Chemistry Related to the Catalytic Cycle of the Antioxidant Ebselen.

The antioxidant drug ebselen has been widely studied in both laboratories and in clinical trials. The catalytic mechanism by which it destroys hydrogen peroxide via reduction with glutathione or other thiols is complex and has been the subject of considerable debate. During reinvestigations of several key steps, we found that the seleninamide that comprises the first oxidation product of ebselen underwent facile reversible methanolysis to an unstable seleninate ester and two dimeric products. In its reaction with benzyl alcohol, the seleninamide produced a benzyl ester that reacted readily by selenoxide elimination, with formation of benzaldehyde. Oxidation of ebselen seleninic acid did not afford a selenonium seleninate salt as previously observed with benzene seleninic acid, but instead generated a mixture of the seleninic and selenonic acids. Thiolysis of ebselen with benzyl thiol was faster than oxidation by ca. an order of magnitude and produced a stable selenenyl sulfide. When glutathione was employed, the product rapidly disproportionated to glutathione disulfide and ebselen diselenide. Oxidation of the S-benzyl selenenyl sulfide, or thiolysis of the seleninamide with benzyl thiol, afforded a transient thiolseleninate that also readily underwent selenoxide elimination. The S-benzyl derivative disproportionated readily when catalyzed by the simultaneous presence of both the thiol and triethylamine. The phenylthio analogue disproportionated when exposed to ambient or UV (360 nm) light by a proposed radical mechanism. These observations provide additional insight into several reactions and intermediates related to ebselen.

Liposomal amphotericin B-the future.

Advances in medicine have led to a growing number of people with compromised or suppressed immune systems who are susceptible to invasive fungal infections. In particular, severe fungal infections are becoming increasingly common in ICUs, affecting people within and outside of traditional risk groups alike. This is exemplified by the emergence of severe viral pneumonia as a significant risk factor for invasive pulmonary aspergillosis, and the recognition of influenza-associated pulmonary aspergillosis and, more recently, COVID-19-associated pulmonary aspergillosis. The treatment landscape for haematological malignancies has changed considerably in recent years, and some recently introduced targeted agents, such as ibrutinib, are increasing the risk of invasive fungal infections. Consideration must also be given to the risk of drug-drug interactions between mould-active azoles and small-molecule kinase inhibitors. At the same time, infections caused by rare moulds and yeasts are increasing, and diagnosis continues to be challenging. There is growing concern about azole resistance among both moulds and yeasts, mandating continuous surveillance and personalized treatment strategies. It is anticipated that the epidemiology of fungal infections will continue to change and that new populations will be at risk. Early diagnosis and appropriate treatment remain the most important predictors of survival, and broad-spectrum antifungal agents will become increasingly important. Liposomal amphotericin B will remain an essential therapeutic agent in the armamentarium needed to manage future challenges, given its broad antifungal spectrum, low level of acquired resistance and limited potential for drug-drug interactions.

Triazavirin-A Novel Effective Antiviral Drug.

This review outlines the data of numerous studies relating to the broad-spectrum antiviral drug Triazavirin that was launched on the Russian pharmaceutical market in 2014 as an anti-influenza drug (the international non-patented name is Riamilovir). The range of antiviral activity of Triazavirin has been significantly expanded during recent years; in particular, it has been shown that Triazavirin exhibits activity against tick-borne encephalitis, Rift Valley fever, West Nile fever, and other infections of viral etiology. This drug has been approved for treatment of influenza and acute respiratory infections by the Russian Ministry of Health on the basis of comprehensive clinical trials involving over 450 patients. Triazavirin was found to be a highly effective and well-tolerated drug, allowing its over-the-counter sale. The recently published data on the use of Triazavirin in clinical practice for the treatment of patients with COVID-19 are discussed, with special attention paid to potential biological targets for this drug.

Effectiveness of front line and emerging fungal disease prevention and control interventions and opportunities to address appropriate eco-sustainable solutions.

