In vitro evaluation of novel mefenamic acid derivatives as potential α-glucosidase and urease inhibitors: Design,synthesis, in silico and cytotoxic studies

This study aim to synthesize new 1,3,4-oxadiazole derivatives incorporating mefenamic acid as promising α-glucosidase and urease inhibitors, potentially leading to the treatment of postprandial hyperglycemia as well as H. pylori related disorders. In this regards, we have designed a series of Mefenamic acid derivatives. The synthetic compounds were structurally elucidated through ¹H NMR, ¹³C NMR and HR-EIMS analysis. The biological evaluation of these derivatives against α-glucosidase and urease enzyme depicted some novel derivatives with potent inhibition against the said enzymes. All the derivatives exhibited potent inhibition against α-glucosidase enzymes with IC₅₀ ranging from 25.81 ± 1.63–113.61 ± 1.31 µM against standard drug acarbose (IC₅₀ = 375.82 ± 1.76 µM) while with respect to urease these derivatives possessed inhibitory potential varied between IC₅₀ = 8.04 ± 1.01–58.18 ± 1.03 µM against the standard thiourea (IC₅₀ = 21.0 ± 1.76 µM). The cell viability results revealed that all of the derivatives were found least cytotoxic. Furthermore, molecular docking studies of the most potent derivatives identify number of key features involved in binding interactions between potential inhibitors and the enzyme's active site.     

Phloroglucinol Derivatives from Dryopteris crassirhizoma as Potent Xanthine Oxidase Inhibitors

Dryopteris crassirhizoma rhizomes are used as a traditional medicine in Asia. The EtOAc extract of these roots has shown potent xanthine oxidase (XO) inhibitory activity. However, the main phloroglucinols in D. crassirhizoma rhizomes have not been analyzed. Thus, we investigated the major constituents responsible for this effect. Bioassay-guided purification isolated four compounds: flavaspidic acid AP (1), flavaspidic acid AB (2), flavaspidic acid PB (3), and flavaspidic acid BB (4). Among these, 1 showed the most potent inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) value of 6.3 µM, similar to that of allopurinol (IC₅₀ = 5.7 µM) and better than that of oxypurinol (IC₅₀ = 43.1 µM), which are XO inhibitors. A comparative activity screen indicated that the acetyl group at C3 and C3′ is crucial for XO inhibition. For example, 1 showed nearly 4-fold higher efficacy than 4 (IC₅₀ = 20.9 µM). Representative inhibitors (1–4) in the rhizomes of D. crassirhizoma showed reversible and noncompetitive inhibition toward XO. Furthermore, the potent inhibitors were shown to be present in high quantities in the rhizomes by a UPLC-QTOF-MS analysis. Therefore, the rhizomes of D. crassirhizoma could be used to develop nutraceuticals and medicines for the treatment of gout.     

Design and Synthesis of Non-Covalent Imidazo[1,2-a]quinoxaline-Based Inhibitors of EGFR and Their Anti-Cancer Assessment

A series of 30 non-covalent imidazo[1,2-a]quinoxaline-based inhibitors of epidermal growth factor receptor (EGFR) were designed and synthesized. EGFR inhibitory assessment (against wild type) data of compounds revealed 6b, 7h, 7j, 9a and 9c as potent EGFR^WT inhibitors with IC₅₀ values of 211.22, 222.21, 193.18, 223.32 and 221.53 nM, respectively, which were comparable to erlotinib (221.03 nM), a positive control. Furthermore, compounds exhibited excellent antiproliferative activity when tested against cancer cell lines harboring EGFR^WT; A549, a non-small cell lung cancer (NSCLC), HCT-116 (colon), MDA-MB-231 (breast) and gefitinib-resistant NSCLC cell line H1975 harboring EGFR^L858R/T790M. In particular, compound 6b demonstrated significant inhibitory potential against gefitinib-resistant H1975 cells (IC₅₀ = 3.65 μM) as compared to gefitinib (IC₅₀ > 20 μM). Moreover, molecular docking disclosed the binding mode of the 6b to the domain of EGFR (wild type and mutant type), indicating the basis of inhibition. Furthermore, its effects on redox modulation, mitochondrial membrane potential, cell cycle analysis and cell death mode in A549 lung cancer cells were also reported.     

