Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways

Characterization of nuclear and mitochondrial G-Quadruplex binding compounds in human Liposarcoma cell lines

G-quadruplexes (G4s) are non-canonical DNA secondary structures that can form in G-rich sequences, which are not randomly distributed along the genome but clustered in defined regions according to recent bioinformatic analyses. The fact that the majority of G4s were found around transcription start sites (TSS) led to the hypothesis that G4s take part in gene regulation. Recent findings confirmed that G4s are implicated in the transcription of genes involved in differentiation, cancer progression and metabolic regulation. In the first part of the study, I focused on characterizing unreported G4s present at relevant genes associated with Liposarcoma (LPS), in particular CDK4 gene. Being LPS a class of soft tissue sarcomas particularly resistant to current chemotherapy, new therapeutic strategies are strongly needed. The most frequent subtypes of LPS are well- and de-differentiated liposarcomas (WD/DDLS). Both WD/DDLS are characterized by the amplification of chromosome segment 12q13-15, which carries the oncogenes MDM2 and CDK4. In the promoters of MDM2 and CDK4, our group has identified G4s by Chromatin Immunoprecipitation with an anti-G-Quadruplex antibody (G4 ChIP). Here we evaluated the activity of the recently emerged G4-binder, named SOP1812, against a panel of Liposarcoma cell lines. SOP1812 displayed an antiproliferative activity in the nanomolar concentrations and induced significant down-regulation of MDM2 and CDK4 transcription and protein expression levels. Intriguingly, the decrease of MDM2 inhibited the ubiquitin-mediated degradation of the tumor suppressor p53, restoring the signal of programmed cell death. Thus, SOP1812 was able to counteract the effects of MDM2 and CDK4 amplification in WD/DDLS, acting at G4s in their promoters. This work presents an efficient compound and highlights a possible new therapeutic strategy against WD/DDLS. Formation of G4 hybrid structure was identified within the mitochondrial DNA non-coding regulatory region, which takes part in the premature termination of mitochondrial RNA polymerase at the conserved sequence box II (CSBII), necessary for the switch between mitochondrial DNA (mtDNA) transcription and replication. In the second part of the project, we developed and evaluated a novel mitochondrial G4-ligand (mtPhamPEG) characterized by a well-known G4-ligand moiety functionalized with the (triphenylphosphonium) TPP+ mitochondrial moiety. We first validated the mitochondrial localization of mtPhamPEG into mitochondria and its cytotoxicity in a panel of cancer and non-cancer cells. mtPhamPEG induced significant reduction of cancer cells growth without affecting normal cells growth. In addition, we observed that the stabilization of mitochondrial CSB II-G4 by mtPhamPEG led to decrease of both mitochondrial transcription and replication in different cancer cells. In conclusion, here we present an alternative antiproliferative strategy based on dysregulation of mitochondrial metabolism of cancer cells.G-quadruplexes (G4s) are non-canonical DNA secondary structures that can form in G-rich sequences, which are not randomly distributed along the genome but clustered in defined regions according to recent bioinformatic analyses. The fact that the majority of G4s were found around transcription start sites (TSS) led to the hypothesis that G4s take part in gene regulation. Recent findings confirmed that G4s are implicated in the transcription of genes involved in differentiation, cancer progression and metabolic regulation. In the first part of the study, I focused on characterizing unreported G4s present at relevant genes associated with Liposarcoma (LPS), in particular CDK4 gene. Being LPS a class of soft tissue sarcomas particularly resistant to current chemotherapy, new therapeutic strategies are strongly needed. The most frequent subtypes of LPS are well- and de-differentiated liposarcomas (WD/DDLS). Both WD/DDLS are characterized by the amplification of chromosome segment 12q13-15, which carries the oncogenes MDM2 and CDK4. In the promoters of MDM2 and CDK4, our group has identified G4s by Chromatin Immunoprecipitation with an anti-G-Quadruplex antibody (G4 ChIP). Here we evaluated the activity of the recently emerged G4-binder, named SOP1812, against a panel of Liposarcoma cell lines. SOP1812 displayed an antiproliferative activity in the nanomolar concentrations and induced significant down-regulation of MDM2 and CDK4 transcription and protein expression levels. Intriguingly, the decrease of MDM2 inhibited the ubiquitin-mediated degradation of the tumor suppressor p53, restoring the signal of programmed cell death. Thus, SOP1812 was able to counteract the effects of MDM2 and CDK4 amplification in WD/DDLS, acting at G4s in their promoters. This work presents an efficient compound and highlights a possible new therapeutic strategy against WD/DDLS. Formation of G4 hybrid structure was identified within the mitochondrial DNA non-coding regulatory region, which takes part in the premature termination of mitochondrial RNA polymerase at the conserved sequence box II (CSBII), necessary for the switch between mitochondrial DNA (mtDNA) transcription and replication. In the second part of the project, we developed and evaluated a novel mitochondrial G4-ligand (mtPhamPEG) characterized by a well-known G4-ligand moiety functionalized with the (triphenylphosphonium) TPP+ mitochondrial moiety. We first validated the mitochondrial localization of mtPhamPEG into mitochondria and its cytotoxicity in a panel of cancer and non-cancer cells. mtPhamPEG induced significant reduction of cancer cells growth without affecting normal cells growth. In addition, we observed that the stabilization of mitochondrial CSB II-G4 by mtPhamPEG led to decrease of both mitochondrial transcription and replication in different cancer cells. In conclusion, here we present an alternative antiproliferative strategy based on dysregulation of mitochondrial metabolism of cancer cells

