Modulation of ATP Production Influences Inorganic Polyphosphate Levels in Non-Athletes' Platelets at the Resting State.

Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4',6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes.

Virtual Screening and Quantum Chemistry Analysis for SARS-CoV-2 RNA-Dependent RNA Polymerase Using the ChEMBL Database: Reproduction of the Remdesivir-RTP and Favipiravir-RTP Binding Modes Obtained from Cryo-EM Experiments with High Binding Affinity.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the pathogenic cause of coronavirus disease 2019 (COVID-19). The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a potential target for the treatment of COVID-19. An RdRp complex:dsRNA structure suitable for docking simulations was prepared using a cryo-electron microscopy (cryo-EM) structure (PDB ID: 7AAP; resolution, 2.60 Å) that was reported recently. Structural refinement was performed using energy calculations. Structure-based virtual screening was performed using the ChEMBL database. Through 1,838,257 screenings, 249 drugs (37 approved, 93 clinical, and 119 preclinical drugs) were predicted to exhibit a high binding affinity for the RdRp complex:dsRNA. Nine nucleoside triphosphate analogs with anti-viral activity were included among these hit drugs, and among them, remdesivir-ribonucleoside triphosphate and favipiravir-ribonucleoside triphosphate adopted a similar docking mode as that observed in the cryo-EM structure. Additional docking simulations for the predicted compounds with high binding affinity for the RdRp complex:dsRNA suggested that 184 bioactive compounds could be anti-SARS-CoV-2 drug candidates. The hit bioactive compounds mainly consisted of a typical noncovalent major groove binder for dsRNA. Three-layer ONIOM (MP2/6-31G:AM1:AMBER) geometry optimization calculations and frequency analyses (MP2/6-31G:AMBER) were performed to estimate the binding free energy of a representative bioactive compound obtained from the docking simulation, and the fragment molecular orbital calculation at the MP2/6-31G level of theory was subsequently performed for analyzing the detailed interactions. The procedure used in this study represents a possible strategy for discovering anti-SARS-CoV-2 drugs from drug libraries that could significantly shorten the clinical development period for drug repositioning.

Caged Dexamethasone/Quercetin Nanoparticles, Formed of the Morphogenetic Active Inorganic Polyphosphate, are Strong Inducers of MUC5AC.

Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.

Nudix hydrolase 18 catalyzes the hydrolysis of active triphosphate metabolites of the antivirals remdesivir, ribavirin, and molnupiravir.

Remdesivir and molnupiravir have gained considerable interest because of their demonstrated activity against SARS-CoV-2. These antivirals are converted intracellularly to their active triphosphate forms remdesivir-TP and molnupiravir-TP. Cellular hydrolysis of these active metabolites would consequently decrease the efficiency of these drugs; however, whether endogenous enzymes that can catalyze this hydrolysis exist is unknown. Here, we tested remdesivir-TP as a substrate against a panel of human hydrolases and found that only Nudix hydrolase (NUDT) 18 catalyzed the hydrolysis of remdesivir-TP with notable activity. The kcat/Km value of NUDT18 for remdesivir-TP was determined to be 17,700 s-1M-1, suggesting that NUDT18-catalyzed hydrolysis of remdesivir-TP may occur in cells. Moreover, we demonstrate that the triphosphates of the antivirals ribavirin and molnupiravir are also hydrolyzed by NUDT18, albeit with lower efficiency than Remdesivir-TP. Low activity was also observed with the triphosphate forms of sofosbuvir and aciclovir. This is the first report showing that NUDT18 hydrolyzes triphosphates of nucleoside analogs of exogenous origin, suggesting that NUDT18 can act as a cellular sanitizer of modified nucleotides and may influence the antiviral efficacy of remdesivir, molnupiravir, and ribavirin. As NUDT18 is expressed in respiratory epithelial cells, it may limit the antiviral efficacy of remdesivir and molnupiravir against SARS-CoV-2 replication by decreasing the intracellular concentration of their active metabolites at their intended site of action.

Polyphosphate in Antiviral Protection: A Polyanionic Inorganic Polymer in the Fight Against Coronavirus SARS-CoV-2 Infection.