Potential Xanthine Oxidase Inhibitory Activity of Endophytic Lasiodiplodia pseudotheobromae

Xanthine oxidase is considered as a potential target for treatment of hyperuricemia. Hyperuricemia is predisposing factor for gout, chronic heart failure, atherosclerosis, tissue injury, and ischemia. To date, only two inhibitors of xanthine oxidase viz. allopurinol and febuxostat have been clinically approved for used as drugs. In the process of searching for new xanthine oxidase inhibitors, we screened culture filtrates of 42 endophytic fungi using in vitro qualitative and quantitative XO inhibitory assays. The qualitative assay exhibited potential XO inhibition by culture filtrates of four isolates viz. #1048 AMSTITYEL, #2CCSTITD, #6AMLWLS, and #96 CMSTITNEY. The XO inhibitory activity was present only in the chloroform extract of the culture filtrates. Chloroform extract of culture filtrate #1048 AMSTITYEL exhibited the highest inhibition of XO with an IC₅₀ value of 0.61 μg ml^?1 which was better than allopurinol exhibiting an IC₅₀ of 0.937 μg ml^?1 while febuxostat exhibited a much lower IC₅₀ of 0.076 μg ml^?1. Further, molecular phylogenetic tools and morphological studies were used to identify #1048 AMSTITYEL as Lasiodiplodia pseudotheobromae. This is the first report of an endophytic Lasiodiplodia pseudotheobromae from Aegle marmelos exhibiting potential XO Inhibitory activity.     

Screening and evaluation of α-glucosidase inhibitors from indigenous medicinal plants in Dak Lak Province,Vietnam

α-Glucosidase inhibitors have received much attention due to their important use in treating diabetes mellitus. Although some synthetic α-glucosidase inhibitors have been available for a long time, they often cause various unexpected side effects. Thus, the present study was aimed at finding a safe, natural source of α-glucosidase inhibitors. Twenty-six samples of 22 medicinal plants were collected in the Dak Lak province of Vietnam and evaluated for α-glucosidase inhibitory activity. Trunk bark extract from Euonymus laxiflorus Champ (ELC extract) was selected as the best α-glucosidase inhibitor with the smallest IC₅₀ = 0.36 mg/mL against rat-derived α-glucosidase. This extract had a stronger inhibitory activity against α-glucosidase from Saccharomyces cerevisiae (IC₅₀ = 1.32 µg/mL) and Bacillus stearothermophilus (IC₅₀ = 5.15 µg/mL). The potential inhibition against some other enzymes were tested, and the results showed that the ELC extract did not inhibit fungal cellulase but strongly inhibited porcine α-amylase (IC₅₀ = 6.7 µg/mL). The ELC extract also inhibited the proteases papain and bromelain, with IC₅₀ = 339 µg/mL and IC₅₀ = 226 µg/mL, respectively. The thermal and pH stabilities of the ELC extract were also investigated.     

Cinnamides Target Leishmania amazonensis Arginase Selectively

Caffeic acid and related natural compounds were previously described as Leishmania amazonensis arginase (L-ARG) inhibitors, and against the whole parasite in vitro. In this study, we tested cinnamides that were previously synthesized to target human arginase. The compound caffeic acid phenethyl amide (CAPA), a weak inhibitor of human arginase (IC₅₀ = 60.3 ± 7.8 μM) was found to have 9-fold more potency against L-ARG (IC₅₀ = 6.9 ± 0.7 μM). The other compounds that did not inhibit human arginase were characterized as L-ARG, showing an IC₅₀ between 1.3–17.8 μM, and where the most active was compound 15 (IC₅₀ = 1.3 ± 0.1 μM). All compounds were also tested against L. amazonensis promastigotes, and only the compound CAPA showed an inhibitory activity (IC₅₀ = 80 μM). In addition, in an attempt to gain an insight into the mechanism of competitive L-ARG inhibitors, and their selectivity over mammalian enzymes, we performed an extensive computational investigation, to provide the basis for the selective inhibition of L-ARG for this series of compounds. In conclusion, our results indicated that the compounds based on cinnamoyl or 3,4-hydroxy cinnamoyl moiety could be a promising starting point for the design of potential antileishmanial drugs based on selective L-ARG inhibitors.     

