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Cyclosporin A showed lipid bilayer permeability even after the deletion of a side chain.

We developed a label-free lipid bilayer permeability assay, which realized the measurement of the penetration speed of cyclosporin A.

De novo design of peptide nanoshapes is of great interest in biomolecular science since the local peptide nanoshapes formed by a short peptide chain in the proteins are often key to the biological activities. Here, we show that the de novo design of peptide nanoshapes with sub-nanometer conformational control can be realized using peptides consisting of N-methyl-L-alanine and N-methyl-D-alanine residues as studied by NMR, X-ray and XFEL crystallographic and computational analyses as well as by direct imaging of the dynamics of the peptide’s nanoshape using cinematographic electron microscopic technique. The conformation of N-methyl-L/D-alanine residue is largely fixed because of the restricted bond rotation, and hence can serve as a scaffold on which we can build a peptide into a designed nanoshape. The local shape control by per-residue conformational restriction by torsional strains starkly contrasts with the global shape stabilization of proteins based on many remote interactions. The oligomers allow the bottom-up design of diverse peptide nanoshapes with a small number of amino acid residues and would offer unique opportunities to realize the de novo design of biofunctional molecules, such as catalysts and drugs.

The first design strategy for a preorganized β-peptoid monomer is described. A cyclopentane constraint realized the preorganized monomer and led to a β-peptoid with a stable twisted strand shape.

Artificial ligands that recognize membrane receptors with high affinity and specificity provide a means for the precise control of receptor activity and the resulting biological functions. In the p...

Elucidation of biological phenomena requires imaging of microenvironments in vivo. Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has a great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we for the first time report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.

Recently, targeted protein degradation (TPD) has attracted much attention as a powerful strategy for effective inhibition of disease-related proteins. However, development of ligands with high affinity and specificity for a target protein is still a demanding task and poses a particular challenge for designing TPD therapeutics. In this work, we report a novel TPD strategy called aptamer-mediated cleavage of extracellular antigen (Apt-clean), where oligonucleotide-based affinity agents are used for selective recruitment of proteases to target membrane proteins. Our data demonstrate that Apt-clean induces selective degradation of the target protein both

We developed a photoreactive molecular glue, BPGlue-N3, which can provide a universal strategy to enhance the efficacy of DNA aptamers by temporary-to-permanent stepwise stabilization of their conjugates with target proteins. As a proof-of-concept study, we applied BPGlue-N3 to the SL1 (DNA aptamer)/c-Met (target protein) conjugate system. BPGlue-N3 can adhere to and temporarily stabilize this aptamer/protein conjugate multivalently using its guanidinium ion (Gu+) pendants that form a salt bridge with oxyanionic moieties (e.g., carboxylate and phosphate) and benzophenone (BP) group that is highly affinitive to DNA duplexes. BPGlue-N3 is designed to carry a dual-mode photoreactivity; upon exposure to UV light, the temporarily stabilized aptamer/protein conjugate reacts with the photoexcited BP unit of adhering BPGlue-N3 and also a nitrene species, possibly generated by the BP-to-N3 energy transfer in BPGlue-N3. We confirmed that SL1, covalently conjugated with c-Met, hampered the binding of hepatocyte growth factor (HGF) onto c-Met, even when the SL1/c-Met conjugate was rinsed prior to the treatment with HGF, and suppressed cell migration caused by HGF-induced c-Met phosphorylation.

Recently, targeted protein degradation (TPD) has attracted much attention as a powerful strategy for effective inhibition of disease-related proteins. However, development of ligands with high affinity and specificity for a target protein is still a demanding task and poses a particular challenge for designing TPD therapeutics. In this work, we report a novel TPD strategy called aptamer-mediated cleavage of extracellular antigen (Apt-clean), where oligonucleotide-based affinity agents are used for selective recruitment of proteases to target membrane proteins. Our data demonstrate that Apt-clean induces selective degradation of the target protein both in vitro and in cellulo. In addition, potential of Apt-clean was demonstrated through the inhibition of a tumor-related growth factor signaling. This novel TPD modality may serve as an efficient and flexible strategy for targeting membrane proteins.

