Hematopoietic progenitor kinase 1 (HPK1) is an intracellular negative regulator of immune responses, particularly in T cell receptor (TCR) signaling. Compelling genetic evidence indicates that HPK1 impairs multiple stages of antitumor immunity largely via its kinase activity, thereby positioning it as a promising target for cancer immunotherapy. Starting from two 3-aminopyrazole hits identified through in-house screening, we initiated a structure-guided medicinal chemistry campaign. Through multi-stage optimization to enhance potency by engaging the Asp101 residue and to improve kinase selectivity via molecular hybridization, we developed a novel series of 1H-pyrazolo[3,4-c]pyridin-3-amine derivatives, from which D5 emerged as a key representative. Compound D5 exhibited potent HPK1 inhibitory activity (IC50 = 26.3 nM), and inhibited SLP76 phosphorylation and promoted IL-2 secretion in cell-based assays. Importantly, D5 showed favorable selectivity across diversity and immune-focused kinase panels, representing a marked improvement over its earlier analogue C4. Furthermore, D5 significantly suppressed tumor growth in the CT26 syngeneic mouse model without causing observable body weight loss. Overall, this work provides both a promising lead and a novel chemical scaffold, offering valuable insights and a concrete starting point for HPK1-targeted drug discovery.

Protein-based probes constructed via genetically encoding acetyl lysine (AcK) or its close analogs represent an important way to detect protein lysine deacetylases. Existing reported probes exhibit excellent sensitivity to NAD+-dependent sirtuins but lack responsiveness to Zn2+-dependent histone deacetylases (HDACs). Herein, we reformed the probe design by replacing the genetically encoded AcK with trifluoroacetyl lysine (TfAcK) and generated fluorescent and bioluminescent probes that could respond specifically to HDAC8 recombinantly expressed in E. coli and to endogenous HDACs in mammalian cells. We believe these probes would benefit the biological investigation of HDAC8 and promisingly some other HDACs, as well as the discovery of innovative HDAC inhibitors.

Isoaspartate (isoAsp) is a β‐linked residue in proteins spontaneously generated through Asn deamidation or Asp dehydration and significantly affects protein properties. However, the sluggish and site‐nonselective generation of isoAsp residues in proteins severely impedes in‐depth biological investigations as well as the exploitation of its unique β‐linkage features. Herein, we introduce a method that allows site‐selective and rapid generation of isoAsp residues in proteins. This method leverages the genetic incorporation of a side‐chain‐esterified Asp derivative (BnD), which undergoes facile intramolecular arrangement to form the key intermediate, aspartyl succinimide (Suc); subsequent hydrolysis of Suc gives rise to isoAsp as the major product. On native sites of proteins, including Cu/Zn superoxide dismutase and calmodulin, we demonstrate that BnD‐mediated isoAsp formation is faster than Asn deamidation generally by three orders of magnitude.

Fried centipede, a popular food in southern China and Vietnam, is well known for its notable health benefits owing to its unique chemical composition. This study focused on acetylcholinesterase (AChE) inhibitor screening, bioactivity evaluation, and the extraction of bioactive compounds derived from fried centipedes. To determine the accuracy of in vitro AChE inhibitor screening, receptor-ligand affinity ultrafiltration and enzymatic reaction kinetics technologies were employed for the rapid screening and structural identification of bioactive compounds and to verify the enzyme inhibition activity and mechanism of the compounds. Moreover, molecular docking and dynamic simulations were utilized to assess the effectiveness of the target, and the integration of experimental and computational approaches facilitated a comprehensive analysis of the active compounds and their activities at four levels. To address the issues of low content and low extraction and separation efficiencies of bioactive compounds derived from centipedes, an activity-oriented complex preparation method (consecutive ultrasonicassisted centrifugal extraction coupled online with UNIFAC countercurrent chromatography and semi-highperformance liquid chromatography (HPLC) was developed for efficient extraction, online concentration, and complex separation of the identified AChE ligands. As a result, six active compounds, including cytidine, guanine, uracil, hypoxanthine, xanthine, and beta -thymidine demonstrated significant binding affinity to the AChE active sites within 3 h. This study provided valuable insights into the active compounds of fried centipedes and their potential anti-Alzheimer ' s disease properties. These findings contribute to the advancement of animal food preparation methodologies by combining cutting-edge extraction techniques, biological activity screening, and the development of AChE inhibitors from fried centipedes. Our method serves as a valuable reference for exploring and developing functional animal materials from diverse medicinal resources.

