Lineweaver-Burk Research Articles

Investigation of the Interaction Between Angiotensin-Converting Enzyme (ACE) and ACE-Inhibitory Tripeptide from Casein

Angiotensin-converting enzyme (ACE) inhibitory peptides exhibit antihypertensive effects by inhibiting ACE activity, and the study of the interaction between ACEs and inhibitory peptides is important for exploring new therapeutic strategies. In this study, the ACE-inhibitory peptide isolated from casein hydrolysate with the amino acid sequence Leu–Leu–Tyr (LLY) exhibited high ACE-inhibitory activity and stability, which holds significant implications for biochemistry and pharmaceutical applications. Furthermore, systematic investigations were conducted on the interaction between ACE and LLY through various approaches. The Lineweaver–Burk plot indicated the non-competitive inhibition pattern of LLY, suggesting that it binds to the enzyme at the non-active site, and the results were further validated by a molecular docking study. Additionally, multispectral experiments and atomic force microscopy were conducted to further elucidate the underlying mechanism of peptide activity. The findings indicated that LLY could induce a conformational change in ACE, thereby inhibiting its activity. This study contributes to a deeper understanding of the mechanism of action of ACE-inhibitory peptides and bears important significance for drug development in hypertension.

Meroterpenoids with BACE1-inhibitory activity from the fruiting bodies of Suillus bovinus and Boletinus cavipes.

Alzheimer disease (AD) is the most common type of dementia and accounts for the largest proportion of dementia cases. The amyloid cascade hypothesis is known for the pathogenesis of AD, in which excessive accumulation of amyloid-β (Aβ) leads to the formation of senile plaques and ultimately to AD. Inhibition of β-secretase (BACE1) may contribute to the treatment of AD by suppressing Aβ production. In this study, we isolated and characterized the activity of new and known BACE1-inhibiting compounds from two mushrooms of the Boletales order, Suillus bovinus and Boletinus cavipes, using a BACE1-inhibitory activity-guided separation approach. Three compounds (1-3) were isolated from Suillus bovinus CHCl3 extract and three compounds (4-6) were isolated from Boletinus cavipes CHCl3 extract. Compound 1 was a new compound. The structures were elucidated using MS, IR, and NMR. Compounds 1-6 showed BACE1-inhibitory activity (IC50; 21.2, 17.8, 1.0, 1.6, 23.7, and 22.8 μM, respectively). To examine the structure-activity relationship, we also evaluated the activity of geranylgerniol, farnesol, 2,5-dihydroxy-1,4-benzoquinone and mesaconic acid. These compounds showed no activity, and these results indicate that chain terpenes alone do not show BACE1-inhibitory activity, but only when mesaconic acid or a quinone with a hydroxyl group is bound. In addition, the mode of inhibition of 2 and 3 were competitive and 4 was uncompetitive inhibition, respectively, as determined by analysis of Lineweaver-Burk and Dixon plots.

Convergent Synthesis, Kinetics, and Computational Studies of Indole(Phenyl)Triazole Bi-Heterocycles Modified With Propanamides as Elastase Inhibitors.

Biological screening combined with the synthesis of heterocyclic compounds with numerous functions is the most effective approach available for pharmacological assessment of potential future medications. In the under taken research that is presented here, 4-(1H-indol-3-yl)butanoic acid was sequentially converted into 4-(1H-indol-3-yl)butanoate, 4-(1H-indol-3-yl)butanohydrazide, and 5-[3-(1H-indol-3-yl)propyl]-1,2,4-triazole-2-thiol as a nucleophile. By treating aryl amines with 3-bromopropanoyl chloride in a series of parallel reactions, different electrophiles were created, leading to the formation of N-(aryl)-3-bromopropanamides. After that, several electrophiles were used in the nucleophilic substitution process of 5 to produce the final bi-heterocyclic derivative. The structural confirmation of all the synthesized compounds was done by IR, 1H-NMR, 13C-NMR, and CHN analysis data. The enzyme inhibitory effects of these bi-heterocyclic propanamides were evaluated against elastase, and all these molecules were identified as potent inhibitors relative to the standard oleanolic acid with IC50 value 13.453±0.015µM used. The kinetics mechanism was ascribed by evaluating the Lineweaver-Burk plots, which revealed that compound 9d inhibited elastase competitively to form an enzyme-inhibitor complex. The inhibition constant Ki calculated from Dixon plots for this compound was 0.51µM. Compound 9d's activity (IC50=0.142±0.014µM) significantly increased when a slightly bulky ethyl group was replaced for the solitary methyl group in 9c at the para-position. However, compound 9e's activity was significantly lower (IC50=38.338±0.993µM) when a more polar ethoxy group was replaced at the same para-position. This was likely because of electronic considerations. These molecules also exhibited mild cytotoxicity toward red blood cell membranes, when analyzing through hemolysis. So, these molecules might be deliberated as nontoxic medicinal scaffolds for dealing with the elastase-related ailments such as lung diseases, cyclic neutropenia, pruritic skin disease, and liver infection.

