Impairment of SARS-CoV-2 spike glycoprotein maturation and fusion activity by nitazoxanide: an effect independent of spike variants emergence.

SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here, we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell-cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections.

Explorative genetic association study of GSTT2B copy number variant in complex disease risks.

BACKGROUND: Glutathione S-transferases (GSTs) are the main phase II enzymes involved in cellular detoxification. Through phase I and phase II detoxification reactions, the cell is able to detoxify endogenous and exogenous toxic compounds. AIMS: This study focused attention on the GSTT2B copy number variant (CNV) in order to explore its involvement in the genetic pre-disposition to asthma, Alzheimer's disease (AD), allergic rhinitis (AR), essential hypertension (EH), hypothyroidism and recurrent miscarriage (RM). METHODS: The study population consists of 1225 individuals divided into six case-control groups. The genotyping of the GSTT2B CNV was performed by using a duplex-PCR. Odds Ratios (ORs) were calculated, adjusting for the confounding variables, to estimate the association between GSTT2B CNV and the disease status. RESULTS: The χ(2)-test and ORs did not show any association between this genetic marker and pathological phenotypes. CONCLUSION: The data highlights that GSTT2B CNV is not associated with the investigated complex diseases in Italian patients. However, further investigations are necessary to replicate these findings in larger sample sizes and to explore other health-related phenotypes.

GSTA1*-69C/T and GSTO2*N142D as asthma- and allergy-related risk factors in Italian adult patients.

1. Asthma and allergies are characterized by variable and subjective symptoms influenced by many genes, molecular mechanisms and environmental factors. The presence of inflammation and oxidative stress in the airways are important biochemical features of asthma and respiratory allergies. Glutathione S-transferase (GSTs) enzymes play an important role in cellular protection against inflammation, and functional genetic polymorphisms in GST genes show a significant association with asthma and allergy risk. Specifically, our previous study on asthmatic children highlighted GSTA1 and GSTO2 as novel susceptibility loci for asthma. 2. In the present study we focused our attention on GSTA1*-69C/T (rs3957357) and GSTO2*N142D (rs156697) polymorphisms to confirm our previous results in an independent adult study population and to clarify whether GSTA1 and GSTO2 gene polymorphisms are involved in a non-discriminative pathway towards asthma and respiratory allergy. 3. To accomplish this, we recruited 103 patients with respiratory allergies, 199 patients with asthma and 200 healthy controls. Genomic DNA extracted from buccal cells was screened for GSTA1*-69C/T and GSTO2*N142D single nucleotide polymorphisms. 4. The GSTA1*-69T and GSTO2*D142 variants are both associated with a significantly increased risk of asthma, whereas only GSTA1*-69C/T is significantly associated with allergies. These outcomes confirm the involvement of GSTO2 loci in asthma and suggest that GSTA1 is a common risk factor for asthma and allergies.

Functional variability of glutathione S-transferases in Basque populations.

OBJECTIVES: Glutathione S-transferases (GSTs) are enzymes involved in Phase II reactions. They play a key role in cellular detoxification. Various studies have shown that genes coding for the GST are highly polymorphic and some of these variants are directly associated with a decrease of enzyme activity making individuals more susceptible to different clinical phenotypes. The aim of this study is to investigate the genetic variability of GST genes among human populations. We have focused our attention on the polymorphic variants of the GSTA1, GSTM1, GSTO1, GSTO2, GSTP1, GSTT1, and GSTT2B genes. METHODS: These polymorphisms were analyzed in a whole sample of 151 individuals: 112 autochthonous Navarrese Basques, and 39 non-autochthonous Navarrese Basques. DNA extraction from plasma was performed by using the phenol:chloroform:isoamylic alcohol method. Genotyping of the gene polymorphisms was performed by PCR Multiplex and the PCR-RFLP method. We applied correspondence analysis and built frequency-maps to compare the genetic structure in worldwide populations. RESULTS: Our results were compared with data available on the Human Genome Diversity Project (HGDP) and on the 1,000 Genomes Project to obtain information on the functional variability of GSTs in Basques. Our data indicated that Basque communities showed a higher differentiation of certain functional GST variants (i.e., GSTM1-positive/null genotype, GSTP1*I105V, and GSTT2B*1/0) than other European and Mediterranean populations. CONCLUSIONS: This might account for epidemiological differences in the predisposition to diseases and drug response among Basques and could be used to design and interpret genetic association studies for this particular population.

