Key Amino Acid Residues Research Articles

Nitrilase GiNIT from Gibberella intermedia Efficiently Degrades Nitriles Derived from Rapeseed Meal Glucosinolate

Rapeseed meal is severely restricted in its utilization as unconventional animal feed due to anti-nutritive compounds, such as glucosinolate, that are degraded to toxic nitriles such as 3-butenenitrile and 4-pentenenitrile in animals. Few studies on nitrilases that can degrade glucosinolate-derived nitriles have been reported thus far. In the present study, a nitrilase gene GiNIT from Gibberella intermedia was over-expressed in Escherichia coli and the purified recombinant nitrilase rGiNIT showed specific activities of 134.48 U/mg and 122.16 U/mg when using 3-butenenitrile and 4-pentenenitrile as substrates at the optimal pH, 7.5, and temperature, 45 °C, which is the highest reported in the literature. The conversion of 3-butenenitrile and 4-pentenenitrile by rGiNIT reached 81.89% and 80.23% after hydrolysis for 15 min and 300 min, respectively. Site-directed mutagenesis and molecular docking analysis revealed that the catalytic ability of rGiNIT depended on the substrate binding pocket comprising 13 key amino acid residues. These results provide a potential enzyme resource for rapeseed meal detoxification and theoretical guidance for protein engineering.

Structure and function of the proton channel OTOP1

OTOP1 belongs to the otopetrin family of membrane proteins that form proton channels in cells of diverse types. In mammals, OTOP1 is involved in sour transduction in taste cells and contributes to otoconia formation in the inner ear. From the structural point of view, otopetrins, including OTOP1, represent a quasi-tetramer consisting of four α-barrels. The exact transport pathways mediating proton flux through the OTOP1 channel and gating units modulating its activity are still a matter of debate. This review discusses current data on structural and functional features of OTOP1. Suggested proton transport pathways, regulatory mechanisms, and key amino acid residues determining functionality of the otopetrins are considered. The existing kinetic models of OTOP1 are discussed as well. Based on revealed functional properties, OTOP1 is suggested to operate as a logical XOR element that allows for proton flux only if transmembrane pH gradient exists.

Efficient screening and discovery of umami peptides in Douchi enhanced by molecular dynamics simulations

In this study, a partial least squares discriminant analysis (PLS-DA) discriminant model for umami peptides was constructed based on molecular dynamics simulation data, achieving a R2 value of 0.949 and a Q2 value of 0.558. Using this novel model and bioinformatics screening methods, five new umami peptides (EALEATAQ, SPPTEE, SEEG, KEE, and FEE, with umami taste thresholds of 0.139, 0.085, 0.096, 0.060, and 0.079 mg/mL, respectively) were identified in Douchi. Molecular docking revealed that the residues ASN150 of T1R1, as well as SER170, GLU301 and GLN389 of T1R3, might be key amino acid residues for the binding of umami peptides to T1R1/T1R3. Molecular dynamics simulations revealed significant differences in the root-mean-square fluctuation (RMSF) values between the two complex systems of umami peptides-T1R1/T1R3 and non-umami peptides-T1R1/T1R3. The newly constructed umami peptide discriminant model can improve the accuracy of umami peptide screening and enhance the efficiency of discovering new umami peptides.

Synthesis, Characterization, and Therapeutic Potential of Novel Schiff Base Metal Complexes: Nickel(II) and Copper(II) Complexes Based 2‐[(2‐Chloroquinolin‐3‐yl)methylidene]aminobenzenethiol (CQAT)