Novel angiotensin-converting enzyme inhibitory peptides derived from tuna (Thunnus maccoyii): The integration of computer tools with in vitro experiments

With the rapid development of bioinformatics and the establishment of various databases for bioactive peptides, they provided a time-efficient method for discovering novel bioactive peptides from diverse protein sources. Peptides with angiotensin converting enzyme (ACE) inhibitory ability were considered potential dietary interventions for hypertension. The aim of this study was to discovery novel potential ACE inhibitory peptides from four tuna (Thunnus maccoyii) protein by the integration of computer tools with in vitro experiments. In this context, eight novel peptides were synthesized and the WHR (IC50 = 1383.2 μM) and LGR (IC50 = 115.4 μM) showed better ACE inhibitory ability. The interaction mechanism of LGR with ACE was investigated. The corresponding results showed that mixed-type was the kinetic pattern by Lineweaver-Burk plot, LGR formed ten hydrogen bonds with ACE by molecular docking and they had strong affinity by molecular dynamic simulation. Furthermore, LGR may regulate blood pressure through AKT1, MAPK1, HRAS, EGFR and SRC targets by network pharmacology predicting. Additionally, LGR exhibited excellent stability in temperature, pH and gastrointestinal digestion. Finally, the apparent permeability coefficient of LGR was measured to be 6.38 × 10−6 cm/s. These results suggested that the integration of computer tools with in vitro experiments was a promising approach to discovery novel potential bioactive peptides.

Synthesis and Evaluation of Phenyltriazole-Deoxynojirimycin Hybrids as Potent α-Glucosidase Inhibitors.

1-deoxynojirimycin (DNJ) is a well-known α-glucosidase inhibitor. A series of phenyltriazole-deoxynojirimycin hybrids containing C4 and C6 (4 and 6 methylenes, respectively) linkers were synthesized. These novel compounds were assessed for preliminary glucosidase inhibition and cytotoxicity tests in vitro. Among them, compounds 12-14 and 16-20 (IC50: 105 ± 9-11 ± 1 μM) were more active than deoxynojirimycin (DNJ, IC50 = 155 ± 15 μM). The kinetics of enzyme inhibition measured by using Lineweaver-Burk plots indicated that compounds 18 and 19 were competitive inhibitors. In addition, a molecular docking study of α-glucosidase revealed that the interaction modes and the orientations of compound 18 and DNJ were clearly different. Furthermore, in tissue culture, HL60 cell compounds showed no cytotoxicity at low concentrations. When the concentration reached 50 µM, only compound 20 exhibited cytotoxicity. The structure-activity relationships exhibit that the length of the linker and the nature of 4-position substituents on the phenyl have a significant effect on the inhibitory potency of glucosidases and cytotoxicity.

Porous Pd-Loaded IRMOF-9 as Highly Efficient Recyclable Material Towards the Reduction of Nitroaromatics in Aqueous Media.

Nitroaromatic compounds (NACs) cause severe hazardous impacts on human health as well as on the environment. Therefore, there is dire need to develop a robust material to reduce the toxicity of these organic pollutants. In this regard, our group developed a series of porous MOF materials viz., Pdx@IRMOF-9 (x=2 %, 5 % and 10 %) by loading different concentration of Pd(II) on IRMOF-9 and explored them towards reduction of different nitroaromatic compounds. Pd10%@IRMOF-9showed ~30 % greater efficiency for the reduction of 4-NP as compared to Pd2%@IRMOF-9. Pd10%@IRMOF-9showed excellent reduction ability (>85 %) towards 4-NP, 2-NP, 2-NA, 3-NA and 2,4-DNPH. The kinetic studies indicates that the reduction follows the pseudo-first-order kinetics. Moreover, the rate constant value for reduction of 3-NA was ~9 times higher than that of 2-NP. Based on the kinetic parameters, the t1/2 values for all the nitroaromatics have been calculated. The kinetic parameters, Km and Vmax have been calculated from double reciprocal Lineweaver-Burk plot and found to be 65.984 μM and 116×10-6 Mmin-1 respectively. Pd10%@IRMOF-9showed excellent recyclability towards the reduction of 4-NP for few consecutive cycles without any remarkable loss in its activity. Thus, highly efficient, porous and robust material for the reduction of nitroaromatic compounds in aqueous media have been demonstrated.

