Enzymes are closely associated with the onset and progression of numerous diseases, making enzymes a primary target in innovative drug development. However, the challenge remains in identifying compounds that exhibit potent inhibitory effects on the target enzymes. With the continuous expansion of the total number of natural products and increasing difficulty in isolating and enriching new compounds, traditional high-throughput screening methods are finding it increasingly challenging to meet the demands of new drug development. Virtual screening, characterized by its high efficiency and low cost, has gradually become an indispensable technology in drug development. It represents a prominent example of the integration of artificial intelligence with biopharmaceuticals and is an inevitable trend in the rapid development of innovative drug screening in the future. Therefore, this article primarily focused on systematically reviewing the recent applications of virtual screening technology in the development of enzyme inhibitors and explored the prospects and advantages of using this technology in developing new drugs, aiming to provide essential theoretical insights and references for the application of related technologies in the field of new drug development.
Artificial Intelligence ; Enzyme Inhibitors/pharmacology* ; High-Throughput Screening Assays ; Molecular Docking Simulation

In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.
Carcinoma, Non-Small-Cell Lung ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; High-Throughput Screening Assays ; Humans ; Lung Neoplasms ; Transcription Factor 4/genetics* ; beta Catenin/genetics*

Salivary alpha-amylase (sAA) is a stress biomarker in human diseases, but there are no reports of sAA measurements in diseased dogs. This study measured the sAA and serum alpha-amylase (AA) levels in 16 healthy dogs and 31 diseased dogs using a kinetic enzyme assay to assess the stress status. The sAA and serum AA levels were significantly higher in the diseased dogs than in healthy dogs (p < 0.05), but there was no correlation between the 2 groups (r = 0.251, p = 0.089). This suggests that sAA can be useful as a stress biomarker in diseased dogs.
alpha-Amylases ; Animals ; Dogs ; Enzyme Assays ; Humans ; Saliva

BACKGROUND: Low temperature plasma (LTP) was recently shown to be potentially useful for biomedical applications such as bleeding cessation, cancer treatment, and wound healing, among others. Keratinocytes are a major cell type that migrates directionally into the wound bed, and their proliferation leads to complete wound closure during the cutaneous repair/regeneration process. However, the beneficial effects of LTP on human keratinocytes have not been well studied. Therefore, we investigated migration, growth factor production, and cytokine secretion in primary human keratinocytes after LTP treatment.METHODS: Primary cultured keratinocytes were obtained from human skin biopsies. Cell viability was measured with the EZ-Cytox cell viability assay, cell migration was evaluated by an in vitro wound healing assay, gene expression was analyzed by quantitative real-time polymerase chain reaction, and protein expression was measured by enzyme-linked immunosorbent assays and western blotting after LTP treatment.RESULTS: Cell migration, the secretion of several cytokines, and gene and protein levels of angiogenic growth factors increased in LTP-treated human keratinocytes without associated cell toxicity. LTP treatment also significantly induced the expression of hypoxia inducible factor-1α (HIF-1α), an upstream regulator of angiogenesis. Further, the inhibition of HIF-1α expression blocked the production of angiogenic growth factors induced by LTP in human keratinocytes.CONCLUSION: Our results suggest that LTP treatment is an effective approach to modulate wound healing-related molecules in epidermal keratinocytes and might promote angiogenesis, leading to improved wound healing.
Anoxia ; Biopsy ; Blotting, Western ; Cell Migration Assays ; Cell Movement ; Cell Survival ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Hemorrhage ; Humans ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Keratinocytes ; Plasma ; Real-Time Polymerase Chain Reaction ; Skin ; Wound Healing ; Wounds and Injuries

