Publications by authors named "Alessandro Desideri"

161 Publications

Ruthenium(II) Diphosphine Complexes with Mercapto Ligands That Inhibit Topoisomerase IB and Suppress Tumor Growth In Vivo.

Inorg Chem 2021 Sep 3. Epub 2021 Sep 3.

Departamento de Química, Universidade Federal de São Carlos, CP 676, CEP 13565-905 São Carlos, São Paulo, Brazil.

Ruthenium(II) complexes (-), with the general formula [Ru(N-S)(dppe)]PF, bearing two 1,2-(diphenylphosphino)ethane (dppe) ligands and a series of mercapto ligands (N-S), have been developed. The combination of these ligands in the complexes endowed hydrophobic species with high cytotoxic activity against five cancer cell lines. For the A549 (lung) and MDA-MB-231 (breast) cancer cell lines, the IC values of the complexes were 288- to 14-fold lower when compared to cisplatin. Furthermore, the complexes were selective for the A549 and MDA-MB-231 cancer cell lines compared to the MRC-5 nontumor cell line. The multitarget character of the complexes was investigated by using DNA (CT DNA), human serum albumin, and human topoisomerase IB (hTopIB). The complexes potently inhibited hTopIB. In particular, complex [Ru(dmp)(dppe)]PF (), bearing the 4,6-diamino-2-mercaptopyrimidine (dmp) ligand, effectively inhibited hTopIB by acting on both the cleavage and religation steps of the catalytic cycle of this enzyme. Molecular docking showed that the - complexes have binding affinity by active sites on the hTopI and hTopI-DNA, mainly via π-alkyl and alkyl hydrophobic interactions, as well as through hydrogen bonds. Complex displayed significant antitumor activity against murine melanoma in mouse xenograph models, but this complex did not damage DNA, as revealed by Ames and micronucleus tests.
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http://dx.doi.org/10.1021/acs.inorgchem.1c01539DOI Listing
September 2021

GATA6 deficiency leads to epithelial barrier dysfunction and enhances susceptibility to gut inflammation.

J Crohns Colitis 2021 Aug 10. Epub 2021 Aug 10.

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.

Background & Aims: Intestinal barrier dysfunction is a hallmark of inflammatory bowel diseases (IBD), but the mechanisms that lead to such a defect are not fully understood. This study was aimed at characterizing the factors involved in the defective barrier function in IBD.

Methods: Transcriptome analysis was performed on colon samples taken from healthy controls (CTR) and IBD patients. Expression of GATA-binding factor 6 (GATA6), a transcription factor involved in intestinal epithelial cell differentiation, was evaluated in colon samples taken from CTR and IBD patients by real-time PCR and immunohistochemistry. Intestinal sections of wild-type and Gata6del mice, which exhibit a conditional Gata6 deletion in intestinal epithelial cells, either left untreated or receiving subcutaneous indomethacin or rectal trinitrobenzene sulfonic acid, were stained with hematoxylin and eosin. In parallel, some Gata6del mice received antibiotics to deplete intestinal flora. Mucosal inflammatory cell infiltration and cytokine production were evaluated by flow cytometry and real-time PCR respectively while tight junction proteins were examined by immunofluorescence. Intestinal barrier integrity was assessed by FITC-dextran assay.

Results: Multiple genes involved in cell commitment/proliferation and wound healing were differentially expressed in IBD compared to CTR. Among these, GATA6 was significantly decreased in the IBD epithelium compared to CTR. In mice, conditional deletion of GATA6 in the intestinal epithelium induced primarily epithelial damage, diminished Zonula Occludens-1 expression and enhanced intestinal permeability, ultimately resulting in bacteria-driven local immune response and enhanced susceptibility to gut inflammation.

Conclusions: Reduced expression of GATA6 promotes intestinal barrier dysfunction thus amplifying intestinal inflammatory pathology.
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http://dx.doi.org/10.1093/ecco-jcc/jjab145DOI Listing
August 2021

In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB.

Int J Mol Sci 2021 Jul 12;22(14). Epub 2021 Jul 12.

Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy.

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.
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http://dx.doi.org/10.3390/ijms22147455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8306514PMC
July 2021

Natural Compounds as Therapeutic Agents: The Case of Human Topoisomerase IB.

Int J Mol Sci 2021 Apr 16;22(8). Epub 2021 Apr 16.

Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, 00133 Rome, Italy.

Natural products are widely used as source for drugs development. An interesting example is represented by natural drugs developed against human topoisomerase IB, a ubiquitous enzyme involved in many cellular processes where several topological problems occur due the formation of supercoiled DNA. Human topoisomerase IB, involved in the solution of such problems relaxing the DNA cleaving and religating a single DNA strand, represents an important target in anticancer therapy. Several natural compounds inhibiting or poisoning this enzyme are under investigation as possible new drugs. This review summarizes the natural products that target human topoisomerase IB that may be used as the lead compounds to develop new anticancer drugs. Moreover, the natural compounds and their derivatives that are in clinical trial are also commented on.
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http://dx.doi.org/10.3390/ijms22084138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073192PMC
April 2021

Plant microRNAs from Regulate Immune Response and HIV Infection.

Front Pharmacol 2020 11;11:620038. Epub 2021 Feb 11.

Department of Biology, University of Rome "Tor Vergata", Rome, Italy.

