The pharmaceutical research and development sector has been confronted with the challenges of protracted timelines (10-15 years), substantial costs (approximately $2.6 billion), and low success rates (with clinical success rates below 10%). In recent years, artificial intelligence (AI) technologies such as deep learning, natural language processing, and large language models have been reshaping the entire drug design process through a data-driven, model-based, and computationally intensive “AI for Science” paradigm, thereby promoting the rapid advancement of AI-assisted drug design (AIDD). This paper summaries the key technologies, core applications, challenges, and limitations of AI in transforming drug design, and offers a perspective on the future directions and opportunities for AI-led drug design.
Objective: To investigate the sex disparity of general transcription factor II subunit 2 (GTF2H2) in regulating the role of receptor-type protein tyrosine phosphatase O (PTPRO) in hepatocellular carcinoma (HCC). Methods: The expression of GTF2H2 in liver cancer tissue microarrays was detected by immunohistochemistry, and its correlation with the malignancy of the tumor was analyzed. The regulatory effect of estrogen receptor (ERα) expression on GTF2H2 was tested by Western blot. The effect of estrogen at different concentrations on PTPRO expression in HCC lines was examined by Western blot. The number of tumors in male and female spontaneous liver cancer models was observed, and the PTPRO expression was detected. The role of GTF2H2 in the regulation of estrogen-mediated PTPRO was analyzed by constructing a GTF2H2 knockdown HCC cell model. The HCC cell model with stable GTF2H2 expression was constructed, and the expression and localization of PTPRO was observed by Western blot and immunofluorescence. Results: The liver cancer tissue microarrays shows that the expression levels of GTF2H2 in liver cancer tissues were lower than those in adjacent tissues, and this was inversely proportional to the malignancy of the tumor. The in vitro study showed that the expression of ERα increased the level of GTF2H2. Estrogen upregulated the expression of PTPRO in HCC in a gradient manner. In the spontaneous liver cancer mouse model, the number of tumors in male mice was significantly increased compared to female mice, and the expression of PTPRO was relatively reduced. The upregulation of PTPRO by estrogen was significantly inhibited in GTF2H2 knockdown HCC cells. Overexpression of GTF2H2 resulted in up-regulation of PTPRO expression and an increase in membrane localization. Conclusion: GTF2H2 participates in the gender difference of HCC by mediating the upregulation of PTPRO expression.
Objective: Vaccination represents the most cost-effective strategy for the prevention and control of Influenza A virus (IAV). This study aimed to develop a multi-epitope vaccine targeting conserved epitopes to activate both humoral and cellular immunity synergistically. Methods: Bioinformatics prediction tools were employed to screen antigenic epitopes for constructing the multi-epitope antigen Flu A, which was fused with flagellin adjuvant to form the fusion protein Flu A-ND. The immunogenicity of the multi-epitope antigens was evaluated using ELISA, splenic lymphocyte proliferation assays, cytokine detection, and flow cytometry. Results: Bioinformatics analysis identified conserved epitopes in the following proteins: HA2 (12-53, 76-130, 113-131), M2e (1-24), and NP (5-47, 304-320, 392-407, 418-426) for the construction of the multi-epitope antigen Flu A. The Flu A-ND group demonstrated a serum IgG titer of 1∶256 000, representing a twofold increase compared to the Flu A group (1∶128 000), with significantly elevated mucosal IgA levels. Assessment of cellular immunity revealed enhanced secretion of Th1-type cytokines in the Flu A-ND group, accompanied by increased proportions of CD4+ and CD8+ T cells compared to the Flu A group. The flagellin adjuvant significantly enhanced T-cell activation. Conclusions:This study successfully developed a multi-epitope antigen, which demonstrated robust humoral and cellular immune responses in mice. The study has demonstrated that Flagellin, functioning as an intramolecular adjuvant, significantly improved immunogenicity. This finding provides critical experimental evidence for the development of safe and effective multi-epitope vaccines against IAV.
