Cyclodextrin-based tailored polyrotaxanes for highly efficient delivery of the genome-editing molecule.

Direct cytosolic delivery of the Cas9 ribonucleoprotein is the most promising method for inducing CRISPR-Cas9 genome editing in mammalian cells. Recently, we focused the movable properties of cyclodextrin-based polyrotaxanes (PRXs), which consist of numerous cyclodextrins threaded onto the axile molecule with bulky endcaps at both ends of the axile molecule, and developed aminated PRXs as multistep transformable carriers for Cas9 ribonucleoprotein, ensuring efficient complexation, cellular internalization, endosomal escape, release, and nuclear localization. This study reports the structural fine-tuning and structure-property relationship of multistep transformable PRXs for more efficient Cas9 ribonucleoprotein delivery. Among various PRXs, PRX derivatives with a longer molecular length (35 kDa polyethylene glycol as the axile molecule) and a low total degree of substitution (1.5 amino groups/α-cyclodextrins), as well as the modified ratio of two modified amines (cystamine and diethylenetriamine) = ≈1:1, exhibited the highest genome-editing efficacy and intracellular dynamics control. These structural properties are important for efficient endosomal escape and Cas9 RNP release. Furthermore, ligand-modified-ß-CD, which can endow the ligand through complexation with PRX termini, improved the cellular uptake and genome-editing effects of the optimized PRX/Cas9 RNP in target cells. Thus, structural fine-tuning and the addition of ligand-modified-ß-cyclodextrin enabled efficient genome editing by the Cas9 RNP.

Surface Plasmon Resonance: A Sensitive Tool to Study Protein-Protein Interactions.

Surface plasmon resonance (SPR) is one of the most commonly used techniques to study protein-protein interactions. The main advantage of SPR is the ability of measuring binding affinities and association/dissociation kinetics of complexes in real time, in a label-free environment, and using relatively small quantities of materials. The method is based on the immobilization of one of the binding partners, called the "ligand," on a dedicated sensor surface. Immobilization is followed by the injection of the other partner, called the "analyte," over the surface containing the ligand. The binding is monitored by following changes in the refractive index of the medium close to the sensor surface upon injection of the analyte. During the last 15 years, SPR has been intensively used in the study of bacterial secretion systems due to its ability of detecting highly dynamic complexes, which are difficult to investigate by other techniques. This chapter will guide users in setting up SPR experiments in order to identify protein complexes and to assess their binding affinity and/or kinetics. It will include detailed protocols for (i) immobilization of proteins with the amine coupling capture method, (ii) analyte-binding analysis, (iii) affinity/kinetics measurements, and (iv) data analysis.

CryoEM Data Analysis of Membrane Proteins. Practical Considerations on Amphipathic Belts, Ligands, and Variability Analysis.

Membrane proteins data analysis by cryoEM shows some specificities, as can be found in other typical investigations such as biochemistry, biophysics, or X-ray crystallography. Membrane proteins are typically surrounded by an amphipathic belt that will have some degree of influence on the 3D reconstruction and analysis. In this chapter, we review our experience with the ABC transporter BmrA, as well as our statistical analysis of amphipathic belts around membrane proteins, to bring awareness on some particular features of membrane protein investigations by cryoEM.

Fluorescence "light-up" sensor based on ligand/SiO2@NH2@cyanuric chloride nanoparticle interactions in alliance with salt dehydration for berberine detection.

