A Review of the Current Landscape of SARS-CoV-2 Main Protease Inhibitors: Have We Hit the Bullseye Yet?

In this review, we collected 1765 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M-pro inhibitors from the bibliography and other sources, such as the COVID Moonshot project and the ChEMBL database. This set of inhibitors includes only those compounds whose inhibitory capacity, mainly expressed as the half-maximal inhibitory concentration (IC50) value, against M-pro from SARS-CoV-2 has been determined. Several covalent warheads are used to treat covalent and non-covalent inhibitors separately. Chemical space, the variation of the IC50 inhibitory activity when measured by different methods or laboratories, and the influence of 1,4-dithiothreitol (DTT) are discussed. When available, we have collected the values of inhibition of viral replication measured with a cellular antiviral assay and expressed as half maximal effective concentration (EC50) values, and their possible relationship to inhibitory potency against M-pro is analyzed. Finally, the most potent covalent and non-covalent inhibitors that simultaneously inhibit the SARS-CoV-2 M-pro and the virus replication in vitro are discussed.

Anti-Toxoplasma Effect of Hydroalchohlic Extract of Terminalia chebula Retz in Cell Culture and Murine Model

Background: We examined anti-Toxoplasma effect of hydroalcoholic extract of Terminalia chebula Retz (T. chebula) in cell culture and murine model. Methods: The study was conducted in Shahrekord University of Medical Sciences, Iran in 2017. Half maximal effective (concentration (EC50) of T. chebula extract and pyrimethamine was determined in infected Hela cells by using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) method. In the animal model, BALB/c mice were injected with tachyzoites (104) of T. RH strain intraperitoneally. 24h after the injection, the test groups were orally treated with 100, 200, 400 and 800 mg/kg of T. chebula extract for 7 days. The survival rate of the mice was determined and blood samples were collected to determine the amount of serum Malondialdehyde (MDA) and antioxidant capacity. Then peritoneal fluid of the mice was collected to count the number of tachyzoites and after necropsy, the pathologic changes, including the weight of liver, spleen and kidneys were investigated. The analysis of data was accomplished using SPSS. Results: EC50 values were 94.7μg/mL and 290.50μg/mL for T. chebula and pyrimethamine respectively. In the animal model, the extract of T. chebula in concentration of 100 mg/kg showed the same anti-Toxoplasma effect as pyrimethamine. This concentration of the extract decreased number of intraperitoneal tachyzoites and increased the survival rate of the mice. This extract reduced the levels of serum MDA and tissue inflammation and increased serum antioxidant capacity. Conclusion: Regarding the positive effect of extract, after more clinical trials in the animal model and standardization of the extract, it can be used as an alternative or complementary therapy for toxoplasmosis.

A novel definition and treatment of hyperinflammation in COVID-19 based on purinergic signalling

Hyperinflammation plays an important role in severe and critical COVID-19. Using inconsistent criteria, many researchers define hyperinflammation as a form of very severe inflammation with cytokine storm. Therefore, COVID-19 patients are treated with anti-inflammatory drugs. These drugs appear to be less efficacious than expected and are sometimes accompanied by serious adverse effects. SARS-CoV-2 promotes cellular ATP release. Increased levels of extracellular ATP activate the purinergic receptors of the immune cells initiating the physiologic pro-inflammatory immune response. Persisting viral infection drives the ATP release even further leading to the activation of the P2X7 purinergic receptors (P2X7Rs) and a severe yet physiologic inflammation. Disease progression promotes prolonged vigorous activation of the P2X7R causing cell death and uncontrolled ATP release leading to cytokine storm and desensitisation of all other purinergic receptors of the immune cells. This results in immune paralysis with co-infections or secondary infections. We refer to this pathologic condition as hyperinflammation. The readily available and affordable P2X7R antagonist lidocaine can abrogate hyperinflammation and restore the normal immune function. The issue is that the half-maximal effective concentration for P2X7R inhibition of lidocaine is much higher than the maximal tolerable plasma concentration where adverse effects start to develop. To overcome this, we selectively inhibit the P2X7Rs of the immune cells of the lymphatic system inducing clonal expansion of Tregs in local lymph nodes. Subsequently, these Tregs migrate throughout the body exerting anti-inflammatory activities suppressing systemic and (distant) local hyperinflammation. We illustrate this with six critically ill COVID-19 patients treated with lidocaine.

