Constant Ki Research Articles

The pharmacological basis for nonpeptide agonism of the GLP-1 receptor by orforglipron.

Orally bioavailable, synthetic nonpeptide agonists (NPAs) of the glucagon-like peptide-1 receptor (GLP-1R) may offer an effective, scalable pharmacotherapy to address the metabolic disease epidemic. One of the first molecules in the emerging class of GLP-1R NPAs is orforglipron, which is in clinical development for treating type 2 diabetes and obesity. Here, we characterized the pharmacological properties of orforglipron in comparison with peptide-based GLP-1R agonists and other NPAs. Competition binding experiments using either [125I]GLP-1(7-36)NH2 or [3H]orforglipron indicated that orforglipron is a high-affinity [inhibition constant (Ki) = 1 nM], selective ligand of the human GLP-1R. Signal transduction assays showed that orforglipron has low intrinsic efficacy for effector activation and negligible β-arrestin recruitment. To evaluate GLP-1R engagement in vivo, mice expressing the human GLP-1R were administered orforglipron and subjected to a glucose tolerance test. Predicted receptor occupancy was calculated using the receptor Ki value of orforglipron and its unbound concentration in vivo that reduces hyperglycemia. These experiments revealed that low GLP-1R occupancy by orforglipron is sufficient to yield a full biological response. Moreover, in a model where CRISPR-Cas9 gene editing was used to sensitize the rat GLP-1R (Glp1rS33W) to GLP-1R NPAs, target engagement by orforglipron in the pancreas and brain was consistent with peptide-based GLP-1R agonists. Diet-induced obesity in Glp1rS33W rats enabled studies showing weight loss in animals orally administered orforglipron versus subcutaneous injection of GLP-1R agonist semaglutide. Furthermore, crossover studies indicated oral orforglipron can sustain efficacy initiated by parenteral semaglutide. The pharmacological properties of orforglipron may inform targeting of other peptide receptors with NPAs.

Free amino acids accelerate the time-dependent inactivation of rat liver nucleotide pyrophosphatase/phosphodiesterase Enpp3 elicited by EDTA

Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn2+-metallohydrolases of nucleoside-5´-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (ki(ap)). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased ki(ap). The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2´,3´-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2´,3´-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2´,3´-cGAMP, and that Enpp1 and Enpp3 account for all the 2´,3´-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2´,3´-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3.

Assessment of Punicalagin activity Against Influenza a Virus Proteins via In-silico Approaches

Background: To investigate the anti-influenza mechanism of punicalagin and its inhibitory effects on influenza virus proteins, we conducted molecular docking studies targeting 11 viral proteins. Additionally, molecular dynamics simulations were performed for the protein with the lowest free energy and the minimum concentration required for binding in a simulated environment. Methods: The molecular structure and data of punicalagin were obtained from the PubChem database and converted into a PDB file. FASTA sequences of viral proteins were retrieved from UniProt, and their PDB structures were predicted using the I-TASSER server. Molecular docking was performed using AutoDock 4.2 software, while molecular dynamics simulations were conducted with Gromacs 2022 software. Results: The PB1-F2 protein exhibited the best inhibitory performance, with a binding energy of -5.76 kcal/mol and the lowest inhibition constant (Ki). Docking of punicalagin to the PB1-F2 protein led to a significant decrease in the average total energy (TE), radius of gyration (Rg), and root mean square deviation (RMSD), as well as alterations in the secondary structure of the protein (P < 0.001). The most prominent secondary structural change was a reduction in coil structures and an increase in turn structures. Conclusions: Punicalagin displayed a strong binding affinity for the PB1-F2 protein compared to other influenza viral proteins. Considering the role of PB1-F2 in exacerbating inflammation caused by influenza, punicalagin may mitigate inflammation in patients by modulating the activity of this protein.