Mechanistic in vitro study of the effect of Cucurbita moschata (Cucurbitaceae) on carbohydrate digestive enzymes.

Diabetes is marked by postprandial hyperglycemia (PHG), an abnormal rise in blood glucose after meals. A key therapeutic goal to reduce PHG is the inhibition of α-amylase (αAM) and α-glucosidase (αGL), enzymes that break down carbohydrates into sugars. Cucurbita moschata has been shown to inhibit both enzymes. However, its inhibition mechanism has not been explored. This study investigated the in vitro inhibition mechanisms of αAM and αGL and conducted a metabolomic analysis of C. moschata (edible part) water-extract (CME), aiming to preliminarily identify its bioactive compounds (BCs). The inhibitory mechanisms were determined using Lineweaver-Burk plots. The BCs were identified and quantified using HPLC-QTOF-MS, employing both targeted and untargeted metabolomic approaches. CME had a significant higher effect (p<0.05) on αAM activity than against αGL with IC50 of 28.99 and 698.42mg/mL, respectively. The extract showed mixed and uncompetitive type inhibitions on αAM and αGL, respectively. The lowest inhibition constant (Ki) was 47.68mg/mL on αAM activity at 20mg/mL. Untargeted metabolic profiling by UPLC-MS-ESI-QTOF putatively identified 30 compounds in CME, such as amino acids, vitamins, phytohormones, fatty acids, cucurbitacins and phenolic acids, and flavonoids. Functional analysis of CME identified significant pathways, including pantothenate and CoA biosynthesis and phenylpropanoids, among others. The targeted analysis by UPLC-MS-ESI-QqQ allowed us to identify 12 compounds, with l-phenylalanine, p-hydroxybenzoic, and p-coumaric acid as majors. This study demonstrated the inhibitory potential of CME on αAM and αGL activities, which may be attributed to its metabolites. Thus, this plant represents a valuable source of BC against PHG. Practical Application: The research highlights that Cucurbita moschata has significant potential in managing postprandial hyperglycemia in diabetic patients by inhibiting enzymes like α-amylase and α-glucosidase. In addition, the identification of its compounds emphasizes its importance as a source of bioactive compounds. Therefore, C. moschata could be effectively utilized in the development of nutraceuticals or as an ingredient in functional foods specifically designed for postprandial hyperglycemia management. Thus, integrating C. moschata as part of the daily diet could offer patients with diabetes a natural alternative to control their blood glucose levels after eating.

Research on the Synergistic Inhibition of Angiotensin-Converting Enzyme (ACE) by the Gastrointestinal Digestion Products of the ACE Inhibitory Peptide FPPDVA.

To gain a deeper understanding of the ACE inhibition effect, the inhibitory effect of ACE-inhibiting peptide (ACEIP) FPPDVA's digestive products on ACE was further investigated. Two novel peptides, PD (IC50 = 161.1 ± 1.10 μM) and DV (IC50 = 66.51 ± 0.99 μM) were identified in the digestive products of FPPDVA using LC-MS/MS. The Peptide Mix (FPPDVA, PD, and DV) exhibited a remarkable synergistic effect on ACE inhibition by significantly enhancing it by up to 508% compared to the individual peptides alone. Furthermore, theoretical simulations suggest that the Peptide Mix synergistically inhibits ACE activity by forming more stable complexes with the active site of ACE, facilitated by an increased number of hydrogen bonds. Additionally, Lineweaver-Burk plot analysis and spectroscopic studies further verified the presence of these stable complexes. ITC results show that the combination of Peptides Mix and ACE is a spontaneous exothermic process driven by entropy. The study showed that FPPDVA has a stronger inhibitory effect on ACE after digestion, making it suitable as an antihypertensive peptide in functional foods.

Radical Scavenging Capacity and In Vitro Cytoprotective Effects of Great Salt Lake-Derived Processed Mineral Water.