Fungal pathogens contribute to significant disease burden globally; however, the fact that fungi are eukaryotes has greatly complicated their role in fungal-mediated infections and alleviation. Antifungal drugs are often toxic to host cells and there is increasing evidence of adaptive resistance in animals and humans. Existing fungal diagnostic and treatment regimens have limitations that has contributed to the alarming high mortality rates and prolonged morbidity seen in immunocompromised cohorts caused by opportunistic invasive infections as evidenced during HIV and COVID-19 pandemics. There is a need to develop real-time monitoring and diagnostic methods for fungal pathogens and to create a greater awareness as to the contribution of fungal pathogens in disease causation. Greater information is required on the appropriate selection and dose of antifungal drugs including factors governing resistance where there is commensurate need to discover more appropriate and effective solutions. Popular azole fungal drugs are widely detected in surface water and sediment due to incomplete removal in wastewater treatment plants where they are resistant to microbial degradation and may cause toxic effects on aquatic organisms such as algae and fish. UV has limited effectiveness in destruction of anti-fungal drugs where there is increased interest in the combination approaches such as novel use of pulsed-plasma gas-discharge technologies for environmental waste management. There is growing interest in developing alternative and complementary green eco-biocides and disinfection innovation. Fungi present challenges for cleaning, disinfection and sterilization of reusable medical devices such as endoscopes where they (example, Aspergillus and Candida species) can be protected when harboured in build-up biofilm from lethal processing. Information on the efficacy of established disinfection and sterilization technologies to address fungal pathogens including bottleneck areas that present high risk to patients is lacking. There is a need to address risk mitigation and modelling to inform efficacy of appropriate intervention technologies that must consider all contributing factors where there is potential to adopt digital technologies to enable real-time analysis of big data, such as use of artificial intelligence and machine learning. International consensus on standardised protocols for developing and reporting on appropriate alternative eco-solutions must be reached, particularly in order to address fungi with increasing drug resistance where research and innovation can be enabled using a One Health approach.

Six Feet under Microbiota: Microbiologic Contamination and Toxicity Profile in Three Urban Cemeteries from Lisbon, Portugal.

Cemeteries are potential environmental reservoirs of pathogenic microorganisms from organic matter decomposition. This study aimed to characterize the microbial contamination in three cemeteries, and more specifically in grave diggers' facilities. One active sampling method (impingement method) and several passive sampling methods (swabs, settled dust, settled dust filters and electrostatic dust cloths-EDC) were employed. The molecular detection of Aspergillus sections and SARS-CoV-2, as well as mycotoxin analysis, screening of azole resistance, and cytotoxicity measurement were also conducted. Total bacteria contamination was 80 CFU·m-2 in settled dust samples, reached 849 CFU·m-2 in EDC and 20,000 CFU·m-2 in swabs, and ranged from 5000 to 10,000 CFU·m-2 in filters. Gram-negative bacteria (VRBA) were only observed in in settled dust samples (2.00 × 105 CFU·m-2). Regarding Aspergillus sp., the highest counts were obtained in DG18 (18.38%) and it was not observed in azole-supplemented SDA media. SARS-CoV-2 and the targeted Aspergillus sections were not detected. Mycophenolic acid was detected in one settled dust sample. Cytotoxic effects were observed for 94.4% filters and 5.6% EDC in A549 lung epithelial cells, and for 50.0% filters and 5.6% EDC in HepG2 cells. Future studies are needed in this occupational setting to implement more focused risk management measures.

Molecular interactions and inhibition of the SARS-CoV-2 main protease by a thiadiazolidinone derivative.

We report molecular interactions and inhibition of the main protease (MPro ) of SARS-CoV-2, a key enzyme involved in the viral life cycle. By using a thiadiazolidinone (TDZD) derivative as a chemical probe, we explore the conformational dynamics of MPro via docking protocols and molecular dynamics simulations in all-atom detail. We reveal the local and global dynamics of MPro in the presence of this inhibitor and confirm the inhibition of the enzyme with an IC50 value of 1.39 ± 0.22 µM, which is comparable to other known inhibitors of this enzyme.

Microbial contamination in waste collection: Unveiling this Portuguese occupational exposure scenario.

