Molecular modelling studies and in vitro enzymatic assays identified A 4-(nitrobenzyl)guanidine derivative as inhibitor of SARS-CoV-2 Mpro.

Scientists and researchers have been searching for drugs targeting the main protease (Mpro) of SARS-CoV-2, which is crucial for virus replication. This study employed a virtual screening based on molecular docking to identify benzoylguanidines from an in-house chemical library that can inhibit Mpro on the active site and three allosteric sites. Molecular docking was performed on the LaSMMed Chemical Library using 88 benzoylguanidine compounds. Based on their RMSD values and conserved pose, three potential inhibitors (BZG1, BZG2, and BZG3) were selected. These results indicate that BZG1 and BZG3 may bind to the active site, while BZG2 may bind to allosteric sites. Molecular dynamics data suggest that BZG2 selectively targets allosteric site 3. In vitro tests were performed to measure the proteolytic activity of rMpro. The tests showed that BZG2 has uncompetitive inhibitory activity, with an IC50 value of 77 µM. These findings suggest that benzoylguanidines possess potential as Mpro inhibitors and pave the way towards combating SARS-Cov-2 effectively.

An Enzyme Assay Kit for GABA Quantification in Plant Tissues.

Gamma-aminobutyric acid (GABA) is an amino acid that has a role as a signaling molecule. In plants, its involvement in stress responses is widely investigated. A newly developed method of quantification of GABA is described in this chapter. The assay kit consisting of three bacterial enzymes enables easy but accurate measurement of GABA (~200 mg/mL) based on the serial enzymatic reaction leading to dye formation. The method was successfully applied to measure the GABA content in several plant tissues.

Prolidase activity assays. A survey of the reported literature methodologies.

Prolidase (EC.3.4.13.9) is a dipeptidase known nowadays to play a pivotal role in several physiological and pathological processes. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), thus finely regulating the homeostasis of free proline and hydroxyproline. Abnormally high or low levels of prolidase have been found in numerous acute and chronic syndromes affecting humans (chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection, and others) for which the content of proline is well recognized as a clinical marker. As a consequence, the accurate analytical determination of prolidase activity is of greatly significant importance in clinical diagnosis and therapy. Apart from the Chinard's assay, some other more sensitive and well validated methodologies have been published. These include colorimetric and spectrophotometric determinations of free proline produced by enzymatic reactions, capillary electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, electrochemoluminescence, thin layer chromatography, and HPLC. The aim of this comprehensive review is to make a detailed survey of the in so far reported analytical techniques, highlighting their general features, as well as their advantages and possible drawbacks, providing in the meantime suggestions to stimulate further research in this intriguing field.

Biogenic Polymer-Based Fluorescent Assemblies: Versatile Platforms for Ultrasensitive ATP Detection and Enzyme Assay.

Here, we investigated the optical properties of biocompatible supramolecular assemblies formed through electrostatic interactions between anionic fluorescent dyes and biogenic polymers. The dynamic equilibrium between the monomeric form (fluorescent) and aggregates (nonfluorescent) of dye molecules is responsible for the stimuli-responsive behavior of these polymer composites, which can respond to changes in pH, temperature, and ionic strength. Furthermore, we employed supramolecular assemblies for the purpose of turn-on fluorescence sensing of adenosine triphosphate (ATP) at physiological pH. Notably, no interference was observed even in the presence of well-known competing analytes such as pyrophosphate. In addition to its outstanding selectivity, the present system can detect ATP at concentrations as low as 4.8 nM. The superior detection capabilities are achieved through multiple interactions with biogenic polymers, involving the adenine ring, ribose unit (through hydrogen bonding), and phosphate groups (via charge pairing) of ATP. Given the remarkable sensitivity to ATP, we have applied the present system for the detection of a dephosphorylating enzyme, alkaline phosphatase.

Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.

Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Preparation of ER Microsomes from Arabidopsis thaliana.

Microsomes are vesicles derived from the endoplasmic reticulum (ER) when cells are broken down in the lab. These microsomes are a valuable tool to study a variety of ER functions such as protein and lipid synthesis in vitro.Here we describe a protocol to isolate ER-derived microsomes Arabidopsis thaliana seedlings and exemplify the use of these purified microsomes in enzyme assays with the auxin precursors tryptophan (Trp) or indole-3-pyruvic acid (IPyA) to quantify auxin synthetic capacity in microsomal and cytosolic fractions.

Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

Newborn screening for the full set of mucopolysaccharidoses in dried blood spots based on first-tier enzymatic assay followed by second-tier analysis of glycosaminoglycans.

Newborn screening (NBS) for the full set of mucopolysaccharidoses (MPSs) is now possible by either measuring all of the relevant enzymatic activities in dried blood spots (DBS) using tandem mass spectrometry followed by measurement of accumulated glycosaminoglycans (GAGs) or the vice-versa approach. In this study we considered multiple factors in detail including reagent costs, time per analysis, false positive rates, instrumentation requirements, and multiplexing capability. Both NBS approaches are found to provide acceptable solutions for comprehensive MPS NBS, but the enzyme-first approach allows for better multiplexing to include numerous additional diseases that are appropriate for NBS expansion. By using a two-tier NBS approach, the false positive and false negatives rates are expected to acceptably low and close to zero.