Traditional medicine is often chosen due to its affordability, its familiarity with patient's cultural practices, and its wider access to the local community. Plants play an important role in providing indispensable nutrients, while specific small RNAs can regulate human gene expression in a cross-kingdom manner. The aim of the study was to evaluate the effects of plant-enriched purified extract microRNAs from seeds (MO) on the immune response and on HIV infection. Bioinformatic analysis shows that plant microRNAs (-miRs) from MO belonging to 18 conserved families, including miR160h, -miR166, miR482b, miR159c, miR395d, miR2118a, miR393a, miR167f-3p, and miR858b are predicted to target with high affinity BCL2, IL2RA, TNF, and VAV1, all these being involved in the cell cycle, apoptosis, immune response and also in the regulation of HIV pathogenesis. The effects of MO -miRs transfected into HIV+ PBMCs were analyzed and revealed a decrease in viability associated with an increase of apoptosis; an increase of T helper cells expressing Fas and a decrease of intracellular Bcl2 protein expression. Meanwhile no effects were detected in PBMCs from healthy donors. In CD4 T cells, transfection significantly reduced cell activation and modified the T cell differentiation, thereby decreasing both central and effector memory cells while increasing terminal effector memory cells. Interestingly, the -miRs transfection induces a reduction of intracellular HIV p24 protein and a reduction of viral DNA integration. Finally, we evaluated the effect of synthetic (mimic) -miR858b whose sequence is present in the MO miR pool and predicted to target VAV1, a protein involved in HIV-Nef binding. This protein plays a pivotal role in T cell antigen receptor (TCR) signaling, so triggering the activation of various pathways. The transfection of HIV+ PBMCs with the synthetic miR858b showed a reduced expression of VAV1 and HIV p24 proteins. Overall, our evidence defines putative mechanisms underlying a supplementary benefit of traditional medicine, alongside current antiretroviral therapy, in managing HIV infection in resource-limited settings where MO remains widely available.
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http://dx.doi.org/10.3389/fphar.2020.620038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905167PMC
February 2021

Combined and selective miR-21 silencing and doxorubicin delivery in cancer cells using tailored DNA nanostructures.

Cell Death Dis 2021 01 7;12(1). Epub 2021 Jan 7.

Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy.

MicroRNAs play an important role in tumorigenesis and, among them, miR-21 is found to be aberrantly up-regulated in various tumors. The tumor-associated antigen, folate receptor alpha is a GPI-membrane protein overexpressed in many malignant tumors of epithelial origin, including ovarian and cervical cancers. Covalently bound octahedral DNA nanocages were functionalized with folate molecules and utilized as scaffolds to engineer four sequestering units with a miR-21 complementary sequence for obtaining biocompatible Fol-miR21-NC non-toxic nanostructures, to be able to selectively recognize folate receptor alpha-overexpressing cancer cells and sequester the oncogenic miR-21. qPCR assays showed that Fol-miR21-NCs reduce the miR-21 expression up to 80% in cancer cells in the first 2 days of treatment. Functional assays demonstrated that miR-21 sequestering leads to up-regulation of miR-21 tumor suppressor targets (i.e., PTEN and Pdcd4), reduction in cancer cell migration, reduction in proliferation, and increase in cell death. Fol-miR21-NCs can be efficiently loaded with the chemotherapeutic agent doxorubicin. Co-delivery of anti-miR-21 and doxorubicin showed additive cytotoxic effects on tumor cells, paving the way for their use as selective nucleic acid drugs.
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http://dx.doi.org/10.1038/s41419-020-03339-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791072PMC
January 2021

Can Gut Microbiota Be a Good Predictor for Parkinson's Disease? A Machine Learning Approach.

Brain Sci 2020 Apr 19;10(4). Epub 2020 Apr 19.

Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy.

The involvement of the gut microbiota in Parkinson's disease (PD), investigated in several studies, identified some common alterations of the microbial community, such as a decrease in and an increase in families in PD patients. However, the results of other bacterial families are often contradictory. Machine learning is a promising tool for building predictive models for the classification of biological data, such as those produced in metagenomic studies. We tested three different machine learning algorithms (random forest, neural networks and support vector machines), analyzing 846 metagenomic samples (472 from PD patients and 374 from healthy controls), including our published data and those downloaded from public databases. Prediction performance was evaluated by the area under curve, accuracy, precision, recall and F-score metrics. The random forest algorithm provided the best results. Bacterial families were sorted according to their importance in the classification, and a subset of 22 families has been identified for the prediction of patient status. Although the results are promising, it is necessary to train the algorithm with a larger number of samples in order to increase the accuracy of the procedure.
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http://dx.doi.org/10.3390/brainsci10040242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226159PMC
April 2020

In Silico and In Cell Analysis of Openable DNA Nanocages for miRNA Silencing.

Int J Mol Sci 2019 Dec 20;21(1). Epub 2019 Dec 20.

Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.

