Amino Acid Residues Research Articles

Development and assessment of novel pyrazole-thiadiazol hybrid derivatives as VEGFR-2 inhibitors: design, synthesis, anticancer activity evaluation, molecular docking, and molecular dynamics simulation.

Cancer remains a significant health challenge globally, requiring the development of targeted chemotherapeutics capable of specifically inhibiting cancer cell growth. Angiogenesis is one of the key features of tumor growth and metastasis and is, therefore, an important target for the treatment of many tumors. The vascular endothelial growth factor (VEGF) signaling pathway has proven to be a promising lead in anticancer therapy due to the central role it plays in tumor angiogenesis. Vascular endothelial growth factor receptor-2 (VEGFR-2) is a key mediator in the signaling pathway regulating angiogenesis. Targeting VEGFR-2 may disrupt angiogenesis, leading to a reduction in tumor blood supply and tumor progression. The design, synthesis, and assessment of novel VEGFR-2 inhibitor derivatives are the focus of this study, with particular emphasis on incorporating the pyrazole-thiadiazol pharmacophore into the molecular structure. Taking advantage of the pharmacophoric properties of pyrazole and 1,3,4-thiadiazol, compounds with different substituents in the main structure were designed and synthesized. The compounds were also evaluated for antiproliferative activity against cancer cell lines. Compound 4e demonstrated the highest activity among all compounds, with an IC50 of 9.673±0.399 μM against HT-29 cells and 23.081±0.400 μM against NIH3T3 cells. To further support the inhibitory activity of compound 4e, an in silico study was performed. Compound 4e demonstrated strong binding to the active site of VEGFR-2 in molecular docking studies, forming hydrogen bonds with key amino acid residues. The stability of the compound in the enzyme's active site was demonstrated through molecular dynamics simulations.

Effects of Phosphorylation on Protein Backbone Dynamics and Conformational Preferences.

Phosphorylations are the most common and extensively studied post-translational modification (PTM) of proteins in eukaryotes. They constitute a major regulatory mechanism, modulating protein function, protein-protein interactions, as well as subcellular localization. Phosphorylation sites are preferably located in intrinsically disordered regions and have been shown to trigger structural rearrangements and order-to-disorder transitions. They can therefore have a significant effect on protein backbone dynamics or conformation, but only sparse experimental data are available. To obtain a more general description of how and when phosphorylations have a significant effect on protein behavior, molecular dynamics (MD) currently provides the only suitable framework to study these effects at a large scale in atomistic detail. This study develops a systematic MD simulation framework to explore the influence of phosphorylations on the local backbone dynamics and conformational propensities of proteins. Through a series of glycine-backbone peptides, we studied the effects of amino acid residues including the three most common phosphorylations (Ser, Thr, and Tyr), on local backbone dynamics and conformational propensities. We further extended our study to investigate the interactions of all such residues between position i to positions i + 1, i + 2, i + 3, and i + 4 in such peptides. The final data set comprises structural ensembles for 3393 sequences with more than 1 μs of sampling for each ensemble. To validate the relevance of the results, the structural and conformational properties extracted from the MD simulations are compared to NMR data from the Biological Magnetic Resonance Data Bank. The systematic nature of this study enables the projection of the gained knowledge onto any phosphorylation site in the proteome and provides a general framework for the study of further PTMs. The full data set is publicly available, as a training and reference set.

Enzymes in Synergy: Bacteria Specific Molecular Probe for Locoregional Imaging of Urinary Tract Infection in vivo

Uropathogenic Escherichia coli (UPECs) is a leading cause for urinary tract infections (UTI), accounting for 70‐90% of community or hospital‐acquired bacterial infections owing to high recurrence, imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor‐intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first‐in‐class near‐infrared (NIR) UPECs fluorescent probe (NO‐AH) capable of specifically targeting UPECs through its collaborative response to bacterial enzymes, enabling locoregional imaging of UTIs both in vitro and in vivo. Our NO‐AH probe incorporates a dual protease activatable moiety, which first reacts with OmpT, an endopeptidase abundantly present on outer membrane of UPECs, releasing an intermediate amino acid residue conjugated with a NIR hemicyanine fluorophore. Such liberated fragment would be subsequently recognized by aminopeptidase (APN) within periplasm of UPECs, activating localized fluorescence for precise imaging of UTIs in complex living environments. The peculiar specificity and selectivity of NO‐AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs‐infected UTIs, which could overcome misdiagnosis in conventional urine tests, thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.