Berberine (BBR) is an important anti-inflammatory drug for the treatment of intestinal diseases. The quantification of BBR is required in clinical medicine because long-term or excessive intake can lead to drug resistance and adverse effects. In this study, SiO2@NH2@cyanuric chloride (CNCl) nanoparticles (NPs) were successfully prepared by covalently incorporating CNCl onto the surface of SiO2 NPs. Furthermore, a novel fluorescence "light-up" sensor for assaying BBR was established based on the interaction between BBR and SiO2@NH2@CNCl NPs. Although BBR was non-emissive in aqueous media, its fluorescence was considerably augmented because of the interaction with the as-prepared SiO2@NH2@CNCl NPs, and the enhancement factor was approximately three times larger than that of pure SiO2 NPs. Compared with SiO2 NPs, SiO2@NH2@CNCl NPs can interact with BBR through electrostatic interactions and π-π stacking. These interactions restricted the intramolecular motion and charge transfer of BBR, resulting in fluorescence enhancement. The sensor was sensitive, with a linear response over a concentration range of 25-2500 nM (R2 = 0.9905) and a detection limit (3σ/k) of 4.7 nM, and it had good selectivity for BBR in the presence of bovine serum albumin, amino acids, and metal ions. When the sensor was applied to real serum samples, rapid extraction and salt dehydration occurred to improve the efficiency of pretreatment, and satisfactory standard recovery rates (95%-96%) were achieved even when only small amounts of acetonitrile was used for protein precipitation. This strategy could serve as a reference for other studies requiring the analysis of drugs in biological samples.

Ratiometric fluorescence aptasensor for the detection of patulin in apple juice based on the octahedral UiO-66-TCPP metal-organic framework and aptamer systems.

Patulin (PAT) is a potentially harmful mycotoxin to human health and is known to contaminate apple juice. In this work, we developed a ratiometric fluorescence aptasensor using tetrakis(4-carboxyphenyl)porphyrin (H2TCPP)-treated octahedral UiO-66-NH2 (defined as UiO-66-TCPP) to detect PAT. This 2-aminoterephthalic acid and H2TCPP functionalized metal-organic framework showed multiple adsorption effects (hydrogen bonding and π-π stacking) on the aptamer (Apt) and served as a quenching material. When the target PAT bound specifically to the Apt, the fluorescence of the 6-carboxyfluorescein-labeled Apt would recover, and the fluorescence of the H2TCPP ligand remained unchanged. This ratiometric fluorescence property improved the accuracy of PAT detection. Moreover, the introduction of the H2TCPP ligand enhanced the quenching efficiency of UiO-66-NH2, thus improving the sensitivity of the fluorescent aptasensor (UiO-66-TCPP vs. UiO-66-NH2: 0.0162 ng/mL vs. 1.8 ng/mL). In addition, we used UiO-66-TCPP to detect PAT in apple juice samples. This work provides a good paradigm for the construction of ratiometric fluorescence aptasensors with high sensitivity and accuracy.

Application and evaluation of molecular docking for aptamer and small molecular interaction - A case study with tetracycline antibiotics.

Molecular docking (MD) analysis is currently the most commonly used theoretical simulation method to investigate the interaction of aptamers (receptors) and small molecules (ligands) and understand the recognition mechanism between them at a molecular level. Using the specific aptamers of tetracycline antibiotics (tetracycline (TET), oxytetracycline (OTC), doxycycline (DOC)) as the docking models, three steady-state aptamers of tertiary structures (SATS) were established for each aptamer with the UNAFold and RNAComposer tools. The binding free energy (BFE), docking score (DS), and binding site (base) of the specific ligands (TET, OTC, and DOC) with their respective SATS were obtained by molecular docking. The results revealed one or more binding sites in the established SATS of the aptamers. The BFE and DS of different binding sites of one specific SATS varied significantly. The results also revealed that the site with the highest BFE represented the most dominant binding site, even if it was not the SATS with minimum energy. The BFE values could also be used to evaluate the affinity and specificity of the aptamer to its target. For the first time, this study proposes a method for MD analysis of the aptamer and its target based on different SATS, clarification of the binding mode, and prediction of the binding sites (bases). This study provides a theoretical basis for tailoring; structural optimization; and base modification of aptamers; identifying aptamers with high affinity and specificity.

One master and two servants: One Zr(â £) with two ligands of TCPP and NH2-BDC form the MOF as the electrochemiluminescence emitter for the biosensing application.

