Screening Method For Inhibitors Research Articles

Rapid screening of xanthine oxidase inhibitors from Ligusticum wallichii by using xanthine oxidase functionalized magnetic metal-organic framework.

In this study, xanthine oxidase was immobilized for the first time using a novel magnetic metal-organic framework material (Fe3O4-SiO2-NH2@MnO2@ZIF-8-NH2). A ligand fishing method was established to rapidly screen XOD inhibitors from Ligusticum wallichii based on the immobilized XOD. Characterization and properties of the immobilized enzyme revealed its excellent stability and reusability. A ligand was screened from Ligusticum wallichii and identified as ligustilide by ultra-high performance liquid chromatography tandem mass spectrometry. The IC50 value of ligustilide was determined to be 27.70 ± 0.13μM through in vitro inhibition testing. Furthermore, molecular docking verified that ligustilide could bind to amino acid residues at the active site of XOD. This study provides a rapid and effective method for thepreliminary screening of XOD inhibitors from complex natural products and has great potential for further discovery of anti-hyperuricemic compounds.

Development of an Ultrasensitive Competitive Double-Enzyme Cascade Fluorescence Signal Amplification Method for Factor XIa Inhibitor Screening.

Coagulation factor XIa (FXIa) is associated with a low risk of bleeding and has been identified as an effective and safe target for the development of novel anticoagulant drugs. In this study, we established an ultrasensitive competitive dual-enzyme cascade signal amplification method for the quantitative analysis and screening of FXIa inhibitors. Due to the specific recognition of FXIa's active site by the aptamer AptE40, the AptE40-QDs-EK recognition probe modified with enterokinase (EK) and the aptamer AptE40, was attached to the MNPs-FXIa capture probe. When FXIa inhibitor was present, it competed with AptE40 for binding to FXIa, resulting in the detachment of AptE40-QDs-EK from MNPs-FXIa. After magnetic separation, the enterokinase of AptE40-QDs-EK in the supernatant hydrolyzed N-terminal hexapeptide of trypsinogen, leading to the production of a large amount of trypsin as part of the first-stage signal cascade amplification. Next, trypsin could hydrolyze the hexameric arginine peptide (RRRRRR, R6), leading to the dissociation of RQDs from the R6-RQDs signal probe; this resulted in a dramatic increase in the fluorescence intensity of the supernatant as the second-stage signal cascade was amplified. The feasibility of the method was investigated using the FXIa inhibitor aptamer FELIAP as a positive model drug. Furthermore, the method was applied to screen the FXIa inhibitors in Eupolyphaga sinensis Walker. Two fractions with more active anticoagulated ingredients were successfully identified and validated via the conventional method, and the results were consistent. The established method provides a key technique for the sensitive detection, high-throughput analysis, and screening of the FXIa inhibitors.

Single G-quadruplex-based fluorescence method for the uracil-DNA glycosylase inhibitor screening

Uracil-DNA glycosylase (UDG) plays a pivotal role in the base repair system. Through bioinformatics, we found that the expression of the UDG enzyme in many cancer cells is increased, and its high expression is not conducive to the prognosis of lung cancer patients. The development of analytical techniques for the quantification of UDG activity and the identification of UDG inhibitors is of paramount importance. We found that when the T base in the G4 loop region mutated to uracil, the G4 structure was not disrupted and still retained the characteristics of a G4 structure (emitting strong fluorescence after binding with ThT (Thioflavin T). Inspired by this phenomenon, we developed a detection method for UDG and its inhibitors utilizing a single DNA strand engineered to form a G-quadruplex structure, containing uracil residues within the loop region, designated as G4-dU. The inclusion of uracil-DNA glycosylase (UDG) in the assay environment induces the removal of uracil, resulting in the formation of apurinic sites (AP) within the G4-dU sequence. Subsequent thermal denaturation leads to strand cleavage at AP sites, precluding the reformation of the G-quadruplex configuration and abrogating fluorescence emission. The detection process in this study can be completed in only 30 min to 1 h, offers a straightforward, expedient, and efficacious modality for assessing UDG activity and UDG inhibitor potency, thereby facilitating the discovery of novel therapeutic agents for cancer treatment.