Competitive inhibition of family GH11 Aspergillus fumigatus endo-xylanase A by Oryza sativa xylanase inhibitor protein: Investigating key interface residues and non-covalent interactions

Glycoside hydrolase (GH) family 11 Aspergillus fumigatus endo-xylanase A (AfXylA11) was recombinantly expressed in Pichia pastoris X33 and secreted into culture medium. Hydrolysis of beechwood xylan by recombinant AfXylA11 (reAfXylA11) predominantly generated xylooligosaccharides (XOs), chiefly comprising xylotriose (X2) and xylotetraose (X3). These hydrolysates demonstrated significant antioxidant capabilities. Competitive inhibition of reAfXylA11 was observed in the presence of the Oryza sativa xylanase inhibitor protein (OsXIP), with an inhibition constant (Ki) of 120.98 nM. Molecular dynamics (MD) simulations and conformational analyses of the AfXylA11-OsXIP complex revealed that the extended loop (Lα4β5, K150-V158) of OsXIP penetrated the active groove of AfXylA11, thereby obstructing xylan from interacting with catalytic sites. The R153 at the bottom of the U-shaped loop, was found to form durable hydrogen bonds with E197 (acid/base catalyst) and Y97 of AfXylA11. Additionally, OsXIP established stable interactions with the “cord” and “thumb” regions of AfXylA11 during MD simulations. The binding free energy of the AfXylA11-OsXIP complex was determined to be -74.3 ± 7.6 kcal/mol, with electrostatic interactions and van der Waals forces being the predominant contributors to enzyme-inhibitor interaction. Further MD simulations demonstrated that the mutation of Y116A or S120A in the “cord” region of AfXylA11 significantly reduced its binding affinity to OsXIP by weakening interface residue interactions. Delving into the conformational dynamics of the AfXylA11-OsXIP complex yields insights into the molecular mechanisms dictating their competitive inhibition. This investigation offers useful basis for engineering mutant xylanases that resist to the inhibitory protein yet retain high catalytic efficacy.

Quassinoids from Eurycoma longifolia as Potential Dihydrofolate Reductase Inhibitors: A Computational Study.

Quassinoids are degraded triterpene compounds that can be obtained from various species of the Simaroubaceae plant family, including Eurycoma longifolia. Quassinoids are the major compounds in E. longifolia, and they are known to have various medicinal potentials, such as anticancer and antimalarial properties. Dihydrofolate reductase (DHFR) was reported to be one of the important targets for certain anticancer and antimalarial drugs. Twelve quassinoids from E. longifolia were identified to have anticancer effects based on their IC50 values. This study aimed to evaluate the interactions of these twelve quassinoids with DHFR via Autodock 4.2 software and Biovia Discovery Studio Visualiser. Twelve quassinoids from E. longifolia and their interactions with DHFR were evaluated via Autodock 4.2 software and Biovia Discovery Studio Visualiser. Their drug-likeness and pharmacokinetic properties were also assessed using the ADMETlab 2.0 program. The molecular docking results showed that eleven quassinoids showed better docking scores than methotrexate, in which the binding energy (BE) of these quassinoids ranged from - 7.87 to -9.58 kcal/mol. Their inhibition constant (Ki) ranged from 0.095 to 1.71 μM. At the same time, the BE and Ki values for methotrexate were -7.80 kcal/mol and 1.64 μM, respectively. From the analysis, 6-dehydrolongilactone and eurycomalide B are among the twelve compounds that showed great potential as hit-to-lead compounds based on the docking score on DHFR, drug-likeness, and ADMET properties. These results suggest a great potential to pursue validation studies via in vitro and in vivo models.

Enzymatic, structural, and biophysical characterization of a single-domain antibody (VHH) selectively and tightly inhibiting neutrophil elastase and exhibiting favorable developability properties.