Metal-organic framework materials (MOFs) and their derivatives have been widely used in the field of photocatalytic water decomposition for hydrogen production. In this study, NiS/CdS was initially acquired and subsequently combined with DUT-67 via ultrasound to create a unique ternary photocatalyst NiS/CdS@DUT-67. The rate of hydrogen production for NiS/CdS@DUT-67 is 9618 mu molg (-1)(NiS/CdS)h(-1) for NiS/CdS@DUT-67, which is 32 times and 2.5 times higher than that for CdS and NiS/CdS, respectively. Of particular interest is the fact that even after 50 h of photocatalysis, the hydrogen production rate did not show a significant decrease, demonstrating its excellent stability compared to CdS and NiS/CdS. In this ternary system, NiS and DUT-67 function as dual co-catalysts for CdS, collaborating to enhance charge separation during the photocatalysis. This study presents a clear demonstration of the advantages of utilizing metal-organic framework derivatives (MOF-derivatives) cophotocatalysts and their synergistic effect, resulting in improved photocatalytic activity and stability of semiconductors. This innovative approach provides a new perspective on constructing photocatalytic materials with exceptional performance.

A food-grade Pickering emulsifier was fabricated using heat-assisted pH-shifting (HP) method for enhancing the emulsion stability. The emulsification performance of the modified pumpkin seed protein isolate (PSPI) nanoparticles was initially tested. The results showed that the modified PSPI had substantially improved emulsifying capacity, with the internal oil phase volume fraction reaching up to 80%, and the emulsion remaining stable after centrifugation at 10,000 g for 60 min and 30 days of storage. The data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), UV-visible, intrinsic fluorescence and fourier transform infrared spectroscopy (FTIR) indicated that the primary, secondary and tertiary structures of PSPI were disrupted, resulting in changes in interfacial tension, wettability, particle size, and dispersibility of the protein, which promoted the adsorption of modified PSPI nanoparticles to the surface of oil droplets and improved the emulsion stability. Overall, the HP modified PSPI is a promising Pickering emulsifier for various applications.

N-6 methyladenosine is the most abundant nucleic acid modification in eukaryotes and plays a crucial role in gene regulation. The AlkB family of alpha-ketoglutarate-dependent dioxygenases is responsible for nucleic acid demethylation. Recent studies have discovered that a chemical demethylation system using hydrogen peroxide and ammonium bicarbonate can effectively demethylate nucleic acids. The addition of ferrous ammonium sulfate boosts the oxidation rate by forming a Fenton reagent with hydrogen peroxide. However, the specific mechanism and key steps of this process remain unclear. In this study, we investigate the influence of ferrous ammonium sulfate concentration on the kinetic isotope effect (KIE) of the chemical demethylation system using LC-MS. As the concentration of ferrous ions increases, the observed KIE decreases from 1.377 ± 0.020 to 1.120 ± 0.016, indicating a combination of the primary isotope effect and inverse α-secondary isotope effect with the ion pairing effect. We propose that the initial hydrogen extraction is the rate-limiting step and observe a tight transition state structure in the formation of the hm6A process through the analysis of KIE trends. The concentration-dependent KIE provides a novel perspective on the mechanism of chemical demethylation and offers a chemical model for enzyme-catalyzed demethylation.