Discovery of putative inhibitors of human Pkd1 enzyme: Molecular docking, dynamics and simulation, QSAR, and MM/GBSA

Polycystic kidney disease is the most prevalent hereditary kidney disease globally and is mainly linked to the overexpression of a gene called PKD1. To date, there is no effective treatment available for polycystic kidney disease, and the practicing treatments only provide symptomatic relief. Discovery of the compounds targeting the PKD1 gene by inhibiting its expression under the disease condition could be crucial for effective drug development. In this study, a molecular docking and molecular dynamic simulation, QSAR, and MM/GBSA-based approaches were used to determine the putative inhibitors of the Pkd1 enzyme from a library of 1379 compounds. Initially, fourteen compounds were selected based on their binding affinities with the Pkd1 enzyme using MOE and AutoDock tools. The selected drugs were further investigated to explore their properties as drug candidates and the stability of their complex formation with the Pkd1 enzyme. Based on the physicochemical and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties, and toxicity profiling, two compounds including olsalazine and diosmetin were selected for the downstream analysis as they demonstrated the best drug-likeness properties and highest binding affinity with Pkd1 in the docking experiment. Molecular dynamic simulation using Gromacs further confirmed the stability of olsalazine and diosmetin complexes with Pkd1 and establishing interaction through strong bonding with specific residues of protein. High biological activity and binding free energies of two complexes calculated using 3D QSAR and Schrodinger module, respectively further validated our results. Therefore, the molecular docking and dynamics simulation-based in-silico approach used in this study revealed olsalazine and diosmetin as potential drug candidates to combat polycystic kidney disease by targeting Pkd1 enzyme.

Enzymes in Synergy: Bacteria Specific Molecular Probe for Locoregional Imaging of Urinary Tract Infection in vivo.

Uropathogenic Escherichia coli(UPECs)is a leading cause for urinary tract infections (UTI), accounting for 70-90% of community or hospital-acquired bacterial infections owing to high recurrence,imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor-intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first-in-class near-infrared (NIR) UPECsfluorescent probe (NO-AH) capable of specifically targetingUPECsthrough itscollaborativeresponse to bacterial enzymes,enabling locoregional imaging of UTIs both in vitroand in vivo.Our NO-AH probe incorporatesa dual protease activatable moiety, which first reactswithOmpT, an endopeptidase abundantly present on outer membrane of UPECs, releasing an intermediate aminoacid residue conjugatedwith a NIR hemicyanine fluorophore. Suchliberated fragment would be subsequentlyrecognized by aminopeptidase (APN) within periplasm of UPECs, activating localized fluorescence forprecise imagingof UTIs in complex living environments.The peculiar specificity and selectivity of NO-AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs-infected UTIs, which could overcomemisdiagnosis inconventional urine tests,thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.

Production of antigens expressed in Nicotiana benthamiana plant and Escherichia coli for the SARS-CoV-2 IgG antibody detection by ELISA

The recent COVID-19 pandemic disclosed a critical shortage of diagnostic kits worldwide, emphasizing the urgency of utilizing all resources available for the development and production of diagnostic tests. Different heterologous protein expression systems can be employed for antigen production. This study assessed novel SARS-CoV-2 proteins produced by a transient expression system in Nicotiana benthamiana utilizing an infectious clone vector based on pepper ringspot virus (PepRSV). These proteins included the truncated S1-N protein (spike protein N-terminus residues 12–316) and antigen N (nucleocapsid residues 37–402). Two other distinct SARS-CoV-2 antigens expressed in Escherichia coli were evaluated: QCoV9 chimeric antigen protein (spike protein residues 449–711 and nucleocapsid protein residues 160–406) and QCoV7 truncated antigen (nucleocapsid residues 37–402). ELISAs using the four antigens individually and the same panel of samples were performed for the detection of anti-SARS-CoV-2 IgG antibodies. Sensitivity was evaluated using 816 samples from 351 COVID-19 patients hospitalized between 5 and 65 days after symptoms onset; specificity was tested using 195 samples collected before 2018, from domiciliary contacts of leprosy patients. Our findings demonstrated consistent test sensitivity, ranging from 85 % to 88 % with specificity of 97.5 %, regardless of the SARS-CoV2 antigen and the expression system used for production. Our results highlight the potential of plant expression systems as useful alternative platforms to produce recombinant antigens and for the development of diagnostic tests, particularly in resource-constrained settings.

