Solving the puzzle of 2-hydroxypropyl ß-cyclodextrin: Detailed assignment of the substituent distribution by NMR spectroscopy.

2-Hydroxypropyl-ß-cyclodextrin (HPBCD) is one of the most important cyclodextrin derivatives, finding extensive applications in the pharmaceutical sector. Beyond its role as an excipient, HPBCD achieved orphan drug status in 2015 for Niemann-Pick type C disease treatment, prompting research into its therapeutic potential for various disorders. However, the acceptance of HPBCD as an active pharmaceutical ingredient may be impeded by its complex nature. Indeed, HPBCD is not a single entity with a well-defined structure, instead, it is a complex mixture of isomers varying in substituent positions and the degree of hydroxypropylation, posing several challenges for unambiguous characterization. Pharmacopoeias' methods only address the average hydroxypropylation extent, lacking a rapid approach to characterize the substituent positions on the CD scaffold. Recognizing that the distribution of substituents significantly influences the complexation ability and overall activity of the derivative, primarily by altering cavity dimensions, we present a straightforward and non-destructive method based on liquid state NMR spectroscopy to analyze the positions of the hydroxypropyl sidechains. This method relies on a single set of routine experiments to establish quantitative assignment and it provides a simple yet effective tool to disclose the substitution pattern of this complex material, utilizing easily accessible (400 MHz NMR) instrumentation.

Autophagy-mediated control of ribosome homeostasis in oncogene-induced senescence.

Oncogene-induced senescence (OIS) is a persistent anti-proliferative response that acts as a barrier against malignant transformation. During OIS, cells undergo dynamic remodeling, which involves alterations in protein and organelle homeostasis through autophagy. Here, we show that ribosomes are selectively targeted for degradation by autophagy during OIS. By characterizing senescence-dependent alterations in the ribosomal interactome, we find that the deubiquitinase USP10 dissociates from the ribosome during the transition to OIS. This release of USP10 leads to an enhanced ribosome ubiquitination, particularly of small subunit proteins, including lysine 275 on RPS2. Both reinforcement of the USP10-ribosome interaction and mutation of RPS2 K275 abrogate ribosomal delivery to lysosomes without affecting bulk autophagy. We show that the selective recruitment of ubiquitinated ribosomes to autophagosomes is mediated by the p62 receptor. While ribophagy is not required for the establishment of senescence per se, it contributes to senescence-related metabolome alterations and facilitates the senescence-associated secretory phenotype.

Post-transcriptional dynamics and RNA homeostasis in autophagy and cancer.

Autophagy is an essential recycling and quality control pathway which preserves cellular and organismal homeostasis. As a catabolic process, autophagy degrades damaged and aged intracellular components in response to conditions of stress, including nutrient deprivation, oxidative and genotoxic stress. Autophagy is a highly adaptive and dynamic process which requires an intricately coordinated molecular control. Here we provide an overview of how autophagy is regulated post-transcriptionally, through RNA processing events, epitranscriptomic modifications and non-coding RNAs. We further discuss newly revealed RNA-binding properties of core autophagy machinery proteins and review recent indications of autophagy's ability to impact cellular RNA homeostasis. From a physiological perspective, we examine the biological implications of these emerging regulatory layers of autophagy, particularly in the context of nutrient deprivation and tumorigenesis.

Chemical Conjugation to Less Targeted Proteinogenic Amino Acids.

Protein bioconjugates are in high demand for applications in biomedicine, diagnostics, chemical biology and bionanotechnology. Proteins are large and sensitive molecules containing multiple different functional groups and in particular nucleophilic groups. In bioconjugation reactions it can therefore be challenging to obtain a homogeneous product in high yield. Numerous strategies for protein conjugation have been developed, of which a vast majority target lysine, cysteine and to a lesser extend tyrosine. Likewise, several methods that involve recombinantly engineered protein tags have been reported. In recent years a number of methods have emerged for chemical bioconjugation to other amino acids and in this review, we present the progress in this area.