Characterization of an influenza B virus isolated from a fatal case of myocarditis in a pediatric patient in Italy.

Influenza B is one of the infective agents that can cause rapid and fatal myocarditis in children. Here, we describe a fatal case of myocarditis in a previously healthy child, after infection with an influenza B/Victoria-lineage virus during the 2022-23 epidemic season in Italy. Influenza B virus was isolated also in a second case, a younger family member showing only a mild influenza-like illness. Genotypic and phenotypic analyses have been performed on both virus samples and results showed that HA1 sequences were identical and genetically and antigenically related to other B viruses circulating in 2022-23 season in Italy. However, a D129N substitution was found in the receptor binding domain of the HA of the two viruses, not detected in other circulating viruses in Italy but only in a proportion of those circulating in other European countries. Phenotypic analyses assessed the susceptibility towards either neuraminidase inhibitors and baloxavir. Annual influenza vaccination remains one of the best interventions to prevent complications such as myocarditis, particularly in children.

Phenotype versus genotype methods for copy number variant analysis of glutathione S-transferases M1.

Several variants have been identified for genes encoding Glutathione S-transferase (GST) enzymes; some are associated with significant alteration of protein function. One of the most extensively studied is a copy number variant (CNV) in the GSTM1 gene. In this study, we compared phenotype (positive, null) and genotype (1/1, 1/0, 0/0) methods in order to assess dissimilarities obtained using these two different approaches to evaluate possible methodology-related bias. We analyzed a sample of 1947 individuals belonging to 18 human populations with different ethnic origins. We also evaluated whether the presence of missense substitutions in the GSTM1 gene might influence the association of the CNV with phenotype distribution. Through the comparison of GSTM1 CNV frequencies in phenotype and genotype among human populations, we observed that differences increase in high heterogeneous populations. Furthermore, we identified two missense variants (rs199816990 and rs202002774) that may distort the outcome of genetic association studies on Asian populations. These results indicate that the phenotype analysis may strongly alter the genetic association. Therefore, genotype discrimination analysis should be used to analyze GSTM1 CNV. To understand the role of GSTM1 in human health, the analysis of CNV should be combined with the investigation of single nucleotide polymorphisms with functional effect.

GSTO2*N142D gene polymorphism associated with hypothyroidism in Italian patients.

Hypothyroidism is a multifactorial endocrinal disease characterized by abnormally low thyroid hormone production. Thyroiditis is one of the primary causes of hypothyroidism, as it is an increasing level of inflammation in the thyroid gland that could be due to a failure of the anti-inflammatory response. Glutathione S-transferases are biomarkers of inflammation and oxidative stress. These phase II enzymes play a relevant role in detoxifying xenobiotic compounds. Particular attention has been focused on GSTA1, GSTM1, GSTO2, GSTP1, and GSTT1 genes to evaluate if GST gene polymorphisms are associated with hypothyroidism. We screened a case-control population (patients with hypothyroidism n = 110, controls n = 122) to analyze GST gene polymorphisms. GST SNPs were determined using the PCR-RFLP method, while GST null polymorphisms were determined using a Multiplex PCR. In this study, we found differences in genotype distribution between hypothyroid individuals and controls only for the GSTO2*N142D polymorphism. Logistic regression analysis, after adjustment for age and sex, confirmed this positive association (OR = 4.56; 95 % CI 1.22-17.00; p = 0.009). The GSTO2 enzyme can catalyze several reactions important for countering oxidative stress: subjects with the D142 allele may have a deficiency in the antioxidant enzymatic system. A decrease in antioxidant capacity may trigger increased oxidative stress. Previous studies have highlighted the role of GST enzymes in inflammation disorders, but no data are available on their role in hypothyroidism. Our results suggest that GSTO2 could increase disease risk susceptibility and could act as a risk factor for hypothyroidism in Italian patients.

Glutathione S-transferase polymorphisms, asthma susceptibility and confounding variables: a meta-analysis.

