Publications by authors named "Federico Iacovelli"

36 Publications

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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 Jan 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-020-03339-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791072PMC
January 2021

Antifungal Effect of All- Retinoic Acid against Aspergillus fumigatus and in a Pulmonary Aspergillosis Model.

Antimicrob Agents Chemother 2021 02 17;65(3). Epub 2021 Feb 17.

Anatomic Pathology, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.

is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. The fungistatic activity of all- retinoic acid (ATRA) has been recently described We evaluated the efficacy of ATRA and its potential synergistic interaction with other antifungal drugs. A rat model of IPA and experiments were performed to assess the efficacy of ATRA against in association with classical antifungal drugs and studies used to clarify its mechanism of action. ATRA (0.5 and 1 mM) displayed a strong fungistatic activity in cultures, while at lower concentrations, synergistically potentiated fungistatic efficacy of subinhibitory concentration of amphotericin B (AmB) and posaconazole (POS). ATRA also enhanced macrophagic phagocytosis of conidia. In a rat model of IPA, ATRA reduced mortality similarly to posaconazole. Fungistatic efficacy of ATRA alone and synergistically with other antifungal drugs was documented , likely by inhibiting fungal heat shock protein 90 () expression and Hsp90-related genes. ATRA treatment reduced mortality in a model of IPA Those findings suggest ATRA as a suitable fungistatic agent that can also reduce dosage and adverse reactions of classical antifungal drugs and add to the development of new therapeutic strategies against IPA and systemic fungal infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01874-20DOI Listing
February 2021

Dynamical Behavior of the Human Ferroportin Homologue from : Insight into the Ligand Recognition Mechanism.

Int J Mol Sci 2020 Sep 16;21(18). Epub 2020 Sep 16.

Department of Sciences, Roma Tre University, 00146 Rome, Italy.

Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer's disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein's dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21186785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555787PMC
September 2020

Targeting the SARS-CoV-2 spike glycoprotein prefusion conformation: virtual screening and molecular dynamics simulations applied to the identification of potential fusion inhibitors.

Virus Res 2020 09 18;286:198068. Epub 2020 Jun 18.

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

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a renewed interest in studying the role of the spike S glycoprotein in regulating coronavirus infections in the natural host. Taking advantage of the cryo-electron microscopy structure of SARS-CoV-2 S trimer in the prefusion conformation, we performed a virtual screening simulation with the aim to identify novel molecules that could be used as fusion inhibitors. The spike glycoprotein structure has been completed using modeling techniques and its inner cavity, needful for the postfusion transition of the trimer, has been scanned for the identification of strongly interacting available drugs. Finally, the stability of the protein-drug top complexes has been tested using classical molecular dynamics simulations. The free energy of interaction of the molecules to the spike protein has been evaluated through the MM/GBSA method and per-residue decomposition analysis. Results have been critically discussed considering previous scientific knowledge concerning the selected compounds and sequence alignments have been carried out to evaluate the spike glycoprotein similarity among the betacoronavirus family members. Finally, a cocktail of drugs that may be used as SARS-CoV-2 fusion inhibitors has been suggested.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2020.198068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301794PMC
September 2020

Correction: Frataxin deficiency induces lipid accumulation and affects thermogenesis in brown adipose tissue.

Cell Death Dis 2020 Mar 3;11(3):165. Epub 2020 Mar 3.

Department Biology, University of Rome Tor Vergata, via della Ricerca Scientifica, Rome, Italy.

Since online publication of this article, the authors noticed that there was a basic citation error in PubMed citation data. Specifically, the name of the author "Piergiorgio La Rosa" is cited as "Rosa P" in the PubMed citation, when it should be "La Rosa P", "La Rosa" being the surname and "Piergiorgio" the name of the author.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-020-2347-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054354PMC
March 2020

Frataxin deficiency induces lipid accumulation and affects thermogenesis in brown adipose tissue.

Cell Death Dis 2020 01 23;11(1):51. Epub 2020 Jan 23.

Department Biology, University of Rome Tor Vergata, via della Ricerca Scientifica, Rome, Italy.

