Shedding light on designing potential meprin β inhibitors through ligand-based robust validated computational approaches: A proposal to chemists!

<p>Human meprin (EC 3.4.24.18) is a member of the metzincin superfamily. It correlates with matrix metalloproteinases and ADAMs (a disintegrin and metalloproteinase). Overexpression of meprin β is implicated in fibrosis, inflammatory diseases and cancers. However, selective meprin β inhibition is crucial to reduce cancer metastasis and adverse effects in inflammation. It also plays critical roles in modulating several interleukins and growth factors. Moreover, meprin β cleaves amyloid precursor protein, thought to be involved in the progression of Alzheimer’s disease. Therefore, meprin β inhibitors are considered to be emerging therapeutics with paramount importance in the treatment of kidney failure, fibrosis, inflammatory bowel diseases and cancer. Despite its crucial implication in several diseases, no meprin β inhibitors are available as drug candidates till date. Therefore, it is an urgent need to identify new potential meprin β inhibitors as prospective therapeutics. In this article, a series of meprin β inhibitors has been analysed through multiple molecular modelling studies as the first initiative to get an idea about their structural, physicochemical and pharmacophoric requirements for higher activity. All <i>in silico</i> approaches performed here are statistically validated and subsequently adjudicated each other. Compounds with <i>p</i>-carboxylic acid substituted arylsulphonamide moiety attached with <i>m</i>-carboxylic acid substituted benzyl group along with a methylene hydroxamate function may be crucial for imparting potential meprin β inhibition. Depending on the results obtained, 14 molecules have been proposed by QSAR model that predicted a minimum of 4-fold higher activity compared to these compounds of the current study.</p

Designing potential HDAC3 inhibitors to improve memory and learning

<p>The work presented here explores the structural and physicochemical features important for benzamide-based HDAC3 inhibitors to get an idea about the design aspect of potential inhibitors. A number of molecular modeling studies (3D-QSAR CoMFA and CoMSIA, Bayesian classification modeling) were performed on 113 diverse set of benzamide-based HDAC3 inhibitors. All these models developed are statistically reliable and correlate the SAR observations. Electron withdrawing substitution is favorable but the bulky hydrophobic group at the cap region reduces HDAC3 inhibition. Hydrophobicity and steric feature of the aryl linker function favor the activity. Aryl group substituted benzamide functionality is not favorable for HDAC3 inhibition. The amide function of the benzamide moiety is essential for Zn²⁺ chelation and the carboxylic acid function may serve as a hydrogen bond acceptor (HBA) feature. Moreover, electron withdrawing substituent at the benzamide moiety influences activity whereas steric and hydrophobic substituents reduce HDAC3 inhibition. Overall, this study may provide a valuable insight on the design of better active HDAC3 inhibitors in future.</p> <p>Communicated by Ramaswamy H. Sarma</p

Octa-arginine modified poly(amidoamine) dendrimers for improved delivery and cytotoxic effect of paclitaxel in cancer

<p>Cell penetrating peptides (CPP) have the ability to penetrate the cell membrane and have been associated with various cargos for their facile intracellular translocation. The current study involves the synthesis of a CPP, octa-arginine (R8)-modified poly(amidoamine) dendrimer of generation 4 (G4), which has additionally been PEGylated and conjugated to the poorly soluble anticancer drug, paclitaxel (PTX). The synthesized dendrimer conjugates were characterized by proton nuclear magnetic resonance (1H-NMR) Spectroscopy and zeta potential measurements and evaluated <i>in vitro</i> in cell monolayers and 3D spheroids. Cellular uptake study in human cervical cancer cell line (HeLa) revealed that R8 modification significantly improved the cell association of conjugates. G4-PTX- polyethylene glycol (PEG)-R8 conjugate demonstrated enhanced cytotoxic potential and higher induction of apoptosis compared to free PTX and G4-PTX-PEG. Further, the penetrability of fluorescently labeled F-G4-PTX-PEG-R8 was evaluated in 3D spheroids of HeLa at various depths by using confocal microscopy. G4-PTX-PEG-R8 induced cell death and inhibited the growth in 3D spheroids as competently as in monolayers. The enhanced intracellular translocation of R8-modified dendrimers resulted in improved anticancer efficacy of PTX. Therefore, the newly developed dendrimer system is efficient for the intracellular delivery of PTX in cancer cells and has a strong potential to be utilized as an effective chemotherapeutic agent for cancer.</p