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis

"Delirium Day": A nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool

Background: To date, delirium prevalence in adult acute hospital populations has been estimated generally from pooled findings of single-center studies and/or among specific patient populations. Furthermore, the number of participants in these studies has not exceeded a few hundred. To overcome these limitations, we have determined, in a multicenter study, the prevalence of delirium over a single day among a large population of patients admitted to acute and rehabilitation hospital wards in Italy. Methods: This is a point prevalence study (called "Delirium Day") including 1867 older patients (aged 65 years or more) across 108 acute and 12 rehabilitation wards in Italian hospitals. Delirium was assessed on the same day in all patients using the 4AT, a validated and briefly administered tool which does not require training. We also collected data regarding motoric subtypes of delirium, functional and nutritional status, dementia, comorbidity, medications, feeding tubes, peripheral venous and urinary catheters, and physical restraints. Results: The mean sample age was 82.0 ± 7.5 years (58 % female). Overall, 429 patients (22.9 %) had delirium. Hypoactive was the commonest subtype (132/344 patients, 38.5 %), followed by mixed, hyperactive, and nonmotoric delirium. The prevalence was highest in Neurology (28.5 %) and Geriatrics (24.7 %), lowest in Rehabilitation (14.0 %), and intermediate in Orthopedic (20.6 %) and Internal Medicine wards (21.4 %). In a multivariable logistic regression, age (odds ratio [OR] 1.03, 95 % confidence interval [CI] 1.01-1.05), Activities of Daily Living dependence (OR 1.19, 95 % CI 1.12-1.27), dementia (OR 3.25, 95 % CI 2.41-4.38), malnutrition (OR 2.01, 95 % CI 1.29-3.14), and use of antipsychotics (OR 2.03, 95 % CI 1.45-2.82), feeding tubes (OR 2.51, 95 % CI 1.11-5.66), peripheral venous catheters (OR 1.41, 95 % CI 1.06-1.87), urinary catheters (OR 1.73, 95 % CI 1.30-2.29), and physical restraints (OR 1.84, 95 % CI 1.40-2.40) were associated with delirium. Admission to Neurology wards was also associated with delirium (OR 2.00, 95 % CI 1.29-3.14), while admission to other settings was not. Conclusions: Delirium occurred in more than one out of five patients in acute and rehabilitation hospital wards. Prevalence was highest in Neurology and lowest in Rehabilitation divisions. The "Delirium Day" project might become a useful method to assess delirium across hospital settings and a benchmarking platform for future surveys

Understanding Factors Associated With Psychomotor Subtypes of Delirium in Older Inpatients With Dementia

Objectives: Few studies have analyzed factors associated with delirium subtypes. In this study, we investigate factors associated with subtypes of delirium only in patients with dementia to provide insights on the possible prevention and treatments. Design: This is a cross-sectional study nested in the \u201cDelirium Day\u201d study, a nationwide Italian point-prevalence study. Setting and Participants: Older patients admitted to 205 acute and 92 rehabilitation hospital wards. Measures: Delirium was evaluated with the 4-AT and the motor subtypes with the Delirium Motor Subtype Scale. Dementia was defined by the presence of a documented diagnosis in the medical records and/or prescription of acetylcholinesterase inhibitors or memantine prior to admission. Results: Of the 1057 patients with dementia, 35% had delirium, with 25.6% hyperactive, 33.1% hypoactive, 34.5% mixed, and 6.7% nonmotor subtype. There were higher odds of having venous catheters in the hypoactive (OR 1.82, 95% CI 1.18-2.81) and mixed type of delirium (OR 2.23, CI 1.43-3.46), whereas higher odds of urinary catheters in the hypoactive (OR 2.91, CI 1.92-4.39), hyperactive (OR 1.99, CI 1.23-3.21), and mixed types of delirium (OR 2.05, CI 1.36-3.07). We found higher odds of antipsychotics both in the hyperactive (OR 2.87, CI 1.81-4.54) and mixed subtype (OR 1.84, CI 1.24-2.75), whereas higher odds of antibiotics was present only in the mixed subtype (OR 1.91, CI 1.26-2.87). Conclusions and Implications: In patients with dementia, the mixed delirium subtype is the most prevalent followed by the hypoactive, hyperactive, and nonmotor subtype. Motor subtypes of delirium may be triggered by clinical factors, including the use of venous and urinary catheters, and the use of antipsychotics. Future studies are necessary to provide further insights on the possible pathophysiology of delirium in patients with dementia and to address the optimization of the management of potential risk factors

