Efficient Binding Research Articles

Enhancing preventive and therapeutic cancer vaccine efficacy through biotherapeutic ligand-associated extracellular vesicles

Extracellular vesicles (EVs), secreted by almost all living cells, have gained significant attention for their role in intercellular communication and their potential as versatile carriers for biotherapeutics. However, the clinical translation of EV-based therapies faces significant challenges, primarily due to the lack of efficient methods for loading biotherapeutic agents into EVs. This study introduces a simple, reproducible strategy for the simultaneous incorporation of various biotherapeutics within EVs. The process is gentle and preserves the essential physicochemical and biological characteristics of EVs, thereby protecting labile ligands from premature degradation and elimination. The binding and uptake efficiency of EVs by target cells reached approximately 97 % within 24 h of incubation. Administration of EVs loaded with oligodeoxynucleotides (ODN) resulted in a 4-fold increase in IFNγ+ CD4+ T cells and a 5-fold increase in IFNγ+ CD8+ T cells in the spleens and lymph nodes. Additionally, the co-administration of EVs with ODN and ovalbumin (OVA) induced elevated Th1-biased antibody responses and antigen-specific cytotoxic T-cell responses, providing long-lasting complete protection in 60 % of mice against T-cell thymoma challenge. Furthermore, EVs associated with three different ligands (OVA, CpG-ODN, and α-GalCer) effectively regressed established murine melanoma and significantly improved survival rates in mice. This study presents a powerful and promising approach to overcoming the limitations of EV-based cancer vaccines, advancing the development of effective cancer immunotherapies. SummaryImmunization with EVs that are co-associated with antigen and biotherapeutic cargo through a lyophilization-based technique elicits potent anti-cancer immunity.

Re-engineering of a carotenoid-binding protein based on NMR structure.

Recently, a number of message passing neural network (MPNN)-based methods have been introduced that, based on backbone atom coordinates, efficiently recover native amino acid sequences of proteins and predict modifications that result in better expressing, more soluble, and stable variants. However, usually, X-ray structures, or artificial structures generated by algorithms trained on X-ray structures, were employed to define target backbone conformations. Here, we show that commonly used algorithms ProteinMPNN and SolubleMPNN display low sequence recovery on structures determined using NMR. We subsequently propose a computational approach that we successfully apply to re-engineer AstaP, a protein that natively binds a large hydrophobic ligand astaxanthin (C40H52O4), and for which only a structure determined using NMR is currently available. The engineered variants, designated NeuroAstaP, are 51 amino acid shorter than the 22 kDa parent protein, have 38%-42% sequence identity to it, exhibit good yields, are expressed in a soluble, mostly monomeric form, and demonstrate efficient binding of carotenoids in vitro and in cells. Altogether, our work further tests the limits of using machine learning for protein engineering and paves the way for MPNN-based modification of proteins based on NMR-derived structures.

Identification of Potential Inhibitors of Histone Deacetylase 6 Through Virtual Screening and Molecular Dynamics Simulation Approach: Implications in Neurodegenerative Diseases

Background: Histone deacetylase 6 (HDAC6) plays a crucial role in neurological, inflammatory, and other diseases; thus, it has emerged as an important target for therapeutic intervention. To date, there are no FDA-approved HDAC6-targeting drugs, and most pipeline candidates suffer from poor target engagement, inadequate brain penetration, and low tolerability. There are a few HDAC6 clinical candidates for the treatment of mostly non-CNS cancers as their pharmacokinetic liabilities exclude them from targeting HDAC6-implicated neurological diseases, urging development to address these challenges. They also demonstrate off-target toxicity due to limited selectivity, leading to adverse effects in patients. Selective inhibitors have thus been the focus of development over the past decade, though no selective and potent HDAC6 inhibitor has yet been approved. Methods: This study involved an integrated virtual screening against HDAC6 using the DrugBank database to identify repurposed drugs capable of inhibiting HDAC6 activity. The primary assessment involved the determination of the ability of molecules to bind with HDAC6. Subsequently, interaction analyses and 500 ns molecular dynamics (MD) simulations followed by essential dynamics were carried out to study the conformational flexibility and stability of HDAC6 in the presence of the screened molecules, i.e., penfluridol and pimozide. Results: The virtual screening results pinpointed penfluridol and pimozide as potential repurposed drugs against HDAC6 based on their binding efficiency and appropriate drug profiles. The docking results indicate that penfluridol and pimozide share the same binding site as the reference inhibitor with HDAC6. The MD simulation results showed that stable protein–ligand complexes of penfluridol and pimozide with HDAC6 were formed. Additionally, MMPBSA analysis revealed favorable binding free energies for all HDAC6–ligand complexes, confirming the stability of their interactions. Conclusions: The study implies that both penfluridol and pimozide have strong and favorable binding with HDAC6, which supports the idea of repositioning these drugs for the management of neurodegenerative disorders. However, further in-depth studies are needed to explore their efficacy and safety in biological systems.

