Claudia Immacolata Trivisani’s research while affiliated with University of Siena and other places

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Publications (13)


Kinetic growth of SARS-CoV-2 lineages bearing Nsp1 partial deletion. (A) Vero E6 cells were infected with Omicron BA.2 and the relative ∆82–85 and ∆141–143 lineages, or with the BQ.1.1 and the ∆82–86 variants at a multiplicity of infection (MOI) of 0.01. Cell culture supernatants were collected at 24 h, 48 h, and 72 h p.i. and viable progeny virus content was assessed by a microtitration assay. The results are presented as Log10 of the mean viral titer expressed as plaque forming units (PFUs) ± standard deviations (SDs) from at least three separate experiments. (B) The expression of Nsp1 was determined by Western blotting on 50 µg of whole cell lysates (WCLs) of Vero E6 cells infected with the indicated virus lineages and collected at 24 h and 48 h p.i. The loading control is represented by the immuno-detection of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. (C) The kinetic growth of the previously described SARS-CoV-2 lineages was also investigated in type I interferon-competent Calu-3 cells. Cell culture supernatants were collected at the indicated time points and assessed for virus replication by microtitration assay. The results are plotted as Log10 of the mean PFU ± standard deviations (SDs) from at least three separate experiments. Significance is reported as ** p < 0.001, *** p < 0.0005, and **** p < 0.0001 with respect to the parental strains. On the contrary, the deletion of the 141–143 domain located at the C-terminus of BA.2 Nsp1 did not affect virus replication in Vero E6 cells, leading to virus titers similar to the parental (BA.2) strain (p > 0.05) (Figure 1A). Meanwhile, cells infected with original SARS-CoV-2 lineages (BA.2 and BQ1.1) and with the Δ141–143 virus variant showed similar levels of Nsp1 protein over time. Indeed, at 24 h and 48 h p.i., Nsp1 was substantially expressed by these virus lineages, while a different behavior was observed in CoV2-Δ82–85- and CoV2-Δ82–86-deleted viruses, which showed a low or undetectable production of Nsp1, indicating a possible role of this non-structural protein in reducing virus progeny release (Figure 1B). Moreover, replication kinetics of selected virus lineages were investigated in Calu-3 cells, which represent a well-defined pulmonary cell system to investigate SARS-CoV-2 fitness in a human, IFN-β-competent environment. The results are comparable to those observed in Vero E6 cells, confirming that the 82–85 domain is critical for viral fitness (Figure 1C). At late times of infections, CoV2-Δ141–143 also showed a reduced growth in Calu-3 cells with respect to BA.2 (p < 0.0001), albeit at a lesser extent when compared to CoV2-Δ82–85 (p = 0.001) and CoV2-Δ82–86 (p = 0.0009) (Figure 1C). Additionally, it seems that the replication kinetics of these virus strains remain unaffected in IFN-β-competent cells, suggesting that the reduced virus fitness in Calu-3 cells was not a consequence of the IFN-β susceptibility antiviral activity.
Induction of IFN-β release by partially deleted Nsp1 SARS-CoV-2 lineages. Secreted IFN-β was assessed in Calu-3 cells infected (MOI = 0.01) with BA.2 and the relative ∆82–85 and ∆141–143 lineages, or with the BQ.1.1 and the ∆82–86 variants by enzyme-linked immunoassay (ELISA) at 48 h and 72 h p.i.. Negative control (Ctr-) was represented by uninfected Calu-3 cells. Quantitative evaluation, based on relative standard curves, was performed and the results are reported as mean concentration (pg/mL) ± standard deviations (SDs) from at least three independent experiments (n ≥ 3). Significance is reported as ns, not significant, * p < 0.05 and ** p < 0.001 with respect to the mock-infected control (Ctr-).