Decreased expression of mitochondrial frataxin (FXN) causes Friedreich's ataxia (FRDA), a neurodegenerative disease with type 2 diabetes (T2D) as severe comorbidity. Brown adipose tissue (BAT) is a mitochondria-enriched and anti-diabetic tissue that turns excess energy into heat to maintain metabolic homeostasis. Here we report that the FXN knock-in/knock-out (KIKO) mouse shows hyperlipidemia, reduced energy expenditure and insulin sensitivity, and elevated plasma leptin, recapitulating T2D-like signatures. FXN deficiency leads to disrupted mitochondrial ultrastructure and oxygen consumption as well as lipid accumulation in BAT. Transcriptomic data highlights cold intolerance in association with iron-mediated cell death (ferroptosis). Impaired PKA-mediated lipolysis and expression of genes controlling mitochondrial metabolism, lipid catabolism and adipogenesis were observed in BAT of KIKO mice as well as in FXN-deficient T37i brown and primary adipocytes. Significant susceptibility to ferroptosis was observed in adipocyte precursors that showed increased lipid peroxidation and decreased glutathione peroxidase 4. Collectively our data point to BAT dysfunction in FRDA and suggest BAT as promising therapeutic target to overcome T2D in FRDA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-020-2253-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978516PMC
January 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21010061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981788PMC
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2019.197805DOI Listing
January 2020

Potential of Curcumin in Skin Disorders.

Nutrients 2019 09 10;11(9). Epub 2019 Sep 10.

Dermatology Unit, Department of "Medicina dei Sistemi", University of Rome Tor Vergata, Via Montpellier, 1-00133 Rome, Italy.

Curcumin is a compound isolated from turmeric, a plant known for its medicinal use. Recently, there is a growing interest in the medical community in identifying novel, low-cost, safe molecules that may be used in the treatment of inflammatory and neoplastic diseases. An increasing amount of evidence suggests that curcumin may represent an effective agent in the treatment of several skin conditions. We examined the most relevant in vitro and in vivo studies published to date regarding the use of curcumin in inflammatory, neoplastic, and infectious skin diseases, providing information on its bioavailability and safety profile. Moreover, we performed a computational analysis about curcumin's interaction towards the major enzymatic targets identified in the literature. Our results suggest that curcumin may represent a low-cost, well-tolerated, effective agent in the treatment of skin diseases. However, bypass of limitations of its in vivo use (low oral bioavailability, metabolism) is essential in order to conduct larger clinical trials that could confirm these observations. The possible use of curcumin in combination with traditional drugs and the formulations of novel delivery systems represent a very promising field for future applicative research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/nu11092169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770633PMC
September 2019

Adipocyte metabolism is improved by TNF receptor-targeting small RNAs identified from dried nuts.

Commun Biol 2019 21;2:317. Epub 2019 Aug 21.

1Department of Biology, University of Rome Tor Vergata, Rome, Italy.

There is a growing interest in therapeutically targeting the inflammatory response that underlies age-related chronic diseases including obesity and type 2 diabetes. Through integrative small RNA sequencing, we show the presence of conserved plant miR159a and miR156c in dried nuts having high complementarity with the mammalian TNF receptor superfamily member 1a (Tnfrsf1a) transcript. We detected both miR159a and miR156c in exosome-like nut nanovesicles (NVs) and demonstrated that such NVs reduce Tnfrsf1a protein and dampen TNF-α signaling pathway in adipocytes. Synthetic single-stranded microRNAs (ss-miRs) modified with 2'--methyl group function as miR mimics. In plants, this modification naturally occurs on nearly all small RNAs. 2'--methylated ss-miR mimics for miR156c and miR159a decreased Tnfrsf1a protein and inflammatory markers in hypertrophic as well as TNF-α-treated adipocytes and macrophages. miR156c and miR159a mimics effectively suppress inflammation in mice, highlighting a potential role of plant miR-based, single-stranded oligonucleotides in treating inflammatory-associated metabolic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-019-0563-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704100PMC
April 2020

Luteolin-7--β-d-Glucoside Inhibits Cellular Energy Production Interacting with HEK2 in Keratinocytes.

Int J Mol Sci 2019 May 31;20(11). Epub 2019 May 31.