A computational and experimental integrated approach was applied in order to study the effect of engineering four DNA hairpins into an octahedral truncated DNA nanocage, to obtain a nanostructure able to recognize and bind specific oligonucleotide sequences. Modeling and classical molecular dynamics simulations show that the new H4-DNA nanocage maintains a stable conformation with the closed hairpins and, when bound to complementary oligonucleotides produces an opened conformation that is even more stable due to the larger hydrogen bond number between the hairpins and the oligonucleotides. The internal volume of the open conformation is much larger than the closed one, switching from 370 to 650 nm, and the predicted larger conformational change is experimentally detectable by gel electrophoresis. H4-DNA nanocages display high stability in serum, can efficiently enter the cells where they are stable and maintain the ability to bind, and sequester an intracellular-specific oligonucleotide. Moreover, H4-DNA nanocages, modified in order to recognize the oncogenic miR21, are able to seize miRNA molecules inside cells in a selective manner.
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http://dx.doi.org/10.3390/ijms21010061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981788PMC
December 2019

Effect of Low-Protein Diet and Inulin on Microbiota and Clinical Parameters in Patients with Chronic Kidney Disease.

Nutrients 2019 Dec 9;11(12). Epub 2019 Dec 9.

Department of Biology, Tor Vergata University of Rome, 00133 Rome, Italy.

Introduction: The gut microbiota has coevolved with humans for a mutually beneficial coexistence and plays an important role in health and disease. A dysbiotic gut microbiome may contribute to progression to chronic kidney disease (CKD) and CKD-related complications such as cardiovascular disease. Microbiota modulation through the administration of prebiotics may represent an important therapeutic target.

Aim: We sought to evaluate the effects of a low-protein diet (LPD) (0.6 g/kg/day) with or without the intake of the prebiotic inulin (19 g/day) on microbiota and clinical parameters in CKD patients.

Materials And Methods: We performed a longitudinal, prospective, controlled, and interventional study on 16 patients: 9 patients treated with LPD (0.6 g/kg/day) and inulin (19 g/day) and 7 patients (control group) treated only with LPD (0.6 g/kg/day). Clinical evaluations were performed and fecal samples were collected for a subsequent evaluation of the intestinal microbiota in all patients. These tests were carried out before the initiation of LPD, with or without inulin, at baseline (T0) and at 6 months (T2). The microbiota of 16 healthy control (HC) subjects was also analyzed in order to identify potential dysbiosis between patients and healthy subjects.

Results: Gut microbiota of CKD patients was different from that of healthy controls. The LPD was able to significantly increase the frequencies of Akkermansiaceae and Bacteroidaceae and decrease the frequencies of Christensenellaceae, Clostridiaceae, Lactobacillaceae, and Pasteurellaceae. Only Bifidobacteriaceae were increased when the LPD was accompanied by oral inulin intake. We showed a significant reduction of serum uric acid (SUA) and C-reactive protein (CRP) in patients treated with LPD and inulin ( = 0.018 and = 0.003, respectively), an improvement in SF-36 (physical role functioning and general health perceptions; = 0.03 and = 0.01, respectively), and a significant increase of serum bicarbonate both in patients treated with LPD ( = 0.026) or with LPD and inulin ( = 0.01). Moreover, in patients treated with LPD and inulin, we observed a significant reduction in circulating tumor necrosis factor alpha (TNF-α) ( = 0.041) and plasma nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2) ( = 0.027) levels. We did not find a significant difference in the circulating levels of Interleukin (IL)-1β ( = 0.529) and IL-6 ( = 0.828) in the two groups.

Conclusions: LPD, associated or not with inulin, modified gut microbiota and modulated inflammatory and metabolic parameters in patients with CKD. Our results suggest that interventions attempting to modulate the gut microbiome may represent novel strategies to improve clinical outcomes in CKD patients and may provide useful therapeutic effects.
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http://dx.doi.org/10.3390/nu11123006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950025PMC
December 2019

Quercetin pentaacetate inhibits in vitro human respiratory syncytial virus adhesion.

Virus Res 2020 01 9;276:197805. Epub 2019 Nov 9.

Universidade Estadual Paulista, UNESP (FCLAssis), Brazil; Universidade Estadual Paulista, UNESP IBILCE, São José do Rio Preto, Brazil. Electronic address:

Human respiratory syncytial virus (hRSV) is one of the main etiological agents of diseases of the lower respiratory tract and is often responsible for the hospitalization of children and the elderly. To date, treatments are only palliative and there is no vaccine available. Natural products show exceptional structural diversity and they have played a vital role in drug research. Several investigations focused on applied structural modification of natural products to improved metabolic stability, solubility and biological actions them. Quercetin is a flavonoid that presents several biological activities, including anti-hRSV role. Some works criticize the pharmacological use of Quercetin because it has low solubility and low specificity. In this sense, we acetylated Quercetin structure and we used in vitro and in silico assays to compare anti-hRSV function between Quercetin (Q0) and its derivative molecule (Q1). Q1 shows lower cytotoxic effect than Q0 on HEp-2 cells. In addition, Q1 was more efficient than Q0 to protect HEp-2 cells infected with different multiplicity of infection (0.1-1 MOI). The virucidal effects of Q0 and Q1 suggest interaction between these molecules and viral particle. Dynamic molecular results suggest that Q0 and Q1 may interact with F-protein on hRSV surface in an important region to adhesion and viral infection. Q1 interaction with F-protein showed ΔG= -14.22 kcal/mol and it was more stable than Q0. Additional, MTT and plate assays confirmed that virucidal Q1 effects occurs during adhesion step of cycle hRSV replication. In conclusion, acetylation improves anti-hRSV Quercetin effects because Quercetin pentaacetate could interact with F-protein with lower binding energy and better stability to block viral adhesion. These results show alternative anti-hRSV strategy and contribute to drug discovery and development.
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http://dx.doi.org/10.1016/j.virusres.2019.197805DOI Listing
January 2020

Palladium(ii) complexes with thiosemicarbazones derived from pyrene as topoisomerase IB inhibitors.