The Great Salt Lake, located in Utah, USA, is a saltwater lake with no outlet and is surrounded by vast mountains and salt deserts. We aimed to use Great Salt Lake-derived processed mineral water (hereafter termed as GSL-MW) for maintaining oral health. Therefore, we examined its radical scavenging activity as an antioxidant and its cytoprotective effect on human gingival fibroblasts (hGFs). The scavenging activity against O2•- radicals was determined by an electron spin resonance (ESR)-spin trapping technique using two kinds of O2•- generation systems; however, we could not reach any concrete conclusion because of the interference caused by GSL-MW in both systems. Detection of ·OH radicals using the ESR-spin trapping technique and kinetic analyses using double-reciprocal plots (corresponding to Lineweaver-Burk plots that are used to represent enzyme kinetics) revealed that GSL-MW has the ability to scavenge ·OH radicals. GSL-MW also showed a weak 2,2-diphenyl-1-picrylhydrazyl (DPPH; a stable radical)-scavenging activity. Regarding the cytoprotective effects, subconfluent hGFs pretreated with 10× and 100× dilutions of GSL-MW for 3 min and then exposed to harsh environmental conditions, such as pure water or 100 μM H2O2 for 3 min, showed enhanced cell viability rate. Moreover, 10× and 100× dilutions of GSL-MW reduced oxidative damage in confluent hGFs exposed to 12.5 and 25 mM H2O2. Our findings show that GSL-MW has antioxidant potential and cytoprotective effects on hGFs, suggesting that GSL-MW can be used to maintain oral health.

Synthesis, Urease Inhibition, Molecular Docking, and Optical Analysis of a Symmetrical Schiff Base and Its Selected Metal Complexes.

Designing and developing small organic molecules for use as urease inhibitors is challenging due to the need for ecosystem sustainability and the requirement to prevent health risks related to the human stomach and urinary tract. Moreover, imaging analysis is widely utilized for tracking infections in intracellular and in vivo systems, which requires drug molecules with emissive potential, specifically in the low-energy region. This study comprises the synthesis of a Schiff base ligand and its selected transition metals to evaluate their UV/fluorescence properties, inhibitory activity against urease, and molecular docking. Screening of the symmetrical cage-like ligand and its metal complexes with various eco-friendly transition metals revealed significant urease inhibition potential. The IC50 value of the ligand for urease inhibition was 21.80 ± 1.88 µM, comparable to that of thiourea. Notably, upon coordination with transition metals, the ligand-nickel and ligand-copper complexes exhibited even greater potency than the reference compound, with IC50 values of 11.8 ± 1.14 and 9.31 ± 1.31 µM, respectively. The ligand-cobalt complex exhibited an enzyme inhibitory potential comparable with thiourea, while the zinc and iron complexes demonstrated the least activity, which might be due to weaker interactions with the investigated protein. Meanwhile, all the metal complexes demonstrated a pronounced optical response, which could be utilized for fluorescence-guided targeted drug delivery applications in the future. Molecular docking analysis and IC50 values from in vitro urease inhibition screening showed a trend of increasing activity from compounds 7d to 7c to 7b. Enzyme kinetics studies using the Lineweaver-Burk plot indicated mixed-type inhibition against 7c and non-competitive inhibition against 7d.

Phytochemistry, antioxidant and anti-diabetic activities of Sterculia villosa in-vitro