Synthesis, DFT investigation, ADME-T properties, molecular docking and molecular dynamics simulation of new α-aminophosphonate inhibitor targeting Mpro and RdRp enzymes in SARS-CoV-2

A specific α-aminophosphonate molecule, namely diethyl (phenyl (4-pyridinylamino) methyl) phosphonate (α-DAP), as a potential SARS-CoV-2 inhibitor, has been synthesized via a Kabachnick-Fields reaction, a three-component, dalfampridine, benzenecarbaldehyde and triethoxyphosphine, and its molecular structure was confirmed by spectroscopic and physicochemical methods such FT-IR, UV–vis, melting point, 1H NMR, 13C NMR and 31P NMR spectra. The Density Functional Theory (DFT) at the B3LYP/6–31 G (d, p) level was used to calculate the optimized structure, reactivity, stability of the target molecule. In this context, several characteristics have been calculated and the obtained theoretical results are in good agreement with the experimental ones. Also, predicted ADME-T were performed and the obtained parameters indicated that the compound under investigation should have good oral bioavailability. In addition, the molecular docking has been used, further, the compound showed high potential docking with SARS-CoV-2 major protease (Mpro) and RNA dependent RNA polymerase (RdRp). Finally, the molecular dynamics simulation demonstrated the stability of the protein-ligand interaction and offered insights into the specific amino acid residues participating in the binding process.

Synthesis, structural, spectral, Anticancer activity, and density functional theory investigations of 2- [hydrazinylidene(phenyl)methyl]pyridine

The condensation reaction of equimolar amounts of 2-benzoylpyridine and hydrazine hydrate to produce hydrazone, 2- [hydrazinylidene(phenyl)methyl]pyridine (HPMP) with a mass spectral peak at m/z = 197. The compound showed 15N NMR peaks at 330.79, 306.64, and 110.45 ppm due to CN (azomethine), pyridine nitrogen and —NH2 nitrogen atoms. The crystal structure data, IR, and Raman studies were correlated with DFT/B3LYP/6–311++G(d,p) calculations. The compound exhibited strong fluorescence at 592 and 481 nm when excited at 290 and 210 nm, respectively. NBO Fock matrix analyses indicated the existence of different π and π* energy levels, and the molecule can undergo internal charge transfer and vibrational relaxation. The IC-50 value for HPMP was found to be 62.5 µg/ml against the MCF-7 breast cancer cell line as shown by the MTT assay. The IC-50 value for HPMP was higher than the standard doxorubicin but expected to have fewer side effects. Fluorescence microscopy analyses showed significant cell mortality compared to that of the control. The NH2 group in HPMP has been shown to interact with Asp73 (A) (2.89 Å) and ASM46 (A) (2.90 Å) through H-bonding with the DNA gyrase protein (Protein ID: 1KZN). The other selected protein residues were made hydrophobic by pyridine and phenyl rings to inactivate DNA gyrase protein. Therefore, HPMP can be used as an anticancer drug and for optical LED applications.

Two novel angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibiting peptides from tilapia (Oreochromis mossambicus) skin and their molecular docking mechanism.

