Molecular Binding Mechanism Research Articles

Constructing Innovative Covalent and Noncovalent Compound Libraries: Insights from 3D Protein-Ligand Interactions.

Noncovalent interactions between small-molecule drugs and protein targets assume a pivotal role in drug design. Moreover, the design of covalent inhibitors, forming covalent bonds with amino acid residues, requires rational reactivity for their covalent warheads, presenting a key challenge as well. Understanding the intricacies of these interactions provides a more comprehensive understanding of molecular binding mechanisms, thereby guiding the rational design of potent inhibitors. In this study, we adopted the fragment-based drug design approach, introducing a novel methodology to extract noncovalent and covalent fragments according to distinct three-dimensional (3D) interaction modes from noncovalent and covalent compound libraries. Additionally, we systematically replaced existing ligands with rational fragment substitutions, based on the spatial orientation of fragments in 3D space. Furthermore, we adopted a molecular generation approach to create innovative covalent inhibitors. This process resulted in the recombination of a noncovalent compound library and several covalent compound libraries, constructed by two commonly encountered covalent amino acids: cysteine and serine. We utilized noncovalent ligands in KLIFS and covalent ligands in CovBinderInPDB as examples to recombine noncovalent and covalent libraries. These recombined compound libraries cover a substantial portion of the chemical space present in the original compound libraries and exhibit superior performance in terms of molecular scaffold diversity compared to the original compound libraries and other 11 commercial libraries. We also recombined BTK-focused libraries, and 23 compounds within our libraries have been validated by former researchers to possess potential biological activity. The establishment of these compound libraries provides valuable resources for virtual screening of covalent and noncovalent drugs targeting similar molecular targets.

Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region.

Intrinsically disordered regions of proteins are responsible for many biological processes such as in the case of liver kinase B1 (LKB1)-a serine/threonine kinase relevant for cell proliferation and cell polarity. LKB1 becomes fully activated upon recruitment to the plasma membrane by binding of its disordered C-terminal polybasic motif consisting of eight lysines/arginines to phospholipids. Here, we present extensive molecular dynamics (MD) simulations of the polybasic motif interacting with a model membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl phosphatidic acid (PA) and cell culture experiments. Protein-membrane binding effects are due to the electrostatic interactions between the polybasic amino acids and PAs. For significant binding, the first three lysines turn out to be dispensable, which was also recapitulated in cell culture using transfected GFP-LKB1 variants. LKB1-membrane binding results in nonmonotonous changes in the structure of the protein as well as the membrane, in particular, accumulation of PAs and reduced thickness at the protein-membrane contact area. The protein-lipid binding turns out to be highly dynamic due to an interplay of PA-PA repulsion and protein-PA attraction. The thermodynamics of this interplay is captured by a statistical fluctuation model, which allows the estimation of both energies. Quantification of the significance of each polar amino acid in the polybasic provides detailed insights into the molecular mechanism of protein-membrane binding of LKB1. These results can likely be transferred to other proteins, which interact by intrinsically disordered polybasic regions with anionic membranes.

Unifying Theory and Experiments: Multi-Target Pharmacology of Dajihan Pill Against Hyperlipidemia

