Search Results (439)

Fritillaria unibracteata is a rare and endangered medicinal plant in the Liliaceae family, whose bulbs have been used in traditional Chinese traditional medicine for over 2000 years. The mevalonate (MVA) pathway is involved in the growth, development, response to environmental stress, and active ingredient production of plants; however, the functional characterization of MVA-pathway genes in the Liliaceae family remains poorly documented. In this study, an Acetyl-CoA C-acetyltransferase gene (FuAACT) was first cloned from F. unibracteata. It exhibited structural features of the thiolase family and showed the highest sequence identity with the Dioscorea cayenensis homolog. The K_m, V_max, and K_cat of the recombinant FuAACT were determined to be 3.035 ± 0.215 μM, 0.128 ± 0.0058 μmol/(min·mg), and 1.275 ± 0.0575 min⁻¹, respectively. The optimal catalytic conditions for FuAACT were ascertained to be 30 °C and pH 8.9. It was stable below 50 °C. His361 was confirmed to be a key amino acid residue to enzymatic catalysis by site-directed mutagenesis. Subsequent subcellular localization experiments demonstrated that FuAACT was localized in chloroplasts and cytoplasm. FuAACT-overexpressing transgenic Arabidopsis thaliana plants showed higher drought tolerance than wild-type plants. This phenotypic difference was corroborated by significant differences in seed germination rate, lateral root number, plant height, and leaf number (p < 0.05). Furthermore, the FuAACT transgenic plants resulted in the formation of a more developed fibrous root system. These results indicated that the FuAACT gene revealed substantial biological activity in vitro and in vivo, hopefully providing the basis for its further research and application in liliaceous ornamental and medicinal plants. Full article

The barbel chub (Squaliobarbus curriculus) exhibits remarkable resistance to grass carp reovirus (GCRV), a devastating pathogen in aquaculture. To reveal the molecular basis of this resistance, we investigated complement factor D (DF)—a rate-limiting serine protease governing alternative complement pathway activation. Molecular cloning revealed that the barbel chub DF (ScDF) gene encodes a 1251-bp cDNA sequence translating into a 250-amino acid protein. Crucially, bioinformatic characterization identified a unique N-glycosylation site at Asn¹³⁹ in ScDF, representing a structural divergence absent in grass carp (Ctenopharyngodon idella) DF (CiDF). While retaining a conserved Tryp_SPc domain harboring the catalytic triad (His⁶¹, Asp¹⁰⁹, and Ser²⁰⁴) and substrate-binding residues (Asp¹⁹⁸, Ser²¹⁹, and Gly²²¹), sequence and phylogenetic analyses confirmed ScDF’s evolutionary conservation, displaying 94.4% amino acid identity with CiDF and clustering within the Cyprinidae. Expression profiling revealed constitutive ScDF dominance in the liver, and secondary prominence was observed in the heart. Upon GCRV challenge in S. curriculus kidney (SCK) cells, ScDF transcription surged to a 438-fold increase versus uninfected controls at 6 h post-infection (hpi; p < 0.001)—significantly preceding the 168-hpi response peak documented for CiDF in grass carp. Functional validation showed that ScDF overexpression suppressed key viral capsid genes (VP2, VP5, and VP7) and upregulated the interferon regulator IRF9. Moreover, recombinant ScDF protein incubation induced interferon pathway genes and complement C3 expression. Collectively, ScDF’s rapid early induction (peaking at 6 hpi) and multi-pathway coordination may contribute to barbel chub’s GCRV resistance. These findings may provide molecular insights into the barbel chub’s high GCRV resistance compared to grass carp and novel perspectives for anti-GCRV breeding strategies in fish. Full article

Background: Protein kinase R (PKR) inhibits general mRNA translation by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). PKR also modulates NF-κB signaling during viral infections, but comparative studies of PKR-mediated NF-κB responses across mammalian species and their regulation by viral inhibitors remain largely unexplored. This study aimed to characterize the conserved antiviral and inflammatory roles of mammalian PKR orthologs and investigate their modulation by poxviral inhibitors. Methods: Using reporter gene assays and quantitative RT-PCR, we assessed the impact of 17 mammalian PKR orthologs on general translation inhibition, stress-responsive translation, and NF-κB-dependent induction of target genes. Congenic human and rabbit cell lines infected with a myxoma virus strain lacking PKR inhibitors were used to compare the effects of human and rabbit PKR on viral replication and inflammatory responses. Site-directed mutagenesis was employed to determine key residues responsible for differential sensitivity to the viral inhibitor M156. Results: All 17 mammalian PKR orthologs significantly inhibited general translation, strongly activated stress-responsive ATF4 translation, and robustly induced NF-κB target genes. Inhibition of these responses was specifically mediated by poxviral K3 orthologs that effectively suppressed PKR activation. Comparative analyses showed human and rabbit PKRs similarly inhibited virus replication and induced cytokine transcripts. Amino acid swaps between rabbit PKRs reversed their sensitivity to viral inhibitor M156 and NF-κB activation. Conclusions: Our data show that the tested PKR orthologs exhibit conserved dual antiviral and inflammatory regulatory roles, which can be antagonized by poxviral K3 orthologs that exploit eIF2α mimicry to modulate the PKR-NF-κB axis. Full article

