Protease Inhibition Assay Research Articles

Sulforaphane's Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-Dependent and -Independent Mechanism of Anti-SARS-CoV-2 Activity.

It is well established that Nrf2 plays a crucial role in anti-oxidant and anti-inflammatory functions. However, its antiviral capabilities remain less explored. Despite this, several Nrf2 activators have demonstrated anti-SARS-CoV-2 properties, though the mechanisms behind these effects are not fully understood. In this study, using two mouse models of SARS-CoV-2 infection, we observed that the absence of Nrf2 significantly increased viral load and altered inflammatory responses. Additionally, we evaluated five Nrf2 modulators. Notably, epigallocatechin gallate (EGCG), sulforaphane (SFN), and dimethyl fumarate (DMF) exhibited significant antiviral effects, with SFN being the most effective. SFN did not impact viral entry but appeared to inhibit the main protease (MPro) of SARS-CoV-2, encoded by the Nsp5 gene, as indicated by two protease inhibition assays. Moreover, using two Nrf2 knockout cell lines, we confirmed that SFN's antiviral activity occurs independently of Nrf2 activation in vitro. Paradoxically, in vivo tests using the MA30 model showed that SFN's antiviral function was completely lost in Nrf2 knockout mice. Thus, although SFN and potentially other Nrf2 modulators can inhibit SARS-CoV-2 independently of Nrf2 activation in cell models, their Nrf2-dependent activities might be crucial for antiviral defense under physiological conditions.

A high-throughput cell-based screening method for Zika virus protease inhibitor discovery

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

Potential inhibitors isolated from Curcuma aeruginosa against dengue virus type 2 (DENV-2) NS2B-NS3 protease activity

Dengue virus (DENV) infection remains a formidable public health concern, causing significant fatalities worldwide. The unavailability of specific anti-DENV therapeutics and poor response towards dengue vaccine have impeded efforts to prevent and control this tropical disease, thereby, there is a pressing need for effective alternative therapeutics. Curcuma species including Curcuma aeruginosa have a long history of traditional use for the prevention and treatment of various ailments, including viral infections. While it is evident that several compounds isolated from these plants have antiviral activity against various viruses, their specific effects against DENV have not been thoroughly explored. The study aimed to investigate the potential of major compounds isolated from C. aeruginosa (demethoxycurcumin, curcumenol, furanodienone germacrone and zederone) in inhibiting DENV-2 NS2B-NS3 protease activity both in vitro and in silico. The results demonstrated that demethoxycurcumin (at 100 μM), furanodienone and germacrone (both at 25 μM) exhibited inhibitory effects on NS2B-NS3 protease activity comparable to aprotinin, a potent NS2B-NS3 protease inhibitor, albeit to a lesser extent. Importantly, these inhibitory concentrations did not adversely affect the viability of normal fibroblast cell line, L-929 cells, suggesting that the compounds’ effects were specific to the protease inhibition. Further in silico molecular docking analyses indicated that demethoxycurcumin, furanodienone and germacrone displayed significant binding affinities with both DENV E protein and DENV NS2B-NS3 protease, corroborating their inhibitory activity observed in the protease inhibition assay. These promising findings collectively suggest that demethoxycurcumin, furanodienone and germacrone from C. aeruginosa merit further development as distinctive inhibitors for treating DENV infection.

Expression of BmSPI38 tandem multimers in Escherichia coli and its antifungal activity

The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His6-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His6-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.

Finding Lead Compounds for Dengue Antivirals from a Collection of Old Drugs through In Silico Target Prediction and Subsequent In Vitro Validation.