Indole- and Pyrazole-Glycyrrhetinic Acid Derivatives as PTP1B Inhibitors: Synthesis,In Vitro and In Silico Studies

Regulating insulin and leptin levels using a protein tyrosine phosphatase 1B (PTP1B) inhibitor is an attractive strategy to treat diabetes and obesity. Glycyrrhetinic acid (GA), a triterpenoid, may weakly inhibit this enzyme. Nonetheless, semisynthetic derivatives of GA have not been developed as PTP1B inhibitors to date. Herein we describe the synthesis and evaluation of two series of indole- and N-phenylpyrazole-GA derivatives (4a–f and 5a–f). We measured their inhibitory activity and enzyme kinetics against PTP1B using p-nitrophenylphosphate (pNPP) assay. GA derivatives bearing substituted indoles or N-phenylpyrazoles fused to their A-ring showed a 50% inhibitory concentration for PTP1B in a range from 2.5 to 10.1 µM. The trifluoromethyl derivative of indole-GA (4f) exhibited non-competitive inhibition of PTP1B as well as higher potency (IC₅₀ = 2.5 µM) than that of positive controls ursolic acid (IC₅₀ = 5.6 µM), claramine (IC₅₀ = 13.7 µM) and suramin (IC₅₀ = 4.1 µM). Finally, docking and molecular dynamics simulations provided the theoretical basis for the favorable activity of the designed compounds.     

Synthesis and anticancer activity of benzotriazole derivatives

A series of benzotriazole (BTA) derivatives were synthesized as tyrosine protein kinase inhibitors using fragment-based design strategy. All desired compounds were synthesized with the reaction of benzotriazole, chloroacetonitrile and aromatic aldehyde using Ultrasonic-Microwave method and characterized by IR, ¹H and ¹³C-NMR, mass spectrometry (MS) and elemental analysis. The anticancer activity of these compounds was evaluated by CCK-8 method against carcinoma VX2, lung cancer A549, stomach cancer cell lines MKN45 and MGC in vitro. The results showed that all compounds showed good antiproliferative activity. In particular, compound 2.1 showed the most prominent inhibition of VX2 cell lines with IC₅₀ of 3.80 ± 0.75 μM. Compound 2.2 exhibited highly potent anticancer activity of stomach MGC cell lines with IC₅₀ of 3.72 ± 0.11 μM. A549 and MKN45 cell lines were sensitive to compound 2.5 with IC₅₀ of 5.47 ± 1.11 and 3.04 ± 0.02 μM, respectively.     

Design,synthesis, in vitro and in silico studies of naproxen derivatives as dual lipoxygenase and α-glucosidase inhibitors

