Protein-arginine methyltransferase 1 suppresses megakaryocytic differentiation via modulation of the p38 MAPK pathway in K562 cells.

Protein-arginine methyltransferase 1 (PRMT1) plays pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has yet to be investigated. Human leukemia K562 cells have been used as a model to study hematopoietic differentiation. In this study, we report that ectopic expression of HA-PRMT1 in K562 cells suppressed phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation as demonstrated by changes in cytological characteristics, adhesive properties, and CD41 expression, whereas knockdown of PRMT1 by small interference RNA promoted differentiation. Impairment of the methyltransferase activity of PRMT1 diminished the suppressive effect. These results provide evidence for a novel role of PRMT1 in negative regulation of megakaryocytic differentiation. Activation of ERK MAPK has been shown to be essential for megakaryocytic differentiation, although the role of p38 MAPK is still poorly understood. We show that knockdown of p38alpha MAPK or treatment with the p38 inhibitor SB203580 significantly enhanced PMA-induced megakaryocytic differentiation. Further investigation revealed that PRMT1 promotes activation of p38 MAPK without inhibiting activation of ERK MAPK. In p38alpha knockdown cells, PRMT1 could no longer suppress differentiation. In contrast, enforced expression of p38alpha MAPK suppressed PMA-induced megakaryocytic differentiation of parental K562 as well as PRMT1-knockdown cells. We propose modulation of the p38 MAPK pathway by PRMT1 as a novel mechanism regulating megakaryocytic differentiation. This study thus provides a new perspective on the promotion of megakaryopoiesis.

Aurora kinase A inhibitors: identification, SAR exploration and molecular modeling of 6,7-dihydro-4H-pyrazolo-[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione scaffold.

Tricyclic 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione was identified as a novel scaffold for Aurora kinase A inhibition through virtual screening. SAR exploration coupled with molecular modeling of 8a reveals the minimum pharmacophore requirements for Aurora kinase A inhibition.

Cellulose-based diagnostic devices for diagnosing serotype-2 dengue fever in human serum.

Here, two types of cellulose-based in vitro diagnostic devices are demonstrated for the diagnosis of dengue virus infection in both buffer system and human serum: 1) paper-based ELISA for providing the semiquantitative information of the disease activity of serotype-2 dengue fever to healthcare persons (i.e., monitoring the disease activity with a specific serotype in single patients); 2) lateral flow immunoassays to screen for infection with serotype-2 dengue fever (i.e., rapid YES or NO diagnosis prepared for large populations, in terms of global public health). Paper-based ELISA (specific to serotype-2 dengue fever), which builds off of our previous studies and a revised previous ELISA procedure, owns multiple advantages: 1) high sensitivity (about 40 times higher than the current ELISA-based approaches, due to our therapeutic-based monoclonal antibody) and specificity (specific to dengue virus serotype-2 nonstructural protein-1 antigens); 2) tiny amount of sample and reagent used for single tests; 3) short operating duration (i.e., rapid diagnostic device); and, 4) inexpensiveness (appropriate for use in all developing and underdeveloped nations of the world). Due to the higher sensitivity and shorter operating duration of paper-based ELISA (compared with conventional ELISA, and lateral flow immunoassays also performed in this study), this study has not only been able to perform the diagnosis of dengue virus serotype-2 nonstructural protein-1 antigens in both buffer system and human serum but also to evaluate dengue virus serotype-2 envelope proteins in the buffer system, thus successfully achieving the first such use of these proteins as the target antigen for the development of diagnostic tools. These results provide a more comprehensive understanding for the genesis of dengue fever diagnostic tools (through antibody-antigen recognition).

Identification, SAR studies, and X-ray co-crystallographic analysis of a novel furanopyrimidine aurora kinase A inhibitor.

Herein we reveal a simple method for the identification of novel Aurora kinase A inhibitors through substructure searching of an in-house compound library to select compounds for testing. A hydrazone fragment conferring Aurora kinase activity and heterocyclic rings most frequently reported in kinase inhibitors were used as substructure queries to filter the in-house compound library collection prior to testing. Five new series of Aurora kinase inhibitors were identified through this strategy, with IC(50) values ranging from approximately 300 nM to approximately 15 microM, by testing only 133 compounds from a database of approximately 125,000 compounds. Structure-activity relationship studies and X-ray co-crystallographic analysis of the most potent compound, a furanopyrimidine derivative with an IC(50) value of 309 nM toward Aurora kinase A, were carried out. The knowledge gained through these studies could help in the future design of potent Aurora kinase inhibitors.