The enzyme ABH2, one of nine human DNA dioxygenases of the AlkB family, belongs to the superfamily of Fe(II)/α-ketoglutarate-dependent dioxygenases and plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases. ABH2 has broad substrate specificity, directly oxidizing DNA damages such as N¹-methyladenine, N³-methylcytosine, 1,N⁶-ethenoadenine, 3,N⁴-ethenocytosine, and a number of others. In our investigation, we sought to uncover the subtleties of the mechanisms governing substrate specificity in ABH2 by focusing on several critical amino acid residues situated in its active site. To gain insight into the function of this enzyme, we performed a functional mapping of its active site region, concentrating on pivotal residues, participating in forming a damaged binding pocket of the enzyme (Val99 and Ser125), as well as the residues directly involved in interactions with damaged bases, namely Arg110, Phe124, Arg172, and Glu175. To support our experimental data, we conducted a series of molecular dynamics simulations, exploring the interactions between the ABH2 mutant forms, bearing corresponding substitutions and DNA substrates, and harboring various types of methylated bases, specifically N¹-methyladenine or N³-methylcytosine. The comparative studies revealed compelling data indicating that alterations in most of the studied amino acid residues significantly influence both the binding affinity of the enzyme for DNA and its catalytic efficiency. Intriguingly, the findings suggest that the mutations impact the catalytic activity of ABH2 to a greater extent than its ability to associate with DNA strands. Collectively, these results show how changes to the active site affect molecular dynamics and reaction kinetics, improving our understanding of the substrate recognition process in this pivotal enzyme. Full article

Despite current vaccines and therapeutics targeting SARS-CoV-2, the causative agent of the COVID-19 pandemic, cases remain high causing a burden on health care systems. Spike-protein mediated membrane fusion of SARS-CoV-2 is a critical step in viral entry. Herein, we describe entry inhibitors identified by first screening a library of about 160 compounds and then analogue synthesis. Specifically, compound 261 was found to inhibit SARS-CoV-2 infection in a tissue model with IC₅₀ of 0.3 µM. Using NMR, we found that 261 interacts with key residues in the aromatic-rich region of the spike protein directly next to the transmembrane domain. Molecular dynamic simulations of the 261 binding pocket in the spike protein was also mapped to the transmembrane domain, consistent with NMR findings. The amino acids in the binding site are conserved among different coronaviruses known to infect humans; therefore, inhibitors targeting this conserved binding site could be a useful addition to current therapeutics and may have pan-coronavirus antiviral activities. Full article

Infections with cytomegalovirus (CMV) can result in increased morbidity and mortality in immunocompromised patients. The pUL97 kinase is a critical enzyme in the regulation of CMV replication. Although it does not phosphorylate deoxynucleosides, this enzyme is involved in the first phosphorylation step of ganciclovir (GCV), a viral DNA polymerase inhibitor. In contrast, maribavir (MBV) is a specific inhibitor of pUL97 kinase activity. In this paper, we analyzed the already-reported amino acid changes, conferring resistance to MBV and cross-resistance to GCV, in the pUL97 protein structure, predicted with AlphaFold2. Docking experiments suggest that MBV is a dual-site inhibitor, targeting ATP binding and substrate phosphorylation. Substitutions that confer resistance to MBV only may directly or indirectly alter the shape of the cavity in the vicinity of the invariant K355 in the putative ATP binding site, without affecting the viral growth. The most frequently encountered T409M substitution may correspond to a gatekeeper mutation. Substitutions that induce cross-resistance to MBV and GCV may directly or indirectly affect the environment of D456 and N461 residues in the catalytic loop, with reduced viral replicative capacity. These results have implications for the clinical use of MBV as well as for the design of novel pUL97 kinase inhibitors. Full article