Human neutrophil elastase (hNE), a serine protease released by neutrophils during inflammation, plays a major role in the pathophysiology of several conditions especially in inflammatory lung diseases. Its inhibition constitutes, therefore, a promising therapeutic strategy to combat these diseases. In this work, we characterized the in vitro properties of a VHH (i.e., the antigen binding domain of camelid heavy chain-only antibodies), referred to as NbE201. This VHH is able to inhibit tightly, selectively and competitively both human and murine elastases with the inhibition constants (Ki) of 4.1 ± 0.9 nM and 36.8 ± 3.9 nM, respectively. The IC50 for the inhibition of the hydrolysis of elastin is in the same range to that of alpha-1 antitrypsin (i.e., the main endogenous inhibitor of hNE also used in the clinic) and 14 times better than that of Sivelestat (i.e., the 2nd clinically approved hNE inhibitor). The X-ray crystal structure of the NbE201-hNE complex reveals that the Complementarity Determining Regions CDR1 and CDR3 of the VHH bind into the substrate binding pocket of hNE and prevent the access to small or macromolecular substrates. They do not, however, bind deep enough into the pocket to be hydrolyzed. NbE201 is highly stable towards oxidation, deamidation, and chemical or thermal denaturation. NbE201 is therefore likely to tolerate manufacturing processes during drug development. These results highlight the high potential of NbE201 as a (pre)clinical tool to diagnose and treat diseases associated with excessive hNE activity, and for fundamental research to better understand the role of hNE in these conditions.

In vitro analysis of a competitive inhibition model for T7 RNA polymerase biosensors

T7 RNA polymerase (T7 RNAP) biosensors, in which T7 RNAP transcribes some reporter gene or signal in response to external stimuli, have wide applications in synthetic biology and metabolic engineering. We adapted a biochemical reaction network model and used an in vitro transcription assay to determine network parameters for different T7 RNAP constructs. Under conditions where template DNA is limiting, the EC50 values of native and engineered T7 RNAPs ranged from 33 nM (29–37 95 % c.i.) to 570 nM (258–714 95 % c.i.) (wild-type T7 RNAP). The measured EC50 values were largely insensitive to free magnesium, pH, or other buffer conditions. Many biosensor configurations use a split RNAP construct, where the C-terminal (CT7) and N-terminal T7 (NT7) are fused to proximity induced dimerization modules. We used proteolysis and ion exchange chromatography to prepare a CT7 (80 kDa) product. The impact of free CT7 on T7 RNAP transcriptional activity was well described by a competitive inhibition model, with an inhibitory constant KI = 23 nM (18–28 95 % c.i.) of the sensor. These model parameters will be useful for forward modeling and design of T7 RNAP-based genetic circuits.

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 Inhibitory Actions of Neuroactive Steroid [3α,5α]-3-Hydroxypregnan-20-One on Toll-like Receptor 4-Dependent Neuroimmune Signaling.

The endogenous neurosteroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) modulates inflammatory and neuroinflammatory signaling through toll-like receptors (TLRs) in human and mouse macrophages, human blood cells and alcohol-preferring (P) rat brains. Although it is recognized that 3α,5α-THP inhibits TLR4 activation by blocking interactions with MD2 and MyD88, the comprehensive molecular mechanisms remain to be elucidated. This study explores additional TLR4 activation sites, including TIRAP binding to MyD88, which is pivotal for MyD88 myddosome formation, as well as LPS interactions with the TLR4:MD2 complex. Both male and female P rats (n = 8/group) received intraperitoneal administration of 3α,5α-THP (15 mg/kg; 30 min) or a vehicle control, and their hippocampi were analyzed using immunoprecipitation and immunoblotting techniques. 3α,5α-THP significantly reduces the levels of inflammatory mediators IL-1β and HMGB1, confirming its anti-inflammatory actions. We found that MyD88 binds to TLR4, IRAK4, IRAK1, and TIRAP. Notably, 3α,5α-THP significantly reduces MyD88-TIRAP binding (Males: -31 ± 9%, t-test, p < 0.005; Females: -53 ± 15%, t-test, p < 0.005), without altering MyD88 interactions with IRAK4 or IRAK1, or the baseline expression of these proteins. Additionally, molecular docking and molecular dynamic analysis revealed 3α,5α-THP binding sites on the TLR4:MD2 complex, targeting a hydrophobic pocket of MD2 usually occupied by Lipid A of LPS. Surface plasmon resonance (SPR) assays validated that 3α,5α-THP disrupts MD2 binding of Lipid A (Kd = 4.36 ± 5.7 μM) with an inhibition constant (Ki) of 4.5 ± 1.65 nM. These findings indicate that 3α,5α-THP inhibition of inflammatory mediator production involves blocking critical protein-lipid and protein-protein interactions at key sites of TLR4 activation, shedding light on its mechanisms of action and underscoring its therapeutic potential against TLR4-driven inflammation.