A series of 28 novel naproxen derivatives (4a-f, 5a-f, 6a-d, 7a-f, and 8a-f) have been designed, synthesized, and characterized. The synthesized derivatives were assessed as dual inhibitors for 15-lipoxygenase (LOX) and α-glucosidase enzymes and checked for cytotoxicity and ADME studies. The inhibitory potential of naproxen derivatives for 15- LOX was checked through two different methods, the UV absorbance method and the Chemiluminescence method. The biological activities result revealed that through the UV absorbance method, compound 4f (IC₅₀ 21.31 ± 0.32 µM) was found potent among the series followed by compounds 4e (IC₅₀ 36.53 ± 0.51 µM) and 4d (IC₅₀ 49.62 ± 0.12 µM) against standard drug baicalein (IC₅₀ 22.46 ± 1.32 µM) and quercetin (IC₅₀ 2.34 ± 0.35 µM), while through chemiluminescence method tested compounds showed significant 15-LOX inhibition at the range of IC₅₀ 1.13 ± 0.62 µM ?123.47 ± 0.37 µM. Among these compounds, 4e (IC₅₀ 1.13 ± 0.62 µM), 5b (IC₅₀ 1.19 ± 0.43 µM), 8c (IC₅₀ 1.23 ± 0.35 µM) were found most potent inhibitors against quercetin (IC₅₀ 4.86 ± 0.14 µM), and baicalein (IC₅₀ 2.24 ± 0.13 µM). The chemiluminescence method was found more sensitive than the UV method to identify 15-LOX inhibitors. Interestingly all synthesized compounds showed significant α-glucosidase inhibitory activity (IC₅₀ 1.0 ± 1.13 µM ? 367.2 ± 1.23 µM) even better than the standard drug acarbose (IC₅₀ 375.82 ± 1.76 µM), while compound 6c (IC₅₀ 1.0 ± 1.13 µM) and 7c (IC₅₀ 1.1 ± 1.17 µM) were found most potent compounds among the series even many folds better than the standard drug. The cell viability results showed that all compounds were less toxic, maintained cellular viability at the range of 99.8 ± 1.3% to 63.7 ± 1.5%. ADME and molecular docking studies supported drug-likeness and binding interactions of compounds with the targeted enzymes.     

Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells

Background: Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities.Results: Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aidehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called ‘branched chain amino acid preferring’ (BrAAP) and ‘small neutral amino acid preferring’ (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity.Conclusions: The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate,     

Development of gold nanoparticle based colorimetric method for quantitatively studying the inhibitors of Cu/Zn induced β-amyloid peptide assembly

In this paper, a kind of gold nanoparticle (GNP)-based colorimetric assay has been developed for studying the reversible interaction of β-amyloid peptide (Aβ) with Cu²⁺ and Zn²⁺, and quantitatively analyzing four inhibitors (i.e., EDTA, EGTA, histidine and clioquinol) of Cu²⁺/Zn²⁺ induced Aβ assembly. The inhibition efficiencies (e.g., half maximal inhibitory concentration, IC₅₀ value) of these inhibitors could be measured in this work. As far as we know, these IC₅₀ values were reported at the first time. In this assay, the streptavidin conjugated GNPs (SA-GNPs) were employed as indicators to monitor the Cu²⁺/Zn²⁺ induced aggregating/disaggregating behaviors of biotin modified β-amyloid 1–16 peptides (Aβ_1–16(biotin)). Because of high affinity of streptavidin (SA) with biotin, the aggregating/disaggregating of Aβ_1–16(biotin) results in the significant color change of SA-GNPs. Furthermore, we demonstrate that the assay can be used as an effective tool for designing anti-dementia drugs through quantitative analysis of the interactions of four representative inhibitors with Cu²⁺/Zn²⁺ induced Aβ assembly.     

Synthesis of 4‐Heteroaryl–Quinazoline Derivatives as Potential Anti‐breast Cancer Agents

4‐Heteroaryl or heteroalkyl–quinazoline derivatives were prepared as dual epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) inhibitors. The new compounds were tested for their dual enzyme inhibition as well as their cytotoxic activity on MCF7 cell line. The results indicated that almost all the compounds showed moderate dual inhibition of both enzymes. Compound 3 (methyl piperidine‐4‐carboxylate derivative) showed the highest inhibitory activity against both enzymes with IC₅₀ 97.6 and 64.0 µM against EGFR and VEGFR‐2 kinases, respectively. Most of the test compounds showed potent to moderate antitumor activity on MCF7 cell line. Five compounds ( 3 , 9c , 11 , 13 , and 15b ) showed potent cytotoxic activity with IC₅₀ values between 10 and 17 µM .     

