A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucose.

Background: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult.

Results: By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis.

The Nitrobenzoxadiazole Derivative NBDHEX Behaves as Gametocyte Selective Inhibitor with Malaria Parasite Transmission Blocking Activity.

This work describes the activity of 6-((7-nitrobenzo[][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and of its newly identified carboxylic acid metabolite on the human malaria parasite . NBDHEX has been previously identified as a potent cytotoxic agent against murine and human cancer cells as well as towards the protozoan parasite . We show here that NBDHEX is active in vitro against all blood stages of , with the rare feature of killing the parasite stages transmissible to mosquitoes, the gametocytes, with a 4-fold higher potency than that on the pathogenic asexual stages.

Anti-influenza A virus activity and structure-activity relationship of a series of nitrobenzoxadiazole derivatives.

Influenza viruses represent a major threat to human health and are responsible for seasonal epidemics, along with pandemics. Currently, few therapeutic options are available, with most drugs being at risk of the insurgence of resistant strains. Hence, novel approaches targeting less explored pathways are urgently needed.

The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor.

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation.

Characterization of water-soluble esters of nitrobenzoxadiazole-based GSTP1-1 inhibitors for cancer treatment.

The 7-nitrobenzo[c][1,2,5]oxadiazole (NBD) derivative NBDHEX (compound 1) and its analogue MC3181 (compound 2) have been found to be potent inhibitors of tumor cell growth in vitro and therapeutically active and safe in mice bearing human melanoma xenografts. To enhance the aqueous solubility of these compounds, we synthesized the hemisuccinate of 1 (compound 3) and the phosphate monoesters of 1 and 2 (compound 4 and 5, respectively). These novel NBD derivatives displayed a solubility in the conventional phosphate-buffered saline up to 150-fold higher than that of 1, and up to 4-fold higher than that of 2.

Non-symmetrical structural behavior of a symmetric protein: the case of homo-trimeric TRAF2 (tumor necrosis factor-receptor associated factor 2).

The oligomeric state of TRAF2 (tumor necrosis factor-receptor associated factor 2), a TNF (tumor necrosis factor) receptor-associated factor, is crucial for membrane binding and probably plays a fundamental role in regulating the protein function in vivo. In this study we have combined molecular dynamics with the protein contact network approach to characterize the interaction of the three identical subunits of TRAF2. The average structure obtained after a 225 ns simulation reveals that two clusters of different size are formed, one of which includes almost completely two subunits, while the third monomer appears to be more independent.

Effects of Glutathione Transferase-Targeting Nitrobenzoxadiazole Compounds in Relation to PD-L1 Status in Human Melanoma Cells.

Background: PD-L1 is a membrane protein with inhibitory effects on immune responses, whose expression has been correlated with high aggressiveness and the propensity of melanoma to metastasize. The nitrobenzoxadiazole (NBD) NBDHEX and its analog MC3181 are endowed with strong antitumor activity towards melanoma and a significant ability to reduce its adhesion and invasiveness. Therefore, we investigated whether PD-L1 status could affect cell sensitivity to the cytotoxic effects of NBDs.

Activation of retinal Müller cells in response to glucose variability.

Purpose: In the earliest stages of diabetic retinopathy (DR), a dysfunction of Müller cells, characterized by high levels of glial fibrillary acidic protein (GFAP), and aquaporins (AQP), has been observed. Although chronic hyperglycemia causes the activation of Müller cells, the effect of glycemic fluctuations is yet unknown. The aim of the study was to analyze the impact of glucose variability on rat retinal Müller cells (rMC-1) adapted to either normal (5 mM) or high (25 mM) glucose levels.

Synthesis and characterisation of a new benzamide-containing nitrobenzoxadiazole as a GSTP1-1 inhibitor endowed with high stability to metabolic hydrolysis.

The antitumor agent 6-((7-nitrobenzo[][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol () is a potent inhibitor of GSTP1-1, a glutathione -transferase capable of inhibiting apoptosis by binding to JNK1 and TRAF2. We recently demonstrated that, unlike its parent compound, the benzoyl ester of (compound ) exhibits negligible reactivity towards GSH, and has a different mode of interaction with GSTP1-1. Unfortunately, is susceptible to rapid metabolic hydrolysis.

Glutathione transferase P silencing promotes neuronal differentiation of retinal R28 cells.

Glutathione transferases (GSTs) play an important role in retinal pathophysiology. Within this family, the GSTP isoform is known as an endogenous regulator of cell survival and proliferation pathways and of cellular responses to oxidative stress. In the present study we silenced GSTP in R28 cells, a retinal precursor cell line with markers of both glial and neuronal origin, and obtained stable clones which were viable and, unexpectedly, characterized by a more neuronal phenotype.

Studying the TRAF2 binding to model membranes: The role of subunits dissociation.

The ability of a C-terminal truncated form of TRAF2 to bind synthetic vesicles has been quantitatively studied by steady-state fluorescence energy transfer from the protein to large unilamellar vesicles (LUVs) prepared with different lipid mixtures. The dissociation constants, the free energy of binding, and the average number of phospholipids interacting with truncated TRAF2 have been evaluated from the corresponding binding curves. The results indicate that the protein strongly interacts with the lipid bilayer, preferentially in the monomeric state.

New insight into the interaction of TRAF2 C-terminal domain with lipid raft microdomains.