Evaluation of host cell shutoff in Nsp1 variants expressing cells. The impact of Nsp1-deleted mutants on the human beta-globin (HBB) promoter-mediated Green Fluorescent Protein (GFP) was assessed in HEK-293T cells. (A) The transcriptional control of GFP was examined by using total RNA purified from HBB-GFP transfected samples, either in combination with an empty plasmid (Ctr-) or Nsp1-expressing plasmids. Cells were collected at 48 h post-transfection and the specific GFP mRNA content was measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). GAPDH gene expression served for relative quantification based on the 2−ΔΔCt method. At least three independent experiments (n ≥ 3) were conducted, and the representative data are presented as mean values ± standard deviations. (B) The modulation of GFP expression in Nsp1 variants expressing HEK-293T cells was also evaluated as protein levels through Western blotting. Equal amounts of total cell lysates were resolved by SDS-PAGE, and specific antibodies were used for GFP, GAPDH, and Nsp1 proteins, with a representative image provided in the lower panel. Densitometric analysis was conducted using ImageJ software. Graph values are presented as the mean fold change in GFP band intensity ± standard deviations (SDs). Significance is reported as ‘ns’, not significant, ** p < 0.001 and *** p < 0.0005 between each sample and the control (Ctr-).
Evaluation of Nsp1 variants’ modulation of viral genes expression. The impact of Nsp1-deleted mutants on viral 5′-Ld-SL1- or 5′-UTR-mediated GFP expression was evaluated in HEK-293T cells. (A) Transcriptional control of GFP was examined in 5′-Ld-SL1- or 5′-UTR-GFP-transfected samples, either in combination with an empty plasmid (Ctr-) or Nsp1-expressing plasmids. Cells were collected at 48 h, and total RNA was purified. GFP and GAPDH mRNAs were quantified by RT-qPCR using the 2−ΔΔCt analysis. Data were presented as mean values ± standard deviations (SD) from different experiments. (B) GFP protein content was assessed in Nsp1 variants expressing HEK-293T cells by Western blotting. Equal amounts of total cell lysates were resolved by SDS-PAGE, and specific antibodies were used to probe for GFP, GAPDH, and Nsp1 proteins. A representative image is provided in the lower panel. Densitometric analysis was performed using ImageJ software. Graph values are presented as the mean fold change in GFP band intensity ± standard deviations (SDs). Significance is reported as ‘ns’, not significant, * p < 0.05, ** p < 0.001, *** p < 0.0005, and **** p < 0.0001 between each sample and the control (Ctr-).
Nsp1 variants’ activity towards cellular and viral mRNAs. (A) The influence of both original and deleted Nsp1versions on cellular mRNA decay was investigated in HEK-293T cells. A time-course experiment was conducted using actinomycin D (ActD), a transcriptional inhibitor. Cells were collected at indicated time points, and after total RNA isolation, GFP mRNA was quantified by RT-qPCR using the 2−ΔΔCt analysis. (B) The impact of different Nsp1 variants on HBB-mediated GFP protein expression was assessed in HEK-293T cells, either mock-treated or treated with cycloheximide (CHX). Equal amounts of total cell lysates from samples collected at indicated time points after CHX treatment were resolved by SDS-PAGE and specific antibodies were used to probe for GFP and GAPDH. (C) GFP mRNA levels were quantified by RT-qPCR in HEK-293T cells expressing Nsp1 variants along with either 5′-Ld-SL1- or 5′-UTR-GFP and treated with ActD for specified periods. (D) HEK-293T cells expressing the GFP protein downstream of either the 5′-Ld-SL1- or 5′-UTR-GFP promoters were co-transfected in order to express different Nsp1 variants. At 48 h post-transfection, cells were mock- or CHX-treated and collected at indicated times. Equal amounts of total cell lysates were resolved by SDS-PAGE and GFP or GAPDH proteins were probed by Western blotting procedure. The protein stability of Nsp1 variants was determined by Western blotting on total cell lysates of transfected HEK-293T cells (E) or Vero E6 cells infected with indicated virus strains (F) and subjected to a time-chase with CHX. Densitometric analysis of Western blotting images was performed using ImageJ software. Graph bars represent mean values ± standard deviations (SD) from different experiments. Significance is reported as * p < 0.05, ** p < 0.001, *** p < 0.0005, and **** p < 0.0001.