ABSTRACTThis research focused on the design and comprehensive characterization of two novel transition metal complexes derived from the symmetrical Schiff base 2‐[(2‐chloroquinolin‐3‐yl)methylidene]aminobenzenethiol (CQAT), coordinated with nickel(II) (NiCQAT) and copper(II) (CuCQAT) ions. The structural elucidation of these complexes was achieved through a range of advanced analytical techniques. Thermal analysis revealed the stability and decomposition patterns of the complexes, supporting the identification of their coordination geometries. The collective data indicated that both NiCQAT and CuCQAT complexes adopt octahedral coordination, with the specific formulas [Ni(CQAT)2(H2O)2] and [Cu(CQAT)2(H2O)2], respectively. To complement the experimental findings, density functional theory (DFT) calculations were employed. These theoretical calculations confirmed the proposed molecular structures and provided detailed insights into key quantum chemical parameters, such as HOMO–LUMO energies, molecular orbitals, and electronic distributions, which are crucial for understanding the complexes' reactivity and stability. Furthermore, extensive in vitro biological evaluations were conducted to assess the anti‐inflammatory and antioxidant properties of the synthesized complexes. These assays demonstrated that both NiCQAT and CuCQAT exhibited significantly enhanced bioactivity compared to the free CQAT ligand, suggesting a synergistic effect of metal coordination on the ligand's biological efficacy. To further explore the mode of action, molecular docking studies were performed targeting two key proteins—human cyclooxygenase‐2 (PDB ID: 5IKT) and human peroxiredoxin 2 (PDB ID: 5IJT). The docking simulations provided valuable insights into the binding affinities, interaction energies, and key amino acid residues involved in the binding process, offering a molecular‐level understanding of their potential biological mechanisms. The findings from these multidisciplinary studies highlight the therapeutic potential of CQAT and its nickel and copper complexes, positioning them as promising candidates for further development in the field of medicinal chemistry.

Unraveling the molecular mechanism of FgGcn5 inhibition by phenazine-1-carboxamide: combined in silico and in vitro studies.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum (F. graminearum), remains a devastating disease worldwide. The histone acetyltransferase Gcn5 plays a crucial role in epigenetic regulation. Aberrant Gcn5 acetylation activity can result in serious impacts such as impaired growth and development in organisms. The secondary metabolite phenazine-1-carboxamide (PCN) inhibits F. graminearum by blocking the acetylation process of Gcn5 (FgGcn5), and is currently used to control FHB. However, the molecular basis of acetylation inhibition by PCN remains to be further explored. Our molecular dynamics simulations revealed that PCN binds to the cleft in FgGcn5 where histone H3 is bound, with key amino acid residues including Leu96 (L96), Arg121 (R121), Phe133 (F133), Tyr169 (Y169), and Tyr201 (Y201), preventing FgGcn5 from binding to histone H3 and affecting histone H3 from being acetylated. Experimental validation of key amino acid mutations further confirmed the impact of these mutations on the interaction of FgGcn5 with PCN and histone H3 peptide. In summary, our study sheds light on the mechanism by which PCN inhibits the acetylation function of FgGcn5, providing a foundation for the development of drugs or fungicides targeting histone acetyltransferases. © 2024 Society of Chemical Industry.

Biological Characteristics of the Cytochrome P 450 Family and the Mechanism of Terpinolene Metabolism in Hyalomma asiaticum (Acari: Ixodidae).

The CYP450 enzyme is a superfamily enzyme ubiquitously found in nearly all organisms, playing a vital role in the metabolism of both endogenous and exogenous compounds, and in biosynthesis. Unfortunately, an understanding of its classification, functions, expression characteristics, and other biological traits in Hyalomma asiaticum, a vector for Crimean-Congo Hemorrhagic Fever, as well as of the genes implicated in its natural product metabolism, is lacking. Towards this end, this study has identified 120 H. asiaticum CYP450 genes via transcriptome data in the face of a joint genome threat from terpinolene. The proteins these genes encode are of higher molecular weight, devoid of a signal peptide, and composed of unstable hydrophobic proteins principally containing 1-3 variable transmembrane regions. Phylogenetic evolution classifies these H. asiaticum CYP450 genes into four subfamilies. These genes all encompass complete CYP450 conserved domains, and five specific conserved motifs, albeit with different expression levels. GO and KEGG annotation findings suggest a widespread distribution of these CYP450 genes in many physiological systems, predominantly facilitating lipid metabolism, terpenoid compound metabolism, and polyketone compound metabolism, as well as cofactor and vitamin metabolism at a cellular level. Molecular docking results reveal a hydrophobic interaction between the ARG-103, ARG-104, LEU-106, PHE-109, and ILE-119 amino acid residues in CYP3A8, which is primarily expressed in the fat body, and terpinolene, with a notably up-regulated expression, with affinity = -5.6 kcal/mol. The conservation of these five key amino acid residues varies across 12 tick species, implying differences in terpinolene metabolism efficacy among various tick species. This study thereby fills an existing knowledge gap regarding the biological characteristics of H. asiaticum CYP450 genes and paves the way for further research into the functions of these particular genes.

