High Affinity Binding Research Articles

Designing multi-epitope vaccine against human cytomegalovirus integrating pan-genome and reverse vaccinology pipelines

Human cytomegalovirus (HCMV) is accountable for high morbidity in neonates and immunosuppressed individuals. Due to the high genetic variability of HCMV, current prophylactic measures are insufficient. In this study, we employed a pan-genome and reverse vaccinology approach to screen the target for efficient vaccine candidates. Four proteins, envelope glycoprotein M, UL41A, US23, and US28, were shortlisted based on cellular localization, high solubility, antigenicity, and immunogenicity. A total of 29 B-cell and 44 T-cell highly immunogenic and antigenic epitopes with high global population coverage were finalized using immunoinformatics tools and algorithms. Further, the epitopes that were overlapping among the finalized B-cell and T-cell epitopes were linked with suitable linkers to form various combinations of multi-epitopic vaccine constructs. Among 16 vaccine constructs, Vc12 was selected based on physicochemical and structural properties. The docking and molecular simulations of VC12 were performed, which showed its high binding affinity (−23.35 kcal/mol) towards TLR4 due to intermolecular hydrogen bonds, salt bridges, and hydrophobic interactions, and there were only minimal fluctuations. Furthermore, Vc12 eliciting a good response was checked for its expression in Escherichia coli through in silico cloning and codon optimization, suggesting it to be a potent vaccine candidate.

Structural basis for the inhibition of βFXIIa by garadacimab.

Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (βFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to βFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-βFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of βFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-βFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.

Computational Insight of Oleracone L, Portulacatone B, and Portulacatal from Portulaca oleracea L. as Potential Anticholinesterase Inhibitors for Alzheimer’s

Alzheimer’s disease, characterized by a decline in cognitive functions, is frequently associated with decreased levels of acetylcholine due to the overactivity of acetylcholinesterase (AChE). Inhibiting AChE has been a key therapeutic strategy in treating Alzheimer’s disease, yet the search for effective inhibitors, particularly from natural sources, continues due to their potential for fewer side effects. In this context, three new alkaloids—oleracone L, portulacatone B, and portulacatal—extracted from Portulaca oleracea L., have recently shown promising anticholinesterase activity in vitro. However, no experimental or computational studies have yet explored their binding potential. This study represents the first comprehensive in silico analysis of these compounds, employing ADME prediction, molecular docking, molecular dynamics simulations, and MM-PBSA calculations to assess their therapeutic potential. The drug-likeness was evaluated based on Lipinski, Pfizer, Golden Triangle, and GSK rules, with all three alkaloids meeting these criteria. The ADME profiles suggested that these alkaloids can effectively cross the blood–brain barrier, a critical requirement for Alzheimer’s treatment. Molecular docking studies revealed that oleracone L had the highest binding affinity (−10.75 kcal/mol) towards AChE, followed by portulacatal and portulacatone B, demonstrating significant interactions with crucial enzyme residues. Molecular dynamics simulations over 200 ns confirmed the stability of these interactions, with RMSD values below 2 Å for all complexes, indicating stable binding throughout the simulation period. RMSF and the radius of gyration analyses further corroborated the minimal impact of these alkaloids on the enzyme’s overall flexibility and compactness. Moreover, MM-PBSA calculations provided additional support for the binding efficacy, showing that oleracone L, with the most favorable binding energy, could be a superior inhibitor, potentially due to its stronger and more consistent hydrogen bonding and favorable electrostatic interactions compared to the other studied alkaloids. These computational findings highlight the binding efficiency and potential therapeutic viability of these alkaloids as AChE inhibitors, suggesting they could be promising candidates for Alzheimer’s disease treatment. The study underscores the importance of further validation through in vitro and in vivo experiments to confirm these predictions.

Differential effects of the N-terminal helix of FGF8b on the activity of a small-molecule FGFR inhibitor in cell culture and for the extracellular domain of FGFR3c in solution.

