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As the critical driving force for chronic myeloid leukemia (CML), BCR gene fused ABL kinase has been extensively explored as a validated target of drug discovery. Although imatinib has achieved tremendous success as the first-line treatment for CML, the long-term application ultimately leads to resistance, primarily via various acquired mutations occurring in the BCR-ABL kinase. Although dasatinib and nilotinib have been approved as second-line therapies that could overcome some of these mutants, the most prevalent gatekeeper T315I mutant remains unconquered. Here, we report a novel type II kinase inhibitor, CHMFL-48, that potently inhibits the wild-type BCR-ABL (wt) kinase as well as a panel of imatinib-resistant mutants, including T315I, F317L, E255K, Y253F, and M351T. CHMFL-48 displayed great inhibitory activity against ABL wt (IC 50 : 1 nM, 70-fold better than imatinib) and the ABL T315I mutant (IC 50 : 0.8 nM, over 10,000-fold better than imatinib) in a biochemical assay and potently blocked the autophosphorylation of BCR-ABL wt and BCR-ABL mutants in a cellular context, which further affected downstream signalling mediators, including signal transducer and activator of transcription 5 (STAT5) and CRK like proto-oncogene (CRKL), and led to the cell cycle progression blockage as well as apoptosis induction. CHMFL-48 also exhibited great anti-leukemic efficacies in vivo in K562 cells and p210-T315I-transformed BaF3 cell-inoculated murine models. This discovery extended the pharmacological diversity of BCR-ABL kinase inhibitors and provided more potential options for anti-CML therapies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ibrutinib is an FDA-approved drug to treat B-lymphoid malignancies, which functions mechanistically as a covalent inhibitor for Bruton's tyrosine kinase (BTK). During the course of screening more potent and selective BTK inhibitors, we discovered that MM2-48, an ibrutinib analogue that contains the alkynyl amide functional group in place of the acrylamide warhead, exhibits a much stronger cytotoxicity. Comparative chemoproteomic profiling of the targets of ibrutinib and MM2-48 revealed that the alkynyl amide warhead exhibits much higher reactivity in proteomes. Unexpectedly, MM2-48 covalently targets a functional cysteine in a BRCA2 and CDKN1A-interacting protein, BCCIP, and significantly inhibits DNA damage repair. Our findings suggest that simultaneous inhibition of BTK activity and DNA damage repair might be a more effective therapeutic strategy for combating B-cell malignancies., (© 2020 Wiley-VCH GmbH.)

Acute myeloid leukemia (AML) is reported to be vulnerable to transcription disruption due to transcriptional addiction. Cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation, has attracted extensive attention as a drug target. Although several inhibitors, such as alvocidib and dinaciclib, have shown potent therapeutic effects in clinical trials on AML, the lack of high selectivity for CDK9 and other CDKs has limited their optimal clinical efficacy. Therefore, developing highly selective CDK9 inhibitors is still imperative for the efficacy and safety profile in treating AML. Here, we report a novel highly selective CDK9 inhibitor, JSH-009, which exhibited high potency against CDK9 and displayed great selectivity over 468 kinases/mutants. It also demonstrates impressive in vitro and in vivo antileukemic efficacy in preclinical models of AML, which makes JSH-009 a useful pharmacological tool for elucidating CDK9-mediated transcription and a novel therapeutic candidate for AML.

Nanotechnology has demonstrated great promise for the development of more effective and safer cancer therapies. We recently developed a highly selective inhibitor of BCR-ABL fusion tyrosine kinase for chronic myeloid leukemia (CML). However, the poor drug-like properties were hurdles to its further clinical development. Herein, we re-investigate it by conjugating an amphiphilic polymer and self-assembling into a nanoparticle (NP) with a high loading (~10.3%). The formulation greatly improved its solubility and drastically extended its circulation half-life from ~5.3 to ~117 h (>20-fold). In the 150 days long-term engraftment model experiment, long intravenous dosing intervals of the NPs (every 4 or 8 days) exhibited much better survival and negligible toxicities as compared to daily oral administration of the inhibitor. Moreover, the NPs showed excellent inhibition of tumor growth in the subcutaneous xenograft model. All results suggest that the ultra-long circulating pro-drug NP may provide an effective and safe therapeutic strategy for BCR-ABL-positive CML., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The outbreak of COVID-19, the pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred an intense search for treatments by the scientific community. In the absence of a vaccine, the goal is to target the viral life cycle and alleviate the lung-damaging symptoms of infection, which can be life-threatening. There are numerous protein kinases associated with these processes that can be inhibited by FDA-approved drugs, the repurposing of which presents an alluring option as they have been thoroughly vetted for safety and are more readily available for treatment of patients and testing in clinical trials. Here, we characterize more than 30 approved kinase inhibitors in terms of their antiviral potential, due to their measured potency against key kinases required for viral entry, metabolism, or reproduction. We also highlight inhibitors with potential to reverse pulmonary insufficiency because of their anti-inflammatory activity, cytokine suppression, or antifibrotic activity. Certain agents are projected to be dual-purpose drugs in terms of antiviral activity and alleviation of disease symptoms, however drug combination is also an option for inhibitors with optimal pharmacokinetic properties that allow safe and efficacious co-administration with other drugs, such as antiviral agents, IL-6 blocking agents, or other kinase inhibitors.