Previous studies anticipated that microorganisms and their metabolites in waste will increase as a consequence of a decreased collection frequency and due to differences in what kind of waste is bagged before collection leading to an increased exposure of workers handling the waste. This study aim was to investigate the microbial contamination present in the waste collection trucks (WCT) and in the support facilities (waste collection station - WCS). It was applied a multi-approach protocol using active (air sampling by impingement and impaction) and passive (surface swabs, electrostatic dust cloths and settled dust) sampling methods. The screening of azole-resistance, the investigation of mycotoxins and the assessment of the elicited biological responses in vitro were also carried out aiming recognizing the possible health effects of waste collection drivers. SARS-CoV-2 detection was also performed. In WCS only air samples had contamination in all the four sampling sites (canteen, operational removal core, operational removal center, and administrative service). Among all the analyzed matrices from the WCT a higher percentage of total bacterial counts and Gram-was detected in swabs (66.93%; 99.36%). In WCS the most common species were Penicillium sp. (43.98%) and Cladosporium sp. (24.68%), while on WCT Aspergillus sp. (4.18%) was also one of the most found. In the azole resistance screening Aspergillus genera was not observed in the azole-supplemented media. SARS-CoV-2 was not detected in any of the environmental samples collected, but Aspergillus section Fumigati was detected in 5 samples. Mycotoxins were not detected in EDC from WCS, while in WCT they were detected in filters (N = 1) and in settled dust samples (N = 16). In conclusion, our study reveals that a comprehensive sampling approach using active and passive sampling (e.g. settled dust sampling for a representative mycotoxin evaluation) and combined analytic methods (i.e., culture-based and molecular) is an important asset in microbial exposure assessments. Concerning the waste collection exposure scenario, the results of this study unveiled a complex exposure, particularly to fungi and their metabolites. Aspergillus section Fumigati highlight the significance of targeting this section in the waste management industry as an indicator of occupational health risk.

COVID-19-associated pulmonary aspergillosis in ICU patients in a German reference centre: Phenotypic and molecular characterisation of Aspergillus fumigatus isolates.

BACKGROUND: COVID-19-associated invasive pulmonary aspergillosis (CAPA) is associated with increased mortality. Cases of CAPA caused by azole-resistant Aspergillus fumigatus strains have been reported. OBJECTIVES: To analyse the twelve-month CAPA prevalence in a German tertiary care hospital and to characterise clinical A. fumigatus isolates from two German hospitals by antifungal susceptibility testing and microsatellite genotyping. PATIENTS/METHODS: Retrospective observational study in critically ill adults from intensive care units with COVID-19 from 17 February 2020 until 16 February 2021 and collection of A. fumigatus isolates from two German centres. EUCAST broth microdilution for four azole compounds and microsatellite PCR with nine markers were performed for each collected isolate (N = 27) and additional for three non-COVID A. fumigatus isolates. RESULTS: welve-month CAPA prevalence was 7.2% (30/414), and the rate of azole-resistant A. fumigatus isolates from patients with CAPA was 3.7% with detection of one TR34/L98H mutation. The microsatellite analysis revealed no major clustering of the isolates. Sequential isolates mainly showed the same genotype over time. CONCLUSIONS: Our findings demonstrate similar CAPA prevalence to other reports and a low azole-resistance rate. Genotyping of A. fumigatus showed polyclonal distribution except for sequential isolates.

Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives.

The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent Mpro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses.

Toxicology and pharmacology of synthetic organoselenium compounds: an update.

Here, we addressed the pharmacology and toxicology of synthetic organoselenium compounds and some naturally occurring organoselenium amino acids. The use of selenium as a tool in organic synthesis and as a pharmacological agent goes back to the middle of the nineteenth and the beginning of the twentieth centuries. The rediscovery of ebselen and its investigation in clinical trials have motivated the search for new organoselenium molecules with pharmacological properties. Although ebselen and diselenides have some overlapping pharmacological properties, their molecular targets are not identical. However, they have similar anti-inflammatory and antioxidant activities, possibly, via activation of transcription factors, regulating the expression of antioxidant genes. In short, our knowledge about the pharmacological properties of simple organoselenium compounds is still elusive. However, contrary to our early expectations that they could imitate selenoproteins, organoselenium compounds seem to have non-specific modulatory activation of antioxidant pathways and specific inhibitory effects in some thiol-containing proteins. The thiol-oxidizing properties of organoselenium compounds are considered the molecular basis of their chronic toxicity; however, the acute use of organoselenium compounds as inhibitors of specific thiol-containing enzymes can be of therapeutic significance. In summary, the outcomes of the clinical trials of ebselen as a mimetic of lithium or as an inhibitor of SARS-CoV-2 proteases will be important to the field of organoselenium synthesis. The development of computational techniques that could predict rational modifications in the structure of organoselenium compounds to increase their specificity is required to construct a library of thiol-modifying agents with selectivity toward specific target proteins.

