Publications by authors named "Giovanni Maga"

194 Publications

Targeting DDX3X Helicase Activity with BA103 Shows Promising Therapeutic Effects in Preclinical Glioblastoma Models.

Cancers (Basel) 2021 Nov 7;13(21). Epub 2021 Nov 7.

Department of Biotechnology, Chemistry & Pharmacy, University of Siena, I-53100 Siena, Italy.

DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.
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http://dx.doi.org/10.3390/cancers13215569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8582824PMC
November 2021

Bithiazole Inhibitors of Phosphatidylinositol 4-Kinase (PI4KIIIβ) as Broad-Spectrum Antivirals Blocking the Replication of SARS-CoV-2, Zika Virus, and Human Rhinoviruses.

ChemMedChem 2021 Aug 11. Epub 2021 Aug 11.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Parco Area delle Scienze, 27/A, 43124, Parma, Italy.

Over half a century since the description of the first antiviral drug, "old" re-emerging viruses and "new" emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS-CoV-2 entry/replication is debated.
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http://dx.doi.org/10.1002/cmdc.202100483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8427023PMC
August 2021

System-oriented optimization of multi-target 2,6-diaminopurine derivatives: Easily accessible broad-spectrum antivirals active against flaviviruses, influenza virus and SARS-CoV-2.

Eur J Med Chem 2021 Nov 5;224:113683. Epub 2021 Jul 5.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Viale delle Scienze, 27/A, 43124, Parma, Italy. Electronic address:

The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC = 0.5-5.3 μM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC = 0.5 μM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets.
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http://dx.doi.org/10.1016/j.ejmech.2021.113683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255191PMC
November 2021

High Flexibility of RNaseH2 Catalytic Activity with Respect to Non-Canonical DNA Structures.

Int J Mol Sci 2021 May 14;22(10). Epub 2021 May 14.

Institute of Molecular Genetics IGM-CNR "Luigi Luca Cavalli-Sforza", via Abbiategrasso 207, I-27100 Pavia, Italy.

Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2'-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi-Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences.
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http://dx.doi.org/10.3390/ijms22105201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155979PMC
May 2021

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Int J Mol Sci 2021 Feb 27;22(5). Epub 2021 Feb 27.

Institute of Molecular Genetics IGM-CNR "Luigi Luca Cavalli-Sforza", Via Abbiategrasso 207, I-27100 Pavia, Italy.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.
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http://dx.doi.org/10.3390/ijms22052365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956399PMC
February 2021

Host DDX Helicases as Possible SARS-CoV-2 Proviral Factors: A Structural Overview of Their Hijacking Through Multiple Viral Proteins.

Front Chem 2020 10;8:602162. Epub 2020 Dec 10.

Institute of Biostructures and Bioimaging (IBB-CNR), Naples, Italy.

As intracellular parasites, viruses hijack the host cell metabolic machinery for their replication. Among other cellular proteins, the DEAD-box (DDX) RNA helicases have been shown to be hijacked by coronaviruses and to participate in essential DDX-mediated viral replication steps. Human DDX RNA helicases play essential roles in a broad array of biological processes and serve multiple roles at the virus-host interface. The viral proteins responsible for DDX interactions are highly conserved among coronaviruses, suggesting that they might also play conserved functions in the SARS-CoV-2 replication cycle. In this review, we provide an update of the structural and functional data of DDX as possible key factors involved in SARS-CoV-2 hijacking mechanisms. We also attempt to fill the existing gaps in the available structural information through homology modeling. Based on this information, we propose possible paths exploited by the virus to replicate more efficiently by taking advantage of host DDX proteins. As a general rule, sequestration of DDX helicases by SARS-CoV-2 is expected to play a pro-viral role in two ways: by enhancing key steps of the virus life cycle and, at the same time, by suppressing the host innate immune response.
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http://dx.doi.org/10.3389/fchem.2020.602162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769135PMC
December 2020

Plant Molecular Farming as a Strategy Against COVID-19 - The Italian Perspective.

Front Plant Sci 2020 14;11:609910. Epub 2020 Dec 14.