Development of surface droplet evaporation-based sensing platform for screening lipase inhibitors

The development of new types of sensing platforms with excellent performance is always appealing. In this report, we demonstrate a low-cost and user-friendly sensing platform based on drying of the aqueous droplet on the surface. The principle relies on the transduction of the surfactant concentration into the area of the dried droplet on the surface. The screening of a lipase inhibitor is demonstrated as an example. When triacylglycerols (GT) and lipase are mixed, the aqueous droplet produces a large dried area on the surface. This phenomenon is attributed to the production of sodium oleate due to the enzymatic digestion of GT by lipase. It results in reduction of the surface tension and causes wetting and spreading of the droplet, leaving a large dried area on the surface. However, in the presence of orlistat, a lipase inhibitor, a small dried area is obtained due to the inhibition of lipase activity by orlistat. Using this method, the lipase inhibitor can be readily and effectively screened. Compared to previous studies, this method is simple, rapid, low-cost and avoids the use of labelled molecules and complex instrumentation, with an IC50 value of 0.59 ± 0.09 μM for orlistat. In addition, it also works well with a portable microscope and a smartphone interface for data analysis. Overall, this study provides a new method for rapid and cost-effective screening of lipase inhibitors.

The Construction and Application of a New Screening Method for Phosphodiesterase Inhibitors.

Phosphodiesterases (PDEs), a superfamily of enzymes that hydrolyze cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), are recognized as a therapeutic target for various diseases. However, the current screening methods for PDE inhibitors usually experience problems due to complex operations and/or high costs, which are not conducive to drug development in respect of this target. In this study, a new method for screening PDE inhibitors based on GloSensor technology was successfully established and applied, resulting in the discovery of several novel compounds of different structural types with PDE inhibitory activity. Compared with traditional screening methods, this method is low-cost, capable of dynamically detecting changes in substrate concentration in live cells, and can be used to preliminarily determine the type of PDEs affected by the detected active compounds, making it more suitable for high-throughput screening for PDE inhibitors.

Rational Design of a Highly Sensitive Carboxylesterase Probe and Its Application in High-Throughput Screening for Uncovering Carboxylesterase Inhibitors.

Tracking carboxylesterases (CESs) through noninvasive and dynamic imaging is of great significance for diagnosing and treating CES-related metabolic diseases. Herein, three BODIPY-based fluorescent probes with a pyridine unit quaternarized via an acetoxybenzyl group were designed and synthesized to detect CESs based on the photoinduced electron transfer process. Notably, among these probes, BDPN2-CES exhibited a remarkable 182-fold fluorescence enhancement for CESs within 10 min. Moreover, BDPN2-CES successfully enabled real-time imaging of endogenous CES variations in living cells. Using BDPN2-CES, a visual high-throughput screening method for CES inhibitors was established, culminating in the discovery of an efficient inhibitor, WZU-13, sourced from a chemical library. These findings suggest that BDPN2-CES could provide a new avenue for diagnosing CES-related diseases, and WZU-13 emerges as a promising therapeutic candidate for CES-overexpression pathological processes.

Rational design of a near-infrared fluorescent probe for monitoring butyrylcholinesterase activity and its application in development of inhibitors.