Molecular Engineering L-Aspartate-Alpha-Decarboxylase to Enhance Catalytic Stability and Performance.

L-aspartate-alpha-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the decisive step in the biosynthesis of β-alanine. However, the low catalytic stability and efficiency of ADC limit its industrial applications. In this study, a variant of ADC from Bacillus subtilis were used as a starting point for engineering. After constructing a random mutagenesis library by error-prone PCR, followed by high-throughput screening,four substitutions (S7 N, K63 N, A99T, and K113R) were identified. By screening saturation mutagenesis libraries on these positions and computational analysis, two recombined variants N3(S7 N/K63 N/I88 M/A99E/K113R/I126*) and Y1(S7Y/K63 N/I88 M/A99E/K113R/I126*) with improved performance were obtained. Compared to the wild type, the catalytic efficiency and catalytic stability of the best two variants were enhanced up to 95 %(variant N3) and up to 89 %(variant Y1), respectively. In addition, Y1 exhibited 3.37 times improved half-life and 2-fold improved total turnover number. Hydrophilicity analysis and molecular dynamics (MD) simulation revealed that the increased hydrophilicity and steric hindrance of key amino acid residues would affect the catalytic activity and stability. The improved catalytic performance of the variants could be attributed to their enhanced binding capacity to the substrate within the active pocket and the alleviation of mechanism-based inactivation.

Genetically encoded green fluorescent sensor for probing sulfate transport activity of solute carrier family 26 member a2 (Slc26a2) protein

Genetically encoded fluorescent biosensors became indispensable tools for biological research, enabling real-time observation of physiological processes in live cells. Recent protein engineering efforts have resulted in the generation of a large variety of fluorescent biosensors for a wide range of biologically relevant processes, from small ions to enzymatic activity and signaling pathways. However, biosensors for imaging sulfate ions, the fourth most abundant physiological anion, in mammalian cells are still lacking. Here, we report the development and characterization of a green fluorescent biosensor for sulfate named Thyone. Thyone, derived through structure-guided design from bright green fluorescent protein mNeonGreen, exhibited a large negative fluorescence response upon subsecond association with sulfate anion with an affinity of 11 mM in mammalian cells. By integrating mutagenesis analyses with molecular dynamics simulations, we elucidated the molecular mechanism of sulfate binding and revealed key amino acid residues responsible for sulfate sensitivity. High anion selectivity and sensitivity of Thyone allowed for imaging of sulfate anion transients mediated by sulfate transporter heterologously expressed in cultured mammalian cells. We believe that Thyone will find a broad application for assaying the sulfate transport in mammalian cells via anion transporters and exchangers.

Impact of the key residues of 1-Deoxy-D-Xylulose-5-Phosphate synthase on its catalysis

1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in the 2-methyl-D-erythritol-4-phosphate (MEP) terpenoid biosynthetic pathway. To explore the active sites of the protein, we made a series of mutations to the key amino acid residues of Escherichia coli DXS including H49, R420, D427, and R478. The results show that several mutants including H49Q lose almost all their catalytic activity in DXP synthesis, but the double-point mutants H49QD427H and H49QD427 N recover 100 % activity of the wild type enzyme. The kinetic characterization displays that the affinity of the double-point mutants for pyruvate and D-glyceraldehyde-3-phosphate only slightly decreases. We also assessed impact of the mutation on the oxidative decarboxylation activity of all the mutants. Despite the fact that H49QD427H and H49QD427 N get back the full activity with regard to DXP synthesis, their oxidase activity remains unrestored. Furthermore, we assayed the acceptor substrate spectrum of the mutants and the results show that a large portion of them catalyzes ligation between pyruvate and nitrosobenzene to form N-phenyl-N-hydroxyacetamide, whereas R420D, R420F, and D427H exhibit activity in connection of pyruvate and benzaldehyde to afford phenylacetocarbinol (PAC), with the first two mutants yielding (S)-PAC (yields 21 %-25 %, ee values 71–75 %) while D427H furnishing (R)-PAC (yield 41 %, ee value 91 %), implying the key role of D427 for the acceptor substrate recognition and the stereoselectivity of the enzyme. It is worth noting that this is the first report of DXS mutants that can recognize benzaldehyde as an acceptor substrate and generate both enantiomers of PAC.