Structural basis for the improved potency of peroxisome proliferator-activated receptor (PPAR) agonists.

The need to develop safer and more effective antidiabetic drugs is essential owing to the growth worldwide of the diabetic population. Targeting the PPAR receptor is one strategy for the treatment of diabetes; the PPAR agonists rosiglitazone and pioglitazone are already on the market. Here we report the identification of a potent PPAR agonist, 15, whose PPARγ activation was more than 20 times better than that of rosiglitazone. Compound 15 was designed to incorporate an indole head with a carboxylic acid group, and 4-phenylbenzophenone tail to achieve a PPARγ EC(50) of 10 nM. Compound 15 showed the most potent PPARγ agonist activity among the compounds we investigated. To gain molecular insight into the improved potency of 15, a structural biology study and binding energy calculations were carried out. Superimposition of the X-ray structures of 15 and agonist 10 revealed that, even though they have the same indole head part, they adopt different conformations. The head part of 15 showed stronger interactions toward PPARγ; this could be due to the presence of the novel tail part 4-phenylbenzophenone, which could enhance the binding efficiency of 15 to PPARγ.

Fast-forwarding hit to lead: aurora and epidermal growth factor receptor kinase inhibitor lead identification.

A focused library of furanopyrimidine (350 compounds) was rapidly synthesized in parallel reactors and in situ screened for Aurora and epidermal growth factor receptor (EGFR) kinase activity, leading to the identification of some interesting hits. On the basis of structural biology observations, the hit 1a was modified to better fit the back pocket, producing the potent Aurora inhibitor 3 with submicromolar antiproliferative activity in HCT-116 colon cancer cell line. On the basis of docking studies with EGFR hit 1s, introduction of acrylamide Michael acceptor group led to 8, which inhibited both the wild and mutant EGFR kinase and also showed antiproliferative activity in HCC827 lung cancer cell line. Furthermore, the X-ray cocrystal study of 3 and 8 in complex with Aurora and EGFR, respectively, confirmed their hypothesized binding modes. Library construction, in situ screening, and structure-based drug design (SBDD) strategy described here could be applied for the lead identification of other kinases.

Structure-based drug design of novel Aurora kinase A inhibitors: structural basis for potency and specificity.

Aurora kinases have emerged as attractive targets for the design of anticancer drugs. Through structure-based virtual screening, novel pyrazole hit 8a was identified as Aurora kinase A inhibitor (IC(50) = 15.1 microM). X-ray cocrystal structure of 8a in complex with Aurora A protein revealed the C-4 position ethyl carboxylate side chain as a possible modification site for improving the potency. On the basis of this insight, bioisosteric replacement of the ester with amide linkage and changing the ethyl substituent to hydrophobic 3-acetamidophenyl ring led to the identification of 12w with a approximately 450-fold improved Aurora kinase A inhibition potency (IC(50) = 33 nM), compared to 8a. Compound 12w showed selective inhibition of Aurora A kinase over Aurora B/C, which might be due to the presence of a unique H-bond interaction between the 3-acetamido group and the Aurora A nonconserved Thr217 residue, which in Aurora B/C is Glu and found to sterically clash with the 3-acetamido group in modeling studies.

Generation of ligand-based pharmacophore model and virtual screening for identification of novel tubulin inhibitors with potent anticancer activity.

A pharmacophore model, Hypo1, was built on the basis of 21 training-set indole compounds with varying levels of antiproliferative activity. Hypo1 possessed important chemical features required for the inhibitors and demonstrated good predictive ability for biological activity, with high correlation coefficients of 0.96 and 0.89 for the training-set and test-set compounds, respectively. Further utilization of the Hypo1 pharmacophore model to screen chemical database in silico led to the identification of four compounds with antiproliferative activity. Among these four compounds, 43 showed potent antiproliferative activity against various cancer cell lines with the strongest inhibition on the proliferation of KB cells (IC(50) = 187 nM). Further biological characterization revealed that 43 effectively inhibited tubulin polymerization and significantly induced cell cycle arrest in G(2)-M phase. In addition, 43 also showed the in vivo-like anticancer effects. To our knowledge, 43 is the most potent antiproliferative compound with antitubulin activity discovered by computer-aided drug design. The chemical novelty of 43 and its anticancer activities make this compound worthy of further lead optimization.