Discovery of a New-Generation S-Adenosylmethionine-Noncompetitive Covalent Inhibitor Targeting the Lysine Methyltransferase Enhancer of Zeste Homologue 2.

The first-generation enhancer of zeste homologue 2 (EZH2) inhibitors suffer from several limitations, such as high dosage, cofactor S-adenosylmethionine (SAM) competition, and acquired drug resistance. Development of covalent EZH2 inhibitors that are noncompetitive with cofactor SAM offers an opportunity to overcome these disadvantages. The structure-based design of compound 16 (BBDDL2059) as a highly potent and selective covalent inhibitor of EZH2 is presented in this context. 16 inhibits EZH2 enzymatic activity at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition. The kinetic assay revealed that 16 is noncompetitive with the cofactor SAM, providing the basis for its superior activity over noncovalent and positive controls by reducing competition with cofactor SAM and offering a preliminary proof for its covalent inhibition nature. Mass spectrometric analysis and washout experiments firmly establish its covalent inhibition mechanism. This study demonstrates that covalent inhibition of EZH2 can offer a new opportunity for the development of promising new-generation drug candidates.

Computer aided drug discovery of highly ligand efficient, low molecular weight imidazopyridine analogs as FLT3 inhibitors.

The FLT3 kinase represents an attractive target to effectively treat AML. Unfortunately, no FLT3 targeted therapeutic is currently approved. In line with our continued interests in treating kinase related disease for anti-FLT3 mutant activity, we utilized pioneering synthetic methodology in combination with computer aided drug discovery and identified low molecular weight, highly ligand efficient, FLT3 kinase inhibitors. Compounds were analyzed for biochemical inhibition, their ability to selectively inhibit cell proliferation, for FLT3 mutant activity, and preliminary aqueous solubility. Validated hits were discovered that can serve as starting platforms for lead candidates.

Discovery of novel c-Met kinase inhibitors bearing a thieno[2,3-d]pyrimidine or furo[2,3-d]pyrimidine scaffold.

A series of thieno[2,3-d]pyrimidines and furo[2,3-d]pyrimidines were synthesized and evaluated for the c-Met inhibition. Thieno[2,3-d]pyrimidine 6b stood out as the most potent showing an IC(50) of 35.7 nM. This compound displayed high inhibitory effect on cell proliferation in BaF3-TPR-Met cells and showed high selectivity for c-Met family against other 14 tested kinases. However, compound 6b was found ineffective in the c-Met-dependent U-87MG human gliobastoma xenograft model that may be relevant to its poor PK profile.