Bioassay-guided isolation of human carboxylesterase 2 inhibitory and antioxidant constituents from Laportea bulbifera: Inhibition interactions and molecular mechanism

Laportea bulbifera (Sieb. et. Zucc.) Wedd has long been utilized in Traditional Chinese Medicines (TCM) for the treatment of rheumatoid arthritis. However, the study of systematic anti-inflammatory chemical constituents in L. bulbifera has never been reported. Thus, bioassay-guided isolation for its roots part led to 46 compounds, including 38 phenolic derivatives. Their structures were determined on the basis of ¹H and ¹³C NMR and MS spectra. All compounds were isolated from L. bulbifera for the first time except for 13 compounds. Most of the compounds showed good COX-2 inhibitory activity (IC₅₀: 0.13–3.94 μM) and DPPH radical-scavenging activity (IC₅₀: 1.57–9.55 μM). Four compounds (4, 17, 35, and 43) with different skeletons showed preferential COX-2 over COX-1 inhibition with selective indices ranging from 12 to 171. High content active compounds are important for elucidating the basis of the active substance of TCM. Compound 4 (COX-2, IC₅₀ 0.24 μM), a high content compound, represented one of the best selective COX-2 inhibitors. Another high content active compound (35) with a different skeleton might have different mechanism. Further study for the inhibition kinetics against COX-2 indicated compounds 4 and 35 were noncompetitive and competitive COX-2 inhibitors, respectively. Moreover, molecular docking and molecular dynamics simulation data further indicated that compound 4 could bind in the cavity of COX-2 and interacted with key residues VAL-538, PHE-142, and GLY-225 of COX-2 through hydrogen bonds. The results indicated that L. bulbifera roots could be applied as antioxidant and anti-inflammatory agents due to their potent selective COX-2 inhibitory and antioxidant activity of phenolic compounds.     

Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with Staphylococcus aureus

With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC₅₀ values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extracts. While commonly used antimicrobial assays visualize cell viability using dyes, the developed and validated assay conveniently uses OD₆₀₀ measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against Staphylococcus aureus, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution S. aureus growth inhibition profiling was evaluated on a crude extract of an inactive Alternaria sp., spiked with ciprofloxacin.     

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their Na_V-inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX ( 3 a ) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC₅₀ and IC₅₀ values of 8.5 and 30.7 nm , respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of Na_V1.5, with an IC₅₀ value of 94.1 nm . Derivatives 3 a – d and 3 f showed low recovery rates from Na_V1.2 subtype (ca 45–79 %) compared to natural dcSTX ( 2 ), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with Na_V in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp¹⁷¹⁷.     

Phenolic composition,antioxidant and enzyme inhibitory activities of Parkia biglobosa (Jacq.) Benth., Tithonia diversifolia (Hemsl) A. Gray,and Crossopteryx febrifuga (Afzel.) Benth