All-trans retinoic acid (ATRA) is known to be a potent inhibitor of FLT3-ITD acute myeloid leukemia (AML) cells, although the exact mechanism remains unclear. In this work, we report that ATRA causes fatal mitotic catastrophe in FLT3-ITD AML cells by degrading Chk1 kinase, and therefore preventing DNA damage repair. In order to explore a further enhancement in the inhibitory effect of ATRA on FLT3-ITD AML cells, we investigated the suitability of a combination of ATRA and DNA damage drug SN38. In vitro experiments showed that this combinatorial approach effectively inhibited the proliferation of FLT3-ITD cells and induced cell apoptosis in AML. In vivo experiments confirmed that the combination could substantially improve the anti-tumor effect of SN38. Taken together, our results indicate that ATRA down-regulates Chk1 in FLT3-ITD AML cells, and the combination of ATRA and SN38 significantly improves the anti-tumor effect of either ATRA or SN38 when used alone., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Starting from our previously developed c-KIT kinase inhibitor CHMFL-KIT-8140, through a type II kinase inhibitor binding element hybrid design approach, we discovered a novel c-KIT kinase inhibitor compound 18 (CHMFL-KIT-64), which is potent against c-KIT wt and a broad spectrum of drug-resistant mutants with improved bioavailability. 18 exhibits single-digit nM potency against c-KIT kinase and c-KIT T670I mutants in the biochemical assay and displays great potencies against most of the gain-of-function mutations in the juxtamembrane domain, drug-resistant mutations in the ATP binding pocket (except V654A), and activation loops (except D816V). In addition, 18 exhibits a good in vivo pharmacokinetic (PK) profile in different species including mice, rats, and dogs. It also displays good in vivo antitumor efficacy in the c-KIT T670I, D820G, and Y823D mutant-mediated mice models as well as in the c-KIT wt patient primary cells which are known to be imatinib-resistant. The potent activity against a broad spectrum of clinically important c-KIT mutants combining the good in vivo PK/pharmacodynamic properties of 18 indicates that it might be a new potential therapeutic candidate for gastrointestinal stromal tumors.

Gain-of-function mutations of c-KIT kinase play crucial pathological roles for the gastrointestinal stromal tumors (GISTs). Despite the success of imatinib as the first-line treatment of GISTs, dozens of drug-acquired resistant mutations emerge, and c-KIT T670I is one of the most common mutants among them. Although several kinase inhibitors are capable of overcoming the T670I mutant, none of them can achieve the selectivity over the c-KIT wild-type (wt), which also plays important roles in a variety of physiological functions such as hematopoiesis. Starting from axitinib, through fragment hybrid type II kinase inhibitor design approach, we have discovered a novel inhibitor 24, which not only exhibits potent activity to c-KIT T670I mutant but also achieves 12-fold selectivity over c-KIT wt. Compound 24 displays good antiproliferative effects against c-KIT T670I mutant-driven GIST cell lines (GIST-T1/T670I and GIST-5R) and also exhibits suitable in vivo pharmacokinetic profiles as well as dose-dependent antitumor efficacy. This study provides a proof of concept for developing a c-KIT mutant selective inhibitor that theoretically can render a better therapeutic window.

Most of the current FMS-like tyrosine kinase 3 (FLT3) inhibitors lack selectivity between FLT3 kinase and cKIT kinase as well as the FLT3 wt and internal tandem duplication (ITD) mutants. We report a new compound 27, which displays GI 50 values of 30-80 nM against different ITD mutants and achieves selectivity over both FLT3 wt (8-fold) and cKIT kinase in the transformed BaF3 cells (>300-fold). 27 potently inhibits the proliferation of the FLT3-ITD-positive acute myeloid leukemia cancer lines through suppression of the phosphorylation of FLT3 kinase and downstream signaling pathways, induction of apoptosis, and arresting the cell cycle into the G0/G1 phase. 27 also displays potent antiproliferative effect against FLT3-ITD-positive patient primary cells, whereas it does not apparently affect FLT3 wt primary cells. In addition, it also exhibits a good therapeutic window to PBMC compared to PKC412. In the in vivo studies, 27 demonstrates favorable PK profiles and suppresses the tumor growth in the MV4-11 cell inoculated mouse xenograft model.