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

Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems. Their functional phenolic groups are able to mediate antioxidant effects by scavenging free radicals. Molecules of this class have been found able to modulate the activity of kinases, phospholipase A2, cyclooxygenases, lipoxygenase, glutathione S-transferase, and many others. Recently, it has been demonstrated that luteolin, in the form of Luteolin-7--β-d-glucoside (LUT-7G) is able to induce the keratinocyte differentiation process in vitro. This flavonoid is able to counteract the proliferative effects of IL-22/IL6 pathway by the inhibition of STAT3 activity also in vivo in a psoriatic mouse model. Observations on energy metabolism changes of differentiating cells led us to perform a complete metabolomics analysis using human primary keratinocytes treated with LUT-7G. Our results show that LUT-7G, is not only able to impair the nuclear translocation of STAT3, but it also blocks the energy metabolism pathway, depressing the glycolytic and Krebs pathway by the inhibition of hexokinase 2 activity. These data confirm that LUT-7G can be proposed as a potential candidate for the treatment of inflammatory and proliferative diseases, but its role as a hexokinase 2 (HEK2) inhibitor opens new perspectives in nutritional science, and especially in cancer therapy, in which the inhibition of the Warburg effect could be relevant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms20112689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600217PMC
May 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212788PMC
November 2018

Antifungal activity of L. (Sapindaceae) against occurs through molecular interaction with fungal Hsp90.

Drug Des Devel Ther 2018 12;12:2185-2193. Epub 2018 Jul 12.

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

Introduction: Dermatophytosis is a superficial fungal infection limited to the stratum corneum of the epidermis, or to the hair and nails, and constitutes an important public health problem because of its high prevalence and associated morbidity. Dermatophyte fungi, especially 2 species, and , are the predominant pathogens. Topical antifungal drugs, mainly azoles or allyamines, are currently used for the treatment of dermatophytoses, although in some cases, such as in nail and hair involvement, systemic treatment is required. However, therapeutic efficacy of current antifungal agents can be limited by their side effects, costs, and the emergence of drug resistance among fungi. Plant extracts represent a potential source of active antimicrobial agents, due to the presence of a variety of chemical bioactive compounds. In the present work, we evaluated in silico and in vitro the antifungal activity of an extract of the medicinal plant against suggesting a potential interaction with Hsp90 as playing an important role in both pathogenicity and drug susceptibility of .

Methods: We investigated in vitro the effect of different concentrations of (from 500 to 31.25 µg) against a clinical isolate of . Furthermore, using a computational assessment, the interaction between different active compounds and the fungal Hsp90 was also investigated.

Results: Our results indicate a clear-cut antifungal activity of the total plant extract at the highest concentrations (500 and 250 µg). Among all tested compounds, the luteolin and rutin molecules have been identified in silico as the most important potential inhibitors of Hsp90. Based on these data, luteolin and rutin were also individually assessed for their antifungal activity. Results demonstrate that both substances display an antifungal effect, even if lower than that of the total plant extract.

Conclusion: Our data indicate a strong fungistatic effect of against , suggesting its potential therapeutic efficacy in the treatment of dermatophytoses. Additionally, compounds, and particularly luteolin and rutin, are all possible Hsp90 interactors, explaining their fungistatic activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2147/DDDT.S155610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047602PMC
December 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/19420862.2018.1494105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204841PMC
October 2018

Succinic semialdehyde dehydrogenase deficiency: The combination of a novel ALDH5A1 gene mutation and a missense SNP strongly affects SSADH enzyme activity and stability.

Mol Genet Metab 2018 07 2;124(3):210-215. Epub 2018 Jun 2.

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

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder of GABA catabolism. SSADH is a mitochondrial homotetrameric enzyme encoded by ALDH5A1 gene. We report the molecular characterization of ALDH5A1 gene in an Italian SSADHD patient, showing heterozygosity for four missense mutations: c.526G>A (p.G176R), c.538C>T (p.H180Y), c.709G>T (p.A237S) and c.1267A>T (p.T423S), the latter never described so far. The patient inherited c.526A in cis with c.538T from the mother and c.709T in cis with c.1267T from the father. To explore the effects of the two allelic arrangements on SSADH activity and protein level, wild type, single or double mutated cDNA constructs were expressed in a cell system. The p.G176R change, alone or in combination with p.H180Y, causes the abolishment of enzyme activity. Western blot analysis showed a strongly reduced amount of the p.176R-p.180Y double mutant protein, suggesting increased degradation. Indeed, in silico analyses confirmed high instability of this mutant homotetramer. Enzyme activity relative to the other p.423S-p.237S double mutant is around 30% of wt. Further in silico analyses on all the possible combinations of mutant monomers suggest the lowest stability for the tetramer constituted by p.176R-p.180Y monomers and the highest stability for that constituted by p.237S-p.423S monomers. The present study shows that when a common SNP, associated with a slight reduction of SSADH activity, is inherited in cis with a mutation showing no consequences on the enzyme function, the activity is strongly affected. In conclusion, the peculiar arrangement of four missense mutations occurring in this patient is responsible for the SSADHD phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymgme.2018.05.006DOI Listing
July 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nano.2018.02.002DOI Listing
June 2018