Background: The increasing incidence of hyperlipidemia (HLP) is attributed to the imbalance in redox homeostasis, aberrant lipid metabolism, and the excessive intake of empty calories. Dajihan Pill (DJHP) is a Traditional Chinese Medicine (TCM) formula composed of Zingiberis Rhizoma (ZR), Piperis Longi Fructus (PLF), Alpiniae Officinarum Rhizome (AOR), and Cinnamomi Cortex (CC) in a ratio of 3:2:3:2. It exhibits a significant preventive effect on HLP. Certainly, the active components and the precise mechanism of action are not fully understood. Therefore, this study aims to elucidate the preventive and ameliorative mechanisms of DJHP against HLP by integrating network pharmacology, molecular docking, and experimental validation. Method: Based on the pharmacological method, active ingredients in DJHP and targets were extracted from Traditional Chinese Medicine System Pharmacology (TCMSP) and UniProt. Then core compounds and targets were obtained by constructing “compounds-targets-disease” and proteinprotein interaction (PPI) network. Gene Ontology (GO) function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to elucidate further the associated action mechanism. The molecular binding mechanisms between the core ingredients and targets were elucidated through molecular docking. Additionally, the antioxidant capacities of DJHP extracts were investigated by assessing their DPPH, hydroxyl, and ABTS radical scavenging activities. Results: A total of 45 active compounds and 258 targets were identified in DJHP. Network analysis indicated that quercetin, beta-sitosterol, kaempferol, and oleic acid might serve as core bioactive compounds. Seven core targets, including AKT1, INS, and TNF, were identified as potential preventive targets. GO analysis suggested the improvement of HLP by DJHP may be related to the lipid metabolic process, high-density lipoprotein particle, triglyceride binding, and inflammatory response. The KEGG analysis indicated TNF, HIF-1, and AMPK signaling pathways were involved. The observations of active compounds binding with core targets indicated an excellent combination. Additionally, antioxidant results showed that DJHP exhibited significant DPPH, hydroxyl, and ABTS radical scavenging activities. Conclusion: Theoretical and experimental investigations indicate that DJHP can effectively modulate various signaling pathways and enhance the redox system, thus mitigating HLP. Our work provided a basis for the pharmacological study of DJHP in preventing HLP and further research.

Tetrandrine inhibits aldosterone synthesis by covalently targeting CYP11A1 to attenuate hypertension.

Tetrandrine (Tet) is the main pharmacological component of Stephania tetrandra S. Moore, which is a well-documented traditional Chinese medicine known for its diuretic and antihypertensive properties. Unraveling the specific targets and mechanisms of Tet involved in inducing diuresis and mitigating hypertension can provide valuable insights into its therapeutic effects. This study aimed to explore the diuretic and antihypertensive targets and mechanisms of Tet using chemical biology coupled with activity analyses in vivo and in vitro. The diuretic effects of Tet were evaluated using a water-loaded mouse model. The direct target proteins for the diuretic and antihypertensive effects of Tet were determined using chemical biology. Furthermore, the molecular mechanism of Tet binding to target proteins was analyzed using a multidisciplinary approach based on the structure and function of the proteins. Finally, the effects of the Tet-targeted protein on downstream signaling pathways and blood pressure were evaluated in hypertensive model rats. Tet exhibited significant antihypertensive and potassium-preserving diuretic effects. The mechanism underlying these effects involves the modulation of the enzyme activity by covalent binding of Tet to Cys423 of CYP11A1. This interaction alters the stability of heme within CYP11A1, subsequently impeding electron transfer and inhibiting aldosterone biosynthesis. This study not only revealed the mechanism of the diuretic and antihypertensive effects of Tet but also discovered a novel covalent inhibitor of CYP11A1. These findings contribute significantly to our understanding of the therapeutic potential of Tet and provide a foundation for future research in the development of targeted treatments for hypertension.

A highly specific aptamer for the SARS-CoV-2 spike protein from the authentic strain.

DNA aptamers are oligonucleotides that specifically bind to target molecules, similar to how antibodies bind to antigens. We identified an aptamer named MEZ that is highly specific to the receptor-binding domain, RBD, of the SARS-CoV-2 spike protein from the Wuhan-Hu-1 strain. The SELEX procedure was utilized to enrich the initial 31-mer oligonucleotide library with the target aptamer. The aptamer identification was performed using the novel protocol based on nanopore sequencing developed in this study. The MEZ aptamer was chemically synthesized and tested for binding with the SARS-CoV-2 RBD of the spike protein from different strains. The Kd is 6.5 nM for the complex with the RBD from the Wuhan-Hu-1 strain, which is comparable with known aptamers; the advantage is that the MEZ aptamer is smaller than known analogs. The proposed aptamer is highly selective for the RBD protein from the Wuhan-Hu-1 strain and does not form complexes with the RBD from Beta, Delta and Omicron strains. Experimental and theoretical studies together revealed the molecular mechanism of aptamer binding. The aptamer occupies the same binding site as ACE2 when bound to the RBD. The 3'-end of the MEZ aptamer is important for complex formation and is responsible for the discrimination of the RBD protein from a specific strain. The 5'-end is responsible for the formation of a loop in the 3D structure of the aptamer, which is important for proper binding.