Oxidative stress is one of the main risk factors for asthma development. Glutathione S-transferases play an important role in antioxidant defences and may influence asthma susceptibility. In particular, GSTM1 and GSTT1 positive/null genotypes and the GSTP1 Ile105 Val polymorphism have been analyzed in a number of genetic association studies, with conflicting outcomes. Two previous meta-analyses have attempted to clarify the associations between GST genes and asthma, but these studies have also showed contrasting results. Our aim was to perform a meta-analysis that included independent genetic association studies on GSTM1, GSTP1, and GSTT1, evaluating also the effect of potential confounding variables (i.e. ethnicity, population age, and urbanization). Systematic review and meta-analysis of the effects of GST genes on asthma were conducted. The meta-analyses were performed using a fixed or, where appropriate, random effects model. The meta-analysis of the GSTM1 (n = 35), GSTT1 (n = 31) and GSTP1 (n = 28) studies suggests that no significant associations with asthma susceptibility were observed for GSTM1 and GSTP1 gene polymorphisms, whereas a significant outcome was detected for the GSTT1 positive/null genotype (pooled OR = 1.33, 95 %CI = 1.10-1.60). However, high between-study heterogeneity was identified in all the general analyses (p heterogenetity < 0.05). The stratification analysis seems to explain the heterogeneity only in few cases. This picture is probably due to the interactive process of genetics and environment that characterizes disease pathogenesis. Further studies on interactions of GST genes with the potential oxidative stress sources and with other antioxidant genes are needed to explain the role of GST enzymes in asthma.

The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein.

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections and life-threatening diseases in a subset of patients. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 µg/mL and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

Thiazolide Prodrug Esters and Derived Peptides: Synthesis and Activity.

Amino acid ester prodrugs of the thiazolides, introduced to improve the pharmacokinetic parameters of the parent drugs, proved to be stable as their salts but were unstable at pH > 5. Although some of the instability was due to simple hydrolysis, we have found that the main end products of the degradation were peptides formed by rearrangement. These peptides were stable solids: they maintained significant antiviral activity, and in general, they showed improved pharmacokinetics (better solubility and reduced clearance) compared to the parent thiazolides. We describe the preparation and evaluation of these peptides.

HapMap-based study of human soluble glutathione S-transferase enzymes: the role of natural selection in shaping the single nucleotide polymorphism diversity of xenobiotic-metabolizing genes.

OBJECTIVE: Glutathione S-transferase enzymes (GSTs; EC: 2.5.1.18) constitute the principal phase II superfamily, which plays a key role in cellular detoxification. GST genes are organized in chromosomal clusters; most of these genes are polymorphic, mainly due to single nucleotide substitutions. Different studies proved significant interethnic differences in GST allelic frequencies but, at present, the role of natural selection in human genetic variability of GSTs is poorly understood. The aim of this study is to investigate the role of natural selection in shaping single nucleotide polymorphism (SNP) diversity of soluble GST genes. METHODS: Using the HapMap database, we analyzed the population differences in the soluble GST genes using the phasing data from unrelated individuals shared among 11 populations in the International HapMap project. A Fst-based selection test was applied to HapMap data to detect soluble GST loci under selection. RESULTS: Comparisons between GST gene polymorphisms among HapMap populations highlight that ethnicity is an influencing factor of GST genetic variability. By applying a genome scan based on F-statistics, we identified nine SNPs that present F-coefficients significantly more different than those expected under neutrality (rs2239892, rs3814309, rs7483, rs1571858, rs929166, rs11807, rs4715344, rs4715354, and rs3734431). CONCLUSION: Our study confirms that GST gene variation reflects human demographic history, but it also demonstrates that natural selection could shape the genetic profile of some GST SNPs. Moreover, the identification of human genome regions and targets of natural selection may have detected candidate genes for complex diseases. In analyzing the literature, we provide complex disease hypothesis (male infertility, embryotoxicity) for the identified GST SNPs.

Glutathione S-transferase variants as risk factor for essential hypertension in Italian patients.

Involvement of genetic polymorphisms in arterial hypertension has already been reported, including GST genes, with contrasting results. The present research evaluates the possible association between GST gene polymorphisms and essential hypertension (EH) in an Italian population sample. 193 hypertensive subjects and 210 healthy controls were recruited. Buccal cells were collected from each subject using an oral swab and DNA was extracted using the phenol:chloroform:isoamilic alcohol method. GST SNPs were determined using the PCR-RFLP method, while GST null polymorphisms were determined using a Multiplex PCR. Among GST polymorphisms, only the frequency of the GSTT1 null phenotype was significantly higher in hypertensive patients than in normotensive participants. GSTT1 null individuals were significantly associated with increased risk of hypertension [P < 0.001; adjusted OR 2.24 (1.43-3.50)]. In sex-based analysis, the risk was significantly higher in female hypertensives [P < 0.001; adjusted OR 3.25 (1.78-5.95)] but not in male subjects. This study analyzed all GST gene that, in other research, have been studied in relation to arterial hypertension and the GSTO polymorphisms, showing an association only with GSTT1. The results for the GSTO genes represent the first analysis of this GST class in relation to blood pressure regulation. The association between the GSTT1 null phenotype and EH was confirmed in the overall population and in women, but not in men. These data suggest that GSTT1 could be a sex-specific candidate gene for EH.