DNA or RNA methylation is a significant dynamic regulation process in regulating human gene expression which could be generated by endogenous substances, exogenous reagents, or radiation. The damaged DNA or RNA may lead to mutations that compromise epigenetic patterns, block DNA and RNA synthesis, and affect the coding of mRNA transcription and translation. Interestingly, it is a reversal process that could be modified by demethylases. Scientists have discovered that ten-eleven translocation proteins (TET) and AlkB family enzymes could repair these damaged DNA or RNA, meanwhile, they are both α-ketoglutarate (α-KG)/Fe(II)-dependent proteins. The detailed mechanism of the reaction is still unknown although intermediates in some nucleobases demethylation reactions have been measured experimentally. Recently, quantum mechanics/ molecular mechanics (QM/MM) methods are becoming a hot research topic to predict mechanisms of catalytic reactions. This review summarizes and comments on many cases in which computational methods have been used to predict the detailed mechanisms of the demethylation of nucleobases.

BACKGROUND:Calcium is important in the formation of bones and teeth, cell metabolism, and other physiological activities. In this work, casein phosphopeptide-calcium chelate (CPP-Ca) was synthesized and the optimal process parameters for the chelation reaction were obtained. The bioavailability of calcium in CPP-Ca was investigated by in vitro gastrointestinal simulated digestion. The existence of phytic acid and oxalic acid in the digestion system was evaluated to clarify the calcium holding ability of casein phosphopeptide (CPP). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify oligopeptides from CPP-Ca. RESULTS:The optimal process parameters for the chelation reaction were: peptide concentration 7.76 mgmL-1 , pH 8.54, and reaction temperature 43.3 °C. The digestion in vitro results indicated that the calcium release rate of CPP-Ca in the stomach for 2 h reached 85%, and about 50% of the ionized calcium was re-chelated with CPP in the intestine. Phytic acid and oxalic acid could lead to a sharp decrease in soluble calcium but around 50% of the calcium was still retained in the form of chelates in the presence of CPP. The LC-MS/MS identified 19 casein-derived oligopeptides after digestion, and calcium modifications were found on eight peptides derived from β-casein and αs2 -casein. CONCLUSIONS:This study clarified the excellent calcium holding capacity of CPP in the presence of phytic acid and oxalic acid. Liquid chromatography-tandem mass spectrometry also revealed peptide changes, and identified peptides that chelate with calcium. These findings provided significant insights that could be relevant to the further utilization and product development of peptide-calcium chelate in the food industry. © 2023 Society of Chemical Industry.

In this work, CPP-Ca chelate was synthesized by chelating casein phosphopeptide (CPP) and calcium and characterized by Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) Energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). The antioxidant activity and calcium holding capacity of CPP-Ca were evaluated and its secondary structure transition was monitored during gastrointestinal digestion by in situ Raman spectroscopy. The results demonstrated that calcium chelating rate reached 40 % and calcium ion was bound to CPP mainly through the interaction of carboxyl and amino groups. The result of calcium holding capacity confirmed the formation of calcium phosphate precipitates could be delayed by 10-15 min with increasing CPP concentration. In vitro simulated digestion revealed CPP-Ca exhibited excellent calcium solubility and its secondary structural changes occurred, especially alpha-helix and beta-sheet content. These findings provided significant insights into enhancing bioavailability of calcium supplements and developing of calcium functional foods for human and animals.

Ambient particulate matter (APM) is extremely harmful to life's health. In this study, we investigated cellular injury in cat (Felix catus) lung cells (FCA-L2) exposed to organic and water-soluble extracts from APM. As well, the protective effect of vitamin E (VE), lycopene and a mixture of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (molar concentration ratio of 2:1) against this damage was evaluated. Organic and water-soluble extracts induced oxidative stress in FCA-L2 cells, as evidenced by excess reactive oxygen species production and mitochondrial damage, while treatment with VE, lycopene and EPA: DHA remarkably alleviated these indices. It was further found that treatment with EPA: DHA decreased lactate dehydrogenase and malondialdehyde, as well as increased activities of superoxide dismutase, glutathione peroxidase and catalase. Our study confirmed that nutrients mediates APM-induced oxidative stress via antioxidant proteins. Also, these findings could provide new insights into reducing APM-induced cytotoxicity by nutritional supplementation based on antioxidant compounds for animals.

