Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy

Sonic hedgehog (Shh), both as a mitogen and as a morphogen, plays an important role in cell proliferation and differentiation during early development. Here, we show that Shh inhibits glutamate transporter activities in neurons, rapidly enhances extracellular glutamate levels, and affects the development of epilepsy. Shh is quickly released in response to epileptic, but not physiological, stimuli. Inhibition of neuronal glutamate transporters by Shh depends on heterotrimeric G protein subunit Gα_i and enhances extracellular glutamate levels. Inhibiting Shh signaling greatly reduces epileptiform activities in both cell cultures and hippocampal slices. Moreover, pharmacological or genetic inhibition of Shh signaling markedly suppresses epileptic phenotypes in kindling or pilocarpine models. Our results suggest that Shh contributes to the development of epilepsy and suppression of its signaling prevents the development of the disease. Thus, Shh can act as a modulator of neuronal activity, rapidly regulating glutamate levels and promoting epilepsy.     

Structural role of the T94I rhodopsin mutation in congenital stationary night blindness

Congenital stationary night blindness (CSNB) is an inherited and non‐progressive retinal dysfunction. Here, we present the crystal structure of CSNB‐causing T94I^2.61 rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin‐II state by establishing a direct van der Waals contact with K296^7.43, the site of retinal attachment. This is in stark contrast to the light‐activated state of the CSNB‐causing G90D^2.57 mutation, where the charged mutation forms a salt bridge with K296^7.43. To find the common denominator between these two functional modifications, we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D^2.57 and the hydrophobic T94I^2.61 mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113^3.28. We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.     

Design,synthesis and biological evaluation of 4-benzoyl-1-dichlorobenzoylthiosemicarbazides as potent Gram-positive antibacterial agents

Twelve 4-benzoyl-1-dichlorobenzoylthiosemicarbazides have been tested as potential antibacterials. All the compounds had MICs between 0.49 and 15.63?µg/ml toward Micrococcus luteus, Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis indicating, in most cases, equipotent or even more effective action than cefuroxime. In order to clarify if the observed antibacterial effects are universal, further research were undertaken to test inhibitory potency of two most potent compounds 3 and 11 on clinical isolates of Staphylococcus aureus. Compound 11 inhibited the growth of methicillin-sensitive S. aureus (MSSA) at MICs of 1.95–7.81?µg/ml, methicillin-resistant S. aureus (MRSA) at MICs of 0.49–1.95?µg/ml and MDR–MRSA at MIC of 0.98 and 3.90?µg/ml, respectively. Finally, inhibitory efficacy of 3 and 11 on planktonic cells and biofilms formation in clinical isolates of S. aureus and Haemophilus parainfluenzae was tested. The majority of cells in biofilm populations of MSSA and MRSA were eradicated at low level of 3, with MBICs in the range of 7.82–15.63?µg/ml.     

Design,synthesis and preliminary structure–activity relationship investigation of nitrogen-containing chalcone derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors: a further study based on Flavokawain B Mannich base derivatives

In order to study the structure–activity relationship of Flavokawain B Mannich-based derivatives as acetylcholinesterase (AChE) inhibitors in our recent investigation, 20 new nitrogen-containing chalcone derivatives (4?a–8d) were designed, synthesized, and evaluated for AChE inhibitory activity in vitro. The results suggested that amino alkyl side chain of chalcone dramatically influenced the inhibitory activity against AChE. Among them, compound 6c revealed the strongest AChE inhibitory activity (IC₅₀ value: 0.85?μmol/L) and the highest selectivity against AChE over BuChE (ratio: 35.79). Enzyme kinetic study showed that the inhibition mechanism of compound 6c against AChE was a mixed-type inhibition. The molecular docking assay showed that this compound can both bind with the catalytic site and the peripheral site of AChE.     

Cinnoline derivatives as human neutrophil elastase inhibitors

Compounds that can effectively inhibit the proteolytic activity of human neutrophil elastase (HNE) represent promising therapeutics for treatment of inflammatory diseases. We present here the synthesis, structure–activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with a cinnoline scaffold. These compounds exhibited HNE inhibitory activity but had lower potency compared to N-benzoylindazoles previously reported by us. On the other hand, they exhibited increased stability in aqueous solution. The most potent compound, 18a, had a good balance between HNE inhibitory activity (IC₅₀ value?=?56?nM) and chemical stability (t_1/2?=?114?min). Analysis of reaction kinetics revealed that these cinnoline derivatives were reversible competitive inhibitors of HNE. Furthermore, molecular docking studies of the active products into the HNE binding site revealed two types of HNE inhibitors: molecules with cinnolin-4(1H)-one scaffold, which were attacked by the HNE Ser195 hydroxyl group at the amido moiety, and cinnoline derivatives containing an ester function at C-4, which is the point of attack of Ser195.     

Synthesis and anticancer and lipophilic properties of 10-dialkylaminobutynyl derivatives of 1,8- and 2,7-diazaphenothiazines

New derivatives of two isomeric types of azaphenothiazines, 1,8- and 2,7-diazaphenothiazine, containing the triple bond substituents and additionally tertiary cyclic and acyclic amine groups, were synthesized and tested for their anticancer activity. The compounds exhibited differential inhibitory activities. Better results were obtained when the acetylenic group was transformed via the Mannich reaction to the dialkylaminobutynyl groups. The most active was 2,7-diazaphenothiazine with the N-methylpiperazine-2-butynyl substituent against the human ductal breast epithelial tumor cell line T47D, more potent than cisplatin. The 2,7-diazaphenothiazine system turned out to be more active than isomeric 1,8-diaza one. For the most active compound, the expression of TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The gene expression ratio BACL-2/BAX suggests the mitochondrial apoptosis in T47D cells. The synthesis makes possible to obtain many new bioactive phenothiazines with the dialkylaminoalkynyl substituents inserting various tertiary cyclic and acyclic amine moieties to the substituents.     

Isolation and biological activities of 3-hydroxy-4(1H)-pyridone

3-Hydroxy-4(4H)-pyridone (3,4-DHP), a degraded product of mimosine [β-[N-(3-hydroxy-4-oxypyridyl)]-α-aminopropionic acid], is known to cause goiters, loss of hair, and infertility in animals, but limits of 3,4-DHP on separation and purification have prevented efforts on investigating other toxicity and biological properties of 3,4-DHP. By this study, a novel and simple isolation of 3,4-DHP was developed either from Leucaena leaves using an ion-exchanged resin or mimosine degraded in high temperature (110°C, 6?h). The inhibition of mimosine on the growth of barnyardgrass was approximately fourfold higher (IC₅₀?=?0.04?mg?g^?1) than that of 3,4-DHP (IC₅₀?=?0.15?mg?g^?1). In general, the antifungal activity of mimosine is much stronger than that of 3,4-DHP, but it differs depending on the kind of fungi. The 1,1-diphyenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of 3,4-DHP, in contrast with the growth inhibitory activity, is about fourfold stronger [EC₅₀?=?2.4?mg?g^?1 gallic acid equivalent (GAE)] than that of mimosine [EC₅₀?=?10.3?mg?g^?1 GAE]. This study is the first to report on the herbicidal, antifungal, and antioxidant activities of 3,4-DHP.     

Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP⁺/NBT/PMS (β-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H₃₇Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H₃₇Ra PGI was 226?μg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H₃₇Ra PGI inhibition by PEP.