BCR fused ABL kinase is the critical driving oncogene for chronic myeloid leukemia (CML) and has been extensively studied as the drug discovery target in the past decade. The successful introduction of tyrosine kinase inhibitors (TKI) such as Imatinib, Dasatinib and Bosutinib has greatly improved the CML patient survival rate. However, upon the chronic treatment, a variety of TKI resistant mutants, such as the V299L mutant which has been found in more and more patients with the high-throughput sequencing technology, are observed, although the incidence is still considered rare compared to the more prevalent gatekeeper T315I mutant. However, with the progress of the precision medicine concept, the rare mutation (or the orphan drug target) has attracted more and more attention. Here we report a novel type II BCR-ABL kinase inhibitor, CHMFL-ABL-039, which not only displayed great potency (IC 50 : 7.9 nM) and selectivity (S score (1) = 0.02) against native ABL kinase among other kinases in the kinome, but also exhibited great potency (IC 50 : 27.9 nM) and selectivity against Imatinib-resistant V299L mutant among other frequently observed ABL kinase mutants. CHMFL-ABL-039 has demonstrated greater efficacies than Imatinib regarding to the anti-proliferation, inhibition of the signaling pathway, arrest of cell cycle progression, induction of apoptosis in vitro and suppression of the tumor progression in vivo in the native and V299L mutated BCR-ABL kinase-driven cells/xenograft models. It would be a useful pharmacological tool to study the TKI resistant ABL V299L mutant-mediated pathology and provide a potential precise treatment approach for this orphan CML subtype in the precision medicine era.

There is still a great demand in the clinic for the drugs which can overcome a variety of imatinib resistant ABL mutants. Starting from a type I inhibitor axitinib, which has been reported to overcome ABL-T315I mutant induced resistance, through a structure guided drug design approach and binding mode switch strategy, we have discovered a novel type II ABL inhibitor 24 (CHMFL-ABL-121), which significantly improved the inhibitory activity against ABL wt and a broad spectrum of mutants including the most prevalent imatinib-resistant gatekeeper mutant T315I. 24 exhibited IC 50 values of 2 nM and 0.2 nM against purified inactive ABL wt and T315I kinase protein respectively and inhibited the proliferation of the established CML cell lines with GI 50 at single digit nM. In cellular context, 24 strongly affected BCR-ABL mediated signaling pathways and induced apoptosis as well as arrested cell cycle at G0/G1 phase. In the in vivo study, 50 mg/kg/day dosage of 24 displayed TGI of 52% in the TEL-ABLT315I-BaF3 cell inoculated allograft mouse model without obvious toxicity., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Through a structure-guided rational drug design approach, we have discovered a highly selective inhibitor compound 40 (JSH-150), which exhibited an IC 50 of 1 nM against CDK9 kinase in the biochemical assay and achieved around 300-10000-fold selectivity over other CDK kinase family members. In addition, it also displayed high selectivity over other 468 kinases/mutants (KINOMEscan S score(1) = 0.01). Compound 40 displayed potent antiproliferative effects against melanoma, neuroblastoma, hepatoma, colon cancer, lung cancer as well as leukemia cell lines. It could dose-dependently inhibit the phosphorylation of RNA Pol II, suppress the expression of MCL-1 and c-Myc, arrest the cell cycle and induce the apoptosis in the leukemia cells. In the MV4-11 cell-inoculated xenograft mouse model, 10 mg/kg dosage of 40 could almost completely suppress the tumor progression. The high selectivity and good in vivo PK/PD profile suggested that 40 would be a good pharmacological tool to study CDK9-mediated physiology and pathology as well as a potential drug candidate for leukemia and other cancers., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

PI3Kδ, which is mainly expressed in leukocytes, plays a critical role in B-cell receptor mediated signaling pathway and has been extensively studied as a drug discovery target for B cell malignances such as AML, CLL etc. In this manuscript, we report the discovery, SAR optimization and pharmacological evaluation of a novel series of aminothiazole-pyridine containing PI3Kδ inhibitors. Among them compound 15i (CHMFL-PI3KD-317) displays an IC 50 of 6 nM against PI3Kδ in the ADP-Glo biochemical assays. It also exhibits over 10-1500 fold selectivity over other class I, II and III PIKK family isoforms. In addition, in the cellular context, 15i can selectively and potently inhibit PI3Kδ mediated phosphorylation of Akt T308 but not PI3Kα, β, γ mediated Akt phosphorylation. 15i also exhibits an excellent selectivity profile in the protein kinases including 468 kinases/mutants at the concentration of 1 μM. 15i has acceptable pharmacokinetic properties and can dose-dependently inhibit the tumor growth of AML cell line MOLM14 inoculated xenograft mouse model. The high selectivity and potency makes 15i a potential valuable addition to the current PI3Kδ armory., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

