IOP-lowering effect of isoquinoline-5-sulfonamide compounds in ocular normotensive monkeys

Rho-associated coiled coil-formed protein kinase (ROCK) inhibitors are under development as a new class of antiglaucoma agents. Based on the potent ROCK inhibitor H-1152, previously developed by us, we explored the possibility of related compounds as antiglaucoma agents and synthesized seven types of H-1152-inspired isoquinoline-5-sulfonamide compounds (H-0103–H-0107, H-1001, H-1005). Although all of these compounds potently inhibited ROCK (IC₅₀ = 18–48 nM), only H-0104 and H-0106 exerted strong intraocular pressure (IOP)-lowering effects into the eyes of monkeys. These results suggested the possibility that there is no direct relationship between ROCK inhibition and IOP-lowering effects, indicating that the initial screening of compounds based on ROCK inhibitory activity may be an unsuitable strategy for developing antiglaucoma agents with potent IOP-lowering effects.     

Reaction intermediate analogues as bisubstrate inhibitors of pantothenate synthetase

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β-alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the intermediate pantoyl adenylate. These inhibitors are competitive inhibitors with respect to pantoic acid and possess submicromolar to micromolar inhibition constants. The observed SAR is rationalized through molecular docking studies based on the reported co-crystal structure of 1a with PanC. Finally, whole cell activity is assessed against wild-type Mtb as well as a PanC knockdown strain where PanC is depleted to less than 5% of wild-type levels.     

Factor VIII-hydrolyzing IgG in acquired and congenital hemophilia

Anti-factor VIII (FVIII) inhibitory IgG may arise as alloantibodies to therapeutic FVIII in patients with congenital hemophilia A, or as autoantibodies to endogenous FVIII in individuals with acquired hemophilia. We have described FVIII-hydrolyzing IgG both in hemophilia A patients with anti-FVIII IgG and in acquired hemophilia patients. Here, we compared the properties of proteolytic auto- and allo-antibodies. Rates of FVIII hydrolysis differed significantly between the two groups of antibodies. Proline-phenylalanine-arginine-methylcoumarinamide was a surrogate substrate for FVIII-hydrolyzing autoantibodies. Our data suggest that populations of proteolytic anti-FVIII IgG in acquired hemophilia patients are different from that of inhibitor-positive hemophilia A patients.     

Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10^–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.     

益智有效抗氧化成分的分离条件的研究

探讨了干柱层析法分离益智渣及益智乙醇提取物的抗氧化成分。实验中采用乙酸乙酯/环己烷(3∶7)、氯仿/甲醇(9∶1)为展开剂依次二次干柱层析分离出抗氧化物质。结果表明,益智渣及益智乙醇提取物均有较强的抗氧化性,益智渣的H2O2清除能力强于益智乙醇提取物。     

The type III TGF-β receptor betaglycan transmembrane-cytoplasmic domain fragment is stable after ectodomain cleavage and is a substrate of the intramembrane protease γ-secretase

The Type III TGF-β receptor, betaglycan, is a widely expressed proteoglycan co-receptor for TGF-β superfamily ligands. The full-length protein undergoes ectodomain cleavage with release of a soluble ectodomain fragment. The fate of the resulting transmembrane-cytoplasmic fragment, however, has never been explored. We demonstrate here that the transmembrane-cytoplasmic fragment is stable in transfected cells and in cell lines expressing endogenous betaglycan. Production of this fragment is inhibited by the ectodomain shedding inhibitor TAPI-2. Treatment of cells with inhibitors of the intramembrane protease γ-secretase stabilizes this fragment, suggesting that it is a substrate of γ-secretase. Expression of the transmembrane-cytoplasmic fragment as well as γ-secretase inhibitor stabilization are independent of TGF-β1 or -β2 and are unaffected by mutation of the cytoplasmic domain serines that undergo phosphorylation. γ-Secretase inhibition or the expression of a transmembrane-cytoplasmic fragment in HepG2 cells blunted TGF-β2 signaling. Our findings thus suggest that the transmembrane-cytoplasmic fragment remaining after betaglycan ectodomain cleavage is stable and a substrate of γ-secretase, which may have significant implications for the TGF-β signaling response.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

SAR optimization studies on a novel series of 2-anilinopyrimidines as selective inhibitors against triple-negative breast cancer cell line MDA-MB-468

Triple-negative breast cancers (TNBCs) account for approximately 15% of breast cancer cases and exhibit an aggressive clinical behavior. In this study, we designed and synthesized two series of 2-anilinopyrimidines based on the structure of our previously reported compound 1 that act as a selective inhibitor of the basal-like TNBC cell line MDA-MB-468. Through the fine-tuning of 1, cyclic and acyclic amines at 4-position of the pyrimidine core were turned out to be crucial for the selectivity. An extensive analysis of structure-activity relationships of the analogs revealed that aminoalkyl groups at the end of the propyl chain are amenable to modification. Among the newly synthesized analogs, compound 38, bearing 4-chloropiperidinyl and cyclohexyl groups, was found to be the most potent and selective, and was about three times more potent and selective than 1 was against the TNBC cells.     

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 μg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 μg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.     

糠醛和5-羟甲基糠醛对大肠杆菌产丁二酸的影响

利用可再生生物质特别是木质纤维素水解液来生产平台化合物丁二酸,是目前研究的热点。虽然许多研究者相继报道了木质纤维素水解液对菌株生长和丁二酸生产存在一定抑制作用,但并没有水解液中各种抑制物对菌株影响的相关动力学研究及机理研究。我们选择了两种代表性木质纤维素水解液抑制物,即糠醛和5-羟甲基糠醛,系统研究了它们对大肠杆菌的生长和丁二酸生产的影响。结果表明：糠醛和5-羟甲基糠醛的初始抑制浓度均为0.8 g/L。当糠醛浓度大于6.4 g/L,5-羟甲基糠醛浓度大于12.8 g/L时,菌株生长完全受到抑制。在最高耐受浓度下,糠醛的存在使菌株生物量比对照菌株下降77.8%,丁二酸产量下降36.1%。5-羟甲基糠醛的存在使菌株生物量比对照菌株降低13.6%,丁二酸产量降低18.3%。糠醛和5-羟甲基糠醛具有明显的协同作用。体外酶活测定表明丁二酸生产途径中关键酶磷酸烯醇式丙酮酸羧化酶、苹果酸脱氢酶、富马酸还原酶均受糠醛和5-羟甲基糠醛抑制。研究结果对丁二酸生产用纤维素水解液的预处理和脱毒工艺开发具有指导作用,有利于实现丁二酸发酵生产的工业化。