Department of Biotechnology, University of Verona, Verona, Italy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 37,000 people in Italy and has caused widespread socioeconomic disruption. Urgent measures are needed to contain and control the virus, particularly diagnostic kits for detection and surveillance, therapeutics to reduce mortality among the severely affected, and vaccines to protect the remaining population. Here we discuss the potential role of plant molecular farming in the rapid and scalable supply of protein antigens as reagents and vaccine candidates, antibodies for virus detection and passive immunotherapy, other therapeutic proteins, and virus-like particles as novel vaccine platforms. We calculate the amount of infrastructure and production capacity needed to deal with predictable subsequent waves of COVID-19 in Italy by pooling expertise in plant molecular farming, epidemiology and the Italian health system. We calculate the investment required in molecular farming infrastructure that would enable us to capitalize on this technology, and provide a roadmap for the development of diagnostic reagents and biopharmaceuticals using molecular farming in plants to complement production methods based on the cultivation of microbes and mammalian cells.
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http://dx.doi.org/10.3389/fpls.2020.609910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768017PMC
December 2020

Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases.

Nucleic Acids Res 2020 11;48(20):11551-11565

Institute of Molecular Genetics IGM-CNR 'Luigi Luca Cavalli-Sforza', via Abbiategrasso 207, I-27100 Pavia, Italy.

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5' of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols β and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.
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http://dx.doi.org/10.1093/nar/gkaa948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672437PMC
November 2020

Novel Insights into the Biochemical Mechanism of CK1ε and its Functional Interplay with DDX3X.

Int J Mol Sci 2020 Sep 3;21(17). Epub 2020 Sep 3.

Institute of Molecular Genetics IGM-CNR "Luigi Luca Cavalli-Sforza", via Abbiategrasso 207, 27100 Pavia, Italy.

Casein Kinase 1 epsilon (CK1ε) is a member of the serine (Ser)/threonine (Thr) CK1 family, known to have crucial roles in several biological scenarios and, ever more frequently, in pathological contexts, such as cancer. Recently, the human DEAD-box RNA helicase 3 X-linked (DDX3X), involved in cancer proliferation and viral infections, has been identified as one of CK1ε substrates and its positive regulator in the Wnt/β-catenin network. However, the way by which these two proteins influence each other has not been fully clarified. In order to further investigate their interplay, we defined the kinetic parameters of CK1ε towards its substrates: ATP, casein, Dvl2 and DDX3X. CK1ε affinity for ATP depends on the nature of the substrate: increasing of casein concentrations led to an increase of Km, while increasing DDX3X reduced it. In literature, DDX3X is described to act as an allosteric activator of CK1ε. However, when we performed kinase reactions combining DDX3X and casein, we did not find a positive effect of DDX3X on casein phosphorylation by CK1ε, while both substrates were phosphorylated in a competitive manner. Moreover, CK1ε positively stimulates DDX3X ATPase activity. Our data provide a more detailed kinetic characterization on the functional interplay of these two proteins.
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http://dx.doi.org/10.3390/ijms21176449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503845PMC
September 2020

New indolylarylsulfone non-nucleoside reverse transcriptase inhibitors show low nanomolar inhibition of single and double HIV-1 mutant strains.

Eur J Med Chem 2020 Dec 11;208:112696. Epub 2020 Aug 11.

Laboratory Affiliated to Istituto Pasteur Italia - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie Del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185, Rome, Italy. Electronic address:

We designed and synthesized 21 new indolylarylsulfones (IASs) as new HIV-1 NNRTIs. Among these, IAS 12 exhibited a remarkable antiviral activity against single and double mutants (K103N EC = <0.7 nM; Y181C EC = <0.7 nM; Y188L EC = 21.3 nM; K103N-Y181C EC = 6.2 nM), resulting equally or more active than previuosly reported IAS 6 and some approved anti-HIV-1 drugs. Docking and molecular dynamics simulations of compound 12 in complex with WT, Y181C, Y188L, K103N and K103N-Y181C RTs clarified a general binding mode that was consistent with biological results. Kinetic experiments disclosed that derivative 12 preferentially binds WT and K103N-Y181C RTs to binary and ternary complexes, respectively.
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http://dx.doi.org/10.1016/j.ejmech.2020.112696DOI Listing
December 2020

Unique Domain for a Unique Target: Selective Inhibitors of Host Cell DDX3X to Fight Emerging Viruses.

J Med Chem 2020 09 18;63(17):9876-9887. Epub 2020 Aug 18.

Istituto di Genetica Molecolare IGM-CNR "Luigi Luca Cavalli-Sforza", Via Abbiategrasso 207, I-27100 Pavia, Italy.

