Room-Temperature CuI-Catalyzed N-Arylation of Cyclopropylamine.

A general and efficient CuI/N-carbazolyl-1H-pyrrole-2-carbohydrazide catalyst system was developed for the N-arylation of cyclopropylamine using aryl bromides at room temperature. Herein, 5 mol % CuI and 5 mol % of the ligand were used to synthesize N-aryl cyclopropylamines in moderate to excellent yields. This protocol was scaled up to produce the desired product at gram levels and has been generalized for C-N coupling between aryl bromides and amines at room temperature.

Novel partially reversible NDM-1 inhibitors based on the naturally occurring houttuynin.

Discovering novel NDM-1 inhibitors is an urgent task for treatment of 'superbug' infectious diseases. In this study, we found that naturally occurring houttuynin and its sulfonate derivatives might be effective NDM-1 inhibitors with novel mechanism, i.e. the attribute of partially covalent inhibition of sulfonate derivatives of houttuynin against NDM-1. Primary structure-activity relationship study showed that both the long aliphatic side chain and the warhead of aldehyde group are vital for the efficiency against NDM-1. The homologs with longer chains (SNH-2 to SNH-5) displayed stronger inhibitory activities with IC50 range of 1.1-1.5 µM, while the shorter chain the weaker inhibition. Further synergistic experiments in cell level confirmed that all these 4 compounds (at 32 µg/mL) recovered the antibacterial activity of meropenem (MER) against E. coli BL21/pET15b-blaNDM-1.

Magnaporthe oryzae endoplasmic reticulum membrane complex regulates the biogenesis of membrane proteins for pathogenicity.

In eukaryotes, the majority of newly synthesized integral membrane proteins are inserted into the endoplasmic reticulum (ER) membrane before transferred to their functional sites. The conserved ER membrane complex (EMC) takes part in the insertion process for tail-anchored membrane proteins. However, the function of EMC in phytopathogenic fungi has not been characterized. Here, we report the identification and functional characterization of two EMC subunits MoEmc5 and MoEmc2 in Magnaporthe oryzae. The knockout mutants ΔMoemc5 and ΔMoemc2 exhibit substantial defect in autophagy, pathogenicity, cell wall integrity, and magnesium ion sensitivity. We demonstrate that the autophagy process was severely impaired in the ΔMoemc5 and ΔMoemc2 mutants because of the low-protein steady-state level of Atg9, the sole membrane-associated autophagy protein. Furthermore, the protein level of membrane proteins Chs4, Fks1, and MoMnr2 is also significantly reduced in the ΔMoemc5 and ΔMoemc2 strains, leading to their supersensitivity to Calcofluor white, Congo red, and magnesium. In addition, MoEmc5, but not MoEmc2, acts as a magnesium transporter independent of its EMC function. Magnaporthe oryzae EMC regulates the biogenesis of membrane proteins for autophagy and virulence; therefore, EMC subunits could be potential targets for fungicide design in the future.

PHB3 Is Required for the Assembly and Activity of Mitochondrial ATP Synthase in Arabidopsis.

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits ß and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

GRGDS-functionalized chitosan nanoparticles as a potential intravenous hemostat for traumatic hemorrhage control in an animal model.

Hemostats, which are used for immediate intervention during internal hemorrhage in order to reduce resulting mortality and morbidity, are relatively rare. Here, we describe novel intravenous nanoparticles (CPG-NPs-2000) with chitosan succinate (CSS) as cores, polyethylene glycol (PEG-2000) as spacers and a glycine-arginine-glycine-aspartic acid-serine (GRGDS) peptide as targeted, active hemostatic motifs. CPG-NPs-2000 displayed significant hemostatic efficacy, compared to the saline control, CSS nanoparticles, and tranexamic acid in liver trauma rat models. Further studies have demonstrated that CPG-NPs-2000 are effectively cleared from organs and blood, within 2 and 48 h, respectively. In addition, administration of CPG-NPs-2000 does not affect clotting function under normal physiological conditions, indicating their potential safety in vivo. CPG-NPs-2000 exhibit excellent thermal stability, good solubility, and redistribution ability, in addition to being low cost. These characteristics indicate that CPG-NPs-2000 may have strong potential as effective intravenous hemostats for treating severe internal bleeding.

