Caterina Frezza

Characterization of the enhanced apoptotic response to azidothymidine by pharmacological inhibition of NF-kB.

AIMS The present study addresses the issue of enhanced apoptotic response to AZT following co-treatment with an NF-kB inhibitor. MAIN METHODS To investigate this issue, different cell lines were assayed for susceptibility to AZT-mediated apoptosis without or with the addition of the NF-kB inhibitor Bay-11-7085. For further investigation, U937 cells were selected as good-responder cells to the combination treatment with 32 or 128 μM AZT, and 1 μM Bay-11-7085. Inhibition of NF-kB activation by Bay-11-7085 in cells treated with AZT was assayed through Western blot analysis of p65 expression and by EMSA. Involvement of the mitochondrial pathway of apoptosis in mechanisms underlying the improved effect of AZT following Bay-11-7085 co-treatment, was evaluated by assaying the cytochrome c release and the mitochondrial membrane potential (MMP) status using the JC-1 dye. Moreover, the transcriptional activity of both anti- and pro-apoptotic genes in U937 cells after combination treatment was quantitatively evaluated through real-time PCR. KEY FINDINGS We found that the combined treatment induced high levels of cytochrome c release and of MMP collapse in association with evident changes in the expression of both anti- and pro-apoptotic genes of the Bcl-2 family. Overexpression of Bcl-2 significantly suppressed the sensitization of U937 cells to an enhanced apoptotic response to AZT following co-treatment with the NF-kB inhibitor. SIGNIFICANCE The new findings suggest that a combination regimen based on AZT plus an NF-kB inhibitor could represent a new chemotherapeutic tool for retrovirus-related pathologies.

Inflammation and Programmed Cell Death in Alzheimer’s Disease: Comparison of the Central Nervous System and Peripheral Blood

Although the central nervous system (CNS) has been defined as a privileged site in Alzheimer’s disease (AD), periphery can be more than simply witness of events leading to neurodegeneration. The CNS and peripheral blood can mutually communicate through cells and factors trafficking from the circulation into the brain and vice versa. A number of articles have reviewed inflammatory profiles and programmed cell death (PCD) in AD, separately in the CNS and at the peripheral level. This review does not provide an exhaustive account of what has been published on inflammation and PCD in AD. Rather, the aim of this review is to focus on possible linkages between the central and the peripheral compartments during AD progression, by critically analyzing, in a comparative manner, phenomena occurring in the CNS as well as the peripheral blood. In fact, growing evidence suggests that CNS and peripheral inflammation might present common features in the disease. Microarrays and metabolomics revealed that dysfunction of the glycolytic and oxidative pathways is similar in the brain and in the periphery. Moreover, dysregulated autophagosome/lysosomal molecular machinery, both at the CNS and the peripheral level, in AD-related cell damage, has been observed. Possible implications of these observations have been discussed.

A Novel, Cell-Free PCR-Based Assay for Evaluating the Inhibitory Activity of Antiretroviral Compounds Against HIV Reverse Transcriptase

This study describes a novel, PCR‐based assay that evaluates the ability of compounds to inhibit cDNA generation by HIV reverse transcriptase (RT), of both commercial and viral lysate origin, from a known RNA template. The template consisted of RNA from stable transfectants ectopically expressing the US6 gene of herpes simplex virus‐1, coding for glycoprotein D. Controls were carried out to demonstrate that no residual DNA polymerase activity or DNA contamination was responsible for the amplified DNA in the tested, control samples. In this assay, 0.1 μM nevirapine totally inhibited the RT activity of 0.5 U commercial HIV RT, while 10 nM inhibited it by only 10%. Conversely, 10 pM efavirenz completely inhibited 0.5 U HIV RT. Similar results were obtained when a self‐prepared viral lysate was used as a source of HIV RT. A reference commercial kit directly measuring HIV RT activity, without amplification, was less sensitive than the new assay. As a consequence, the HIV RT 50% inhibitory concentration of nevirapine and efavirenz in the newly described assay was 8 and 5 × 103 times lower, respectively, than in the commercial assay. In conclusion, this novel method was sensitive, reproducible, and sufficiently rapid for screening in vitro the functional activity of known or potential antiretroviral compounds against HIV RT. J. Med. Virol. 86:1–7, 2014.