Discovery of potential natural therapeutics targeting cell wall biosynthesis in multidrug-resistant Enterococcus faecalis: a computational perspective.

Identifying therapeutic inhibitors of crucial enzymes involved in the peptidoglycan biosynthesis pathway is pivotal for developing new treatments against multidrug-resistant Enterococcus faecalis V583. MurM, an essential enzyme in this pathway, plays a significant role in the bacterium's cell wall synthesis, making it an attractive druggable target for novel antimicrobial strategies. This study explored the potential of natural compounds as inhibitors of MurM, aiming to discover promising drug candidates that could serve as the foundation for future therapeutic development. The three-dimensional structure of MurM was predicted, optimized, and its binding pocket was analyzed by comparing it with related structures. Over 4,70,000 natural compounds from the COCONUT database were subjected to virtual high-throughput screening (vHTS). The top lead candidates were selected based on their Lipinski's profile, ADME profile, toxicity profile, estimated binding free energy (ΔG) and estimated inhibition constant (Ki). Interaction pattern analysis was used to evaluate the non-covalent interactions between the inhibitors and key residues in MurM's binding pocket. Molecular dynamics simulations were performed over 300 ns to assess the structural stability and impact of these inhibitors on MurM's enzyme. Three lead compounds-CNP0056520, CNP0126952, and CNP0248480-were identified and prioritized with estimated ΔG ranging from - 9.35 to -7.9kcal/mol. Molecular dynamics simulations revealed minimal impact on MurM's overall structure and dynamics, with the candidate inhibitors forming stable protein-ligand complexes. These interactions were supported by several non-covalent interactions between the candidate inhibitors and key residues within MurM's binding pocket. These findings suggest that the identified natural product candidates could serve as promising inhibitors of MurM, potentially leading to novel therapeutics targeting cell wall biosynthesis in multidrug-resistant E. faecalis.

Synthesis, Crystal Structure, Intermolecular Interactions, HOMO-LUMO, MEP, NLO Properties, and DFT/TD-DFT Investigation of (Z)-5-(4-Nitrobenzylidene)-3-N(3-Chlorophenyl)-2-Thioxothiazolidin-4-One

A novel heterocyclic compound named (Z)-5-(4-nitrobenzylidene)-3-N(3-chlorophenyl)-2-thioxothiazolidin-4-one (NCTh) was synthesized and analyzed using various techniques, including single crystal X-ray diffraction, FT-IR, UV-Vis, NMR spectroscopy, and mass spectral data methods. NCTh was observed to crystallize in the triclinic crystal system P-1 space group. To validate the experimental findings, density functional theory (DFT) calculations were performed using the B3LYP functional with the 6-311 G(d,p) basis set. The results indicated good agreement in geometrical parameters between the experimental and theoretical outcomes. The study also calculated HOMO-LUMO energies, molecular electrostatic potential (MEP), and global chemical reactivity descriptors to evaluate the compound’s reactivity. Additionally, the study conducted reduced density gradient and Hirshfeld surface analyses to reveal attractive, repulsive, and van der Waals strong and weak interactions. The calculation of thermodynamic parameters was also included. The study focused on investigating the nonlinear optical (NLO) properties of NCTh, which were characterized through key parameters such as the electric dipole moment (µ), polarizability (α), first-order hyperpolarizability (β), and second-order hyperpolarizability (γ). These properties provide insights into the material’s potential applications in optical and photonic technologies. Additionally, a molecular docking study was performed to further explore the structure-activity relationship, particularly in a biological context. The docking results revealed a strong ligand-protein interaction, with a binding energy of −10.3 kcal/mol, indicating significant affinity. Moreover, the inhibition constant (Ki) was calculated to be 0.0281 μM, suggesting a highly potent interaction, which could be relevant for drug design or biochemical applications.