Butyrylcholinesterase (BChE) is widely expressed in multiple tissues and has a vital role in several key human disorders, such as Alzheimer's disease and tumorigenesis. However, the role of BChE in human disorders has not been investigated. Thus, to quantitatively detect and visualize dynamical variations in BChE activity is essential for exploring the biological roles of BChE in the progression of a number of human disorders. Herein, based on the substrate characteristics of BChE, we customized and synthesized three near-infrared (NIR) fluorescent probe substrates with cyanine-skeleton, and finally selected a NIR fluorescence probe substrate named CYBA. The CYBA demonstrated a significant increase in fluorescence when interacting with BChE, but mainly avoided AChE. Upon the addition of BChE, CYBA could be specifically hydrolyzed to TBO, resulting in a significant NIR fluorescence signal enhancement at 710nm. Systematic evaluation revealed that CYBA exhibited exceptional chemical stability in complex biosamples and possessed remarkable selectivity and sensitivity towards BChE. Moreover, CYBA was successfully applied for real-time imaging of endogenous BChE activity in two types of nerve-related living cells. Additionally, CYBA demonstrated exceptional stability in the detection of complex biological samples in plasma recovery studies (97.51%-104.01%). Furthermore, CYBA was used to construct a high-throughput screening (HTS) method for BChE inhibitors using human plasma as the enzyme source. We evaluated inhibitory effects of a series of natural products and four flavonoids were identified as potent inhibitors of BChE. Collectively, CYBA can serve as a practical tool to track the changes of BChE activity in complicated biological environments due to its excellent capabilities.

A fast and efficient method for screening and evaluation of hypoglycemic ingredients of Traditional Chinese Medicine acting on PTP1B by capillary electrophoresis

As a pivotal enzyme that regulates dephosphorylation in cell activities and participates in the insulin signaling pathway, protein tyrosine phosphatase 1B (PTP1B) is considered to be an important target for the therapy of diabetes. In this work, a rapid and efficient inhibitor screening method of PTP1B was established based on capillary electrophoresis (CE), and used for screening and evaluating the inhibition effect of Traditional Chinese Medicine on PTP1B. Response Surface Methodology was used for optimizing the conditions of analysis. After method validation, the enzyme kinetic study and inhibition test were performed. As a result, the IC50 of PTP1B inhibitors Ⅳ and ⅩⅧ were consistent with reported values measured by a conventional method. It was found that the extracts of Astragalus membranaceus (Fisch) Bunge and Morus alba L. showed prominent inhibition on the activity of PTP1B, which were stronger than the positive controls. Meanwhile, on top of the excellent advantages of CE, the whole analysis time is less than 2 min. Thus, the results demonstrated that a fast and efficient screening method was successfully developed. This method could be a powerful tool for screening inhibitors from complex systems. It can also provide an effective basis for lead compound development in drug discovery.

Establishment and application of a screening method for α-glucosidase inhibitors based on dual sensing and affinity chromatography

α-Glucosidase plays a direct role in the metabolic pathways of starch and glycogen, any dysfunction in its activity could result in metabolic disease. Concurrently, this enzyme serves as a target for diverse drugs and inhibitors, contributing to the regulation of glucose metabolism in the human body. Here, an integrated analytical method was established to screen inhibitors of α-glucosidase. This step-by-step screening model was accomplished through the biosensing and affinity chromatography techniques. The newly proposed sensing program had a good linear relationship within the enzyme activity range of 0.25 U mL−1 to 1.25 U mL−1, which can quickly identify active ingredients in complex samples. Then the potential active ingredients can be captured, separated, and identified by an affinity chromatography model. The combination of the two parts was achieved by an immobilized enzyme technology and a microdevice for reaction, and the combination not only ensured efficiency and accuracy for inhibitor screening but also eliminated the occurrence of false positive results in the past. The emodin, with a notable inhibitory effect on α-glucosidase, was successfully screened from five traditional Chinese medicines using this method. The molecular docking results also demonstrated that emodin was well embedded into the active pocket of α-glucosidase. In summary, the strategy provided an efficient method for developing new enzyme inhibitors from natural products.