An alternative medicinal chemistry approach was conducted on Bruton's tyrosine kinase (BTK) inhibitor 1 (ibrutinib) by merging the pyrazolo[3,4- d]pyrimidine component into a tricyclic skeleton. Two types of compounds were prepared, and their biochemical activities on BTK as well as stereochemistry effects were determined. Structural optimization focusing on the reactive binding group to BTK Cys481 and on the metabolic site guided by metabolic study were conducted. 7S was identified as the most potent showing an IC 50 value of 0.4 nM against BTK and 16 nM against BTK-dependent TMD8 cells. Compared to 1, 7S was slightly more selective with strong inhibition on the B-cell receptor signaling pathway. In a TMD8 cell-derived animal xenograft model, 7S showed a relative tumor volume of 5.3 at 15 mg/kg QD dosage that was more efficacious than 1 (RTV 6.6) at a higher dose of 25 mg/kg QD. All these results suggest 7S as a new BTK inhibitor worthy of further profiling.

FLT3-ITD mutant has been observed in about 30% of AML patients and extensively studied as a drug discovery target. On the basis of our previous study that ibrutinib (9) exhibited selective and moderate inhibitory activity against FLT3-ITD positive AML cells, through a structure-guided drug design approach, we have discovered a new type II FLT3 kinase inhibitor, compound 14 (CHMFL-FLT3-213), which exhibited highly potent inhibitory effects against FLT3-ITD mutant and associated oncogenic mutations (including FLT3-D835Y/H/V, FLT3-ITD-D835Y/I/N/A/G/Del, and FLT3-ITD-F691L). In the cellular context 14 strongly affected FLT3-ITD mediated signaling pathways and induced apoptosis by arresting cell cycle into G0/G1 phase. In the in vivo studies 14 demonstrated an acceptable bioavailability (F = 19%) and significantly suppressed the tumor growth in MV4-11 cell inoculated xenograft model (15 mg kg -1 day -1 , TGI = 97%) without exhibiting obvious toxicity. Compound 14 might be a potential drug candidate for FLT3-ITD positive AML.

Recently, more and more concomitant EGFR mutations and ALK rearrangement are observed from the clinic, which still lacks effective single-agent therapy. Starting from ALK inhibitor 14 (TAE684), we have developed a highly potent EGFR/ALK dual kinase inhibitor compound 18 (CHMFL-ALK/EGFR-050), which potently inhibited EGFR L858R, del 19 and T790M mutants as well as EML4-ALK, R1275Q, L1196M, F1174L and C1156Y mutants biochemically. Compound 18 significantly inhibited the proliferation of EGFR mutant and EML4-ALK driven NSCLC cell lines. In the cellular context it strongly affected EGFR and ALK mediated signaling pathways, induced apoptosis and arrested cell cycle at G0/G1 phase. In the in vivo studies, 18 significantly suppressed the tumor growth in H1975 cell inoculated xenograft model (40 mg/kg/d, TGI: 99%) and H3122 cell inoculated xenograft model (40 mg/kg/d, TGI: 78%). Compound 18 might be a potential drug candidate for EGFR- or ALK-individual as well as concomitant EGFR/ALK NSCLC., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

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 (k inact /K i ) of 0.01 μM -1 s -1 . Compound 18 potently inhibited BTK kinase Y223 auto-phosphorylation (EC 50  < 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., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Currently there are several irreversible BTK inhibitors targeting Cys481 residue under preclinical or clinical development. However, most of these inhibitors also targeted other kinases such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a structure-based drug design approach, we discovered a highly potent (IC 50 : 7 nM) irreversible BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in KINOMEscan (S score (35) = 0.00) among 468 kinases/mutants at the concentration of 1 μM. Compound 9 completely abolished BMX, JAK3 and EGFR's activity. Both X-ray crystal structure and cysteine-serine mutation mediated rescue experiment confirmed 9's irreversible binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC 50 : <30 nM), arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe these features would make 9 a good pharmacological tool to study the BTK related pathology., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

On the basis of Ibrutinib's core pharmacophore, which was moderately active to EGFR T790M mutant, we discovered novel epidermal growth factor receptor (EGFR) inhibitor compound 19 (CHMFL-EGFR-202), which potently inhibited EGFR primary mutants (L858R, del19) and drug-resistant mutant L858R/T790M. Compound 19 displayed a good selectivity profile among 468 kinases/mutants tested in the KINOMEscan assay (S score (1) = 0.02). In particular, it did not exhibit apparent activities against INSR and IGF1R kinases. The X-ray crystal structure revealed that this class of inhibitors formed a covalent bond with Cys797 in a distinct "DFG-in-C-helix-out" inactive EGFR conformation. Compound 19 displayed strong antiproliferative effects against EGFR mutant-driven nonsmall cell lung cancer (NSCLC) cell lines such as H1975, PC9, HCC827, and H3255 but not the wild-type EGFR expressing cells. In the H1975 and PC9 cell-inoculated xenograft mouse models, compound 19 exhibited dose-dependent tumor growth suppression efficacy without obvious toxicity. Compound 19 might be a potential drug candidate for EGFR mutant-driven NSCLC.