Elucidating molecular mechanisms of protoxin-II state-specific binding to the human NaV1.7 channel.

Human voltage-gated sodium (hNaV) channels are responsible for initiating and propagating action potentials in excitable cells, and mutations have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels are expressed in peripheral neurons and are promising targets for pain therapy. The tarantula venom peptide protoxin-II (PTx2) has high selectivity for hNaV1.7 and is a valuable scaffold for designing novel therapeutics to treat pain. Here, we used computational modeling to study the molecular mechanisms of the state-dependent binding of PTx2 to hNaV1.7 voltage-sensing domains (VSDs). Using Rosetta structural modeling methods, we constructed atomistic models of the hNaV1.7 VSD II and IV in the activated and deactivated states with docked PTx2. We then performed microsecond-long all-atom molecular dynamics (MD) simulations of the systems in hydrated lipid bilayers. Our simulations revealed that PTx2 binds most favorably to the deactivated VSD II and activated VSD IV. These state-specific interactions are mediated primarily by PTx2's residues R22, K26, K27, K28, and W30 with VSD and the surrounding membrane lipids. Our work revealed important protein-protein and protein-lipid contacts that contribute to high-affinity state-dependent toxin interaction with the channel. The workflow presented will prove useful for designing novel peptides with improved selectivity and potency for more effective and safe treatment of pain.

In Silico Activity Prediction and Docking Studies of the Binding Mechanisms of Levofloxacin Structure Derivatives to Active Receptor Sites of Bacterial Type IIA Topoisomerases

The need for new antimicrobial agents (AntAg) is driven by the persistent antibiotic resistance in microorganisms, as well as the increasing frequency of pandemics. Due to the deficiency of AntAg, research aimed at developing speedy approaches to find new drug candidates is relevant. This study aims to conduct an in silico study of the biological activity spectrum as well as the molecular binding mechanisms of four structurally different forms of levofloxacin (Lvf) with bacterial topoisomerases targets of type IIA (DNA gyrase and topoisomerase IV) to enable the development of drugs with an improved characterization of the safety profile. To achieve this goal, a number of software products were used, such as ChemicPen v. 2.6, PyMol 2.5, Avogadro 1.2.0, PASS, AutoDockTools 1.5.7 with the new generation software Autodock Vina. These software products are the first to be made available for visualization of clusters with determination of ligand-receptor pair binding affinity, as well as clustering coordinates and proposed mechanisms of action. One of the real structures of Lvf, a decarboxylated derivative, was obtained with tribochemical (TrbCh) exposure. The action spectrum of molecular ligands is described based on a Bayesian probability activity prediction model (PASS software Version 2.0). Predicted and real (PMS and RMS) molecular structures of Lvf, with decreasing levels of structural complexity, were translated into descriptors via Wiener (W), Balaban (Vs), Detour (Ip), and Electropy € indices. The 2D «structure-activity» diagrams were used to differentiate closely related structures of levofloxacin. PMS and RMS were visualized as 3D models of the ligand-receptor complexes. The contact regions of RMS and PMS with key amino acid residues—SER-79, DT-15, DG-1, DA-1—were demonstrated. The intra- and inter-molecular binding sites, data on free energy (affinity values, kcal/mol), the binding constant Kb (M−1), and the number of clusters are presented. The research results obtained from the presented in silico approach to explore the spectrum of action find quantitative “structure-activity” correlations, and predict molecular mechanisms may be of applied interest for directed drug discovery.