Here we put forward an innovative "one master and two servants" strategy for enhancing the ECL performance. A novel ECL luminophore named Zr-TCPP/NH2-BDC (TCPP@UiO-66-NH2) was synthesized by self-assembly of meso-tetra(4-carboxyphenyl)porphine (TCPP) and 4-aminobenzoic acid (NH2-BDC) with Zr clusters. TCPP@UiO-66-NH2 has a porous structure and a highly ordered structure, which allows the molecular motion of TCPP to be effectively confined, thereby inhibiting nonradiative energy transfer. Importantly, TCPP@UiO-66-NH2 has a higher and more stable ECL signal. To further improve the sensitivity of the sensor, we use polydopamine-coated manganese dioxide (PDA@MnO2), which has a double quenching effect, as the quencher. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-N) is one of the ideal markers for the early diagnosis of COVID-19, and its sensitivity detection is of great significance for the prevention and treatment of COVID-19. Thus, we constructed a quenching-type ECL sensor for the ultrasensitive detection of the SARS-CoV-2-N. Its linear range is 10 fg/mL∼1 µg/mL and the calculated detection limit is 1.4 fg/mL (S/N = 3). The spiked recoveries are 97.40-103.8%, with the relative standard deviations (RSD) under 3.0%. More importantly, the technique offers a viable way to identify and diagnose viral infections early.

Recent progress of SELEX methods for screening nucleic acid aptamers.

Nucleic acid aptamers are oligonucleotide sequences screened by an in vitro methodology called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Known as "chemical antibodies", aptamers can achieve specific recognition towards the targets through conformational changes with high affinity, and possess multiple attractive features including, but not limited to, easy and inexpensive to prepare by chemical synthesis, relatively stable and low batch-to-batch variability, easy modification and signal amplification, and low immunogenicity. Now, aptamers are attracting researchers' attentions from more than 25 disciplines, and have showed great potential for application and economic benefits in disease diagnosis, environmental detection, food security, drug delivery and discovery. Although some aptamers exist naturally as the ligand-binding elements of riboswitches, SELEX is a recognized method for aptamers screening. After thirty-two years of development, a series of SELEX methods have been investigated and developed, as well as have shown unique advantages to improve sequence performances or to explore screening mechanisms. This review would mainly focus on the novel or improved SELEX methods that are available in the past five years. Firstly, we present a clear overview of the aptamer's history, features, and SELEX development. Then, we highlight the specific examples to emphasize the recent progress of SELEX methods in terms of carrier materials, technical improvements, real sample-improved screening, post-SELEX and other methods, as well as their respects of screening strategies, implementation features, screening parameters. Finally, we discuss the remaining challenges that have the potential to hinder the success of SELEX and aptamers in practical applications, and provide the suggestions and future directions for developing more convenient, efficient, and stable SELEX methods in the future.

Antiviral Drug Target Identification and Ligand Discovery.

This chapter intends to provide a general overview of web-based resources available for antiviral drug discovery studies. First, we explain how the structure for a potential viral protein target can be obtained and then highlight some of the main considerations in preparing for the application of receptor-based molecular docking techniques. Thereafter, we discuss the resources to search for potential drug candidates (ligands) against this target protein receptor, how to screen them, and preparing their analogue library. We make specific reference to free, online, open-source tools and resources which can be applied for antiviral drug discovery studies.

Protein-Ligand Blind Docking Using CB-Dock2.

Protein-ligand blind docking is a widely used method for studying the binding sites and poses of ligands and receptors in pharmaceutical and biological research. Recently, our new blind docking server named CB-Dock2 has been released and is currently being utilized by researchers worldwide. CB-Dock2 outperforms state-of-the-art methods due to its accuracy in binding site identification and binding pose prediction, which are enabled by its knowledge-based docking engine. This highly automated server offers interactive and intuitive input and output web interfaces, making it an efficient and user-friendly tool for the bioinformatics and cheminformatics communities. This chapter provides a brief overview of the methods, followed by a detailed guide on using the CB-Dock2 server. Additionally, we present a case study that evaluates the performance of protein-ligand blind docking using this tool.

Molecular Dynamics as a Tool for Virtual Ligand Screening.