Spin hyperpolarization enables real-time metabolic imaging of carbon-13-labeled substrates. While hyperpolarized l-(1

Designing synthetic surrogates of functional proteins is an important, albeit challenging, task in the field of chemistry. A strategy toward the design of synthetic agonists for growth factor or cytokine receptors that elicit a desired signal activity has been in high demand, as such ligands hold great promise as safer and more effective therapeutics. In the present study, we used a DNA aptamer as a building block and described the strategy-guided design of a synthetic receptor agonist with fine-tuned agonism. The developed synthetic partial agonist can regulate therapeutically relevant cellular activities by eliciting fine-tuned receptor signaling.

Growth factor receptors are activated through dimerization by the binding of their ligands and play pivotal roles in normal cell function. However, the aberrant activity of the receptors has been associated with cancer malignancy. One of the main causes of the aberrant receptor activation is the overexpression of receptors and the resultant formation of unliganded receptor dimers, which can be activated in the absence of external ligand molecules. Thus, the unliganded receptor dimer is a promising target to inhibit aberrant signaling in cancer. Here, we report an aptamer that specifically binds to fibroblast growth factor receptor 2b and inhibits the aberrant receptor activation and signaling. Our investigation suggests that this aptamer inhibits the formation of the receptor dimer occurring in the absence of external ligand molecules. This work presents a new inhibitory function of aptamers and the possibility of oligonucleotide-based therapeutics for cancer.

Nuclear hyperpolarization has emerged as a method to dramatically enhance the sensitivity of NMR spectroscopy. By application of this powerful tool, small molecules with stable isotopes have been used for highly sensitive biomedical molecular imaging. The recent development of molecular probes for hyperpolarized in vivo analysis has demonstrated the ability of this technique to provide unique metabolic and physiological information. This review presents a brief introduction of hyperpolarization technology, approaches to the rational design of molecular probes for hyperpolarized analysis, and examples of molecules that have met with success in vitro or in vivo.

Receptor dimerization geometry plays a significant role in signal transduction induced by growth factors and cytokines. A chemical strategy capable of controlling dimerization geometry provides a means for studying receptor activation mechanisms and designing novel agonists transducing fine-tuned receptor signaling. However, a generalized approach that can be applied to given receptors is still limited. In the present study, we propose a strategy using CiD agonists (circular DNA aptamer-based agonists), where circularized DNA is used as a rigid scaffold to present two receptor-binding aptamers from the duplex linker domain in a fixed distance and orientation. We targeted Met, a receptor for hepatocyte growth factor (HGF), and designed Met-binding CiD agonists with variable linker length. The designed CiD agonists demonstrated a distinctive periodic change in the receptor activation potential dependent on their linker length. This strategy represents a useful approach for the rational design of partial agonists that transduce fine-tuned receptor signaling and exert moderate biological activity.

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo. We recently developed γ-glutamyl-[1-13C]glycine (γ-Glu-[1-13C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo. However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu–Gly scaffold and develop a deuterated probe, γ-Glu-[1-13C]Gly-d2, that can realize a longer lifetime of the hyperpolarized signal. We initially evaluated the GGT-mediated enzymatic conversion of γ-Glu–Gly and the magnetic properties of 13C-enriched γ-Glu–Gly (γ-Glu-[1-13C]Gly and γ-[5-13C]Glu–Gly) to support the validity of γ-Glu-[1-13C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time (T1) of γ-Glu-[1-13C]Gly and γ-Glu-[1-13C]Gly-d2 under various conditions (D2O, PBS, and serum) and confirmed that the T1 of γ-Glu-[1-13C]Gly and γ-Glu-[1-13C]Gly-d2 was maintained for 30 s (9.4 T) and 41 s (9.4 T), respectively, even in serum. Relaxation analysis of γ-Glu-[1-13C]Gly revealed a significant contribution of the dipole–dipole interaction and the chemical shift anisotropy relaxation pathway (71% of the total relaxation rate at 9.4 T), indicating the potential of deuteration and the use of a lower magnetic field for realizing a longer T1. In fact, by using γ-Glu-[1-13C]Gly-d2 as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T.

Dynamic nuclear polarization (DNP) is a cutting-edge technique that markedly enhances the detection sensitivity of molecules using nuclear magnetic resonance (NMR)/magnetic resonance imaging (MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging because of the lack of established design guidelines. Here, we report Ala-[1- 13 C]Gly- d 2 -NMe 2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.