In this study we provide the first evidence of the interaction of a truncated-TRAF2 with lipid raft microdomains. We have analyzed this interaction by measuring the diffusion coefficient of the protein in large and giant unilamellar vesicles (LUVs and GUVs, respectively) obtained both from synthetic lipid mixtures and from natural extracts. Steady-state fluorescence measurements performed with synthetic vesicles indicate that this truncated form of TRAF2 displays a tighter binding to raft-like LUVs with respect to the control (POPC-containing LUVs), and that this process depends on the protein oligomeric state.

The nitrobenzoxadiazole derivative MC3181 blocks melanoma invasion and metastasis.

The novel nitrobenzoxadiazole (NBD) derivative MC3181 is endowed with remarkable therapeutic activity in mice bearing both sensitive and vemurafenib-resistant human melanoma xenografts. Here, we report that subtoxic concentrations of this compound significantly reduced invasiveness of BRAF-V600D mutated WM115 and WM266.4 melanoma cell lines derived from the primary lesion and related skin metastasis of the same patient, respectively.

A new nitrobenzoxadiazole-based GSTP1-1 inhibitor with a previously unheard of mechanism of action and high stability.

Context: The nitrobezoxadiazole derivative NBDHEX is a potent inhibitor of glutathione transferase P1-1 (GSTP1-1) endowed with outstanding anticancer activity in different tumor models.

Objective: To characterize by in vitro biochemical and in silico studies the NBDHEX analogues named MC2752 and MC2753.

Materials And Methods: Synthesis of MC2752 and MC2753, biochemical assays and in silico docking and normal-mode analyses.

Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function.

Negative cooperativity in enzyme reactions, in which the first event makes subsequent events less favorable, is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear.

Thiol-Activated Anticancer Agents: The State of the Art.

The thiol or sulfhydryl group, as part of low molecular weight non-peptide biomolecules, as well as part of the cysteine residues in peptides and proteins, is known to play extremely important roles in several aspects of cellular function. Glutathione (γ-Glu-Cys-Gly; GSH) is the most abundant thiol-containing peptide in mammals, being present intracellularly in the low millimolar concentration range, but only in the low micromolar concentration range in the majority of extracellular fluids. Notably, intracellular levels of GSH have been found to be significantly upregulated in a number of human cancers, a phenomenon thought to contribute, in concert with overexpression of some GSHassociated enzymes, to the development of tumor cell chemo- and radioresistance.

c-Jun N-terminal kinase activation by nitrobenzoxadiazoles leads to late-stage autophagy inhibition.

Background: Nitrobenzoxadiazole derivatives (NBDs), including NBDHEX and the recently developed MC3181, are promising anticancer agents able to target glutathione transferase and inhibit both its catalytic activity and ability to sequester TNF-receptor associated factor 2 (TRAF2) and c-Jun N-terminal kinase (JNK). NBDs have been shown to impair the growth and survival of a broad-spectrum of tumor types, in vitro and in vivo. Herein, we evaluated the effects of the new compound MC3181 on U-2OS osteosarcoma cells and investigated the impact of both NBDHEX and MC3181 on autophagy.

TNFR-Associated Factor-2 (TRAF2): Not Only a Trimer.

TNF receptor-associated factors (TRAFs) are characterized by an oligomeric structure that plays a fundamental role in the binding process with membrane receptors. In this work, we studied the trimer-to-monomer (T ↔ 3M) equilibrium transition of the TRAF2 C-terminal domain using both chemical (dilution/guanidinium hydrochloride) and mechanical stress (high pressure) to induce the dissociation of the native protein into subunits. The experimental results and computer simulations indicate that stable monomers exist and that their population accounts for 15% of the total TRAF2 molecules already at a physiological intracellular concentration (≈1 μM), being instead the predominant species in the nanomolar concentration range.

Nitrobenzoxadiazole-based GSTP1-1 inhibitors containing the full peptidyl moiety of (pseudo)glutathione.

Context: The inhibition of glutathione S-transferase P1-1 (GSTP1-1) is a sound strategy to overcome drug resistance in oncology practice.

Objective: The nitrobenzoxadiazolyl (NBD) S-conjugate of glutathione and the corresponding γ-oxa-glutamyl isostere (compounds 1 and 5, respectively) have been disclosed as GST inhibitors. The rationale of their design is discussed in juxtaposition to non-peptide NBD thioethers.

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib.

A novel orally active water-soluble inhibitor of human glutathione transferase exerts a potent and selective antitumor activity against human melanoma xenografts.

We designed and synthesized two novel nitrobenzoxadiazole (NBD) analogues of the anticancer agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX). The new compounds, namely MC3165 and MC3181, bear one and two oxygen atoms within the hydroxy-containing alkyl chain at the C4 position of the NBD scaffold, respectively. This insertion did not alter the chemical reactivity with reduced glutathione, while it conferred a remarkable increase in water solubility.

Glutathione S-transferase P1-1 as a target for mesothelioma treatment.

Malignant pleural mesothelioma is a poorly responsive tumor known to overexpress the phase II detoxification enzyme glutathione-S-transferase, which catalyzes the conjugation between glutathione and platinum(II)-containing drugs. Therefore, we evaluated the effect of the strong glutathione S-transferase inhibitor NBDHEX on human mesothelioma cell lines (MSTO-211H, MPP89, MM-B1 and Mero 48a) featuring the most common mesothelioma phenotypes: epithelioid and biphasic. Even though a different response to NBDHEX was observed, the molecule was very effective on all cell lines tested, triggering a sustained activation of both JNK and p38, followed by caspase activation and apoptosis.