ObjectiveAs ongoing research continues, many anti-diabetic agents still have a multitude of side effects which is the reason why more and more people resort to traditional medicines such as Traditional Chinese Medicine (TCM). There are many Therapeutic Species of Sterculia spp. that are commonly used in TCM practices. Here, we have studied one of such TCM herbal plants, namely Sterculia villosa Roxb. (Chinese: 绒毛苹婆), for its anti-diabetic potential. In this way, the study intends to assess its effectiveness and examine its suitability as a raw natural medicine for diabetes management in traditional medicine. MethodIn-vitro anti-diabetic property was evaluated by α-amylase and α-glucosidase enzyme inhibition and glucose uptake assays. Metabolomic profiling by GC–MS was conducted to determine the probable phyto-constituents in S. villosa. These phyto-compounds were further subjected to in-silico molecular docking studies, identifying stigmasterol as the most potent anti-diabetic phyto-compound. Glucose uptake efficacy was estimated by a fluorescence microplate reader employing glucose analog, 2-NBDG. Molecular dynamic studies (MDS) were carried out with GROMACS to comprehend the protein-ligand binding stability. ResultsStigmasterol showed enhanced enzyme inhibition for both α-amylase (IC50 23.84±1.37 μM) and α-glucosidase (IC50 39.56±1.5 μM). Line-weaver Burk plot revealed competitive and mixed modes of inhibition for α-amylase and α-glucosidase, respectively. The GC-–MS analysis identified 23 vital phyto-compounds present in the methanolic extract of the S. villosa leaves. Upon screening against crucial anti-diabetic proteins by in-silico molecular docking, stigmasterol followed by lupeol, Stigmasta-3,5-diene, and γ-sitosterol were found to be the most effective lead compounds. Stigmasterol also showed potent hypoglycaemic efficacy, as evidenced by augmentation of 2-NBDG glucose uptake in 3T3-L1 cells. The docking and simulation studies against four crucial anti-diabetic targets, α-amylase, α-glucosidase, GLUT 4, and IRS-1, predicted that stigmasterol exhibits a multi-pronged anti-diabetic activity mediated by α-amylase inhibition and activation of IRS1 pathway, simultaneously. The MDS studies highlighted that stigmasterol showed stable interactions with these four proteins, as evidenced by the RMSD, RMSF and hydrogen bond analysis. ConclusionThe results of this study are intriguing, as they identify stigmasterol, sourced from the medicinal plant S. villosa, as a potential breakthrough in the field of diabetic remediation. This discovery paves the way for further research and development of stigmasterol as a more effective and safer treatment for diabetes.

Mechanistic insights into β-glucuronidase inhibition by isoprenylated flavonoids from Centaurea scoparia: Bridging experimental and computational approaches

Exploring the intricate mechanisms underlying the inhibition of β-glucuronidase, particularly the enzyme produced by gastrointestinal bacteria, is essential for the development of novel therapeutic interventions and pharmaceutical advancements. Inhibiting bacterial β-glucuronidase can prevent the reactivation of harmful drug metabolites in the gut, thereby reducing associated toxicities. The inhibitory potential of four isoprenylated flavonoids from Centaurea scoparia against β-glucuronidase was intensively investigated by combined in vitro and in silico tools. The results of in vitro inhibitory activity showed that Compounds 1 and 3 exhibited the highest inhibitory activity, as evidenced by their low IC50 values of 3.65 ± 0.28 and 3.97 ± 0.11 μM, respectively. The enzyme kinetics analysis revealed that Compound 1 and the positive control drug (EGCG) follow a mixed inhibition mechanism. In contrast, compounds 3 and 4 exhibited a noncompetitive inhibition mode, as suggested by the intersection patterns observed in the Lineweaver-Burk plots. The docking studies corroborated the in vitro inhibitory assay results, with Compounds 1 and 3 demonstrating the lowest binding affinities and the highest extent of polar and hydrophobic interactions with residues in the enzyme's binding site. Utilizing a 200 ns molecular dynamics (MD) simulation, we examined the interaction dynamics between isolated flavonoids and β-glucuronidase. The analysis of multiple MD parameters revealed that compounds 1 and 3 maintained consistent trajectories and demonstrated significant energy stabilization when interacting with β-glucuronidase. These computational findings align with experimental results, highlighting the potential of compounds 1 and 3 as potent inhibitors for β-glucuronidase.

Exploration of catalytic activities of Caesalpinia bonduc (L.) Roxb. ash on green-oxidation of small organic substrates: an effective way to reduce the use of harsh chemicals

In this modern era, large-scale industrial use of harsh chemicals is an alarming problem as it can cause serious environmental damage. Among industrial pollutants, harsh chemical oxidizing agents are one of the leading chemicals that can cause severe environmental threats. These harmful harsh chemicals are practically omnipresent in industries and are used to oxidize numerous small organic substrates. Replacement of these harsh chemicals with some plant-based products might be helpful to safeguard our environmental damage. In this study, Caesalpinia bonduc (L.) Roxb. ash is used as a catalyst to activate aerial oxygen for promoting the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) and o-aminophenol (OAPH) like small organic substrates. Detailed kinetic studies for these catalytic oxidation reactions have been performed spectrophotometrically, which confirms that catalytic reactions follow Michaelis–Menten kinetics. Several enzyme-kinetics plots such as the Michaelis–Menten plot, Lineweaver–Burk plot, Hanes–Woolf plot, and Eadie–Hofstee plot have been used to determine the maximum rate achieved by the reaction (Vmax) and Michaelis constant (KM) like kinetic parameters. The average values of Vmax and KM for catalytic oxidation of 3,5-DTBC are (8.6669 ± 0.6519) × 10–5 M S−1 and (21.4289 ± 0.4741) × 10–4 M, respectively. Similarly, the average values of Vmax and KM for catalytic oxidation of OAPH are (2.1781 ± 0.2071) × 10–5 M S−1 and (20.1062 ± 1.2690) × 10–3 M, respectively. The mineral composition of Caesalpinia bonduc (L.) Roxb. ash has also been evaluated by using inductively coupled plasma optical emission spectroscopy (ICP-OES).Graphical

Biogenic synthesis of AgNPs via polyherbal formulation: Mechanistic neutralization and toxicological impact on acetylcholinesterase from Bungarus sindanus venom.