Polyanions as polymers carrying multiple negative charges have been extensively studied with regard to their potential antiviral activity. Most studies to date focused on organic polyanionic polymers, both natural and synthetic. The inorganic polymer, polyphosphate (polyP), despite the ubiquitous presence of this molecule from bacteria to man, has attracted much less attention. More recently, and accelerated by the search for potential antiviral agents in the fight against the pandemic caused by the coronavirus SARS-CoV-2, it turned out that polyP disrupts the first step of the viral replication cycle, the interaction of the proteins in the virus envelope and in the cell membrane that are involved in the docking process of the virus with the target host cell. Experiments on a molecular level using the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the cellular angiotensin converting enzyme 2 (ACE2) receptor revealed that polyP strongly inhibits the binding reaction through an electrostatic interaction between the negatively charged centers of the polyP molecule and a cationic groove, which is formed by positively charged amino acids on the RBD surface. In addition, it was found that polyP, due to its morphogenetic and energy delivering activities, enhances the antiviral host innate immunity defense of the respiratory epithelium. The underlying mechanisms and envisaged application of polyP in the therapy and prevention of COVID-19 are discussed.

The Anti-Viral Applications of Marine Resources for COVID-19 Treatment: An Overview.

The ongoing pandemic has led to an urgent need for novel drug discovery and potential therapeutics for Sars-CoV-2 infected patients. Although Remdesivir and the anti-inflammatory agent dexamethasone are currently on the market for treatment, Remdesivir lacks full efficacy and thus, more drugs are needed. This review was conducted through literature search of PubMed, MDPI, Google Scholar and Scopus. Upon review of existing literature, it is evident that marine organisms harbor numerous active metabolites with anti-viral properties that serve as potential leads for COVID-19 therapy. Inorganic polyphosphates (polyP) naturally found in marine bacteria and sponges have been shown to prevent viral entry, induce the innate immune response, and downregulate human ACE-2. Furthermore, several marine metabolites isolated from diverse sponges and algae have been shown to inhibit main protease (Mpro), a crucial protein required for the viral life cycle. Sulfated polysaccharides have also been shown to have potent anti-viral effects due to their anionic properties and high molecular weight. Likewise, select marine sponges produce bromotyrosines which have been shown to prevent viral entry, replication and protein synthesis. The numerous compounds isolated from marine resources demonstrate significant potential against COVID-19. The present review for the first time highlights marine bioactive compounds, their sources, and their anti-viral mechanisms of action, with a focus on potential COVID-19 treatment.

Long-chain polyphosphates impair SARS-CoV-2 infection and replication.

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

Triple-target stimuli-responsive anti-COVID-19 face mask with physiological virus-inactivating agents.

Conventional face masks to prevent SARS-CoV-2 transmission are mostly based on a passive filtration principle. Ideally, anti-COVID-19 masks should protect the carrier not only by size exclusion of virus aerosol particles, but also be able to capture and destroy or inactivate the virus. Here we present the proof-of-concept of a filter mat for such a mask, which actively attracts aerosol droplets and kills the virus. The electrospun mats are made of polycaprolactone (PCL) a hydrophilic, functionalizable and biodegradable polyester, into which inorganic polyphosphate (polyP) a physiological biocompatible, biodegradable and antivirally active polymer (chain length, ∼40 Pi units) has been integrated. A soluble Na-polyP as well as amorphous calcium polyP nanoparticles (Ca-polyP-NP) have been used. In this composition, the polyP component of the polyP-PCL mats is stable in aqueous protein-free environment, but capable of transforming into a gel-like coacervate upon contact with divalent cations and protein like mucin present in (virus containing) aerosol droplets. In addition, the Ca-polyP-NP are used as a carrier of tretinoin (all-trans retinoic acid) which blocks the function of the SARS-CoV-2 envelope (E) protein, an ion channel forming viroporin. The properties of this novel mask filter mats are as follows: First, to attract and to trap virus-like particles during the polyP coacervate formation induced in situ by aerosol droplets on the spun PCL fibers, as shown here by using SARS-CoV-2 mimicking fluorescent nanoparticles. Second, after disintegration the NP by the aerosol-mucus constituents, to release polyP that binds to and abolishes the function of the receptor binding domain of the viral spike protein. Third, to destroy the virus by releasing tretinoin, as shown by the disruption of virus-mimicking liposomes with the integrated recombinant viral viroporin. It is proposed that these properties, which are inducible (stimuli responsive), will allow the design of antiviral masks that are smart.