Supramolecular Complexes of Plant Neurotoxin Veratridine with Cyclodextrins and Their Antidote-like Effect on Neuro-2a Cell Viability.

Veratridine (VTD) is a plant neurotoxin that acts by blocking the voltage-gated sodium channels (VGSC) of cell membranes. Symptoms of VTD intoxication include intense nausea, hypotension, arrhythmia, and loss of consciousness. The treatment for the intoxication is mainly focused on treating the symptoms, meaning there is no specific antidote against VTD. In this pursuit, we were interested in studying the molecular interactions of VTD with cyclodextrins (CDs). CDs are supramolecular macrocycles with the ability to form host-guest inclusion complexes (ICs) inside their hydrophobic cavity. Since VTD is a lipid-soluble alkaloid, we hypothesized that it could form stable inclusion complexes with different types of CDs, resulting in changes to its physicochemical properties. In this investigation, we studied the interaction of VTD with ß-CD, γ-CD and sulfobutyl ether ß-CD (SBCD) by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. Docking and molecular dynamics studies confirmed the most stable configuration for the inclusion complexes. Finally, with an interest in understanding the effects of the VTD/CD molecular interactions, we performed cell-based assays (CBAs) on Neuro-2a cells. Our findings reveal that the use of different amounts of CDs has an antidote-like concentration-dependent effect on the cells, significantly increasing cell viability and thus opening opportunities for novel research on applications of CDs and VTD.

Canonical and non-canonical roles for ATG8 proteins in autophagy and beyond.

During autophagy, the ATG8 family proteins have several well-characterized roles in facilitating early, mid, and late steps of autophagy, including autophagosome expansion, cargo recruitment and autophagosome-lysosome fusion. Their discovery has importantly allowed for precise experimental monitoring of the pathway, bringing about a huge expansion of research in the field over the last decades. In this review, we discuss both canonical and non-canonical roles of the autophagic lipidation machinery, with particular focus on the ATG8 proteins, their post-translational modifications and their increasingly uncovered alternative roles mediated through their anchoring at different membranes. These include endosomes, macropinosomes, phagosomes and the plasma membrane, to which ATG8 proteins can bind through canonical or alternative lipidation. Beyond new ATG8 binding partners and cargo types, we also explore several open questions related to alternative outcomes of autophagic machinery engagement beyond degradation. These include their roles in plasma membrane repair and secretion of selected substrates as well as the physiological implications hereof in health and disease.

Undefeated-Changing the phenamacril scaffold is not enough to beat resistant Fusarium.

Filamentous fungi belonging to the genus Fusarium are notorious plant-pathogens that infect, damage and contaminate a wide variety of important crops. Phenamacril is the first member of a novel class of single-site acting cyanoacrylate fungicides which has proven highly effective against important members of the genus Fusarium. However, the recent emergence of field-resistant strains exhibiting qualitative resistance poses a major obstacle for the continued use of phenamacril. In this study, we synthesized novel cyanoacrylate compounds based on the phenamacril-scaffold to test their growth-inhibitory potential against wild-type Fusarium and phenamacril-resistant strains. Our findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity.

Disulphide-mediated site-directed modification of proteins.

Methods for chemical modification of native proteins in a controlled fashion are in high demand. Here, a novel protocol that exploits bifunctional reagents for transient targeting of solvent exposed disulphides to direct the introduction of a single exogenous reactive thiol handle at a lysine side chain has been developed. The protocol has successfully been applied to functionalize six different Fabs and human growth hormone.

Introduction of an Aldehyde Handle on Nanobodies by Affinity-Guided Labeling.