Therapeutic approaches against coronaviruses acute respiratory syndrome.

Coronaviruses represent global health threat. In this century, they have already caused two epidemics and one serious pandemic. Although, at present, there are no approved drugs and therapies for the treatment and prevention of human coronaviruses, several agents, FDA-approved, and preclinical, have shown in vitro and/or in vivo antiviral activity. An in-depth analysis of the current situation leads to the identification of several potential drugs that could have an impact on the fight against coronaviruses infections. In this review, we discuss the virology of human coronaviruses highlighting the main biological targets and summarize the current state-of-the-art of possible therapeutic options to inhibit coronaviruses infections. We mostly focus on FDA-approved and preclinical drugs targeting viral conserved elements.

Photochemistry of the Organoselenium Compound Ebselen: Direct Photolysis and Reaction with Active Intermediates of Conventional Reactive Species Sensitizers and Quenchers.

Ebselen (EBS), 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is an organoselenium pharmaceutical with antioxidant and anti-inflammatory properties. Furthermore, EBS is an excellent scavenger of reactive oxygen species. This property complicates conventional protocols for sensitizing and quenching reactive species because of potential generation of active intermediates that quickly react with EBS. In this study, the photochemical reactivity of EBS was investigated in the presence of (1) 1O2 and •OH sensitizers [rose Bengal (RB), perinaphthanone, and H2O2] and (2) reactive species scavenging and quenching agents (sorbic acid, isopropanol, sodium azide, and tert-butanol) that are commonly employed to study photodegradation mechanisms and kinetics. The carbon analogue of EBS, namely, 2-phenyl-3H-isoindol-1-one, was included as a reference compound to confirm the impact of the selenium atom on EBS photochemical reactivity. EBS does not undergo acid dissociation, but pH-dependent kinetics were observed in RB-sensitized solutions, suggesting EBS reaction with active intermediates (3RB2-*, O2•-, and H2O2) that are not kinetically relevant for other compounds. In addition, the observed rate constant of EBS increased in the presence of sorbic acid, isopropanol, and sodium azide. These findings suggest that conventional reactive species sensitizers, scavengers, and quenchers need to be carefully applied to highly reactive organoselenium compounds to account for reactions that are typically slow for other organic contaminants.

Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance.

Aspergillus fumigatus is an opportunistic human pathogen that causes aspergillosis, a spectrum of environmentally acquired respiratory illnesses. It has a cosmopolitan distribution and exists in the environment as a saprotroph on decaying plant matter. Azoles, which target Cyp51A in the ergosterol synthesis pathway, are the primary class of drugs used to treat aspergillosis. Azoles are also used to combat plant pathogenic fungi. Recently, an increasing number of azole-naive patients have presented with pan-azole-resistant strains of A. fumigatus. The TR34/L98H and TR46/Y121F/T289A alleles in the cyp51A gene are the most common ones conferring pan-azole resistance. There is evidence that these mutations arose in agricultural settings; therefore, numerous studies have been conducted to identify azole resistance in environmental A. fumigatus and to determine where resistance is developing in the environment. Here, we summarize the global occurrence of azole-resistant A. fumigatus in the environment based on available literature. Additionally, we have created an interactive world map showing where resistant isolates have been detected and include information on the specific alleles identified, environmental settings, and azole fungicide use. Azole-resistant A. fumigatus has been found on every continent, except for Antarctica, with the highest number of reports from Europe. Developed environments, specifically hospitals and gardens, were the most common settings where azole-resistant A. fumigatus was detected, followed by soils sampled from agricultural settings. The TR34/L98H resistance allele was the most common in all regions except South America where the TR46/Y121F/T289A allele was the most common. A major consideration in interpreting this survey of the literature is sampling bias; regions and environments that have been extensively sampled are more likely to show greater azole resistance even though resistance could be more prevalent in areas that are under-sampled or not sampled at all. Increased surveillance to pinpoint reservoirs, as well as antifungal stewardship, is needed to preserve this class of antifungals for crop protection and human health.