SSR128129E (SSR) is a unique small-molecule inhibitor of fibroblast growth factor receptors (FGFRs). SSR is a high-affinity allosteric binder that selectively blocks one of the two major FGFR-mediated pathways. The mechanisms of SSR activity were studied previously in much detail, allowing the identification of its binding site, located in the hydrophobic groove of the receptor D3 domain. The binding site overlaps with the position of an N-terminal helix, an element exclusive for the FGF8b growth factor, which could potentially convert SSR from an allosteric inhibitor into an orthosteric blocker for the particular FGFR/FGF8b system. In this regard, we report here on the structural and functional investigation of FGF8b/FGFR3c system and the effects imposed on it by SSR. We show that SSR is equally or more potent in inhibiting FGF8b-induced FGFR signaling compared to FGF2-induced activation. On the other hand, when studied in the context of separate extracellular domains of FGFR3c in solution with NMR spectroscopy, SSR is unable to displace the N-terminal helix of FGF8b from its binding site on FGFR3c and behaves as a weak orthosteric inhibitor. The substantial inconsistency between the results obtained with cell culture and for the individual water-soluble subdomains of the FGFR proteins points to the important role played by the cell membrane.

Ligand-Based Virtual Screening for Discovery of Indole Derivatives as Potent DNA Gyrase ATPase Inhibitors Active against Mycobacterium tuberculosis and Hit Validation by Biological Assays.

Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 μM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 μg/mL, respectively, superior to novobiocin (MIC > 100.0 μg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.

Ocr-mediated suppression of BrxX unveils a phage counter-defense mechanism.

The burgeoning crisis of antibiotic resistance has directed attention to bacteriophages as natural antibacterial agents capable of circumventing bacterial defenses. Central to this are the bacterial defense mechanisms, such as the BREX system, which utilizes the methyltransferase BrxX to protect against phage infection. This study presents the first in vitro characterization of BrxX from Escherichiacoli, revealing its substrate-specific recognition and catalytic activity. We demonstrate that BrxX exhibits nonspecific DNA binding but selectively methylates adenine within specific motifs. Kinetic analysis indicates a potential regulation of BrxX by the concentration of its co-substrate, S-adenosylmethionine, and suggests a role for other BREX components in modulating BrxX activity. Furthermore, we elucidate the molecular mechanism by which the T7 phage protein Ocr (Overcoming classical restriction) inhibits BrxX. Despite low sequence homology between BrxX from different bacterial species, Ocr effectively suppresses BrxX's enzymatic activity through high-affinity binding. Cryo-electron microscopy and biophysical analyses reveal that Ocr, a DNA mimic, forms a stable complex with BrxX, highlighting a conserved interaction interface across diverse BrxX variants. Our findings provide insights into the strategic counteraction by phages against bacterial defense systems and offer a foundational understanding of the complex interplay between phages and their bacterial hosts, with implications for the development of phage therapy to combat antibiotic resistance.

Cluster of Differentiation 36 (CD36) Preferentially Mediates Intestinal Absorption of Dietary Z-Astaxanthin and Especially 9-Z-Isomer via Higher Binding Affinity.

Variances in the biological functions of astaxanthin geometric isomers (i.e., all-E, Z) are related to their intestinal absorption, but the mechanism of isomer absorption mediated by transporters remains unclear. Here, models of in vitro cell overexpression, in situ intestinal perfusion, and in vivo mouse inhibition were employed to investigate the impact of cluster of differentiation 36 (CD36) on the absorption of astaxanthin isomers. Cells overexpressing CD36 notably enhanced the uptake of Z-astaxanthin, particularly the 9-Z-isomer (47.76%). The absorption rate and permeability of Z-astaxanthin surpassed that of the all-E-isomer by the in situ model. Furthermore, the addition of the CD36-specific inhibitor sulfo-N-succinimidyl oleate significantly reduced the absorption of Z-astaxanthin in the mouse duodenum and jejunum, especially the 9-Z-isomer (57.66%). Molecular docking and surface plasmon resonance techniques further validated that 9-Z-astaxanthin binds to more amino acids of CD36 with higher affinity and in a fast-binding, fast-dissociating mode, thus favoring transport. Our findings elucidate, for the first time, the mechanism of the CD36-mediated transmembrane transport of astaxanthin geometric isomers.

Structure-based investigation of a DNA aptamer targeting PTK7 reveals an intricate 3D fold guiding functional optimization

DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules.

Harnessing the potential of microbial keratinases for bioconversion of keratin waste.