Chemically modified antigen-binding proteins are widely applied for their targeting abilities in the fields of biotechnology, medicine, and diagnostics. However, the production of site-selectively modified proteins remains a challenge. Here, we have designed a chemical probe for the introduction of a reactive aldehyde on nanobodies by metal-complex-guided conjugation. The probe design allows for purification of the conjugates, and the aldehyde constitutes an efficient handle for further modification of the nanobodies. In vitro experiments confirmed the binding activity and selectivity of fluorescent conjugates toward the native antigen. Furthermore, the modification strategy allowed for production of a nanobody-drug conjugate that was active in vitro.

Efficient Grafting of Cyclodextrin to Alginate and Performance of the Hydrogel for Release of Model Drug.

Controlling the rate of release of molecules from a hydrogel is of high interest for various drug delivery systems and medical devices. A strategy to alter the release profiles of soluble and poorly soluble active ingredients from hydrogels can be to combine the hydrogel forming ability of alginate with the inclusion forming ability of cyclodextrins (CyD). Here, ß-CyD was grafted to alginate in a three-step synthesis using periodate oxidation, reductive amination and copper(I)-catalyzed azide-alkyne cycloaddition. A grafting degree of 4.7% mol ß-CyD/mol sugar residues was obtained. The grafting degree was controlled by varying the reaction parameters where the amount of linker used in reductive amination was especially influential. Ca-alginate gel beads grafted with ß-CyD showed increased uptake of the model molecule methyl orange. Release experiments showed that the grafted material had a prolonged release of methyl orange and an increased total amount of released methyl orange. These results show that the ß-CyD grafted alginate is still able to form a hydrogel while the grafted cyclodextrins retain their ability to form inclusion complex with methyl orange. Further testing should be done with this system to investigate capability for drug delivery applications.

Peptide-Directed DNA-Templated Protein Labelling for The Assembly of a Pseudo-IgM.

The development of methods for conjugation of DNA to proteins is of high relevance for the integration of protein function and DNA structures. Here, we demonstrate that protein-binding peptides can direct a DNA-templated reaction, selectively furnishing DNA-protein conjugates with one DNA label. Quantitative conversion of oligonucleotides is achieved at low stoichiometries and the reaction can be performed in complex biological matrixes, such as cell lysates. Further, we have used a star-like pentameric DNA nanostructure to assemble five DNA-Rituximab conjugates, made by our reported method, into a pseudo-IgM antibody structure that was subsequently characterized by negative-stain transmission electron microscopy (nsTEM) analysis.

Self-assembled nanoparticles based on cyclodextrin-modified pullulan: Synthesis, and structural characterization using SAXS.

Synthesis of novel host-guest functionalized polymers is presented along with structural characterization using small-angle X-ray scattering (SAXS) of the resulting nanoparticles. Mono-6-deoxy-mono-6-azidoßCD (N3ßCD) was grafted onto alkyne-functionalized pullulan via the "click" reaction copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and an adamantane-modified dextran was prepared via the same strategy. Characterization of the polymers was carried out using nuclear magnetic resonance (NMR) spectroscopy, gel filtration chromatography (GFC), isothermal titration calorimetry (ITC) and SAXS. Nanoparticles were created via host-guest interactions between the well-defined ßCD-pullulans and adamantane-modified dextran. Characterization was carried out using dynamic light scattering (DLS) and SAXS, which revealed spherical particles in the sub-100 nm range. The studies shed light on the importance of molecular structure and host-guest ratio on crucial properties such as particle size, size distribution, porosity and stability towards aggregation.

Publisher Correction: Development of a genetically encodable FRET system using fluorescent RNA aptamers.

In the original version of this Article the last section of the Methods describing Fluorescence microscopy was inadvertently omitted during the production process. This has now been corrected in the PDF and HTML versions of the Article.

Development of a genetically encodable FRET system using fluorescent RNA aptamers.

Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Förster resonance energy transfer (FRET) based on fluorescent proteins has been used to detect conformational changes, however, such FRET devices have not yet been produced using fluorescent RNA aptamers. Here we develop an RNA aptamer-based FRET (apta-FRET) system using single-stranded RNA origami scaffolds. To obtain FRET, the fluorescent aptamers Spinach and Mango are placed in close proximity on the RNA scaffolds and a new fluorophore is synthesized to increase spectral overlap. RNA devices that respond to conformational changes are developed, and finally, apta-FRET constructs are expressed in E. coli where FRET is observed, demonstrating that the apta-FRET system is genetically encodable and that the RNA nanostructures fold correctly in bacteria. We anticipate that the RNA apta-FRET system could have applications as ratiometric sensors for real-time studies in cell and synthetic biology.

Therapeutic efficacy of folate receptor-targeted amphiphilic cyclodextrin nanoparticles as a novel vehicle for paclitaxel delivery in breast cancer.

PURPOSE: The aim of this study is to test folate-conjugated cyclodextrin nanoparticles (FCD-1 and FCD-2) as a vehicle for reducing toxicity and increasing the antitumor efficacy of paclitaxel especially for metastatic breast cancer. METHODS: For the evaluation of PCX-loaded FCD nanoparticles, animal studies were realised in terms of survival rate, tumour size, weight change, metastazis and histopathological examination. RESULTS: FCD-1 displayed significant advantages such as efficient targeting of folate receptor positive breast cancer cells and having considerably lower toxicity compared to that of Cremophor®. When loaded with paclitaxel, FCD-1 nanoparticles, which have smaller particle size, neutral zeta potential, high encapsulation efficiency and better loading capacity for controlled release, emerged as an effective formulation in terms of cytotoxicity and high cellular uptake. In an experimental breast cancer model, anticancer activity of these nanoparticles were compatible with that of paclitaxel in Cremophor® however repeated administrations of FCD-1 nanoparticles were better tolerated by the animals. These nanoparticles were able to localise in tumour site. Both paclitaxel-loaded FCD-1 and FCD-2 significantly reduced tumour burden while FCD-1 significantly improved the survival. CONCLUSIONS: Folate-conjugated amphiphilic cyclodextrin nanoparticles can be considered as promising Cremophor®-free, low-toxicity and efficient active drug delivery systems for paclitaxel.

Host-guest interaction and structural ordering in polymeric nanoassemblies: Influence of molecular design.

Host-guest nanoassemblies made from spontaneous self-association of host and guest polymers in aqueous solutions have been studied. The specific motivation behind this work was to clarify the impact of the molecular design of the polymers on the interactions between them and on the inner structure of the resulting nanoassemblies. The polymers were composed of a dextran backbone, functionalized with either pendant ß-cyclodextrin (CD) or adamantyl (Ada). Those groups were connected to the backbone either directly or with hydrophilic polyethylene glycol (PEG) spacers. To study the impact of those spacers we have proposed a synthetic pathway to new guest polymers. The latter relied on the use of thiol-substituted dextrans as a scaffold, which is subsequently transformed into PEG-Ada grafted guest polymers via nucleophile-mediated thiol-click reaction. Surface plasmon resonance (SPR) studies evidenced strong mutual affinities between the host and guest polymers and showed that the stoichiometry was close to the ideal one (CD/Ada = 1/1) when PEG spacers were introduced. The structure of the nanoassemblies was studied by a combination of dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). The nature of the individual host or guest polymers has a strong impact on the size and internal structure of the resulting nanoassemblies. The presence of PEG spacers in the polymers led to smaller and less compact nanoassemblies, as evidenced by their large correlation length values (4-20nm compared to 2nm without PEG spacers). At the same time, all types of nanoassemblies appear to have radial density distribution with denser cores and pending polymer chains at the periphery. This study, centered on the influence of the molecular design on the host-guest interactions and structural ordering in polymeric nanoassemblies, will help to tailor host-guest nanoassemblies with attractive drug delivery profiles.

Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics.

Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized ß-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.