6-hydroxy-3-succinoylsemialdehyde-pyridine as a potential inhibitor of schizophrenia-associated enzyme

Schizophrenia’s cognitive deficits limit quality of life. Current drugs are ineffective. This study investigates 6-hydroxy-3-succinoylsemialdehyde-pyridine (6H3SAP), a nicotine derivative, as a potential inhibitor of KATII, an enzyme in the kynurenine pathway linked to cognitive dysfunction. Molecular docking was employed to assess the binding affinity of 6H3SAP to the KATII enzyme. The results indicated that 6H3SAP interacted with the KATII active site, demonstrating a binding affinity comparable to nicotine (previously suggested as a potential KATII blocker) but lower than NS1502, a well-established KATII inhibitor. Subsequent molecular dynamics simulations provided further insights into the interaction details between 6H3SAP and KATII. These simulations revealed the formation of stable interactions between 6H3SAP and key amino acid residues within the KATII binding pocket. Though promising, further research is needed to assess 6H3SAP’s efficacy and safety. Surprisingly, the molecular dynamics data also proposed that the nicotine-KATII tie is weak.

Molecular insights and functional analysis of isocitrate dehydrogenase in two gram-negative pathogenic bacteria.

Klebsiella pneumoniae and Legionella pneumophila are common Gram-negative bacteria that can cause lung infections. The multidrug resistance of K. pneumoniae presents a significant challenge for treatment. This study focuses on isocitrate dehydrogenase (IDH), a key enzyme in the oxidative metabolic pathway of these two bacteria. KpIDH and LpIDH were successfully overexpressed and purified, and their biochemical characteristics were thoroughly investigated. The study revealed that KpIDH and LpIDH are homodimeric enzymes with molecular weights of approximately 70kDa. They are completely dependent on the coenzyme NADP+ and are inactive towards NAD+. KpIDH exhibits the highest catalytic activity at pH 8.0 in the presence of Mn2+ and at pH 7.8 in the presence of Mg2+. Its optimal catalytic performance is achieved with both ions at 55°C. LpIDH exhibited its highest activity at pH 7.8 in the presence of Mn2+ and Mg2+, respectively, and exhibits optimal catalytic performance at 45°C. Heat inactivation studies showed that KpIDH and LpIDH retained over 80% of their activity after being exposed to 45°C for 20min. Furthermore, we successfully altered the coenzyme specificity of KpIDH and LpIDH from NADP+ to NAD+ by replacing four key amino acid residues. This study provides a comprehensive biochemical characterization of two multidrug-resistant bacterial IDHs commonly found in hospital environments. It enhances our understanding of the characteristics of pathogenic bacteria and serves as a reference for developing new therapeutic strategies.

Molecular Mechanisms for the Selective Transport of Dichlorofluorescein by Human Organic Anion Transporting Polypeptide 1B1.

Human organic anion transporting polypeptide (OATP) 1B1 and 1B3 are two highly homologous liver-specific uptake transporters. However, 2',7'-dichlorofluorescein (DCF) is preferably transported by OATP1B1. In the present study, the molecular mechanisms for the selective transport of DCF by OATP1B1 were investigated by constructing and characterizing an array of OATP1B1/1B3 chimeras and site-directed mutagenesis. Our results show that transmembrane domain (TM) 10 is crucial for the surface expression and function of OATP1B1, in which Q541 and L545 play the most important roles in DCF transport. Replacement of TM10 in OATP1B1 with its OATP1B3 counterpart led to OATP1B1's complete intracellular retention. Q541 and L545 may interact with DCF directly via hydrogen bonding and hydrophobic interactions. The decrease of DCF uptake by Q541A and L545S was due to their reduced binding affinity for DCF as compared with OATP1B1. In addition, Q541 and L545 are also crucial for the transport of estradiol-17β-glucuronide (E17βG) but not for the transport of estrone-3-sulfate (E3S), indicating different interaction modes between DCF/E17βG and E3S in OATP1B1. Taken together, Q541 and L545 in TM10 are critical for OATP1B1-mediated DCF uptake, but their effect is substrate-dependent. SIGNIFICANCE STATEMENT: The key TMs and amino acid residues for the selective transport of DCF by OATP1B1 were identified. TM10 is crucial for the surface expression and function of OATP1B1. Within TM10, Q541 and L545 played the most significant roles and affected the function of OATP1B1 in a substrate-dependent manner. This information is crucial for a better understanding of the mechanism of the multispecificity of OATP1B1 and as a consequence the mechanism of OATP1B1-mediated drug-drug interactions.