Dalton Trans 2019 Nov;48(44):16509-16517

São Carlos Institute of Chemistry, University of São Paulo, 13560-970, São Carlos, Brazil.

New palladium complexes with thiosemicarbazonate ligands derived from pyrene exhibit potent antiproliferative activity against A2780 and cisplatin-resistant A2780Cis human ovarian cancer cells, which is dependent on substituent groups of the thiosemicarbazone ligands. Cellular accumulation and distribution studies confirmed that palladium enters the cell nucleus. DNA and topoisomerase IB studies show that one complex is a potent TopIB inhibitor, with selectivity for cancer versus normal cells.
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http://dx.doi.org/10.1039/c9dt02570gDOI Listing
November 2019

Non-mutagenic Ru(ii) complexes: cytotoxicity, topoisomerase IB inhibition, DNA and HSA binding.

Dalton Trans 2019 Oct;48(39):14885-14897

Dipartimentodi Biologia, UniversitàTorVergatadi Roma, 00133 Rome, Italy.

Herein we discuss five ruthenium(ii) complexes with good cytotoxicity against cancer cells. These complexes are named [Ru(tzdt)(bipy)(dppb)]PF6 (1), [Ru(mmi)(bipy)(dppb)]PF6 (2), [Ru(dmp)(bipy)(dppb)]PF6 (3), [Ru(mpca)(bipy)(dppb)]PF6 (4) and [Ru(2mq)(bipy)(dppb)]PF6 (5), where tzdt = 1,3-thiazolidine-2-thione, mmi = mercapto-1-methyl-imidazole, dmp = 4,6-diamino-2-mercaptopyrimidine, mpca = 6-mercaptopyridine-3-carboxylic acid, 2mq = 2-mercapto-4(3H)-quinazolinone, bipy = 2,2'-bipyridine and dppb = 1,4-bis(diphenylphosphino)butane. In vitro cell culture experiments revealed significant cytotoxic activity for 1-5 against MDA-MB-231, MCF-7, A549, DU-145 and HepG2 tumor cells, higher than that for the standard anticancer drug cisplatin. Compound/DNA interaction studies were carried out showing that 1-5 interact with DNA by electrostatic force of attraction or by hydrogen bonding. Moreover, the complexes interact, moderately and spontaneously, with human serum albumin (HSA) through the hydrophobic region. The five complexes are able to inhibit the DNA supercoiled relaxation mediated by human topoisomerase IB (TopIB), and complex 1 is found to be the most efficient TopIB inhibitor among the five compounds. The inhibitory effect and analysis of different steps of the TopIB catalytic cycle indicate that complex 1 inhibits the cleavage reaction impeding the binding of the enzyme to DNA and has no effect on the religation step. Complexes 1, 2 and 3 did not show mutagenic activity when they were evaluated by the cytokinesis-block micronucleus cytome assay in HepG2 cells and the Ames test in the presence and absence of mouse liver S9 metabolic activation. Therefore, it is necessary to perform further in-depth analysis of the therapeutic potential of these promising ruthenium complexes as anticancer drugs.
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http://dx.doi.org/10.1039/c9dt01905gDOI Listing
October 2019

Dysbiosis of gut microbiota in a selected population of Parkinson's patients.

Parkinsonism Relat Disord 2019 08 3;65:124-130. Epub 2019 Jun 3.

Department of Biology, University of Rome Tor Vergata, Rome, Italy. Electronic address:

Introduction: In recent years the hypothesis that gut microbiota associates with Parkinson's disease (PD) has gained importance, although it has not been possible to define a specific microbiota composition as a predictive biomarker of this disease. We have investigated dysbiosis of gut microbiota in a selected population of PD patients from Central Italy, and examined the weight of specific confounders and predictors, in order to identify potential correlations with clinical phenotypes.

Methods: 152 fecal samples were collected from 80 patients and 72 healthy controls. Patients were enrolled according to tight inclusion criteria. Microbiota composition was studied through 16s ribosomal RNA gene amplicon sequencing analysis in combination with data on dietary/life habits. Age, loss of weight, and sex were recognized as confounding factors, whereas PD-status, age, Body Mass Index, "eat cereals", "gain of weigth" and "physical activity" as predictors. The presence of Lactobacillaceae, Enterobacteriaceae and Enterococcaceae families was significantly higher in feces from PD patients compared to healthy controls, while Lachnospiraceae were significantly reduced. Lower levels of Lachnospiraceae and higher levels of Enterobacteriaceae families also correlated with increased disease severity and motor impairment (Hoehn & Yahr stage, MDS-UPDRS Part III). Predictive metagenomics indicated a significant variation of genes involved in the metabolism of short chain fatty acids and amino acids, and in lipopolysaccharide biosynthesis.

Conclusions: PD showed a distinctive microbiota composition. Functional predictions suggest changes in pathways favoring a pro-inflammatory environment in the gastrointestinal tract, and a reduction in the biosynthesis of amino acids acting as precursors of physiological transmitters.
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http://dx.doi.org/10.1016/j.parkreldis.2019.06.003DOI Listing
August 2019

Cellular uptake of covalent and non-covalent DNA nanostructures with different sizes and geometries.