Effect of prenatal tetrandrine administration on transforming growth factor-beta1 level in the lung of nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: Tetrandrine (Tet) is a bisbenzylisoquinoline alkaloid isolated from the root of Stephania tetrandra, which has been used in traditional Chinese medicine to treat patients with silicosis, asthma, and pulmonary hypertension, and others and can be used as a pulmonary therapeutic agent. We hypothesized that it can also improve the lung growth in congenital diaphragmatic hernia (CDH) for its multiple biological effects. There are increasing evidences that suggest transforming growth factor beta1(TGF-beta1) plays a crucial role in fetal lung growth and morphogenesis. The aim of this study was to evaluate the effect of prenatal administration of Tet and to investigate its possible mechanism on the expression of TGF-beta1 in the lung of nitrofen-induced CDH rat model. METHODS: A CDH model was induced in pregnant Sprague-Dawley rats by administration of nitrofen on day 9.5 of gestation (Ed9.5 term, day 22). Tetrandrine (30 mg/kg) was given through gavage (once a day, for 3 days) on Ed11.5. Accordingly, there were 3 groups as follows: control (n = 9), CDH (n = 9), and CDH + Tet (n = 9). All the fetuses were delivered by cesarean delivery on Ed16.5, 18.5, and 21.5, respectively, to check if diaphragmatic hernia existed on each fetus, then the lung tissue weight (LW) and body weight (BW) of each fetus were recorded. Histologic evaluations and TGF-beta1 immunohistochemistry staining in the lung sample were performed for image analysis. RESULTS: Diaphragmatic hernia was observed in 95 of the 112 rat fetuses in CDH and CDH + Tet groups on Ed18.5 and Ed21.5 (84.8%), the incidence between the 2 groups had no statistical significance (P = .642). Lung weight/body weight in the CDH group and the CDH + Tet group were lower than that in the control group (P < .01), and LW/BW in the CDH group was lower than that in the CDH + Tet group (P < .05). Observed under the light microscope and electron microscope, marked hypoplasia of the lungs in fetuses among the CDH groups was observed, in contrast to improvement of the lungs in CDH + Tet fetuses. Statistical differences in morphological parameters (percentage of alveoli area, counting bronchus) were found even on Ed16.5 when diaphragm had not closed (P < .01). The number of type II pneumocytes and lamellar bodies in each group had no significant difference (P > .05). The immunoreactivity of TGF-beta1 in CDH group and CDH + Tet group were markedly stronger than that in the control group (P < .01). In addition, TGF-beta1 expression in the CDH group was stronger than that in the CDH + Tet group (P < .01). CONCLUSION: Nitrofen can interfere with lung development early in the fetal rat development before and separate from diaphragm development, and increased expression of TGF-beta1 in the lung of CDH rat model may suppress lung growth and development. Prenatal treatment with Tet can improve the growth of the lung of the nitrofen-induced CDH fetuses and its mechanism seems to be involved in downregulating the expression of TGF-beta1. It is a likely new approach to treat CDH and its coexistent lung hypoplasia by maternal Tet administration.

Prenatal tetrandrine treatment can reverse the abnormal conditions in the lung of newborn with congenital diaphragmatic hernia.

Pulmonary hypoplasia and persistent pulmonary hypertension are the most important reasons for the high morbidity and mortality of congenital diaphragmatic hernia (CDH). Despite surgical advances and advances in neonatal intensive care, the mortality still remains high. Then the research on how to improve prenatal fetal lung growth has become a focus. Some researches involved in fetal surgery, tracheal occlusion, prenatal use of corticosteroids etc., have been carried out in CDH animal models and humans. But the results either showed no benefit for the outcome of CDH or were unproved. Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from the root of Stephania tetrandra. It has been used in traditional Chinese medicine for several decades to treat patients with silicosis, asthma and pulmonary hypertension etc. Some researches showed that prenatal tetrandrine administration can improve the lung development in CDH rat models. We hypothesize that prenatal treatment with tetrandrine can reverse the abnormal condition in the lung of newborn with CDH, and thus decrease the mortality.

[Effect of tetrandrine on lung development: experiment of nitrofen-induced congenital diaphragmatic hernia fetal rat model].

OBJECTIVE: To assess the efficacy of prenatal administration of tetrandrine (TET) on pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) fetal rat model. METHODS: Six timed-pregnant female Sprague-Dawley rats were randomly divided into 3 equal groups: CDH group (receiving gavage of nitrofen 125 mg dissolved in seed fat on day 9.5), and TET group (receiving gavage of nitrofen 125 mg on day 9.5 and then gavage of TET 30 mg/kg on days 11.5 - 14.5), and control group (given the same dose of peanut oil on day 9.5 and the same dose of normal saline on days 11.5 - 14.5). The fetuses were delivered by cesarean section on day 21 to undergo light microscopy and electron microscopy. The numbers of type II pneumocytes were recorded and compared. RESULTS: CDH were detected in 32 of the 41 fetuses from the CDH and TET groups with a teratogenic rate of 78%, however, without a significant difference between the CDH and TET groups (P = 0.645). Microscopy showed significant lung hypoplasia in both histologic structure and cellular structure in the CDH group; however the lung development of the TET group was improved in comparison to the CDH group. There was no significant difference in numbers of type II pneumocytes among the 3 groups (P = 0.779). CONCLUSION: Prenatal administration of TET can improve the lung development of CDH rats in both histological structure and cellular structure. This may provide a new idea for the clinical treatment of CDH.