NnM469, a novel recombinant jellyfish venom metalloproteinase from Nemopilema nomurai, disrupted the cell matrix

Molecular cloning and functional characterization of Nemopilema nomurai venom metalloproteinases have provided deeper insights into the pathogenesis of jellyfish dermatitis. This study reports a new cDNA clone from N. nomurai tentacle venom (Transcript sequence: ID469) encoding 362 amino acid residues, belonged to astacin family and capable of disrupting the cell matrix. The N. nomurai metalloproteinase 469 (NnM469) comprises a signal peptide and propeptide, followed by metalloproteinase domain containing a zinc-binding motif, and two ShKT domains. Notably, NnM469 features a zinc-binding motif (HEXXH) at the active site, within an extended sequence of HEXXHXXGFXHE, which is unique to astacin. Immunocytochemistry revealed that NnM469 is located in the stab tube and envelope of jellyfish nematocysts. Western blot and LC-MS/MS analysis confirmed that the NnM469 protein was successfully expressed using the Pichia pastoris expression system. The recombinant NnM469 could degrade the cell matrix, resulting in the death of HaCaT cells with an IC50 of 26.34 μg/mL. Finally, I-TASSER-generated structure and function predictions indicated that conserved Asp53, His168, His172, His178, and Tyr227 serve as key amino acid residues for the Zn2+ ion binding in the catalytic center. In summary, the study of the molecular characteristics and function of NnM469 presents an opportunity to develop therapeutic interventions for jellyfish venom-induced dermatitis.

Malate dehydrogenase: a story of diverse evolutionary radiation.

Malate dehydrogenase (MDH) is a ubiquitous enzyme involved in cellular respiration across all domains of life. MDH's ubiquity allows it to act as an excellent model for considering the history of life and how the rise of aerobic respiration and eukaryogenesis influenced this evolutionary process. Here, we present the diversity of the MDH family of enzymes across bacteria, archaea, and eukarya, the relationship between MDH and lactate dehydrogenase (LDH) in the formation of a protein superfamily, and the connections between MDH and endosymbiosis in the formation of mitochondria and chloroplasts. The development of novel and powerful DNA sequencing techniques has challenged some of the conventional wisdom underlying MDH evolution and suggests a history dominated by gene duplication, horizontal gene transfer, and cryptic endosymbiosis events and adaptation to a diverse range of environments across all domains of life over evolutionary time. The data also suggest a superfamily of proteins that do not share high levels of sequential similarity but yet retain strong conservation of core function via key amino acid residues and secondary structural components. As DNA sequencing and 'big data' analysis techniques continue to improve in the life sciences, it is likely that the story of MDH will continue to refine as more examples of superfamily diversity are recovered from nature and analyzed.

SARS-CoV-2 ORF 3a-mediated currents are inhibited by antiarrhythmic drugs.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to cardiovascular complications, notably cardiac arrhythmias. The open reading frame (ORF) 3a of the coronavirus genome encodes for a transmembrane protein that can function as an ion channel. The aim of this study was to investigate the role of the SARS-CoV-2 ORF 3a protein in COVID-19-associated arrhythmias and its potential as a pharmacological target. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and cultured human fibroblasts were infected with SARS-CoV-2. Subsequent immunoblotting assays revealed the expression of ORF 3a protein in hiPSC-CM but not in fibroblasts. After intracytoplasmic injection of RNA encoding ORF 3a proteins into Xenopus laevis oocytes, macroscopic outward currents could be measured. While class I, II, and IV antiarrhythmic drugs showed minor effects on ORF 3a-mediated currents, a robust inhibition was detected after application of class III antiarrhythmics. The strongest effects were observed with dofetilide and amiodarone. Finally, molecular docking simulations and mutagenesis studies identified key amino acid residues involved in drug binding. Class III antiarrhythmic drugs are potential inhibitors of ORF 3a-mediated currents, offering new options for the treatment of COVID-19-related cardiac complications.

