response, and cell proliferation. Modulation of IKK-ß kinase activity could be useful for treatment and management of such diseases. Starting from a discovered weakly active hit compound, twenty four thiazolidinedione-scaffold based chemical entities belonging to five series have been designed, synthesized and evaluated as potential IKK-ß modulators. Among them, compounds 6q, 6r and 6u showed low micromolar IC50 values while compounds 6v, 6w, and 6x elicited submicromolar IC50 values equal to 0.4, 0.7 and 0.9 µM respectively. These submicromolar IC50 values are 243, 139 and 105 folds the value of the reported IC50 of the starting hit compound. Kinetic study of compounds 6v and 6w confirmed this class of modulators as irreversible inhibitors. LPS-treated RAW 264.7 macrophages proved the anti-inflammatory activity of compounds 6q and 6v. Assay of hERG inhibition demonstrated a safe profile of compound 6q suggesting it as a lead for further development of IKK-ß modulators.

First SAR Study for Overriding NRAS Mutant Driven Acute Myeloid Leukemia.

GNF-7, a multitargeted kinase inhibitor, served as a dual kinase inhibitor of ACK1 and GCK, which provided a novel therapeutic strategy for overriding AML expressing NRAS mutation. This SAR study with GNF-7 derivatives, designed to target NRAS mutant-driven AML, led to identification of the extremely potent inhibitors, 10d, 10g, and 11i, which possess single-digit nanomolar inhibitory activity against both ACK1 and GCK. These substances strongly suppress proliferation of mutant NRAS expressing AML cells via apoptosis and AKT/mTOR signaling blockade. Compound 11i is superior to GNF-7 in terms of kinase inhibitory activity, cellular activity, and differential cytotoxicity. Moreover, 10k possessing a favorable mouse pharmacokinetic profile prolonged life-span of Ba/F3-NRAS-G12D injected mice and significantly delayed tumor growth of OCI-AML3 xenograft model without causing the prominent level of toxicity found with GNF-7. Taken together, this study provides insight into the design of novel ACK1 and GCK dual inhibitors for overriding NRAS mutant-driven AML.

Amphiphilic small peptides for delivery of plasmid DNAs and siRNAs.

Although various delivery systems for nucleic acids have been reported, development of an efficient and non-toxic delivery carrier is still a key subject for gene therapy. To find new efficient delivery carriers for nucleic acids, we synthesized amphiphilic peptides composed of a guanidino group, an oleyl group, and a cysteine. We prepared both linear and branched types of peptides and found that the linear peptides were superior to the branched peptides as nucleic acid carriers. Our study also suggested that the intermolecular cysteine disulfides might allow the linear peptides to form the optimal particle sizes with nucleic acids for cellular uptake. The incorporation of a benzoyl group to the linear peptide gave rise to smaller, less suitable particle size with plasmid DNA, which greatly reduced the efficiency of plasmid DNA delivery. On the other hand, the benzoyl modification maintained the optimal particle size with siRNA, and interestingly it significantly enhanced the siRNA delivery. The higher efficiency is because the hydrophobicity from the benzoyl group might assist in interacting with the hydrophobic cell membrane. This demonstrates that a small structural change can modulate the preference of the carriers. Our study may provide an insight designing efficient delivery carriers.

Structure-activity relationship investigation for benzonaphthyridinone derivatives as novel potent Bruton's tyrosine kinase (BTK) irreversible inhibitors.

Through a structure-based drug design approach, a tricyclic benzonaphthyridinone pharmacophore was used as a starting point for carrying out detailed medicinal structure-activity relationhip (SAR) studies geared toward characterization of a panel of proposed BTK inhibitors, including 6 (QL-X-138), 7 (BMX-IN-1) and 8 (QL47). These studies led to the discovery of the novel potent irreversible BTK inhibitor, compound 18 (CHMFL-BTK-11). Kinetic analysis of compound 18 revealed an irreversible binding efficacy (kinact/Ki) of 0.01 µM-1s-1. Compound 18 potently inhibited BTK kinase Y223 auto-phosphorylation (EC50 < 100 nM), arrested cell cycle in G0/G1 phase, and induced apoptosis in Ramos, MOLM13 and Pfeiffer cells. We believe these features would make 18 a good pharmacological tool for studying BTK-related pathologies.