In the study, papain was used to hydrolyze tilapia (Oreochromis mossambicus) skin to obtain a tilapia skin hydrolysate (TSH) with dual angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. The resulting TSH was sequentially fractionated by ultrafiltration, size exclusion separation chromatography, and reverse-phase high-performance liquid chromatography. Its inhibitory effects on ACE and DPP-IV were determined by commercial reagent kits. Two peptides purified from TSH were identified as Gly-Pro-Leu-Gly-Ala-Leu (GPLGAL) and Lys-Pro-Ala-Gly-Asn (KPAGN) by the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Inhibitory concentration (IC50) of GPLGAL on ACE and DPP-IV were 117.20±1.69 and 187.10±2.75µM, respectively. IC50 of KPAGN on ACE and DPP-IV were 137.40±2.33 and 259.20±2.85µM, respectively. The molecular simulation demonstrated that the binding affinities of GPLGAL to ACE and DPP-IV proteins were -8.5 and -7.4kcal/mol, respectively, whereas those of KPAGN to ACE and DPP-IV proteins were -7.9 and -6.7kcal/mol, respectively. GPLGAL interacted with 21 amino acid residues of the ACE active site, whereas KPAGN engaged with 19 amino acid residues. Additionally, GPLGAL interacted with 10 amino acid residues of the DPP-IV active site, whereas KPAGN engaged with 13 amino acid residues. The two peptides predominantly occupied the active sites of ACE (His513, Tyr523, and Ala354) and DPP-IV (Tyr662 and Arg125) through hydrogen bonding. This leads to the deactivation of ACE and DPP-IV. PRACTICAL APPLICATION: Accelerate tilapia skin development and high-value utilization; provide foundation for preparing the peptides with dual ACE and DPP-IV inhibiting activity.

Coronavirus Inhibitory Activity of Tamarind Indica

Background: SARS-COVID-19 is an infectious disease, the causative agent Caroni virus. WHO announced the pandemic on 3rd November 2020 to the whole world. Objective: Severe Acute Respiratory Syndrome COVID-19 is an infectious disease globally declared a pandemic by WHO. There is a need to find the proper medication for recovery. The study uses the molecular docking method to predict the anti-covid activity of plant phytoconstituents of Tamarind indica. Methods: Molecular docking techniques were accomplished to search the binding pattern of plant phytoconstituents of T. indica against the crystal structure SARS-CoV-2 enzyme (PDB ID: 6LU7) with the help of PyRx virtual screening software to study the amino acid interaction and inhibitory potential of phytoconstituents of T. indica. In addition, we performed a pharmacokinetic and toxicological study of plant phytoconstituents of T. indica using SwissADME and the pkCSM online server. Results: The phytoconstituents of Plant T. indica docking results proposed that apigenin (-7.8 kcal/mol), epicatechin (-7.1 kcal/mol) and taxifolin (-7.5 kcal/mol) show the best binding energy as compared to favipiravir (-5.2 kcal/mol). The phytoconstituents exposed promising interaction with amino acid residue, leading to an inhibitory effect against the SARS-CoV-2 enzyme (PDB ID: 6LU7). Further, ADMET studies showed that pharmacokinetics and toxicological parameters are within acceptable limits. Conclusion: In silico study revealed that the phytochemicals of T.indica show promising inhibitory results against the SARS-CoV-2 enzyme (PDB ID: 6LU7). Moreover, the traditional benefits of T.indica were clinical treatment and drug discovery.

Discovery of Substituted 2-oxoquinolinylthiazolidin-4-one Analogues as Potential EGFRK Inhibitors in Lung Cancer Treatment.

Cancer is the second leading cause of death globally and is responsible for an estimated 9.6 million deaths in 2018. Globally, about 1 in 6 deaths is due to cancer and the chemotherapeutic drugs available have high toxicity and have reported side effects hence, there is a need for the synthesis of novel drugs in the treatment of cancer. The current research work dealt with the synthesis of a series of 3-(3-acetyl-2-oxoquinolin-1-(2H)-yl-2-(substitutedphenyl)thiazolidin-4-one (Va-j) derivatives and evaluation of their in-vitro anticancer activity. All the synthesized compounds were satisfactorily characterized by IR and NMR data. Compounds were further evaluated for their in-vitro anticancer activity against A-549 (lung cancer) cell lines. The in-vitro anticancer activity was based upon the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay method. The synthesized compounds exhibited satisfactory anticancer properties against the A-549 cell line. The compound (VH): showed the highest potency amongst the tested derivatives against the A-549 cell line with IC50 values of 100 µg/ml respectively and was also found to be more potent than Imatinib (150 µg/ml) which was used as a standard drug. Molecular docking studies of the titled compounds (Va-j) were carried out using AutoDock Vina/PyRx software. The synthesized compounds exhibited well-conserved hydrogen bonds with one or more amino acid residues in the active pocket of the EGFRK tyrosine kinase domain (PDB 1m17). Among all the synthesized analogues, the binding affinity of the compound (Vh) was found to be higher than other synthesized derivatives and a molecular dynamics simulation study explored the stability of the docked complex system.