Emerging viruses like dengue, West Nile, chikungunya, and Zika can cause widespread viral epidemics. Developing novel drugs or vaccines against specific targets for each virus is a difficult task. As obligate parasites, all viruses exploit common cellular pathways, providing the possibility to develop broad-spectrum antiviral agents targeting host factors. The human DEAD-box RNA helicase DDX3X is an essential cofactor for viral replication but dispensable for cell viability. Herein, we exploited the presence of a unique structural motif of DDX3X not shared by other cellular enzymes to develop a theoretical model to aid in the design of a novel class of highly selective inhibitors acting against such specific targets, thus limiting off-targeting effects. High-throughput virtual screening led us to identify hit compound , endowed with promising antienzymatic activity. To improve its aqueous solubility, and its two enantiomers were synthesized and converted into their corresponding acetate salts (compounds , , and ). mutagenesis and biochemical and cellular assays further confirmed that the developed molecules were selective for DDX3X and were able to suppress replication of West Nile and dengue viruses in infected cells in the micromolar range while showing no toxicity for uninfected cells. These results provide proof of principle for a novel strategy in developing highly selective and broad-spectrum antiviral molecules active against emerging and dangerous viral pathogens. This study paves the way for the development of larger focused libraries targeting such domain to expand SAR studies and fully characterize their mode of interaction.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01039DOI Listing
September 2020

DDX3X inhibitors, an effective way to overcome HIV-1 resistance targeting host proteins.

Eur J Med Chem 2020 Aug 7;200:112319. Epub 2020 May 7.

Istituto di Genetica Molecolare "Luigi Luca Cavalli - Sforza", IGM-CNR, Via Abbiategrasso 207, I-27100, Pavia, Italy. Electronic address:

The huge resources that had gone into Human Immunodeficiency virus (HIV) research led to the development of potent antivirals able to suppress viral load in the majority of treated patients, thus dramatically increasing the life expectancy of people living with HIV. However, life-long treatments could result in the emergence of drug-resistant viruses that can progressively reduce the number of therapeutic options, facilitating the progression of the disease. In this scenario, we previously demonstrated that inhibitors of the human DDX3X helicase can represent an innovative approach for the simultaneous treatment of HIV and other viral infections such as Hepatitis c virus (HCV). We reported herein 6b, a novel DDX3X inhibitor that thanks to its distinct target of action is effective against HIV-1 strains resistant to currently approved drugs. Its improved in vitro ADME properties allowed us to perform preliminary in vivo studies in mice, which highlighted optimal biocompatibility and an improved bioavailability. These results represent a significant advancement in the development of DDX3X inhibitors as a novel class of broad spectrum and safe anti-HIV-1 drugs.
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http://dx.doi.org/10.1016/j.ejmech.2020.112319DOI Listing
August 2020

A Structural View of SARS-CoV-2 RNA Replication Machinery: RNA Synthesis, Proofreading and Final Capping.

Cells 2020 05 20;9(5). Epub 2020 May 20.

Institute of Biostructures and Bioimaging, IBB, CNR, 80134 Naples, Italy.

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.
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http://dx.doi.org/10.3390/cells9051267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291026PMC
May 2020

Exploring the Implication of DDX3X in DENV Infection: Discovery of the First-in-Class DDX3X Fluorescent Inhibitor.

ACS Med Chem Lett 2020 May 9;11(5):956-962. Epub 2020 Apr 9.

Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, via Aldo Moro 2, 53100 Siena, Italy.

In the absence of effective drugs or vaccines for the treatment of the five Dengue Virus serotypes, the search for novel antiviral drugs is of primary importance for the scientific community. In this context, drug repurposing represents the most used strategy; however, the study of host targets is now attracting attention since it allows identification of broad-spectrum drugs endowed with high genetic barrier. In the last ten years our research group identified several small molecules DDX3X inhibitors and proved their efficacy against different viruses including novel emerging ones. Herein, starting from a screening of our compounds, we designed and synthesized novel derivatives with potent activity and high selectivity. Finally, we synthesized a fluorescent inhibitor that allowed us to study DDX3X cellular localization during DENV infection . Immunofluorescence analysis showed that our inhibitor colocalized with DDX3X, promoting the reduction of infected cells and recovering the number of viable cells.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236276PMC
May 2020

Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach †.