Room-Temperature CuI-Catalyzed Amination of Aryl Iodides and Aryl Bromides.

A general and effective CuI/N',N'-diaryl-1H-pyrrole-2-carbohydrazide catalyst system was developed for the amination of aryl iodides and bromides at room temperature with good chemoselectivity between -OH and -NH2 groups. Only 5 mol % of CuI and ligands was needed in this protocol to effect the amination of various aryl bromides and aryl iodides with a wide range of aliphatic and aryl amines (1.3 equiv).

Structural Asymmetry of Phosphodiesterase-9A and a Unique Pocket for Selective Binding of a Potent Enantiomeric Inhibitor.

Phosphodiesterase-9 (PDE9) inhibitors have been studied as potential therapeutics for treatment of central nervous system diseases and diabetes. Here, we report the discovery of a new category of PDE9 inhibitors by rational design on the basis of the crystal structures. The best compound, (S)-6-((1-(4-chlorophenyl)ethyl)amino)-1-cyclopentyl-1,5,6,7-tetrahydro-4H-pyrazolo[3,4-day]pyrimidin-4-one [(S)-C33], has an IC50 value of 11 nM against PDE9 and the racemic C33 has bioavailability of 56.5% in the rat pharmacokinetic model. The crystal structures of PDE9 in the complex with racemic C33, (R)-C33, and (S)-C33 reveal subtle conformational asymmetry of two M-loops in the PDE9 dimer and different conformations of two C33 enantiomers. The structures also identified a small hydrophobic pocket that interacts with the tyrosyl tail of (S)-C33 but not with (R)-C33, and is thus possibly useful for improvement of selectivity of PDE9 inhibitors. The asymmetry of the M-loop and the different interactions of the C33 enantiomers imply the necessity to consider the whole PDE9 dimer in the design of inhibitors.

Dual specificity and novel structural folding of yeast phosphodiesterase-1 for hydrolysis of second messengers cyclic adenosine and guanosine 3',5'-monophosphate.

Cyclic nucleotide phosphodiesterases (PDEs) decompose second messengers cAMP and cGMP that play critical roles in many physiological processes. PDE1 of Saccharomyces cerevisiae has been subcloned and expressed in Escherichia coli. Recombinant yPDE1 has a KM of 110 µM and a kcat of 16.9 s(-1) for cAMP and a KM of 105 µM and a kcat of 11.8 s(-1) for cGMP. Thus, the specificity constant (kcat/KM(cAMP))/(kcat/KM(cGMP)) of 1.4 indicates a dual specificity of yPDE1 for hydrolysis of both cAMP and cGMP. The crystal structures of unliganded yPDE1 and its complex with GMP at 1.31 Å resolution reveal a new structural folding that is different from those of human PDEs but is partially similar to that of some other metalloenzymes such as metallo-ß-lactamase. In spite of their different structures and divalent metals, yPDE1 and human PDEs may share a common mechanism for hydrolysis of cAMP and cGMP.

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity.

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant-fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.

Pyrrolo[2,3-c]azepine derivatives: a new class of potent protein tyrosine phosphatase 1B inhibitors.

A series of pyrrolo[2,3-c]azepine derivatives was designed, synthesized, and evaluated as a new class of inhibitors against protein tyrosine phosphatase 1B (PTP1B) in vitro. The results demonstrated that compounds bearing a biphenyl moiety were proved to markedly influence the potency of these inhibitors. Particularly, compounds 29, 35 and 36 showed interesting inhibition with IC(50) value of 16.36, 14.93 and 13.92µM, respectively.

Direct Arylation of Pyrroles via Indirect Electroreductive C-H Functionalization Using Perylene Bisimide as an Electron-Transfer Mediator.

