Search Results (95)

Given the limitations of clinical and potent natural α-glucosidase inhibitors, novel selective inhibitors are urgently needed. To accelerate discovery, we employed machine learning-integrated virtual screening to rapidly evaluate a library of 100 K⁺ compounds, identifying a series of selective α-glucosidase inhibitors. Activity validation demonstrated that these inhibitors exhibit significantly enhanced selectivity and potency compared to the positive control acarbose. Mechanistic studies through inhibition kinetics and fluorescence quenching revealed their improved inhibitory profile. Molecular docking indicates that key interactions—hydrogen bonding or salt bridges with the catalytic residue ASP526—strengthen binding within the active site. These interactions competitively obstruct enzyme-substrate binding, thereby amplifying inhibition. In vitro and in vivo starch digestion assays further corroborated these findings. Full article

BACH1 (BTB And CNC Homology 1) and NRF2 (Nuclear Factor Erythroid 2-related Factor 2) are transcription factors that regulate antioxidant and iron metabolism genes by competing for binding to cis-regulatory Maf-binding elements (CsMBEs) as heterodimers with small Maf proteins (sMafs). To dissect the mechanisms underlying this competition, we developed a chimeric tethering system where the DNA-binding domains of BACH1 or NRF2 were covalently linked to sMafG via a flexible, cleavable linker. This design enables efficient heterodimer formation on DNA and circumvents kinetic barriers to partner exchange in the solution. The site-specific fluorescent labelling of proteins allowed for the tracking of complex compositions by electrophoretic mobility shift assays. Both BACH1/sMafG and NRF2/sMafG heterodimers bind CsMBEs with similar affinities. Notably, DNA binding by BACH1 was impaired in a C574-dependent, redox-sensitive manner and promoted the exchange of heterodimer partners. Competition assays demonstrated that BACH1 and NRF2 can displace each other from preformed DNA-bound complexes, with greater efficiency when presented as preassembled heterodimers with sMafG. These findings reveal a redox-sensitive mechanism for regulating transcriptional switches at CsMBEs and highlight how preformed heterodimers facilitate the rapid displacement at target promoters. Full article

Aleurocanthus spiniferus, an invasive pest native to Southeast Asia, exhibits rapid dispersal capacity and high eradication resistance. In recent years, there have been continuous records of its invasion into new host plants. Odorant-binding proteins (OBPs) are essential at the peripheral level of olfaction, and their olfactory function has been partially confirmed by research. This study explores the functions of key OBPs mediating host selection by measuring the in vivo and in vitro binding capabilities of OBPs from A. spiniferus to host volatiles. Under exposure to more than five host volatiles, the two OBPs, AspiOBP1 and AspiOBP2, exhibited significant differential transcriptional regulation. AspiOBP1 exhibited good binding affinity to (Z)-3-hexenol and 3-carene, and with binding energies greater than −3 kcal/mol, ARG-79 might be the critical amino acid site for AspiOBP1 binding to host volatiles. AspiOBP2 exhibited no binding to any of the six tested volatiles in fluorescent competitive binding assays. Adults fed with dsAspiOBP1 showed significantly reduced behavioral and EAG responses to the attractant 3-carene and two repellents [(Z)-3-hexenol and nonanal]. Adults fed with dsAspiOBP2 lost both behavioral and EAG responses to the attractant 3-carene and the repellent (Z)-3-hexenol. The findings of this study not only elucidate the binding mechanisms between OBPs of A. spiniferus and host volatiles but also provide new targets for the future development of novel plant-derived insecticides and RNA-based pesticides to control this pest. Full article