"Pseudosubstrate Envelope"/Free Energy Perturbation-Guided Design and Mechanistic Investigations of Benzothiazole HIV Capsid Modulators with High Ligand Efficiency.

Based on our proposed "pseudosubstrate envelope" concept, 25 benzothiazole-bearing HIV capsid protein (CA) modulators were designed and synthesized under the guidance of free energy perturbation technology. The most potent compound, IC-1k, exhibited an EC50 of 2.69 nM against HIV-1, being 393 times more potent than the positive control PF74. Notably, IC-1k emerged as the highest ligand efficiency (LE = 0.32) HIV CA modulator, surpassing that of the approved drug lenacapavir (LE = 0.21). Surface plasmon resonance assay and crystallographic analysis confirmed that IC-1k targeted HIV-1 CA within the chemical space of the "pseudosubstrate envelope". Further mechanistic studies revealed a dual-stage inhibition profile: IC-1k disrupted early-stage capsid-host-factor interactions and promoted late-stage capsid misassembly. Preliminary pharmacokinetic evaluations demonstrated significantly improved metabolic stability in human liver microsomes for IC-1k (T1/2 = 91.3 min) compared to PF74 (T1/2 = 0.7 min), alongside a favorable safety profile. Overall, IC-1k presents a promising lead compound for further optimization.

Evaluating the insecticidal potential of alkaloids for the management of Thrips palmi: in vivo and in silico perspectives.

Insecticidal potential of seven commonly available alkaloids against melon thrips (Thrips palmi Karny) was investigated through in vivo experiments and the bioactivity was explained via in silico approaches. In vivo screening showed highest mortality of T. plami larvae for reserpine (43%), closely followed by tropinone (41%) after 24h of incubation. After 48h, tropinone surpassed reserpine with 83% mortality, indicating its prolonged insecticidal activity. A detailed bioassay of tropinone revealed its LC50 values as 1187.9 and 686.9µg mL-1 after 24 and 48h, respectively. While studying the molecular interactions between the alkaloids and four physiologically important target proteins of T. palmi, tropinone demonstrated the highest ligand efficiency and lowest predicted inhibitory constant, particularly when forming complexes with CathB protein. However, binding energy calculations of the docked complexes showed most favorable binding of reserpine with CathB. To clear the ambiguity, considering both binding energy and ligand efficiency as the evaluation parameters, a molecular dynamics study was carried out, which predicted higher stability of CathB-tropinone complex than CathB-reserpine complex in terms of the total energy of the system. These in silico findings aligned well with the in vivo results, confirming tropinone as a promising candidate for effective thrips management programs.

Whole-blood model reveals granulocytes as key sites of dengue virus propagation, expanding understanding of disease pathogenesis.