To develop an enzyme-linked immunosorbent assay (ELISA)-based high throughput screening (HTS) method for β-catenin/Lef1 interaction antagonists screening, Escherichia coli Rosetta (DE3) competent cells were transformed with β-catenin-pET-30a(+) plasmid. β-catenin protein was expressed after induction and purified using affinity chromatography. The biological activity of purified β-catenin was further analyzed by GST Pulldown assay. The β-catenin/GST-Lef1 binding model was established using ELISA principle, and the ELISA-based HTS method was further optimized through determination of an optimal coated concentration of GST-Lef1 and working concentration of β-catenin. The results showed that β-catenin protein was successfully expressed and purified. The GST Pulldown assay demonstrated a perfect biological activity for purified β-catenin. Subsequently, the ELISA-based HTS method was performed using 10 μg/mL GST-Lef1 and 6 μg/mL β-catenin, with the Z factor of 0.76. Taken together, we have successfully developed a simple, robust and reliable ELISA-based HTS method for screening of novel Wnt inhibitors targeting β-catenin/Lef1 interaction.
Antineoplastic Agents ; Enzyme-Linked Immunosorbent Assay ; High-Throughput Screening Assays ; Lymphoid Enhancer-Binding Factor 1 ; beta Catenin

An indirect competitive enzyme-linked immunosorbent assay( ic-ELISA) was developed for the rapid detection of ochratoxin A( OTA) in nutmeg( Myristicae Semen),ginger( Zingiberis Rhizoma) and turmeric( Curcumae Longae Rhizoma). The matrix matching standard curve was used instead of the standard curve of sample diluent,and the sample extract and sample diluent were optimized. The sensitivity( IC_(50)) of this method for OTA in nutmeg,ginger and turmeric were determined as 0. 146,0. 157 and 0. 153 ng·m L~(-1),respectively and the limits of detection( LODs) were 0. 040,0. 032 and 0. 031 ng·m L~(-1),respectively. The recovery of samples ranged from 75. 99% to 122. 3%,with RSD<10%. Two positive samples for nutmeg and one positive sample for turmeric occurred in 50 samples,and the highest OTA contamination value was 1 167. 8 μg·kg~(-1). The results were further confirmed by LC-MS/MS. It shows that the developed ic-ELISA method is simple,rapid and sensitive,and can be applied for rapid and high-throughput screening of OTA in nutmeg,ginger and turmeric,as well as some other CHMs.
Chromatography, Liquid ; Drug Contamination ; Drugs, Chinese Herbal/analysis* ; Enzyme-Linked Immunosorbent Assay ; High-Throughput Screening Assays ; Ochratoxins/analysis* ; Tandem Mass Spectrometry

PURPOSE: To evaluate the classification, diagnosis, and natural course of ophthalmoplegia associated with mitochondrial disease. MATERIALS AND METHODS: Among 372 patients with mitochondrial disease who visited our hospital between January 2006 and January 2016, 21 patients with ophthalmoplegia were retrospectively identified. Inclusion criteria included onset before 20 years of age, pigmentary retinopathy, and cardiac involvement. The 16 patients who were finally included in the study were divided into three groups according to disease type: Kearns-Sayre syndrome (KSS), KSS-like, and chronic progressive external ophthalmoplegia (CPEO). RESULTS: The prevalences of clinical findings were as follows: ptosis and retinopathy, both over 80%; myopathy, including extraocular muscles, 75%; lactic acidosis, 71%; and elevated levels of serum creatine kinase, 47%. Half of the patients had normal magnetic resonance imaging findings. A biochemical enzyme assay revealed mitochondrial respiratory chain complex I defect as the most common (50%). The prevalence of abnormal muscle findings in light or electron microscopic examinations was 50% each, while that of large-scale mitochondrial DNA (mtDNA) deletions in a gene study was 25%. We compared the KSS and KSS-like groups with the CPEO patient group, which showed pigmentary retinopathy (p < 0.001), cardiac conduction disease (p=0.013), and large-scale mtDNA deletions (p=0.038). KSS and KSS-like groups also had gastrointestinal tract disorders such as abnormal gastrointestinal motility (p=0.013) unlike the CPEO group. CONCLUSION: Patients with KSS had gastrointestinal symptoms, which may indicate another aspect of systemic involvement. The presence of large-scale mtDNA deletions was an objective diagnostic factor for KSS and a gene study may be helpful for evaluating patients with KSS.
Acidosis, Lactic ; Classification ; Creatine Kinase ; Diagnosis ; DNA, Mitochondrial ; Electron Transport ; Enzyme Assays ; Gastrointestinal Motility ; Gastrointestinal Tract ; Genes, vif ; Humans ; Kearns-Sayre Syndrome ; Magnetic Resonance Imaging ; Mitochondrial Diseases* ; Muscles ; Muscular Diseases ; Ophthalmoplegia* ; Ophthalmoplegia, Chronic Progressive External ; Prevalence ; Retinitis Pigmentosa ; Retrospective Studies