Steroids are found in bacteria and eukaryotes, and genes potentially encoding steroid metabolic enzymes have also been identified in giant viruses. For decades, hydroxylated steroids have been utilized in medicine to treat various human diseases. The hydroxylation of steroids can be achieved using microbial enzymes, especially cytochrome P450 monooxygenases (CYPs/P450s) and is well documented. Understanding the structural determinants that govern the regio- and stereoselectivity of steroid hydroxylation by P450s is essential in order to fully exploit their potential. Herein, we present a comprehensive analysis of the steroid-hydroxylating CYP109 family across the domains of life and delineate the structural determinants that govern steroid hydroxylation. Data mining, annotation, and phylogenetic analysis revealed that CYP109 family members are highly populated in bacteria, and indeed, these members passed from bacteria to archaea by horizontal gene transfer, leading to the evolution of P450s in archaea. Analysis of twelve CYP109 crystal structures revealed large, flexible, and dynamic active site cavities that can accommodate multiple ligands. The correct positioning and orientation of the steroid in the active site cavity and the nature of the C17 substituent on the steroid molecule influence catalysis. In an analogous fashion to the CYP107 family, the amino acid residues within the CYP109 binding pocket involve hydrophilic and hydrophobic interactions, influencing substrate orientations and anchoring and determining the site of hydroxylation and catalytic activity. A handful of amino acids, such as Val84, Val292, and Ser387 in CYP109B4, have been found to play a role in determining the catalytic regiospecificity, and a single amino acid, such as Arg74 in CYP109A2, has been found to be essential for the enzymatic activity. This work serves as a reference for the precise understanding of CYP109 structure–function relationships and for P450 enzymes in general. The findings will guide the genetic engineering of CYP109 enzymes to produce valuable steroid molecules of medicinal and biotechnological importance. Full article

The development of orally bioavailable non-peptidomimetic glucagon-like peptide-1 receptor agonists (GLP-1RAs) offers a promising therapeutic avenue for the treatment of type 2 diabetes mellitus (T2DM) and obesity. An extensive in silico approach combining structure-based drug design and ligand-based strategies together with pharmacokinetic properties and drug-likeness predictions is implemented to identify novel non-peptidic GLP-1RAs from the COCONUT and Marine Natural Products (CMNPD) libraries. More than 700,000 compounds were screened by shape-based similarity filtering in combination with precision docking against the orthosteric site of the GLP-1 receptor (PDB ID: 6X1A). The docked candidates were further assessed with the molecular mechanics MM-GBSA tool to check the binding affinities; the final list of candidates was validated by running a 500 ns long MD simulation. Twenty final hits were identified, ten from each database. The hits contained compounds with reported antidiabetic effects but with no evidence of GLP-1 agonist activity, including hits 1, 6, 7, and 10. These findings proposed a novel mechanism for these hits through GLP-1 activity and positioned the other hits as potential promising scaffolds. Among the studied compounds—especially hits 1, 5, and 9—possessed strong and stable interactions with critical amino acid residues such as TRP-203, PHE-381, and GLN-221 at the active site of the 6X1A-substrate along with favorable pharmacokinetic profiles. Moreover, the RMSF and RMSD plots further suggested the possibility of stable interactions. Specifically, hit 9 possessed the best docking score with a ΔG_bind value of −102.78 kcal/mol, surpassing even the control compound in binding affinity. The ADMET profiling also showed desirable drug-likeness and pharmacokinetic characteristics for hit 9. The pipeline of computational integration underscores the potential of non-peptidic alternatives in natural product libraries to pursue GLP-1-mediated metabolic therapy into advanced preclinical validation. Full article

Heat shock transcription factor (Hsf) plays a pivotal role in regulating plant growth, development, and stress responses. Hsf activates or represses target gene transcription by binding to the heat shock element (HSE) of downstream genes. However, the specific interaction sites between Hsf and the HSE in the promoter remain unclear. In this study, the critical amino acid residues of ZmHsf17 and the paralogous ZmHsf05 involved in DNA binding were identified using molecular docking models, site-directed mutagenesis, and the electrophoretic mobility shift assay (EMSA). The results reveal that both ZmHsf17 and ZmHsf05 bind to the HSE of the ZmPAH1 promoter via a conserved arginine residue located in the α3 helix of their DNA-binding domains. Sequence substitution experiments among distinct HSEs demonstrated that flanking sequences upstream and downstream of the HSE core synergistically contribute to the specificity of DNA-binding domain recognition. Comparative evolutionary analysis of DNA-binding domain sequences from 25 phylogenetically diverse species reveals that the α3 helix constitutes the most conserved structural element. This study elucidates the key interaction sites between maize HsfA2 and its target genes, providing theoretical insights into the binding specificity to the HSEs of the plant’s Hsf family and the functional divergence. Additionally, these findings offer new targets for the precise engineering of Hsf proteins and synthetic HSEs. Full article