Covalently bound protein kinase inhibitors have been frequently designed to target noncatalytic cysteines at the ATP binding site. Thus, it is important to know if a given cysteine can form a covalent bond. Here we combine a function-site interaction fingerprint method and DFT calculations to determine the potential of cysteines to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site cysteine data set was assembled. The orientation of the cysteine thiol group indicates which cysteines can potentially form covalent bonds. These covalent inhibitor easy-available cysteines are located within five regions: P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop, and DFG-3 close to the DFG peptide. In an independent test set these cysteines covered 95% of covalent kinase inhibitors. This study provides new insights into cysteine reactivity and preference which is important for the prospective development of covalent kinase inhibitors.

BTK plays a critical role in the B cell receptor mediated inflammatory signaling in the rheumatoid arthritis (RA). Through a rational design approach we discovered a highly selective and potent BTK kinase inhibitor (CHMFL-BTK-11) which exerted its inhibitory efficacy through a covalent bond with BTK Cys481. CHMFL-BTK-11 potently blocked the anti-IgM stimulated BCR signaling in the Ramos cell lines and isolated human primary B cells. It significantly inhibited the LPS stimulated TNF-α production in the human PBMC cells but only weakly affecting the normal PBMC cell proliferation. In the adjuvant-induced arthritis rat model, CHMFL-BTK-11 ameliorated the inflammatory response through blockage of proliferation of activated B cells, inhibition of the secretion of the inflammatory factors such as IgG1, IgG2, IgM, IL-6 and PMΦ phagocytosis, stimulation of secretion of IL-10. The high specificity of CHMFL-BTK-11 makes it a useful pharmacological tool to further detect BTK mediated signaling in the pathology of RA.

EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct "DFG-in" and "cHelix-out" inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose.

BMX is a member of TEC family nonreceptor tyrosine kinase and is involved in a variety of critical physiological and pathological processes. Through combination of irreversible inhibitor design and type II inhibitor design approaches, we have discovered a highly selective and potent type II irreversible BMX kinase inhibitor compound 41 (CHMFL-BMX-078), which exhibited an IC 50 of 11 nM by formation of a covalent bond with cysteine 496 residue in the DFG-out inactive conformation of BMX. It displayed a high selectivity profile (S score(1) = 0.01) against the 468 kinases/mutants in the KINOMEscan evaluation and achieved at least 40-fold selectivity over BTK kinase. Given the fact that BMX mediated signaling pathway is still not fully understood, compound 41 would serve as a useful pharmacological tool to elucidate the detailed mechanism of BMX mediated signaling pathways.

The discovery of a novel potent type II ABL/c-KIT dual kinase inhibitor compound 34 (CHMFL-ABL/KIT-155), which utilized a hydrogen bond formed by NH on the kinase backbone and carbonyl oxygen of 34 as a unique hinge binding, is described. 34 potently inhibited purified ABL (IC 50 : 46 nM) and c-KIT kinase (IC 50 : 75 nM) in the biochemical assays and displayed high selectivity (S Score (1) = 0.03) at the concentration of 1 μM among 468 kinases/mutants in KINOMEscan assay. It exhibited strong antiproliferative activities against BCR-ABL/c-KIT driven CML/GISTs cancer cell lines through blockage of the BCR-ABL/c-KIT mediated signaling pathways, arresting cell cycle progression and induction of apoptosis. 34 possessed a good oral PK property and effectively suppressed the tumor progression in the K562 (CML) and GIST-T1 (GISTs) cells mediated xenograft mouse model. The distinct hinge-binding mode of 34 provided a novel pharmacophore for expanding the chemical structure diversity for the type II kinase inhibitors discovery.

Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

cKIT kinase inhibitors, e.g., imatinib, could induce drug-acquired mutations such as cKIT T670I that rendered drug resistance after chronic treatment. Through a type II kinase inhibitor design approach we discovered a highly potent type II cKIT kinase inhibitor compound 35 (CHMFL-KIT-8140), which potently inhibited both cKIT wt (IC50 = 33 nM) and cKIT gatekeeper T670I mutant (IC50 = 99 nM). Compound 35 displayed strong antiproliferative effect against GISTs cancer cell lines GIST-T1 (cKIT wt, GI50 = 4 nM) and GIST-5R (cKIT T670I, GI50 = 26 nM). In the cellular context it strongly inhibited c-KIT mediated signaling pathways and induced apoptosis. In the BaF3-TEL-cKIT-T670I isogenic cell inoculated xenograft mouse model, 35 exhibited dose dependent tumor growth suppression efficacy and 100 mg/kg dosage provided 47.7% tumor growth inhibition (TGI) without obvious toxicity. We believe compound 35 would be a good pharmacological tool for exploration of the cKIT-T670I mutant mediated pathology in GISTs.