Dengue virus (DENV) infection poses a significant global health threat, yet our understanding of its immunopathogenesis remains incomplete due to limitations of existing models. Here, we establish an in vitro whole-blood model using hirudin, an anticoagulant that preserves complement activity and cellular interactions, to study DENV infection. Our model reveals the susceptibility of all major leukocyte populations to DENV infection, with monocytes and granulocytes demonstrating high permissiveness and production of infectious virus progeny. Notably, granulocytes emerge as previously unrecognized targets of DENV infection, highlighting the importance of studying viral tropism within a physiologically relevant context. We also observed efficient DENV binding to B cells, but limited production of infectious virus, suggesting a potential role in viral sequestration or immune dysregulation. Interestingly, both NK and T cells, while less permissive, were also found to be susceptible to DENV infection. Our ex vivo analysis of whole blood from DENV-infected patients confirms the susceptibility of granulocytes, monocytes, B cells, natural killer cells, and T cells to infection, further validating the clinical relevance of our model. Additionally, we observed dynamic changes in circulating blood cell populations during acute dengue, potentially reflecting both direct virus-mediated effects and immune responses. This whole-blood model offers a valuable tool for investigating the complex interplay between DENV and host factors, facilitating a deeper understanding of dengue pathogenesis and ultimately contributing to the development of novel therapeutic strategies.IMPORTANCEDengue virus (DENV) infection is a significant global health threat, with increasing incidence in endemic regions and expanding geographic range due to factors like global warming. Current models for studying DENV pathogenesis often lack the complexity of the human immune system, hindering the development of effective therapies and vaccines. To address this, we have established the first in vitro whole-blood model using hirudin, preserving critical immune components and cellular interactions. This model reveals granulocytes as previously unrecognized targets of productive DENV infection, challenging existing paradigms of viral tropism. Our ex vivo analysis of patient blood samples confirms the clinical relevance of this finding and validates our model's utility. This unique model offers a powerful platform for future studies to dissect the complex interactions between DENV and the host immune system, including the roles of different leukocyte populations, ultimately informing the development of novel therapeutic strategies to combat this devastating disease.

Framework Nucleic Acid-Based Selective Cell Catcher for Endogenous Stem Cell Recruitment.

Cell-surface engineering holds great promise in boosting endogenous stem cell attraction for tissue regeneration. However, challenges such as cellular internalization of ligand and the dynamic nature of cell membranes often complicate ligand-receptor interactions. The aim of this study is to harness the innovative potential of programmable tetrahedral framework nucleic acid (tFNA) to enable precise, tunable ligand-receptor interactions, thereby improving stem cell recruitment efficiency. This approach involves experimental screening and theoretical analysis using dissipative particle dynamics. The results demonstrate that altering the flexibility and topology of ligands on tFNA changes their cellular internalization and membrane binding efficiency. Furthermore, optimizing the distribution of the mesenchymal stem cell (MSC)-binding aptamer 19S (Apt19S) on the tFNA enhances the stem cell capture efficiency. Following successful in vitro MSC capture, Apt19S-modified tFNA is chemically linked to a hyaluronic acid hydrogel, forming an efficient "stem cell catcher" system. Subsequent in vivo experiments demonstrate that this system effectively promotes early stem cell recruitment and accelerates bone regeneration in different bone healing scenarios, including cranial and maxillary defects.

Synchronous Modulation of H-bond Interaction and Steric Hindrance via Bio-molecular Additive Screening in Zn Batteries.

In addressing challenges such as side reaction and dendrite formation, electrolyte modification with bio-molecule sugar species has emerged as a promising avenue for Zn anode stabilization. Nevertheless, considering the structural variability of sugar, a comprehensive screening strategy is meaningful yet remains elusive. Herein, we report the usage of sugar additives as a representative of bio-molecules to develop a screening descriptor based on the modulation of the hydrogen bond component and electron transfer kinetics. It is found that xylo-oligosaccharide (Xos) with the highest H-bond acceptor ratio enables efficient water binding, affording stable Zn/electrolyte interphase to alleviate hydrogen evolution. Meanwhile, sluggish reduction originated from the steric hindrance of Xos contributes to optimized Zn deposition. With such a selected additive in hand, the Zn||ZnVO full cells demonstrate durable operation. This study is anticipated to provide a rational guidance in sugar additive selection for aqueous Zn batteries.

Design, synthesis and evaluation of benzothiazole-derived phenyl thioacetamides as dual inhibitors of monoamine oxidases and cholinesterases.

A series of rationally designed benzothiazole-derived thioacetamides was synthesized and investigated for monoamine oxidases (MAO-A and MAO-B) and cholinesterases (AChE and BChE) inhibition properties. The tested compounds 18-31 inhibited MAO-A and MAO-B in the micromolar to nanomolar range and AChE in the submicromolar range. Compound 28 was identified as the most potent MAO-A inhibitor with an IC50 = 0.030 ± 0.008 µM, whereas compound 30 showed the highest potency towards MAO-B and AChE with IC50 values of 0.015 ± 0.007 µM and 0.114 ± 0.003 µM, respectively. Further, compound 30 inhibited BChE at an IC50 value of 4.125 ± 0.143 µM. Among all screened molecules, compound 30 emerged as the lead dual MAO-B and AChE inhibitor that blocked these enzymes in a competitive-reversible and mixed-reversible mode, respectively. Selected compounds have displayed iron-chelation and antioxidant properties. Further, computational assessment of ligand binding affinity and pharmacokinetic parameters of all new compounds and molecular dynamic simulation of compound 30 with MAO-B and AChE were carried out to understand ligand efficiency, pharmacokinetic, and virtual molecular interaction profile, respectively. The in silico ADMET prediction studies revealed a few undesired pharmacokinetic attributes of our compounds. The attempted virtual lead-based library synthesis and subsequent biological investigation produced a new benzothiazole-bearing dual MAO-B and AChE inhibitor as a prospective MTDL candidate for treating neurological disorders.