BRAIN Journal - Suicide: Neurochemical Approaches

<div><i>Abstract</i></div><div><br></div><div>Despite the devastating effect of suicide on numerous lives, there is still a dearth of knowledge concerning its neurochemical aspects. There is increasing evidence that brain-derived neurotrophic factor (BDNF) and Nerve growth factor (NGF) are involved in the pathophysiology and treatment of depression through binding and activating their cognate receptors trk B and trk A respectively. The present study was performed to examine whether the expression profiles of BDNF and/or trk B as well as NGF and/or trk A were altered in postmortem brain in subjects who commit suicide and whether these alterations were associated with specific psychopathologic conditions. These studies were performed in hippocampus obtained 21 suicide subjects and 19 non-psychiatric control subjects. The protein and mRNA levels of BDNF, trk B and NGF, trk A were determined with Sandwich ELISA, Western Blot and RT PCR respectively. Given the importance of BDNF and NGF along with their cognate receptors in mediating physiological functions, including cell survival and synaptic plasticity, our findings of reduced expression of BDNF, Trk B and NGF, Trk A in both protein and mRNA levels of postmortem brain in suicide subjects suggest that these molecules may play an important role in the pathophysiological aspects of suicidal behavior.</div><div><br></div><div><b>Find more at:</b></div><div><b>https://www.edusoft.ro/brain/index.php/brain/article/view/425</b><br></div

Polylactide-Based Block Copolymeric Micelles Loaded with Chlorin e6 for Photodynamic Therapy: <i>In Vitro</i> Evaluation in Monolayer and 3D Spheroid Models

Recently, photodynamic therapy (PDT) has found wide application as a noninvasive treatment modality for several cancers. However, the suboptimal delivery of photosensitizers (PSs) to the tumor site is a drawback, which inhibits the effectiveness of PDT. Hydrophobicity, strong oxygen and light dependence, and limited tissue penetrability of photosensitizers represent the major barriers to the clinical application of PDT. In order to improve biopharmaceutical properties of a clinically approved photosensitizer chlorin e6 (Ce6), we developed a nanoformulation encapsulating Ce6 in methoxy-poly­(ethylene glycol)-poly­(d,l-lactide) (mPEG-PLA) copolymeric micelles. The physicochemical properties, including particle size, zeta potential, encapsulation efficiency, drug loading, generation of reactive oxygen species following near-infrared light illumination (633 nm), and <i>in vitro</i> drug release, were determined. The therapeutic efficacy of Ce6-mPEG-PLA micelles following illumination were evaluated <i>in vitro</i> in both two- and three-dimensional cell culture systems by using human uterine cervix carcinoma (HeLa) and human alveolar adenocarcinoma (A549) cells in monolayers and in A549 spheroids, respectively. The mPEG-PLA micelles were stable with a particle size of 189.6 ± 14.32 nm and loaded Ce6 efficiently (encapsulation efficiency ∼75%). The Ce6-loaded micelles generated singlet oxygen at a higher concentration compared to free Ce6 in aqueous media. Ce6-mPEG-PLA micelle mediated PDT showed improved cellular internalization in both of the cell lines, resulting in enhanced cytotoxicity compared to free Ce6. In contrast, the Ce6-loaded micelles did not show any cytotoxicity in the absence of irradiation. The Ce6-loaded micelles exhibited deep penetration in the spheroids leading to phototoxicity and cellular apoptosis in the A549 spheroidal model. Results from this study indicated that the newly developed nanoformulation of Ce6 could be utilized in PDT as an effective treatment modality for solid tumors

d‑α-Tocopheryl Succinate/Phosphatidyl Ethanolamine Conjugated Amphiphilic Polymer-Based Nanomicellar System for the Efficient Delivery of Curcumin and To Overcome Multiple Drug Resistance in Cancer