Identification of TG100-115 as a new and potent TRPM7 kinase inhibitor, which suppresses breast cancer cell migration and invasion.

BACKGROUND: Transient receptor potential melastatin 7 (TRPM7) regulates breast cancer cell proliferation, migration, invasion and metastasis in its ion channel- and kinase domain-dependent manner. The pharmacological effects of TRPM7 ion channel inhibitors on breast cancer cells have been studied, but little is known about the effects of TRPM7 kinase domain inhibitors due to lack of potent TRPM7 kinase inhibitors. METHODS: Screening was performed by using TRPM7 kinase assay. Effects of TG100-115 on breast cancer cell proliferation, migration, invasion, myosin IIA phosphorylation, and TRPM7 ion channel activity were assessed by using MTT, wound healing, transwell assay, Western blotting, and patch clamping, respectively. RESULTS: We found that CREB peptide is a potent substrate for the TR-FRET based TRPM7 kinase assay. Using this method, we discovered a new and potent TRPM7 kinase inhibitor, TG100-115. TG100-115 inhibited TRPM7 kinase activity in an ATP competitive fashion with over 70-fold stronger activity than that of rottlerin, known as a TRPM7 kinase inhibitor. TG100-115 has little effect on proliferation of MDA-MB-231 cells, but significantly decreases cell migration and invasion. Moreover, TG100-115 inhibits TRPM7 kinase regulated phosphorylation of the myosin IIA heavy chain and phosphorylation of focal adhesion kinase. TG100-115 also suppressed TRPM7 ion channel activity. CONCLUSIONS: TG100-115 can be used as a potent TRPM7 kinase inhibitor and a potent inhibitor of breast cancer cell migration. GENERAL SIGNIFICANCE: TG100-115 could be a useful tool for studying the pharmacological effects of TRPM7 kinase activity aimed at providing insight into new therapeutic approaches to the treatment of breast cancer.

Identification of the First Selective Activin Receptor-Like Kinase 1 Inhibitor, a Reversible Version of L-783277.

We synthesized 1 (San78-130), a reversible version of L-783277, as a selective and potent ALK1 inhibitor. Our study showed that 1 possesses great kinase selectivity against a panel of 342 kinases and more potent activity against ALK1 than L-783277. Among the six ALK isotypes (ALK1-6), ALK1 is most significantly inhibited by compound 1. Compound 1 suppresses the BMP9-induced Smad1/5 pathway by mainly inhibiting ALK1 in C2C12 cells. Our molecular dynamics simulations suggest that H-bonding interaction between the C-4' hydroxyl group of 1 and Arg334 of ALK1 substantially contributes to the ALK1 inhibition. To the best of our knowledge, 1 is the first selective ALK1 inhibitor. Furthermore, compound 1 promoted angiogenesis in both endothelial tube formation and microfluidic chip based 3D angiogenesis assays, suggesting that 1 could be a lead compound for therapeutic angiogenesis agents. Our study may provide an insight into designing selective and potent inhibitors against ALK1.

A benzo[b]thiophene-based selective type 4 S1P receptor agonist.

S1P receptors (S1PR1-5) are a group of GPCRs activated by a high affinity binding with S1P that have important roles in the regulation of the immune system. A potent S1PR agonist FTY720 is an immunomodulator used to treat multiple sclerosis and several 'second generation' drugs are under clinical development. Subtype-selective agonists have been reported for each S1PR isotype, some of which are used as pharmacological tools for functional studies. Here we report the discovery and initial characterization of compound 5c, a benzo[b]thiophene amino carboxylate which exhibits potent and selective agonist activity for S1PR4. Compound 5c has an EC50=200nM as an agonist in GTPγ35S binding assay for S1PR4 and exhibits no activity against S1PR1,2,3,5. We confirmed its potent activity and decent S1PR subtype selectivity using biochemical and cellular assays.

