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Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N 2 -methyl-dG ( N 2 -MedG) and N 2 - n -butyl-dG ( N 2 - n BudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N 2 -MedG and N 2 - n BudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N 2 -MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N 2 -MedG and N 2 - n BuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N 2 -alkyl-dG lesions in mammalian cells.

N 6 -Methyladenosine (m 6 A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m 6 A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m 6 A and other modified nucleosides in RNA. Here, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding m 6 A eraser proteins (i.e., ALKBH5 , FTO ) and the catalytic subunit of the major m 6 A writer complex (i.e., METTL3 ) being individually ablated. Notably, eight proteins, including writer proteins for 5-methylcytidine and pseudouridine, were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of METTL3 and ALKBH5 . Analysis of previously published m 6 A mapping results revealed the presence of m 6 A in the corresponding mRNAs for four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between m 6 A and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.

Drug resistance remains a major challenge in the clinical treatment of gastrointestinal stromal tumours (GISTs). While acquired on-target mutations of mast/stem cell growth factor receptor (KIT) kinase is the major resistance mechanism, activation of alternative signalling pathways may also play a role. Although several second- and third-generation KIT kinase inhibitors have been developed that could overcome some of the KIT mutations conferring resistance, the low clinical responses and narrow safety window have limited their broad application. The present study revealed that nintedanib not only overcame resistance induced by a panel of KIT primary and secondary mutations, but also overcame ERK-reactivation-mediated resistance caused by the upregulation of fibroblast growth factor (FGF) activity. In preclinical models of GISTs, nintedanib significantly inhibited the proliferation of imatinib-resistant cells, including GIST-5R, GIST-T1/T670I and GIST patient-derived primary cells. In addition, it also exhibited dose-dependent inhibition of ERK phosphorylation upon FGF ligand stimulation. In vivo antitumour activity was also observed in several xenograft GIST models. Considering the well-documented safety and pharmacokinetic profiles of nintedanib, this finding provides evidence for the repurposing of nintedanib as a new therapy for the treatment of GIST patients with de novo or acquired resistance to imatinib., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

ALKBH4 is a versatile demethylase capable of catalyzing the demethylation of monomethylated lysine-84 on actin and N 6 -methyladenine in DNA. In this study, we conducted a quantitative proteomic experiment to reveal the altered expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results showed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein., (© 2022 Wiley-VCH GmbH.)

Protein α-N-methylation is an evolutionarily conserved type of post-translational modification; however, little is known about the regulatory mechanisms for this modification. Methylation at the N 6 position of adenosine in mRNAs is dynamic and modulates their stability, splicing, and translational efficiency. Here, we found that the expression of N-terminal methyltransferase 1 (NTMT1) protein is altered by depletion of those genes encoding the reader/writer/eraser proteins of N 6 -methyladenosine (m 6 A). We also observed that MRG15 is N-terminally methylated by NTMT1, and this methylation could also be modulated by reader/writer/eraser proteins of m 6 A. Together, these results revealed a novel m 6 A-based epitranscriptomic mechanism in regulating protein N-terminal methylation., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

BACKGROUND An increasing number of studies have demonstrated that Streptococcus bovis and its concomitant inflammatory factors concentrate in the intestine in colorectal cancer (CRC). However, the molecular mechanism of S. bovis on colorectal tumorigenesis remains unclear. This study aimed to explore the role of S. bovis in carcinogenesis and its potential mechanism in CRC of mice orally pretreated with S. bovis. MATERIAL AND METHODS The colons of experimental mice were collected and evaluated for the extent of neoplasm. In addition, comparative feces DNA sequencing was adopted to verify the abundance change of S. bovis during the progression of CRC in patients. RESULTS The results of this study found that S. bovis is more likely to be present at higher levels in patients with progressive colorectal carcinoma compared to those adenoma patients and healthy volunteers (P<0.05). Pretreatment with S. bovis aggravated tumor formation in mice, resulting in more substantial and a higher number of tumor nodes (P<0.05). A cytokine expression pattern with increased levels of IL-6, Scyb1, Ptgs2, IL-1ß, TNF, and Ccl2 was detected in S. bovis pretreated CRC mice (all P<0.05). Furthermore, S. bovis recruited myeloid cells, especially CD11b⁺TLR-4⁺ cells, which could promote pro-tumor immunity in the tumor microenvironment (P<0.05). CONCLUSIONS Collectively, our study indicates that S. bovis may induce a suppressive immunity that is conducive to CRC by recruiting tumor-infiltrating CD11b⁺TLR-4⁺ cells. In conclusion, S. bovis contributes to colorectal tumorigenesis via recruiting CD11b⁺TLR-4⁺ cells.