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Deletion of 82–85 N-Terminal Residues in SARS-CoV-2 Nsp1 Restricts Virus Replication
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April 2024

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Non-structural protein 1 (Nsp1) represents one of the most crucial SARS-CoV-2 virulence factors by inhibiting the translation of host mRNAs and promoting their degradation. We selected naturally occurring virus lineages with specific Nsp1 deletions located at both the N- and C-terminus of the protein. Our data provide new insights into how Nsp1 coordinates these functions on host and viral mRNA recognition. Residues 82–85 in the N-terminal part of Nsp1 likely play a role in docking the 40S mRNA entry channel, preserving the inhibition of host gene expression without affecting cellular mRNA decay. Furthermore, this domain prevents viral mRNAs containing the 5′-leader sequence to escape translational repression. These findings support the presence of distinct domains within the Nsp1 protein that differentially modulate mRNA recognition, translation and turnover. These insights have implications for the development of drugs targeting viral proteins and provides new evidences of how specific mutations in SARS-CoV-2 Nsp1 could attenuate the virus.

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Figure 3. RMSD plots of imidacloprid (IMI) in structures of human nAChR α7 (7kox, 7koq). (A) Plot of the pairwise RMSD of the 50ns MD trajectory (500 frames) of the common binding mode of IMI in nAChR α7 (PDB-ID:7kox) against itself. After 280 frames, which is equivalent to 28ns simulation time, the only major conformational transition occurs. (B) Plot of the pairwise RMSD of the trajectories of the common binding mode of IMI (IMI_common; 7kox) which was compared to the trajectory of IMI in an inverted binding mode (IMI_inverted; 7koq). IMI_common converges towards the conformational space of IMI_inverted during the last 310 simulation frames. (C) Onedimensional RMSD plot of IMI equivalent to (A); a major conformational transition of the common binding mode of IMI (orange curve) occurs after 280 frames. The inverted binding mode of IMI (purple curve) shows one transition occurring at 180 simulation frames and reports an overall lower mean RMSD than the common binding mode (see also Table 2).
Statistical uncertainty of observable (MM-GBSA delta G (dG) binding energy).
Structural Insights into Neonicotinoids and N-Unsubstituted Metabolites on Human nAChRs by Molecular Docking, Dynamics Simulations, and Calcium Imaging

August 2023

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94 Reads

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4 Citations

International Journal of Molecular Sciences

Neonicotinoid pesticides were initially designed in order to achieve species selectivity on insect nicotinic acetylcholine receptors (nAChRs). However, concerns arose when agonistic effects were also detected in human cells expressing nAChRs. In the context of next-generation risk assessments (NGRAs), new approach methods (NAMs) should replace animal testing where appropriate. Herein, we present a combination of in silico and in vitro methodologies that are used to investigate the potentially toxic effects of neonicotinoids and nicotinoid metabolites on human neurons. First, an ensemble docking study was conducted on the nAChR isoforms α7 and α3β4 to assess potential crucial molecular initiating event (MIE) interactions. Representative docking poses were further refined using molecular dynamics (MD) simulations and binding energy calculations using implicit solvent models. Finally, calcium imaging on LUHMES neurons confirmed a key event (KE) downstream of the MIE. This method was also used to confirm the predicted agonistic effect of the metabolite descyano-thiacloprid (DCNT).


Figure 3. Effects of aluminum-based adjuvants (ABA).
Figure 5. 2D structures of small molecule Toll−like receptors agonists. TLR2 agonists (violet upper panel), TLR4 agonists (cyan, middle panel), TLR 7/8 agonists (green, bottom panel).
Classification of novel delivery systems and immune potentiators adjuvants.
Pathogen-associated molecular patterns (PAMPs) recognized by Toll-like receptors.
Toll-like receptors agonists in clinical trials.
Progress towards Adjuvant Development: Focus on Antiviral Therapy