Nanoscale 2019 Jun;11(22):10808-10818

Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133, Roma, Italy.

DNA nanostructures with different sizes and shapes, assembled through either covalent or non-covalent bonds, namely tetrahedral and octahedral nanocages, rod-shaped chainmails, square box and rectangular DNA origami structures, were compared for their stability in serum, cell surface binding, internalization efficiency, and intracellular degradation rate. For cell internalization a specific cell system, highly expressing the scavenger receptor LOX-1 was used. The results indicate that LOX-1 binds and internalizes a broad family of DNA structures of different sizes that, however, have a different fate and lifetime inside the cells. Covalently linked tetrahedra, octahedra or chainmails are intact inside cells for up to 18 hours whilst the same DNA nanostructures without covalent bonds along with square box and rectangular origami are rapidly degraded. These data suggest that non-covalently linked structures may be useful for fast drug release whilst the covalently-linked structures could be appropriate vehicles for slow release of molecules.
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http://dx.doi.org/10.1039/c9nr02006cDOI Listing
June 2019

Probing the Functional Topology of a pH-Dependent Triple Helix DNA Nanoswitch Family through Gaussian Accelerated MD Simulation.

J Chem Inf Model 2019 06 22;59(6):2746-2752. Epub 2019 May 22.

Department of Biology, Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB) , University of Rome Tor Vergata , Via della Ricerca Scientifica 1 , 00133 Rome , Italy.

The topology of a pH-dependent triple helix DNA nanoswitch family has been characterized through simulative analysis to evaluate the efficiency of the switching mechanism varying the length of the loop connecting the two strands forming the double helix portion. In detail, the system is formed by a double helix made by two six base complementary sequences, connected by one loop having an increasing number of thymidines, namely 5, 7, or 9. The triplex-forming sequence made by six bases, connected to the double helix through a constant 25 base loop, interacts at pH 5.0 through Hoogsteen hydrogen bonds with one strand of the double helical region. We demonstrate, through molecular dynamics simulation, that the thymidine loop length exerts a fine regulatory role for the stability of the triple helix structure and is critical in modulating the switching mechanism triggered by the pH increase.
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http://dx.doi.org/10.1021/acs.jcim.9b00133DOI Listing
June 2019

Engineering a responsive DNA triple helix into an octahedral DNA nanostructure for a reversible opening/closing switching mechanism: a computational and experimental integrated study.

Nucleic Acids Res 2018 11;46(19):9951-9959

Biology Department, University of Rome Tor Vergata, Rome 00133, Italy.

We propose an experimental and simulative approach to study the effect of integrating a DNA functional device into a large-sized DNA nanostructure. We selected, as a test bed, a well-known and characterized pH-dependent clamp-switch, based on a parallel DNA triple helix, to be integrated into a truncated octahedral scaffold. We designed, simulated and experimentally characterized two different functionalized DNA nanostructures, with and without the presence of a spacer between the scaffold and the functional elements. The experimental and simulative data agree in validating the need of a spacer for the occurrence of the pH dependent switching mechanism. The system is fully reversible and the switching can be monitored several times without any perturbation, maintaining the same properties of the isolated clamp switch in solution.
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http://dx.doi.org/10.1093/nar/gky857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212788PMC
November 2018

Design, selection and optimization of an anti-TRAIL-R2/anti-CD3 bispecific antibody able to educate T cells to recognize and destroy cancer cells.

MAbs 2018 10 6;10(7):1084-1097. Epub 2018 Aug 6.

a Molecular Therapies Unit, Department of Experimental Oncology and Molecular Medicine , Fondazione IRCCS Istituto Nazionale dei Tumori , Milan , Italy.

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or TRAIL-receptor agonistic monoclonal antibodies promote apoptosis in most cancer cells, and the differential expression of TRAIL-R2 between tumor and normal tissues allows its exploitation as a tumor-associated antigen. The use of these antibodies as anticancer agents has been extensively studied, but the results of clinical trials were disappointing. The observed lack of anticancer activity could be attributed to intrinsic or acquired resistance of tumor cells to this type of treatment. A possible strategy to circumvent drug resistance would be to strike tumor cells with a second modality based on a different mechanism of action. We therefore set out to generate and optimize a bispecific antibody targeting TRAIL-R2 and CD3. After the construction of different bispecific antibodies in tandem-scFv or single-chain diabody formats to reduce possible immunogenicity, we selected a humanized bispecific antibody with very low aggregates and long-term high stability and functionality. This antibody triggered TRAIL-R2 in an agonistic manner and its anticancer activity proved dramatically potentiated by the redirection of cytotoxic T cells against both sensitive and resistant melanoma cells. The results of our study show that combining the TRAIL-based antitumor strategy with an immunotherapeutic approach in a single molecule could be an effective addition to the anticancer armamentarium.
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http://dx.doi.org/10.1080/19420862.2018.1494105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204841PMC
October 2018

Entry, fate and degradation of DNA nanocages in mammalian cells: a matter of receptors.

Nanoscale 2018 Jul;10(25):12078-12086

Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy.