Size and Morphology Dependent Activity of Cu Clusters for CO2 Activation and Reduction: A First Principles Investigation.

Various Cu-based materials in diverse forms have been investigated as efficient catalysts for electrochemical reduction of CO2; however, they suffer from issues such as higher over potential and poor selectivity. The activity and selectivity of CO2 electro reduction have been shown to change significantly when the surface morphology (steps, kinks, and edges) of these catalysts is altered. In light of this, size and morphology dependent activity of selected copper clusters, Cun (n=2-20) have been evaluated for the activation and reduction of CO2 molecule. The phase-space of these copper clusters is rich in conformations of distinct morphologies starting from planar, 2D geometries to prolate-shaped geometries and also high-symmetry structures. The binding efficiency and the activation of CO2 are highest for medium sized clusters (n=9-17) with prolate-morphologies as compared to small or larger sized CunCO2 clusters that are existing mainly as planar (triangular, tetragonal etc.) or highly-symmetric geometries (icosahedron, capped-icosahedron etc.), respectively. The best performing (prolate-shaped) CunCO2 conformations are quite fluxional and also they are thermally stable, as demonstrated by the molecular dynamics simulations. Furthermore, on these CunCO2 conformations, the step-by-step hydrogenation pathways of CO2 to produce value-added products like methanol, formic acid, and methane are exceptionally favorable and energy-efficient.

Development of Promising CDK5 Inhibitors Using Structure‐Based Pharmacophore Modeling, Molecular Docking, and Molecular Dynamics Approach

AbstractCancer is highlighted as one of the deadliest diseases globally, with CDK5 identified as a key enzyme in cancer progression. Despite its potential as a therapeutic target, developing CDK5 inhibitors has been challenging. We used multicomplex‐based pharmacophore modeling on CDK5 complexes, identifying hydrophobic groups, hydrogen bond donors, and acceptors as crucial inhibition features. Validated models were used for the virtual screening of drug‐like natural product databases. Thereafter, the screened candidates were selected to study their binding pattern and binding efficiency in the enzyme. Four molecules were shortlisted and analyzed for electrostatic potential (ESP) energy maps. Molecular dynamic simulations and free energy calculations on the docked complexes revealed stable behavior for all, with three (CNP0299652, CNP0362830, and CNP0009633) showing higher Poisson Boltzmann surface area continuum solvation (MM‐PBSA) binding scores than the reference. These candidates demonstrated drug‐like characteristics, crucial amino acid interactions, favorable electron potentials in ESP plots, stable dynamicigher free energy, highlighting their potential as CDK5 inhibitors.

Enhancing the viscoelastic properties of bacterial cellulose hydrogels through ultrasonic and enzymatic modification of xyloglucan

Bacterial cellulose (BC) hydrogels exhibit nanofibril porous network with good viscoelasticity for use as food ingredients and medical materials. Xyloglucan (XG), a hemicellulose with branching residues, can hybridize with BC to improve the hydrogel's extensibility. Thus, modifying the molecular structure of XG can fine-tune the viscoelastic properties of BC hydrogels. In this study, tamarind seed XG subjected to ultrasonic and enzymatic treatment was hybridized with BC to form composite materials. The results indicated that incorporating modified XG reduced the modulus and enhanced the viscous behaviour of BC to varying degrees. XG modified via ultrasonic treatment demonstrated a higher binding efficiency (19–22 %) with cellulose compared to enzymatically treated XG (11–13 %). The enzymatically treated XG improved the maximum elongation ratio to 57 %, but reduced the storage modulus to 30 kPa. Although ultrasonic-treated XG had a similar effect on the shear modulus, it had less impact on the extensibility of BC, with an elongation ratio of 38 %. Additionally, the incorporation of modified XG also regulated the nonlinear viscoelasticity of BC. These findings advance our understanding of the application of XG as a regulator of mechanical and rheological properties, broadening its utility in BC hydrogel formulations for the food industry and medical material development.