Functional polymorphisms of GSTA1 and GSTO2 genes associated with asthma in Italian children.

BACKGROUND: Asthma is an airway disorder characterized by bronchial inflammation. An imbalance between the oxidative forces and the antioxidant defense systems has been implicated in the pathogenesis of asthma. Glutathione S-transferases (GSTs) play an important role in cellular protection against inflammation. Several studies have investigated the genetic variability of GSTM1, GSTP1 and GSTT1 enzymes in asthma development with conflicting results. Moreover, in our previous independent case-control study on GSTs and asthma, we have found that GSTA1 and GSTO2 gene polymorphisms are associated with asthma. The aim of the present study is to analyze if some functional polymorphisms of GSTA1, GSTM1, GSTP1, GSTO2 and GSTT1 are associated with asthma in pediatric patients from Chieti (Italy). METHODS: In this study, we performed an association study on 127 asthmatic children and 126 controls. We screened single nucleotide polymorphisms at GSTA1, GSTO2 and GSTP1 loci. The effects of GSTM1 and GSTT1 null genotype were also investigated. RESULTS: The GSTA1*-69T and GSTO2*D142 variants are associated with the significant increased risk of asthma development in our study population, while GSTM1, GSTP1 and GSTT1 genotype distributions were nearly equal between the control group and asthmatics. CONCLUSIONS: Confirming our previous study, these findings suggest that the GSTA1 and the GSTO2 are asthma susceptible genes involved in increasing the risk of asthma development in the Italian population.

GSTT1 and GSTM1 gene polymorphisms in European and African populations.

Glutathione S-transferases (GSTs) are a superfamily of detoxificant enzymes. Pharmacogenomic studies have revealed interethnic differences in GST allelic frequencies. This study is focused on GSTT1 (gene deletion, rs17850155, rs2234953, and rs11550605) and GSTM1 (gene deletion) gene frequency distributions in two population samples of Europe origin (Italy, n = 120; Spain, n = 94) and two population samples of Africa origin (Cameroon, n = 126; Ethiopia, n = 153). Detection of GSTT1 and GSTM1 null genotypes was performed by multiplex PCR analysis, while the other GSTT1 gene polymorphisms were detected using allele specific PCR and sequencing. GSTT1 and GSTM1 null frequencies in the samples analyzed fit with the variability range observed in European and African populations, respectively. The SNP analysis in GSTT1 gene did not highlight any nucleotide substitution in 493 individuals analyzed. The comparisons among GSTM1 and GSTT1 null phenotype frequencies in worldwide populations show different patterns between Asians, Africans, and Europeans. Important insights into the effects of GSTM1 and GSTT1 gene deletions on the pathogenesis of human diseases have been hypothesized. Detailed studies on the geography of GST variants could therefore increase knowledge about the relationship between ethnicity and the prevalence of certain diseases.

Synthesis, antiviral activity, preliminary pharmacokinetics and structural parameters of thiazolide amine salts.

Background: The thiazolides, typified by nitazoxanide, are an important class of anti-infective agents. A significant problem with nitazoxanide and its active circulating metabolite tizoxanide is their poor solubility. Results: We report the preparation and evaluation of a series of amine salts of tizoxanide and the corresponding 5-Cl thiazolide. These salts demonstrated improved aqueous solubility and absorption, as shown by physicochemical and in vivo measurements. They combine antiviral activity against influenza A virus with excellent cell safety indices. We also report the x-ray crystal structural data of the ethanolamine salt. Conclusion: The ethanol salt of thiazolide retains the activity of the parent together with an improved cell safety index, making it a good candidate for further evaluation.

GSTA1, GSTO1 and GSTO2 gene polymorphisms in Italian asthma patients.