The increasing global consumption of poultry meat has led to the generation of a vast quantity of feather keratin waste daily, posing significant environmental challenges due to improper disposal methods. A growing focus is on utilizing keratinous polymeric waste, amounting to millions of tons annually. Keratins are biochemically rigid, fibrous, recalcitrant, physiologically insoluble, and resistant to most common proteolytic enzymes. Microbial biodegradation of feather keratin provides a viable solution for augmenting feather waste's nutritional value while mitigating environmental contamination. This approach offers an alternative to traditional physical and chemical treatments. This review focuses on the recent findings and work trends in the field of keratin degradation by microorganisms (bacteria, actinomycetes, and fungi) via keratinolytic and proteolytic enzymes, as well as the limitations and challenges encountered due to the low thermal stability of keratinase, and degradation in the complex environmental conditions. Therefore, recent biotechnological interventions such as designing novel keratinase with high keratinolytic activity, thermostability, and binding affinity have been elaborated here. Enhancing protein structural rigidity through critical engineering approaches, such as rational design, has shown promise in improving the thermal stability of proteins. Concurrently, metagenomic annotation offers insights into the genetic foundations of keratin breakdown, primarily predicting metabolic potential and identifying probable keratinases. This may extend the understanding of microbial keratinolytic mechanisms in a complex community, recognizing the significance of synergistic interactions, which could be further utilized in optimizing industrial keratin degradation processes.

Development of a subunit vaccine against the cholangiocarcinoma causing Opisthorchis viverrini: a computational approach.

Opisthorchis viverrini is the etiological agent of the disease opisthorchiasis and related cholangiocarcinoma (CCA). It infects fish-eating mammals and more than 10 million people in Southeast Asia suffered from opisthorchiasis with a high fatality rate. The only effective drug against this parasite is Praziquantel, which has significant side effects. Due to the lack of appropriate treatment options and the high death rate, there is a dire need to develop novel therapies against this pathogen. In this study, we designed a multi-epitope chimeric vaccine design against O. viverrini by using immunoinformatics approaches. Non-allergenic and immunogenic MHC-1, MHC-2, and B cell epitopes of three candidate proteins thioredoxin peroxidase (Ov-TPx-1), cathepsin F1 (Ov-CF-1) and calreticulin (Ov-CALR) of O. viverrini, were predicted to construct a potent multiepitope vaccine. The coverage of the HLA-alleles of these selected epitopes was determined globally. Four vaccine constructs made by different adjuvants and linkers were evaluated in the context of their physicochemical properties, antigenicity, and allergenicity. Protein-protein docking and MD simulation found that vaccines 3 was more stable and had a higher binding affinity for TLR2 and TLR4 immune receptors. In-silico restriction cloning of vaccine model led to the formation of plasmid constructs for expression in a suitable host. Finally, the immune simulation showed strong immunological reactions to the engineered vaccine. These findings suggest that the final vaccine construct has the potential to be validated by in vivo and in vitro experiments to confirm its efficacy against the CCA causing O. viverrini.

Antibacterial and Cytotoxicity of Extracts and Isolated Compounds from Artemisia abyssinica: A Combined Experimental and Computational Study.

Artemisia abyssinica is a widely cultivated hedge plant in Ethiopia. Traditionally, they have been used to treat a variety of health conditions, including intestinal problems, infectious diseases, tonsillitis, and leishmaniasis. Silica gel chromatographic separation of the methanol and ethyl acetate extracts of the leaves, roots, and stem barks of A. abyssinica led to the isolation of 12 compounds, labeled as 1-12. Among these, compounds 1, 3, 4, 5, and 7-11 are reported as new to the genus Artemisia. The extracts and isolated compounds from A. abyssinica were evaluated for their in vitro antibacterial activity against four bacterial strains: Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, using the disc diffusion assay. All of the extracts displayed weak antibacterial activity, with inhibition zone diameters (IZDs) ranging from 6.10 ± 0.3 to 9.30 ± 0.20 mm. The isolated compounds, on the other hand, exhibited weak to moderate antibacterial activity, with IZDs ranging from 6.00 ± 0.300 to 13.50 ± 0.50 mm. The most potent antibacterial activity was observed for compound 6, which showed an IZD of 13.30 ± 0.50 mm against E. coli and 13.50 ± 0.50 mm against P. aeruginosa. This activity was comparable to that of the positive control ceftriaxone, which had IZDs of 14.1 ± 0.3 and 13.8 ± 0.5 mm against E. coli and P. aeruginosa, respectively. The in silico molecular docking analysis against DNA gyrase B revealed that compound 5 showed a higher binding affinity (-6.9 kcal/mol), followed by compound 10 (-6.7 kcal/mol) and compound 12 (-6.3 kcal/mol), whereas ciprofloxacin showed -7.3 kcal/mol. The binding affinities of compounds 5, 11, 10, and 9 were found to be -5.0, -4.3, -4.2, and -4.0 kcal/mol against S. aureus Pyruvate kinase, respectively, whereas ciprofloxacin showed a binding affinity of -4.9 kcal/mol, suggesting that compound 5 had a better binding affinity compared with ciprofloxacin. The effect of extracts of A. abyssinica was evaluated for cytotoxic activity against the breast cancer cell line (MCF-7) by the MTT assay. The extracts induced a decrease in cell viability and exerted a cytotoxic effect at a concentration of 20 μg/mL. The highest percent cell viability was observed for the methanol extract of the stem (92.9%), whereas the least was observed for the methanol extract of the root (34.5%). The result of the latter was significant compared with the positive control. The binding affinities of the isolated compounds were also assessed against human topoisomerase inhibitors IIβ. Results showed that compound 5 showed a binding affinity of -6.0 kcal/mol, followed by 11 (-5.4 kcal/mol), 10 (-5.0 kcal/mol), and 11 (-4.9 kcal/mol). Similar to ciprofloxacin, compounds 4, 5, 6, 9, 10, and 12 comply with Lipinski's rule of five. Overall, the comprehensive investigation of the chemical constituents and their biological activities reinforces the traditional medicinal applications of A. abyssinica and warrants further exploration of this plant as a source of novel therapeutic agents.