Dengue virus (DENV) infection is one of the most widely spread flavivirus infections. Despite the fatality it could cause, no antiviral treatment is currently available to treat the disease. Hence, this study aimed to repurpose old drugs as novel DENV NS3 inhibitors. Ligand-based (L-B) and proteochemometric (PCM) prediction models were built using 62,354 bioactivity data to screen for potential NS3 inhibitors. Selected drugs were then subjected to the foci forming unit reduction assay (FFURA) and protease inhibition assay. Finally, molecular docking was performed to validate these results. The in silico studies revealed that both models performed well in the internal and external validations. However, the L-B model showed better accuracy in the external validation in terms of its sensitivity (0.671). In the in vitro validation, all drugs (zileuton, trimethadione, and linalool) were able to moderately inhibit the viral activities at the highest concentration tested. Zileuton showed comparable results with linalool when tested at 2 mM against the DENV NS3 protease, with a reduction of protease activity at 17.89 and 18.42%, respectively. Two new compounds were also proposed through the combination of the selected drugs, which are ziltri (zilueton + trimethadione) and zilool (zileuton + linalool). The molecular docking study confirms the in vitro observations where all drugs and proposed compounds were able to achieve binding affinity ≥ -4.1 kcal/mol, with ziltri showing the highest affinity at -7.7 kcal/mol, surpassing the control, panduratin A. The occupation of both S1 and S2 subpockets of NS2B-NS3 may be essential and a reason for the lower binding energy shown by the proposed compounds compared to the screened drugs. Based on the results, this study provided five potential new lead compounds (ziltri, zilool, zileuton, linalool, and trimethadione) for DENV that could be modified further.

Phyllanthus emblica (Amla) methanolic extract regulates multiple checkpoints in 15-lipoxygenase mediated inflammopathies: Computational simulation and in vitro evidence

Amla (Phyllanthus emblica) has long been used in traditional folk medicine to prevent and cure a variety of inflammatory diseases. In this study, the antioxidant activity (DPPH scavenging and reducing power), anti-inflammatory activity (RBC Membrane Stabilization and 15-LOX inhibition), and anticoagulation activity (Serin protease inhibition and Prothrombin Time assays) of the methanolic extract of amla were conducted. Amla exhibited a substantial amount of phenolic content (TPC: 663.53 mg GAE/g) and flavonoid content (TFC: 418.89 mg GAE/g). A strong DPPH scavenging effect was observed with an IC50 of 311.31 µg/ml as compared to standard ascorbic acid with an IC50 of 130.53 µg/ml. In reducing power assay, the EC50 value of the extract was found to be 196.20 µg/ml compared to standard ascorbic acid (EC50 = 33.83 µg/ml). The IC50 value of the RBC membrane stabilization and 15-LOX assays was observed as 101.08 µg/ml (IC50 of 58.62 µg/ml for standard aspirin) and 195.98 µg/ml (IC50 of 19.62 µg/ml for standard quercetin), respectively. The extract also strongly inhibited serine protease (trypsin) activity with an IC50 of 505.81 µg/ml (IC50 of 295.44 µg/ml for standard quercetin). The blood coagulation time (PTT) was found to be 11.91 min for amla extract and 24.11 min for standard Warfarin. Thus, the findings of an in vitro study revealed that the methanolic extract of amla contains significant antioxidant, anti-inflammatory, and anticoagulation activity. Furthermore, in silico docking and simulation of reported phytochemicals of amla with human 15-LOXA and 15-LOXB were carried out to validate the anti-inflammatory activity of amla. In this analysis, epicatechin and catechin showed greater molecular interaction and were considerably stable throughout the 100 ns simulation with 15-lipoxygenase A (15-LOXA) and 15-lipoxygenase B (15-LOXB) respectively.

Virtual and In Vitro Screening of Natural Products Identifies Indole and Benzene Derivatives as Inhibitors of SARS-CoV-2 Main Protease (Mpro).

The rapid spread of the coronavirus disease 2019 (COVID-19) resulted in serious health, social, and economic consequences. While the development of effective vaccines substantially reduced the severity of symptoms and the associated deaths, we still urgently need effective drugs to further reduce the number of casualties associated with SARS-CoV-2 infections. Machine learning methods both improved and sped up all the different stages of the drug discovery processes by performing complex analyses with enormous datasets. Natural products (NPs) have been used for treating diseases and infections for thousands of years and represent a valuable resource for drug discovery when combined with the current computation advancements. Here, a dataset of 406,747 unique NPs was screened against the SARS-CoV-2 main protease (Mpro) crystal structure (6lu7) using a combination of ligand- and structural-based virtual screening. Based on 1) the predicted binding affinities of the NPs to the Mpro, 2) the types and number of interactions with the Mpro amino acids that are critical for its function, and 3) the desirable pharmacokinetic properties of the NPs, we identified the top 20 candidates that could potentially inhibit the Mpro protease function. A total of 7 of the 20 top candidates were subjected to in vitro protease inhibition assay and 4 of them (4/7; 57%), including two beta carbolines, one N-alkyl indole, and one Benzoic acid ester, had significant inhibitory activity against Mpro protease. These four NPs could be developed further for the treatment of COVID-19 symptoms.