Functionalizable synthetic molecules with nanometer sizes and defined shapes in water are useful as molecular scaffolds to mimic the functions of biomacromolecules and develop chemical tools for manipulating biomacromolecules. Herein, we propose oligo(N-methylalanine) (oligo-NMA) as a peptide-based molecular scaffold with a minimal structure and a high density of functionalizable sites. Oligo-NMA forms a defined shape in water without hydrogen-bonding networks or ring constraints, which enables the molecule to act as a scaffold with minimal atomic composition. Furthermore, functional groups can be readily introduced on the nitrogens and α-carbons of oligo-NMA. Computational and NMR spectroscopic analysis suggested that the backbone structure of oligo-NMA is not largely affected by functionalization. Moreover, the usefulness of oligo-NMA was demonstrated by the design of protein ligands. The ease of synthesis, minimal structure, and high functionalization flexibility makes oligo-NMA a useful scaffold for chemical and biological applications.

Dissolution-dynamic nuclear polarization (d-DNP) is a state-of-the-art technology that can dramatically enhance the detection sensitivity of nuclear magnetic resonance (NMR). DNP NMR has been applied to small molecules with stable isotopes and has been used to obtain metabolic and physiological information in vivo. However, the hyperpolarized state exponentially decays back to the thermal equilibrium state, depending on the spin-lattice relaxation time (T1). This signal decay has remained a major problem associated with this technology. Therefore, DNP NMR molecular probes useful for in vivo analysis have been limited to naturally occurring small molecules that inherently show long T1. While peptides are promising targets for DNP NMR studies, because of the limitation in T1, DNP NMR molecular probes applicable in vivo have been limited to amino acids or dipeptides. Herein we propose a 13C-labeling strategy to utilize the C-terminal [1-13C]Gly-d2 residue for realizing long T1 in peptides. Structure-based T1 relaxation analysis of amino acids and peptides revealed that (1) T1 does not decrease monotonically with increasing molecular weight and (2) T1 is not significantly affected by a side chain on the neighboring amino acid residue. These findings suggest that the C-terminal [1-13C]Gly-d2 residue affords sufficiently long T1 for biological uses, even in oligopeptides, and allowed us to develop 13C-b- casomorphin-5 (Tyr-Pro-Phe-Pro-[1-13C]Gly-d2, T1 = 24 ± 4 s at 3 T in H2O) and 13C-glutathione (g-Glu-Cys-[1-13C]Gly-d2, T1 = 58 ± 3 s at 3 T in H2O) as DNP NMR probes with long T1. We succeeded in in vivo detection of enzymatic conversions of these two probes. These results demonstrate the utility of our strategy and would contribute to further expansion of the substrate scope for DNP applications.

Dissolution-dynamic nuclear polarization (d-DNP) is a state-of-the-art technology that can dramatically enhance the detection sensitivity of nuclear magnetic resonance (NMR). DNP NMR has been applied to small molecules with stable isotopes and has been used to obtain metabolic and physiological information in vivo. However, the hyperpolarized state exponentially decays back to the thermal equilibrium state, depending on the spin-lattice relaxation time (T1). This signal decay has remained a major problem associated with this technology. Therefore, DNP NMR molecular probes useful for in vivo analysis have been limited to naturally occurring small molecules that inherently show long T1. While peptides are promising targets for DNP NMR studies, because of the limitation in T1, DNP NMR molecular probes applicable in vivo have been limited to amino acids or dipeptides. Herein we propose a 13C-labeling strategy to utilize the C-terminal [1-13C]Gly-d2 residue for realizing long T1 in peptides. Structure-based T1 relaxation analysis of amino acids and peptides revealed that (1) T1 does not decrease monotonically with increasing molecular weight and (2) T1 is not significantly affected by a side chain on the neighboring amino acid residue. These findings suggest that the C-terminal [1-13C]Gly-d2 residue affords sufficiently long T1 for biological uses, even in oligopeptides, and allowed us to develop 13C-β-casomorphin-5 (Tyr-Pro-Phe-Pro-[1-13C]Gly-d2, T1 = 24 ± 4 s at 3 T in H2O) and 13C-glutathione (γ-Glu-Cys-[1-13C]Gly-d2, T1 = 58 ± 3 s at 3 T in H2O) as DNP NMR probes with long T1. We succeeded in in vivo detection of enzymatic conversions of these two probes. These results demonstrate the utility of our strategy and would contribute to further expansion of the substrate scope for DNP applications.