Targeting delivery for nutritional factor is increasingly paid in research field of agricultural and food science. In this present work, a novel asialoglycoprotein receptor (ASGPR)-targeted alpha-linolenic acid (ALA)-loaded microemulsions (MEs) for hepatocellular carcinoma (HCC) was designed. The N-octadecyl lactose-amide (NOL) was synthesized and used as active targeting ligand. The structure of NOL was characterized by Fourier transform infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). The load-ALA MEs as delivery carrier with or without NOL were evaluated by in vitro release and viability effect to hepa1-6 cells. Notably, NOL-modified MEs loading ALA (NOL-ALA-ME) consisted of spheroidal droplets with a particle size of 10-20 nm. Compared with the no-targeted ligand MEs, NOL-ALA-ME possessed a sustained release characteristic in vitro. Due to specific ligand-receptor interactions, NOL-ALA-ME demonstrated a more remarkable selective toxicity to Hepa1-6 cells. Molecular docking of ligand and receptor compounds indicated a docking score of -5.4 kcal/mol, meaning a strong targeting binding force between ligand and receptor, theoretically supports the results of cell experiments. These findings could offer significant insight into MEs as targeting delivery carrier, which may be regarded as a promising strategy for functional enhancement of active components in agricultural and food industry. (c) 2022 Elsevier B.V. All rights reserved.

In this work, four hydrophilic ionic liquids (ILs, 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim] BF4), 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), 1-butyl-3-methylimidazolium bromide ([C4mim]Br) and 1,3-dimethylimidazolium iodide ([dmim]I)) were selected to probe their effect on lysozyme activity and the molecular interaction in aqueous solutions. Specifically, the effects of pH and concentration of ILs on lysozyme activity were investigated by using the spectrophotometric turbidity assay. The results demonstrated that the effect of ILs on lysozyme activity depended on the concentration of ILs. Fixing the pH at 6.85, lysozyme activity rose firstly and then reduced with the increase of concentration of four ILs, reaching the maximum at 75 mM. The UV-Vis spectra illustrated that the interaction between ILs and lysozyme was weak. The results of emission and synchronous fluorescence spectra indicated that ILs had a quenching effect on the fluorescence of lysozyme, and the quenching effect increased with the increasing concentration of ILs. Meanwhile, the fluorescent quenching mechanism of ILs on lysozyme was dynamic quenching, and ILs had significant effects on the tryptophan (Trp) residues Trp62 or Trp108 at the active site of lysozyme, which suggested that effects of ILs on lysozyme activity could be attributed to the conformational changes of lysozyme triggered by changes in the microenvironment surrounding Trp62 or Trp108. (C)& nbsp;2022 Elsevier B.V. All rights reserved.

Crocetin, a high-value apocarotenoid in saffron, is widely applied to the fields of food and medicine. However, the existing method of obtaining crocetin through large-scale cultivation is far from meeting the market demand. Microbial synthesis of crocetin is a potential alternative to traditional resources, and it is found that carotenoid cleavage dioxygenase (CCD) is the critical enzyme to synthesize crocetin. So, in this study, we used "hybrid-tunnel" engineering to obtain variants of Crocus sativus-derived CsCCD2, essential for zeaxanthin conversion into crocetin, with a broader substrate specificity and higher catalytic efficiency. Variants including S323A, with a lower charge bias and a larger tunnel size than the wild-type, showed a 5-fold higher crocetin titer in yeast-based fermentations. S323A could also convert the β-carotene substrate to crocetin dialdehyde and exhibited a 12.83-fold greater catalytic efficiency (kcat/Km) toward zeaxanthin than the wild-type in vitro. This strategy enabled the production of 107 mg/L crocetin in 5 L fed-batch fermentation, higher than that previously reported. Our findings demonstrate that engineering access tunnels to expand the substrate profile by in silico protein design represents a viable strategy to refine the catalytic properties of enzymes across a range of applications.