Nanomedicines have emerged as a promising treatment strategy for cancer. Multiple drug resistance due to overexpression of various drug efflux transporters and upregulation of apoptotic inhibitory pathways in cancer cells are major barriers that limit the success of chemotherapy. Here, we developed a d-α-tocopherol (α-TOS)/lipid-based copolymeric nanomicellar system (VPM) by conjugating phosphatidyl ethanolamine (PE) and α-TOS with poly­(ethylene glycol) (PEG) via an amino acid linkage. The synthesized polymers were characterized by Fourier transform IR, gas-phase chromatography, and ¹H and ¹³C NMR spectroscopy. VPM exhibited mean hydrodynamic diameter of 141.0 ± 0.94 nm with low critical micelles concentrations (CMC) of 15 μM compared to plain PEG–PE micelles (PPM) with size of 23.9 ± 0.34 nm and CMC 20 μM. The bigger hydrophobic compartment in VPM resulted in improved loading of a potent chemotherapeutic drug, curcumin (Cur), and increased encapsulation efficiency (EE) (% drug loading 98.3 ± 1.92, and 85.3 ± 3.29; EE 14.8 ± 0.16 and 12.8 ± 0.09 for VPM and PPM, respectively). Curcumin loaded Vitamin E based micelles exhibited higher cytotoxicity compared to Curcumin loaded PEG-PE micelles in tested cancer cell lines. C-VPM demonstrated ∼3.2 and ∼2.7-fold higher ability to reverse multiple drug resistance compared to PPM and verapamil (concentration used 30 μM), respectively. In the in vivo study by using B16F10 implanted C57Bl6/J mice, C-VPM reduced the tumor volume and weight more efficiently than C-PPM by inducing apoptosis as analyzed by TUNEL assay on tumor cryosections. The newly developed polymeric micelles, VPM with improved drug loadability and ability to reverse the drug resistance could successfully be utilized as a nanocarrier system for hydrophobic chemotherapeutic agents for the treatment of drug-resistant solid tumors

MPTAG restores PKA activity in TNFα-stimulated HUVECs.

<p>(A) The cells were treated with media (control), MPTAG, SQ 22536, H-89 or their indicated combinations in absence and presence of TNF-α stimulation. PKA activity was assessed in the cell lysates. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046528#s2" target="_blank">Results</a> are expressed as mean ± sem of three independent experiments. *p<0.05 vs. control. <b>Proposed model of MPTAG action in TNF-α-stimulated HUVECs.</b> (B) In TNF-α-stimulated HUVECs (without MPTAG treatment), PI-3K-regulated Akt is activated (indicated by solid line arrow) resulting in NF-κB activation. On the other hand, PKA activity remained repressed (indicated by broken line arrow) under these conditions. (C) MPTAG pretreatment to TNF-α-stimulated HUVECs restored the repressed activity of PKA. Thus, derepression of PKA activity resulted in inhibition of Akt and overall inhibition of NF-κB in these cells.</p

MPTAG inhibits the TNF-α-induced Akt activation and its association with IKK-β.

<p>(A) The cells were induced with TNF-α (10 ng/ml) for the indicated times. The total cell extracts were prepared and subjected to western blot analysis using anti-phosphoAkt, for both Ser473 and Thr308 residues, and anti-Akt antibodies. (B) The cells were treated with MPTAG (400 µM) and induced with TNF-α for 30 mins followed by western blot analysis as stated above. (C) The intensity of bands were densitometrically scanned and normalized with total Akt levels. The values presented are mean ± SEM. *p<0.05 vs. uninduced cells; **p<0.05 vs. TNF-α-induced cells, statistical significance was set at p<0.05. (D) The cells were induced with TNF-α (10 ng/ml) for the indicated times. The total cell extracts were prepared and immunoprecipitated with anti-IKK-β antibody followed by western blot analysis with anti-Akt and anti-IKK-β antibodies. (E) The cells were pretreated with MPTAG at varying concentrations and induced with TNF-α for 30 mins. The total cell extracts were prepared and processed as stated above and analyzed for western blot as stated above. (F) The intensity of bands were densitometrically scanned and normalized with IKK-β levels. (G) The cells were pretreated with 400 µM MPTAG and then stimulated with 10 ng/ml TNF-α for the indicated times. The total cell extracts were prepared, immunoprecipitated with anti-IKK-β antibody and analyzed by western blot using anti-Akt and anti-IKK-β antibodies. (H) <b>Effect of MPTAG on TNF-α-induced p38 MAPK and ERK1/2 activation.</b> The cells were treated with MPTAG and induced with TNF-α as stated above. The total cell extracts were prepared and analyzed by western blot using anti-phosphospecific p38 MAPK and ERK1/2 antibodies. The same membrane was reblotted with anti-p38 MAPK, ERK 1/2 and β-actin antibodies. The values presented are mean ± SEM. *p<0.05 vs. uninduced cells; **p<0.05 vs. TNF-α-induced cells, statistical significance was set at p<0.05.</p