This study aims to examine the biogenic production, characterization, and anti-acetylcholinesterase (AAChE) properties of polyherbal formulation PHF-extract-synthesized silver nanoparticles (PHF-AgNPs). The Elapidae snake Bungarus sindanus has extremely dangerous venom for humans and contains a high amount of AChE (acetylcholinesterase). Inhibiting AChE leads to acetylcholine buildup, affecting neurotransmission. The study tested silver nanoparticles as AChE inhibitors using kinetics. Their production was confirmed through ultraviolet (UV) spectrometry at 425 nm (SPR peak of 1.94), and stabilizing functional groups were identified via Fourier transform infrared spectroscopy (FT-IR). The average length of 20 nm was confirmed by analyzing the scanning electron microscopy (SEM) data. Energy-dispersive X-ray spectroscopy (EDX) identified silver as the primary component of PHF-AgNPs (26%). Statistical analysis showed that the activity of AChE in krait venom decreased by up to 45% and 37% at a given dose of ACh (0.5 mM) by PHF and AgNPs, respectively. Utilizing the Lineweaver-Burk plot for kinetic analysis, a competitive type of inhibition is found. RESEARCH HIGHLIGHTS: Successfully synthesized PHF-extract-induced silver nanoparticles (PHF-AgNPs) demonstrated through UV spectrometry and characterized as crystalline with an average size of 45 nm by X-ray diffraction. PHF-AgNPs effectively inhibited acetylcholinesterase (AChE), an enzyme critical in neurotransmission, reducing its activity in krait venom by up to 45% at certain concentrations. Kinetic analysis revealed that the inhibition mechanism of AChE by PHF-AgNPs is competitive, offering potential for therapeutic applications in neurologically related conditions.

Novel Alkynylamide-Based Nonpeptidic Allosteric Inhibitors for SARS-CoV-2 3-Chymotrypsin-like Protease.

Although the coronavirus disease 2019 (COVID-19) crisis has passed, there remains a necessity for continuous efforts toward developing more targeted drugs and preparing for potential future virus attacks. Currently, most of the drugs received authorization for the treatment of COVID-19 have exhibited several limitations, such as poor metabolic stability, formidable preparation, and uncertain effectiveness. It is still significant to develop novel, structurally diverse small-molecule antiviral drugs targeting SARS-CoV-2 3-chymotrypsin-like protease (3CLpro). Herein, we report a class of alkynylamide-based nonpeptidic 3CLpro inhibitors that can be prepared conveniently by our previously developed one-pot synthetic method. The structure-activity relationships of alkynylamides as SARS-CoV-2 3CLpro inhibitors have been carefully investigated and discussed in this study. The two stereoisomers of the resulting molecules exhibit stereoselective interaction with 3CLpro, and the optimized compound (S,R)-4y inhibits 3CLpro with high potency (IC50 = 0.43 μM), low cytotoxicity, and acceptable cell permeability. Compound (S,R)-4y presents as a noncovalent inhibitor of 3CLpro against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. The Lineweaver-Burk plots, binding energy, surface plasmon resonance, and molecular docking studies suggest that (S,R)-4y specifically binds to an allosteric pocket of the SARS-CoV-2 3CLpro. These findings provide a novel class of nonpeptidic alkynylamide-based allosteric inhibitors with high selectivity against SARS-CoV-2 3CLpro featured by a simplified one-pot synthesis at room temperature in air.