Fresh fly maggots are characterised by a high protein substrate content, which makes them a high quality raw material for the preparation of active small molecule peptides. In the study, we used alcalase proteinase for the enzyme digestion of fresh fly maggots to obtain fly maggot hydrolysate and explored the antibacterial and antitumor activities of the hydrolysate. Then, the active components were isolated from the hydrolysate by means of ultrafiltration membrane and dextran gel column chromatography and the peptide sequences of the active components were identified by LC-MS/MS. Finally, we screened out potential bioactive peptides by combining with a bioinformatics prediction method and verified their antibacterial, antimicrobial, antioxidant and antitumor activities. The results showed that, the fly maggot alcalase proteinase hydrolysate exhibited specific antibacterial activity and significant antitumor activity. The IC50 values for inhibiting the proliferation of colorectal and gastric cancer cells following a 72-hour cancer cell culture were 0.14 mg/mL and 0.18 mg/mL, respectively. Following isolation and characterization, a total of 58 peptide sequences were identified from the active fractions of the hydrolysate. These peptides were predominantly oligopeptides and were primarily derived from aryl storage proteins. Through bioinformatics prediction combined with activity validation, a novel bioactive peptide from fly maggot with promising antibacterial and antitumor activities for RPPFYH (Arg-Pro-Pro-Phe-Tyr-His), and a novel bioactive peptide with favorable antioxidant activity and anti-tumor activity for HELPYPW (His-Glu-Leu-Pro-Tyr-Pro-Trp) were screened. Among them, the active peptide RPPFYH inhibited the growth of five animal pathogens, with minimal inhibitory concentrations (MICs) ranging from 0.5 to 2 mg/mL, and the IC50 of 2.83 mg/mL and 4.79 mg/mL against the proliferation of colorectal and gastric cancer cells, respectively. It significantly inhibited the migration of the cancer cells within a specific concentration range. The active peptide HELPYPW demonstrated effective scavenging properties against DPPH radicals and ABTS radicals, with IC50 values of 1.15 mg/mL and 0.26 mg/mL, respectively. Additionally, it exhibited significant inhibitory effects on the proliferation of colorectal and gastric cancer cells, with IC50 values of 2.03 mg/mL and 0.89 mg/mL, respectively. It demonstrated a substantial inhibition of cancer cell migration within a specific concentration range. The study provides a theoretical basis for the development and utilization of small molecule active peptides from fly maggots.
Prednisolone, a glucocorticoid drug widely employed in the treatment of rheumatoid arthritis, systemic lupus erythematosus, and related inflammatory diseases, is conventionally synthesized through chemical methods. However, these methods are characterized by high environmental pollution and harsh reaction conditions. In contrast, biocatalytic approaches utilizing 3-ketosteroid-Δ1-dehydrogenase (KstD) offer a sustainable and efficient alternative. However, the industrial-scale application of KstD-mediated bioconversion is hindered by limitations in enzyme catalytic efficiency, which arises from suboptimal gene expression levels, transcriptional regulation, and fermentation process parameters. In this study, the KstD1 gene from Mycobacterium sp. LY-1 was heterologously expressed in Bacillus subtilis WB600, and its transcriptional regulation and fermentation process were optimized. The results demonstrated that, based on the successful heterologous expression of KstD1, the optimal promoter and ribosome binding site (RBS) were identified as P566 and R9A-4, respectively. Compared to the control strain, the recombinant strains exhibited a 14.8% and 51.2% increase in prednisone production, respectively. Subsequent fermentation process optimization focuses on the enhancement of substrate solubility, nutrient availability, and redox cofactor supply. Tween 80, when supplemented at 7% (V/V), has been shown to effectively solubilize the hydrophobic substrate hydrocortisone while maintaining cell viability. A statistically optimized medium formulation comprising 10 g/L maltose, 35.4 g/L beef extract, 9.4 g/L K2HPO4, and 2.2 g/L KH2PO4 has been established to support robust microbial growth and bioconversion activity. Furthermore, the addition of 0.1 mmol/L phenazine methosulfate (PMS) as an artificial electron acceptor has been shown to substantially improve reaction kinetics by facilitating redox cofactor regeneration, thereby alleviating metabolic bottlenecks in the dehydrogenation process. Under optimized conditions with an initial hydrocortisone concentration of 2 g/L, the recombinant B. subtilis strain produced 1 815.7 mg/L prednisolone, a 113.7% increase over the control. This work laid the a foundation for the industrial-scale production of prednisolone via engineered B. subtilis, integrating genetic optimization with process intensification strategies.