DNA has been used to build nanostructures with potential biomedical applications. However, their use is limited by the lack of information on the mechanism of entry, intracellular fate and degradation rate of nanostructures inside cells. We generated octahedral DNA nanocages functionalized with folic acid and investigated the cellular uptake mediated by two distinctive internalization pathways, using two cellular systems expressing the oxidized low-density lipoprotein receptor-1 (LOX-1) and the α isoform of the folate receptor (αFR), respectively. Here, we report that DNA nanocages are very efficiently and selectively internalized by both receptors with an efficiency at least 30 times higher than that observed in cells not expressing the receptors. When internalized by LOX-1, nanocages traffic to lysosomes within 4 hours and are rapidly degraded. When the uptake is mediated by αFR, DNA nanocages are highly stable (>48 hours) and accumulate inside cells in a time-dependent way. These data demonstrate that the selection of the cellular receptor is crucial for targeting specific sub-cellular compartments and for modulating the DNA nanocage intracellular half-life, indicating that vitamin-mediated uptake may constitute a protected pathway for intracellular drug delivery.
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http://dx.doi.org/10.1039/c8nr02411aDOI Listing
July 2018

A Comparative Genomic Analysis Provides Novel Insights Into the Ecological Success of the Monophasic Serovar 4,[5],12:i:.

Front Microbiol 2018 17;9:715. Epub 2018 Apr 17.

Department of Food Safety, National Reference Center for Salmonella, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy.

Over the past decades, 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated . 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene . A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of . 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread.
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http://dx.doi.org/10.3389/fmicb.2018.00715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913373PMC
April 2018

High-Density ZnO Nanowires as a Reversible Myogenic-Differentiation Switch.

ACS Appl Mater Interfaces 2018 Apr 12;10(16):14097-14107. Epub 2018 Apr 12.

Department of Biology , University of Rome Tor Vergata , Via della Ricerca Scientifica 1 , 00133 Rome , Italy.

Mesoangioblasts are outstanding candidates for stem-cell therapy and are already being explored in clinical trials. However, a crucial challenge in regenerative medicine is the limited availability of undifferentiated myogenic progenitor cells because growth is typically accompanied by differentiation. Here reversible myogenic-differentiation switching during proliferation is achieved by functionalizing the glass substrate with high-density ZnO nanowires (NWs). Specifically, mesoangioblasts grown on ZnO NWs present a spherical viable undifferentiated cell state without lamellopodia formation during the entire observation time (8 days). Consistently, the myosin heavy chain, typically expressed in skeletal muscle tissue and differentiated myogenic progenitors, is completely absent. Remarkably, NWs do not induce any damage while they reversibly block differentiation, so that the differentiation capabilities are completely recovered upon cell removal from the NW-functionalized substrate and replating on standard culture glass. This is the first evidence of a reversible myogenic-differentiation switch that does not affect the viability. These results can be the first step toward for the in vitro growth of a large number of undifferentiated stem/progenitor cells and therefore can represent a breakthrough for cell-based therapy and tissue engineering.
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http://dx.doi.org/10.1021/acsami.7b19758DOI Listing
April 2018

Molecular dynamics characterization of the SAMHD1 Aicardi-Goutières Arg145Gln mutant: structural determinants for the impaired tetramerization.

J Comput Aided Mol Des 2018 05 28;32(5):623-632. Epub 2018 Mar 28.

Department of Biology, Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy.

Aicardi-Goutières syndrome, a rare genetic disorder characterized by calcification of basal ganglia, results in psychomotor delays and epilepsy states from the early months of children life. This disease is caused by mutations in seven different genes encoding proteins implicated in the metabolism of nucleic acids, including SAMHD1. Twenty SAMHD1 gene variants have been discovered and in this work, a structural characterization of the SAMHD1 Aicardi-Goutières Arg145Gln mutant is reported by classical molecular dynamics simulation. Four simulations have been carried out and compared. Two concerning the wild-type SAMHD1 form in presence and absence of cofactors, in order to explain the role of cofactors in the SAMHD1 assembly/disassembly process and, two concerning the Arg145Gln mutant, also in presence and absence of cofactors, in order to have an accurate comparison with the corresponding native forms. Results show the importance of native residue Arg145 in maintaining the tetramer, interacting with GTP cofactor inside allosteric sites. Replacement of arginine in glutamine gives rise to a loosening of GTP-protein interactions, when cofactors are present in allosteric sites, whilst in absence of cofactors, the occurrence of intra and inter-chain interactions is observed in the mutant, not seen in the native enzyme, making energetically unfavourable the tetramerization process.
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http://dx.doi.org/10.1007/s10822-018-0115-0DOI Listing
May 2018

Selective targeting and degradation of doxorubicin-loaded folate-functionalized DNA nanocages.

Nanomedicine 2018 06 17;14(4):1181-1190. Epub 2018 Feb 17.

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy. Electronic address:

Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells.
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http://dx.doi.org/10.1016/j.nano.2018.02.002DOI Listing
June 2018

Selective targeting and degradation of doxorubicin-loaded folate-functionalized DNA nanocages.

Nanomedicine 2018 06 17;14(4):1181-1190. Epub 2018 Feb 17.

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy. Electronic address:

Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells.
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http://dx.doi.org/10.1016/j.nano.2018.02.002DOI Listing
June 2018

[Appropriateness in Cardiology: a statement of the ANMCO Veneto Region].

G Ital Cardiol (Rome) 2018 Jan;19(1):24-31

U.O. Cardiologia, Ospedali dell'Ovest Vicentino, Arzignano (VI).