In silico studies of the open form of human tissue transglutaminase

Human tissue transglutaminase (tTG) is an intriguing multifunctional enzyme involved in various diseases, including celiac disease and neurological disorders. Although a number of tTG inhibitors have been developed, the molecular determinants governing ligand binding remain incomplete due to the lack of high-resolution structural data in the vicinity of its active site. In this study, we obtained the complete high-resolution model of tTG by in silico methods based on available PDB structures. We discovered significant differences in the active site architecture between our and known tTG models, revealing an additional loop which affects the ligand binding affinity. We assembled a library of new potential tTG inhibitors based on the obtained complete model of the enzyme. Our library substantially expands the spectrum of possible drug candidates targeting tTG and encompasses twelve molecular scaffolds, eleven of which are novel and exhibit higher binding affinity then already known ones, according to our in silico studies. The results of this study open new directions for structure-based drug design of tTG inhibitors, offering the complete protein model and suggesting a wide range of new compounds for further experimental validation.

In silico homology modeling of dengue virus non-structural 4B (NS4B) protein and its molecular docking studies using triterpenoids

BackgroundDengue fever has become a significant worldwide health concern, because of its high morbidity rate and the potential for an increase in mortality rates due to lack of adequate treatment. There is an immediate need for the development of effective medication for dengue fever.MethodsHomology modeling of dengue virus (DENV) non-structural 4B (NS4B) protein was performed by SWISS-MODEL to predict the 3D structure of the protein. Structure validation was conducted using PROSA, PROCHECK, Ramachandran plot, and VERIFY-3D. MOE software was used to find out the in-Silico inhibitory potential of the five triterpenoids against the DENV-NS4B protein.ResultsThe SWISS-MODEL was employed to predict the three-dimensional protein structure of the NS4B protein. Through molecular docking, it was found that the chosen triterpenoid NS4B protein had a high binding affinity interaction. It was observed that the NS4B protein binding energy for 15-oxoursolic acid, betulinic acid, ursolic acid, lupeol, and 3-o-acetylursolic acid were − 7.18, − 7.02, − 5.71, − 6.67 and − 8.00 kcal/mol, respectively.ConclusionsNS4B protein could be a promising target which showed good interaction with tested triterpenoids which can be developed as a potential antiviral drug for controlling dengue virus pathogenesis by inhibiting viral replication. However, further investigations are necessary to validate and confirm their efficacy.

Bimodal MRI/Fluorescence Nanoparticle Imaging Contrast Agent Targeting Prostate Cancer.