Objective: To overcome the drawbacks associated with free epoxide hydrolase or whole cells, such as poor stability and the difficulties involved in separation and recovery during catalysis, highly stable and reusable catalytic microspheres have been developed for the preparation of (S)-styrene oxide via chiral resolution. Methods: Firstly, the most suitable immobilization material was selected from a range of five different sodium alginate-based carriers, and single-factor experiments were then conducted to optimize the immobilization conditions. The microsphere structure was characterized by scanning electron microscopy, and thermal stability was determined. Secondly, the high-concentration racemic styrene oxide was resolved. Finally, the reusability of the prospoed mehtod was evaluated. Results: Sodium alginate was selected as the optimal carrier. The single-factor experiment indicated that the optimal conditions were as follows: a calcification time of 8 h, and concentrations of calcium chloride, bacteria and sodium alginate of 20 mg/mL, 60 mg/mL and 20 mg/mL, respectively. The immobilized microspheres exhibited a specific activity of 24.59 U/g cells. The half-life of the cells at 40℃ was found to be 3.80 h, which is 1.73 times longer than that of the free cells. Chiral resolution of 500 mmol/L racemic styrene oxide in an n-octane/water biphasic system yielded (S)-styrene oxide within 6 h, with an enantiomeric excess value greater than 99.5% and a space-time yield of 2.146 g/ (L·h). The microspheres retained 80.60% of initial activity following 6 cycles. Conclusion: This study successfully established a catalytic system based on sodium alginate-immobilized recombinant E. coli/sfeh3 whole cells, achieving efficient chiral resolution of high-concentration racemic styrene oxide. This provides a green and economical biocatalytic strategy for the preparation of chiral epoxides.
Objective: The objective of this study is twofold: firstly, to enhance glucose uptake in the high-yielding L-arginine strain, and secondly, to investigate an economically viable mixed-carbon-source utilization approach. Methods: By enhancing the PTS and IPGS systems, the strain’s sugar consumption capacity was improved. We used a mixed carbon source of glucose and sucrose to optimize utilization efficiency, and introduced a heterologous fructokinase to improve metabolism of mixed sugars. Results: Compared to the initial bacteria, the engineered strain with modified sugar transport systems and optimized utilization of the carbon source showed a 31.80% increase in arginine production. Conclusion: The combined approach of sugar transporter optimization and mixed-sugar utilization significantly improved the yield of L-arginine, providing valuable insights for the modification and application of industrial strains.
Objective: Converting CO2-derived substrates into valuable chemicals through microbial cell factories provides a viable, carbon-neutral pathway for biorefineries. Ethanol can be obtained through anaerobic syngas fermentation and electrocatalytic CO2 fixation and itaconic acid, an important chemical, can be synthesized through ethanol metabolism. Therefore, this study is the first to produce itaconic acid using engineered Escherichia coli and ethanol as the sole carbon source, providing a technical reference for exploring the production of itaconic acid using third-generation biorefinery substrates derived from CO2. Methods: The main research methods included the following: (1) Expression of the AdhEA267T/E568K mutant of the endogenous ethanol dehydrogenase (AdhE*) allows E.coli to grow in a medium with ethanol as the sole carbon source. (2) We enabled E. coli to metabolize ethanol and synthesize itaconic acid by expressing cis-aconitic acid decarboxylase (CAD) through three promoters with different expression strengths. (3) We reduced the production of the by-products acetate and lactate to increase the production of itaconic acid. (4) We increased the production of itaconic acid further by using fed-batch fermentation. Results: An engineered E. coli strain was constructed for the efficient production of itaconic acid, with a yield of 4.2 g/L. This was achieved by expressing AdhE* and CAD, and by reducing the synthesis of the by-products acetate and lactate. Conclusion: The efficient metabolism of ethanol for itaconic acid production can be achieved by engineered E. coli, providing a technological reference for the production of itaconic acid using CO2-derived third-generation biorefinery substrates.
Alzheimer’s disease (AD) is a common neurodegenerative disease, with typical pathological hallmarks of β-amyloid protein (Aβ) deposits and abnormal tau protein phosphorylation. Apolipoprotein E (APOE) gene has been identified as the strongest genetic risk factor for AD, with APOE4 having a significantl impact on disease risk. The technology of induced pluripotent stem cells (iPSCs) overcomes the limitations of traditional animal models by reprogramming patient somatic cells into pluripotent stem cells and directing them to differentiate into neurons and glial cells. It can preserve the patient’s genetic background and simulate complex cell-to-cell interactions, showing great potential in exploring the pathological mechanism of APOE4 in Alzheimer’s disease. The latest progress in iPSCs technology in APOE4-related AD research has attracted attention, and its application value is high in analyzing the pathogenesis of APOE4. Using iPSC-derived neuron and glial cell models, related studies have elucidated the multifaceted mechanisms of action of APOE4 in Aβ metabolism, tau pathology, and neuroinflammation, and verified the therapeutic potential of targeting APOE4 through CRISPR gene editing technology. In addition, the multicellular interaction model provides a breakthrough platform for analyzing the role of APOE4 in dynamic interactions between cells, revealing the pathological amplification effect between neurons and glial cells. Although the iPSC model still faces challenges in simulating the complexity and pathological mechanisms of the disease, continued optimization is expected to make it a more accurate tool for studying the mechanism of APOE4 in AD. By summarizing the advantages and limitations of iPSC technology and disucssing future research directions, this study provides new ideas for in-depth understanding of the mechanism of APOE4 in AD and promotes the development of treatment strategies.
