Identification of new pharmacophore against SARS-CoV-2 spike protein by multi-fold computational and biochemical techniques.

COVID-19 appeared as a highly contagious disease after its outbreak in December 2019 by the virus, named SARS-CoV-2. The threat, which originated in Wuhan, China, swiftly became an international emergency. Among different genomic products, spike protein of virus plays a crucial role in the initiation of the infection by binding to the human lung cells, therefore, SARS-CoV-2's spike protein is a promising therapeutic target. Using a combination of a structure-based virtual screening and biochemical assay, this study seeks possible therapeutic candidates that specifically target the viral spike protein. A database of ~ 850 naturally derived compounds was screened against SARS-CoV-2 spike protein to find natural inhibitors. Using virtual screening and inhibitory experiments, we identified acetyl 11-keto-boswellic acid (AKBA) as a promising molecule for spike protein, which encouraged us to scan the rest of AKBA derivatives in our in-house database via 2D-similarity searching. Later 19 compounds with > 85% similarity with AKBA were selected and docked with receptor binding domain (RBD) of spike protein. Those hits declared significant interactions at the RBD interface, best possess and excellent drug-likeness and pharmacokinetics properties with high gastrointestinal absorption (GIA) without toxicity and allergenicity. Our in-silico observations were eventually validated by in vitro bioassay, interestingly, 10 compounds (A3, A4, C3, C6A, C6B, C6C, C6E, C6H, C6I, and C6J) displayed significant inhibitory ability with good percent inhibition (range: > 72-90). The compounds C3 (90.00%), C6E (91.00%), C6C (87.20%), and C6D (86.23%) demonstrated excellent anti-SARS CoV-2 spike protein activities. The docking interaction of high percent inhibition of inhibitor compounds C3 and C6E was confirmed by MD Simulation. In the molecular dynamics simulation, we observed the stable dynamics of spike protein inhibitor complexes and the influence of inhibitor binding on the protein's conformational arrangements. The binding free energy ΔGTOTAL of C3 (-38.0 ± 0.08 kcal/mol) and C6E (-41.98 ± 0.08 kcal/mol) respectively indicate a strong binding affinity to Spike protein active pocket. These findings demonstrate that these molecules particularly inhibit the function of spike protein and, therefore have the potential to be evaluated as drug candidates against SARS-CoV-2.

Benzimidazole-Based Schiff Base Hybrid Scaffolds: A Promising Approach to Develop Multi-Target Drugs for Alzheimer's Disease.

A series of benzimidazole-based Schiff base derivatives (1-18) were synthesized and structurally elucidated through 1H NMR, 13C NMR and HREI-MS analysis. Subsequently, these synthetic derivatives were subjected to evaluation for their inhibitory capabilities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). All these derivatives showed significant inhibition against AChE with an IC50 value in the range of 123.9 ± 10.20 to 342.60 ± 10.60 µM and BuChE in the range of 131.30 ± 9.70 to 375.80 ± 12.80 µM in comparison with standard Donepezil, which has IC50 values of 243.76 ± 5.70 µM (AChE) and 276.60 ± 6.50 µM (BuChE), respectively. Compounds 3, 5 and 9 exhibited potent inhibition against both AChE and BuChE. Molecular docking studies were used to validate and establish the structure-activity relationship of the synthesized derivatives.

Prophylactic effects of dietary caper (Capparis spinosa) extracts on the control of Streptococcus agalactiae infection, growth, immune-antioxidant, and inflammation cytokine responses of Nile tilapia fingerlings.