We developed a novel site-specific bimodal MRI/fluorescence nanoparticle contrast agent targeting gastrin-releasing peptide receptors (GRPrs), which are overexpressed in aggressive prostate cancers. Biocompatible ultra-small superparamagnetic iron oxide (USPIO) nanoparticles were synthesized using glucose and casein coatings, followed by conjugation with a Cy7.5-K-8AOC-BBN [7-14] peptide conjugate. The resulting USPIO(Cy7.5)-BBN nanoparticles were purified by 100 kDa membrane dialysis and fully characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, and magnetic resonance imaging (MRI) relaxivity, as well as evaluated for in vitro and in vivo binding specificity and imaging efficacy in PC-3 prostate cancer cells and xenografted tumor-bearing mice. The USPIO(Cy7.5)-BBN nanoparticles had a core diameter of 4.93 ± 0.31 nm and a hydrodynamic diameter of 35.56 ± 0.58 nm. The r2 relaxivity was measured to be 70.2 ± 2.5 s-1 mM-1 at 7T MRI. The Cy7.5-K-8AOC-BBN [7-14] peptide-to-nanoparticle ratio was determined to be 21:1. The in vitro GRPr inhibitory binding (IC50) value was 2.5 ± 0.7 nM, indicating a very high binding affinity of USPIO(Cy7.5)-BBN to the GRPr on PC-3 cells. In vivo MRI showed significant tumor-to-muscle contrast enhancement in the uptake group at 4 h (31.1 ± 3.4%) and 24 h (25.7 ± 2.1%) post-injection compared to the blocking group (4 h: 15.3 ± 2.0% and 24 h: -2.8 ± 6.8%; p < 0.005). In vivo and ex vivo near-infrared fluorescence (NIRF) imaging revealed significantly increased fluorescence in tumors in the uptake group compared to the blocking group. These findings demonstrate the high specificity of bimodal USPIO(Cy7.5)-BBN nanoparticles towards GRPr-expressing PC-3 cells, suggesting their potential for targeted imaging in aggressive prostate cancer.

Phage display-derived alpaca nanobodies as potential therapeutics for Naja atra snake envenomation.

Naja atra, the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified VHH genes from isolated peripheral blood mononuclear cells and constructed a phage display VHH library of 1.0 × 107 transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed nanobodies specific to the genus Naja. An in vivo mouse venom neutralization assay demonstrated that all nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment.IMPORTANCEChinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.

An αIIbβ3 monoclonal antibody traps a semiextended conformation and allosterically inhibits large ligand binding

AbstractMonoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation and binds to thrombin-activated αIIbβ3. Using human platelets as the immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with thrombin receptor–activating peptide (T6). R21D10 also partially inhibited T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and adenosine 5'-diphosphate–induced platelet aggregation. Mutual competition experiments showed that R21D10 does not inhibit the binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-β3 β-I domain), and immunoblot studies indicated that R21D10 binds to β3. The dissociation of αIIbβ3 by EDTA had a minimal effect on R21D10 binding. Cryogenic electron microscopy of the αIIbβ3-R21D10 Fab complex revealed that R21D10 binds to the β3 integrin-epidermal growth factor 1 (I-EGF1) domain and traps an intermediate conformation of αIIbβ3 with semiextended leg domains. The binding of R21D10 produces a major structural change in the β3 I-EGF2 domain associated with a new interaction between the β3 I-EGF2 and αIIb thigh domains, which may prevent the swing-out motion of the β3 hybrid domain required for high-affinity ligand binding and protect αIIbβ3 from EDTA-induced dissociation. R21D10 partially reversed the ligand binding priming effect of eptifibatide, suggesting that it could convert the swung-out conformation into a semiextended conformation. We concluded that R21D10 inhibits ligand binding to αIIbβ3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.

MoRgs3 functions in intracellular reactive oxygen species perception-integrated cAMP signaling to promote appressorium formation in Magnaporthe oryzae.

We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.

In silico Screening of Plectranthus amboinicus and Hyptis suaveolens Phyto-chemicals: Novel Repellents Targeting Odorant Binding Proteins of Aedes aegypti and Aedes albopictus

Background and Aim: Mosquitoes significantly threaten public health, transmitting dangerous diseases to humans and animals. Conventional insecticide spraying, while common, has limitations in effectively controlling vector-borne diseases. Many chemical pesticides harm humans and animals, and some persist in the environment and cause toxic effects. Recently, there has been renewed interest in plant-based products due to concerns about insecticide re-sistance, cross-resistance, potential toxicity associated with synthetic options, and rising costs. Therefore, this study aimed to screen the Plectranthus amboinicus and Hyptis suaveolens phy-tochemicals targeting the odorant binding proteins (OBPs) of Aedes aegypti and Aedes albopic-tus. Methods: In this study, we conducted molecular docking analyses using specific plant-derived compounds from H. suaveolens and P. amboinicus. Results: We focused on the interaction of these compounds with OBPs from dengue and chikungunya vectors (Aedes aegypti and Aedes albopictus). The selected phytochemical com-pounds exhibited strong binding with the OBP of both Ae. aegypti and Ae. albopictus. Tetrahy-drofuran-2-carboxylic acid, Carvacryl acetate, and Brallobarbital showed high binding affinity and significant interaction with Ae. aegypti. Tetrahydrofuran-2-carboxylic acid and 3-Methyl-4-isopropylphenol also demonstrated substantial binding affinity and effective interaction with Ae. albopictus OBP. Conclusion: These findings suggest that the identified compounds can potentially disrupt the attraction of mosquitoes to humans, thus reducing human-vector contact. They may offer a promising alternative for developing natural and efficient mosquito repellents, surpassing cur-rently used synthetic options like N, N-diethyl-meta-toluamide, and other conventional repel-lents.

