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[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (1) is a potent inhibitor of GSTP1-1, a glutathione S-transferase capable of inhibiting apoptosis by binding to JNK1 and TRAF2. We recently demonstrated that, unlike its parent compound, the benzoyl ester of 1 (compound 3) exhibits negligible reactivity towards GSH, and has a different mode of interaction with GSTP1-1. Unfortunately, 3 is susceptible to rapid metabolic hydrolysis. In an effort to improve the metabolic stability of 3, its ester group has been replaced by an amide, leading to N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexyl)benzamide (4). Unlike 3, compound 4 was stable to human liver microsomal carboxylesterases, but retained the ability to disrupt the interaction between GSTP1-1 and TRAF2 regardless of GSH levels. Moreover, 4 exhibited both a higher stability in the presence of GSH and a greater cytotoxicity towards cultured A375 melanoma cells, in comparison with 1 and its analog 2. These findings suggest that 4 deserves further preclinical testing

Human metapneumovirus establishes persistent infection in lung microvascular endothelial cells and primes a th2-skewed immune response

Human metapneumovirus (HMPV) is a major cause of lower respiratory tract infections. HMPV infection has been hypothesized to alter dendritic cell (DC) immune response; however, many questions regarding HMPV pathogenesis within the infected lung remain unanswered. Here, we show that HMPV productively infects human lung microvascular endothelial cells (L-HMVECs). The release of infectious virus occurs for up to more than 30 days of culture without producing overt cytopathic effects and medium derived from persistently HMPV-infected L-HMVECs (secretome) induced monocyte-derived DCs to prime naïve CD4 T-cells toward a Th2 phenotype. Moreover, we demonstrated that infected secretomes trigger DCs to up-regulate OX40L expression and OX40L neutralization abolished the pro-Th2 effect that is induced by HMPV-secretome. We clarified secretome from HMPV by size exclusion and ultracentrifugation with the aim to characterize the role of viral particles in the observed pro-Th2 effect. In both cases, the percentage of IL-4-producing cells and expression of OX40L returned at basal levels. Finally, we showed that HMPV, per se, could reproduce the ability of secretome to prime pro-Th2 DCs. These results suggest that HMPV, persistently released by L-HMVECs, might take part in the development of a skewed, pro-Th2 lung microenvironment

In vitro and in vivo efficacy of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on human melanoma

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is a powerful inhibitor of the glutathione transferase P1-1 (GSTP1-1) and causes the disruption of the complex between GSTP1-1 and c-Jun N-terminal Kinase (JNK). This induces JNK activation and apoptosis in tumour cells. in the present work we assess the in vitro and in vivo effectiveness of NBDHEX on two human melanoma cell lines, Me501 and A375. NBDHEX shows IC50 values in the low micromolar range (IC50 of 1.2 +/- 0.1 mu M and 2.0 +/- 0.2 mu M for Me501 and A375, respectively) and is over 100 times more cytotoxic to these cell lines than temozolomide. Apoptosis is observed in Me501 cells within 3 h of the addition of NBDHEX, while in A375 cells the apoptotic event is rather late, and is preceded by a G2/M phase arrest. In both melanoma cell lines, INK activity is required for the ability of NBDHEX to trigger apoptosis, confirming that the JNK pathway is an important therapeutic target for this tumour. NBDHEX is also both effective and well tolerated in in vivo tumour models. A tumour inhibition of 70% is observed in vivo against Me501 human melanoma and a similar result is obtained on A375 model, with 63% of turnout inhibition. These findings indicate that the activation of the JNK pathway, through a selective GSTP1-1 targeting, could prove to be a promising new strategy for treating melanoma, which responds poorly to conventional therapies. (C) 2009 Elsevier Ltd. All rights reserved

Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin: IDENTIFICATION OF THE HEPARIN-BINDING MOTIF OF p17 AS A TARGET FOR THE DEVELOPMENT OF MULTITARGET ANTAGONISTS

Once released by HIV cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (Kd 190 nM) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg3Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highlyN,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists

Proapoptotic activity of new glutathione S-transferase inhibitors

Selected 7-nitro-2,1,3-benzoxadiazole derivatives have been recently found very efficient inhibitors of glutathione S-transferase (GST) PI-1,(5) an enzyme which displays antiapoptotic activity and is also involved in the cellular resistance to anticancer drugs. These new inhibitors are not tripeptide glutathione-peptidomimetic molecules and display lipophylic properties suitable for crossing the plasma membrane. In the present work, we show the strong cytotoxic activity of these compounds in the following four different cell lines: K562 (human myeloid leukemia), HepG2 (human hepatic carcinoma), CCRF-CEM (human T-lymphoblastic leukemia), and GLC-4 (human small cell lung carcinoma). The LC50 values are in the micromolar/submicromolar range and are close to the ICs values obtained with GSTPI-1, suggesting that the target of these molecules inside the cell is indeed this enzyme. The cytotoxic mechanism of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, the most effective GSTPI-1 inhibitor, has been carefully investigated in leukemic CCRF-CEM and K562 cell lines. Western blot and immunoprecipitation analyzes have shown that 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanoI promotes in both cell lines the dissociation of the GSTPI-1 in a complex with c-jun NH2-terminal kinase (JNK). This process triggers a reactive oxygen species (ROS) -independent activation of the JNK-mediated pathway that results in a typical process of apoptosis. Besides this main pathway, in K562 cells, a ROS-mediated apoptosis partially occurs (about 30%) which involves the p38(MAPK) signal transduction pathway. The low concentration of this new compound needed to trigger cytotoxic effects on tumor cells and the low toxicity on mice indicate that the new 7-nitro-2,1,3-benzoxadiazole derivatives are promising anticancer agents