May 2023

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131 Reads

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7 Citations

International Journal of Molecular Sciences

In recent decades, vaccines have been extraordinary resources to prevent pathogen diffusion and cancer. Even if they can be formed by a single antigen, the addition of one or more adjuvants represents the key to enhance the response of the immune signal to the antigen, thus accelerating and increasing the duration and the potency of the protective effect. Their use is of particular importance for vulnerable populations, such as the elderly or immunocompromised people. Despite their importance, only in the last forty years has the search for novel adjuvants increased, with the discovery of novel classes of immune potentiators and immunomodulators. Due to the complexity of the cascades involved in immune signal activation, their mechanism of action remains poorly understood, even if significant discovery has been recently made thanks to recombinant technology and metabolomics. This review focuses on the classes of adjuvants under research, recent mechanism of action studies, as well as nanodelivery systems and novel classes of adjuvants that can be chemically manipulated to create novel small molecule adjuvants.


Discovery and Optimization of a Novel Macrocyclic Amidinourea Series Active as Acidic Mammalian Chitinase Inhibitors

March 2023

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42 Reads

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2 Citations

ACS Medicinal Chemistry Letters

Our research group has been involved for a long time in the development of macrocyclic amidinoureas (MCAs) as antifungal agents. The mechanistic investigation drove us to perform an in silico target fishing study, which allowed the identification of chitinases as one of their putative targets, with 1a showing a submicromolar inhibition of Trichoderma viride chitinase. In this work, we investigated the possibility to further inhibit the corresponding human enzymes, acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1), involved in several chronic inflammatory lung diseases. Thus, we first validated the inhibitory activity of 1a against AMCase and CHIT1 and then designed and synthesized new derivatives aimed at improving the potency and selectivity against AMCase. Among them, compound 3f emerged for its activity profile along with its promising in vitro ADME properties. We also gained a good understanding of the key interactions with the target enzyme through in silico studies.


Efficient use of agricultural waste to naturally fortify Tenebrio molitor mealworms and evaluation of their nutraceutical properties

December 2022

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68 Reads

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11 Citations

Journal of Insects as Food and Feed

Circular economy principles aim to maintain resources in the economic circle. Accordingly, the use of agricultural waste and its transformation into novel products is a smart approach that creates benefits for the environment, industries and consumers. Herein, we conjugated the principles of circular economy with the search for novel sustainable functional foods, transforming agricultural food chain by-products into feed supplements for Tenebrio molitor larvae (TML) feeding. Accordingly, tomato peels and seeds (TPS) stemming from the tomato sauce production, as well as olive (Olea europaea) and mastic (Pistacia lentiscus) leaves (OEL and PLL) from pruning were finely pulverised and used as feed supplements. All the diet supplements efficiently supported larval growth, offering optimal values of larvae mean body weight and survival rate. Interestingly, both total phenol content and antioxidant activities increased compared to the control, thanks to the accumulation of active compounds with hydrophilic or lipophilic characteristics. In addition, the fatty acids composition was determined, revealing a beneficial reduction of omega-6 (n-6)/omega-3 (n-3) ratio. Taken together, our results strongly support TPS, OEL and PLL use as smart breeding supplements, able to increase the antioxidant activity and ameliorate fatty acid profile of TML with important applications in human and animal nutrition.


DEAD-Box Helicase DDX3X as a Host Target against Emerging Viruses: New Insights for Medicinal Chemical Approaches

July 2022

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72 Reads

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7 Citations

Journal of Medicinal Chemistry

In recent years, globalization, global warming, and population aging have contributed to the spread of emerging viruses, such as coronaviruses (COVs), West Nile (WNV), Dengue (DENV), and Zika (ZIKV). The number of reported infections is increasing, and considering the high viral mutation rate, it is conceivable that it will increase significantly in the coming years. The risk caused by viruses is now more evident due to the COVID-19 pandemic, which highlighted the need to find new broad-spectrum antiviral agents able to tackle the present pandemic and future epidemics. DDX3X helicase is a host factor required for viral replication. Selective inhibitors have been identified and developed into broad-spectrum antivirals active against emerging pathogens, including SARS-CoV-2 and most importantly against drug-resistant strains. This perspective describes the inhibitors identified in the last years, highlighting their therapeutic potential as innovative broad-spectrum antivirals.


