MRマイクロイメージングとガドリニウム-デンドロン修飾ナノリポソーム造影剤を用いた3D微小血管およびナノ粒子分布の腫瘍内評価

The enhanced permeability and retention (EPR) effect is variable depending on nanoparticle properties and tumor/vessel conditions. Thus, intratumoral evaluations of the vasculature and nanoparticle distribution are important for predicting the therapeutic efficacy and the intractability of tumors. We aimed to develop a tumor vasculature evaluation method and high-resolution nanoparticle delivery imaging using magnetic resonance (MR) micro-imaging technology with a gadolinium (Gd)-dendron assembled liposomal contrast agent. Using the Gd-liposome and a cryogenic receiving coil, we achieved 50-μm isotropic MR angiography with clear visualization of tumor micro-vessel structure. The Gd-liposome-enhanced MR micro-imaging revealed differences in the vascular structures between Colon26- and SU-DHL6-grafted mice models. The vessel volumes and diameters measured for both tumors were significantly correlated with histological observations. The MR micro-imaging methods facilitate the evaluation of intratumoral vascularization patterns, the quantitative assessment of vascular-properties that alter tumor malignancy, particle retentivity, and the effects of treatment

Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface

Biosensor-based high throughput biopanning and bioinformatics analysis strategy for the global validation of drug-protein interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as "broken relics", of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest

Design and development of the molecular probes for application of the hyperpolarized-NMR/MRI

Hyperpolarized-nuclear magnetic resonance and imaging (HP-NMR/MRI) is a promising technique that allows direct monitoring of metabolic reactions in vitro and in vivo. Given the 13C- or 15N-labeled molecular probes whose NMR signal is amplified by the dynamic nuclear polarization (DNP) under a cryogenic magnetic field (3.35T, 1.4K), the sample is rapidly dissolved with a superheated solvent (~458 K), and then quickly reacted with enzymes or cells, or administered in animals put into the magnet. The metabolic reaction is readout onto the NMR spectra or MR image based on the >10,000-fold enhanced signals. By elucidating the extent of the metabolic reaction of the probes, early detection of disease-specific elevation of enzymatic activity, metabolic reprogramming, and alteration of their flux upon the treatment is possible.The development of various molecular probes will be a key factor to expand the utility of this approach. In this presentation, we will concisely introduce the basics of the dis-DNP, design and application of our recent new molecular probes that allow in vitro and in vivo HP-NMR/MRI.The 24th International Spin Symposium (SPIN2021

Hyperpolarized-NMR/MRI in Japan

なしHYPERMAG Center, lab semina

Phage display technology for target determination of small-molecule therapeutics: an update

IntroductionOur understanding of the mechanism of action of bioactive small molecules contributes to the research and development of new medical drugs, as well as elucidating the pathological mechanisms underlying various diseases. Researchers in this field are committed to a very ambitious goal: the discovery of novel therapeutic compounds along with their molecular targets. To achieve this goal, new methodological developments are indispensable.Areas coveredThis review gives an update on the advancements of phage display (PD) technology in the past decade (2011–2020) for determining the targets of the small molecule therapeutics. In particular, other than providing a brief overview of this field of research, we focus on reporting the research trends and the results solely obtained using this strategy.Expert opinionDespite the development of bioinformatics tools and artificial intelligence (AI)-mediated methods, affinity-guided information obtained experimentally are still indispensable to identify drug-protein interactions. By taking advantage of small-molecule-oriented PD methods and their improvements, the extension of the druggable proteome will be further expanded, providing new opportunities to generate small-molecule therapeutics

Using the QCM Biosensor-Based T7 Phage Display Combined with Bioinformatics Analysis for Target Identification of Bioactive Small Molecule

Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used

Hyperpolarized [1-13C]pyruvate MRS reveals early-phase transition of energy metabolism in multicellular spheroid tumors

Most tumor cells rely on aerobic glycolysis instead of mitochondrial oxidative phosphorylation (OXPHOS) to generate energy (ATP production) for cellular activities. This distinct glucose metabolism, known as the Warburg effect, is further amplified by an organization of the cells as solid tumor tissues in vivo. However, much has remained elusive; e.g. when and how the metabolic shift is triggered during tumorigenesis. In the present study, hyperpolarized (HP) [1-13C]pyruvate MRS was employed to directly monitor the transition of energy metabolism in tiny multicellular tumor spheroids of early-phase. The HP [1-13C]pyruvate conversion to [1-13C]lactate in the smaller tumor spheroids was noninvasively monitored on an NMR spectra