A strong glutathione S-transferase inhibitor overcomes the P-glycoprotein-mediated resistance in tumor cells - 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) triggers a caspase-dependent apoptosis in MDR1-expressing leukemia cells

The new glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol- 4-ylthio)hexanol (NBDHEX) is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines, i.e. CEM-VBL10, CEM-VBL100, and U-2 OS/DX580. The mechanism of cell death triggered by NBDHEX has been deeply investigated in leukemia cell lines. Kinetic data indicate a similar NBDHEX membrane permeability between multidrug resistance cells and their sensitive counterpart revealing that NBDHEX is not a substrate of the P-glycoprotein export pump. Unexpectedly, this molecule promotes a caspase-dependent apoptosis that is unusual in the P-glycoprotein-overexpressing cells. The primary event of the apoptotic pathway is the dissociation of glutathione S-transferase P1-1 from the complex with c-Jun N-terminal kinase. Interestingly, leukemia MDR1-expressing cells show lower LC50 values and a higher degree of apoptosis and caspase-3 activity than their drug-sensitive counterparts. The increased susceptibility of the multidrug resistance cells toward the NBDHEX action may be related to a lower content of glutathione S-transferase P1-1. Given the low toxicity of NBDHEX in vivo, this compound may represent an attractive basis for the selective treatment of MDR1 P-glycoproteinpositive tumors

Inhibition of DNA Repair Mechanisms and Induction of Apoptosis in Triple Negative Breast Cancer Cells Expressing the Human Herpesvirus 6 U94

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers. In spite of initial good response to chemotherapy, the prognosis of TNBC remains poor and no effective specific targeted therapy is readily available. Recently, we demonstrated the ability of U94, the latency gene of human herpes virus 6 (HHV-6), to interfere with proliferation and with crucial steps of the metastatic cascade by using MDA-MB 231 TNBC breast cancer cell line. U94 expression was also associated with a partial mesenchymal-to-epithelial transition (MET) of cells, which displayed a less aggressive phenotype. In this study, we show the ability of U94 to exert its anticancer activity on three different TNBC cell lines by inhibiting DNA damage repair genes, cell cycle and eventually leading to cell death following activation of the intrinsic apoptotic pathway. Interestingly, we found that U94 acted synergistically with DNA-damaging drugs. Overall, we provide evidence that U94 is able to combat tumor cells with different mechanisms, thus attesting for the great potential of this molecule as a multi-target drug in cancer therapy

Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and β, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin

p17 from HIV induces brain endothelial cell angiogenesis through EGFR-1-mediated cell signalling activation

© 2018, United States & Canadian Academy of Pathology. HIV-associated neurocognitive disorder in HIV patients substantially reduces their quality of life. We previously showed that the HIV matrix protein, p17 could stimulate lymph-angiogenesis in vitro potentially contributing to lymphoma tumour growth and in addition is associated with vascular activation in neuro-degenerating brain tissue; here, therefore, we have investigated the detailed molecular mechanisms of this action. We performed in vitro cell culture, angiogenesis experiments, phospho-protein microarrays and Western blotting to identify cellular signalling induced by p17 within human brain endothelial cells (HbMEC), and inhibitor studies to block p17-induced vascular growth. We also characterised the effects of hippocampal CA1 injection of p17 on epidermal growth factor receptor-1 (EGFR1) expression linked to our murine model of dementia. p17 strongly induced angiogenesis of HbMEC (migration, tube formation and spheroid growth). p17 concomitantly increased phosphorylation of EGFR1 as well as down-stream intermediates ERK1/2, FAK, PLC-γ and PKC-β whilst an inhibitor peptide of EGFR, blocked cell signalling and angiogenesis. Finally, Mice that showed reduced cognitive function and behavioural deficiencies after p17 injection, demonstrated that p17 localised in cortical microvessels and also neurones many of which stained positive for p-EGFR1 by histology/IHC. This work provides strong support that p17 may be involved in initiating and/or perpetuating vascular tissue pathophysiology associated with comorbidity in HIV patients

HIV-1 matrix protein p17 misfolding forms toxic amyloidogenic assemblies that induce neurocognitive disorders

© 2017 The Author(s). Human immunodeficiency virus type-1 (HIV-1)-Associated neurocognitive disorder (HAND) remains an important neurological manifestation that adversely affects a patient's quality of life. HIV-1 matrix protein p17 (p17) has been detected in autoptic brain tissue of HAND individuals who presented early with severe AIDS encephalopathy. We hypothesised that the ability of p17 to misfold may result in the generation of toxic assemblies in the brain and may be relevant for HAND pathogenesis. A multidisciplinary integrated approach has been applied to determine the ability of p17 to form soluble amyloidogenic assemblies in vitro. To provide new information into the potential pathogenic role of soluble p17 species in HAND, their toxicological capability was evaluated in vivo. In C. elegans, capable of recognising toxic assemblies of amyloidogenic proteins, p17 induces a specific toxic effect which can be counteracted by tetracyclines, drugs able to hinder the formation of large oligomers and consequently amyloid fibrils. The intrahippocampal injection of p17 in mice reduces their cognitive function and induces behavioral deficiencies. These findings offer a new way of thinking about the possible cause of neurodegeneration in HIV-1-seropositive patients, which engages the ability of p17 to form soluble toxic assemblies