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.

Measurement variability of blood-brain barrier permeability using dynamic contrast-enhanced magnetic resonance imaging.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is used to quantify the blood-brain barrier (BBB) permeability-surface area product. Serial measurements can indicate changes in BBB health, of interest to the study of normal physiology, neurological disease, and the effect of therapeutics. We performed a scan-rescan study to inform both sample size calculation for future studies and an appropriate reference change value for patient care. The final dataset included 28 healthy individuals (mean age 53.0 years, 82% female) scanned twice with mean interval 9.9 weeks. DCE-MRI was performed at 3T using a 3D gradient echo sequence with whole brain coverage, T1 mapping using variable flip angles, and a 16-min dynamic sequence with a 3.2-s time resolution. Segmentation of white and grey matter (WM/GM) was performed using a 3D magnetization-prepared gradient echo image. The influx constant Ki was calculated using the Patlak method. The primary outcome was the within-subject coefficient of variation (CV) of Ki in both WM and GM. Ki values followed biological expectations in relation to known GM/WM differences in cerebral blood volume (CBV) and consequently vascular surface area. Subject-derived arterial input functions showed marked within-subject variability which were significantly reduced by using a venous input function (CV of area under the curve 46 vs. 12%, p < 0.001). Use of the venous input function significantly improved the CV of Ki in both WM (30 vs. 59%, p < 0.001) and GM (21 vs. 53%, p < 0.001). Further improvement was obtained using motion correction, scaling the venous input function by the artery, and using the median rather than the mean of individual voxel data. The final method gave CV of 27% and 17% in WM and GM, respectively. No further improvement was obtained by replacing the subject-derived input function by one standard population input function. CV of Ki was shown to be highly sensitive to dynamic sequence duration, with shorter measurement periods giving marked deterioration especially in WM. In conclusion, measurement variability of 3D brain DCE-MRI is sensitive to analysis method and a large precision improvement is obtained using a venous input function.

Visnagin alleviates rheumatoid arthritis via its potential inhibitory impact on malate dehydrogenase enzyme: in silico, in vitro, and in vivo studies

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder. The present study aimed to evaluate the in silico, in vitro, and in vivo inhibitory effect of visnagin on malate dehydrogenase activity and elucidate its inflammatory efficacy when combined with methotrexate in the RA rat model. The molecular docking, ADMET simulations, MDH activity, expression, and X-ray imaging were detected. Moreover, CRP, RF, (anti-CCP) antibody, (TNF-α), (IL-6), (IL-17), and (IL-10) were evaluated. The expression levels of MMP3 and FOXP3 genes and CD4, CD25, and CD127 protein levels were assessed. Histological assessment of ankle joints was evaluated. The results revealed that visnagin showed reversible competitive inhibition on MDH with inhibitory constant (Ki) equal to 141 mM with theoretical IC50 equal to 1202.7 mM, LD50 equal to 155.39 mg/kg, and LD25 equal to 77.69 mg/kg. In vivo studies indicated that visnagin exhibited anti-inflammatory effects through decreasing MDH1 activity and expression and induced proliferation of anti-inflammatory CD4+CD25+FOXP3 regulatory T cells with increasing the anti-inflammatory cytokine IL-10 levels. Moreover, visnagin reduced the levels of inflammatory cytokines and the immuno-markers. Our findings elucidate that visnagin exhibits an anti-inflammatory impact against RA through its ability to inhibit the MDH1 enzyme, improve methotrexate efficacy, and reduce oxidative stress.Graphical