J Med Chem 2019 12 3;62(23):10833-10847. Epub 2019 Dec 3.

Dipartimento di Scienze degli Alimenti e del Farmaco , Università degli Studi di Parma , Viale delle Scienze, 27/A , 43124 Parma , Italy.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients' age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure-activity relationship (SAR) study that led to compound . This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01416DOI Listing
December 2019

Synthesis and Antiviral Activity of Novel 1,3,4-Thiadiazole Inhibitors of DDX3X.

Molecules 2019 Nov 4;24(21). Epub 2019 Nov 4.

Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. Moro 2, I-53100 Siena, Italy.

The human ATPase/RNA helicase X-linked DEAD-box polypeptide 3 (DDX3X) emerged as a novel therapeutic target in the fight against both infectious diseases and cancer. Herein, a new family of DDX3X inhibitors was designed, synthesized, and tested for its inhibitory action on the ATPase activity of the enzyme. The potential use of the most promising derivatives it has been investigated by evaluating their anti-HIV-1 effects, revealing inhibitory activities in the low micromolar range. A preliminary ADME analysis demonstrated high metabolic stability and good aqueous solubility. The promising biological profile, together with the suitable in vitro pharmacokinetic properties, make these novel compounds a very good starting point for further development.
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http://dx.doi.org/10.3390/molecules24213988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6864647PMC
November 2019

Identification of a novel antiviral micro-RNA targeting the NS1 protein of the H1N1 pandemic human influenza virus and a corresponding viral escape mutation.

Antiviral Res 2019 11 27;171:104593. Epub 2019 Aug 27.

Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, 27100, Pavia, Italy. Electronic address:

The influenza A virus (IAV) NS1 protein is one of the major regulators of pathogenicity, being able to suppress innate immune response and host protein synthesis. In this study we identified the human micro RNA hsa-miR-1307-3p as a novel potent suppressor of NS1 expression and influenza virus replication. Transcriptomic analysis indicates that hsa-miR-1307-3p also negatively regulates apoptosis and promotes cell proliferation. In addition, we identified a novel mutation in the NS1 gene of A(H1N1)pdm09 strains circulating in Italy in the 2010-11 season, which enabled the virus to escape the hsa-miR-1307-3p inhibition, conferring replicative advantage to the virus in human cells. To the best of our knowledge, this is the first validation of suppression of IAV H1N1 NS1 by a human micro RNA and the first example of an escape mutation from micro RNA-mediated antiviral response for the A(H1N1)pdm09 virus.
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http://dx.doi.org/10.1016/j.antiviral.2019.104593DOI Listing
November 2019

Identification of a new family of pyrazolo[3,4-d]pyrimidine derivatives as multitarget Fyn-Blk-Lyn inhibitors active on B- and T-lymphoma cell lines.

Eur J Med Chem 2019 Nov 18;181:111545. Epub 2019 Jul 18.

Department of Pharmacy, University of Genoa, Viale Benedetto XV, 3, 16132, Genoa, Italy.

Abnormal activation of B-cell receptor (BCR) signaling plays a key role in the development of lymphoid malignancies, and could be reverted by the simultaneous inhibition of Lyn, Fyn and Blk, three members of the Src family kinase (SFK). Fyn and Blk are also promising targets for the treatment of some forms of T-cell non-Hodgkin lymphoma which point to the druggability of SFKs for the treatment of these cancers. We recently identified Si308 as a potent Fyn inhibitor, while preliminary data showed that it might also inhibit Lyn and Blk. Here, molecular modelling studies were coupled with enzymatic assays to further investigate the effect of Si308 on Lyn and Blk. A small library of pyrazolo[3,4-d]pyrimidines structurally related to Si308 was synthesized and tested on human lymphoma cell lines. Compound 2h emerged as a new multitarget inhibitor of Lyn, Fyn and Blk endowed with remarkable antiproliferative effects on human B and T lymphoma cell lines. Its favorable ADME properties make the compound suitable for further developments.
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http://dx.doi.org/10.1016/j.ejmech.2019.07.048DOI Listing
November 2019

From the magic bullet to the magic target: exploiting the diverse roles of DDX3X in viral infections and tumorigenesis.

Future Med Chem 2019 06 28;11(11):1357-1381. Epub 2019 Feb 28.

Department of DNA Enzymology & Molecular Virology, Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy.