Structure–function characterization of two enzymes from novel subfamilies of manganese peroxidases secreted by the lignocellulose-degrading Agaricales fungi Agrocybe pediades and Cyathus striatus

BackgroundManganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure–function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule.ResultsTwo MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD—referred to as Ape-MnP1 and Cst-MnP1, respectively—were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation.ConclusionsUnderstanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure–function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.

Identification of novel and potential inhibitors against the dengue virus NS2B/NS3 protease using virtual screening and biomolecular simulations

Approximately 3.9 billion individuals are vulnerable to dengue infection, a prevalent cause of tropical diseases worldwide. Currently, no drugs are available for preventing or treating Flavivirus diseases, including Dengue, West Nile, and the more recent Zika virus. The highly conserved Flavivirus NS2B-NS3 protease, crucial for viral replication, is a promising therapeutic target. This study employed in-silico methodologies to identify novel and potentially effective anti-dengue small molecules. A pharmacophore model was constructed using an experimentally validated NS2B-NS3 inhibitor, with the Gunner Henry score confirming the model's validity. The Natural Product Activity and Species Source (NPASS) database was screened using the validated pharmacophore model, yielding a total of 60 hits against the NS2B-NS3 protease. Furthermore, the docking finding reveals that our newly identified compounds from the NPASS database have enhanced binding affinities and established significant interactions with allosteric residues of the target protein. MD simulation and post-MD analysis further validated this finding. The free binding energy was computed in terms of MM-GBSA analysis, with the total binding energy for compound 1 (−57.3 ± 2.8 and − 52.9 ± 1.9 replica 1 and 2) indicating a stronger binding affinity for the target protein. Overall, this computational study identified these compounds as potential hit molecules, and these findings can open up a new avenue to explore and develop inhibitors against Dengue virus infection.

Antibacterial, antibiofilm, and chemical profiles of Ammi visnaga L. and Foeniculum vulgare mill. Essential oils, and ADMET, molecular docking investigation of essential oils major components

This study determined chemical profiles, antibacterial and antibiofilm activities of the essential oils (EOs) obtained by A. visnaga aerial parts and F. vulgare fruits. Butanoic acid, 2-methyl-, 3-methylbutyl ester (38.8%), linalyl propionate (34.7%) and limonene (8.5%) resulted as main constituents of A. visnaga EO. In F. vulgare EO trans-anethole (76.9%) and fenchone (14.1%) resulted as main components. The two EOs were active against five bacterial strains (Acinetobacter baumannii, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus) at different degrees. The MIC values ranged from 5 ± 2 to 10 ± 2 μL/mL except for S. aureus (MIC >20 μL/mL). EOs exhibited inhibitory effect on the formation of biofilm up to 53.56 and 48.04% against E. coli and A. baumannii, respectively and activity against bacterial metabolism against A. baumannii and E. coli, with biofilm-inhibition ranging from 61.73 to 73.55%. The binding affinity of the identified components was estimated by docking them into the binding site of S. aureus gyrase (PDB code 2XCT) and S. aureus tyrosyl-tRNA synthetase (PDB code 1JIJ). trans-Anethole and butanoic acid, 2-methyl-, 3-methylbutyl ester showed relatively moderate binding interactions with the amino acid residues of S. aureus tyrosyl-tRNA synthetase. In addition, almost all predicted compounds possess good pharmacokinetic properties with no toxicity, being inactive for cytotoxicity, carcinogenicity, hepatotoxicity, mutagenicity and immunotoxicity parameters. The results encourage the use of these EOs as natural antibacterial agents in food and pharmaceutical industries.