MPTAG prevents the TNF-α-induced NF-κB transcription and activation in human endothelial cells.

<p>(A) The cells were pretreated with or without 400 µM of MPG before induction with TNF-α (10 ng/ml) for 4 h. The total RNA of the cells was isolated and analysed by RT-PCR. The intensity of transcripts was normalized with that of β-Actin levels expressed under similar conditions. (B) The cells were pretreated with MPTAG at varying concentrations and then induced with TNF-α. The cytoplasmic (CE) and nuclear (NE) extracts were prepared from untreated and MPTAG-treated TNF-α-stimulated cells (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046528#s4" target="_blank">Methods</a>”). The nuclear extracts were analyzed for NF-κB activation by EMSA. (C) The nuclear extracts from unstimulated or TNF-α-stimulated HUVECs were incubated with the indicated antibodies and analyzed for NF-κB activation by EMSA (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046528#s4" target="_blank">Methods</a>”). (D) The cells were transiently transfected by electroporation with a NF-κB-containing luciferase reporter gene followed by treatment with 400 µM MPTAG and TNF-α stimulation. The supernatants after cell lysis were assayed for luciferase activity. The mean value for cells treated with no MPTAG and no TNF-α was set to 1, and -fold differences were determined by comparing values against this set value. *p<0.005 vs. uninduced cells; **p<0.01 vs. TNF-α-induced cells, statistical difference was set at p<0.05.</p

Regulation of NF-κB Activation through a Novel PI-3K-Independent and PKA/Akt-Dependent Pathway in Human Umbilical Vein Endothelial Cells

<div><p>The transcription factor NF-κB regulates numerous inflammatory diseases, and proteins involved in the NF-κB-activating signaling pathway are important therapeutic targets. In human umbilical vein endothelial cells (HUVECs), TNF-α-induced IκBα degradation and p65/RelA phosphorylation regulate NF-κB activation. These are mediated by IKKs (IκB kinases) viz. IKKα, β and γ which receive activating signals from upstream kinases such as Akt. Akt is known to be positively regulated by PI-3K (phosphoinositide-3-kinase) and differentially regulated via Protein kinase A (PKA) in various cell types. However, the involvement of PKA/Akt cross talk in regulating NF-κB in HUVECs has not been explored yet. Here, we examined the involvement of PKA/Akt cross-talk in HUVECs using a novel compound, 2-methyl-pyran-4-one-3-O-β-D-2′,3′,4′,6′-tetra-O-acetyl glucopyranoside (MPTAG). We observed that MPTAG does not directly inhibit IKK-β but prevents TNF-α-induced activation of IKK-β by blocking its association with Akt and thereby inhibits NF-κB activation. Interestingly, our results also revealed that inhibitory effect of MPTAG on Akt and NF-κB activation was unaffected by wortmannin, and was completely abolished by H-89 treatment in these cells. Thus, MPTAG-mediated inhibition of TNF-α-induced Akt activation was independent of PI-3K and dependent on PKA. Most importantly, MPTAG restores the otherwise repressed activity of PKA and inhibits the TNF-α-induced Akt phosphorylation at both Thr308 and Ser473 residues. Thus, we demonstrate for the first time the involvement of PKA/Akt cross talk in NF-κB activation in HUVECs. Also, MPTAG could be useful as a lead molecule for developing potent therapeutic molecules for diseases where NF-κB activation plays a key role.</p> </div