Downregulation of proinflammatory cytokines in HTLV-1-infected T cells by Resveratrol.

BackgroundHuman T-cell leukemia virus (HTLV-1) is a lymphotropic retrovirus associated to adult T cell leukemia (ATL) and to non-neoplastic inflammatory conditions affecting the central nervous system, lung or skin. The inflammatory disorders associated to HTLV-1 are mediated by different proinflammatory cytokines as IL-1α, IL-6, TNF-α. The release and the role of IL-17 is still debated. Aims of this study were to analyze IL-17 induction by HTLV-1 infection and to determine whether resveratrol (RES) is able to down regulate the pathway of cytokines production either in HTLV-1 chronically infected MT-2 cell line or in human CD4+ cells infected in vitro with HTLV-1.MethodsMT-2 and HTLV-1 infected CD4+ cells were analyzed for proinflammatory cytokine production before or after RES treatment. The concentrations of IL-17, IL-1α, IL-6, and TNF-α were measured in cell culture supernatants by ELISA and SearchLight™ technology. The IL-17 mRNA expression was evaluated by RT-PCR. NF-kB activation was detected by non-radioactive, Electro Mobility Shift Assay (EMSA). HTLV-1 RNA expression was detected by Real-time-PCR (RQ-PCR).ResultsWe found that RES is capable of inducing a dose-dependent inhibition of IL-1α, IL-6 and TNF-α production in vitro and can down regulate the expression of IL-17 at both mRNA and protein levels in HTLV-1 infected cells. This effect was associated with a dose-dependent inhibition of the of the nuclear factor kappa-B (NF-kB) activity. Conversely, RES did not apparently affect HTLV-1 proliferation.ConclusionsThese results support the anti-inflammatory properties of RES, suggesting that it might be a useful therapeutic agent for the treatment of HTLV-1 related inflammatory diseases.

Phosphonated Nucleoside Analogues as Antiviral Agents

The present review is focused on the description of synthesis and antiviral activities of both acyclic and carbocyclic nucleoside phosphonates, endowed with an antiviral potential. Despite the outstanding results in antiviral therapy of acyclovir and azidothymidine, a major drawback concerning the use of nucleoside analogues (NA) is the retention of their stability following triphosphorylation within the host cell. The instability of the phosphate forms of NA has been, at least partially, overcome by the introduction of phosphate groups in the molecular structure. This approach gives rise to two main classes of compounds endowed with ascertained or potential antiviral activity, such as acyclic nucleoside phosphonates (ANP) and phosphonated carbocyclic nucleosides (PCN). Regarding ANP, a higher affinity for HIV reverse transcriptase (RT), with respect to NA, and the potent inhibition of HIV and hepatitis B virus (HBV) have been reported for some of them. Regarding PCN, some phosphonated cyclopropyl and cyclopentyl carbanucleosides, characterized by the presence of one or more phosphonic groups and by replacement of the endocyclic oxygen atom with a methylene group, showed to be good inhibitors of HBV and HIV infection. Another class of PCN is represented by phosphonated N,O-nucleosides (PCOAN). PCOAN encompass homo phosphonated-, phosphonated- and truncated phosphonated-N,O-nucleosides. Some PCOAN have been shown to directly inhibit RT activity of both murine and human retroviruses and to block HTLV-1 infection in vitro. The flexibility of the phosphonated NA structure suggests the possibility to develop new analogues endowed with antiviral activity towards a broad range of DNA or RNA viruses.

Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3

An assay, specifically optimized to evaluate the anti‐HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti‐HIV agents, zidovudine (AZT), abacavir (ABC), 2′,3′‐dideoxyinosine (DDI), lamivudine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegravir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV‐1 NL4‐3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen‐capture assay. For infection, CEM‐GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37°C before HIV‐1 NL4‐3 was added to each sample. The adsorption was prolonged for 3 hr at 37°C. After 72 hr of incubation, HIV‐induced GFP expression in infected CEM‐GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC50 values, the anti‐HIV activity, as assayed by this method, was EFV > 3TC > AZT > NVP > DDI > ABC and ETR > RPV > RAL. The comparison between the IC50 values calculated through flow cytometry and p24 production revealed overlapping results, showing that the optimized protocol of CEM‐GFP infection with HIV NL4‐3 is a suitable method to perform quantitative, rapid and low‐expensive screening tests to evaluate the in vitro effect of new candidate anti‐HIV drugs. J. Med. Virol. 88:979–986, 2016.

Synthesis of potential HIV integrase inhibitors inspired by natural polyphenol structures

Abstract Drawing inspiration from the structural features of some natural polyphenols, the synthesis of two different model compounds as potential inhibitors of HIV integrase (IN) has been described. The former was characterised by a diketo acid (DKA) bioisostere, such as a β-hydroxycarbonyl moiety, between two fragments containing aromatic groups, while in the latter an epoxide linked two polyoxygenated aromatic residues. The moieties present in the structures are thought to function by chelating divalent metal ions on the enzyme catalytic site. Overall, 10 compounds were prepared and some of that submitted to molecular modelling studies (to investigate their interactions with the active site of IN), to metal titration studies (to detect their chelating capability) and to biological assays.

Synthesis, molecular modeling and biological evaluation of two new chicoric acid analogs

Abstract Two conformationally constrained compounds similar to chicoric acid but lacking the catechol and carboxyl groups were prepared. In these analogues, the single bond between the two caffeoyl fragments has been replaced with a chiral oxirane ring and both aromatic residues modified protecting completely or partially the catechol moiety as methyl ether. Preliminary molecular modelling studies carried out on the two analogues showed interactions near the active site of HIV integrase; however, in comparison with raltegravir, the biological evaluation confirmed that CAA-1 and CAA-2 were unable to inhibit infection at lower concentration.

Synthesis and Biological Evaluation of Styrylheterocycles Analogs of Resveratrol as Apoptosis-Inducing Agents

Background: Apoptosis is a route of cell death induced by a highly regulated intracellular program. An increase in apoptotic activity could lead to neurodegenerative pathologies, whereas a decrease in apoptotic activity could lead to uncontrolled cellular proliferation. Objective: The aim of this research was to synthesize ten styrylheterocycle compounds, analogs of resveratrol, having a thiophene ring substituted in position 2- or 3- and a benzene ring with one or two hydroxy and methoxy groups. The compounds were evaluated for their cytotoxicity and apoptotic activity against the U-937 cancer cell line. Method: The experiments were conducted at 1000 µM, 250 µM, 100 µM, and 50 µM in order to measure apoptotic activity (% hypodiploid nuclei). The cytotoxicity was expressed as the concentration of a substance able to inhibit 50% of the metabolic activity in an MTS assay (maCC50), and as the percentage of cellular death (% Death Cell.) at 1000 µM and 100 µM. Results: Changing the phenyl group in a thienyl group occupying the 2- or 3-position almost always leads to an improvement in apoptosis. In addition, whereas the presence of a hydroxy group makes the molecules more cytotoxic than resveratrol, it also improves apoptosis. However, the presence of a methoxy group in the phenyl ring leads to collapse of the cytotoxicity, thereby allowing an increase in the analytical concentrations, and verifying that, at 1000 µM, the compounds 3b and 3c show apoptosis levels of 81% and 72%, respectively. Conclusion: We look forward to synthesizing other heterocyclic molecules to find even more active derivatives endowed with anti-cancer potential.

Simple and efficient synthesis of benzofuran derivatives from tyrosol

ABSTRACT A convenient strategy for the preparation of compounds bearing the benzofuran skeleton starting from tyrosol, a phenol largely present in olive oil production waste, with no biological importance, is reported. A bromination/methoxylation sequence, already described for the synthetic transformation of naturally occurring compounds, was exploited. Depending on the solvent used for the methoxylation reaction together with the presence of a 4-phenol moiety respect to the side chain, benzodihydrofurans or a benzofurans derivative can be obtained. GRAPHICAL ABSTRACT