DDX3X is an ATPase/RNA helicase of the DEAD-box family and one of the most multifaceted helicases known up to date, acting in RNA metabolism, cell cycle control, apoptosis, stress response and innate immunity. Depending on the virus or the viral cycle stage, DDX3X can act either in a proviral fashion or as an antiviral factor. Similarly, in different cancer types, it can act either as an oncogene or a tumor-suppressor gene. Accumulating evidence indicated that DDX3X can be considered a promising target for anticancer and antiviral chemotherapy, but also that its exploitation requires a deeper understanding of the molecular mechanisms underlying its dual role in cancer and viral infections. In this Review, we will summarize the known roles of DDX3X in different tumor types and viral infections, and the different inhibitors available, illustrating the possible advantages and potential caveats of their use as anticancer and antiviral drugs.
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http://dx.doi.org/10.4155/fmc-2018-0451DOI Listing
June 2019

DDX3X Helicase Inhibitors as a New Strategy To Fight the West Nile Virus Infection.

J Med Chem 2019 03 21;62(5):2333-2347. Epub 2019 Feb 21.

Dipartimento Biotecnologie, Chimica e Farmacia , Università degli Studi di Siena , Via A. De Gasperi 2 , I-53100 Siena , Italy.

Increased frequency of arbovirus outbreaks in the last 10 years represents an important emergence for global health. Climate warming, extensive urbanization of tropical regions, and human migration flows facilitate the expansion of anthropophilic mosquitos and the emerging or re-emerging of new viral infections. Only recently the human adenosinetriphosphatase/RNA helicase X-linked DEAD-box polypeptide 3 (DDX3X) emerged as a novel therapeutic target in the fight against infectious diseases. Herein, starting from our previous studies, a new family of DDX3X inhibitors was designed, synthesized, validated on the target enzyme, and evaluated against the West Nile virus (WNV) infection. Time of addition experiments after virus infection indicated that the compounds exerted their antiviral activities after the entry process, likely at the protein translation step of WNV replication. Finally, the most interesting compounds were then analyzed for their in vitro pharmacokinetic parameters, revealing favorable absorption, distribution, metabolism, and excretion values. The good safety profile together with a good activity against WNV for which no treatments are currently available, make this new class of molecules a good starting point for further in vivo studies.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01403DOI Listing
March 2019

Effect of α-Methoxy Substitution on the Anti-HIV Activity of Dihydropyrimidin-4(3 H)-ones.

J Med Chem 2019 01 20;62(2):604-621. Epub 2018 Dec 20.

Dipartimento di Chimica e Tecnologie del Farmaco , Università degli Studi di Roma "La Sapienza" , P. le A. Moro 5 , 00185 Roma , Italy.

Conformational restriction applied to dihydrobenzylpyrimidin-4-(3 H)-ones (DABOs) by the intoduction of a methyl group at the α-benzylic position is known to massively improve the anti-HIV-1 activity of these compounds. Here, we report the effects of methoxy substitution at the α-benzylic position in S-, NH-, and N, N-DABOs carrying 2,6-difluoro, 2-chloro-6-fluoro, or 2,6-dichloro substituted benzyl moieties. The various α-methoxy DABO series (12-14) present different SAR at the dihalo benzyl substitution, with the most potent compounds (12d,e and 13c) showing similar (picomolar/nanomolar) anti-HIV-1 potency as the corresponding α-methyl analogues against wt HIV-1, and 10-100-fold increased potency (up to low nanomolar) against clinically relevant K103N, Y181C, Y188L, IRLL98, and K103N+Y181C HIV-1 mutant strains, highlighting the importance of the α-methoxy substitution to provide highly efficient DABOs as "second generation" NNRTIs. HPLC enantioseparation of three of the most potent derivatives (12d, 13c, and 14c) provided single enantiomers with significant enantioselectivity in HIV-1 inhibition. Computational studies allowed to correlate the best antiviral activity with the ( R) absolute configuration at the α-methoxy stereogenic center.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01238DOI Listing
January 2019

Efficient optimization of pyrazolo[3,4-d]pyrimidines derivatives as c-Src kinase inhibitors in neuroblastoma treatment.

Bioorg Med Chem Lett 2018 11 20;28(21):3454-3457. Epub 2018 Sep 20.

Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology Temple University, BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, PA 19122, United States; Lead Discovery Siena S.r.l., Via Vittorio Alfieri 31, 53019 Castelnuovo Berardenga, Siena, Italy. Electronic address:

The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar K values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.
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http://dx.doi.org/10.1016/j.bmcl.2018.09.024DOI Listing
November 2018

p15PAF binding to PCNA modulates the DNA sliding surface.

Nucleic Acids Res 2018 10;46(18):9816-9828

Structural Biology Laboratory, Elettra-Sincrotrone Trieste S.C.p.A., Trieste 34149, Italy.

p15PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF-PCNA-DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15PAF impairs primer synthesis by pol η-PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15PAF in DNA replication and suppression of DNA lesion bypass.
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http://dx.doi.org/10.1093/nar/gky723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182140PMC
October 2018

Identification of Broad-Spectrum Dengue/Zika Virus Replication Inhibitors by Functionalization of Quinoline and 2,6-Diaminopurine Scaffolds.

ChemMedChem 2018 07 1;13(14):1371-1376. Epub 2018 Jun 1.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Viale delle Scienze, 27/A, 43124, Parma, Italy.

Social and demographic changes across the world over the past 50 years have resulted in significant outbreaks of arboviruses such as dengue virus (DENV) and Zika virus (ZIKV). Despite the increased threat of infection, no approved drugs or fully protective vaccines are available to counteract the spread of DENV and ZIKV. The development of "broad-spectrum" antivirals (BSAs) that target common components of multiple viruses can be a more effective strategy to limit the rapid emergence of viral pathogens than the classic "one-bug/one-drug" approach. Starting from previously identified multitarget DENV inhibitors, herein we report the identification of novel 2,6-diaminopurine derivatives that are able to block the replication of both Zika virus and all serotypes of dengue virus (DENV 1-4) in infected cells.
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http://dx.doi.org/10.1002/cmdc.201800178DOI Listing
July 2018

Living on the Edge: DNA Polymerase Lambda between Genome Stability and Mutagenesis.

Chem Res Toxicol 2017 11 8;30(11):1936-1941. Epub 2017 Sep 8.

DNA Enzymology & Molecular Virology and Cell Nucleus & DNA replication Units, Institute of Molecular Genetics IGM-CNR , via Abbiategrasso 207, I-27100 Pavia, Italy.

In human cells, only four DNA polymerases (pols) are necessary and sufficient for the duplication of the genetic information. However, more than a dozen DNA pols are required to maintain its integrity. Such a high degree of specialization makes DNA repair pols able to cope with specific lesions or repair pathways. On the other hand, the same DNA pols can have partially overlapping roles, which could result in possible conflicts of functions, if the DNA pols are not properly regulated. DNA pol λ is a typical example of such an enzyme. It is a multifunctional enzyme, endowed with special structural and biochemical properties, which make it capable of participating in different DNA repair pathways such as base excision repair, nonhomologous end joining, and translesion synthesis. However, when mutated or deregulated, DNA pol λ can also be a source of genetic instability. Its multiple roles in DNA damage tolerance and its ability in promoting tumor progression make it also a possible target for novel anticancer approaches.
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http://dx.doi.org/10.1021/acs.chemrestox.7b00152DOI Listing
November 2017

Chiral Indolylarylsulfone Non-Nucleoside Reverse Transcriptase Inhibitors as New Potent and Broad Spectrum Anti-HIV-1 Agents.

J Med Chem 2017 08 5;60(15):6528-6547. Epub 2017 Jul 5.

Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma , Piazzale Aldo Moro 5, I-00185 Roma, Italy.

We designed and synthesized a series of chiral indolyarylsulfones (IASs) as new HIV-1 NNRTIs. The new IASs 8-37 showed potent inhibition of the HIV-1 WT NL4-3 strain and of the mutant K103N, Y181C, Y188L, and K103N-Y181C HIV-1 strains. Six racemic mixtures, 8, 23-25, 31, and 33, were separated at semipreparative level into their pure enantiomers. The (R)-8 enantiomer bearing the chiral (α-methylbenzyl) was superior to the (S)-counterpart. IAS derivatives bearing the (S) alanine unit, (S)-23, (S,R)-25, (S)-31, and (S)-33, were remarkably more potent than the corresponding (R)-enantiomers. Compound 23 protected hippocampal neuronal cells from the excitotoxic insult, while efavirenz (EFV) did not contrast the neurotoxic effect of glutamate. The present results highlight the chiral IASs as new NNRTIs with improved resistance profile against the mutant HIV-1 strains and reduced neurotoxic effects.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01906DOI Listing
August 2017

How to win the HIV-1 drug resistance hurdle race: running faster or jumping higher?