PI3Kδ has been found to be over-expressed in B-Cell-related malignancies. Despite the clinical success of the first selective PI3Kδ inhibitor, CAL-101, inhibition of PI3Kδ itself did not show too much cytotoxic efficacy against cancer cells. One possible reason is that PI3Kδ inhibition induced autophagy that protects the cells from death. Since class III PI3K isoform PIK3C3/Vps34 participates in autophagy initiation and progression, we predicted that a PI3Kδ and Vps34 dual inhibitor might improve the anti-proliferative activity observed for PI3Kδ-targeted inhibitors. We discovered a highly potent ATP-competitive PI3Kδ/Vps34 dual inhibitor, PI3KD/V-IN-01, which displayed 10-1500 fold selectivity over other PI3K isoforms and did not inhibit any other kinases in the kinome. In cells, PI3KD/V-IN-01 showed 30-300 fold selectivity between PI3Kδ and other class I PI3K isoforms. PI3KD/V-IN-01 exhibited better anti-proliferative activity against AML, CLL and Burkitt lymphoma cell lines than known selective PI3Kδ and Vps34 inhibitors. Interestingly, we observed FLT3-ITD AML cells are more sensitive to PI3KD/V-IN-01 than the FLT3 wt expressing cells. In AML cell inoculated xenograft mouse model, PI3KD/V-IN-01 exhibited dose-dependent anti-tumor growth efficacies. These results suggest that dual inhibition of PI3Kδ and Vps34 might be a useful approach to improve the PI3Kδ inhibitor's anti-tumor efficacy., Competing Interests: Dr. Shanchun Zhang is a shareholder of Hefei Cosource Medicine Technology Co. LTD.

STK16, a serine/threonine protein kinase, is ubiquitously expressed and is conserved among all eukaryotes. STK16 has been implicated to function in a variety of cellular processes such as VEGF and cargo secretion, but the pathways through which these effects are mediated remain to be elucidated. Through screening of our focused library of kinase inhibitors, we discovered a highly selective ATP competitive inhibitor, STK16-IN-1, which exhibits potent inhibitory activity against STK16 kinase (IC50: 0.295 μM) with excellent selectivity across the kinome as assessed using the KinomeScan profiling assay (S score (1) = 0.0). In MCF-7 cells, treatment with STK16-IN-1 results in a reduction in cell number and accumulation of binucleated cells, which can be recapitulated by RNAi knockdown of STK16. Co-treatment of STK16-IN-1 with chemotherapeutics such as cisplatin, doxorubicin, colchicine, and paclitaxel results in a slight potentiation of the antiproliferative effects of the chemotherapeutics. STK16-IN-1 provides a useful tool compound for further elucidating the biological functions of STK16.

PI3Kδ is predominately expressed in leukocytes and has been found overexpressed in B-cell related malignances such as CLL and AML. We have discovered a highly selective ATP competitive PI3Kd inhibitor PI3KD-IN-015, which exhibits a high selectivity among other PI3K isoforms in both biochemical assays and cellular assay, meanwhile did not inhibit most of other protein kinases in the kinome. PI3KD-IN-015 demonstrates moderately anti-proliferation efficacies against a variety of B-cell related cancer cell lines through down-regulate the PI3K signaling significantly. It induced both apoptosis and autophagy in B-cell malignant cell lines. In addition, combination of autophagy inhibitor Bafilomycin could potentiate the moderate anti-proliferation effect of PI3KD-IN-015. PI3KD-IN-015 shows anti-proliferation efficacy against CLL and AML patient primary cells. Collectively, these results indicate that PI3KD-IN-015 may be useful drug candidate for further development of anti-B-cell related malignances therapies., Competing Interests: No potential conflicts of interest were disclosed.

c-KIT kinase is a validated drug discovery target for gastrointestinal stromal tumors (GISTs). Clinically used c-KIT kinase inhibitors, i.e., Imatinib and Sunitinib, bear other important targets such as ABL or FLT3 kinases. Here we report our discovery of a more selective c-KIT inhibitor, compound 13 (CHMFL-KIT-110), which completely abolished ABL and FLT3 kinase activity. KinomeScan selectivity profiling (468 kinases) of 13 exhibited a high selectivity (S score (1) = 0.01). 13 displayed great antiproliferative efficacy against GISTs cell lines GIST-T1 and GIST-882 (GI50: 0.021 and 0.043 μM, respectively). In the cellular context, it effectively affected c-KIT-mediated signaling pathways and induced apoptosis as well as cell cycle arrest. In addition, 13 possessed acceptable bioavailability (36%) and effectively suppressed the tumor growth in GIST-T1 cell inoculated xenograft model without apparent toxicity. 13 currently is undergoing extensive preclinical evaluation and might be a potential drug candidate for GISTs.