In silico Studies on the Interaction between Mpro and PLpro From SARS-CoV-2 and Ebselen, its Metabolites and Derivatives.

The COVID-19 pandemic caused by the SARS-CoV-2 has mobilized scientific attention in search of a treatment. The cysteine-proteases, main protease (Mpro) and papain-like protease (PLpro) are important targets for antiviral drugs. In this work, we simulate the interactions between the Mpro and PLpro with Ebselen, its metabolites and derivatives with the aim of finding molecules that can potentially inhibit these enzymes. The docking data demonstrate that there are two main interactions between the thiol (-SH) group of Cys (from the protease active sites) and the electrophilic centers of the organoselenium molecules, i. e. the interaction with the carbonyl group (O=C… SH) and the interaction with the Se moiety (Se… SH). Both interactions may lead to an adduct formation and enzyme inhibition. Density Functional Theory (DFT) calculations with Ebselen indicate that the energetics of the thiol nucleophilic attack is more favorable on Se than on the carbonyl group, which is in accordance with experimental data (Jin et al. Nature, 2020, 582, 289-293). Therefore, organoselenium molecules should be further explored as inhibitors of the SARS-CoV-2 proteases. Furthermore, we suggest that some metabolites of Ebselen (e. g. Ebselen diselenide and methylebselenoxide) and derivatives ethaselen and ebsulfur should be tested in vitro as inhibitors of virus replication and its proteases.

Ebselen and Analogues: Pharmacological Properties and Synthetic Strategies for Their Preparation.

Ebselen is the leader of selenorganic compounds, and starting from its identification as mimetic of the key antioxidant enzyme glutathione peroxidase, several papers have appeared in literature claiming its biological activities. It was the subject of several clinical trials and it is currently in clinical evaluation for the treatment of COVID-19 patients. Given our interest in the synthesis and pharmacological evaluation of selenorganic derivatives with this review, we aimed to collect all the papers focused on the biological evaluation of ebselen and its close analogues, covering the timeline between 2016 and most of 2021. Our analysis evidences that, even if it lacks specificity when tested in vitro, being able to bind to every reactive cysteine, it proved to be always well tolerated in vivo, exerting no sign of toxicity whatever the administered doses. Besides, looking at the literature, we realized that no review article dealing with the synthetic approaches for the construction of the benzo[d][1,2]-selenazol-3(2H)-one scaffold is available; thus, a section of the present review article is completely devoted to this specific topic.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

Candida auris: A Quick Review on Identification, Current Treatments, and Challenges.

Candida auris is a novel and major fungal pathogen that has triggered several outbreaks in the last decade. The few drugs available to treat fungal diseases, the fact that this yeast has a high rate of multidrug resistance and the occurrence of misleading identifications, and the ability of forming biofilms (naturally more resistant to drugs) has made treatments of C. auris infections highly difficult. This review intends to quickly illustrate the main issues in C. auris identification, available treatments and the associated mechanisms of resistance, and the novel and alternative treatment and drugs (natural and synthetic) that have been recently reported.

Identification of ebselen and its analogues as potent covalent inhibitors of papain-like protease from SARS-CoV-2.

An efficient treatment against a COVID-19 disease, caused by the novel coronavirus SARS-CoV-2 (CoV2), remains a challenge. The papain-like protease (PLpro) from the human coronavirus is a protease that plays a critical role in virus replication. Moreover, CoV2 uses this enzyme to modulate the host's immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs. We used Approximate Bayesian Computation tools, molecular modelling and enzyme activity studies to identify highly active inhibitors of the PLpro. We discovered organoselenium compounds, ebselen and its structural analogues, as a novel approach for inhibiting the activity of PLproCoV2. Furthermore, we identified, for the first time, inhibitors of PLproCoV2 showing potency in the nanomolar range. Moreover, we found a difference between PLpro from SARS and CoV2 that can be correlated with the diverse dynamics of their replication, and, putatively to disease progression.