Proteins From Tenebrio molitor: an Interesting Functional Ingredient and a Source of ACE Inhibitory Peptides

June 2022

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43 Reads

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28 Citations

Food Chemistry

The angiotensin-converting enzyme (ACE) inhibitory potential of the main protein fractions from Tenebrio molitor larvae (TML) was examined to evaluate their use as a novel antihypertensive functional food. Both fractions contained YAN tripeptide, previously reported as responsible for ACE inhibition. Although YAN has been synthesized and was used as a standard for LC-MS/MS quantification and IC50 against ACE was determined, low yields of YAN from TML did not explain adequately the activity of the whole protein fraction. LC-HRMS/MS investigation led to the identification of other three peptides, which were evaluated in silico, synthesized and tested against ACE. Among them, tetrapeptide NIKY showed the most promising activity (52 µM), highlighting once more the potential of TML and paving the way for exploitation in novel foods.


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

November 2021

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185 Reads

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7 Citations

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.


Si113-prodrugs selectively activated by plasmin against hepatocellular and ovarian carcinoma

June 2021

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10 Citations

European Journal of Medicinal Chemistry

Si113, a pyrazolo[3,4-d]pyrimidine derivative, gained more attention as an anticancer agent due to its potent anticancer activity on both in vitro and in vivo hepatocellular carcinomas (HCC) and ovarian carcinoma models. But the drawback is the low water solubility which prevents its further development. In this context, we successfully overcame this limitation by synthesizing two novel prodrugs introducing the amino acid sequence D-Ala-Leu-Lys (TP). Moreover, TP sequence has a high affinity with plasmin, a protease recognized as overexpressed in many solid cancers, including HCC and ovarian carcinoma. The prodrugs were synthesized and fully characterized in terms of in vitro ADME properties, plasma stability and plasmin-induced release of the parent drug. The inhibitory activity against Sgk1 was evaluated and in vitro growth inhibition was evaluated on ovarian carcinoma and HCC cell lines in the presence and absence of human plasmin. In vivo pharmacokinetic properties and preliminary tissue distribution confirmed a better profile highlighting the importance of the prodrug approach. Finally, the prodrug antitumor efficacy was evaluated in an HCC xenografted murine model, where a significant reduction (around 90%) in tumor growth was observed. Treatment with ProSi113-TP in combination with paclitaxel in a paclitaxel-resistant ovarian carcinoma xenografted murine model, resulted in an impressive reduction of tumor volume greater than 95%. Our results revealed a promising activity of Si113 prodrugs and pave the way for their further development against resistant cancer.