The antibacterial activity of aqueous (AE) or ethanolic extracts (EE) of caper (Capparis spinosa) against Streptococcus agalactiae was evaluated in vitro. Both caper extracts showed antagonistic activity against S. agalactiae and the inhibition zones in case of ethanolic extracts were larger than those of aqueous ones. Additionally, TEM investigations show that S. agalactiae cells treated with both C. spinosa extracts were damaged and degraded and this damage was greater in case of ethanolic extract. Another study was done to assess the promotion effects of dietary caper (C. spinosa) extracts on growth, antioxidant and immune activity, and inflammation cytokine responses of Nile tilapia (Oreochromis niloticus) and its resistance to S. agalactiae infection. However, fish (40 ± 2 g) were fed on diets containing 1.0 and 2.0 g/kg feed of each caper extract as well as the control group (free of caper) for 6 weeks. Fish were intraperitoneally injected (IP) with Streptococcus agalactiae at the end of the feeding trial, and fish mortality was tracked for additional ten days. Compared with other treatments, fish fed on 2.0 g EE/kg feed had higher counts of white and red blood cells as well as higher hemoglobin levels accompanied with lower AST and ALT activities. Antioxidant (superoxide dismutase and catalase activities) and immune, total protein, globulin, lysozyme, and immunoglobulin M) indices were increased along with significant decline in MDA levels in both caper extracts treated fish groups compared to the control group. Significant promotion in fish growth was affected positively with the increase in both caper extracts; particularly, the larger fish growth was observed in the treatment of 2.0 g EE/kg feed. Expressions of IL-1ß and IL-8 were declined; meanwhile levels of IL-10, SOD and CAT genes were upregulated in fish fed on 2.0 g EE/kg feed compared to other groups. After being challenged with S. agalactiae infection, fish survival was considerably (P < 0.05) greater in fish groups that fed on diets with caper extracts; particularly 2.0 g EE/kg feed (75%); while all fish fed on the control one were dead. According to these findings, the antioxidant and immune response of Nile tilapia fingerlings is stimulated by ethanolic extract of caper (2.0 g/kg feed), which also enhanced the growth performance and fish resistance to S. agalactiae infection.

The growth performance, antioxidant and immune responses, and disease resistance of Litopenaeus vannamei fed on diets supplemented with Indian ginseng (Withania somnifera).

In the current study, white-leg shrimp (Litopenaeus vannamei) were fed on diets containing varying doses of Withania somnifera aqueous extract (WSAE) at a rate of 0 (control), 0.5, 1.0, and 2.0 g/kg feed for 56 days. After the feeding trial, shrimps in all groups were challenged with the exposure to Vibrio harveyi for ten days during which animals' mortality was observed. It is noted that the dietary WSAE linearly and quadratically stimulated shrimp's growth indices particularly at the treatment of 2.0 g/kg feed. Compared to the control group, the WSAE-fed L. vannamei had significantly higher villi length, villi width, and absorption area particularly in the treatment of 2.0 g/kg feed. Furthermore, L. vannamei fed on WSAE-enriched diets consumed more feed and exhibited higher total proteolytic activity, lipase, and α-amylase activities as compared with the control group. The dietary WSAE at escalating levels linearly and quadratically enhanced the antioxidant activity (serum superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity, and reduced glutathione) and the immune response (total hemocyte counts, total protein, lysozyme, and phagocytic activity). Similarly, the mRNA expression levels of cMn-SOD, CAT, and GPx genes were linearly and quadratically upregulated in the hepatopancreas of L. vannamei fed on WSAE-enriched diets (especially in the 2.0 g/kg feed treatment), while their lowest levels were significantly observed in the control group. On the other hand, malondialdehyde levels were significantly decreased in WSAE-supplemented shrimp groups, and its highest levels were observed in animals fed on the control diet. After the bacterial exposure, the survival rates of L. vannamei fed on 1.0 and 2.0 g WSAE/kg feed (61.3% and 66.7%, respectively) were higher than those in the control animals. Taken together, the results obtained herein indicate that inclusion of WSAE in diets of L. vannamei effectively enhanced the growth, antioxidant biomarkers, immune response, and resistance to the V. harveyi infection, particularly at the treatment of 2.0 g/kg feed.

Pharmacological and ameliorative effects of Withania somnifera against cadmium chloride-induced oxidative stress and immune suppression in Nile tilapia, Oreochromis niloticus.