Rational drug design is essential for new drugs to emerge, especially when the structure of a target protein or nucleic acid is known. To that purpose, high-throughput virtual ligand screening campaigns aim at discovering computationally new binding molecules or fragments to modulate particular biomolecular interactions or biological activities, related to a disease process. The structure-based virtual ligand screening process primarily relies on docking methods which allow predicting the binding of a molecule to a biological target structure with a correct conformation and the best possible affinity. The docking method itself is not sufficient as it suffers from several and crucial limitations (lack of full protein flexibility information, no solvation and ion effects, poor scoring functions, and unreliable molecular affinity estimation).At the interface of computer techniques and drug discovery, molecular dynamics (MD) allows introducing protein flexibility before or after a docking protocol, refining the structure of protein-drug complexes in the presence of water, ions, and even in membrane-like environments, describing more precisely the temporal evolution of the biological complex and ranking these complexes with more accurate binding energy calculations. In this chapter, we describe the up-to-date MD, which plays the role of supporting tools in the virtual ligand screening (VS) process.Without a doubt, using docking in combination with MD is an attractive approach in structure-based drug discovery protocols nowadays. It has proved its efficiency through many examples in the literature and is a powerful method to significantly reduce the amount of required wet experimentations (Tarcsay et al, J Chem Inf Model 53:2990-2999, 2013; Barakat et al, PLoS One 7:e51329, 2012; De Vivo et al, J Med Chem 59:4035-4061, 2016; Durrant, McCammon, BMC Biol 9:71-79, 2011; Galeazzi, Curr Comput Aided Drug Des 5:225-240, 2009; Hospital et al, Adv Appl Bioinforma Chem 8:37-47, 2015; Jiang et al, Molecules 20:12769-12786, 2015; Kundu et al, J Mol Graph Model 61:160-174, 2015; Mirza et al, J Mol Graph Model 66:99-107, 2016; Moroy et al, Future Med Chem 7:2317-2331, 2015; Naresh et al, J Mol Graph Model 61:272-280, 2015; Nichols et al, J Chem Inf Model 51:1439-1446, 2011; Nichols et al, Methods Mol Biol 819:93-103, 2012; Okimoto et al, PLoS Comput Biol 5:e1000528, 2009; Rodriguez-Bussey et al, Biopolymers 105:35-42, 2016; Sliwoski et al, Pharmacol Rev 66:334-395, 2014).

Accelerating Molecular Dynamics Simulations for Drug Discovery.

Accurate prediction of ligand binding thermodynamics and kinetics is crucial in drug design. However, it remains challenging for conventional molecular dynamics (MD) simulations due to sampling issues. Gaussian accelerated MD (GaMD) is an enhanced sampling method that adds a harmonic boost to overcome energy barriers, which has demonstrated significant benefits in exploring protein-ligand interactions. Especially, the ligand GaMD (LiGaMD) applies a selective boost potential to the ligand nonbonded potential energy, significantly improving sampling for ligand binding and dissociation. Furthermore, a selective boost potential is applied to the potential of both ligand and protein residues around binding pocket in LiGaMD2 to further increase the sampling of protein-ligand interaction. LiGaMD and LiGaMD2 simulations could capture repetitive ligand binding and unbinding events within microsecond simulations, allowing to simultaneously characterize ligand binding thermodynamics and kinetics, which is expected to greatly facilitate drug design. In this chapter, we provide a brief review of the status of LiGaMD in drug discovery and outline its usage.

Alchemical Free Energy Workflows for the Computation of Protein-Ligand Binding Affinities.

Alchemical free energy methods can be used for the efficient computation of relative binding free energies during preclinical drug discovery stages. In recent years, this has been facilitated further by the implementation of workflows that enable non-experts to quickly and consistently set up the required simulations. Given the correct input structures, workflows handle the difficult aspects of setting up perturbations, including consistently defining the perturbable molecule, its atom mapping and topology generation, perturbation network generation, running of the simulations via different sampling methods, and analysis of the results. Different academic and commercial workflows are discussed, including FEW, FESetup, FEPrepare, CHARMM-GUI, Transformato, PMX, QLigFEP, TIES, ProFESSA, PyAutoFEP, BioSimSpace, FEP+, Flare, and Orion. These workflows differ in various aspects, such as mapping algorithms or enhanced sampling methods. Some workflows can accommodate more than one molecular dynamics (MD) engine and use external libraries for tasks. Differences between workflows can present advantages for different use cases, however a lack of interoperability of the workflows' components hinders systematic comparisons.

