Kailin Yu

Ibrutinib selectively and irreversibly targets EGFR (L858R, Del19) mutant but is moderately resistant to EGFR (T790M) mutant NSCLC Cells

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 ibrutinibs 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.

Ibrutinib selectively targets FLT3-ITD in mutant FLT3-positive AML.

Ibrutinib (PCI-32765) is an irreversible BTK (Bruton’s tyrosine kinase) kinase inhibitor that has been extensively used as a tool compound to validate the role of BTK kinase in B cell related malignances. Ibrutinib has been shown in preclinical studies to inhibit the proliferation of diffuse large B-cell lymphoma cells, mantle cell lymphoma cells, chronic lymphocytic leukemia cells and multiple myeloma cells by blocking BTK kinase activity; ibrutinib was recently approved for the clinical application on mantle cell lymphoma and chronic lymphocytic leukemia cells. Ibrutinib has also exhibited anti-inflammatory effects in preclinical models. Recently, it has been reported that ibrutinib is also effective against epidermal growth factor receptor mutantpositive non-small cell lung cancers through inhibition of epidermal growth factor receptor kinase activities. In addition, there is evidence showing that BTK is also an important target for Acute Myeloid Leukemia (AML). Despite the evidence that BTK knockdown impaired AML cancer cell growth, which suggested that BTK was important for AML cell proliferation, BTK kinase inhibition through use of a small molecule inhibitor like ibrutinib led only to moderate inhibition of proliferation of U937 cells with no apparent activity against other AML cell lines such as HL-60, TF-1 and THP-1. To further investigate the potency and activity of ibrutinib against AML, we screened a panel of AML cell lines spanning M0–M7 disease stages. Interestingly, we found that only FLT3-internal tandem duplication (ITD) mutant AML cell lines (MOLM13, MOLM14 and MV4-11) were sensitive to ibrutinib (Figure 1a and Supplementary Table 1). This is similar to what has been observed with the highly

Dual inhibition of AKT/FLT3-ITD by A674563 overcomes FLT3 ligand-induced drug resistance in FLT3-ITD positive AML

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.

Discovery of a Highly Selective STK16 Kinase Inhibitor.

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.

Discovery of a highly potent FLT3 kinase inhibitor for FLT3-ITD-positive AML.

TH was responsible for the cytomorphologic analysis and was the principle investigator of the study. AS contributed to the cytogenetics, SE to the cytomorphology, KP to the mutation analyses and TA to the collection of clinical data. WK was involved in the statistical analyses. CH was responsible for the cytogenetics. MM investigated the molecular mutations, analyzed the data and wrote the manuscript. All authors read and contributed to the final version of the manuscript.

Discovery of N-((1-(4-(3-(3-((6,7-Dimethoxyquinolin-3-yl)oxy)phenyl)ureido)-2-(trifluoromethyl)phenyl)piperidin-4-yl)methyl)propionamide (CHMFL-KIT-8140) as a Highly Potent Type II Inhibitor Capable of Inhibiting the T670I “Gatekeeper” Mutant of cKIT Kinase

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.

Discovery of (R)-1-(3-(4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-(dimethylamino)ethanone (CHMFL-FLT3-122) as a Potent and Orally Available FLT3 Kinase Inhibitor for FLT3-ITD Positive Acute Myeloid Leukemia.

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.

Irreversible inhibition of BTK kinase by a novel highly selective inhibitor CHMFL-BTK-11 suppresses inflammatory response in rheumatoid arthritis model

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.

Discovery of 2-((3-acrylamido-4-methylphenyl)amino)-N-(2-methyl-5-(3,4,5-trimethoxybenzamido)phenyl)-4-(methylamino)pyrimidine-5-carboxamide (CHMFL-BMX-078) as a Highly Potent and Selective Type II Irreversible Bone Marrow Kinase in the X Chromosome (BMX) Kinase Inhibitor.

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 IC50 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.

Discovery and characterization of a novel irreversible EGFR mutants selective and potent kinase inhibitor CHMFL-EGFR-26 with a distinct binding mode

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.