This study was carried out to evaluate the effects of dietary supplementation of aqueous extract of Withania somnifera (W. somnifera) against cadmium chloride-induced toxicity in the Nile tilapia, Oreochromis niloticus. Five experimental groups were designed: group (I) was free from cadmium chloride and W. somnifera and served as a control, group (II) was exposed to 1.775 mg L-1 of cadmium chloride only (which is equivalent to 1/4 96-h LC50), while groups (III), (IV), and (V) were exposed to 1.775 mg cadmium chloride L-1 with co-supplementation of dietary W. somnifera in doses of 1.0, 2.0, and 3.0 mL kg-1 body weight (bwt), respectively. The experiment lasted for 4 weeks. In the second and fourth weeks of the experiment, the following indicators were evaluated: hematological (hemogram and blood protein profile), biochemical (activities of serum liver enzymes, namely alanine transaminase (ALT) and aspartate transaminase (AST)), immunological (immunoglobulin M (IgM), serum lysozyme), and tissue antioxidant changes (malondialdehyde (MDA) levels and activities of catalase (CAT) and superoxide dismutase (SOD)). Additionally, gene expressions of glutathione-S-transferase (GST) in the liver were assessed. At the end of the experiment, all fish in all groups were experimentally challenged with Aeromonas hydrophila and the relative protection survival (RPS) was demonstrated. The results revealed that groups exposed to cadmium chloride toxicity and co-supplemented with dietary aqueous extract of W. somnifera at high doses showed significant ameliorative effects in hemogram parameters, total protein, globulin, IgM, and lysozyme against cadmium chloride-induced toxicity compared to the control group and the group exposed to a sublethal dose of cadmium chloride without co-suplemntation of W. somnifera. The results showed also that groups supplemented orally with W. somnifera at high doses have higher antioxidant activities of CAT and SOD and reduction of MDA formation. Levels of gene expressions of GST in the liver were higher in W. somnifera extract-supplemented groups more than those in the group exposed to cadmium chloride-induced toxicity without W. somnifera supplementation. In addition, the results revealed improved RPS with the dietary supply of W. somnifera extract in high doses. In conclusion, this study showed that the dietary supplementation of W. somnifera extract to diets of O. niloticus could be suggested as an effective way to overcome cadmium chloride-induced toxicity because it improves blood parameters and antioxidants, and it can be used as an immunostimulant against the invading bacterial pathogens.

The diamidine diminazene aceturate is a substrate for the high-affinity pentamidine transporter: implications for the development of high resistance levels in trypanosomes.

African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(-/-), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The K(m) for diminazene transport in bloodstream tbat1(-/-) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(-/-), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes.

Symmetrical choline-derived dications display strong anti-kinetoplastid activity.

OBJECTIVES: to investigate the anti-kinetoplastid activity of choline-derived analogues with previously reported antimalarial efficacy. METHODS: from an existing choline analogue library, seven antimalarial compounds, representative of the first-, second- and third-generation analogues previously developed, were assessed for activity against Trypanosoma and Leishmania spp. Using a variety of techniques, the effects of choline analogue exposure on the parasites were documented and a preliminary investigation of their mode of action was performed. RESULTS: the activities of choline-derived compounds against Trypanosoma brucei and Leishmania mexicana were determined. The compounds displayed promising anti-kinetoplastid activity, particularly against T. brucei, to which 4/7 displayed submicromolar EC(50) values for the wild-type strain. Low micromolar concentrations of most compounds cleared trypanosome cultures within 24-48 h. The compounds inhibit a choline transporter in Leishmania, but their entry may not depend only on this carrier; T. b. brucei lacks a choline carrier and the mode of uptake remains unclear. The compounds had no effect on the overall lipid composition of the cells, cell cycle progression or cyclic adenosine monophosphate production or short-term effects on intracellular calcium levels. However, several of the compounds, displayed pronounced effects on the mitochondrial membrane potential; this action was not associated with production of reactive oxygen species but rather with a slow rise of intracellular calcium levels and DNA fragmentation. CONCLUSIONS: the choline analogues displayed strong activity against kinetoplastid parasites, particularly against T. b. brucei. In contrast to their antimalarial activity, they did not act on trypanosomes by disrupting choline salvage or phospholipid metabolism, instead disrupting mitochondrial function, leading to chromosomal fragmentation.

Synthesis of marine-derived 3-alkylpyridinium alkaloids with potent antiprotozoal activity.

Given the pressing need for new antiprotozoal drugs without cross-resistance with current (failing) chemotherapy, we have explored 3-tridecylpyridinium alkaloids (3TPAs), derivatives of viscosamine, as antiparasitic agents. We have developed a simple synthetic route toward viscosamine and related cyclic and linear monomers and oligomers. Evaluation for cytotoxicity on the protozoan parasites Trypanosoma brucei, Leishmania spp., and Plasmodium falciparum revealed several 3TPAs with antiprotozoal activity in the nanomolar range. Their promising selectivity index in vitro prompted us to study the dynamics of cytotoxicity on trypanosomes in more detail. Parasites were killed relatively slowly at therapeutically safe concentrations, in a process that did not target the cell cycle. Clearance of T. brucei cultures was observed at drug concentrations of 1-10 µM.