Phenylpyrazolone-1,2,3-triazole Hybrids as Potent Antiviral Agents with Promising SARS-CoV-2 Main Protease Inhibition Potential

COVID-19 infection is now considered one of the leading causes of human death. As an attempt towards the discovery of novel medications for the COVID-19 pandemic, nineteen novel compounds containing 1,2,3-triazole side chains linked to phenylpyrazolone scaffold and terminal lipophilic aryl parts with prominent substituent functionalities were designed and synthesized via a click reaction based on our previous work. The novel compounds were assessed using an in vitro effect on the growth of SARS-CoV-2 virus-infested Vero cells with different compound concentrations: 1 and 10 μM. The data revealed that most of these derivatives showed potent cellular anti-COVID-19 activity and inhibited viral replication by more than 50% with no or weak cytotoxic effect on harboring cells. In addition, in vitro assay employing the SARS-CoV-2-Main protease inhibition assay was done to test the inhibitors' ability to block the common primary protease of the SARS-CoV-2 virus as a mode of action. The obtained results show that the one non-linker analog 6h and two amide-based linkers 6i and 6q were the most active compounds with IC50 values of 5.08, 3.16, and 7.55 μM, respectively, against the viral protease in comparison to data of the selective antiviral agent GC-376. Molecular modeling studies were done for compound placement within the binding pocket of protease which reveal conserved residues hydrogen bonding and non-hydrogen interactions of 6i analog fragments: triazole scaffold, aryl part, and linker. Moreover, the stability of compounds and their interactions with the target pocket were also studied and analyzed by molecular dynamic simulations. The physicochemical and toxicity profiles were predicted, and the results show that compounds behave as an antiviral activity with low or no cellular or organ toxicity. All research results point to the potential usage of new chemotype potent derivatives as promising leads to be explored in vivo that might open the door to rational drug development of SARS-CoV-2 Main protease potent medicines.

In vitro and in silico investigation of garlic’s (Allium sativum) bioactivity against 15-lipoxygenase mediated inflammopathies

Introduction: Garlic (Allium sativum) is widely used as a flavor-enhancing dietary ingredient and exhibits a wide spectrum of pharmacological effects. This study aimed to investigate the therapeutic effects of aqueous garlic extract to explore the bioactivity against 15-lipoxygenase (15-LOX) mediated inflammopathies. Methods: In this study, the antioxidant (DPPH free radical scavenging assay and reducing power assay), anti-inflammatory (hypotonicity-induced hemolysis assay and 15-LOX inhibition assay) and anticoagulation (serine protease inhibition assay and prothrombin time assay) effects of the aqueous garlic extract were investigated. Furthermore, in silico molecular docking and dynamic simulation analysis of reported small compounds of garlic against 15-LOX1 and 15-LOX2 were performed to figure out the most efficient phytochemical ligands and validate the anti-inflammatory potential. Results: The DPPH scavenging effect and the reducing power of the extract were found with the IC50 of 213.87 ± 1.49 μgmL-1 and EC50 of 124.78 ± 3.39 μgmL-1, respectively. In the hypotonicity-induced hemolysis and 15-LOX inhibition assay, the IC50 values were observed as 147.59 ± 2.98 μgmL-1 and 250.05 ± 8.48 μgmL-1, respectively. The extract inhibited serine protease activity with an IC50 of 301.33 ± 1.31 μgmL-1 and prevented blood coagulation for 10.05 ± 0.35 minutes in prothrombin time assay. The in silico study identified Rhamnetin as a potential 15-LOX1 and 15-LOX2 inhibitor, and it exhibited a stable interaction with the targets throughout the 100 ns dynamic simulation. Conclusion: The findings of this study provide molecular insights into garlic’s medicinal properties as well as its bioactive compounds, which can be potential therapeutic interventions for 15-LOX mediated inflammations.