GAA/(Au–Au/IrO2)@Cu(PABA) reactor with cascade catalytic activity for α-glucosidase inhibitor screening

BackgroundIn vitro screening strategies based on the inhibition of α-glucosidase (GAA) activity have been widely used for the discovery of potential antidiabetic drugs, but they still face some challenges, such as poor enzyme stability, non-reusability and narrow range of applicability. To overcome these limitations, an in vitro screening method based on GAA@GOx@Cu-MOF reactor was developed in our previous study. However, the method was still not satisfactory enough in terms of construction cost, pH stability, organic solvent resistance and reusability. Thence, there is still a great need for the development of in vitro screening methods with lower cost and wider applicability. ResultsA colorimetric sensing strategy based on GAA/(Au–Au/IrO2)@Cu(PABA) cascade catalytic reactor, which constructed through simultaneous encapsulating Au–Au/IrO2 nanozyme with glucose oxidase-mimicking and peroxidase-mimicking activities and GAA in Cu(PABA) carrier with peroxidase-mimicking activity, was innovatively developed for in vitro screening of GAA inhibitors in this work. It was found that the reactor not only exhibited excellent thermal stability, pH stability, organic solvent resistance, room temperature storage stability, and reusability, but also possessed cascade catalytic performance, with approximately 12.36-fold increased catalytic activity compared to the free system (GAA + Au–Au/IrO2). Moreover, the in vitro GAA inhibitors screening method based on this reactor demonstrated considerable anti-interference performance and detection sensitivity, with a detection limit of 4.79 nM for acarbose. Meanwhile, the method owned good reliability and accuracy, and has been successfully applied to the in vitro screening of oleanolic acid derivatives as potential GAA inhibitors. SignificanceThis method not only more effectively solved the shortcomings of poor stability, narrow scope of application, and non-reusability of natural enzymes in the classical method compared with our previous work, but also broaden the application scope of Au–Au/IrO2 nanozyme with glucose oxidase and peroxidase mimicking activities, and Cu(PABA) carrier with peroxidase mimicking activity, which was expected to be a new generation candidate method for GAA inhibitor screening.

Paper-based ligand fishing method for rapid screening and real-time capturing of α-glucosidase inhibitors from the Chinese herbs

Identifying medicinally relevant compounds from natural resources generally involves the tedious work of screening plants for the desired activity before capturing the bioactive molecules from them. In this work, we created a paper-based ligand fishing platform to vastly simplify the discovery process. This paper-based method exploits the enzymatic cascade reaction between α-glucosidase (GAA), glucose oxidase (GOx), and horseradish peroxidase (HRP), to simultaneously screen the plants and capture the GAA inhibitors from them. The designed test strip could capture ligands in tandem with screening the plants, and it features a very simply operation based on direct visual assessment. Multiple acylated flavonol glycosides from the leaves of Quercus variabilis Blume were newly found to possess GAA inhibitory activities, and they may be potential leads for new antidiabetic medications. Our study demonstrates the prospect of the newly discovered GAA ligands as potential bioactive ingredients as well as the utility of the paper-based ligand fishing method.

Bioactive components and mechanisms of Pu-erh tea in improving levodopa metabolism in rats through COMT inhibition.

Catechol-O-methyltransferase (COMT) plays a central role in the metabolic inactivation of endogenous neurotransmitters and xenobiotic drugs and hormones having catecholic structures. Its inhibitors are used in clinical practice to treat Parkinson's disease. In this study, a fluorescence-based visualization inhibitor screening method was developed to assess the inhibition activity on COMT both in vitro and in living cells. Following the screening of 94 natural products, Pu-erh tea extract exhibited the most potent inhibitory effect on COMT with an IC50 value of 0.34 μg mL-1. In vivo experiments revealed that Pu-erh tea extract substantially hindered COMT-mediated levodopa metabolism in rats, resulting in a significant increase in levodopa levels and a notable decrease in 3-O-methyldopa in plasma. Subsequently, the chemical components of Pu-erh tea were analyzed using UHPLC-Q-Exactive Orbitrap HRMS, identifying 24 major components. Among them, epigallocatechin gallate, gallocatechin gallate, epicatechin gallate, and catechin gallate exhibited potent inhibition of COMT activity with IC50 values from 93.7 nM to 125.8 nM and were the main bioactive constituents in Pu-erh tea responsible for its COMT inhibition effect. Inhibition kinetics analyses and docking simulations revealed that these compounds competitively inhibit COMT-mediated O-methylation at the catechol site. Overall, this study not only explained how Pu-erh tea catechins inhibit COMT, suggesting Pu-erh tea as a potential dietary intervention for Parkinson's disease, but also introduced a new strategy for discovering COMT inhibitors more effectively.