Real-time imaging of the growth-inhibitory effect of JS399-19 on Fusarium.

Real-time imaging was used to study the effects of a novel Fusarium-specific cyanoacrylate fungicide (JS399-19) on growth and morphology of four Fusarium sp. This fungicide targets the motor domain of type I myosin. Fusarium graminearum PH-1, Fusarium solani f. sp. pisi 77-13-4, Fusarium avenaceum IBT8464, and Fusarium avenaceum 05001, which has a K216Q amino-acid substitution at the resistance-implicated site in its myosin type I motor domain, were analyzed. Real-time imaging shows that JS399-19 inhibits fungal growth but not to the extent previously reported. The fungicide causes the hypha to become entangled and unable to extend vertically. This implies that type I myosin in Fusarium is essential for hyphal and mycelia propagation. The K216Q substitution correlates with reduced susceptibility in F. avenaceum.

Design and optimization of novel paclitaxel-loaded folate-conjugated amphiphilic cyclodextrin nanoparticles.

As nanomedicines are gaining momentum in the therapy of cancer, new biomaterials emerge as alternative platforms for the delivery of anticancer drugs with bioavailability problems. In this study, two novel amphiphilic cyclodextrins (FCD-1 and FCD-2) conjugated with folate group to enable active targeting to folate positive breast tumors were introduced. The objective of this study was to develop and characterize new folated-CD nanoparticles via 3(2) factorial design for optimal final parameters. Full physicochemical characterization studies were performed. Blank and paclitaxel loaded FCD-1 and FCD-2 nanoparticles remained within the range of 70-275nm and 125-185nm, respectively. Zeta potential values were neutral and -20mV for FCD-1 and FCD-2 nanoparticles, respectively. Drug release studies showed initial burst release followed by a longer sustained release. Blank nanoparticles had no cytotoxicity against L929 cells. T-47D and ZR-75-1 human breast cancer cells with different levels of folate receptor expression were used to assess anti-cancer efficacy. Through targeting the folate receptor, these nanoparticles were efficiently engulfed by the breast cancer cells. Additionally, breast cancer cells became more sensitive to cytotoxic and/or cytostatic effects of PCX delivered by FCD-1 and FCD-2. In conclusion, these novel folate-conjugated cyclodextrin nanoparticles can therefore be considered as promising alternative systems for safe and effective delivery of paclitaxel with a folate-dependent mechanism.

ß-CD-dextran polymer for efficient sequestration of cholesterol from phospholipid bilayers: Mechanistic and safe-toxicity investigations.

The aim of this work was to investigate the suitability of ß-cyclodextrin-dextran (BCD-dextran) polymer as cholesterol sequestering agent in vitro. For this purpose, BCD-dextran-cholesterol complexation was studied by phase solubility studies as well as with a specifically designed in vitro model based on giant unilamellar vesicles (GUVs) to evaluate the ability of this polymer to sequestrate cholesterol from phospholipid bilayers. Cholesterol-sequestering ability of BCD-dextran was also investigated on different cell lines relevant for the hematopoietic system and results were correlated to cells toxicity. BCD-dextran polymer was capable of extracting significant amount of cholesterol from phospholipid bilayers and to a higher extent in comparison to available ß-cyclodextrins (BCDs). The ability of BCD-dextran in sequestering cholesterol resulted also very high on cell lines relevant for the hematopoietic system. Moreover, BCD-dextran resulted less toxic on cell cultures due to higher selectivity in sequestering cholesterol in comparison to MBCD (that sequestrated also significant amounts of cholesteryl esters). In conclusion, BCD-dextran resulted an extremely efficient cholesterol-sequestering agent and BCD-dextran resulted more selective to cholesterol extraction in comparison to other BCDs (therefore of lower cytotoxicity). This phenomenon might play a key role to develop an efficient treatment for hypercholesterolemia based on cholesterol segregation.