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.abb.2017.10.015DOI Listing
December 2017

SSADH deficiency in an Italian family: a novel ALDH5A1 gene mutation affecting the succinic semialdehyde substrate binding site.

Metab Brain Dis 2017 10 29;32(5):1383-1388. Epub 2017 Jun 29.

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

SSADH deficiency (SSADHD) is a rare autosomal recessively inherited metabolic disorder. It is associated with mutations of ALDH5A1 gene, coding for the homotetrameric enzyme SSADH. This enzyme is involved in γ-aminobutyric acid (GABA) catabolism, since it oxidizes succinic semialdehyde (SSA) to succinate. Mutations in ALDH5A1 gene result in the abnormal accumulation of γ-hydroxybutyrate (GHB), which is pathognomonic of SSADHD. In the present report, diagnosis of SSADHD in a three-month-old female was achieved by detection of high levels of GHB in urine. Sequence analysis of ALDH5A1 gene showed that the patient was a compound heterozygote for c.1226G > A (p.G409D) and the novel missense mutation, c.1498G > C (p.V500 L). By ALDH5A1 gene expression in transiently transfected HEK293 cells and enzyme activity assays, we demonstrate that the p.V500 L mutation, despite being conservative, produces complete loss of enzyme activity. In silico protein modelling analysis and evaluation of tetramer destabilizing energies suggest that structural impairment and partial occlusion of the access channel to the active site affect enzyme activity. These findings add further knowledge on the missense mutations associated with SSADHD and the molecular mechanisms underlying the loss of the enzyme activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11011-017-0058-5DOI Listing
October 2017

Multiple molecular dynamics simulations of human LOX-1 and Trp150Ala mutant reveal the structural determinants causing the full deactivation of the receptor.

Proteins 2017 Oct 12;85(10):1902-1912. Epub 2017 Jul 12.

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

Multiple classical molecular dynamics simulations have been applied to the human LOX-1 receptor to clarify the role of the Trp150Ala mutation in the loss of binding activity. Results indicate that the substitution of this crucial residue, located at the dimer interface, markedly disrupts the wild-type receptor dynamics. The mutation causes an irreversible rearrangement of the subunits interaction pattern that in the wild-type protein allows the maintaining of a specific symmetrical motion of the monomers. The subunits dislocation determines a loss of linearity of the arginines residues composing the basic spine and a consequent alteration of the long-range electrostatic attraction of the substrate. Moreover, the anomalous subunits arrangement observed in the mutated receptor also affects the integrity of the hydrophobic tunnel, actively involved in the short-range hydrophobic recognition of the substrate. The combined effect of these structural rearrangements generates the impairing of the receptor function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/prot.25344DOI Listing
October 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.6b11470DOI Listing
April 2017

Intrinsic Dynamics Analysis of a DNA Octahedron by Elastic Network Model.

Molecules 2017 Jan 16;22(1). Epub 2017 Jan 16.

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

DNA is a fundamental component of living systems where it plays a crucial role at both functional and structural level. The programmable properties of DNA make it an interesting building block for the construction of nanostructures. However, molecular mechanisms for the arrangement of these well-defined DNA assemblies are not fully understood. In this paper, the intrinsic dynamics of a DNA octahedron has been investigated by using two types of Elastic Network Models (ENMs). The application of ENMs to DNA nanocages include the analysis of the intrinsic flexibilities of DNA double-helices and hinge sites through the calculation of the square fluctuations, as well as the intrinsic collective dynamics in terms of cross-collective map calculation coupled with global motions analysis. The dynamics profiles derived from ENMs have then been evaluated and compared with previous classical molecular dynamics simulation trajectories. The results presented here revealed that ENMs can provide useful insights into the intrinsic dynamics of large DNA nanocages and represent a useful tool in the field of structural DNA nanotechnology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules22010145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155889PMC
January 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10822-017-0014-9DOI Listing
May 2017

Polymorphism in Mitochondrial Coding Regions of Mediterranean Loggerhead Turtles: Evolutionary Relevance and Structural Effects.