Isatin-Fluorene Schiff base as potent antidiabetic agent: Synthesis, DFT calculations, Topology, in silico ADME analysis and molecular docking studies

Diabetes mellitus is a rapidly evolving planetary challenge with substantial societal, salubrity, and financial connotations. Derivatives of isatin with hydrazides, coumarin and aldehydes have proven to possess anti-diabetic activities. Hence, as a novel perspective, isatin was merged with a polycyclic aromatic compound, 2-amino fluorene to build a Schiff base compound with anti-diabetic activity. The anti-diabetic characteristics of the designed compound were scrutinised through in silico molecular docking studies. The designed compound, (Z)-3-((9H-fluoren-2-yl)imino)indolin-2-one (FII) was synthesised in the laboratory using a simple condensation procedure. NMR, HRMS, and Infrared spectral analyses did the experimental structural characterisation of FII. The theoretical investigations of FII were evaluated via density functional theory (DFT). The DFT studies provide insight into the structure, reactivity, and electronic properties of compound FII. The most stable conformation of FII was identified and confirmed by the B3LYP method. The basis set 6-31+G(d) was used for the calculations. The DFT studies include frontier molecular orbital (FMO) analysis to define the global parameters and molecular orbital energy gap, molecular electrostatic potential (MEP) map to find out the reactive sites and natural bond orbital (NBO) analysis for understanding the electron density and charge delocalisation of the title compound, FII. Topological analyses such as ELF, LOL, RDG, and QTAIM were performed to investigate the electronic bonding parameters. Additionally, UV-visible and photoluminescence spectra were performed to investigate the optical properties of FII. The biological potential of the title compound was validated by calculating the physicochemical properties using Lipinski's rule. The molecular docking of the synthesised compound, FII, has been computed using two different proteins, exhibiting inhibitory activity against the α-Glucosidase enzyme. The binding energy and ligand efficiency of both the selected proteins with the ligand molecule were also calculated.

Can Benzimidazole Resistance Undermine the Philippines’ Success in Controlling and Eliminating Soil-Transmitted Helminth Infections? A Mini-Review

Abstract Soil-transmitted helminth (STH) infections remain a significant global health burden, affecting over 1.5 billion people worldwide. In the Philippines, STH infections continue to be prevalent, despite ongoing control efforts. The Integrated Helminth Control Program (IHCP), whose cornerstone is mass drug administration (MDA) with benzimidazole drugs, has shown some success in reducing STH prevalence in the country. However, the persistence of infection prevalence being above the national and global targets and the potential for benzimidazole resistance have raised concerns about the long-term sustainability of current control strategies. This review examines the development of benzimidazole resistance in STH, focusing on the role of single nucleotide polymorphisms (SNPs) in the β-tubulin isotype that alter the protein’s amino acid constitution, thereby negatively affecting benzimidazole drug binding efficiency. This review discusses the epidemiology of STH infections in the Philippines, the implementation of IHCP and the potential challenges posed by benzimidazole resistance. This review highlights the need for further research to assess the occurrence of benzimidazole resistance in Philippine STH populations and to explore alternative control strategies. Understanding the mechanisms of benzimidazole resistance and developing effective countermeasures is crucial for achieving sustainable STH control and elimination in the Philippines.

2-Methoxy-4-formylphenol suppresses methylglyoxal glycation mediated structural alterations and esterase activity of hemoglobin – A multi spectroscopic, biophysical and in-silico study

Glycation is the non-enzymatic reaction of glucose or its metabolites to proteins, causing irreversible changes. Methylglyoxal, a dicarbonyl, affects the structure and function of physiologically important proteins. Being a major circulatory protein, hemoglobin is highly prone to glycation. Current research focuses on identifying potent glycation inhibitors to prevent glycation and their impact on protein structure and function. The present study investigates the Advanced Glycation Endproducts (AGEs) inhibitory effects of 2-methoxy-4-formylphenol (Vanillin) against methylglyoxal mediated glycation of hemoglobin. The hemoglobin-vanillin glycation model exhibited inhibition of AGE formation, amyloid fibrils, aggregates and reduction in esterase activity. The fluorescence spectroscopic technique revealed efficient binding of vanillin and hemoglobin, with Stern Volmer plot indicating the presence of static quenching. The conformational stability of the vanillin and hemoglobin interaction was also evident from the molecular docking and dynamics studies. The proximal orientation of residues (H2 and K82 associated in esterase activity) of hemoglobin β1 chain and vanillin, supports the noted effect of reduced esterase activity in the presence of vanillin in glycated hemoglobin and the inhibition of the overall formation of AGE of hemoglobin in the presence of vanillin.