Medicinal plants from Chad grow under special climatic conditions in between the equatorial forest of Central Africa and the desert of North Africa and are understudied. Three medicinal plants from Chad (T. diversifolia, P. Biglobosa and C. Febrifuga) were evaluated for their phenolic composition, antioxidant and enzyme inhibition activities. The total phenolic composition varied from 203.19 ± 0.58 mg GAE/g DW in the ethyl acetate extract of P. biglobosa, to 56.41 ± 0.89 mg GAE/g DW in the methanol extract of C. febrifuga while the total flavonoid content varied from 51.85 ± 0.91 mg QE/g DW in the methanol extract of P. biglobosa to 08.56 ± 0.25 mg QE/g DW in the methanol extract of C. febrifuga. HPLC-DAD revealed that rutin, gallic acid and protocatechuic acid were the most abundant phenolics in T. diversifolia, P. Biglobosa and C. Febrifuga respectively. The antioxidant activity assayed by five different methods revealed very good activity especially in the DPPH^?, ABTS^?+ and CUPRAC assays where the extracts were more active than the standard compounds used. Good inhibition was exhibited against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with methanol (IC₅₀: 15.63 ± 0.72 µg/mL), ethyl acetate (IC₅₀: 16.20 ± 0.67 µg/mL) extracts of P. biglobosa, and methanol (IC₅₀: 21.53 ± 0.65 µg/mL) and ethyl acetate (IC₅₀: 30.81 ± 0.48 µg/mL) extracts of T. diversifolia showing higher inhibition than galantamine (IC₅₀: 42.20 ± 0.44 µg/mL) against BChE. Equally, good inhibition was shown on α-amylase and α-glucosidase. On the α-glucosidase, the ethyl acetate (IC₅₀ = 12.47 ± 0.61 µg/mL) and methanol extracts (IC₅₀ = 16.51 ± 0.18 µg/mL) of P. biglobosa showed higher activity compared to the standard acarbose (IC₅₀ = 17.35 ± 0.71 µg/mL) and on α-amylase, the ethyl acetate (IC₅₀ = 13.50 ± 0.90 µg/mL) and methanol (IC₅₀ = 18.12 ± 0.33 µg/mL) extracts of P. biglobosa showed higher activity compared to acarbose (IC₅₀ = 23.84 ± 0.25 µg/mL). The results indicate that these plants are good sources of antioxidant phenolics and can be used to manage oxidative stress linked illnesses such as Alzheimer’s disease and diabetes.     

Gold nanoparticle-based electrochemical detection of protein phosphorylation

In this report, we demonstrate the application of Au nanoparticles in the electrochemical detection of protein phosphorylation. The method is based on the labeling of a specific phosphorylation event with Au nanoparticles, followed by electrochemical detection. The phosphorylation reaction is coupled with the biotinylation of the kinase substrate using a biotin-modified adenosine 5′-triphosphate [γ]-biotinyl-3,6,9-trioxaundecanediamine (ATP) as the co-substrate. When the phosphorylated and biotinylated kinase substrate is exposed to streptavidin-coated Au nanoparticles, the high affinity between the streptavidin and biotin resulted in the attachment of Au nanoparticles on the kinase substrate. The electrochemical response obtained from Au nanoparticles enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. We determined the activity of Src non-receptor protein tyrosine kinase, p60^c-Src and protein kinase A in combination with their highly specific substrate peptides Raytide™ EL and Kemptide, respectively. The detection limits for Raytide™ EL and Kemptide were determined as 5 and 10 μM, (S/N = 3), and the detection limits for the kinase activity of p60^c-Src and protein kinase A (PKA) were determined as 5 and 10 U mL⁻¹, (S/N = 3), respectively. Tyrosine kinase reactions were also performed in the presence of a well-defined inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), and its negative control molecule, 4-amino-7-phenylpyrazol[3,4-d] pyrimidine (PP3), which had no inhibition effect. Based on the dependency of Au nanoparticle signal on inhibitor concentration, IC₅₀ value, half-maximal inhibition of the inhibitors was estimated. IC₅₀ values of PP2, genistein and herbimycin A to p60^c-Src were detected as 5 nM, 25 μM and 900 nM, respectively. The inhibition of PKA activity on Kemptide using ellagic acid was monitored with an IC₅₀ of 3.5 μM. The performance of the biosensor was optimized including the kinase reaction, incubation with streptavidin-coated Au nanoparticles, and the small molecule inhibitors. Kinase peptide-modified electrochemical biosensors are promising candidates for cost-effective kinase activity and inhibitor screening assays.     