Heterotetramerization of Kv1.1 and Kv1.2 α-subunits expands the functional diversity of voltage-gated potassium Kv1 channels in the central nervous system (CNS), thus necessitating the study of the properties of these heterochannels, including their interactions with ligands. We report on the expression, electrophysiological, and ligand-binding properties of human heterochannels Kv(1.1-1.2)₂ formed by dimeric concatemers Kv1.1-Kv1.2 fused with fluorescent protein mKate2 in Neuro-2a cells. Kv(1.1-1.2)₂ is a low-voltage-activated, highly active, non-inactivating channel with a fast activation rate. Its activation rate and half-maximum activation voltage are similar to that of the Kv1.1 channel, but differ from that of Kv1.2. This suggests that the membrane expression of Kv(1.1-1.2)₂ may functionally compensate for the absence of membrane presentation of homotetrameric Kv1.1 channels in CNS. Hongotoxin 1 fused with fluorescent protein GFP (HgTx-G) is shown to be a pore-blocking ligand of Kv(1.1-1.2)₂ with a dissociation constant of 100 pM. Using confocal microscopy and competitive binding assay, HgTx-G and cells expressing Kv(1.1-1.2)₂, the apparent dissociation constants of the complexes between Kv(1.1-1.2)₂ and peptides Ce1, Ce4, hongotoxin 1, MeKTx11-1, agitoxin 2, charybdotoxin, and scyllatoxin were evaluated to be 14, 33, 40, 250, 800, and >>3300 pM, respectively. Heterotetramerization of α-subunits has a different effect on the affinity of ligands compared to those for Kv1.1 and Kv1.2 channels. Full article

Adoxophyes orana (Lepidoptera: Tortricidae) is a significant polyphagous leafroller that damages trees and shrubs in Rosaceae and other families. However, the molecular mechanisms by which this pest recognizes sex pheromones and host plant volatiles remain largely unknown. Tissue expression profiles indicated that two general odorant-binding proteins (AoraGOBP1 and AoraGOBP2) were more abundant in the antennae and wings of both sexes, with AoraGOBP1 being rich in the female head and abdomen. Temporal expression profiles showed that AoraGOBP1 was expressed at the highest level in 5 day-nmated adults, while AoraGOBP2 exhibited high expression in 5 day-unmated, 7 day-unmated, and mated female adults. Fluorescence competitive binding assays of heterologous expressed AoraGOBPs demonstrated that AoraGOBP2 strongly bound to the primary sex pheromone Z9-14:Ac, and two minor sex pheromones Z9-14:OH and Z11-14:OH, whereas AoraGOBP1 only showed a high binding affinity to Z9-14:Ac. What is more, AoraGOBP1 exhibited a broader binding spectrum for host plant volatiles than AoraGOBP2. Molecular dockings, molecular dynamic simulations, and per-residue binding free decompositions indicated that the van der Waals interaction was the predominant contributor to the binding free energy. Electrostatic interactions between aldehydes, or alcohols and AoraGOBPs stabilized the conformational structures. Phe12 from AoraGOBP1, and Phe13 from AoraGOBP2 were identified as the most important residues that contributed to bind free energy. Our findings provide a comprehensive insight into the molecular mechanisms of olfactory recognition in A. orana, facilitating the development of chemical ecology-based approaches for the control. Full article

The tomato leafminer (Tuta absoluta), a globally invasive pest, poses a major economic threat to tomato production. Although chemical control remains the primary management method, sustainable alternatives are urgently needed. Sex pheromone communication is critical for moth courtship and mating, with pheromone-binding proteins (PBPs) playing a key role in this process. In this study, we identified a PBP gene, TabsPBP2, from the T. absoluta transcriptome. Real-time quantitative PCR (RT-qPCR) revealed that TabsPBP2 is highly expressed in the antennae, with a strong male-biased expression pattern. Ligand-binding assays demonstrated that TabsPBP2 has the highest affinity for the sex pheromones (3E, 8Z, 11Z)-tetradecatrienyl acetate (TDTA) and (3E, 8Z)-tetradecadienyl acetate (TDDA). It also demonstrated a moderate-to-strong binding affinity to several tomato volatiles, including 2-carene, myrcene, α-pinene, cis-3-hexen-l-ol, methyl salicylate, sabinene, and α-terpinene. Molecular docking suggested that hydrophobic interactions predominantly stabilize the TabsPBP2–ligand complexes, with PHE118, PHE12, LEU90, LEU68, and ALA73 identified as key interacting residues. Electroantennogram (EAG) and Y-tube olfactometer assays confirmed that TDTA and TDDA act as strong attractants for male T. absoluta. This study enhances our understanding of the pheromone recognition in T. absoluta and provides a foundation for developing novel, pheromone-based pest control strategies. Full article