Inappropriate tests are responsible for longer waiting lists, higher economical costs for the National Health System and major clinical risks due to radiation exposure from prescription abuse of diagnostic testing. Clinical inappropriateness frequently derives from poor knowledge of guidelines, "defensive medicine" approach and/or repeat requests of patients and family members. About one third of non-invasive imaging tests are considered inappropriate.In order to define the most appropriate instruments for the follow-up of the most common cardiovascular diseases with the highest risk of inappropriateness, all the cardiologists of the Veneto Region (Italy), along with the local chapters of the main national cardiology societies and general practitioners have been involved by the Regional Section of the Italian Association of Hospital Cardiologists (ANMCO) in several scientific meetings on the following topics: hypertension, chronic ischemic heart disease, valvular heart disease, heart failure, and atrial fibrillation. This has led to the present document where: (i) the most appropriate clinical and diagnostic strategies are taken into account, and (ii) the most robust scientific evidence is provided for the regulatory commission of the Veneto Region Health Service to identify inappropriateness, prescription unsuitability, and economical sustainability.
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http://dx.doi.org/10.1714/2852.28776DOI Listing
January 2018

Accrual monitoring in cardiovascular trials.

Open Heart 2017;4(2):e000720. Epub 2017 Dec 17.

Department of Clinical and Biological Sciences, University of Torino, Torino, Italy.

Objective: To provide brief guidance on how to design accrual monitoring activities in a clinical trial protocol.

Setting: Two completed clinical trials that did not achieve the planned sample size, the Cost of Strategies After Myocardial Infarction (COSTAMI) trial and the Biventricular Pacing After Cardiac Surgery (BiPACS) trial.

Design: A Bayesian monitoring tool, the constant accrual model, is applied retrospectively to accrual data from each case study to illustrate how the tool could be used to identify problems with accrual early in the trial period and to frame the conditions in which the approach can be used in practice.

Results: After 312 days and 155 patients enrolled in the COSTAMI trial, accrual could be classified as 'off target' on the basis of statistical criteria outlined in the protocol. As for the BiPACS trial, after 2 years, it was already evident that the accrual was 'considerably off target'.

Conclusions: Prompt awareness of a high risk of accrual failure could trigger different interventions to overcome protocol-related, patient-related or investigator-related barriers to recruitment or ultimately contribute to an early stopping decision due to recruitment futility.Accrual prediction models should be included as standard tools for routine monitoring activities in cardiovascular research. Among them, methods relying on the Bayesian approach are particularly attractive, as they can naturally update past evidence when actual accrual data becomes available.
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http://dx.doi.org/10.1136/openhrt-2017-000720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761309PMC
December 2017

Ru/Fe bimetallic complexes: Synthesis, characterization, cytotoxicity and study of their interactions with DNA/HSA and human topoisomerase IB.

Arch Biochem Biophys 2017 12 28;636:28-41. Epub 2017 Oct 28.

Department of Biology, University of Rome Tor Vergata, 00133, Rome, Italy. Electronic address:

Three ruthenium/iron-based compounds, 1: [Ru(MIm)(bipy)(dppf)]PF (MIm = 2-mercapto-1-methylimidazole anion), 2: [RuCl(Im)(bipy)(dppf)]PF (Im = imidazole), and 3: [Ru(tzdt)(bipy)(dppf)]PF (tzdt = 1,3-thiazolidine-2-thione anion) (dppf = 1,1'-bis(diphenylphosphine)ferrocene and bipy = 2,2'-bipyridine), were synthesized, and characterized by elemental analyses, conductivity, UV/Vis, IR, H, C and P{1H} NMR spectroscopies, and by electrochemical technique. The complex 3 was also characterized by single-crystal X-ray. The three ruthenium(II) complexes show cytotoxicity against DU-145 (prostate carcinoma cells) and A549 (lung carcinoma cells) tumor cells. The free ligands do not exhibit any cytotoxic activity, such as evident by the IC values higher than 200 μM. UV/Vis and viscosity experiments showed that the complexes interact weakly with the DNA molecule, via electrostatic forces. The interaction of the complexes 1-3 with the HSA is moderate, with K values in range of 10-10 M, presenting a static mechanism of interaction stabilized by hydrophobic. Complexes 2 and 3 showed high affinity for the FA7 HSA site as evidenced by fluorescence spectroscopy and molecular docking. Complexes 1-3 were tested as potential human Topoisomerase IB inhibitors by analysing the different steps of the enzyme catalytic cycle. The results indicate that all compounds efficiently inhibit the DNA relaxation and the cleavage reaction, in which the effect increases upon pre-incubation. Complexes 1 and 2 are also able to slow down the religation reaction.
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http://dx.doi.org/10.1016/j.abb.2017.10.015DOI Listing
December 2017

Efficacy of a Binuclear Cyclopalladated Compound Therapy for Cutaneous Leishmaniasis in the Murine Model of Infection with Leishmania amazonensis and Its Inhibitory Effect on Topoisomerase 1B.

Antimicrob Agents Chemother 2017 08 25;61(8). Epub 2017 Jul 25.