Eriodictyol Flavanones Based Virtual Screening of Bioactive Compounds from ChEMBL 2D Database with Classic 3-point Pharmacophore Screening Method for HER2 Inhibitors for Breast Cancer

Understanding binding interactions between flavanones and human epidermal growth factor receptor 2 (HER2) is an important step in developing effective treatments for breast cancer, and this study applies computational methods to do just that. This research presents a comprehensive computational methodology for identifying potential HER2 inhibitors with a focus on breast cancer treatment. The study leverages a combination of structural and pharmacophore-based approaches, starting with bioactive compound selection from the ChEMBL 2D database. The PDB-REDO refined crystal structure of Kinase domain of Human HER2 (erbB2) was used to conduct molecular docking simulations with the identified drugs. The Kleywegt-like plot analysis demonstrates the improved structural quality of the HER2 kinase domain after refinement, showing enhanced agreement with experimental data. Molecular docking simulations, conducted using the AutoDock tool, reveal the binding affinity and interaction patterns of selected compounds with the HER2 receptor. Virtual screening results highlight compounds with high binding affinity, favorable interaction patterns, and structural compatibility as potential lead candidates. To ensure safety and efficacy, ADMETox filtering was employed, providing insights into the compound’s toxicity profile and pharmacokinetic attributes. The selected compound, Eriodictyol (C20H20O6), exhibits a generally favorable safety profile, with predicted inactivity across multiple toxicity classifications and endpoints. While immunotoxicity is predicted, the overall low probabilities suggest a relatively low risk. Physicochemical and pharmacokinetic assessments indicate Eriodictyol’s potential for drug development. With a molecular weight of 356.37 g/mol, balanced lipophilicity, and high gastrointestinal absorption, the compound aligns with drug-likeness criteria. However, careful consideration is warranted due to its inhibitory effects on certain enzymes and alerts for catechol_A and isolated_alkene. In conclusion, this integrated computational approach streamlines the identification of potential HER2 inhibitors, offering a systematic strategy for drug discovery. Eriodictyol emerges as a promising candidate, demonstrating a favorable safety profile and pharmacokinetic attributes, paving the way for further in-depth studies and development as a potential therapeutic agent for breast cancer.

Screening of Acetylcholinesterase Inhibitors by Capillary Electrophoresis with Oriented-Immobilized Enzyme Microreactors Based on Gold Nanoparticles.

A facial and efficient method for the screening of acetylcholinesterase (AChE) inhibitors by capillary electrophoresis was developed. Based on the specific affinity of concanavalin A (Con A) for binding to the glycosyl group of AChE, enzyme molecules were oriented-immobilized on the surface of gold nanoparticles (AuNPs@Con A@AChE). Then, these modified nanoparticles were bounded to the capillary inlet (about 1.0 cm) by electrostatic self-assembly to obtain the oriented-immobilized enzyme microreactor (OIMER). Compared to an IMER with a free enzyme, the peak area of the product obtained by the OIMER increased by 52.6%. The Michaelis-Menten constant (Km) was as low as (0.061 ± 0.003) mmol/L. The method exhibits good repeatability with a relative standard deviation (RSD) of 1.3% for 100 consecutive runs. The system was successfully applied to detect the IC50 values of donepezil and four components from Chinese medicinal plants. This work demonstrates the potential of this method as a low cost, simple, and accurate screening method for other enzyme inhibitors.