Physiol Biochem Zool 2016 Nov/Dec;89(6):473-486. Epub 2016 Sep 15.

We sequenced coding portions (1.6 kb) of the mtDNA in 170 loggerhead (Caretta caretta) turtles sampled in the central Mediterranean. The sequences spanned the entire ND1 and ND3 genes, the tRNAGly and tRNAArg, plus the 3' and 5' termini of COXIII and ND4L genes, respectively. Based on our sequencing results and published complete mitogenomes, we constructed a maximum parsimony phylogeny of C. caretta matrilines that sheds new light on the evolutionary relationships within the collection of lineages found in the Mediterranean and so far recognized by D-loop haplotypes only. We show that the new variants are useful to understand the ancestry of extant haplotypes, to improve genetically based studies on the philopatry and migratory behavior of the species, and for conservation purposes. In order to better understand the biological significance of the observed variation, we addressed intraspecific nonsynonymous substitutions in the context of the three-dimensional modeled structures of ND1 and ND3. The positions of variant amino acids within the folded subunits are consistent with a coadaptation with the restructuring of membrane thickness, fluidity, and lipid composition, a well-known response mechanism to thermal conditions. The pattern of amino acid substitutions departs from neutrality, suggesting local adaptation and/or polymorphism-based local selection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1086/688679DOI Listing
June 2017

DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family.

Nanoscale 2016 Jul 24;8(27):13333-41. Epub 2016 Jun 24.

Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.

In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c6nr01806hDOI Listing
July 2016

A Simple and Fast Semiautomatic Procedure for the Atomistic Modeling of Complex DNA Polyhedra.

J Chem Inf Model 2016 05 18;56(5):941-9. Epub 2016 Apr 18.

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

A semiautomatic procedure to build complex atomistic covalently linked DNA nanocages has been implemented in a user-friendly, free, and fast program. As a test set, seven different truncated DNA polyhedra, composed by B-DNA double helices connected through short single-stranded linkers, have been generated. The atomistic structures, including a tetrahedron, a cube, an octahedron, a dodecahedron, a triangular prism, a pentagonal prism, and a hexagonal prism, have been probed through classical molecular dynamics and analyzed to evaluate their structural and dynamical properties and to highlight possible building faults. The analysis of the simulated trajectories also allows us to investigate the role of the different geometries in defining nanocages stability and flexibility. The data indicate that the cages are stable and that their structural and dynamical parameters measured along the trajectories are slightly affected by the different geometries. These results demonstrate that the constraints imposed by the covalent links induce an almost identical conformational variability independently of the three-dimensional geometry and that the program presented here is a reliable and valid tool to engineer DNA nanostructures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jcim.5b00586DOI Listing
May 2016

Virtual Screening for the Development of Dual-Inhibitors Targeting Topoisomerase IB and Tyrosyl-DNA Phosphodiesterase 1.

Curr Drug Targets 2017 ;18(5):544-555

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

Human topoisomerase IB is an important target in cancer therapy and drugs selectively stabilizing the topoisomerase IB-DNA covalent complex are in clinical use for several cancer types. Tyrosyl- DNA phosphodiesterase 1 is involved in the DNA repair resolving the topoisomerase IB-DNA covalent complex that is extremely dangerous for the survival of the cells since it produces an irreversible DNA damage. Given the close biological relationship between these two enzymes, the development of synergistic inhibitors, called dual-inhibitors, is an important challenge in cancer therapy and computer-aided drug design may help in the identification of the best compounds. In this review, an overview of the compounds inhibiting one of the two enzymes or acting as dual inhibitors is provided. Moreover, the general procedures of the virtual screening approach, providing a description of two widely used opensource programs, namely AutoDock4 and AutoDock Vina, are described. Finally, an application of the two programs on a selected number of dual inhibitors for tyrosyl-DNA phosphodiesterase 1 and topoisomerase IB and their performance is briefly discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1389450116666150727114742DOI Listing
September 2017