Analysis of continuous enzyme kinetic data using ICEKAT.

ICEKAT (Interactive Continuous Enzyme Analysis Tool) is an interactive web-based program for calculating initial rates and kinetic parameters (e.g., Vmax, kcat, KM, EC50, IC50) from continuous enzyme kinetic assay data that satisfy Michaelis-Menten and steady-state kinetic assumptions. ICEKAT is valuable in educational and research settings to consistently and accurately calculate initial rates and kinetic parameters, increasing assay veracity and reproducibility. Provided freely online to the scientific community, ICEKAT has been cited in at least 26 publications, and the initial journal article has been accessed nearly 9000 times since its debut in 2020 (Olp et al., 2020). Here, we provide in-depth instructions for software use, offer vital considerations for data analysis, and highlight updated software features for new and existing users. Through ICEKAT, we aim for the analysis of data from continuous enzyme kinetic studies worldwide to become more rapid, reliable, and repeatable. ICEKAT remains free of charge and available to all scientists at https://icekat.herokuapp.com/icekat; the source code for local use is found at https://github.com/SmithLabMCW/icekat.

17ß-Hydroxysteroid dehydrogenases types 1 and 2: Enzymatic assays based on radiometric and mass-spectrometric detection.

The 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1) has a key role in estrogen biosynthesis as it catalyzes the reduction of estrone to the most potent estrogen, estradiol. Estradiol has a high affinity for estrogen receptors and thus stimulates their transactivation, which leads to cell proliferation and numerous other effects. HSD17B2 catalyzes the oxidation of estradiol to the less potent estrone, thereby decreasing estrogen receptor activation, which results in reduction of estrogen-associated effects. HSD17B1 and HSD17B2 overexpressing E.coli homogenates or recombinant enzymes can be used for screening and development of drugs against various pathologies such as cancer, endometriosis or osteoporosis. Here we describe the preparation of HSD17B1 and HSD17B2 bacterial homogenates and purified recombinant HSD17B1 protein as enzyme sources as well as enzymatic assays based on radiometric and mass-spectrometric detection for enzyme characterization.

Aldo keto-reductase family 1C members 1 through 4 recombinant enzyme purification and enzyme assay.

Aldo-keto reductase family 1C (AKR1C) members transform steroids via their 3-, 17-, and 20-ketosteroid reductase activities. The biochemical study of these enzymes can help to inform their roles in hormone-dependent diseases and develop therapeutic inhibitors. This work describes a protocol to purify AKR1C1-4 members from a bacterial expression system using two chromatography steps. It also describes the basis of discontinuous assays to measure steroid conversion.

Sensitive Silver-Enhanced Microplate Apta-Enzyme Assay of Sb3+ Ions in Drinking and Natural Waters.

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb3+ detection was developed. The biotinylated aptamer A10 was hybridized with its complementary biotinylated oligonucleotide T10 and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3',5,5'-tetramethylbenzidine substrate, generating an optical signal. Sb3+ presenting in the sample bounded to an A10 aptamer, thus releasing T10, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb3+ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag+ at the stage of A10/T10 complex formation promoted dehybridization of the aptamer A10 and the formation of the A10/Sb3+ complex. The working range of the Ag+-enhanced microplate apta-enzyme assay for Sb3+ was determined to be 8-135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb3+ in the range of 109.0-126.2% and 99.6-106.1%, respectively.

The cotton miR171a-SCL6 module mediates plant resistance through regulating GhPR1 expression.

Plants have developed intricate defense mechanisms in response to fluctuating environmental cues, including the use of microRNA (miRNA) as post-transcriptional regulators. However, the specific mechanisms through which miRNA contributes to disease resistance remain largely elusive. While the miR171-SCLs have been investigated in an eclectic array of plants, there has been a notable scarcity of research specifically focused on cotton (Gossypium hirsutum). In our previous miRNA-sequencing analysis, we found that ghr-miR171a displayed a differential response to infections by Verticillium dahliae. In this study, we further investigated the function of the miR171a-SCL6 module in cotton during V. dahliae infection. The ghr-miR171a was confirmed to direct the cleavage of GhSCL6 mRNA in the post-transcriptional process, as evidenced by 5' RLM-RACE, ß-glucuronidase (GUS) histochemical staining and enzyme activity assay. Interestingly, we found that overexpressing ghr-miR171a reduced cotton plants' resistance to V. dahliae, while suppressing ghr-miR171a increased the plants' defense capacity. The GhSCL6 protein, when fused with green fluorescent protein (GFP), localizes in the cell nucleus, indicating its potential role in gene regulation. This was further corroborated by yeast two-hybrid assays, which verified GhSCL6's transcriptional activation ability. Through quantitative reverse transcriptase PCR (qRT-PCR), luciferase (LUC) fluorescence, and yeast one-hybrid assays, we found that GhSCL6 binds to the GT-box element of the GhPR1 promoter, activating its expression and thereby enhancing plant disease resistance. Taken together, our findings demonstrate that the cotton miR171a-SCL6 module regulates Verticillium wilt resistance in plants through the post-transcriptional process. This insight may offer new perspectives for disease resistance strategies in cotton.