Tandem Multimerization Can Enhance the Structural Homogeneity and Antifungal Activity of the Silkworm Protease Inhibitor BmSPI39

Previous studies have shown that BmSPI39, a serine protease inhibitor of silkworm, can inhibit virulence-related proteases and the conidial germination of insect pathogenic fungi, thereby enhancing the antifungal capacity of Bombyx mori. The recombinant BmSPI39 expressed in Escherichia coli has poor structural homogeneity and is prone to spontaneous multimerization, which greatly limits its development and application. To date, the effect of multimerization on the inhibitory activity and antifungal ability of BmSPI39 remains unknown. It is urgent to explore whether a BmSPI39 tandem multimer with better structural homogeneity, higher activity and a stronger antifungal ability can be obtained by protein engineering. In this study, the expression vectors of BmSPI39 homotype tandem multimers were constructed using the isocaudomer method, and the recombinant proteins of tandem multimers were obtained by prokaryotic expression. The effects of BmSPI39 multimerization on its inhibitory activity and antifungal ability were investigated by protease inhibition and fungal growth inhibition experiments. In-gel activity staining and protease inhibition assays showed that tandem multimerization could not only greatly improve the structural homogeneity of the BmSPI39 protein, but also significantly increase its inhibitory activity against subtilisin and proteinase K. The results of conidial germination assays showed that tandem multimerization could effectively enhance the inhibitory ability of BmSPI39 on the conidial germination of Beauveria bassiana. A fungal growth inhibition assay showed that BmSPI39 tandem multimers had certain inhibitory effects on both Saccharomyces cerevisiae and Candida albicans. The inhibitory ability of BmSPI39 against these the above two fungi could be enhanced by tandem multimerization. In conclusion, this study successfully achieved the soluble expression of tandem multimers of the silkworm protease inhibitor BmSPI39 in E. coli and confirmed that tandem multimerization can improve the structural homogeneity and antifungal ability of BmSPI39. This study will not only help to deepen our understanding of the action mechanism of BmSPI39, but also provide an important theoretical basis and new strategy for cultivating antifungal transgenic silkworms. It will also promote its exogenous production and development and application in the medical field.

A Comparative Study of Anti-Inflammatory Properties and Activities of Green and Red Christia vespertilionis Leaves

Christia vespertilionis is a plant traditionally used to treat various diseases, including inflammation. This study aims to compare and analyze the anti-inflammatory properties and activities of green and red C. vespertilionis leaves and to determine the correlation between the protease inhibition assay and phenolic content. Preliminary phytochemical analysis showed the presence of alkaloids, tannins, saponins, cardenolides, phenolics, cardiac glycosides, flavonoids, and steroids in green and red leaves. Green leaf had the highest anti-inflammation potential, with a high extraction yield (6.39 ± 0.01%), phenolic content (29.25 ± 0.50 mg) gallic acid equivalent [GAE]/mL), flavonoid content (1.57 ± 0.03 mg quercetin acid equivalent [QE]/mL) and tannin content (22.70 ± 3.15 mg tannic acid equivalent [TAE]/mL). Green leaf contained more anti-inflammatory compounds, including n-hexadecanoic acid, phytol, 9, 12, 15-octadecatrienoic acid (Z, Z, Z)- and squalene. The protease inhibition assay showed that green leaf had anti-inflammatory activity at 66.55 ± 2.59% with the half-maximal inhibitory concentration (IC50) of 284.59 ± 10.63 µg/mL. The green leaf showed a significant positive correlation between the protease inhibition assay and phenolic content, with R2 = 0.6821. We conclude that both green and red C. vespertilionis types have the potential to act as anti-inflammatory agents, but green leaves are preferable.

Pedoclimatic Conditions Influence the Morphological, Phytochemical and Biological Features of Mentha pulegium L.

In this study, Mentha pulegium leaves and flowers harvested in three different Sicilian areas were investigated from a micromorphological, phytochemical and biological point of view. Light and scanning electron microscopy showed the presence of spherocrystalline masses of diosmin both in the leaf epidermal cells and in thin flower petals. Two different chemotypes were identified (I, kaempferide/rosmarinic acid; II, jaceidin isomer A). Phytochemical screening identified plant from collection site II as the richest in total phenolics (16.74 g GAE/100 g DE) and that from collection site I as the richest in flavonoids (46.56 g RE/100 g DE). Seventy-seven metabolites were identified both in flower and leaf extracts. Plant from site II showed the best antioxidant (0.90-83.72 µg/mL) and anti-inflammatory (27.44-196.31 µg/mL) activity expressed as half-maximal inhibitory concentration (IC50) evaluated by DPPH, TEAC, FRAP, ORAC, BSA denaturation and protease inhibition assays. These data were also corroborated by in vitro cell-based assays on lymphocytes and erythrocytes. Moreover, plant of site II showed the best antiangiogenic properties (IC50 33.43-33.60 µg/mL) in vivo on a chick chorioallantoic membrane. In conclusion, pedoclimatic conditions influence the chemotype and the biological activity of M. pulegium, with chemotype I showing the most promising biological properties.