Niacinamide was recently shown to directly interact with bacterial DNA and interfere with cell replication; niacinamide mode of interaction and efficacy as a natural anti-microbial molecule were also described. The aim of this study is to elucidate the exact binding mechanism of niacinamide to microbial DNA. Intercalation is a binding mode where a small planar molecule, such as niacinamide, is inserted between base pairs, causing structural changes in the DNA. Melting curve analysis with various intercalating dyes demonstrated that niacinamide interaction with bacterial DNA reduces its melting temperature in a linear dose-dependent manner. Niacinamide’s effect on the melting temperature was found to be % GC-dependent, while purine stretches were also found to influence the binding kinetics. Finally, fluorescent intercalator displacement (FID) assays demonstrated that niacinamide strongly reduces SYBR Safe signal in a dose-dependent manner. Interestingly, competition assays with a minor groove binder also reduced Hoechst signal but in a non-linear manner, which can be attributed to strand lengthening and unwinding following niacinamide intercalation. Taken altogether; our results suggest a “disruptive intercalation” as the mode of interaction of niacinamide with bacterial DNA. Formation of locally destabilized DNA portions by niacinamide might interfere with protein–DNA interaction and potentially affect several crucial bacterial cellular processes, e.g., DNA repair and replication, subsequently leading to cell death. Full article

The tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae), is a destructive pest of Solanaceae crops worldwide. Its olfactory system plays an important role in locating mating partners and recognizing host plants. Understanding its olfactory recognition mechanism, particularly the function of odorant-binding proteins (OBPs), may reveal potential targets for pest management. In this study, we characterized two antenna-specific OBPs, TabsOBP45 and TabsOBP46, which were identified from the T. absoluta genome. Sequence analysis revealed that both TabsOBPs belong to the classic OBP subfamily, which is characterized by the presence of six conserved cysteine residues and an N-terminal signal peptide. Both TabsOBPs showed predominant antennal expression in quantitative real-time PCR (qRT-PCR) assays, suggesting their key roles in olfactory perception. Fluorescence competitive binding assays with a total of 63 tested volatiles revealed that 13 compounds exhibited strong binding affinities (Ki < 22 µM) to TabsOBP45, with the highest binding affinity to β-ionone, β-caryophyllene, terpinolene, and cinnamaldehyde. Nine compounds showed strong binding affinities to TabsOBP46, with the strongest binding to 4-anisaldehyde, 4-methoxybenzaldehyde, cinnamaldehyde, and β-ionone. Molecular docking analysis revealed the key residues involved in β-ionone binding: TabsOBP45 interacted with ILE8, ALA9, PHE12, TRP37, ILE92, PHE94, THR115, and PHE118, while TabsOBP46 interacted with ILE8, PHE12, PHE36, TRP37, ILE92, LEU94, PHE118, and VAL134. These results provide new insights into the olfactory mechanism of T. absoluta and potential molecular targets for the development of olfactory-based pest control strategies. Full article

Peroxisome proliferator-activated receptors (PPARs), composed of the α/δ/γ subtypes, are ligand-activated nuclear receptors/transcription factors that sense endogenous fatty acids or therapeutic drugs to regulate lipid/glucose metabolism and oxidative stress. PPAR forms a multiprotein complex with a retinoid X receptor and corepressor complex in an unliganded/inactive state, and ligand binding induces the replacement of the corepressor complex with the coactivator complex to initiate the transcription of various genes, including the metabolic and antioxidant ones. We investigated the processes by which the corepressor is replaced with the coactivator or in which two coactivators compete for the PPARα/δ/γ-ligand-binding domains (LBDs) using single- and dual-emission fluorescence resonance energy transfer (FRET) assays. Single-FRET revealed that the respective PPARα/δ/γ-selective agonists (pemafibrate, seladelpar, and pioglitazone) induced the dissociation of the two corepressor peptides, NCoR1 and NCoR2, from the PPARα/δ/γ-LBDs and the recruitment of the two coactivator peptides, CBP and TRAP220. Meanwhile, dual-FRET demonstrated that these processes are simultaneous and that the four coactivator peptides, CBP, TRAP220, PGC1α, and SRC1, were competitively recruited to the PPARα/δ/γ-LBDs with different preferences upon ligand activation. Furthermore, the five newly obtained cocrystal structures using X-ray diffraction, PPARα-LBDs–NCoR2/CBP/TRAP220/PGC1α and PPARγ-LBD–NCoR2, were co-analyzed with those from our previous studies. This illustrates that these coactivators bound to the same PPARα-LBD loci via their consensus LXXLL motifs in the liganded state; that NCoR1/NCoR2 corepressors bound to the same loci via the IXXXL sequences within their consensus LXXXIXXXL motifs in the unliganded state; and that ligand activation induced AF-2 helix 12 formation that interfered with corepressor binding and created a binding space for the coactivator. These PPARα/γ-related biochemical and physicochemical findings highlight the coregulator dynamics on limited PPARα/δ/γ-LBDs loci. Full article