Understanding the molecular mechanisms of odorant binding and activation of the human OR52 family

Structural and mechanistic studies on human odorant receptors (ORs), key in olfactory signaling, are challenging because of their low surface expression in heterologous cells. The recent structure of OR51E2 bound to propionate provided molecular insight into odorant recognition, but the lack of an inactive OR structure limited understanding of the activation mechanism of ORs upon odorant binding. Here, we determined the cryo-electron microscopy structures of consensus OR52 (OR52cs), a representative of the OR52 family, in the ligand-free (apo) and octanoate-bound states. The apo structure of OR52cs reveals a large opening between transmembrane helices (TMs) 5 and 6. A comparison between the apo and active structures of OR52cs demonstrates the inward and outward movements of the extracellular and intracellular segments of TM6, respectively. These results, combined with molecular dynamics simulations and signaling assays, shed light on the molecular mechanisms of odorant binding and activation of the OR52 family.

DNA Aptamer Binding Octapeptide Repeat Region of Cellular Prion Protein.

Cellular prion protein (PrPC) is highly expressed in a variety of tumor cells and plays a crucial role in neurodegenerative diseases. Its N-terminal domain contains a conserved octapeptide (PHGGGWGQ) repeat sequence. The number of repeats has been correlated with the species as well as the development of associated diseases. Herein, PrPC was identified to be the molecular target of a high-affinity DNA aptamer HA5-68 obtained by cell-SELEX. Aptamer HA5-68 was further optimized to two short sequences (HA5-40-1 and HA5-40-2), and its binding site to PrPC was identified to be located in the loop-stem-loop region of the head of its secondary structure. HA5 series aptamers were demonstrated to bind the octapeptide repeat region of PrPC, as well as the synthesized peptides containing different numbers of octapeptide repeats. The PrPC expression on 42 cell lines was measured by using aptamer HA5-68 as a molecular probe. The clear understanding of the molecular structure and binding mechanism of this set of aptamers will provide information for the design of diagnostic methods and therapeutic drugs targeting PrPC.

Identification and molecular binding mechanism of novel pancreatic lipase and cholesterol esterase inhibitory peptides from heat-treated adzuki bean protein hydrolysates

Heat-treated adzuki bean protein hydrolysates exhibit lipid-reducing properties; however, few studies have reported pancreatic lipase (PL) and cholesterol esterase (CE) inhibitory effects and elucidated the underlying mechanisms. In this study, we accomplished the identification of antiobesity peptides through peptide sequencing, virtual screening, and in vitro experiments. Furthermore, the mechanisms were investigated via molecular docking. The findings reveal that the action of pepsin and pancreatin resulted in the transformation of intact adzuki bean protein into smaller peptide fragments. The < 3 kDa fraction exhibited a high proportion of hydrophobic amino acids and displayed superior inhibitory properties for both PL and CE. Five novel antiobesity peptides (LLGGLDSSLLPH, FDTGSSFYNKPAG, IWVGGSGMDM, YLQGFGKNIL, and IFNNDPNNHP) were identified as PL and CE inhibitors. Particularly, IFNNDPNNHP exhibited the most robust biological activity. These peptides exerted their inhibitory action on PL and CE by occupying catalytic or substrate-binding sites through hydrogen bonds, hydrophobic interactions, salt bridges, and π-π stacking.

Obtaining New Candidate Peptides for Biological Anticancer Drugs from Enzymatic Hydrolysis of Human and Bovine Hemoglobin.