Structure-based Virtual Screening from Natural Products as Inhibitors of SARS-CoV-2 Spike Protein and ACE2-h Receptor Binding and their Biological Evaluation In vitro.

In the last years, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused more than 760 million infections and 6.9 million deaths. Currently, remains a public health problem with limited pharmacological treatments. Among the virus drug targets, the SARS-CoV-2 spike protein attracts the development of new anti-SARS-CoV-2 agents. The aim of this work was to identify new compounds derived from natural products (BIOFACQUIM and Selleckchem databases) as potential inhibitors of the spike receptor binding domain (RBD)-ACE2h binding complex. Molecular docking, molecular dynamics simulations, and ADME-Tox analysis were performed to screen and select the potential inhibitors. ELISA-based enzyme assay was done to confirm our predictive model. Twenty compounds were identified as potential binders of RBD of the spike protein. In vitro assay showed compound B-8 caused 48% inhibition at 50 μM, and their binding pattern exhibited interactions via hydrogen bonds with the key amino acid residues present on the RBD. Compound B-8 can be used as a scaffold to develop new and more efficient antiviral drugs.

C–3 alkoxymethylation of 4-oxo-1,4-dihydroquinoline 2-carboxylic acid esters via organic additives

Esters of kynurenic acid, a known neuroprotective agent were reacted with cyclic amino acids to yield novel alkoxymethylated products under optimized reaction conditions. The importance of amino acid based (primary, secondary, biogenic and synthetic) organic additives was proven by the conduction of numerous test reactions. Thoroughly extended investigations, directly focusing on amino acid catalysis, which is an emerging and up-to-date field of catalysis and green chemical processes, have been conducted. The mechanism of the alkoxymethylation reaction was proposed and later the findings supported the hypothesis of the first retro-Mannich step (formation of the ortho-quinone methide intermediate) and subsequent formation of the alkoxymethylated derivatives. As a preparative result, two novel kynurenic acid derivatives bearing an alkoxymethyl moiety and two additional derivatives having amino acid residues at the site C-3 were synthesized, thus setting the scope and limitations of the modified Mannich reaction of kynurenic acid derivatives using amino acid nucleophiles. The mechanistic investigations highlighted the significant physicochemical effects of used nucleophiles on the amino-acid driven one-pot retro-Mannich initiated alkoxylation of kynurenic acid.

Ultrasound-assisted modification of soybean protein isolate with L-histidine: Relationship between structure and function

Herein, the effects of ultrasound-assisted L-histidine (L-His) on the physicochemical properties and conformation of soybean protein isolate (SPI) were investigated. Particle size, zeta potential, turbidity, and solubility were used to evaluate protein aggregation, and the relationship between structural and functional changes of the proteins was characterized using spectral analysis, surface hydrophobicity, emulsification, and antioxidant properties. After ultrasound-assisted L-His treatment, SPI exhibited a smaller particle size, higher solubility, and more homogeneous micromorphology owing to the decrease in alpha-helix content and subsequent increases in zeta potential and active sulfhydryl content. In addition, spectral analysis showed that L-His and SPI could form a complex, which changed the microenvironment of the amino acid residues in SPI, thus improving its emulsification and antioxidant properties. At the concentration of L-His was 0.3 % w/w, the nanocomplex had a smaller particle size (140.03 nm), higher ζ-potential (–23.63 mV), and higher emulsification stability (22.48 min).

Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Pisum sativum L

Squamous promoter binding protein-like (SPL) genes encode plant-specific transcription factors (TFs) that play essential roles in modulating plant growth, development, and stress response. Pea (Pisum sativum L.) is a coarse grain crop of great importance in food production, biodiversity conservation and molecular genetic research, providing genetic information and nutritional resources for improving agricultural production and promoting human health. However, only limited researches on the structure and functions of SPL genes exist in pea (PsSPLs). In this study, we identified 22 PsSPLs and conducted a genome-wide analysis of their physical characteristics, chromosome distribution, gene structure, phylogenetic evolution and gene expression patterns. As a result, the PsSPLs were unevenly distributed on the seven chromosomes of pea and harbored the SBP domain, which is composed of approximately 76 amino acid residues. The phylogenetic analysis revealed that the PsSPLs clustered into eight subfamilies and showed high homology with SPL genes in soybean. Further analysis showed the presence of segmental duplications in the PsSPLs. The expression patterns of 22 PsSPLs at different tissues, developmental stages and under various stimulus conditions were evaluated by qRT-PCR method. It was found that the expression patterns of PsSPLs from the same subfamily were similar in different tissues, the transcripts of most PsSPLs reached the maximum peak value at 14 days after anthesis in the pod. Abiotic stresses can cause significantly up-regulated PsSPL19 expression with spatiotemporal specificity, in addition, four plant hormones can cause the up-regulated expression of most PsSPLs including PsSPL19 in a time-dependent manner. Therefore, PsSPL19 could be a key candidate gene for signal transduction during pea growth and development, pod formation, abiotic stress and plant hormone response. Our findings should provide insights for the elucidating of development regulation mechanism and breeding for resistance to abiotic stress pea.

High-efficient depletion and separation of histidine-rich proteins via Cu2+-chelated porous polymer microspheres

Depletion and separation of histidine-rich proteins from complicated biosamples are crucial for various downstream applications in proteome research and clinical diagnosis. Herein, porous polymer microspheres coated with polyacrylic acid (SPSDVB-PAA) were fabricated through double emulsion interfacial polymerization technique and followed by immobilization of Cu2+ ions on the surface of SPSDVB-PAA. The as-prepared SPSDVB-PAA-Cu with uniform size and nanoscale pore structure enabled coordination interaction of Cu2+ with histidine residues in his-rich proteins, resulting in high-performance adsorption. As metal affinity adsorbent, the SPSDVB-PAA-Cu exhibited favorable selectivity for adsorbing hemoglobin (Hb) and human serum albumin (HSA) with the maximum adsorption capacities of 152.2 and 100.7 mg g−1. Furthermore, the polymer microspheres were used to isolate histidine-rich proteins from human whole blood and plasma, underscoring their effectiveness. The liquid chromatography tandem mass spectrometry (LC-MS/MS) results indicated that the content of 14 most abundant proteins in human plasma was depleted from 81.6 % to 30.7 % and low-abundance proteins were enriched from 18.4 % to 69.3 % after treatment with SPSDVB-PAA-Cu, illustrating potential application of SPSDVB-PAA-Cu in proteomic research.

Evaluating Chemical Footprinting-Induced Perturbation of Protein Higher Order Structure.

Specific amino acid footprinting mass spectrometry (MS) is an increasingly utilized method for elucidating protein higher order structure (HOS). It does this by adding to certain amino acid residues a mass tag, whose reaction extent depends on solvent accessibility and microenvironment of the protein. Unlike reactive free radicals and carbenes, these specific footprinters react slower than protein unfolding. Thus, their footprinting, under certain conditions, provokes structural changes to the protein, leading to labeling on non-native structures. It is critical to establish conditions (i.e., reagent concentrations, time of reaction) to ensure that the structure of the protein following footprinting remains native. Here, we compare the efficacy of five methods in assessing protein HOS following footprinting at the intact protein level and then further localize the perturbation at the peptide level. Three are MS-based methods that provide dose-response plot analysis, evaluation of Poisson distributions of precursor and products, and determination of the average number of modifications. These MS-based methods reliably and effectively indicate HOS perturbation at the intact protein level, whereas spectroscopic methods (circular dichroism (CD) and dynamic light scattering (DLS)) are less sensitive in monitoring subtle HOS perturbation caused by footprinting. Evaluation of HOS at the peptide level indicates regions that are sensitive to localized perturbations. Peptide-level analysis also provides higher resolution of the HOS perturbation, and we recommend using it for future footprinting studies. Overall, this work shows conclusive evidence for HOS perturbation caused by footprinting. Implementation of quality control workflows can identify conditions to avoid the perturbation, for footprinting, allowing accurate and reliable identification of protein structural changes that accompany, for example, ligand interactions, mutations, and changes in solution environment.