Pharmacokinetic modelling to estimate intracellular favipiravir ribofuranosyl-5'-triphosphate exposure to support posology for SARS-CoV-2.

OBJECTIVES: Favipiravir has discrepant activity against SARS-CoV-2 in vitro, concerns about teratogenicity and pill burden, and an unknown optimal dose. This analysis used available data to simulate the intracellular pharmacokinetics of the favipiravir active metabolite [favipiravir ribofuranosyl-5'-triphosphate (FAVI-RTP)]. METHODS: Published in vitro data for intracellular production and elimination of FAVI-RTP in Madin-Darby canine kidney cells were fitted with a mathematical model describing the time course of intracellular FAVI-RTP as a function of favipiravir concentration. Parameter estimates were then combined with a published population pharmacokinetic model in Chinese patients to predict human intracellular FAVI-RTP. In vitro FAVI-RTP data were adequately described as a function of concentrations with an empirical model, noting simplification and consolidation of various processes and several assumptions. RESULTS: Parameter estimates from fittings to in vitro data predict a flatter dynamic range of peak to trough for intracellular FAVI-RTP (peak to trough ratio of ∼1 to 1) when driven by a predicted free plasma concentration profile, compared with the plasma profile of parent favipiravir (ratio of ∼2 to 1). This approach has important assumptions, but indicates that, despite rapid clearance of the parent from plasma, sufficient intracellular FAVI-RTP may be maintained across the dosing interval because of its long intracellular half-life. CONCLUSIONS: Population mean intracellular FAVI-RTP concentrations are estimated to be maintained above the Km for the SARS-CoV-2 polymerase for 9 days with a 1200 mg twice-daily regimen (following a 1600 mg twice-daily loading dose on day 1). Further evaluation of favipiravir as part of antiviral combinations for SARS-CoV-2 is warranted.

The therapeutic potential of inorganic polyphosphate: A versatile physiological polymer to control coronavirus disease (COVID-19).

Rationale: The pandemic caused by the novel coronavirus SARS-CoV-2 is advancing rapidly. In particular, the number of severe courses of the disease is still dramatically high. An efficient drug therapy that helps to improve significantly the fatal combination of damages in the airway epithelia, in the extensive pulmonary microvascularization and finally multiorgan failure, is missing. The physiological, inorganic polymer, polyphosphate (polyP) is a molecule which could prevent the initial phase of the virus life cycle, the attachment of the virus to the target cells, and improve the epithelial integrity as well as the mucus barrier. Results: Surprisingly, polyP matches perfectly with the cationic groove on the RBD. Subsequent binding studies disclosed that polyP, with a physiological chain length of 40 phosphate residues, abolishes the binding propensity of the RBD to the ACE2 receptor. In addition to this first mode of action of polyP, this polymer causes in epithelial cells an increased gene expression of the major mucins in the airways, of MUC5AC and MUC1, as well as a subsequent glycoprotein production. MUC5AC forms a gel-like mucus layer trapping inhaled particles which are then transported out of the airways, while MUC1 constitutes the periciliary liquid layer and supports ciliary beating. As a third mode of action, polyP undergoes enzymatic hydrolysis of the anhydride bonds in the airway system by alkaline phosphatase, releasing metabolic energy. Conclusions: This review summarizes the state of the art of the biotherapeutic potential of the polymer polyP and the findings from basic research and outlines future biomedical applications.

Morphogenetic (Mucin Expression) as Well as Potential Anti-Corona Viral Activity of the Marine Secondary Metabolite Polyphosphate on A549 Cells.

The mucus layer of the nasopharynx and bronchial epithelium has a barrier function against inhaled pathogens such as the coronavirus SARS-CoV-2. We recently found that inorganic polyphosphate (polyP), a physiological, metabolic energy (ATP)-providing polymer released from blood platelets, blocks the binding of the receptor binding domain (RBD) to the cellular ACE2 receptor in vitro. PolyP is a marine natural product and is abundantly present in marine bacteria. Now, we have approached the in vivo situation by studying the effect of polyP on the human alveolar basal epithelial A549 cells in a mucus-like mucin environment. These cells express mucins as well as the ectoenzymes alkaline phosphatase (ALP) and adenylate kinase (ADK), which are involved in the extracellular production of ATP from polyP. Mucin, integrated into a collagen-based hydrogel, stimulated cell growth and attachment. The addition of polyP to the hydrogel significantly increased cell attachment and also the expression of the membrane-tethered mucin MUC1 and the secreted mucin MUC5AC. The increased synthesis of MUC1 was also confirmed by immunostaining. This morphogenetic effect of polyP was associated with a rise in extracellular ATP level. We conclude that the nontoxic and non-immunogenic polymer polyP could possibly also exert a protective effect against SARS-CoV-2-cell attachment; first, by stimulating the innate antiviral response by strengthening the mucin barrier with its antimicrobial proteins, and second, by inhibiting virus attachment to the cells, as deduced from the reduction in the strength of binding between the viral RBD and the cellular ACE2 receptor.