Computational discovery of AKT serine/threonine kinase 1 inhibitors through shape screening for rheumatoid arthritis intervention.

Rheumatoid Arthritis (RA) is a chronic, symmetrical inflammatory autoimmune disorder characterized by painful, swollen synovitis and joint erosions, which can cause damage to bone and cartilage and be associated with progressive disability. Despite expanded treatment options, some patients still experience inadequate response or intolerable adverse effects. Consequently, the treatment options for RA remain quite limited. The enzyme AKT1 is crucial in designing drugs for various human diseases, supporting cellular functions like proliferation, survival, metabolism, and angiogenesis in both normal and malignant cells. Therefore, AKT serine/threonine kinase 1 is considered crucial for targeting therapeutic strategies aimed at mitigating RA mechanisms. In this context, directing efforts toward AKT1 represents an innovative approach to developing new anti-arthritis medications. The primary objective of this research is to prioritize AKT1 inhibitors using computational techniques such as molecular modeling and dynamics simulation (MDS) and shape-based virtual screening (SBVS). A combined SBVS approach was employed to predict potent inhibitors against AKT1 by screening a pool of compounds sourced from the ChemDiv and IMPPAT databases. From the SBVS results, only the top three compounds, ChemDiv_7266, ChemDiv_2796, and ChemDiv_9468, were subjected to stability analysis based on their high binding affinity and favorable ADME/Tox properties. The SBVS findings have revealed that critical residues, including Glu17, Gly37, Glu85, and Arg273, significantly contribute to the successful binding of the highest-ranked lead compounds at the active site of AKT1. This insight helps to understand the specific binding mechanism of these leads in inhibiting RA, facilitating the rational design of more effective therapeutic agents.

Computational and in-vitro Investigation of Phytochemicals from Allamanda cathartica as a Potential Candidate for the Treatment of Type 2 Diabetes mellitus

Diabetes, a chronic metabolic disease, has become a severe health problem worldwide. According to the latest data from the International Diabetes Federation (IDF), there were 537 million diabetic mellitus (DM) patients worldwide in 2021, expected to increase to 783 million by 2045. Over 90% of people with diabetes have type 2 diabetes, which is driven by socio-economic, demographic, environmental, and genetic factors. Diabetes is still a major global health issue, with prevalence rates rising all across the world. This not only imposes a significant burden on healthcare systems but also impacts the quality of life of affected individuals and their families. Therefore, there is a continuous high demand worldwide to develop novel, more effective, and easily affordable medications against diabetes. This paper illustrates the in-vitro antidiabetic activity and probable binding mechanism prediction of phytoconstituents of Allamanda cathartica against type 2 diabetes molecular targets. Through the application of bioactivity score prediction, molecular docking, and ADMET prediction approach, the potential and selective phytoconstituents with the highest binding affinity and lower toxicity were gained from the curated datasets. Further molecular dynamics simulations and DFT calculations were carried out to identify the favorable binding conformations when the top-scored phytoconstituents bind with the PPAR[Formula: see text] receptors compared to the rosiglitazone. Compound AC2 interacts with the PPAR[Formula: see text] proteins by forming seven hydrogen bonds with the amino acid residues Phe282, His449, Tyr327, His323, and Ser289. The ligand was bound to the protein during the simulation since none of the complex conformations was unstable, and no unfolding or folding occurred. Our results provided an approach to further design and optimize the natural product-inspired small druglike molecules as potential antidiabetic agents. This study highlighted that the phytoconstituents of Allamanda cathartica have antidiabetic potential through the binding of PPAR[Formula: see text], [Formula: see text]-amylase and [Formula: see text]-glucosidase proteins that can be further explored for novel antidiabetic drug development.