DFT, Molecular Docking, Bioactivity and ADME Analyses of Vic-dioxim Ligand Containing Hydrazone Group and its Zn(II) Complex.

BACKGROUND: Cancer is one of the diseases affecting a large population worldwide and resulting in death. Finding new anti-cancer drugs that are target-focused and have low toxicity is of great importance. OBJECTIVE: This study aimed to investigate the effects of vic-dioxime derivatives carrying hydrazone group and its Zn(II) complex on cancer using molecular docking, bioactivity and quantum chemical calculations. METHODS: Molecular docking studies were performed on epidermal growth factor receptor and vascular endothelial growth factor receptor 2 target proteins. Furthermore, molecular geometry was performed, and the frontier molecular orbitals, Mulliken charges and molecular electron density distribution were evaluated using density functional theory. Also, the bioactivity parameters of the compounds were evaluated, and ADME analysis was performed using web-based tools. RESULTS: Higher binding affinity was observed for Zn(II) complex with target proteins vascular endothelial growth factor receptor 2 and against epidermal growth factor receptor when compared with LH2. Only the Zn(II) complex against the epidermal growth factor receptor had ligand efficiency and fit quality in the valid range. Furthermore, LH2 has the most potent electrophilic ability (acceptor) among other compounds. Moreover, both LH2 and Zn(II) complexes strongly satisfy Lipinski's rule of five. CONCLUSION: In conclusion, these novel compounds, especially Zn(II) complex, can be new candidates for anticancer drug development studies which are target-focused and have low toxicity.

Binding studies of potential amyloid-ß inhibiting chalcone derivative with bovine serum albumin.

Chalcones (α-phenyl-ß-benzoylethylene) and their natural-source derivatives have been investigated for their remarkable biological activities, like neuroprotective, anti-inflammatory, and anti-tumor properties. A triazole chalcone ligand (E)-3-(4-(dimethylamino)phenyl)-1-(4-((1-(2-(4-((E)-3-(4(dimethylamino)phenyl)acryloyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)prop-2-en-1-one (L1) was synthesized by Cu(I)- catalysed click reaction. The mechanistic properties of L1 for therapy were evaluated by analyzing the binding interactions between L1 and bovine serum albumin (BSA) through photophysical and computational studies. The structural elucidation of ligand L1 was carried out by NMR and mass spectrometry. The Aß inhibitory activity of L1 was studied by thioflavin T assay and transmission electron microscopy. The biomolecular interaction of L1 with bovine serum albumin was examined through multi-spectroscopic techniques in combination with in silico studies. UV-Visible absorption, fluorescence spectroscopy, circular dichroism, Förster resonance energy transfer, and three-dimensional fluorescence studies confirmed the formation of a BSA-L1 complex. The potential binding sites, mechanism of interactions, and variations in the environment of tyrosine and tryptophan amino acid residues of BSA were assessed at different temperatures. The binding constant for the Static quenching mechanism of intrinsic fluorescence of BSA was of the order of 105 M-1. The esterase enzyme activity assay in the presence of L1 revealed an increase in the protein enzyme activity. Molecular docking studies suggested L1 was predominantly bound to BSA by hydrogen bonds and Van der Waals forces.

Cadmium speciation in cacao beans changes during a fermentation-like incubation.

Cadmium (Cd) concentrations in cacao often exceed food limits. Recently, it was shown that cacao bean fermentation enhances Cd solubility, opening potential for Cd mitigation in cacao products. This study was set-up to identify changes in Cd speciation during fermentation. X-Ray absorption spectroscopy (XAS) complemented with speciation calculations, were used on samples collected from high and low Cd farms, that were subjected to a fermentation-like incubation that reached high temperatures (>45 °C) and acidic pH (<5). Incubation decreased nib Cd concentration up to a factor 1.5 and changed Cd complexation in high Cd beans from sulphur to oxygen ligands, likely due to pH changes. In beans with lower Cd concentrations, Cd was complexed before and after incubation with oxygen-ligands. A combination of pH changes and/or phytate breakdown may explain the migration of Cd outward from the nib. XAS and speciation calculations proved complimentary techniques and indicated similar speciation changes during fermentation.