Ultra-highly selective recognition of nucleosides over nucleotides by rational modification of tetralactam macrocycle and its application in enzyme assay

Artificial macrocycle with high binding selectivity in water is often challenging but urgently needed in various research and application areas. Herein, we report a new water-soluble biomimetic tetralactam macrocycle and realize the ultra-high selectivity to nucleosides over corresponding monophosphate nucleotides by rational modification. The introduction of charged groups at the periphery of endo-functionalized cavity makes the selectivity (guanosine to guanosine 5′-monophosphate) increase remarkably from 100 to 1119. Based on the ultra-high selectivity of biomimetic tetralactam macrocycle, the sensitive CD73 enzyme activity assay was then achieved through product-selective fluorescence indicator displacement assay. Furthermore, the capability of the proposed method for inhibitor screening was successfully displayed.

Identification of new drug candidates against Trichomonas gallinae using high-throughput screening

Trichomonas gallinae is a protozoan parasite that is the causative agent of trichomoniasis, and infects captive and wild bird species throughout the world. Although metronidazole has been the drug of choice against trichomoniasis for decades, most Trichomonas gallinae strains have developed resistance. Therefore, drugs with new modes of action or targets are urgently needed. Here, we report the development and application of a cell-based CCK-8 method for the high-throughput screening and identification of new inhibitors of Trichomonas gallinae as a beginning point for the development of new treatments for trichomoniasis. We performed the high-throughput screening of 173 anti-parasitic compounds, and found 16 compounds that were potentially effective against Trichomonas gallinae. By measuring the median inhibitory concentration (IC50) and median cytotoxic concentration (CC50), we identified 3 potentially safe and effective compounds against Trichomonas gallinae: anisomycin, fumagillin, and MG132. In conclusion, this research successfully established a high-throughput screening method for compounds and identified 3 new safe and effective compounds against Trichomonas gallinae, providing a new treatment scheme for trichomoniasis.

Effects of Computer Virtual Screening and Treatment Based on Pharmacophore BRD4-Targeted Small-Molecule Inhibitors on NSE and CA19-9 Levels in Patients with Liver Cancer

Objective: Neuron-specific enolase (NSE) and carbohydrate antigen 19-9 (CA19-9) levels were compared before and after treatment with brd4-targeting small-molecule inhibitors based on pharmacophores and computer technology to investigate an efficient virtual screening method for bromodomain protein 4 (brd4)-targeted small-molecule inhibitors. The purpose of this research was to examine the efficacy of pharmacophore screening and BRD4 targeting by small-molecule inhibitors in the management of liver cancer. Methods: Subjects included 106 individuals with hepatocellular cancer who were hospitalised between July 2019 and December 2021. Using a receiver operating characteristic (ROC) curve, we assessed the clinical relevance and utility of pharmacophore-based virtual screening for brd4-targeted small-molecule inhibitors. The levels of the tumour markers NSE and CA19-9 were analysed for changes using computational methods. Results: The pharmacophore model was built using the HipHop technique, and its results were validated and optimised utilising computational technology analysis. Twenty-four small-molecule classes were covered by the pharmacophore, and the number of mismatched pharmacodynamic characteristics was 0. The first eight pharmacophores each had two clusters of hydrophobic atoms, a hydrogen bond receptor, and an aromatic ring at its core. There were no changes to the pharmacodynamic characteristics, although their locations varied. False-positive and true-positive rates are depicted on the horizontal and vertical axes, respectively, in the figure below; the other two pharmacophore groups contained a hydrophobic cluster, a hydrogen bond receptor, and the centre of an aromatic ring. The ROC curve was quite consistent, as indicated by an AUC value close to 1, which was between 0 and 1. For pharmacophores 07 and 06, the area under the curve (AUC) was 0.949 and 0.955, respectively, very near to 1. Both the hydrophobic cluster and the hydrogen bond receptor are required for a pharmacophore to exist. The examination of pharmacodynamic definitions revealed a positive correlation between database reliability and element type (P <0.05). Compared to pre-healing levels, post-healing CA19-9 and NSE levels were statistically considerably reduced (P < 0.05). Conclusions: The assessment approach was highly accurate and specific, and the pharmacophore-based virtual screening method and computer technology for brd4-targeting small-molecule inhibitors were of great benefit in the regulation of the cell cycle and the process of cell proliferation. Effective treatment modalities can be created to increase levels of tumour markers NSE and CA19-9, and pharmacophore and computational-based small-molecule inhibitors of brd4 can be employed as crucial virtual screening criteria for the evaluation of liver cancer.