The biomaterial polyphosphate blocks stoichiometric binding of the SARS-CoV-2 S-protein to the cellular ACE2 receptor.

The effect of the polyanionic polymer of inorganic polyphosphate (polyP) involved in innate immunity on the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the cellular ACE2 receptor was studied. The RBD surface comprises a basic amino acid stretch of four arginine residues which interact with the physiological polyP (polyP40) and polyP3. Subsequently, the interaction of RBD with ACE2 is sensitively inhibited. After the chemical modification of arginine, an increased inhibition by polyP, at a 1 : 1 molar ratio (polyP : RBP), is measured already at 0.1 µg mL-1. Heparin was ineffective. The results suggest a potential therapeutic benefit of polyP against SARS-CoV-2 infection.

Pharmacokinetics and tissue distribution of remdesivir and its metabolites nucleotide monophosphate, nucleotide triphosphate, and nucleoside in mice.

Remdesivir (RDV) exerts anti-severe acute respiratory coronavirus 2 activity following metabolic activation in the target tissues. However, the pharmacokinetics and tissue distributions of the parent drug and its active metabolites have been poorly characterized to date. Blood and tissue levels were evaluated in the current study. After intravenous administration of 20 mg/kg RDV in mice, the concentrations of the parent drug, nucleotide monophosphate (RMP) and triphosphate (RTP), as well as nucleoside (RN), in the blood, heart, liver, lung, kidney, testis, and small intestine were quantified. In blood, RDV was rapidly and completely metabolized and was barely detected at 0.5 h, similar to RTP, while its metabolites RMP and RN exhibited higher blood levels with increased residence times. The area under the concentration versus time curve up to the last measured point in time (AUC0-t) values of RMP and RN were 4558 and 136,572 h∙nM, respectively. The maximum plasma concentration (Cmax) values of RMP and RN were 2896 nM and 35,819 nM, respectively. Moreover, RDV presented an extensive distribution, and the lung, liver and kidney showed high levels of the parent drug and metabolites. The metabolic stabilities of RDV and RMP were also evaluated using lung, liver, and kidney microsomes. RDV showed higher clearances in the liver and kidney than in the lung, with intrinsic clearance (CLint) values of 1740, 1253, and 127 mL/(min∙g microsomal protein), respectively.

The inorganic polymer, polyphosphate, blocks binding of SARS-CoV-2 spike protein to ACE2 receptor at physiological concentrations.

Inorganic polyphosphate (polyP) is a morphogenetically active and metabolic energy-delivering physiological polymer that is released from blood platelets. Here, we show that polyP efficiently inhibits the binding of the envelope spike (S)-protein of the coronavirus SARS-CoV-2, the causative agent of COVID-19, to its host cell receptor ACE2 (angiotensin-converting enzyme 2). To stabilize polyP against the polyP-degrading alkaline phosphatase, the soluble polymer was encapsulated in silica/polyP nanoparticles. Applying a binding assay, soluble Na-polyP (sizes of 40 Pi and of 3 Pi units) as well as silica-nanoparticle-associated polyP significantly inhibit the interaction of the S-protein with ACE2 at a concentration of 1 µg/mL, close to the level present in blood. This inhibition is attributed to an interaction of polyP with a basic amino acid stretch on the surface of the receptor binding domain of S-protein. PolyP retains its activity in a flushing solution, opening a new strategy for the prevention and treatment of SARS-CoV-2 infection in the oropharyngeal cavity. The data suggest that supplementation of polyP might contribute to a strengthening of the human innate immunity system in compromised, thrombocytopenic COVID-19 patients.