BACKGROUND AND PURPOSE: The pathogenesis of mitochondrial disease (MD) involves the disruption of cellular energy metabolism, which results from defects in the mitochondrial respiratory chain complex (MRC). We investigated whether infants with MRC I defects showed ultrastructural changes in skeletal muscle. METHODS: Twelve infants were enrolled in this study. They were initially evaluated for unexplained neurodegenerative symptoms, myopathies, or other progressive multiorgan involvement, and underwent muscle biopsies when MD was suspected. Muscle tissue samples were subjected to biochemical enzyme assays and observation by transmission electron microscopy. We compared and analyzed the ultrastructure of skeletal muscle tissues obtained from patients with and without MRC I defects. RESULTS: Biochemical enzyme assays confirmed the presence of MRC I defects in 7 of the 12 patients. Larger mitochondria, lipid droplets, and fused structures between the outer mitochondrial membrane and lipid droplets were observed in the skeletal muscles of patients with MRC I defects. CONCLUSIONS: Mitochondrial functional defects in MRC I disrupt certain activities related to adenosine triphosphate synthesis that produce changes in the skeletal muscle. The ultrastructural changes observed in the infants in this study might serve as unique markers for the detection of MD.
Adenosine Triphosphate ; Biopsy ; Electron Transport* ; Energy Metabolism ; Enzyme Assays ; Humans ; Infant* ; Lipid Droplets ; Microscopy, Electron, Transmission ; Mitochondria ; Mitochondrial Diseases ; Mitochondrial Membranes ; Muscle, Skeletal* ; Muscular Diseases

PURPOSE: To report ocular findings of a mucolipidosis type II patient with novel mutation. CASE SUMMARY: A 10-year-old boy visited our pediatric genetic metabolic clinic for evaluation of his overall developmental delay and short stature. The boy was diagnosed with mucolipidosis type II (I-cell disease) using plasma enzyme assay and DNA sequencing of the GNPTAB gene mutation. An ophthalmologic investigation was then performed, and a depressed nasal bridge, broad nose, and swelling in the upper lid of both eyes were noted. The best corrected visual acuity was 0.32 and 0.1 and the intraocular pressure was 35 mmHg and 24 mmHg in the right and left eyes, respectively. The anterior chamber angles of both eyes were normal and mild cornea opacity in both eyes was observed. Fundus examination revealed retinal atrophy with folds in both eyes, as well as optic disc edema and optic atrophy in the right and left eyes, respectively. Atherosclerotic changes in the retinal vessels and cystoid macular edema in the left eye were observed, and ocular ultrasound revealed increased posterior sclera thickness in both eyes. CONCLUSIONS: Ocular manifestations of mucolipidosis type II are not currently well-known, and differentiation from other metabolic disorders may be difficult. An ophthalmic work-up can assist in diagnosis, and regular ophthalmic examinations should be used to maintain visual function in mucolipidosis patients.
Anterior Chamber ; Atrophy ; Child ; Cornea ; Diagnosis ; Edema ; Enzyme Assays ; Humans ; Intraocular Pressure ; Lysosomal Storage Diseases ; Macular Edema ; Male ; Mucolipidoses* ; Nose ; Optic Atrophy ; Plasma ; Retinal Vessels ; Retinaldehyde ; Sclera ; Sequence Analysis, DNA ; Ultrasonography ; Visual Acuity

No abstract available.
Aged, 80 and over ; Ascorbate Oxidase/*metabolism ; Ascorbic Acid/administration & dosage/blood/*chemistry ; Breast Neoplasms/pathology ; Cholesterol/*blood ; *Colorimetry ; Enzyme Assays ; Female ; Humans ; Injections, Intravenous ; Intestine, Small/surgery ; Kidney/physiopathology ; Male ; Middle Aged ; Palliative Care ; Recurrence