Enzymatic hydrolysis of bovine and human hemoglobin generates a diversity of bioactive peptides, mainly recognized for their antimicrobial properties. However, antimicrobial peptides stand out for their ability to specifically target cancer cells while preserving rapidly proliferating healthy cells. This study focuses on the production of bioactive peptides from hemoglobin and evaluates their anticancer potential using two distinct approaches. The first approach is based on the use of a rapid screening method aimed at blocking host cell protein synthesis to evaluate candidate anticancer peptides, using Lepidium sativum seed germination as an indicator. The results show that: (1) The degree of hydrolysis (DH) significantly influences the production of bioactive peptides. DH levels of 3 to 10% produce a considerably stronger inhibition of radicle growth than DH 0 (the native form of hemoglobin), with an intensity three to four times greater. (2) Certain peptide fractions of bovine hemoglobin have a higher activity than those of human hemoglobin. (3) The structural characteristics of peptides (random coil or alpha helix) play a crucial role in the biological effects observed. (4) The α137-141 peptide, the target of the study, was the most active of the fractions obtained from bovine hemoglobin (IC50 = 29 ± 1 µg/mL) and human hemoglobin (IC50 = 48 ± 2 µg/mL), proving to be 10 to 15 times more potent than the other hemoglobin fractions, attributed to its strong antimicrobial potential. The second approach to assessing anticancer activity is based on the preliminary in vitro analysis of hydrolysates and their peptide fractions, with a focus on the eL42 protein. This protein is of major interest due to its overexpression in all cancer cells, making it an attractive potential target for the development of anticancer molecules. With this in mind, astudy was undertaken using a method for labeling formylase (formyl-methionyl-tRNA transformylase (FMTS)) with oxidized tRNA. This approach was chosen because of the similarities in the interaction between formylase and the eL42 protein with oxidized tRNA. The results obtained not only confirmed the previous conclusions but also reinforced the hypothesis that the inhibition of protein synthesis plays a key role in the anticancer mechanism of these peptides. Indeed, the data suggest that samples containing α137-141 peptide (NKT) and total hydrolysates may have modulatory effects on the interaction between FMTS and oxidized tRNA. This observation highlights the possibility that the latter could influence molecular binding mechanisms, potentially resulting in a competitive situation where the ability of substrate tRNA to bind efficiently to ribosomal protein is compromised in their presence. Ultimately, these results suggest the feasibility of obtaining candidate peptides for biological anticancer drugs from both human and bovine hemoglobin sources. These scientific advances show new hope in the fight against cancer, which affects a large number of people around the world.

The SET and ankyrin domains of the secreted Legionella pneumophila histone methyltransferase work together to modify host chromatin.

Legionella pneumophila is an intracellular bacterium responsible of Legionnaires' disease, a severe pneumonia that is often fatal when not treated promptly. The pathogen's ability to efficiently colonize the host resides in its ability to replicate intracellularly. Essential for intracellular replication is translocation of many different protein effectors via a specialized secretion system. One of them, called RomA, binds and directly modifies the host chromatin at a unique site (tri-methylation of lysine 14 of histone H3 [H3K14me]). However, the molecular mechanisms of binding are not known. Here, we resolve this question through structural characterization of RomA together with the H3 peptide. We specifically reveal an active role of the ankyrin repeats located in its C-terminal in the interaction with the histone H3 tail. Indeed, without the ankyrin domains, RomA loses its ability to act as histone methyltransferase. These results discover the molecular mechanisms by which a bacterial histone methyltransferase that is conserved in L. pneumophila strains acts to modify chromatin.

Molecular binding mechanism and novel antidiabetic and anti-hypertensive bioactive peptides from fermented camel milk with anti-inflammatory activity in raw macrophages cell lines.