Multi-step synthesis, kinetics and in silico explorations of indole-Phenyl-1,2,4-triazole Bi-heterocyclic hybrids unified with 3-substituted benzamides as elastase inhibitors

In the present research work, a library of unique indole-phenyltriazole hybrids comprising 3-substituted-benzamide moiety was synthesized through convergent multi-step strategy. The structural confirmation of all synthesized molecules was corroborated by IR, 1H NMR, 13C MMR, EI-MS and CHN analysis data. The results of in vitro inhibitory potential of novel benzamides against elastase enzyme revealed that all molecules were potent inhibitors and 10d was most active compound among them having IC50 value of (0.197 ± 0.027 µM), relative to the standard (13.421 ± 0.016 µM). The Kinetics mechanism analyzed by Lineweaver-Burk plots which exposed that 10d inhibited this enzyme competitively by forming an enzyme-inhibitor complex. The inhibition constant Ki determined from Dixon plot for compound 10d was 0.85 µM. Moreover, cytotoxicity of these compounds was also profiled by hemolytic activity and it was observed that almost all these benzamides compounds displayed low cytotoxicity. These molecules also exhibited mild cytotoxicity toward red blood cell membrane. In addition, the in silico computational explorations fully supported the in vitro enzyme inhibitory results. The binding energy ranges from -7.1 to -9.1 kcal/mol, which showed that compound possessed good interaction tendencies to the pancreatic elastase target protein. So, it was anticipated from the experimental results and computational investigations of the current research that these derivatives might lead to further research gateways for obtaining better and safe nontoxic medicinal scaffolds for dealing with the elastase related ailments such as lungs diseases, pruritic skin disease and liver infection.

Optimization of the production of a new milk clotting enzyme by Aspergillus flavus thermophile on agro-industrial residues medium and characterization of the enzyme

This study focused on the optimization of the production of a milk clotting protease by Aspergillus flavus, isolated from a thermal station. The fermentation was carried out on wheat bran as a medium, followed by a partial characterization of the enzyme. The fungal isolate was screened for protease production based on clear zone hydrolysis on skim milk agar, and then it was identified as belonging to the genus Aspergillus based on its morphological characteristics. This identification was confirmed by molecular analyzes with 100% similarity with the A. flavus strain. After 7 days of fermentation, the crude extract is recovered by simple filtration. The production and optimization of the milk coagulant protease using wheat bran and agro-industrial waste as a medium was carried out using a statistical method of experimental design based on the Plackett and Burman matrices. The results showed that the optimal fermentation medium consisted of wheat bran (4% w/w), incubated at a temperature of 30 °C, with a pH of 5.0 after 6 days of fermentation. The enzyme was partially purified by ammonium sulfate precipitation, yielding an estimated proteolytic activity of 2,985 U. The partially purified protease exhibits optimal activity at pH 5 with hemoglobin as a substrate and an optimal temperature of 50 °C. The Lineweaver-Burk plot showed a KM value of 4.83 g/L and a Vm of 1,560 U. This enzyme causes very rapid coagulation of fresh cow’s milk with an optimum at 45 °C, a pH 7 and a concentration of 50 mM of calcium.

Description of a non-competitive ELISA based on time course analysis of ligand binding at saturation, and a direct method for calculating the affinity of monoclonal antibodies

We present a time-course saturation ELISA for measuring the equilibrium constant of the monoclonal antibody (mAb) SIM 28 against horse radish peroxidase (HRP). The curves of HRP binding to a series of fixed mAb dilutions were plotted to completion, and the Kt (= Ks) value (time to occupy 50 % of the mAb paratopes) was determined for each mAb dilution and HRP concentration. Analysis of the kinetic mechanism of the reaction by Lineweaver-Burk and Hanes plots showed that the slope and y-intercept were affected, indicating that mAb ligand saturation follows non-competitive inhibition kinetics in this assay format. In this kinetics, the inhibition constant Ki (= Kd) is the time required to double the slope or halve the Vmax of the Lineweaver-Burk plot. The Kt values of the time courses were doubled (2 x Kt) and normalized by dividing by the total reaction time to obtain a unitless factor which, when multiplied by the concentration of HRP, gives the Ki. The affinity constant of mAb SIM 28 was determined from ELISA data (n = 16) by three methods: i) doubling of Kt, ii) Beatty equation (Kaff = (n-1)/2 (n [HRP’]t - [HRP]t), and iii) SPR (Biacore) analysis. The calculated affinities (mean ± 95 % confidence limits) were i) 4.6 ± 0.67 × 10−9 M, ii) Kaff = 0.23 ± 0.03 × 109 M−1 (Kd = 4.8 ± 0.81 × 10−9 M), and iii) 4.3 ± 0.57 × 10−9 M, respectively. The similar results obtained with the three different techniques indicate that this time-course saturation ELISA, combined with the double Kt method, is a repeatable and direct approach to mAb affinity determination.