Methoxy-Substituted Tyramine Derivatives Synthesis,Computational Studies and Tyrosinase Inhibitory Kinetics

Targeting tyrosinase for melanogenesis disorders is an established strategy. Hydroxyl-substituted benzoic and cinnamic acid scaffolds were incorporated into new chemotypes that displayed in vitro inhibitory effects against mushroom and human tyrosinase for the purpose of identifying anti-melanogenic ingredients. The most active compound 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2,4-dihydroxyphenyl) acrylate (Ph9), inhibited mushroom tyrosinase with an IC₅₀ of 0.059 nM, while 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) had an IC₅₀ of 2.1 nM compared to the positive control, kojic acid IC₅₀ 16700 nM. Results of human tyrosinase inhibitory activity in A375 human melanoma cells showed that compound (Ph9) and Ph6 exhibited 94.6% and 92.2% inhibitory activity respectively while the positive control kojic acid showed 72.9% inhibition. Enzyme kinetics reflected a mixed type of inhibition for inhibitor Ph9 (K_i 0.093 nM) and non-competitive inhibition for Ph6 (K_i 2.3 nM) revealed from Lineweaver–Burk plots. In silico docking studies with mushroom tyrosinase (PDB ID:2Y9X) predicted possible binding modes in the catalytic site for these active compounds. Ph9 displayed no PAINS (pan-assay interference compounds) alerts. Our results showed that compound Ph9 is a potential candidate for further development of tyrosinase inhibitors.     

Structural Bases for Hesperetin Derivatives: Inhibition of Protein Tyrosine Phosphatase 1B,Kinetics Mechanism and Molecular Docking Study

In the present study, we investigated the structure-activity relationship of naturally occurring hesperetin derivatives, as well as the effects of their glycosylation on the inhibition of diabetes-related enzyme systems, protein tyrosine phosphatase 1B (PTP1B) and α-glycosidase. Among the tested hesperetin derivatives, hesperetin 5-O-glucoside, a single-glucose-containing flavanone glycoside, significantly inhibited PTP1B with an IC₅₀ value of 37.14 ± 0.07 µM. Hesperetin, which lacks a sugar molecule, was the weakest inhibitor compared to the reference compound, ursolic acid (IC₅₀ = 9.65 ± 0.01 µM). The most active flavanone hesperetin 5-O-glucoside suggested that the position of a sugar moiety at the C-5-position influences the PTP1B inhibition. It was observed that the ability to inhibit PTP1B is dependent on the nature, position, and number of sugar moieties in the flavonoid structure, as well as conjugation. In the kinetic study of PTP1B enzyme inhibition, hesperetin 5-O-glucoside led to mixed-type inhibition. Molecular docking studies revealed that hesperetin 5-O-glucoside had a higher binding affinity with key amino residues, suggesting that this molecule best fits the PTP1B allosteric site cavity. The data reported here support hesperetin 5-O-glucoside as a hit for the design of more potent and selective inhibitors against PTP1B in the search for a new anti-diabetic treatment.     

5-Bromo- and 3,5-dibromo-2-hydroxy-N-phenylbenzamides — inhibitors of photosynthesis

5-Bromo-(Br-PBA) and 3,5-dibromo-2-hydroxy-N-phenylbenzamides (Br₂-PBA) inhibited photosynthetic electron transport (PET) and their inhibitory efficiency depended on the compound lipophilicity as well as on the electronic properties of the R substituent in the N-phenyl moiety. Br-PBA showed higher PET inhibiting activity than Br₂-PBA with the same R substituent. The most effective inhibitors in the tested series were the derivatives with R = 3-F (Br-PBA; IC₅₀ = 4.3 μmol dm^?3) and R = 3-Cl (Br₂-PBA; IC₅₀ = 8.6 μmol dm^?3). Bilinear dependence of the PET inhibiting activity on the lipophilicity of the compounds as well as on the Hammett constant, σ, of the R substituent was observed for both investigated series. Using EPR spectroscopy it was found that the site of action of the tested compounds in the photosynthetic apparatus is situated on the donor side of PS 2, in D^· or in the Z^·/D^· intermediates. Interaction of the studied compounds with chlorophyll a and aromatic amino acids present in the pigment-protein complexes mainly in photosystem 2 was documented by fluorescence spectroscopy.