Interaction of some chalcone derivatives with calcium channels using a theoretical model

For several years, different drugs have been used to treat heart failure, such as digoxin, captopril, spironolactone, milrinone, levosimedam, dobutamine, and others. However, some of these drugs can produce secondary effects such as arrhythmia, cough, hyperkalemia, and others. Analyzing these data, this study aimed to evaluate the interaction of some chalcone derivatives (1-17) with calcium channels using theoretical models. It is important to mention that 7pjx protein, nifedipine, amlodipine, diltiazem, and verapamil were used as theoretical tools in the DockingServer program. The results showed differences in the interaction of chalcone derivatives compared with nifedipine, amlodipine, diltiazem, and verapamil drugs. Other data indicate that the inhibition constant (Ki) for chalcone analog 1 was lower compared with nifedipine, amlodipine, verapamil, and diltiazem. Besides, other results suggest that Ki for compound 11 was lower compared with nifedipine, verapamil, and diltiazem. All these data suggest that chalcone derivatives 1 and 11 could act as calcium channel inhibitors; this phenomenon could be translated into changes in blood pressure through a decrease in calcium intracellular levels. These data suggest that chalcone derivatives 1 and 11 could be good therapeutic alternatives to treat heart failure.

Development of novel dual-target drugs against visceral leishmaniasis and combinational study with miltefosine

The dual-target inhibitors (ZINC000008876351 and ZINC000253403245) were identified by utilizing an advanced computational drug discovery method by targeting two critical enzymes such as FeSODA (Iron superoxide dismutase) and TryR (Trypanothione reductase) within the antioxidant defense system of Leishmania donovani (Ld). In vitro enzyme inhibition kinetics reveals that both the compound's ability to inhibit the function of enzyme LdFeSODA and LdTryR with inhibition constant (Ki) value in the low μM range. Flow cytometry analysis, specifically at IC50 and 2X IC50 doses of both the compounds, the intracellular ROS was significantly increased as compared to the untreated control. The compounds ZINC000253403245 and ZINC000008876351 exhibited strong anti-leishmanial activity in a dose-dependent manner against both the promastigote and amastigote stages of the parasite. The data indicate that these molecules hold promise as potential anti-leishmanial agents for developing new treatments against visceral leishmaniasis, specifically targeting the LdFeSODA and LdTryR enzymes. Additionally, the in vitro MTT assay shows that combining these compounds with miltefosine produces a synergistic effect compared to miltefosine alone. This suggests that the compounds can boost miltefosine's effectiveness by synergistically inhibiting the growth of L. donovani promastigotes. Given the emergence of miltefosine resistance in some Leishmania strains, these findings are particularly significant.

Cysteine protease I29 propeptide from Calotropis procera R. Br. As a potent cathepsin L inhibitor and its suppressive activity in breast cancer metastasis.

Breast cancer metastasis is associated with a poor prognosis and a high rate of mortality. Cathepsin L (CTSL) is a lysosomal cysteine protease that promotes tumor metastasis by degrading the extracellular matrix. Gene set enrichment analysis revealed that CTSL expression was higher in tumorous than in non-tumorous tissues of breast cancer patients and that high-level CTSL expression correlated positively with the epithelial-mesenchymal transition. Therefore, we hypothesized that inhibiting CTSL activity in tumor cells would prevent metastasis. In this study, we characterized the inhibitory activity of SnuCalCpI15, the I29 domain of a CTSL-like cysteine protease from Calotropis procera R. Br., and revealed that the propeptide stereoselectively inhibited CTSL in a reversible slow-binding manner, with an inhibitory constant (Ki) value of 1.38 ± 0.71 nM, indicating its potency as an exogenous inhibitor in anti-cancer therapy. SnuCalCpI15 was localized intracellularly in MDA-MB-231 breast cancer cells and suppressed tumor cell migration and invasion. These results demonstrate the potential of SnuCalCpI15 as a novel agent to prevent breast cancer metastasis.