Isolation of antibodies to hypochlorite-modified low-density lipoproteins from human serum and study of their specificity

BACKGROUND: Modified low-density lipoproteins have immunogenic properties and induce the production of antibodies. In this case, HOCl promotes the formation of subsequent active halogen-containing compounds interacting with proteins and lipid parts of low-density lipoproteins, which leads to their modification and the production of antibodies to them. AIM: The aim of this work is to isolate antibodies to hypochlorite modified low-density lipoproteins from human blood sera and study their specificity. MATERIALS AND METHODS: Malondialdehyde, acetic anhydride and sodium hypochlorite were used to obtain modified low-density lipoproteins. IgG antibodies to hypochlorite modified low-density lipoproteins were isolated by affinity chromatography. The total IgG fraction antibodies was previously isolated from human blood serum using MabSelect Xtra. The specific antibodies to hypochlorite modified low-density lipoproteins were isolated from this IgG pool by affinity chromatography. CNBr-Sepharose 4B conjugated with human serum albumin modified with NaOCl was used as a sorbent. The specificity of antibodies against hypochlorite modified low-density lipoproteins was tested using a competitive enzyme-linked immunosorbent assay. The competitors were hypochlorite modified low-density lipoproteins, acetic anhydride modified low-density lipoproteins and malondialdehyde modified low-density lipoproteins in concentrations (1–250 μg/ml). RESULTS: IgG antibodies against hypochlorite-modified proteins that interact with hypochlorite modified low-density lipoproteins were detected in human blood. According to ELISA date the binding of the isolated antibodies to hypochlorite modified low-density lipoproteins was almost completely inhibited only by appropriately modified low-density lipoproteins, that is, hypochlorite modified low-density lipoproteins, but not native low-density lipoproteins or acetic anhydride modified low-density lipoproteins. Malondialdehyde modified low-density lipoproteins also showed some competitive activity, but much weaker than hypochlorite modified low-density lipoproteins. Hypochlorite modified low-density lipoproteins itself and, to a lesser extent, malondialdehyde modified low-density lipoproteins competed for binding with antibodies of human serum to hypochlorite modified low-density lipoproteins. Acetic anhydride modified low-density lipoproteins and native low-density lipoproteins did not reduce the efficiency of antibody binding to their antigen. CONCLUSIONS: Hypochlorite modified low-density lipoproteins forms epitopes independent of other low-density lipoproteins modifications studied. These epitopes are responsible for the formation of specific antibodies.

Tuning the properties of peptide imprinted nanoparticles for protein immunoprecipitation using magnetic streptavidin beads

Maximizing the binding properties of thermoresponsive molecularly imprinted nanoparticles (MIN) was aimed to explore their feasibility as antibody substitutes in protein immunoprecipitation (IPP) with magnetic streptavidin beads (MSB). Thermoresponsive MIN targeting the cannabinoid CB1 receptor were produced by epitope imprinting through solid-phase synthesis. It was intended to determine how different variables influenced physicochemical features, binding behaviour and immunoprecipitation of the target recombinant glutathione S-transferase tagged fusion protein (GST-CTer). Such variables included the cross-linking degree of MIN, and variables like pH, temperature or the use of Tween-20 for binding and IPP experiments. The cross-linker (CL) amount influenced the coil-to-globule transition of thermoresponsive MIN, making the lower critical solution temperature (LCST) decrease from 37.2 °C using 5% of CL, to 29.0 °C using 25%, also suggesting higher plasticity on the former. Temperature influence on size was corroborated by dynamic light scattering, observing size reductions from 250–450 nm (RT) to 70–100 nm (> LCST) for MIN produced with 5–15% of CL. However, binding behaviour did not clearly improve for more than 10% CL. Further experiments revealed that temperature and pH control were critical for efficient binding and release, selecting 40 °C and pH 5 as appropriate. Following binding experiments, the GST-CTer-MIN complex was successfully immunoprecipitated using MSB, achieving an IPP efficiency of 11.48% over the initial input protein concentration, which was calculated after SDS-PAGE separation and Western blot analysis. The methodology may be exploited for selective protein extraction and quantification from complex tissue homogenates.Graphical

Marker-dependent cleavage of RNA by binary (split) DNAzyme (BiDz) and binary DNA machines (biDNM).