The investigation was to determine the effect of camel milk fermented with Limosilactobacillus fermentum KGL4 (MTCC 25515) on ACE-inhibiting, anti-inflammatory, and diabetes-preventing properties and also to release the novel peptides with antidiabetic and anti-hypertensive attributes with molecular interaction studies. Growth conditions were optimised on the basis of total peptide production by inoculating the culture in camel milk at different rates (1.5, 2.0, and 2.5%) along with different incubation periods (12, 24, 36, and 48h). However, after 48h of fermentation with a 2.5% rate of inoculum, the highest proteolytic activity was obtained. Reverse phase high-pressure liquid chromatography (RP-HPLC) was used to calculate the % Rpa from permeates of 3kDa and 10kDa fractions. Molecular weight distributions of fermented and unfermented camel milk protein fractions were compared using SDS-PAGE. Spots obtained from 2D gel electrophoresis were separated on the basis of pH and molecular weight. Spots obtained from 2D gel were digested with trypsin, and the digested samples were subjected to RP-LC/MS for the generation of peptide sequences. The inhibition of tumour necrosis factor alpha, interleukin-6, and interleukin-1 during fermentation was studied using RAW 264.7 macrophages. In the study, fermented camel milk with KGL4 (CMKGL4) inhibited LPS-induced nitric oxide (NO) production and pro-inflammatory cytokine production (TNF-α, IL-6, and IL-1β) by the murine macrophages. The results showed that the peptide structures (YLEELHRLNK and YLQELYPHSSLKVRPILK) exhibited considerable binding affinity against hPAM and hMGA during molecular interaction studies.

The mechanism of UP1 binding and unfolding of human telomeric DNA G-quadruplex

The human telomere contains multiple copies of the DNA sequence d(TTAGGG) which can fold into higher order intramolecular G-quadruplexes and regulate the maintenance of telomere length and chromosomal integrity. The nucleic acid binding protein heteronuclear ribonucleoprotein A1 (hnRNP A1) and its N-terminus proteolytic product UP1 have been shown to efficiently bind and unfold telomeric DNA G-quadruplex. However, the understanding of the molecular mechanism of the UP1 binding and unfolding telomeric G-quadruplexes is still limited. Here, we performed biochemical and biophysical characterizations of UP1 binding and unfolding of human telomeric DNA G-quadruplex d[AGGG(TTAGGG)3], and in combination of systematic site-direct mutagenesis of two tandem RNA recognition motifs (RRMs) in UP1, revealed that RRM1 is responsible for initial binding and unfolding, whereas RRM2 assists RRM1 to complete the unfolding of G-quadruplex. Isothermal titration calorimetry (ITC) and circular dichroism (CD) studies of the interactions between UP1 and DNA G-quadruplex variants indicate that the “TAG” binding motif in Loop2 of telomeric G-quadruplex is critical for UP1 recognition and G-quadruplex unfolding initiation. Together we depict a model for molecular mechanism of hnRNP A1 (UP1) binding and unfolding of the human telomeric DNA G-quadruplex.

Structural basis of white-tailed deer, Odocoileus virginianus, ACE2 recognizing all the SARS-CoV-2 variants of concern with high affinity.

SARS-CoV-2 has been expanding its host range, among which the white-tailed deer (WTD), Odocoileus virginianus, became the first wildlife species infected on a large scale and might serve as a host reservoir for variants of concern (VOCs) in case no longer circulating in humans. In this study, we comprehensively assessed the binding of the WTD angiotensin-converting enzyme 2 (ACE2) receptor to the spike (S) receptor-binding domains (RBDs) from the SARS-CoV-2 prototype (PT) strain and multiple variants. We found that WTD ACE2 could be broadly recognized by all of the tested RBDs. We further determined the complex structures of WTD ACE2 with PT, Omicron BA.1, and BA.4/5 S trimer. Detailed structural comparison revealed the important roles of RBD residues on 486, 498, and 501 sites for WTD ACE2 binding. This study deepens our understanding of the interspecies transmission mechanisms of SARS-CoV-2 and further addresses the importance of constant monitoring on SARS-CoV-2 infections in wild animals. IMPORTANCE Even if we manage to eliminate the virus among humans, it will still circulate among wildlife and continuously be transmitted back to humans. A recent study indicated that WTD may serve as reservoir for nearly extinct SARS-CoV-2 strains. Therefore, it is critical to evaluate the binding abilities of SARS-CoV-2 variants to the WTD ACE2 receptor and elucidate the molecular mechanisms of binding of the RBDs to assess the risk of spillback events.

Expression Pattern and Ligand Binding Characteristics Analysis of Chemosensory Protein SnitCSP2 from Sirex nitobei.