A robust luminescent assay for screening alkyladenine DNA glycosylase inhibitors to overcome DNA repair and temozolomide drug resistance

Temozolomide (TMZ) is an anticancer agent used to treat glioblastoma, typically following radiation therapy and/or surgical resection. However, despite its effectiveness, at least 50% of patients do not respond to TMZ, which is associated with repair and/or tolerance of TMZ-induced DNA lesions. Studies have demonstrated that alkyladenine DNA glycosylase (AAG), an enzyme that triggers the base excision repair (BER) pathway by excising TMZ-induced N3-methyladenine (3meA) and N7-methylguanine lesions, is overexpressed in glioblastoma tissues compared to normal tissues. Therefore, it is essential to develop a rapid and efficient screening method for AAG inhibitors to overcome TMZ resistance in glioblastomas. Herein, we report a robust time-resolved photoluminescence platform for identifying AAG inhibitors with improved sensitivity compared to conventional steady-state spectroscopic methods. As a proof-of-concept, this assay was used to screen 1440 food and drug administration-approved drugs against AAG, resulting in the repurposing of sunitinib as a potential AAG inhibitor. Sunitinib restored glioblastoma (GBM) cancer cell sensitivity to TMZ, inhibited GBM cell proliferation and stem cell characteristics, and induced GBM cell cycle arrest. Overall, this strategy offers a new method for the rapid identification of small-molecule inhibitors of BER enzyme activities that can prevent false negatives due to a fluorescent background.

High-throughput screening of lipase inhibitors as anti-obesity drugs on liquid crystal-infused porous surfaces

High-throughput screening of lipase inhibitors is critical for developing anti-obesity drugs. Herein, a robust and versatile sensing platform based on the difference in the sliding time of droplets on liquid crystal-infused porous surfaces (LCIPS) is developed to detect lipase inhibitors. Lipase catalyzes glycerol trioleate (GT) to produce oleate that can adsorb at the solution/liquid crystal (LC) interface due to its high amphiphilicity, which impedes the sliding of droplets on LCIPS, resulting in an extended sliding time. However, in the presence of lipase inhibitors, the enzymatic hydrolysis of GT by lipase is inhibited, which shortens the sliding time. The screening of three lipase inhibitors, orlistat, cetilistat, and epigallocatechin gallate (EGCG), popular anti-obesity drugs, is demonstrated to have IC50 values of 17.45 ± 3.15 nM, 72.15 ± 6.32 nM, and 2.19 ± 0.24 μM, respectively. This study provides a simple, inexpensive, portable, user-friendly, and instrument-free method for rapid, accurate, and high-throughput screening of lipase inhibitors. The LCIPS-based sensing platform is up-and-coming in developing advanced bioanalytical sensors.

Label-Free Detection of T4 Polynucleotide Kinase Activity and Inhibition via Malachite Green Aptamer Generated from Ligation-Triggered Transcription.

Polynucleotide kinase (PNK) is a key enzyme that is necessary for ligation-based DNA repair. The activity assay and inhibitor screening for PNK may contribute to the prediction and improvement of tumor treatment sensitivity, respectively. Herein, we developed a simple, low-background, and label-free method for both T4 PNK activity detection and inhibitor screening by combining a designed ligation-triggered T7 transcriptional amplification system and a crafty light-up malachite green aptamer. Moreover, this method successfully detected PNK activity in the complex biological matrix with satisfactory outcomes, indicating its great potential in clinical practice.