Starting from a dihydropyrimidopyrimidine core scaffold based compound 27 (GNF-7), we discovered a highly potent (ABL1: IC50 of 70 nM) and selective (S score (1) = 0.02) BCR-ABL inhibitor 18a (CHMFL-ABL-053). Compound 18a did not exhibit apparent inhibitory activity against c-KIT kinase, which is the common target of currently clinically used BCR-ABL inhibitors. Through significant suppression of the BCR-ABL autophosphorylation (EC50 about 100 nM) and downstream mediators such as STAT5, Crkl, and ERK's phosphorylation, 18a inhibited the proliferation of CML cell lines K562 (GI50 = 14 nM), KU812 (GI50 = 25 nM), and MEG-01 (GI50 = 16 nM). A pharmacokinetic study revealed that 18a had over 4 h of half-life and 24% bioavailability in rats. A 50 mg/kg/day dosage treatment could almost completely suppress tumor progression in the K562 cells inoculated xenograft mouse model. As a potential useful drug candidate for CML, 18a is under extensive preclinical safety evaluation now.

Through comprehensive comparison study, we found that ibrutinib, a clinically approved covalent BTK kinase inhibitor, was highly active against EGFR (L858R, del19) mutant driven NSCLC cells, but moderately active to the T790M 'gatekeeper' mutant cells and not active to wild-type EGFR NSCLC cells. Ibrutinib strongly affected EGFR mediated signaling pathways and induced apoptosis and cell cycle arrest (G0/G1) in mutant EGFR but not wt EGFR cells. However, ibrutinib only slowed down tumor progression in PC-9 and H1975 xenograft models. MEK kinase inhibitor, GSK1120212, could potentiate ibrutinib's effect against the EGFR (L858R/T790M) mutation in vitro but not in vivo. These results suggest that special drug administration might be required to achieve best clinical response in the ongoing phase I/II clinical trial with ibrutinib for NSCLC.

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

The ATP site of kinases displays remarkable conformational flexibility when accommodating chemically diverse small molecule inhibitors. The so-called activation segment, whose conformation controls catalytic activity and access to the substrate binding pocket, can undergo a large conformational change with the active state assuming a 'DFG-in' and an inactive state assuming a 'DFG-out' conformation. Compounds that preferentially bind to the DFG-out conformation are typically called 'type II' inhibitors in contrast to 'type I' inhibitors that bind to the DFG-in conformation. This review surveys the large number of type II inhibitors that have been developed and provides an analysis of their crystallographically determined binding modes. Using a small library of type II inhibitors, we demonstrate that more than 200 kinases can be targeted, suggesting that type II inhibitors may not be intrinsically more selective than type I inhibitors.

The DDR1 receptor tyrosine kinase is activated by matrix collagens and has been implicated in numerous cellular functions such as proliferation, differentiation, adhesion, migration, and invasion. Here we report the discovery of a potent and selective DDR1 inhibitor, DDR1-IN-1, and present the 2.2 Å DDR1 co-crystal structure. DDR1-IN-1 binds to DDR1 in the 'DFG-out' conformation and inhibits DDR1 autophosphorylation in cells at submicromolar concentrations with good selectivity as assessed against a panel of 451 kinases measured using the KinomeScan technology. We identified a mutation in the hinge region of DDR1, G707A, that confers >20-fold resistance to the ability of DDR1-IN-1 to inhibit DDR1 autophosphorylation and can be used to establish what pharmacology is DDR1-dependent. A combinatorial screen of DDR1-IN-1 with a library of annotated kinase inhibitors revealed that inhibitors of PI3K and mTOR such as GSK2126458 potentiate the antiproliferative activity of DDR1-IN-1 in colorectal cancer cell lines. DDR1-IN-1 provides a useful pharmacological probe for DDR1-dependent signal transduction.

The interaction between the HSP90 chaperone and its client kinases is sensitive to the conformational status of the kinase, and stabilization of the kinase fold by small molecules strongly decreases chaperone interaction. Here we exploit this observation and assay small-molecule binding to kinases in living cells, using chaperones as 'thermodynamic sensors'. The method allows determination of target specificities of both ATP-competitive and allosteric inhibitors in the kinases' native cellular context in high throughput. We profile target specificities of 30 diverse kinase inhibitors against >300 kinases. Demonstrating the value of the assay, we identify ETV6-NTRK3 as a target of the FDA-approved drug crizotinib (Xalkori). Crizotinib inhibits proliferation of ETV6-NTRK3-dependent tumor cells with nanomolar potency and induces the regression of established tumor xenografts in mice. Finally, we show that our approach is applicable to other chaperone and target classes by assaying HSP70/steroid hormone receptor and CDC37/kinase interactions, suggesting that chaperone interactions will have broad application in detecting drug-target interactions in vivo.