N 6 -Methyladenosine (m 6 A) in messenger RNA (mRNA) regulates its stability, splicing, and translation efficiency. Here, we explored how the expression levels of small GTPase proteins are regulated by m 6 A modulators. We employed a high-throughput scheduled multiple-reaction monitoring (MRM)-based targeted proteomic approach to quantify systemically the changes in expression of small GTPase proteins in cells upon genetic ablation of METTL3 (the catalytic subunit of the major m 6 A methyltransferase complex), m 6 A demethylases (ALKBH5 and FTO), or m 6 A reader proteins (YTHDF1, YTHDF2, and YTHDF3). Depletions of METTL3 and ALKBH5 resulted in substantially diminished and augmented expression, respectively, of a subset of small GTPase proteins, including RHOB and RHOC. Our results also revealed that the stability of RHOB mRNA is significantly increased in cells depleted of METTL3, suggesting an m 6 A-elicited destabilization of this mRNA. Those small GTPases that are targeted by METTL3 and/or ALKBH5 also displayed higher discrepancies between protein and mRNA expression in paired primary/metastatic melanoma or colorectal cancer cells than those that are not. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome regulated by epitranscriptomic modulators of m 6 A, and our study suggests the potential of an alternative therapeutic approach to target the currently "undruggable" small GTPases.

Isoprenoid pyrophosphates are involved in protein prenylation and assume regulatory roles in cells; however, little is known about the cellular proteins that can interact with isoprenoid pyrophosphates. Here, we devised a chemical proteomic strategy, capitalizing on the use of a desthiobiotin-geranyl pyrophosphate (GPP) acyl phosphate probe for the enrichment and subsequent identification of GPP-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By combining stable isotope labeling by amino acids in cell culture (SILAC) and competitive labeling with low vs high concentrations of GPP probe, with ATP vs GPP acyl phosphate probes, or with the GPP probe in the presence of different concentrations of free GPP, we uncovered a number of candidate GPP-binding proteins. We also discovered, for the first time, histone deacetylase 1 (HDAC1) as a GPP-binding protein. Furthermore, we found that the enzymatic activity of HDAC1 could be modulated by isoprenoid pyrophosphates. Together, we developed a novel chemical proteomic method for the proteome-wide discovery of GPP-binding proteins, which sets the stage for a better understanding about the biological functions of isoprenoids.

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.)

Angiogenesis is an essential process in tumor growth, invasion and metastasis. VEGF receptor 2 (VEGFR2) inhibitors targeting tumor angiogenic pathway have been widely used in the clinical cancer treatment. However, most of currently used VEGFR2 kinase inhibitors are multi-target inhibitors which might result in target-associated side effects and therefore limited clinical toleration. Highly selective VEGFR inhibitors are still highly demanded from both basic research and clinical application point of view. Here we report the discovery and characterization of a novel VEGFR2 inhibitor (CHMFL-VEGFR2-002), which exhibited high selectivity among structurally closed kinases including PDGFRs, FGFRs, CSF1R, etc. CHMFL-VEGFR2-002 displayed potent inhibitory activity against VEGFR2 kinase in the biochemical assay (IC 50  = 66 nmol/L) and VEGFR2 autophosphorylation in cells (EC 50 s ∼100 nmol/L) as well as potent anti-proliferation effect against VEGFR2 transformed BaF3 cells (GI 50  = 150 nmol/L). In addition, CHMFL-VEGFR2-002 also displayed good anti-angiogenesis efficacy in vitro and exhibited good in vivo PK (pharmacokinetics) profile with bioavailability over 49% and anti-angiogenesis efficacy in both zebrafish and mouse models without apparent toxicity. These results suggest that CHMFL-VEGFR2-002 might be a useful research tool for dissecting new functions of VEGFR2 kinase as well as a potential anti-angiogenetic agent for the cancer therapy., (© 2019 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

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.