Figure 1. Human DDX3X has RNaseH2-like activity. (A) Time course of DDX3X (lanes 1-5) and RNaseH2 (lanes 6-10) digestions of Substrate *D 39 R 1 D 15 : D 55 . (B) Apparent digestion rates for DDX3X and RNaseH2 enzymes on Substrate *D 39 R 1 D 15 : D 55 . Values are the means of three independent estimates ± S.D. (C) Digestion by DDX3X (lanes 2 and 5) and RNaseH2 (lanes 3 and 6) on Substrate *D 19 R 1 D 4 : D 24 and Substrate *D 18 R 1 D 5 : D 24 respectively. Lanes 1 and 4: Substrates *D 19 R 1 D 4 : D 24 and *D 18 R 1 D 5 : D 24 alone, respectively. (D) Substrate *D 19 R 4 D 18 : D 41 in the presence of RNaseH2 (lanes 4-6) or DDX3X (lanes 7-9). Lanes 1-2 control reactions with RNaseH2 and Substrate *D 19 R 1 D 4 : D 24 . Lane 3 Substrate *D 19 R 4 D 18 : D 41 alone. Lanes 10, 11 oligonucleotide size markers of defined lengths, used to better identify the different digestion products. (E) Time course of DDX3X digestion of Substrate *D 39 R 1 D 15 : D 55 (lanes 1-5) and Substrate *D 39 R 1 D 15 : D 39 8oxoG 1 D 15 (lanes 7-11). Control reactions with RNaseH2 of Substrate *D 39 R 1 D 15 : D 55 (lane 6) and Substrate *D 39 R 1 D 15 : D 39 8oxoG 1 D 15 (lane 12) respectively.
Figure 2. DDX3X RNaseH2-like activity is not dependent by its helicase activity and is inhibited by ATP. (A) Titration of DDX3X wild type (lanes 2-6) in helicase reaction with a dsRNA substrate 18/38mer with a 20 nt 3'-ss overhang (Substrate *R 18 : R 38 ). Lane 1: Substrate *R 18 : R 38 alone. Lane 7: 18mer single strand. (B) Native PAGE of the products of DDX3X helicase reaction with Substrate *D 18 R 1 D 5 : D 24 in the presence (lanes 1-5) or in the absence (lanes 7-11) of ATP. Lanes 6, 12: ss24mer as marker. (C) Dependence of the DDX3X helicase activity from the enzyme concentration. (D). Dependence of the DDX3X nuclease activity from the enzyme concentration. (E) Inhibition of DDX3X nuclease activity by ATP. All values are the means of three independent estimates ± S.D.
Figure 3. Identification of DDX3X residues important for its RNaseH2-like activity. (A) Sequence alignment showing amino acid (aa) similarities between the conserved -DEAD-(box II) helicase motif of DDX3X and the catalytic site of human RNaseH2. Red letters indicate catalytically important residues. Green shading indicated identical and cyan shading similar aa. The numbers of the aa residues corresponding to each selected region are boxed. (B) Structure comparison between human DDX3X in open conformation (PDB ID: 5e7I) on the left and mouse RNaseH2 (PDB ID: 3KIO) of the region surrounding Arg351 of DDX3X (left) and Arg38 of RNaseH2 (right), showing the hydrogen bond formed by these residues with the RNA substrate. Arg351 in DDX3X and Arg38 in RNase H2 were similarly positioned within these regions. (C) Graphical representation of all the DDX3X mutants and DDX5 protein. N and C indicate the N-terminal and C-terminal of the proteins respectively. Boxes represent all the conserved motifs of DEAD-box proteins (with the addition of DDX3 unique motif). Red letters indicate single aa mutations. (D) Nuclease activities of the DDX3X mutants and DDX5 protein relative to DDX3X wild type (taken as 100%). All catalytic activities were assessed on Substrate *D 39 R 1 D 15 : D 55 . Values are the mean of four independent experiments ± S.D. All proteins were tested at 2 M. (E) Summary table of the catalytic activities shown in panel D.
Figure 4. Different DNA polymerases support full ribonucleotide excision repair together with RNaseH2. (A) RER reactions in the presence of RNaseH2, Pol and dNTPs alone (lane 3), or with DNA ligase 1 and Fen-1 (lanes 4-5). Lane 5: re-digestion by RNaseH2 of the ligated products after heat inactivation of the reaction. RER reactions in the presence of RNaseH2, Pol , PCNA and dNTPs (lane 6), or with added DNA ligase 1 and Fen-1 (lanes 7-8). Lane 8: re-digestion by RNaseH2 as in Lane 5. Lane 1: Substrate *D 39 R 1 D 15 : D 55 alone. (B) RER reactions in the presence of RNaseH2, Pol , dNTPs, Fen-1 and DNA ligase 1 (lanes 2-5). RER reactions in the presence of RNaseH2, Pol , PCNA and dNTPs (lanes 6-8), DNA ligase 1 and Fen-1 (lanes 7-8). Lanes 5 and 8: re-digestion by RNaseH2. Lane 1: Substrate *D 19 R 4 D 18 : D 41 alone. (C) RER reactions in the presence of RNaseH2 (lanes 2-3), Pol , dNTPs (lanes 4-5), Fen-1 and DNA ligase 1 (lanes 6-7). Lanes 3, 5 and 7: re-digestion by RNaseH2. Lane 1: Substrate *D 39 R 1 D 15 : D 55 alone. (D) Lane 1: Substrate *D 19 R 4 D 18 : D 41 alone. RER reactions in the presence of RNaseH2 (lanes 2-3), Pol , dNTPs (lanes 4-5), Fen-1 and DNA ligase 1 (lanes 6-7). Lanes 3, 5 and 7: re-digestion by RNaseH2.
Figure 5. A role of DDX3X in ribonucleotide excision repair. (A) RER reactions in the presence of DDX3X (lanes 2-3), Pol , dNTPs (lanes 4-5), Fen-1 and DNA ligase 1 (lanes 6-7). Lanes 3, 5, 7: re-digestion by RNaseH2 of the heat inactivated reactions. RER reactions in the presence of DDX3X, Pol , PCNA, dNTPs (lanes 8-9), Fen-1 and DNA ligase 1 (lanes 10-11). Lanes 9 and 11: re-digestion by RNaseH2. Lane 1: Substrate *D 39 R 1 D 15 : D 55 alone. (B) RER reactions in the presence of DDX3X (lanes 2-3), Pol , dNTPs (lanes 4-5), Fen-1 and DNA ligase 1 (lanes 6-7). Lanes 3, 5 and 7: re-digestion by RNaseH2. Lane 1: Substrate *D 39 R 1 D 15 : D 55 alone. (C) Western blot for DDX3X and -actin. L, molecular weight markers. NSC, non-silencing vector control cells; shDDX3X, stable DDX3X-silenced cells. (D) Quantification of genomic rNMPs by the riboassay. NSC untreated cells signal was set as 1 fluorescence arbitrary unit. Different treatments were: untreated genomic DNA (-), genomic DNA treated with 1 U of E. coli RNaseH2 or with 50 nM of human recombinant RNaseH2 (+). Data represent mean ± SEM of at least four independent experiments. (E) Western blot for vinculin, DDX3X and RNaseH2A. L, molecular weight markers. siCNT, non-silencing smart-pool siRNAs; siDDX3X, DDX3X-silenced cells, siRH2A, RNaseH2A-silenced cells. (F) Quantification of genomic rNMPs by the riboassay. siCNT (control) cells signal was set as 1 fluorescence arbitrary unit. As above, different treatments were: untreated genomic DNA (-) and genomic DNA treated with 50 nM of human recombinant RNaseH2 (+). Data represent mean ± SEM of at least four independent experiments.
Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases

November 2020

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171 Reads

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12 Citations

Nucleic Acids Research

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.


Citations (11)


... These results are similar to those of a previous study where re-docked native ligands interacted with residues Ile347, Asp348, Ala262, Phe273, Ile279, Phe297, Ile411, and Phe413 [21]. The interactions of these compounds with amino acids present in the binding site may decrease enzymatic activity and inhibit the SIRT1 as a functional protein [22][23]. Ten compounds in the extract had more negative binding energies and lower inhibition constant values than those control inhibitors, indicating that the compounds exhibited promising inhibitory activity towards SIRT1. ...

Reference:

Chemical Profiling Ethyl Acetate Extract of Basilicum polystachyon Leaves and Exploration of Anticancer SIRT1 Inhibitors Using In Silico Approach
Structural Insights into Neonicotinoids and N-Unsubstituted Metabolites on Human nAChRs by Molecular Docking, Dynamics Simulations, and Calcium Imaging

International Journal of Molecular Sciences

... Adjuvants, substances added to vaccines, have long been known to boost immune responses to antigens and extend their longevity [15]. Adjuvants minimize the amount of antigen required for each vaccination dose and the frequency of vaccinations, while simultaneously improving antigen stability or sustained antigen release and, eventually, increasing immunogenicity [16]. ...

Progress towards Adjuvant Development: Focus on Antiviral Therapy

International Journal of Molecular Sciences

... Each experiment was performed in triplicate. At 7,15,21,28,34,42, and 48, survival rates, and biometric data were made. The larvae were freeze-dried with an Edward MOD. freeze-dryer (Edward and Co., Ltd., London, UK) and kept at −20 °C prior to analysis. ...