Scaffold hopping approach to the novel hexacyclic pyrazol-3-amide derivatives as potential multi-target insect growth regulators candidates

Ecdysone receptor (EcR) and three insect chitinases (OfChtI, OfChtII, and OfChi-h) are considered as attractive targets for the development of novel insect growth regulators (IGRs) since they are closely related to the insect molting. In this study, to develop potent multi-target IGRs, a series of hexacyclic pyrazol-3-amide derivatives were rationally designed by utilizing the scaffold hopping strategy with the previously reported compound 6j (N-(4-bromobenzyl)-2-phenyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide) as a lead compound. The bioassay results indicated that most of the target compounds exhibited obvious insecticidal activity. Especially, compounds a5 and a21 displayed excellent insecticidal activities against P. xylostella with LC50 values of 82.29 and 69.45 mg/L, respectively, exceeding that of 6j (263.78 mg/L). Compounds a5 and a21 also dramatically disturbed the growth and development of O. furnacalis larvae, and their LC50 values were 124.71 and 127.54 mg/L, respectively, superior to the lead 6j (267.33 mg/L). The action mechanism study revealed that the most active compound a21 could act simultaneously on EcR (21.4 % binding activity at 8 mg/L), OfChtI (94.9 % inhibitory at 10 μM), OfChtII (23.1 % inhibitory at 10 μM), and OfChi-h (94.3 % inhibitory at 10 μM), significantly higher than that of the lead compound 6j. The result of molecular docking indicated that transferring the carboxamide group from pyrazole position 5 to 3 enhanced the interactions of a21 with the key amino acid residues of the OfChtI, OfChtII, and OfChi-h, resulting in stronger affinity to the three targets than 6j. The present work offers a useful guidance for the further development of novel multi-target IGRs.

Exploring the salty peptides of enzymatically hydrolyzed Volvariella volvacea protein: Structural analysis and taste perception insights

This study aimed to prepare and screen salty peptides and elucidate the factors triggering their saltiness properties and saltiness-enhancing effects. Electronic tongue results indicated that samples treated with Alcalase (AJ) exhibited the highest saltiness intensity (5.05 ± 0.11) and greater saltiness-enhancing compared to those treated with Flavourzyme (AF), Neutrase (AZ), and Papain (AM). Peptides identified by LC-MS/MS were docked to the TMC4 receptor, resulting in 23 peptides with good docked results. The key amino acid residues (Thr431, Arg433, Phe440, Phe432, and Ser273) were discovered. The salty peptides WPGFK (633.75 Da), YFDWPGFK (1059.17 Da), and YFDWPGFKT (1160.27 Da) were selected for salt reduction and molecular characterization studies. Electronic tongue results showed that the 0.01 % WPGFK solution matched the saltiness of the 0.24 % NaCl solution and demonstrated superior saltiness-enhancing compared to YFDWPGFK and YFDWPGFKT. Primary sequence analysis of three salty peptides suggested that the WPG amino acid segment might play a crucial role in generating saltiness. Circular dichroism (CD) analysis of the secondary structure indicated that increased random coil and decreased β-turn contents resulted in higher saltiness perception and increased random coil content led to greater saltiness-enhancing ability.