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

Assessment of toxicity and kinetic effects of erythromycin on activated sludge consortium by fast respirometry method

Background: The present study aimed to assess the acute impact of erythromycin (ERY) as an inhibitor on peptone mixture utilization of activated sludge (AS) consortium. Methods: For this purpose, the inhibition of oxygen consumption was used based on the ISO 8192:2007 procedure. In this method, the AS consortium (10-day age) was extracted from lab-scale membrane bioreactor, then, percentage inhibition for total, heterotrophic, and nitrifying microorganisms, in separate batch respirometric tests were calculated in the absence and presence of N-allylthiourea (ATU) as a specific Nitrification inhibitor. Results: The obtained data showed that the height of oxygen uptake rate (OUR) profiles and amount of oxygen consumption reduced with increasing ERY dose. The half-maximal effective concentration (EC50) of ERY for heterotrophic and nitrifier microorganisms were 269.4 and 1243.1 mg/L, respectively. In Run 1, the kinetic coefficients bH, fA,H, YH, and µH were calculated as 2.61 d-1, 0.44, 0.4945 mg VSS/mg COD, and 0.047 d-1, respectively. Also, for maximum ERY concentration (1000 mg/L), the kinetic coefficients bH., fA,H, YH, and µH were calculated as 2.27 d-1, 0.3, 0.4983 mg VSS/mg COD, and 0.0049 d-1, respectively. Conclusion: The findings showed that the inhibitory impact of ERY was observed as a decrease in the amount of oxygen consumption by OUR profiles in rapid respirometric method (ISO 8192), which offered a novel insight for the acute inhibitory impact of this antibiotic. Also, chemical oxygen demand (COD) as an overall substrate parameter is most helpful in interpreting the behavior and the metabolic functions of AS systems.

Comparative Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii in vitro and in Pregnant Mice Infected with T. gondii Oocysts

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacte-rial and parasitic infections, most notably coccidiosis in poultry and in ruminants. We have in-vestigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. It induced distinct alterations in the parasite mitochondrion within 24h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selec-tivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ antici-pated to have better physicochemical properties than DCQ were assessed in vitro. One such com-pound RMB060 displayed an exceedingly low IC50 of 0.07 nM when applied concomitantly with infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6h after commencement of treatment. After 48h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features remi-niscent of bradyzoites. Exposure of infected cultures to 300 nM RMB060 for 52 days did not re-sult in complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. Treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

Griffithsin and Carrageenan Combination Results in Antiviral Synergy against SARS-CoV-1 and 2 in a Pseudoviral Model

Over 182 million confirmed cases of COVID-19 and more than 4 million deaths have been reported to date around the world. It is essential to identify broad-spectrum antiviral agents that may prevent or treat infections by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but also by other coronaviruses that may jump the species barrier in the future. We evaluated the antiviral selectivity of griffithsin and sulfated and non-sulfated polysaccharides against SARS-CoV-1 and SARS-CoV-2 using a cytotoxicity assay and a cell-based pseudoviral model. The half-maximal cytotoxic concentration (CC50) and half-maximal effective concentration (EC50) were determined for each compound, using a dose-response-inhibition analysis on GraphPad Prism v9.0.2 software (San Diego, CA, USA). The therapeutic index (TI = CC50/EC50) was calculated for each compound. The potential synergistic, additive, or antagonistic effect of different compound combinations was determined by CalcuSyn v1 software (Biosoft, Cambridge, UK), which estimated the combination index (CI) values. Iota and lambda carrageenan showed the most potent antiviral activity (EC50 between 3.2 and 7.5 µg/mL). Carrageenan and griffithsin combinations exhibited synergistic activity (EC50 between 0.2 and 3.8 µg/mL; combination index <1), including against recent SARS-CoV-2 mutations. The griffithsin and carrageenan combination is a promising candidate to prevent or treat infections by SARS-CoV-1 and SARS-CoV-2.