Lurasidone (LUR) is an antipsychotic drug whose interaction with human serum albumin (HSA) plays a crucial role in its pharmacokinetic and pharmacodynamic properties. A thorough understanding of LUR’s binding mechanism to HSA is crucial for predicting its transport, distribution, and potential drug interactions. Methods: The interaction between LUR and HSA was investigated using fluorescence and circular dichroism (CD) spectroscopy, followed by molecular docking simulations. Binding characteristics were analyzed through quenching mechanisms, thermodynamic parameters, and competitive site marker experiments. Results: This study revealed a systematic decrease in HSA fluorescence intensity with increasing LUR concentration, indicating a static quenching mechanism driven by non-fluorescent complex formation. Binding constants suggest enhanced complex stability at higher temperatures, with thermodynamic analysis confirming an endothermic, hydrophobic interaction. Competitive site marker assays and synchronous fluorescence spectra confirmed that LUR primarily binds to site I (subdomain IIA) near tryptophan residues. Conformational changes in HSA, observed as a decrease in α-helix content, further demonstrate the structural impact of LUR binding. Conclusions: These findings offer key insights into the molecular interactions between LUR and HSA, enhancing our understanding of LUR’s pharmacokinetics and its potential interactions with other drugs. Understanding these binding characteristics can aid in optimizing LUR’s clinical application and predicting possible interactions with other biomolecules. Full article

Boosting the accuracy and speed of cancer detection is highly desirous in tumor detection, and sensors capable of detecting carcinoembryonic antigen (CEA) have great application prospects in this field. A highly sensitive sensor is constructed based on the fluorescence resonance energy transfer (FRET) with heavily rare-earth-doped upconversion nanoparticles (UCNPs) as energy donors and polydopamine nanoparticles (PDA NPs) as energy acceptors. This sensor detects the fluctuations in CEA molecules via luminescence quenching and recovery resulting from a competitive binding assay between CEA and PDA NPs. The high-level-doped design of UCNPs (i.e., NaYF₄@NaYbF₄:1%Tm@NaYF₄) is beneficial, providing upconversion luminescence intensity that is more than 10 times higher than that of the conventional low-level-doped UCNPs (i.e., NaYF₄@NaYF₄:20%Yb, 0.2%Tm@NaYF₄). The sensor exhibits impressive sensitivity. Specifically, in diluted fetal bovine serum, the detection limit reaches 0.013 ng/mL in the range of 0–1.5 ng/mL (S/N = 3), while the detection limit is 1.38 ng/mL in the range of 1.5–250 ng/mL (S/N = 3). This method has great potential for future applications in the rapid and early diagnosis and treatment of cancer. Full article

Moths use pheromones to ensure intraspecific communication. Nevertheless, few studies are focused on both intra- and intersexual communication based on pheromone recognition. Pheromone-binding proteins (PBPs) are generally believed pivotal for male moths in recognizing female pheromones. Our research revealed that PBP1 of Agriphila aeneociliella (AaenPBP1) serves a dual function in both intra- and intersexual pheromone recognition. Here, a total of 20 odorant-binding protein (OBP) family genes from A. aeneociliella were identified and subjected to transcriptional analysis. Among these, AaenPBP1 was primarily highly expressed in the antennae. Competitive fluorescence binding assays and molecular docking analyses demonstrated that AaenPBP1 exhibits a strong binding affinity for the female sex pheromone (Z)-9-Hexadecenyl acetate and the male pheromone 1-Nonanal. Notably, hydrogen bonds were observed between Ser56 and the ligands. The analysis of pheromone components and PBPs in lepidopteran lineage suggested that their strong and precise interactions, shaped by coevolution, may play a crucial role in facilitating reproductive isolation in moths. Our findings provide valuable insight into the functional significance of PBPs in invertebrates and support the development of behavioral regulation tools as part of an integrated pest management strategy targeting crambid pests. Full article