Single time-point analysis of product and substrate inhibition.

When enzyme inhibition by either the product or excess substrate occurs, it is possible to determine the characteristic kinetic parameters based on [P]/t measurements, even when a large proportion of the substrate is converted. The advantages of various approaches are discussed. Most of them allow a good estimation of the V and Km values. Conversely, the determination of Kp (product inhibition) and Ki (inhibition by excess substrate) can be more challenging. In the first case, determination of the type of inhibition requires more complex experiments that are beyond the scope of the present contribution. In the second, the inhibition constant Ki can only be roughly estimated. In an experimental approach, we compared the results obtained either with initial rate measurements or with 50 to 60% conversion of the substrate. Similar values of V and Km were obtained. Measurements involving the conversion of a large proportion of substrate are particularly advantageous when the assay method is difficult or time-consuming, or when obtaining the substrate presents experimental difficulties or involves substantial costs.

PENGEMBANGAN SISTEM PENGEREMAN TURBIN ANGIN SUMBU HORIZONTAL MENGGUNAKAN KONTROL PID

Horizontal axis wind turbines will generate voltage if the turbine rotates due to wind gusts. The bigger the wind blows, the faster the turbine spins and the greater the voltage generated. The braking system is needed for wind turbine protection when it receives very large wind speeds. This system is built using PID control based on microcontroller with reference to the output voltage. There are 2 units resistor designed as voltage sensors to the wind turbine output voltage. In testing, the wind turbine is rotated using artificial wind which is generated through a blower measuring 1.3 x 1.3 x 0.3 m, with a maximum wind speed is 11 m/s. The sistem uses PID kontrol which is used to brake wind turbine rotation and regulate its output voltage. Determination of constant parameters for proportional constant (Kp), integral constant (Ki), and Derivative constant (Kd) were obtained for Kp = 0.139, Ki = 0.715, and Kd = 0.006 respectively. The output voltage of the wind turbine can reach 60 volts and then controlled up to 24 volts according to the battery voltage requirements. In the test results, the control system managed to regulate the wind turbine voltage at 24 volts with a steady state error of 6.62%

Probe substrates assay estimates the effect of polyphyllin H on the activity of cytochrome P450 enzymes in human liver microsomes.

Cytochrome P450 enzymes (CYPs) play a crucial role in phase I metabolic reactions. The activity of CYPs would affect therapeutic efficacy and may even induce toxicity. Given the complex components of traditional Chinese medicine, it is important to understand the effect of active ingredients on CYPs activity to guide their prescription. This study aimed to evaluate the effect of polyphyllin H on the activity of CYPs major isoforms providing a reference for the clinical prescription of polyphyllin H and its source herbs. The effects of polyphyllin H were evaluated in pooled human liver microsomes using probe substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to determine their activities. The Lineweaver-Burk was used to model the inhibition, and a time-dependent inhibition experiment was performed to understand the characteristics of the inhibition. Polyphyllin H significantly suppressed the activity of CYP1A2, 2D6, and 3A4 with IC50 values of 6.44, 13.88, and 4.52 μM, respectively. The inhibition of CYP1A2 and 2D6 was best fitted with a competitive model, yielding the inhibition constant (Ki) values of 3.18 and 6.77 μM, respectively. The inhibition of CYP3A4 was fitted with the non-competitive model with the Ki value of 2.38 μM. Moreover, the inhibition of CYP3A4 was revealed to be time-dependent with the inhibition parameters inhibition constant (KI) and inactivation rate constant (Kinact) values of 2.26 μM-1 and 0.045 min-1. Polyphyllin H acted as a competitive inhibitor of CYP1A2 and 2D6 and a non-competitive and time-dependent inhibitor of CYP3A4.