Acetone carboxylase (AC) from Xanthobacter autotrophicus is a 360 KDa α₂β₂γ₂ heterohexamer that catalyzes the ATP-dependent formation of phosphorylated acetone and bicarbonate intermediates that react at Mn(II) metal active sites to form acetoacetate. Structural models of X. autotrophicus AC (XaAC) with and without nucleotides reveal that the binding and phosphorylation of the two substrates occurs ~40 Å from the Mn(II) active sites where acetoacetate is formed. Based on the crystal structures, a significant conformational change was proposed to open and close a tunnel that facilitates the passage of reaction intermediates between the sites for nucleotide binding and phosphorylation of substrates and Mn(II) sites of acetoacetate formation. We have employed electron paramagnetic resonance (EPR), kinetic assays, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) of poised ligand-bound states and site-specific amino acid variants to complete an in-depth analysis of Mn(II) coordination and allosteric communication throughout the catalytic cycle. In contrast with the established paradigms for carboxylation, our analyses of XaAC suggested a carboxylate shift that couples both local and long-range structural transitions. Shifts in the coordination mode of a single carboxylic acid residue (αE89) mediate both catalysis proximal to a Mn(II) center and communication with an ATP active site in a separate subunit of a 180 kDa α₂β₂γ₂ complex at a distance of 40 Å. This work demonstrates the power of combining structural models from X-ray crystallography with solution-phase spectroscopy and biophysical techniques to elucidate functional aspects of a multi-subunit enzyme. Full article

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation. Full article

The human coagulation pathway orchestrates a complex series of events vital for maintaining vascular integrity, in which the intrinsic pathway plays a pivotal role in amplifying and propagating the coagulation response. Dysregulation of this pathway can lead to various bleeding disorders and thrombotic complications, posing significant health risks. In this pathway, the activation of Factor (F) X zymogen is catalyzed by the FVIIIa-FIXa binary complex, but knowledge about this is still incomplete. Understanding the structural and functional intricacies of the FVIIIa-FIXa-FX (zymogen) complex is imperative for unraveling the molecular mechanisms underlying coagulation regulation and guiding the development of targeted therapeutic interventions. In this study, utilizing Alphafold-Multimer and molecular dynamics (MD) simulations, we provide insights into factor interactions within the ternary complex and propose novel functional mechanisms contributing to the functional defects inflicted by their cross-reactive material (CRM) positive mutations. The amino acid residue replacement impairs the coagulation function by interfering with structure elements, including the following: (1) a knot-like structure between Arg-562 of FVIIIa’s 558-Loop (residue 555–571) and the 333-Loop of FIXa (residue 333–346) contributes to FVIIIa-FIXa binding; (2) the a2 region of FVIIIa (residue 716–740) opens the lid of active site (FIXa’s 266-Loop, residue 256–270) and facilitates substrate binding; (3) the activation peptide (AP) of FX zymogen (residue 143–194) not only assists in the activation of itself but also adheres the interface of the three factors like a double-sided tape. Our work provides novel insights for the pathogenesis of a number of reported clinical CRM-positive mutations and may lay the groundwork for the structure-based development of therapeutic interventions. Full article