Antitumor Effects and Mechanisms of AZD4547 on FGFR2-Deregulated Endometrial Cancer Cells.

Uncontrolled activation of FGFRs induces the progression of various cancers. It was recently reported that FGFR2-activating mutants are implicated in about 12% of endometrial carcinomas. AZD4547, a potent pan-FGFR inhibitor, is currently being evaluated in clinical trials for several FGFR-driven cancers. However, AZD4547 has not been examined yet against FGFR2 mutant-driven endometrial cancers. Thus, we evaluated the activity of AZD4547 against four different endometrial cancer cells, including AN3-CA, MFE296, MFE280, and HEC1A, where all but HEC1A cells express distinctive FGFR2 mutations. We found that AZD4547 exhibits potent antiproliferative activity (EC50 = 31 nmol/L) against AN3-CA cells harboring FGFR2-K310R/N550K mutant. Analysis using a phospho-kinase array revealed that AZD4547 blocks FGFR2 downstream signaling, such as p38, ERK1/2, JNK, p70S6K, and PLCγ. Moreover, oral administration of AZD4547 (30 mg/kg, every day) remarkably delayed tumor growth in a mouse xenograft model of AN3-CA cells. Unbiased reporter gene assay showed that AZD4547 antagonizes the aFGF-induced activation of several transcription factors, including EGR1, ELK-1/SRF, AP-1, and NFκB. Genome-wide transcriptome analysis revealed that AZD4547 perturbs a number of transcriptions, and EGR1 was identified as one of the major targets of AZD4547. The significance of the FGFR2-EGR1 axis in endometrial cancer progression has not been reported. In addition, using kinome-wide inhibition profiling analysis, we first identified potential new target kinases of AZD4547, including MAP4K3, MAP4K5, IRR, RET, and FLT3. Our study demonstrated that AZD4547 exhibits its therapeutic activity against endometrial cancer cells by perturbing various regulatory mechanisms related to FGFR signaling.

Identification of Novel ROS Inducers: Quinone Derivatives Tethered to Long Hydrocarbon Chains.

We performed the first synthesis of the 17-carbon chain-tethered quinone moiety 22 (SAN5201) of irisferin A, a natural product exhibiting anticancer activity, and its derivatives. We found that 22 is a potent ROS inducer and cytotoxic agent. Compound 25 (SAN7401), the hydroquinone form of 22, induced a significant release of intracellular ROS and apoptosis (EC50 = 1.3-2.6 µM) in cancer cell lines, including A549 and HCT-116. Compared with the activity of a well-known ROS inducer, piperlongumine, 22 and 25 showed stronger cytotoxicity and higher selectivity over noncancerous cells. Another hydroquinone tethering 12-carbon chain, 26 (SAN4601), generated reduced levels of ROS but showed more potent cytotoxicity (EC50 = 0.8-1.6 µM) in cancer cells, although it lacked selectivity over noncancerous cells, implying that the naturally occurring 17-carbon chain is also crucial for ROS production and a selective anticancer effect. Both 25 and 26 displayed strong, equipotent activities against vemurafenib-resistant SK-Mel2 melanoma cells and p53-deficient H1299 lung cancer cells as well, demonstrating their broad therapeutic potential as anticancer agents.

A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition.

Patient-derived models of acquired resistance can identify effective drug combinations for cancer.

Targeted cancer therapies have produced substantial clinical responses, but most tumors develop resistance to these drugs. Here, we describe a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance. We established cell culture models derived from biopsy samples of lung cancer patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected these cells to genetic analyses and a pharmacological screen. Multiple effective drug combinations were identified. For example, the combination of ALK and MAPK kinase (MEK) inhibitors was active in an ALK-positive resistant tumor that had developed a MAP2K1 activating mutation, and the combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant resistant cancer with a mutation in FGFR3. Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, this strategy could help direct therapeutic choices for individual patients.

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.