Template design on polymorph control, especially conformational polymorphs, is still in its infancy and the result of polymorph control is often accidental. A method of regulating the crystallization of conformational polymorphs based on the crystal structure similarity of templates and the target crystal form has been developed. Crystal structure similarity was considered to be able to introduce lattice matching (geometric term) with chemical interactions to regulate conformational polymorph nucleation. The method was successfully applied to induce the crystallization of DA7-II [HOOC–(CH 2 ) n −2 –COOH (diacids), named DA n , where n = 7, 9, 15, 17 and II represents the metastable polymorph] on the surface of DA15-II. An analogous two-dimensional plane – the (002) face of both DA15-II and DA7-II – was firstly predicted as the epitaxially attached face with similar lattice parameters and the strongest adsorption energy. The powder DA15-II template with the preferred orientation face in (002) presented much stronger inducing DA7-II ability than the template with other preferred orientation faces. The epitaxial growth of DA7-II on DA15-II through an identical (002) face was clearly observed and verified by the single-crystal inducing experiments. The molecular dynamics simulation results demonstrated that the strong interactions occurred between DA7 molecules and the (002) face of DA15-II. This method has been verified and further applied to the crystallization of DA7-II on the surface of DA17-II and DA9-II on the surface of DA15-II. This study developed a strategy based on structure similarity to regulate the conformational polymorph and verified the significant role of lattice matching and chemical effects on the design and preparation of templates.

DNA and RNA have various methylation modifications or damage that are directly related to some human diseases and physiological regulation. Most of these methylation modifications are reversible and can be dynamically repaired by RNA or DNA demethylases. Over the past few decades, enzymes from the ALKB and TET families have been shown to have the ability to demethylate nucleic acids, which involves intermediates in the oxidative repair process. These intermediates can be accurately captured by advanced methods such as HPLC, LC-MS, TLC, and crystallization, which can significantly promote our understanding of the dynamic mechanism of demethylation. In this review, we discuss recent research advances in this area and raise open questions and constructive opinions about the capture of nucleic acid demethylation intermediates.

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein–protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.

As an ancient and huge family of membrane proteins, ATP-binding cassette transporter (ABC transporter) plays an important physiological role in most organisms. Herein, we introduce the research progress in ABC transporters on the aspects of structural characteristic, transport mechanism and physiological functions. We also focus on the application of ABC transporters in the field of synthetic biology in recent years. Finally, we propose future research needs.

Pyrene-based dyes were synthesized by a clean, efficient Diels-Alder [4 + 2] click reaction where no catalyst adding or side reactions occurring. The optical behaviors, optoelectronic properties and supramolecular donor/acceptor dipolar interactions of these new dyes were investigated by UV-vis absorption, fluorescence spectroscopy, electrostatic potential and frontier molecular orbital calculations as well as electrochemical cyclic voltammetry measurements. Two pyrene-based dyes can be mutually transformed by reversible formation or cleavage of Diels-Alder covalent bonds through click/declick reaction. During the reversible reaction, switchable fluorescence ON/OFF behaviors were interestingly observed. X-ray single crystal diffraction revealed that supramolecular donor/acceptor dipolar and pi-pi stacking interactions work cooperatively to form stronger interaction and the shorter distance (3.38 angstrom) between donor and acceptor than general pi-pi stacking distance (3.54 angstrom). An amphiphilic dye is self-assembled in water into blue fluorescent, hollow vesicular aggregates with the average diameter of 115-120 nm and a narrow size distribution as revealed by dynamic light scattering, scanning electron microscopy and transmission electron microscopy.