Oligonucleotide gene therapy (OGT) can be used to suppress specific RNA in cells and thus have been explored for gene therapy. Despite extensive effort, there is no clinically significant OGT for treating cancer. Low efficiency of OGT is one of the problems. Earlier, we proposed to address this problem by suppressing most vital genes in cancer cells e.g. housekeeping genes. To achieve specific activation of the OGT agents in cancer but not in normal cells, we designed binary (split) DNAzyme (BiDz), which is activated by cancer-related nucleic acid sequences. This work is devoted to BiDz optimization using cancer marker-related sequence as an activator and three folded RNA targets. To achieve efficient binding of folded RNA, the BiDz design was equipped with RNA binding/unwinding arms, a construction that was dabbed 'BiDz machines' (BiDM). BiDM was designed to have improved both iRNA cleavage rates and RNA recognition in comparison with BiDz. Further development of DNA nanotechnology-inspired agents can advance OGT technology in treating cancer, viral infections, and genetic disorders.

Binding of a Tricationic meso-Substituted Porphyrin to poly(A)⋅poly(U): an Experimental Study.

The porphyrins are macrocyclic compounds widely used as photosensitizers in anticancer photodynamic therapy. The binding of a tricationic meso-tris(N-methylpyridinium)-porphyrin, TMPyP3+, to poly(A)⋅poly(U) polynucleotide has been studied in neutral buffered solution, pH6.9, of low and near-physiological ionic strength in a wide range of molar phosphate-to-dye ratios (P/D). Effective TMPyP3+ binding to the biopolymer was established using absorption spectroscopy, polarized fluorescence, fluorimetric titration and resonance light scattering. We propose a model in which TMPyP3+ binds to the polynucleotide in two competitive binding modes: at low P/D ratios (< 4) external binding of the porphyrin to polynucleotide backbone without self-stacking dominates, and at higher P/D (> 30) the partially stacked porphyrin J-dimers are embedded into the polymer groove. Enhancement of the porphyrin emission was observed upon binding in all P/D range, contrasting the binding of this porphyrin to poly(G)⋅poly(C) with significant quenching of the porphyrin fluorescence at low P/D ratios. This observation indicates that TMPyP3+ can discriminate between poly(A)⋅poly(U) and poly(G)⋅poly(C) polynucleotides at low P/D ratios. Formation of highly scattering extended porphyrin aggregates was observed near the stoichiometric in charge binding ratio, P/D = 3. It was revealed that the efficiency of the porphyrin external binding and aggregation is reduced in the solution of near-physiological ionic strength.

Structure‐Based Computational Approach in Search of the Potent Molecules Targeting Phosphoglycerate Dehydrogenase (PHGDH) Enzyme for Cancer Treatment

AbstractThis work aims to target phosphoglycerate dehydrogenase (PHGDH), a promising druggable target, that is overexpressed in various types of cancer. A structure‐based approach was employed to identify novel inhibitors against the enzyme. A common five‐feature pharmacophore model (RRHDA) was constructed using the active site co‐crystalized ligands. These chemical features were responsible for showing inhibition. The generated models were subsequently subjected to the validation method using a test set, receiver‐operator characteristic analysis, enrichment factor, and Güner–Henry studies. The validated models were subjected to the screening of a dataset of natural compounds. The screened unique natural compounds (1795) were further selected for the interaction analysis and study of ligand binding affinity considering the effect of the hydrogen bonding and desolvation and hydrophobic interactions contribution to the binding. The natural compounds which exhibited good ligand binding efficiency were selected and further subjected to pharmacokinetics and pharmacodynamic study. The natural compounds which exhibited good ligand binding efficiency were selected and further subjected to pharmacokinetics and pharmacodynamic study. The finalized complexes were selected for the simulation studies and MM/PBSA‐based binding free energy calculations. This study expands the possibilities for the development of shortlisted molecules as novel anti‐cancer compounds.