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC(50) of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC(50), 28 nmol/L), ATR (EC(50), 35 nmol/L), and DNA-PK (EC(50), 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role., (©2013 AACR.)

Protein kinases are a large family of approximately 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues, such as tyrosine, serine, and threonine, that serves as a ubiquitous mechanism for cellular signal transduction. The clinical success of a number of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large number of kinases may represent therapeutically relevant targets. To date, the majority of clinical and preclinical kinase inhibitors are ATP competitive, noncovalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently, there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a number of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency, and ability to validate pharmacological specificity through mutation of the reactive cysteine residue. Here, we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biological activity profiles. In addition, we provided an informatics assessment of the potential "kinase cysteinome" and discuss strategies for the efficient development of new covalent inhibitors., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objectives: Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells., Methods: We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy., Results and Conclusions: Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment.

Anaplastic lymphoma kinase (ALK) is an important drug target in many cancers, including lymphoma, neuroblastoma, and lung cancer. Here, we demonstrate proof-of-principle for a novel and inexpensive assay for ALK inhibitor activity and identification in zebrafish. We demonstrate that the human oncogenic ALK fusion, NPM-ALK, drives overproduction of iridophores, a highly visible, shiny pigment cell-type in zebrafish. Treatment with the potent ALK inhibitor, TAE684, fully inhibits production of ALK-dependent iridophores. Using our assay, we test multiple properties of TAE684 in vivo, including efficacy, specificity, and toxicity. We note that TAE684 also inhibits the closely related leukocyte tyrosine kinase (Ltk) that is required for endogenous iridophore development. Similar effects are observed with an independent inhibitor, Crizotinib. Our assay can thus be utilized to identify ALK or LTK inhibitors. Importantly, the natural reflectivity of iridophores lends itself to automation for high throughput assessment of ALK and LTK inhibitor compounds in vivo.

Natural products have been widely used to dissect the basic mechanisms of fundamental life science and as clinical therapeutics. Recently, there has been significant interest in discovering new chemical pharmacophores in natural products to fulfil the vast demand for novel kinase inhibitors and address critical unmet medical needs with respect to signal transduction pathways. In this review, we summarize the history of several different classes of natural product-derived kinase inhibitors, discuss their kinome-wide target profiles and examine their structural binding modes based on available 3D X-ray structures. In particular, their origin, target activity, selectivity, scope and potential therapeutic development are highlighted against the backdrop of medicinal chemistry.

The mammalian Target of Rapamycin (mTOR)-mediated signaling transduction pathway has been observed to be deregulated in a wide variety of cancer and metabolic diseases. Despite extensive clinical development efforts, the well-known allosteric mTOR inhibitor rapamycin and structurally related rapalogs have failed to show significant single-agent antitumor efficacy in most types of cancer. This limited clinical success may be due to the inability of the rapalogs to maintain a complete blockade mTOR-mediated signaling. Therefore, numerous efforts have been initiated to develop ATP-competitive mTOR inhibitors that would block both mTORC1 and mTORC2 complex activity. Here, we describe our experimental approaches to develop Torin1 using a medium throughput cell-based screening assay and structure-guided drug design.

The management of neuropathic pain is unsatisfactory, and new treatments are required. Because the sensitivity of a subset of fast-conducting primary afferent nociceptors is thought to be regulated by the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, selectively targeting mTORC1 represents a new strategy for the control of chronic pain. Here we show that activated mTOR was expressed largely in myelinated sensory fibers in mouse and that inhibiting the mTORC1 pathway systemically alleviated mechanical hypersensitivity in mouse models of inflammatory and neuropathic pain. Specifically, systemic administration of mTORC1 inhibitor temsirolimus (CCI-779), both acutely (25 mg/kg i.p.) and chronically (4 daily 25 mg/kg i.p.), inhibited the mTORC1 pathway in sensory axons and the spinal dorsal horn and reduced mechanical and cold hypersensitivity induced by nerve injury. Moreover, systemic treatment with CCI-779 also reduced mechanical but not heat hypersensitivity in an inflammatory pain state. This treatment did not influence nociceptive thresholds in naive or sham-treated control animals. Also, there was no evidence for neuronal toxicity after repeated systemic treatment with CCI-779. Additionally, we show that acute and chronic i.p. administration of Torin1 (20 mg/kg), a novel ATP-competitive inhibitor targeting both mTORC1 and mTORC2 pathways, reduced the response to mechanical and cold stimuli in neuropathic mice. Our findings emphasize the importance of the mTORC1 pathway as a regulator of nociceptor sensitivity and therefore as a potential target for therapeutic intervention, particularly in chronic pain., (Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)