Bioactive compounds, anti-inflammatory, anti-nociceptive and antioxidant potentials of ethanolic leaf fraction of Sida linifolia L. (Malvaceae)

This study investigated the bioactive compounds, anti-inflammatory, anti-nociceptive, and antioxidant properties of the ethanolic leaf fraction of Sida linifolia (ELFSL). The in vitro anti-inflammatory study employed membrane stabilization, phospholipase A2, platelet aggregation, albumin denaturation, and protease inhibition assays. Intraperitoneal injection of freshly prepared carrageenan solution (0.1 mL of 0.01 g/mL), undiluted egg albumin (0.1 mL), acetic acid (0.6 % (v/v) (10 mL/kg bw), and formalin solution (0.02 mL of 1 % v/v) into mice hind paw, were used to evaluate the anti-inflammatory and anti-nociceptive mechanisms, respectively. In vitro antioxidant potentials were determined using 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), nitric oxide (NO), ferric reducing power (FRAP), and total antioxidant capacity (TAC) assays. Varying quantities of flavonoids, phenols, tannins, saponins, terpenoids, steroids, and alkaloids, were detected in the fraction. GC-FID phytochemical profiling of ELFSL revealed a high level of epicatechin, moderate levels of catechin, kaempferol, flavone, naringenin, rutin flavanones, tannins, sapogenins, proanthocyanidin, and steroids, and small amounts of sparteine, resveratrol, and lunamarine. The ELFSL exerted excellent dose-dependent in vitro anti-inflammatory activities comparable with standard drugs (aspirin/prednisolone). The LD50 test showed safety up to 5000 mg/kg body weight (per oral) ELFSL. Interestingly, mice pre-administered various doses (200, 400, 600 mg/kg bw, po) of ELFSL showed significant (P < 0.05) reduction in edema, writhing, and time spent licking paw in all phases compared with control and were at par with 100 mg/kg bw (po) aspirin. The result also registered good concentration-dependent antioxidant potentials for ELFSL and was comparable to standards (gallic acid, butylated hydroxytoluene, and ascorbic acid). These imply that ELFSL possesses excellent antioxidant, anti-inflammatory, and anti-nociceptive potentials mediated by peripheral and central mechanisms.

Identification of Doxorubicin as Repurposing Inhibitory Drug for MERS-CoV PLpro.

Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the betacoronavirus genus can cause severe respiratory illnesses, accompanied by pneumonia, multiorgan failure, and ultimately death. CoVs have the ability to transgress species barriers and spread swiftly into new host species, with human-to-human transmission causing epidemic diseases. Despite the severe public health threat of MERS-CoV, there are currently no vaccines or drugs available for its treatment. MERS-CoV papain-like protease (PLpro) is a key enzyme that plays an important role in its replication. In the present study, we evaluated the inhibitory activities of doxorubicin (DOX) against the recombinant MERS-CoV PLpro by employing protease inhibition assays. Hydrolysis of fluorogenic peptide from the Z-RLRGG-AMC-peptide bond in the presence of DOX showed an IC50 value of 1.67 μM at 30 min. Subsequently, we confirmed the interaction between DOX and MERS-CoV PLpro by thermal shift assay (TSA), and DOX increased ΔTm by ~20 °C, clearly indicating a coherent interaction between the MERS-CoV PL protease and DOX. The binding site of DOX on MERS-CoV PLpro was assessed using docking techniques and molecular dynamic (MD) simulations. DOX bound to the thumb region of the catalytic domain of the MERS-CoV PLpro. MD simulation results showed flexible BL2 loops, as well as other potential residues, such as R231, R233, and G276 of MERS-CoV PLpro. Development of drug repurposing is a remarkable opportunity to quickly examine the efficacy of different aspects of treating various diseases. Protease inhibitors have been found to be effective against MERS-CoV to date, and numerous candidates are currently undergoing clinical trials to prove this. Our effort follows a in similar direction.