Drug Prescription and Delirium in Older Inpatients: Results From the Nationwide Multicenter Italian Delirium Day 2015-2016

Objective: This study aimed to evaluate the association between polypharmacy and delirium, the association of specific drug categories with delirium, and the differences in drug-delirium association between medical and surgical units and according to dementia diagnosis. Methods: Data were collected during 2 waves of Delirium Day, a multicenter delirium prevalence study including patients (aged 65 years or older) admitted to acute and long-term care wards in Italy (2015-2016); in this study, only patients enrolled in acute hospital wards were selected (n = 4,133). Delirium was assessed according to score on the 4 "A's" Test. Prescriptions were classified by main drug categories; polypharmacy was defined as a prescription of drugs from 5 or more classes. Results: Of 4,133 participants, 969 (23.4%) had delirium. The general prevalence of polypharmacy was higher in patients with delirium (67.6% vs 63.0%, P =.009) but varied according to clinical settings. After adjustment for confounders, polypharmacy was associated with delirium only in patients admitted to surgical units (OR = 2.9; 95% CI, 1.4-6.1). Insulin, antibiotics, antiepileptics, antipsychotics, and atypical antidepressants were associated with delirium, whereas statins and angiotensin receptor blockers exhibited an inverse association. A stronger association was seen between typical and atypical antipsychotics and delirium in subjects free from dementia compared to individuals with dementia (typical: OR = 4.31; 95% CI, 2.94-6.31 without dementia vs OR = 1.64; 95% CI, 1.19-2.26 with dementia; atypical: OR = 5.32; 95% CI, 3.44-8.22 without dementia vs OR = 1.74; 95% CI, 1.26-2.40 with dementia). The absence of antipsychotics among the prescribed drugs was inversely associated with delirium in the whole sample and in both of the hospital settings, but only in patients without dementia. Conclusions: Polypharmacy is significantly associated with delirium only in surgical units, raising the issue of the relevance of medication review in different clinical settings. Specific drug classes are associated with delirium depending on the clinical setting and dementia diagnosis, suggesting the need to further explore this relationship

Drug prescription and delirium in older inpatients: Results from the nationwide multicenter Italian Delirium Day 2015-2016

Objective: This study aimed to evaluate the association between polypharmacy and delirium, the association of specific drug categories with delirium, and the differences in drug-delirium association between medical and surgical units and according to dementia diagnosis. Methods: Data were collected during 2 waves of Delirium Day, a multicenter delirium prevalence study including patients (aged 65 years or older) admitted to acute and long-term care wards in Italy (2015-2016); in this study, only patients enrolled in acute hospital wards were selected (n = 4,133). Delirium was assessed according to score on the 4 "A's" Test. Prescriptions were classified by main drug categories; polypharmacy was defined as a prescription of drugs from 5 or more classes. Results: Of 4,133 participants, 969 (23.4%) had delirium. The general prevalence of polypharmacy was higher in patients with delirium (67.6% vs 63.0%, P =.009) but varied according to clinical settings. After adjustment for confounders, polypharmacy was associated with delirium only in patients admitted to surgical units (OR = 2.9; 95% CI, 1.4-6.1). Insulin, antibiotics, antiepileptics, antipsychotics, and atypical antidepressants were associated with delirium, whereas statins and angiotensin receptor blockers exhibited an inverse association. A stronger association was seen between typical and atypical antipsychotics and delirium in subjects free from dementia compared to individuals with dementia (typical: OR = 4.31; 95% CI, 2.94-6.31 without dementia vs OR = 1.64; 95% CI, 1.19-2.26 with dementia; atypical: OR = 5.32; 95% CI, 3.44-8.22 without dementia vs OR = 1.74; 95% CI, 1.26-2.40 with dementia). The absence of antipsychotics among the prescribed drugs was inversely associated with delirium in the whole sample and in both of the hospital settings, but only in patients without dementia. Conclusions: Polypharmacy is significantly associated with delirium only in surgical units, raising the issue of the relevance of medication review in different clinical settings. Specific drug classes are associated with delirium depending on the clinical setting and dementia diagnosis, suggesting the need to further explore this relationship