Sirex nitobei is an important wood-boring wasp to conifers native to Asia, causing considerable economic and ecological damage. However, the current control means cannot achieve better efficiency, and it is expected to clarify the molecular mechanism of protein-ligand binding for effective pest control. This study analyzed the expression pattern of CSP2 in S. nitobei (SnitCSP2) and its features of binding to the screened ligands using molecular docking and dynamic simulations. The results showed that SnitCSP2 was significantly expressed in female antennae. Molecular docking and dynamic simulations revealed that SnitCSP2 bound better to the host plant volatile (+)-α-pinene and symbiotic fungal volatiles terpene and (-)-globulol than other target ligands. By the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, the free binding energies of the three complexes were calculated as -44.813 ± 0.189 kJ/mol, -50.446 ± 0.396 kJ/mol, and -56.418 ± 0.368 kJ/mol, and the van der Waals energy was found to contribute significantly to the stability of the complexes. Some key amino acid residues were also identified: VAL13, GLY14, LYS61, MET65, and LYS68 were important for the stable binding of (+)-α-pinene by SnitCSP2, while for terpenes, ILE16, ALA25, TYR26, CYS29, GLU39, THR37, and GLY40 were vital for a stable binding system. We identified three potential ligands and analyzed the interaction patterns of the proteins with them to provide a favorable molecular basis for regulating insect behavioral interactions and developing new pest control strategies.

Study of molecular mechanisms of herbicide binding t o target proteins of weeds by in silico methods

The aim of this study was to study of the molecular mechanisms of herbicide binding (pinoxaden, quizalofop- P-tephuryl, prometryn, metribuzin, metsulfuron- methyl) to target proteins (acetyl- CoA carboxylase, acetolactate synthase, cytochrome P450, p-hydroxyphenylpyruvate dioxygenase) of the weed species growing in Siberia: wild oat Avena fatua (L.), wheatgrass Elymus repens (L.), amaranth Amaranthus retro exus (L.), common lamb's quarters Chenopodium album (L.), bristlegrass Setaria viridis (L.). The work was carried out in the Novosibirsk region in 2022-2023. Using bioinformatic methods, virtual three- dimensional complexes of pesticides and their targets were created. A new approach has been proposed to identify the parameters of the cavity of binding sites, which consists in combining the alignment method and machine learning technology. Docking of pesticides with targets was carried out. The calculation of the binding energy indicates a high degree of stability of the ligand- protein complexes. The in silico approach we proposed may be useful for elucidating the behavior of herbicides when bound to weed enzymes. This approach allows a deeper understanding of the mechanism of action and ecotoxicological aspects of the use of pesticides. The studied complexes of herbicides and proteins have a potential high stability due to the low binding energy with receptors, which varies from -5.60 to -13.24 kcal/mol. The binding energy value did not show a direct dependence on the number of amino acid residues of the ligand- binding site. In general, the study supplemented some aspects of the molecular mechanisms of binding herbicides to target proteins of weeds, however, a more in-depth study is required in order to localize critical amino acid points involved in the biological functions of target proteins.

A Novel Blood-Brain Barrier-Penetrating and Vascular-Targeting Chimeric Peptide Inhibits Glioma Angiogenesis.