Synthesis, enzyme inhibitory kinetics, & computational studies of N-(substituted phenyl)-(5-(3,4-dichlorobenzyl)-4-(4-chlorophenyl)-4H-1,2,4-triazol-3-ylthio)methylbenzamides: As potent alkaline phosphatase inhibitors

The present research work encompass an innovative approach towards the synthesis of potent series of target compounds (8a-j) having 1,2,4-triazole and benzamide moieties as alkaline phosphatase inhibitors. The synthetic methodology was initiated by Fischer's esterification of 3,4-dichlorophenylacetic acid (1) to achieve ethyl 2-(3,4-dichlorophenyl)acetate (2) which underwent hydrazinolysis using hydrazine hydrate under reflux to 2-(3,4-dichlorophenyl)acetohydrazide (3). The compound (3) with 4-chlorophenyl isothiocyanate (4) was base-catalyzed cyclization (10% aqueous NaOH) to 4-(4-chlorophenyl)-5-(3,4-dichlorobenzyl)-4H-1,2,4-triazole-3-thiol (5) under reflux via N-(4-chlorophenyl)-2-(2-(3,4-dichlorophenyl)acetyl)hydrazinecarbocarbothioamide, as an intermediate. Finally, a series of derivatives (8a-j) was synthesized by reacting (5) with different electrophiles; N-(aryl)-4-(chloromethyl)benzamides (7a-j) which were obtained by the reaction of substituted aryl amines (6a-j) with 4-(chloromethyl)benzoyl chloride in aqueous alkaline medium. The structural confirmation of all these novel derivatives was corroborated by contemporary spectral analysis i.e., IR, EI-MS, 1H and 13CNMR . The in vitro inhibitory potential of these benzamides against alkaline phosphatase enzyme disclosed that nine out of ten exhibited potent inhibition relative to standard used. Among these, 8i was identified as most potent molecule with IC50 value of (0.046 ± 0.013 μM), comparative to standard (5.241 ± 0.471 μM). The Kinetics mechanism examined by Lineweaver-Burk Plots (LBP), which revealed that 8i inhibited alkaline phosphatase enzyme competitively by forming an enzyme-inhibitor complex. The inhibition constant Ki determined from Dixon plots of this compound was 0.02 µM. The results of computational study were in full agreement with experimental proceedings and these ligands showed good interactions with the active site of enzyme. Based on the current investigations, these potent inhibitotrs might lead to further research gateways for the discovery of non-toxic medicinal scaffolds for dealing with the alkaline phosphatase ailments such as bone diseases and liver dysfunction. DFT analysis was also performed.

Unraveling the mechanism of carbonic anhydrase IX inhibition by alkaloids from Ruta chalepensis: A synergistic analysis of in vitro and in silico data

Due to the pivotal role of carbonic anhydrase IX (CA IX) in pathological conditions, there's a pressing need for novel inhibitors to improve patient outcomes and clinical management. Herein, we investigated the inhibitory efficacy of six alkaloids from Ruta chalepensis against CA IX through in vitro inhibition assay and computational modeling. Skimmianine and maculosidine displayed significant inhibitory activity in vitro, with low IC50 values of 105.2 ± 3.2 and 295.7 ± 14.1 nM, respectively. Enzyme kinetics analyses revealed that skimmianine exhibited a mixed inhibition mode, contrasting with the noncompetitive inhibition mechanism observed for the reference drug (acetazolamide), as indicated by intersecting lines in the Lineweaver-Burk plots. The findings of docking calculations revealed that skimmianine and maculosidine exhibited extensive polar interactions with the enzyme. These alkaloids demonstrate substantial binding interactions and occupy identical binding site as acetazolamide, thereby enhancing their efficacy as inhibitors of CA IX. Utilizing a 100 ns molecular dynamics (MD) simulation, the dynamic interactions between isolated alkaloids and CA IX were intensively assessed. Analysis of diverse MD parameters revealed that skimmianine and maculosidine displayed consistent trajectories and notable energy stabilization during their interaction with CA IX. The findings of MM/PBSA analysis depicted the minimum binding free energy for skimmianine and maculosidine. In addition, the Potential Energy Landscape (PEL) analysis revealed distinct and stable conformational states for the CA IX-ligand complexes, with Skimmianine showing the most stable and lowest energy configuration. These computational findings align with experimental results, emphasizing the potential efficacy of skimmianine and maculosidine as inhibitors of CA IX.