1. Previous studies have established that genetic alterations in glutathione S-transferase enzymes may change the ability of the airway to deal with toxic substances and increase the risk of asthma. The present study analysed the association between asthma and GSTA1, GSTO1 and GSTO2 gene polymorphisms. 2. The GSTA1*-69C/T, GSTO1*A140D and GSTO2*N142D polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism, whereas the GSTO1*E155del polymorphism was detected using the confronting two-pair primer method. 3. Distribution of the GSTA1*-69C/T genotype differed significantly between asthmatics and controls. Subjects with at least one allele -69T in the GSTA1 genotype have an increased risk of asthma (odds ratio (OR) 3.45; 95% confidence interval (CI) 1.80-6.62). The distribution of the GSTO1 genotype was nearly equal between the control group and asthmatics, however, the distribution of the GSTO2 gene differed significantly between asthmatics and controls (Chi-squared test). Subjects who had the GSTO2 homozygous D142 genotype were found to have an increased risk of asthma (OR 5.91; 95% CI 1.80-19.42). 4. The results show a potential association between the GST genes and asthma. This is particularly significant given that, in the literature, there are no epidemiological studies on alpha and omega classes of glutathione transferases in asthma.

Glutathione S-transferase ω class (GSTO) polymorphisms in a sample from Rome (Central Italy).

Glutathione S-transferases are a superfamily of enzymes that are involved in biotransformation of drugs, xenobiotics and play a fundamental role in the protection of cells from oxidative stress. In humans, the recently described GST Omega class contains two expressed genes GSTO1 and GSTO2, located on chromosome 10 (10q24.3). Four polymorphisms in GSTO genes have been identified in ethnic groups: GSTO1*A140D (rs4925), GSTO1*E155del (rs56204475), GSTO1*E208K (rs11509438) and GSTO2*N142D (rs156697). This study provides the allele frequencies of GSTO polymorphism in a sample consisting of 116 apparently healthy individuals of both sexes from Rome (Central Italy). Detection of GSTO1*A140D and GSTO2*N142D alleles was performed by PCR-RFLP analysis, while GSTO1*E155del and GSTO1*E208K alleles were detected using the Confronting Two-Pair Primers analysis (PCR-CTPP) and allele specific PCR, respectively. The GSTO allele frequencies found in the Italian sample were included in the variability range observed in European populations. Comparison between the data presented in this study and data in previous studies showed different patterns among European, Asian and African populations.

Serum proteins and work habits in a group of farm-workers exposed to EBDCs.

BACKGROUND: Study of the association between genetic variability and individual susceptibility can help to characterize occupational or environmental risks due to xenobiotics. AIM: This study evaluates the influence of genetic components and environmental factors in relation to pesticide exposure. SUBJECTS AND METHODS: The study population consisted of 37 non-occupationally exposed workers and 74 farm-workers exposed to pesticide. Exposure was assessed through the measurement of urine concentration of ethylenethiourea (ETU). Genetic differences in drug metabolism were detected by a qualitative variability in serum proteins. The environmental factors were recorded by using a questionnaire. RESULTS: The results show a difference between ETU levels in farm-workers and in non-occupationally exposed workers. In the non-exposed group a relationship between ETU urinary concentration and lifestyle habits is present. In farm-workers ETU urinary concentration is less correlated with lifestyle habits, but is associated, rather, with their work. In the exposed individuals the serum protein analyses show a possible link between ETU urinary concentration and the polymorphism of group-specific component (Gc). CONCLUSIONS: The association between Gc polymorphism and ETU urinary concentration of subjects exposed to EBDCs could be due to the immunological function of Gc and the effects on the immune system of EBDCs.

Modulation of the GSTT1 activity by the GSTM1 phenotype in a sample of Italian farm-workers.

Glutathione S-transferase (GST) isozymes catalyze nucleophilic attack by reduced Glutathione (GSH) on a variety of electrophilic compounds and play a central role in biotransformation of xenobiotics (Hayes et al., Annu Rev Pharmacol Toxicol 45:51-88, 2005). We performed a case-control study to evaluate the GSTM1 and GSTT1 polymorphisms and to investigate if exposure to pesticides conditions the GSTT1 activity level in 115 healthy controls and 90 farm-workers exposed to pesticides. Polymorphisms were investigated using a GSTM1 or a GSTT1-specific PCR. Enzyme activity was measured by means of DCM as co-substrate, as described by Bruhn et al. (Biochem Pharmacol 56:1189-1193, 1998). There was no significant difference between the farm-workers and the healthy controls regarding the distribution of various alleles of the GSTM1 and GSTT1 genes and the GSTT1 enzyme activity. In farm-workers, the GSTM1 null genotype was associated with a significant increase of GSTT1 activity, suggesting a regulative mechanism common to GSTM1 and GSTT1 enzymes after exposure to xenobiotics.