Fluorescence Spectroscopy: A Useful Method to Explore the Interactions of Small Molecule Ligands with DNA Structures.

Small molecule ligands-DNA interactions have recently received a lot of attention in the fields of life sciences, medicine, and chemical sciences. To decode these interactions, many strategies have been developed. DNA is the primary target for a wide range of drugs that may interact with DNA in particular or non-specific ways and impact its activities. Fluorescence spectroscopy is a highly advanced and non-invasive technology for measuring the concentrations of substrates and products or identifying characteristic processing states. Small molecule ligands-DNA interaction studies are beneficial not only in comprehending the method of interaction, but also in synthesizing DNA-targeted particular drugs. Several small compounds that bind to DNA are clinically established therapeutic medicines, while their specific mechanism of action is unknown. Figuring out their molecular recognizing patterns is the only way to construct innovative compounds that can target specific DNA sequences with strong affinities. This book chapter will mostly explore several fluorescence spectroscopic methodologies used to investigate interactions between small molecule ligands and DNA. In addition, we provide many approaches for determining a drug's binding mode with DNA. These strategies produce data that is both trustworthy and easy to comprehend. All of the knowledge gained by studying these fluorescence spectroscopies are supposed to lead to the development of more efficient new pharmaceuticals that might aid in the treatment of diseases.

Molecular Modeling Techniques and In-Silico Drug Discovery.

Molecular modeling is the technique to determine the overall structure of an unknown molecule, be it a small one or a macromolecule. The technique encompasses the method of screening ligand libraries for the development of new candidate drug molecules. All these aspects have become an essential topic of research. This field is truly interdisciplinary and finds its applications in almost all fields of life science research. In this chapter, an overview of the protocol associated with molecular modeling techniques will be discussed.

Adaptive evolution of the Clf-Sdr subfamily contributes to Staphylococcus aureus musculoskeletal infection: Evidence from comparative genomics.

Persistent Staphylococcus aureus infections of the musculoskeletal system are a challenge in clinical practice. Although extensive studies on the genotypic changes in S. aureus in soft tissue and blood system infections have been conducted, little is known about how S. aureus adapts to the microenvironment of the musculoskeletal system. Here, we used comparative genomics to analyze the isolates from patients with an S. aureus infection of the musculoskeletal system. We observed that mutations in the Clf-Sdr subfamily proteins frequently occurred during persistent infections. Furthermore, these mutations were primarily located in the non-active site (R region), rather than in the active site (A region). Mechanistically, the clfA/B mutation enhanced the S. aureus biofilm formation ability through the binding to fibrinogen and intercellular adhesion. Complementation studies using the USA300-ΔMSCRAMMs strains clfA and clfB revealed that mutations in both the A and R regions could enhance their corresponding function. The results of protein structure prediction and ligand-binding simulations suggest that these mutations influence the protein structure and ligand binding. In conclusion, our study suggests that the Clf-Sdr subfamily mutations may be one of the mechanisms contributing to persistent S. aureus infections of the musculoskeletal system.

Theoretical models of staurosporine and analogs uncover detailed structural information in biological solution.

Staurosporine and its analogs (STA-analogs) are indolocarbazoles (ICZs) compounds able to inhibit kinase proteins in a non-specific way, while present antimicrobial and cytostatic properties. The knowledge of molecular features associated to the complexation, including the ligand shape in solution and thermodynamics of complexation, is substantial to the development of new bioactive ICZs with improved therapeutic properties. In this context, the empirical approach of GROMOS force field is able to accurately reproduce condensed phase physicochemical properties of molecular systems after parameterization. Hence, through parameterization under GROMOS force field and molecular simulations, we assessed STA-analogs dynamics in aqueous solution, as well as its interaction with water to probe conformational and structural features involved in complexation to therapeutic targets. The coexistence of multiple conformers observed in simulations, and confirmed by metadynamics calculations, expanding the conformational space knowledge of these ligands with potential implications in understanding the ligand conformational selection during complexation. Also, changes in availability to H-bonding concerning the different substituents and water can reflect on effects at complexation free energy due to variation at the desolvation energetic costs. Based on these results, we expect the obtained structural data provide systemic framework for rational chemical modification of STA-analogs.