CircRNA 06209 inhibits cataract development by sponging miR-6848-5p and regulating ALOX15 expression.

Cataract is the leading cause of blindness in the world, and there is a lack of effective treatment drugs. CircRNA plays an important part in a variety of diseases, however, the role of circRNA in cataracts remains largely unknown. In this study, we constructed a cataract model of rats and obtained the circRNAs related to cataracts by whole transcriptome sequencing and circRNA-mRNA co-expression network. To investigate the effect and mechanism of circRNA 06209 on cataracts, we performed several in vivo and in vitro experiments, including CCK8 assay, flow cytometry, dual luciferase reporter assay, RIP assay, actinomycin D assay, and Western blot analysis. We identify that a necroptosis-related circRNA, circRNA 06209, is down-regulated in cataracts. Vitro experiments showed that up-regulation of circRNA 06209 could promote cell proliferation and inhibit cell apoptosis. Vivo experiments revealed that circRNA 06209 overexpression could inhibit the development of cataracts. Mechanistically, circRNA 06209 acts as a miRNA sponge and competitively binds to miR-6848-5p to curb the inhibitory effect of miR-6848-5p on ALOX15, thereby affecting cell viability and apoptosis. This study found that circRNA 06209 plays a critical part in inhibiting cataracts through the miR-6848-5p/ALOX15 pathway, suggesting that circRNA 06209 may be a promising therapeutic target for cataracts.

11,12-seco-Abietane-type diterpene lactones with potential antiplatelet activity from Salvia prattii.

Eleven new abietane-type diterpene lactones, salpratlactones D-N (1-11), including five 11,12-seco-11-nor-abietane diterpenes (1-5), four 11,12-seco-abietane diterpenes (6-9), two 20(10 â†’ 5)-abeo-4,5;11,12-bis-seco-abietane diterpenes (10-11), and two known analogues (12-13), were characterized from Salvia prattii. Notably, compounds 1-3 were characterized by a unique linear 6/6/6 tricyclic skeleton. The structures were established by spectroscopic data interpretation, calculated NMR-DP4+ and electronic circular dichroism analysis, as well as single-crystal X-ray diffraction. A bioactivity study showed that 1, 2, 5, 11, and 12 can potently inhibit platelet aggregation induced by arachidonic acid (AA), with IC50 values of 5.66-16.10 µg/ml, stronger than aspirin. In addition, the lactate dehydrogenase assay showed that they had no effect on platelet integrity. Structurally, the same 1,2-benzopyrone fragments of 1, 2, and 5 should be the important pharmacophore for antiplatelet activity.

In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site.

APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is a central enzyme of the base excision repair (BER) pathway playing a pivotal role in protecting mammalian cells against genotoxins and in safeguarding genome stability. Recently, we demonstrated the APE1 ability to process abasic ribonucleotides embedded in DNA. Here, we provide a pipeline of protocols to quantify endodeoxyribonuclease activity by APE1 on these substrates, by using recombinant protein and whole-cell extracts. The repair capacity is measured by using fluorescent oligonucleotide substrates, which are then separated by polyacrylamide gel electrophoresis and detected by imaging scanning. The specificity of APE1 action is demonstrated using specific APE1 enzymatic inhibitors.

Discovery of (S)-N1-(thiazol-2-yl) pyrrolidine-1,2-dicarboxamide derivatives targeting PI3Ka/HDAC6 for the treatment of cancer.

Recently, PI3K and HDAC have been considered as promising targets for the cancer therapy. A couple of pan-PI3K/HDAC dual inhibitors have been developed as a new class of anticancer agents. Herein, we discovered a new series of (S)-N1-(thiazol-2-yl) pyrrolidine-1,2-dicarboxamide derivatives targeting PI3Kα/HDAC6. All the derivatives exerted dual-target inhibitory activities. Particularly, in the enzymatic selectivity assay, compound 21j was identified as a subtype-selective PI3Kα/HDAC6 dual inhibitor (IC50 = 2.9 and 26 nM against PI3Kα and HDAC6, respectively), which displayed high potency against L-363 cell line with IC50 value of 0.17 µM. In addition, 21j significantly inhibited phosphorylation of pAkt(Ser473) and induced accumulation of acetylated α-tubulin while having a negligible effect on the levels of acetylated Histone H3 and H4 at nanomolar level. Attributed to its favorable in vitro performance, 21j has the potential to alleviate the adverse effects resulted from pan-PI3K inhibition and pan-HDAC inhibition. It is valuable for further functional investigation as an anti-cancer agent.