Biochem J 2017 04 26;474(10):1559-1577. Epub 2017 Apr 26.

DNA Enzymology & Molecular Virology Unit, Institute of Molecular Genetics - CNR, via Abbiategrasso 207, Pavia 27100, Italy

Infections by the human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), are still totaling an appalling 36.7 millions worldwide, with 1.1 million AIDS deaths/year and a similar number of yearly new infections. All this, in spite of the discovery of HIV-1 as the AIDS etiological agent more than 30 years ago and the introduction of an effective combinatorial antiretroviral therapy (cART), able to control disease progression, more than 20 years ago. Although very effective, current cART is plagued by the emergence of drug-resistant viral variants and most of the efforts in the development of novel direct-acting antiviral agents (DAAs) against HIV-1 have been devoted toward the fighting of resistance. In this review, rather than providing a detailed listing of all the drugs and the corresponding resistance mutations, we aim, through relevant examples, at presenting to the general reader the conceptual shift in the approaches that are being taken to overcome the viral resistance hurdle. From the classic 'running faster' strategy, based on the development of novel DAAs active against the mutant viruses selected by the previous drugs and/or presenting to the virus a high genetic barrier toward the development of resilience, to a 'jumping higher' approach, which looks at the cell, rather than the virus, as a source of valuable drug targets, in order to make the cellular environment non-permissive toward the replication of both wild-type and mutated viruses.
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http://dx.doi.org/10.1042/BCJ20160772DOI Listing
April 2017

Discovery of Multitarget Agents Active as Broad-Spectrum Antivirals and Correctors of Cystic Fibrosis Transmembrane Conductance Regulator for Associated Pulmonary Diseases.

J Med Chem 2017 02 10;60(4):1400-1416. Epub 2017 Feb 10.

P4T Group, Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma , Viale delle Scienze, 27/A, 43124 Parma, Italy.

Enteroviruses (EVs) are among the most frequent infectious agents in humans worldwide and represent the leading cause of upper respiratory tract infections. No drugs for the treatment of EV infections are currently available. Recent studies have also linked EV infection with pulmonary exacerbations, especially in cystic fibrosis (CF) patients, and the importance of this link is probably underestimated. The aim of this work was to develop a new class of multitarget agents active both as broad-spectrum antivirals and as correctors of the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding defect responsible for >90% of CF cases. We report herein the discovery of the first small molecules able to simultaneously act as correctors of the F508del-CFTR folding defect and as broad-spectrum antivirals against a panel of EVs representative of all major species.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01521DOI Listing
February 2017

Identification of new pyrrolo[2,3-d]pyrimidines as Src tyrosine kinase inhibitors in vitro active against Glioblastoma.

Eur J Med Chem 2017 Feb 19;127:369-378. Epub 2016 Dec 19.

Dipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy. Electronic address:

In the last few years, several pyrrolo-pyrimidine derivatives have been either approved by the US FDA and in other countries for the treatment of different diseases or are currently in phase I/II clinical trials. Herein we present the synthesis and the characterization of a novel series of pyrrolo[2,3-d]pyrimidines, compounds 8a-j, and their activity against Glioblastoma multiforme (GBM). Docking studies and MM-GBSA analysis revealed the ability of such compounds to efficiently interact with the ATP binding site of Src. Enzymatic assays against a mini-panel of kinases (Src, Fyn, EGFR, Kit, Flt3, Abl, AblT315I) have been performed, showing an unexpected selectivity of our pyrrolo[2,3-d]pyrimidines for Src. Finally, the derivatives were tested for their antiproliferative potency on U87 GBM cell line. Compound 8h showed a considerable cytotoxicity effect against U87 cell line with an IC value of 7.1 μM.
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http://dx.doi.org/10.1016/j.ejmech.2016.12.036DOI Listing
February 2017

Ribonucleotide incorporation by human DNA polymerase η impacts translesion synthesis and RNase H2 activity.

Nucleic Acids Res 2017 03;45(5):2600-2614

DNA Enzymology & Molecular Virology and Cell Nucleus & DNA replication Units, Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy.

Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.
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http://dx.doi.org/10.1093/nar/gkw1275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389505PMC
March 2017
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