Reactivity of (3‐(4‐Fluorophenyl)oxyran‐2‐yl)(naphthalene‐2‐yl)methanone Toward Some Nucleophiles for Preparing Promising Agents with Antimicrobial and Antioxidant Activities

Abstract(3‐(4‐Fluorophenyl)oxyran‐2‐yl)(naphthalene‐2‐yl)methanone 2 was prepared from the reaction of 3‐(4‐fluorophenyl)‐1‐(naphthalen‐2‐yl)prop‐2‐en‐1‐one (1) with hydrogen peroxide and reacted with different nucleophiles such as hydrazine hydrate, phenyl hydrazine, thiosemicarbazide to give 4‐hydroxy‐1H‐pyrazole derivatives 3a,b‐4. Also, compound 2 was treated with hydroxylamine hydrochloride to give the substituted 2,3‐dihydroisoxazole 5. Similarly, compound 2 was treated with carbon disulfide, phenyl isothiocyanate, and phenyl isocyanate to give the corresponding compounds 6–8. Various spectroscopic techniques and elemental analysis were used to determine the molecular structures of the new derivatives. All the new analogs were screened against five human pathogenic microbial strains: Escherichia coli and Pseudomonas aeruginosa as Gram‐negative bacteria, Bacillus subtilis and Staphylococcus aureus as Gram‐positive bacteria and the yeast Candida albicans using agar diffusion method. Compound 4 exhibited moderate antimicrobial activity against all the tested microorganisms compared to positive control ciprofloxacin and fluconazole. On the other hand, the free radical scavenging activity of the new compounds using DPPH assay protocol revealed that most of the compounds have a weak reducing activity. Moreover, compounds 4 and 8 were docked against 1KZN protein (DNA gyrase), 3FYV and 5V5Z showing the ability to interact with key amino acid residues and the potential to act as inhibitors.

PocketDTA: an advanced multimodal architecture for enhanced prediction of drug-target affinity from 3D structural data of target binding pockets.

Accurately predicting the drug-target binding affinity (DTA) is crucial to drug discovery and repurposing. Although deep learning has been widely used in this field, it still faces challenges with insufficient generalization performance, inadequate use of 3D information, and poor interpretability. To alleviate these problems, we developed the PocketDTA model. This model enhances the generalization performance by pre-trained models ESM-2 and GraphMVP. It ingeniously handles the first 3 (top-3) target binding pockets and drug 3D information through customized GVP-GNN Layers and GraphMVP-Decoder. In addition, it uses a bilinear attention network to enhance interpretability. Comparative analysis with state-of-the-art (SOTA) methods on the optimized Davis and KIBA datasets reveals that the PocketDTA model exhibits significant performance advantages. Further, ablation studies confirm the effectiveness of the model components, whereas cold-start experiments illustrate its robust generalization capabilities. In particular, the PocketDTA model has shown significant advantages in identifying key drug functional groups and amino acid residues via molecular docking and literature validation, highlighting its strong potential for interpretability. Code and data are available at: https://github.com/zhaolongNCU/PocketDTA.

Daphnetin weakened the pathogenicity of methicillin-resistant Staphylococcus aureus by inhibiting Sortase A and α-hemolysin

The increasing prevalence of antibiotic-resistant bacteria, represented by Methicillin-resistant Staphylococcus aureus (MRSA), has necessitated a shift towards anti-virulence strategies in treatment approaches. This research demonstrated that daphnetin effectively disrupted MRSA virulence by targeting Sortase A (SrtA), an enzyme in Staphylococcus aureus (S. aureus) responsible for adhesion and invasion, as well as the toxin α-hemolysin (Hla) that leads to cell lysis. Utilizing Fluorescence Resonance Energy Transfer, daphnetin showed direct inhibitory effect on SrtA activity, with an IC50 of 25.98 μg/mL. Additionally, daphnetin hindered various SrtA-mediated processes in S. aureus, such as fibronectin adherence, A549 cell invasion, biofilm formation, and bacterial motility. Daphnetin inhibited S. aureus-induced hemolysis and reduced Hla expression as confirmed by Western blot analysis. Molecular docking studies identified specific binding sites of daphnetin with SrtA, highlighting key amino acid residues like GLU-77, TYR-75, and LYS-145, with a docking score of −7.139 kcal/mol. Besides that, daphnetin exhibited a protective effect on MRSA-induced pneumonia in vivo. In summary, daphnetin, a natural compound, effectively inhibited SrtA and Hla activities, attenuating MRSA virulence and showcasing potential for treating bacterial infections.