Evaluation of Antioxidant and Anticoagulation Activity of Piper chaba Hunter Stem

Background: Piper chaba Hunter, a flowering vine of the Piperaceae family, has long been used in South Asian countries for culinary purposes and traditionally in fat-rich meat preparation. The curative potential of this herb is of great interest to be studied.&#x0D; Objectives: The antioxidant and anticoagulation potential, as well as total phenolic and flavonoid content, were evaluated using cold and boiled water extract separately from the dried and ground stem.&#x0D; Methods: Antioxidant potential was evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power (FRAP) assay. The anticoagulation activity was evaluated by serine protease inhibition assay and prothrombin time (PT) assay. Folin–Ciocalteu (FC) reagent and aluminum complex (AlCl3 ) were used to assess total phenolic content and total flavonoid content, respectively.&#x0D; Results: DPPH scavenging assay revealed the IC50 value of 125.52 µg.mL-1 and 157.94 µg.mL-1 for boiled and cold water extract, respectively. Potent ferric reducing potential (FRAP) was observed as 142.87 µM and 135.37 µM of ferrous equivalent per 100 µg for boiled and cold water extract, respectively. The IC50 value of serine protease inhibitory activity was found as 182 µg.mL-1 and 161.12 µg.mL-1 for cold and boiled water extract, respectively. The PT time was 27.00 min for boiled water extract and 24.68 min for cold water extract. Significant phenolic and flavonoid content was also found in the test sample.&#x0D; Conclusion: P. chaba stem extract possesses potent antioxidant and anticoagulation activity, which can neutralize oxidative free radicals and have a vasodilation effect in oxidative and inflammatory diseases.

Evaluation of Anti-Inflammatory and Wound Healing Potential of Sodium Glucose Co-Transporter2 (SGLT2) Inhibitors

Wound healing is a very complex process involving many stages out of which inflammatory stage remains as a rate limiting phase. Most of the anti-inflammatory drugs were proven to speed up the wound healing process which is majorly achieved by migration of monocytes and neutrophils to the healing site along with leukocytes. The presence of inflammatory mediators like interleukins, TNF delay the healing process and so the drug that actively combats these mediators will prevent the prolongation of wound healing. On the other hand SGLT2 inhibitors otherwise called as Gliflozins are extensively used for their antidiabetic potential. These drugs were also proven to exhibit antihyperlipidemic, antioxidant, cardio-protective, antihypertensive activities. Considering this, Gliflozins like Canagliflozin, Dapagliflozin and Empagliflozin were employed to investigate their anti-inflammatory profile and their wound healing ability in vitro on VERO cell lines. Anti-inflammatory activity was investigated in vitro using protease inhibition assay at drug concentration of 200, 400, 600, 800 and 1000 𝜇g/ml. This showed that the selected Gliflozins exhibited good activity compared to that of the standard drug. Invitro cytotoxicity was determined using MTT assay on VERO cell lines and Canagliflozin showed a significant viability at 250𝜇g/ml compared to control. Similarly wound healing activity was determined invitro using scratch assay method. Dapagliflozin showed a significant activity of over 60% wound closure compared to the standard drug Phenytoin. Overall this work opens up a new route for exploring other medications in the SGLT2 inhibitor family for the aforementioned actions, as well as creating alternatives to the medication's principal action. This also allows for the avoidance and limitation of the use of multiple drugs for various pharmacological activities.

In vitro Anti-cancer, Anti-inflammatory and Anti-arthritic activity of Ethanolic extract of Ochna obtusata leaves

Objective: The objective behind this study was to expose the prospective of ethanolic extract of Ochna obtusata (EEOO) for its in vitro anti-cancer anti-inflammatory and anti-arthritic activities. Methods: The fresh leaves were used to prepare the extracts using petroleum ether, chloroform, ethanol, methanol and water in increasing order of the polarity of solvents. The EEOO was reported to have the presence of a few major phytoconstituents such as, Flavonoids, Phenolics, Glycosides, and Reducing Sugars which was confirmed by preliminary phytochemical screening. The in vitro MTT assay method has been performed with EEOO to evaluate the anti-cancer activity. Similarly, the HRBC Membrane Stabilization assay, Protein denaturation assay, and Protease inhibition assay methods were performed to evaluate the anti-inflammatory and anti-arthritic activity of EEOO. Results and Discussion: From the study, it was confirmed that the various phytoconstituents such as Flavonoids, Phenolics, and Terpenoids were responsible for the anti-cancer activity. Similarly, the prevention of RBC Membrane from its lysis, decrease in albumin denaturation, and protease inhibition was observed which significantly confirmed the anti-inflammatory and anti-arthritic activity of EEOO. Conclusion: It was finally concluded that the phytoconstituents present in the extract were responsible for its anti-cancer, anti-inflammatory, and anti-arthritic activities.