The high vascularization of glioma highlights the potential value of anti-angiogenic therapeutics for glioma treatment. Previously, we designed a novel vascular-targeting and blood-brain barrier (BBB)-penetrating peptide, TAT-AT7, by attaching the cell-penetrating peptide TAT to a vascular-targeting peptide AT7, and we demonstrated that TAT-AT7 could target binding to the vascular endothelial growth factor receptor 2 (VEGFR-2) and Neuropilin-1 (NRP-1), which are both highly expressed in endothelial cells. TAT-AT7 has been proven to be a good targeting peptide which could effectively deliver the secretory endostatin gene to treat glioma via the TAT-AT7-modified polyethyleneimine (PEI) nanocomplex. In the current study, we further explored the molecular binding mechanisms of TAT-AT7 to VEGFR-2 and NRP-1 and its anti-glioma effects. Accordingly, TAT-AT7 was proven to competitively bind to VEGFR-2 and NRP-1 and prevent VEGF-A165 binding to the receptors by the surface plasmon resonance (SPR) assay. TAT-AT7 inhibited endothelial cells' proliferation, migration, invasion, and tubule formation, as well as promoted endothelial cells' apoptosis in vitro. Further research revealed that TAT-AT7 inhibited the phosphorylation of VEGFR-2 and its downstream PLC-γ, ERK1/2, SRC, AKT, and FAK kinases. Additionally, TAT-AT7 significantly inhibited angiogenesis of zebrafish embryo. Moreover, TAT-AT7 had a better penetrating ability and could penetrate the BBB into glioma tissue and target glioma neovascularization in an orthotopic U87-glioma-bearing nude mice model, and exhibited the effect of inhibiting glioma growth and angiogenesis. Taken together, the binding and function mechanisms of TAT-AT7 were firstly revealed, and TAT-AT7 was proven to be an effective and promising peptide for the development of anti-angiogenic drugs for targeted treatment of glioma.

Adsorption of SARS-CoV-2 Spike (N501Y) RBD to Human Angiotensin-Converting Enzyme 2 at a Lipid/Water Interface.

The receptor binding domain (RBD) of spike proteins plays a crucial role in the process of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) attachment to the human angiotensin-converting enzyme 2 (ACE2). The N501Y mutation and later mutations introduced extra positive charges on the spike RBD and resulted in higher transmissibility, likely due to stronger binding with the highly negatively charged ACE2. Consequently, many studies have been devoted to understanding the molecular mechanism of spike protein binding with the ACE2 receptor. Most of the theoretical studies, however, have been done on isolated proteins. ACE2 is a transmembrane protein; thus, it is important to understand the interaction of spike proteins with ACE2 in a lipid matrix. In this study, the adsorption of ACE2 and spike (N501Y) RBD at a lipid/water interface was studied using the heterodyne-detected vibrational sum frequency generation (HD-VSFG) technique. The technique is a non-linear optical spectroscopy which measures vibrational spectra of molecules at an interface and provides information on their structure and orientation. It is found that ACE2 is effectively adsorbed at the positively charged 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) lipid monolayer via electrostatic interactions. The adsorption of ACE2 at the DPTAP monolayer causes a reorganization of interfacial water (D2O) from the D-down to the D-up orientation, indicating that the originally positively charged DPTAP interface becomes negatively charged due to ACE2 adsorption. The negatively charged interface (DPTAP/ACE2) allows further adsorption of positively charged spike RBD. HD-VSFG spectra in the amide I region show differences for spike (N501Y) RBD adsorbed at D2O, DPTAP, and DPTAP/ACE2 interfaces. A red shift observed for the spectra of spike RBD/DPTAP suggests that spike RBD oligomers are formed upon contact with DPTAP lipids.

Molecular Mechanism of Ligand Binding to the Minor Groove of DNA

Although DNA–ligand binding is pervasive in biology, little is known about molecular-level binding mechanisms. Using all-atom, explicit-solvent molecular dynamics simulations in conjunction with weighted ensemble (WE)-enhanced sampling, an ensemble of 2562 binding trajectories of Hoechst 33258 (H33258) to d(CGC AAA TTT GCG) was generated from which the binding mechanism was extracted. In particular, the electrostatic interaction between the positively charged H33258 and the negatively charged DNA backbone drives the formation of initial H33258–DNA contacts. After this initial contact, a hinge-like intermediate state is formed in which one end of H33258 inserts into the minor groove of DNA. Following hinge state formation is a concerted motion whereby the second end of H33258 swings into the minor groove and the spine of hydration along the minor groove causing dehydration. This study illustrates how WE-enhanced simulations of biomolecular ligation processes can offer novel mechanistic insights by generating ensembles of binding events.