Efficient use of agricultural waste to naturally fortify Tenebrio molitor mealworms and evaluation of their nutraceutical properties
  • Citing Article
  • December 2022

Journal of Insects as Food and Feed

... The RecA-like domains I and II show more than 96% and 99% sequence similarity between DDX3X and DDX3Y respectively, but all motifs identified within these two domains are completely identical between the two proteins with the exception of the motif Va in the RecA-like domain II, which has a single conservative lysine to arginine substitution (5,56). The other motifs, namely the RS-like sequence, the CTE and the NTE, all have near 100% sequence similarity and over 80% sequence identity. ...

DEAD-Box Helicase DDX3X as a Host Target against Emerging Viruses: New Insights for Medicinal Chemical Approaches
  • Citing Article
  • July 2022

Journal of Medicinal Chemistry

... ACE-inhibitory peptides have been identified and assessed in both in vivo and in vitro settings, originating from a variety of distinct species. Brai et al. (2022) conducted a study to assess the ACE inhibitory capabilities of the primary protein fractions extracted from Tenebrio molitor larvae to determine their suitability as a novel functional food for managing hypertension. While YAN was synthesized and employed as a reference standard for LC-MS/MS quantification, and the determination of IC50 against ACE was carried out, the limited yields of YAN from TML did not provide a complete explanation for the activity of the entire protein fraction. ...

Proteins From Tenebrio molitor: an Interesting Functional Ingredient and a Source of ACE Inhibitory Peptides
  • Citing Article
  • June 2022

Food Chemistry

... 61 Recently, Brai and co-workers identified BA103 as a micromolar CC 50 anti-GBM agent blocking the helicase activity of DDX3X, further underscoring DDX3X-targeted small molecules as promising drug leads for GBM. 62 Additionally, Kerr and co-workers reported that DDX3 was highly expressed in pluripotent stem cells, such as embryonic stem cells (ESCs) and embryonal carcinoma cells (ECCs), and that inhibition of DDX3 using the DDX3X inhibitor RK-33 decreased the proliferation of undifferentiated stem cells. 63 In agreement with previous literature, we showed that RK-33 had effects on GBM CSC neurospheres, albeit at much higher drug concentrations than for Roc ASFs. ...

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

... In addition to the above mentioned, small molecule prodrugs that can be activated by other kinds of enzymes have also been developed (Zhang H. et al., 2018;Sun et al., 2019;Fukai et al., 2021;Rango et al., 2021;Liu et al., 2023;Tyagi et al., 2023). They are highly effective in targeting tumors and enhancing therapeutic efficacy. ...

Si113-prodrugs selectively activated by plasmin against hepatocellular and ovarian carcinoma

European Journal of Medicinal Chemistry

... Low ERCC1 expression has been associated with prolonged survival in lung adenocarcinoma patients treated with PMX as first line therapy 66 . A novel DDX3Xmediated NMP removal pathway has been reported, though its contribution to cytotoxicity in vivo requires further investigation 67 . ...

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

Nucleic Acids Research

... Most of the structures reported for STING and TLR small molecule agonists are heterocyclic compounds, mimic of the nucleobases. Even in this case, many structurally related compounds have been already developed with different applications, including kinase inhibitors with broad applications in cancer disorders and poorly known mechanisms of action [227] or ligands of human cofactors [228][229][230]. The existence of high-throughput fully automated instruments for the synthesis of peptides is another aspect that should be taken into consideration. ...

Unique Domain for a Unique Target: Selective Inhibitors of Host Cell DDX3X to Fight Emerging Viruses
  • Citing Article
  • July 2020

Journal of Medicinal Chemistry

... Differences in RAS prevalence may result from various factors, including differential selective pressures exerted by antiviral treatments, variability in sample sizes, intrinsic genetic differences between HCV subtypes, or disparities in treatment access [127]. Additionally, patients previously treated with less effective medications may have developed resistant variants, and co-infections with other viruses, such as HIV, could alter viral dynamics, facilitating the emergence of resistance mutations due to changes in the immunological or pharmacological environment [128]. ...

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

European Journal of Medicinal Chemistry