The rapid and accurate detection of animal pathogens is of great significance for maintaining animal health, preventing disease transmission and ensuring food safety. Electrochemical immunoassay (ECIA), an emerging bioassay technology, has shown significant application potential in the detection of animal pathogens by combining the advantages of ECIA with the specificity of antigen-antibody recognition in immunology and highly sensitive signal transduction in the electrochemical field. This paper presents a systematic review of the technical principles, methodological classification and signal amplification strategies of ECIA, and the latest developments in the application of ECIA in animal disease detection are discussed. The development prospects of ECIA are also presented to provide theoretical and technical references for the construction of an efficient, portable and low-cost next-generation animal disease monitoring platform.
As a cyclic sesquiterpene compound with a three-dimensional conjugated double bond structure, farnesene has demonstrated diverse application scenarios in the fields of clean energy, biomedicine, and new materials. The advancement of the global carbon neutrality strategy has resulted in a marked increase in market demand for farnesene, thereby giving rise to a significant contradiction with the technical limitations of traditional production methods. The extraction process from plant sources is constrained by the low content of the target component in natural raw materials and the periodic fluctuations in raw material supply. Moreover, despite the chemical synthesis route’s capacity for large-scale production, it involves complex side reaction systems and ecological toxicity risks when using petroleum-derived isoprene as the raw material. This complicates the fulfilment of sustainable development requirements. Against this backdrop, synthetic biology technology, by reconstructing the genetic circuits of microbial metabolic mechanisms, offers a disruptive solution for establishing an environmentally friendly biomanufacturing system. Although initial engineering modifications have confirmed the technical feasibility of microbial farnesene synthesis, the strain production capacity is generally below 10 g/L, with the fundamental constraints being the shortage of precursor substances, metabolic bypass competition, and low efficiency of enzymatic reactions. To overcome these limitations, there has been a shift in the research paradigm from single-target optimization to system-level design in recent years. Through the multi-level synergy of genetic modification of host strains, reconstruction of metabolic pathways, and directed evolution of enzyme molecules, the product concentration has been successfully increased to over 130 g/L. This review systematically summarizes the key progress in the field from the perspective of metabolic engineering. Firstly, we analyze the metabolic characteristics and modification strategies of different microbial chassis, comparing their performance differences in precursor flux regulation and product tolerance. Secondly, we explore the optimization paths of the isoprene unit synthesis module, with a focus on the innovative application of dynamic regulation technology in balancing metabolic flux distribution. Finally, we focus on the rational design of farnesene synthase, revealing the intrinsic connection between the evolution of enzyme molecular domains and the improvement of catalytic performance. In response to the core challenges existing in the current technical system, including the limitations of enzyme catalytic kinetics, the cytotoxicity of intermediate metabolites, and the cost of large-scale production, this paper proposes three groundbreaking paths. Firstly, subcellular structure-directed localization strategies should be developed to utilize the ultra-high precursor concentration in peroxisomes and other compartments to enhance reaction efficiency. Secondly, the construction of dynamic regulation networks is imperative to achieve NADPH/ATP cofactor homeostasis and optimize carbon source conversion rates through the process of energy metabolism reprogramming. Thirdly, there is a need to integrate artificial intelligence-driven enzyme structure-activity relationship analysis and high-throughput screening platforms should be integrated to accelerate the discovery process of high-performance mutants. These solutions not only provide theoretical support for industrial production but also mark the transformation of terpene biosynthesis from a trial-and-error empirical process to a computationally-driven intelligent design paradigm. This paradigm has significant guiding value for the promotion of the iteration of green biomanufacturing technology.
Nonribosomal peptide synthetases (NRPSs), characterized by their modular and linear organization, synthesize a vast array of structurally complex and functionally diverse peptide-based natural products. The adenylation (A) domain functions as the “initiation switch” of each module and serves as the core unit for substrate recognition and activation. Therefore, elucidating its structure-activity relationships and enabling its rational engineering are essential for expanding the chemical diversity of nonribosomal peptides. This review highlights the dynamic catalytic mechanisms of A domains and their substrate-specific recognition, and summarizes current engineering strategies, such as site-directed mutagenesis, subdomain substitution or insertion, and domain recombination. These approaches have significantly increased the potential for the biosynthesis of non-natural peptides, which could overcome the inherent substrate limitations of native A domains. However, despite this progress, challenges such as poor module compatibility persist. This work provides theoretical insights and practical strategies for the rational design and artificial reprogramming of NRPS systems.