Background: cKIT kinase overexpression and gain-of-function mutations are the critical pathogenesis of gastrointestinal stromal tumors (GISTs). Although the multiple kinase inhibitors such as imatinib, sunitinib, and regorafenib have been approved for GISTs, the acquisition of polyclonal secondary resistance mutations in KIT is still a limitation for GIST treatment. Here we explored the KIT inhibitory activity of axitinib in preclinical models and describe initial characterization of its activity in GIST patient-derived primary cells., Methods: The activities of axitinib against mutant KIT were evaluated using protein-based assay and a panel of engineered and GIST-derived cell lines. The binding modes of axitinib-KIT/KIT mutants were analyzed. Four primary cells derived from GIST patients were also used to assess the drug response of axitinib., Results: Axitinib exhibited potent activities against a variety of cKIT associated primary and secondary mutations. It displayed better activity against cKIT wild-type, cKIT V559D/A/G, and L576P primary gain-of-function mutations than imatinib, sunitinib, and regorafenib. In addition, it could inhibit imatinib resistant cKIT T670I and V654A mutants in vitro and in vivo GIST preclinical models., Conclusion: Our results provide the basis for extending the application of axitinib to GISTs patients who are unresponsive or intolerant to the current therapies., Competing Interests: Conflict of interest statement: The authors declare that there are no conflicts of interest.

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.

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.)

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC 50 : 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC 50 : 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan ™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

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.

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.)

Background: For colorectal liver metastasis (CRLM) patients, hepatic resection is currently the sole cure offering the chance of long-term survival. Tumor shrinkage and planned liver remnant hypertrophy are the two key strategies for conversion of initially unresectable CRLM. First conducted in 2012, associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) allows rapid liver growth. As a means to induce hypertrophy, portal vein embolization (PVE) has been widely applied before extending hepatectomy. Recently, Peng et al. present a new approach of terminal branches portal vein embolization (TBPVE), offering an efficient way to amplify FLR and making chances for surgery in 2 weeks., Case Presentation: We reported a 61-year-old woman with synchronous hepatic metastasized carcinoma of the colon sigmoideum underwent TBPVE after 6 cycles of neoadjuvant therapy in order to perform a planned right trisectionectomy. Rapid liver remnant hypertrophy and remarkable tumor shrinkage were achieved, and laparoscopic sigmoidectomy and right trisectionectomy were successfully performed. The postsurgical course was uneventful and 7 months of recurrence-free survival have been witnessed., Conclusions: The dual tactics of tumor shrinkage and planned rapid liver remnant hypertrophy will make concerted efforts to further increase the clinical candidacy for curative resection, which are valuable for further investigation.

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.

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.

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.

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.

The FLT3-ITD mutation is one of the most prevalent oncogenic mutations in AML. Several FLT3 kinase inhibitors have shown impressive activity in clinical evaluation, however clinical responses are usually transient and clinical effects are rapidly lost due to drug resistance. One of the resistance mechanisms in the AML refractory patients involves FLT3-ligand induced reactivation of AKT and/or ERK signaling via FLT3 wt kinase. Via a screen of numerous AKT kinase inhibitors, we identified the well-established orally available AKT inhibitor, A674563, as a dual suppressor of AKT and FLT3-ITD. A674563 suppressed FLT3-ITD positive AML both in vitro and in vivo. More importantly, compared to other FLT3 inhibitors, A674563 is able to overcome FLT3 ligand-induced drug resistance through simultaneous inhibition of FLT3-ITD- and AKT-mediated signaling. Our findings suggest that A674563 might be a potential drug candidate for overcoming FLT3 ligand-mediated drug resistance in FLT3-ITD positive AML., Competing Interests: Dr. Shanchu Zhang is a shareholder of Hefei Cosource Medicine Technology Co. LTD.

FLT3-ITD mutant has been observed in about 30% of AML patients and extensively studied as a drug discovery target. On the basis of the structure of PCI-32765 (ibrutinib), a BTK kinase inhibitor that was recently reported to bear FLT3 kinase activity through a structure-guided drug design approach, we have discovered compound 18 (CHMFL-FLT3-122), which displayed an IC50 of 40 nM against FLT3 kinase and achieved selectivity over BTK kinase (over 10-fold). It significantly inhibited the proliferation of FLT3-ITD positive AML cancer cell lines MV4-11 (GI50 = 22 nM), MOLM13/14 (GI50 = 21 nM/42 nM). More importantly, 18 demonstrated 170-fold selectivity between FLT3 kinase and c-KIT kinase (GI50 = 11 nM versus 1900 nM) in the TEL-fusion isogenic BaF3 cells indicating a potential to avoid the FLT3/c-KIT dual inhibition induced myelosuppression toxicity. In the cellular context it strongly affected FLT3-ITD mediated signaling pathways and induced apoptosis by arresting the cell cycle into the G0/G1 phase. In the in vivo studies 18 demonstrated a good bioavailability (30%) and significantly suppressed the tumor growth in MV4-11 cell inoculated xenograft model (50 mg/kg) without exhibiting obvious toxicity. Compound 18 might be a potential drug candidate for FLT3-ITD positive AML.

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.