The plum fruit moth (PFM), Grapholita funebrana, and the oriental fruit moth (OFM), G. molesta, are closely related fruit moth species that severely damage fruit trees in Rosaceae. Both species share common primary sex pheromone components Z8-12:Ac and E8-12:Ac. The secondary sex pheromone components of PFMs consist of Z8-12:OH, Z8-14:Ac, and Z10-14:Ac, while those of OFMs include Z8-12:OH and 12:OH. Previous researchers have proved that the inclusion of Z8-14:Ac and Z10-14:Ac did not augment PFM catches but inhibited OFM catches in orchards in Europe, thereby maintaining the species-specificity of the PFM sex attractant. However, which of these components, Z8-14:Ac or Z10-14:Ac, plays the major role in inhibiting OFM attraction remains unclear. In the current study, electroantennogram (EAG) assays indicated that both OFM and PFM males exhibited a moderate EAG response to Z8-14:Ac and Z10-14:Ac. Rubber septa loaded with varying ratios of Z8-14:Ac (1% to 30%) or Z10-14:Ac (5% to 110%) combined with a constant dose of Z8-12:Ac and E8-12:Ac produced diverse trapping effects. Sex attractants containing Z8-14:Ac did not significantly affect the trapping of PFM males but drastically reduced the capture of OFM males, with the reduction reaching up to 96.54%. Attractants containing more than 10% of Z10-14:Ac simultaneously reduced the number of OFM and PFM males captured. Z8-14:Ac was indispensable for maintaining the specificity of sex pheromones. Fluorescence competitive binding assays of recombinant GmolPBP2 showed the lowest K_i value (0.66 ± 0.02 μM) among the PBPs/GOBPs from OFMs, suggesting that it is the most likely target for Z8-14:Ac. Molecular dynamic simulation and site-directed mutagenesis assays confirmed that the Phe12 residue, which forms a π–alkyl interaction with Z8-14:Ac, was crucial for GmolPBP2 binding to Z8-14:Ac. In conclusion, Z8-14:Ac is vital to the specificity of PFM sex pheromones inhibiting OFM attractants when added to Z8-12:Ac and E8-12:Ac. This could be potentially used to develop species-specific sex attractants for the PFM. Full article

Modular nanotransporters (MNTs) are drug delivery systems for targeted cancer treatment. As MNTs are composed of several modules, they offer the advantage of high specificity and biocompatibility in delivering drugs to the target compartment of cancer cells. The large carrier module brings together functioning MNT modules and serves as a platform for drug attachment. The development of smaller-sized MNTs via truncation of the carrier module appears advantageous in facilitating tissue penetration. In this study, two new MNTs with a truncated carrier module containing either an N-terminal (MNT_N) or a C-terminal (MNT_C) part were developed by genetic engineering. Both new MNTs demonstrated a high affinity for target receptors, as revealed by fluorescent-labeled ligand-competitive binding. The liposome leakage assay proved the endosomolytic activity of MNTs. Binding to the importin heterodimer of each truncated MNT was revealed by a thermophoresis assay, while only MNT_N possessed binding to Keap1. Finally, the photodynamic efficacy of the photosensitizer attached to MNT_N was significantly higher than when attached to either MNT_C or the original MNTs. Thus, this work reveals that MNT’s carrier module can be truncated without losing MNT functionality, favoring the N-terminal part of the carrier module due to its ability to bind Keap1. Full article

Saxitoxin (STX), an exceptionally potent marine toxin for which no antidote is currently available, is produced by methanogens and cyanobacteria. This poses a significant threat to both shellfish aquaculture and human health. Consequently, the development of a rapid, highly sensitive STX detection method is of great significance. The objective of this research is to create a novel approach for identifying STX. Therefore, amplified luminescent proximity homogeneous assay (AlphaLISA) was established using a direct competition method based on the principles of fluorescence resonance energy transfer and antigen–antibody specific binding. This method is sensitive, rapid, performed without washing, easy to operate, and can detect 8–128 ng/mL of STX in only 10 min. The limit of detection achieved by this method is as low as 4.29 ng/mL with coefficients of variation for the intra-batch and inter-batch analyses ranging from 2.61% to 3.63% and from 7.67% to 8.30%, respectively. In conclusion, our study successfully establishes a simple yet sensitive, rapid, and accurate AlphaLISA method for the detection of STX which holds great potential in advancing research on marine biotoxins. Full article