Adenosine Receptor Reserve and Long-Term Potentiation: Unconventional Adaptive Mechanisms in Cardiovascular Diseases?

While the concept of a receptor reserve (spare receptors) is old, their presence on human cells as an adaptive mechanism in cardiovascular disease is a new suggestion. The presence of spare receptors is suspected when the activation of a weak fraction of receptors leads to maximal biological effects, in other words, when the half-maximal effective concentration (EC50) for a biological effect (cAMP production, for example) is lower than the affinity (KD) of the ligand for a receptor. Adenosine is an ATP derivative that strongly impacts the cardiovascular system via its four membrane receptors, named A1R, A2AR, A2BR, and A3R, with the A1R being more particularly involved in heart rhythm, while the A2AR controls vasodilation. After a general description of the tools necessary to explore the presence of spare receptors, this review focuses on the consequences of the presence of spare adenosine receptors in cardiovascular physiopathology. Finally, the role of the adenosinergic system in the long-term potentiation and its possible consequences on the physiopathology are also mentioned.

In Vitro Antifungal Activity and Toxicity of Dihydrocarvone-Hybrid Derivatives against Monilinia fructicola

The aim of this study was to synthesize a series of novel and known dihydrocarvone-hybrid derivatives (2–9) and to evaluate mycelial growth activity of hybrid molecules against two strains of Monilinia fructicola, as well as their toxicity. Dihydrocarvone-hybrid derivatives have been synthesized under sonication conditions and characterized by FTIR, NMR, and HRMS. Antifungal efficacy against both strains of M. fructicola was determined by half maximal effective concentration (EC50) and toxicity using the brine shrimp lethality test (BSLT). Among the synthesized compounds, 7 and 8 showed the best activity against both strains of M. fructicola with EC50 values of 148.1 and 145.9 µg/mL for strain 1 and 18.1 and 15.7 µg/mL for strain 2, respectively, compared to BC 1000® (commercial organic fungicide) but lower than Mystic® 520 SC. However, these compounds showed low toxicity values, 910 and 890 µg/mL, respectively, compared to Mystic® 520 SC, which was highly toxic. Based on the results, these hybrid compounds could be considered for the development of more active, less toxic, and environmentally friendly antifungal agents against phytopathogenic fungi.

Comparisons of the Effect of Different Metal Oxide Nanoparticles on the Root and Shoot Growth under Shaking and Non-Shaking Incubation, Different Plants and Binary Mixture Conditions

We evaluated the toxicity of five metal oxide nanoparticles (NPs) in single or binary mixtures based on root and shoot growth of two plant species under non-shaking and shaking conditions. The effects of NPs on root and shoot growth differed depending on the NP type, incubation condition, and plant type. The half maximal effective concentration (EC50) of NPs based on root growth were significantly lower, by 2.6–9.8 times, under shaking than non-shaking conditions (p = 0.0138). The magnitude of the effects of NPs followed the order CuO > ZnO > NiO >> Al2O3, TiO2. In addition, Lactuca sativa L. was more sensitive to the tested NPs than Raphanus sativus L., with an EC50 0.2–0.7 times lower (p = 0.0267). The observed effects of 12 combinations of binary NP mixtures were slightly, albeit non-significantly, lower than expected, indicative of an additive effect of the individual NPs in the mixtures. The results emphasize the importance of careful plant model selection, appropriate application of incubation conditions, and consideration of chemical mixtures rather than single compounds when evaluating the effects of metal oxide NPs.