The indirect electroreductive coupling of aryl halides and pyrroles was successfully conducted using a catalytic amount of perylene bisimide as a mediator in 1-ethyl-3-methylimidazolium bis((trifluoromethyl)sulfonyl)imide ([EMIM]NTf2)/DMSO.

An arch-bridge-type fluorophore for bridging the gap between aggregation-caused quenching (ACQ) and aggregation-induced emission (AIE).

Solution and solid dual photoluminescence (PL) molecules fill the substantial gap between ACQ and AIE molecules to explore the mechanism of molecular luminescence in greater detail and to facilitate practical applications. A unique arch-bridge-like thiazolo[5,4-b]thieno[3,2-e]pyridine moiety is obtained as a stator after the rigidification of rotor 1 by intramolecular H-bonding of ortho -OH or -NH2 to afford two classes of solid and solution dual PL molecules. As a typical example, DF5 is dual PL active. Moreover, the large Stokes shift with high dual PL efficiency (ΦF up to 51% in the solid state, 80% in DMF, 74% in DMSO, and 100% in water), together with the good thermal stability (Tm > 200 °C and T05 > 200 °C), make it more practical for promising optoelectronic and biological applications.

Discovery of 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one as a phosphodiesterase-5 inhibitor and its complex crystal structure.

Phosphodiesterase-5 (PDE5) inhibitors have been approved for the treatment of erectile dysfunction and pulmonary hypertension, but enthusiasm on discovery of PDE5 inhibitors continues for their potential new applications. Reported here is discovery of a series of new PDE5 inhibitors by structure-based design, molecular docking, chemical synthesis, and enzymatic characterization. The best compound, 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one (57), has an IC50 of 17 nM against the PDE5 catalytic domain and good selectivity over other PDE families. The crystal structure of the PDE5 catalytic domain in complex with 57 was determined at 2Å resolution and showed that 57 occupies the same pocket as other PDE5 inhibitors, but has a different binding pattern in detail. On the basis of the binding pattern of 57, a novel scaffold can be proposed as a candidate of PDE inhibitors.

Discovery of a phosphodiesterase 9A inhibitor as a potential hypoglycemic agent.

Phosphodiesterase 9 (PDE9) inhibitors have been studied as potential therapeutics for treatment of diabetes and Alzheimer's disease. Here we report a potent PDE9 inhibitor 3r that has an IC50 of 0.6 nM and >150-fold selectivity over other PDEs. The HepG2 cell-based assay shows that 3r inhibits the mRNA expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase. These activities of 3r, together with the reasonable pharmacokinetic properties and no acute toxicity at 1200 mg/kg dosage, suggest its potential as a hypoglycemic agent. The crystal structure of PDE9-3r reveals significantly different conformation and hydrogen bonding pattern of 3r from those of previously published 28s. Both 3r and 28s form a hydrogen bond with Tyr424, a unique PDE9 residue (except for PDE8), but 3r shows an additional hydrogen bond with Ala452. This structure information might be useful for design of PDE9 inhibitors.

Structure-based discovery of highly selective phosphodiesterase-9A inhibitors and implications for inhibitor design.

A new series of phosphodiesterase-9 (PDE9) inhibitors that contain a scaffold of 6-amino-pyrazolopyrimidinone have been discovered by a combination of structure-based design and computational docking. This procedure significantly saved the load of chemical synthesis and is an effective method for the discovery of inhibitors. The best compound 28 has an IC(50) of 21 nM and 3.3 µM, respectively, for PDE9 and PDE5 and about 3 orders of magnitude of selectivity against other PDE families. The crystal structure of the PDE9 catalytic domain in complex with 28 has been determined and shows a hydrogen bond between 28 and Tyr424. This hydrogen bond may account for the 860-fold selectivity of 28 against PDE1B, in comparison with about 30-fold selectivity of BAY73-6691. Thus, our studies suggest that Tyr424, a unique residue of PDE8 and PDE9, is a potential target for improvement of selectivity of PDE9 inhibitors.