Thiazole-based SARS-CoV-2 protease (COV Mpro ) inhibitors: Design, synthesis, enzyme inhibition, and molecular modeling simulations.

As an attempt to contribute to the efforts of combating the pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) responsible for COVID‐19, new analogs of the repurposed drug nitazoxanide which showed promising inhibitory efficacy on a viral protease enzyme were designed, synthesized and evaluated for their inhibitory activity on the main protease of the SARS‐CoV‐2 virus, using the COV2‐3CL protease inhibition assay. The obtained results showed that the N‐(substituted‐thiazol‐2‐yl)cinnamamide analogs 19, 20, and 21 were the most active compounds with IC50 values of 22.61, 14.7, 21.99 µM, respectively, against the viral protease compared to the reference drugs, nitazoxanide, and lopinavir. Molecular modeling studies showed binding interactions of 19, 20, and 21 with hydrogen bonds to Gln189 and Glu166, arene–arene interaction between the thiazole moiety and His41, and other hydrophobic interactions between the ethene spacer moiety and Asn142. Moreover, an extra arene–arene interaction between substituted benzo[d]thiazole and His41 was observed regarding compounds 19 and 21. Surface mapping and flexible alignment proved the structural similarity between the new drug candidates and nitazoxanide. Compliance of the new compounds to Lipinski's rule of five was investigated and absorption, distribution, metabolism, excretion, and toxicology data were predicted. The newly synthesized compounds are promising template ligands for further development and optimization.

The discovery of Zika virus NS2B-NS3 inhibitors with antiviral activity via an integrated virtual screening approach

With expanding recent outbreaks and a lack of treatment options, the Zika virus (ZIKV) poses a severe health concern. The availability of ZIKV NS2B-NS3 co-crystallized structures paved the way for rational drug discovery. A computer-aided structure-based approach was used to screen a diverse library of compounds against ZIKV NS2B-NS3 protease. The top hits were selected based on various binding free energy calculations followed by per-residue decomposition analysis. The selected hits were then evaluated for their biological potential with ZIKV protease inhibition assay and antiviral activity. Among 26 selected compounds, 8 compounds showed promising activity against ZIKV protease with a percentage inhibition of greater than 25 and 3 compounds displayed ∼50% at 10 µM, which indicates an enrichment rate of approximately 36% (threshold IC50 < 10 µM) in the ZIKV-NS2B-NS3 protease inhibition assay. Of these, only one compound (23) produced whole-cell anti-ZIKV activity, and the binding mode of 23 was extensively analyzed through long-run molecular dynamics simulations. The current study provides a promising starting point for the further development of novel compounds against ZIKV.

Design, Synthesis, and in-vitro Protease Inhibition Assay of Linear Pentapeptides as Potential Dengue Virus NS2B/NS3 Protease Inhibitors.

Nowadays dengue virus infection (DENV) is one of the major health complications in the world. Although DENV is an old and common disease, unfortunately, until now, there are no specific relevant treatments available for it. This study, therefore, aimed to design, as well as synthesize selective peptide inhibitors, and investigate their activity by in-vitro NS2B/NS3 protease inhibition assay. The design of the peptide ligands was based on studying the interactions with the dengue NS2B/NS3 protease using the computational docking technique in the MOE and AutoDock (version 4.2) software. To this end, the researchers designed 26 linear pentapeptides based on previous studies. It was revealed that two linear pentapeptides (i.e., GKRRK and KRRRK) are the best potential inhibitors. Furthermore, based on the findings of the two independent docking programs, the peptide GKRRK was synthesized by solid-phase peptide synthesis and its structure was confirmed. The in-vitro protease inhibitor study was conducted for these two peptides to examine their activity against the dengue virus using a protin in as a control. It was found that the designed potential peptides possess interesting inhibition against the NS2B/NS3 protease. Additionally, the findings showed that the peptide GKRRK had the highest percentage of inhibition (71.11%) at 100 µM with the IC50 of 48.87 µM; therefore, this linear peptide could serve as a good inhibitor for the DENV.