São Paulo State University (UNESP), School of Pharmaceutical Sciences, Araraquara, Brazil

Leishmaniasis is a disease found throughout the (sub)tropical parts of the world caused by protozoan parasites of the genus. Despite the numerous problems associated with existing treatments, pharmaceutical companies continue to neglect the development of better ones. The high toxicity of current drugs combined with emerging resistance makes the discovery of new therapeutic alternatives urgent. We report here the evaluation of a binuclear cyclopalladated complex containing Pd(II) and ,'-dimethylbenzylamine (Hdmba) against The compound [Pd(dmba)(μ-N)] (CP2) inhibits promastigote growth (50% inhibitory concentration [IC] = 13.2 ± 0.7 μM) and decreases the proliferation of intracellular amastigotes in incubated macrophages (IC = 10.2 ± 2.2 μM) without a cytotoxic effect when tested against peritoneal macrophages (50% cytotoxic concentration = 506.0 ± 10.7 μM). In addition, CP2 was also active against intracellular amastigotes (IC = 2.3 ± 0.5 μM, selective index = 225), an indication of its potential for use in Chagas disease therapy. assays using -infected BALB/c showed an 80% reduction in parasite load compared to infected and nontreated animals. Also, compared to amphotericin B treatment, CP2 did not show any side effects, which was corroborated by the analysis of plasma levels of different hepatic and renal biomarkers. Furthermore, CP2 was able to inhibit topoisomerase 1B (topo1B), a potentially important target in this parasite. (This study has been registered at ClinicalTrials.gov under identifier NCT02169141.).
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http://dx.doi.org/10.1128/AAC.00688-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527659PMC
August 2017

Simulative and Experimental Characterization of a pH-Dependent Clamp-like DNA Triple-Helix Nanoswitch.

J Am Chem Soc 2017 04 7;139(15):5321-5329. Epub 2017 Apr 7.

Department of Biology and ‡Department of Chemistry, University of Rome, Tor Vergata , 00173 Rome, Italy.

Here we couple experimental and simulative techniques to characterize the structural/dynamical behavior of a pH-triggered switching mechanism based on the formation of a parallel DNA triple helix. Fluorescent data demonstrate the ability of this structure to reversibly switch between two states upon pH changes. Two accelerated, half microsecond, MD simulations of the system having protonated or unprotonated cytosines, mimicking the pH 5.0 and 8.0 conditions, highlight the importance of the Hoogsteen interactions in stabilizing the system, finely depicting the time-dependent disruption of the hydrogen bond network. Urea-unfolding experiments and MM/GBSA calculations converge in indicating a stabilization energy at pH 5.0, 2-fold higher than that observed at pH 8.0. These results validate the pH-controlled behavior of the designed structure and suggest that simulative approaches can be successfully coupled with experimental data to characterize responsive DNA-based nanodevices.
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http://dx.doi.org/10.1021/jacs.6b11470DOI Listing
April 2017

Human topoisomerase inhibition and DNA/BSA binding of Ru(II)-SCAR complexes as potential anticancer candidates for oral application.

Biometals 2017 06 16;30(3):321-334. Epub 2017 Mar 16.

School of Pharmaceutical Sciences, São Paulo State University, Araraquara, 14800-903, Brazil.

Three ruthenium(II) phosphine/diimine/picolinate complexes were selected aimed at investigating anticancer activity against several cancer cell lines and the capacity of inhibiting the supercoiled DNA relaxation mediated by human topoisomerase IB (Top 1). The structure-lipophilicity relationship in membrane permeability using the Caco-2 cells have also been evaluated in this study. SCAR 5 was found to present 45 times more cytotoxicity against breast cancer cell when compared to cisplatin. SCAR 4 and 5 were both found to be capable of inhibiting the supercoiled DNA relaxation mediated by Top 1. Interaction studies showed that SCAR 4 and 5 can bind to DNA through electrostatic interactions while SCAR 6 is able to bind covalently to DNA. The complexes SCAR were found to interact differently with bovine serum albumin (BSA) suggesting hydrophobic interactions with albumin. The permeability of all complexes was seen to be dependent on their lipophilicity. SCAR 4 and 5 exhibited high membrane permeability (P  > 10 × 10 cm·s) in the presence of BSA. The complexes may pass through Caco-2 monolayer via passive diffusion mechanism and our results suggest that lipophilicity and interaction with BSA may influence the complexes permeation. In conclusion, we demonstrated that complexes have powerful pharmacological activity, with different results for each complex depending on the combination of their ligands.
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http://dx.doi.org/10.1007/s10534-017-0008-zDOI Listing
June 2017

A molecular dynamics simulation study decodes the early stage of the disassembly process abolishing the human SAMHD1 function.

J Comput Aided Mol Des 2017 May 1;31(5):497-505. Epub 2017 Mar 1.

Department of Biology, Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy.

The human sterile alpha motif SAM and HD domain-containing protein 1 (SAMHD1) restricts in non-cycling cells type the infection of a large range of retroviruses including HIV-1, reducing the intracellular pool concentration of deoxynucleoside triphosphates (dNTPs) required for the reverse transcription of the viral genome. The enzyme is in equilibrium between different forms depending on bound cofactors and substrate. In this work, two SAMHD1 three-dimensional models have been investigated through classical molecular dynamics simulation, to define the role of cofactors and metal ions in the association of the tetrameric active form. A detailed analysis of the inter-subunit interactions, taking place at the level of helix 13, indicates that removal of metal ions and cofactors induces an asymmetric loosening of the monomer-monomer interface leading to the formation of a loose tetramer where the two dimeric interfaces are weakened in different way.
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http://dx.doi.org/10.1007/s10822-017-0014-9DOI Listing
May 2017
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