Background/Objectives: Oxidative stress, the Warburg effect, and resistance to apoptosis are key hallmarks driving colorectal tumorigenesis. This study aimed to develop novel multi-target compounds capable of modulating these pathways. Methods: A library of 24 newly synthesized compounds—incorporating annulated thiophene, thiazole, quinazolinone, 2-oxoindoline, and 1,2,3-oxadiazole scaffolds, as well as N-(1-(4-hydroxy-3-methoxyphenyl)-3-oxo-3-(2-(phenylcarbamothioyl)hydrazineyl) prop-1-en-2-yl)benzamide—was evaluated for antioxidant activity (DPPH assay), PDK-1 and LDHA inhibition, cytotoxic effects against LoVo and HCT-116 colon carcinoma cells, with parallel assessment of safety profiles on normal HUVECs. The underlying anticancer mechanism of the most active compound was investigated through analysis of cell cycle distribution, apoptosis induction, intracellular reactive oxygen species levels, mitochondrial membrane potential disruption, and expression levels of apoptosis-related genes. Molecular docking assessed binding interactions within LDHA and PDK-1 active sites. The physicochemical, drug-likeness, and ADMET properties of the multi-bioactive candidates were predicted in silico. Results: Among the synthesized compounds, thiophenes 3b and 3d exhibited potent PDK-1/LDHA and DPPH/LDHA inhibitions, along with significant cytotoxic effects on LoVo/HCT-116 cells (IC₅₀ in µM: 190.30/170.21 and 156.60/160.96, respectively), while showing minimal cytotoxicity toward HUVECs. Molecular docking revealed favorable interactions with key amino acid residues within the LDHA and/or PDK-1 active sites. Compound 3d notably induced G2/M (LoVo) and G1 (HCT-116) arrest and promoted apoptosis via enhancing ROS generation, modulating Bax/Bcl-2 expressions, disrupting mitochondrial membrane potential, and ultimately activating caspses-3. In silico predictions indicated their promising drug-likeness and pharmacokinetics, though high lipophilicity, poor solubility (especially for 3b), and potential toxicity risks were identified as limitations. Conclusions: Thiophenes 3b and 3d emerged as promising multi-target candidates; however, structural optimization is warranted to enhance their solubility, bioavailability, and safety to support further development as lead anti-colon cancer agents. Full article

Background: Inhibition of intestinal α-glucosidase is a key strategy for controlling postprandial hyperglycemia in diabetes. Astragali Radix (AR), a traditional medicinal and dietary herb widely consumed in China, is rich in flavonoids that are believed to exhibit hypoglycemic properties. Methods: A total of 29 AR-related flavonoids, including both original constituents and metabolites, were screened for α-glucosidase inhibitory activity using in vitro enzymatic assays. Mechanistic investigations were conducted through enzyme kinetics, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR), and molecular docking. The in vivo hypoglycemic effects were assessed using a postprandial hyperglycemic mouse model. Additionally, potential mixture effects of flavonoid combinations were evaluated. Results: Of the 29 flavonoids, 16 demonstrated significant α-glucosidase inhibitory activity, with five (C3, C17, C19, C28, and C29) identified as novel inhibitors. Structure–activity relationship (SAR) analysis revealed that hydroxylation, particularly at the C-3 position, enhanced activity, while glycosylation and methoxylation reduced it. Mechanistic studies demonstrated that these compounds bind to distinct amino acid residues within the active site of α-glucosidase, inducing conformational changes and exerting different types of inhibition, leading to varying inhibitory mechanisms. Additionally, 15 compounds reduced postprandial blood glucose levels, with C3, C16, C17, C19, and C28 confirmed as novel in vivo inhibitors. Notably, two compositions of flavonoids combined at their individually ineffective concentrations exhibited significant inhibitory effects. Conclusions: This study provides a comprehensive evaluation of AR-related flavonoids as α-glucosidase inhibitors and offers valuable insights for the development of highly effective, low-toxicity, flavonoid-based, antidiabetic therapeutics and functional foods. Full article

HIV/AIDS and Mycobacterial tuberculosis (Mtb) are the leading cause of deaths worldwide. Thus, better medicaments are required to manage these diseases. Quinolines have shown great potential due to their broad spectrum of biological activity. Thus, quinoline–1,2,3-triazole–aniline hybrids were synthesised in moderate to good yields. Compounds 11g (IC₅₀ = 0.388 µM), 11h (IC₅₀ = 0.01032 µM) and 11i (IC₅₀ = 0.167 µM) exhibited the most promising in vitro activities against the wild-type HIV-1 subtype B, with 11h being 9-fold more active than AZT (IC₅₀ = 0.0909 µM), the reference drug. Furthermore, compound 11h displayed moderate activity, with a MIC₉₀ of 88μM against Mtb’s H37Rv strain. Cytotoxicity studies on TZM-bl cell lines revealed that most of the tested compounds were generally non-cytotoxic; the selectivity index (SI) for 11h, the front runner, is >2472. Molecular docking studies revealed that 11h interacted with Phe112, Tyr108, Glu283 and Trp86 amino acid residues in the active site of HIV-1. DFT studies revealed that 11h has the ability to donate and accept electrons to and from available orbitals. The predicted ADMET studies showed that these compounds possess drug-likeness, and 11h has the potential for further optimisation as an anti-HIV-1 agent. Full article