Rapid cellular proliferation and incomplete neovascularization in solid tumors leads to spatially heterogeneous regions with relatively low oxygen concentrations, called hypoxia. Hypoxia in tumors causes significant changes in various physiological processes such as DNA repair pathways, anaerobic respiration, metabolic reprograming, unregulated angiogenesis, and the spread of cancer stem cells [Chem. Soc. Rev. 2019, 48, 771–813]. Consequently, the plasticity of these processes results in the acquisition of the malignant phenotype in tumors. Therefore, effective imaging methods that allow for the detailed analysis of hypoxia are needed to elucidate tumor pathophysiology. To date, a variety of imaging techniques for tumor hypoxia have been developed. Magnetic resonance imaging (MRI)- and positron emission tomography (PET)-based methods permit real-time monitoring and give three-dimensional (3D) information about tumor hypoxia without the limitation of imaging depth in vivo [Mol. Imaging Biol. 2010, 13, 399–410]. However, these imaging methods have poor resolution, restricting the detailed visualization of hypoxia-related biological events in cells or tissues. The imaging methods for tumor hypoxia with cellular resolution rely on tissue sectioning [Nat. Nanotechnol. 2016, 11, 724–730]. Although the reconstruction of serial sections theoretically enables 3D imaging of tumor hypoxia, it is impractical for multiple samples and difficult to obtain accurate data with the original tissue morphology. Recently, optical tissue-clearing techniques have been developed for depth-independent visualization. These techniques enable 3D imaging with fluorescent probes including genetically encoded proteins and chemical dyes in entire tissues without the preparation of tissue sections [Cell 2014, 157, 726–739]. However, the application of these techniques with conventional hypoxia probes suffers from some challenges. For example, a well-known hypoxia probe (pimonidazole) and hypoxia makers (e.g. HIF1-) need specific antibodies to be visualized [Nat. Commun. 2012, 3, 710–783]. Due to the low tissue permeability of antibodies [Mol. Cancer Ther. 2019, 18, 213–226], the existence of these probes deep inside tumors cannot be detected. Additionally, hypoxia probes lacking the ability to covalently bind to cellular components can be washed from tumors during tissue-clearing treatments. To achieve 3D and high-resolution imaging of tumor hypoxia throughout an entire tissue, we developed a fluorescent molecular probe for tumor hypoxia which is compatible with tissue-clearing. The fluorescent probe has low cytotoxicity, high fluorescence intensity in tissue-clearing solution, high tissue permeability, and the ability to covalently bind to cellular proteins only under hypoxic conditions. The designed probe was applied for the 3D imaging of spatially heterogeneous tumor hypoxia in combination with tissue-clearing., PACIFICHEM2021

Detecting activities of peptidases in vivo is an attractive challenge in chemical biology because peptidase-activities are correlated strongly with various biological phenomena and diseases. Nuclear magnetic resonance (NMR) is a powerful technique for this purpose due to its advantages in high tissue penetration and low invasiveness, allowing a molecular detection at deep area. However, the sensitivity of NMR is severely low for in vivo application. To overcome this problem, dynamic nuclear polarization (DNP) has emerged. NMR signals are dramatically enhanced by using this method. However, there are few molecular probes to detect peptidase-activity by using DNP technique to date. Herein, we have developed a practical molecular probe for DNP-NMR to detect the activity of Aminopeptidase N (APN), which is a representative peptidase, in vivo through rational molecular design. It is demonstrated that this probe has many advantages for in vivo application and enables detection of APN-activity in xenograft tumors bearing mice. Starting with this success, we developed some DNP-NMR molecular probes for detecting peptidase-activities based on chemical design., PACIFICHEM2021

Backbone stereochemistry of cyclic peptides has been reported to have a great influence on microsomal stability and membrane permeability, two important factors that determine oral bioavailability. Here, we comprehensively investigated the correlation between the backbone stereochemistry of cyclic hexapeptide stereoisomers and their stability in liver microsomes, as well as passive membrane permeability.

Here, we investigated the effect of CH3 to CF3 substitution on the membrane permeability of peptides. We synthesized a series of peptides with CF3 groups and corresponding nonfluorinated peptides and measured the membrane permeability of the peptides. As a result, we demonstrated that CH3 to CF3 substitution is useful for increasing the membrane permeability of di-/tri-peptides.

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the process of X dimerization. Our findings will impact on a strategy for regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules., Senoo et al. describe a chemical fragment that disrupts dimerization of P-cadherin. In doing so, this fragment inhibits cadherin-mediated cell-adhesion and aggregation.

The term "peptoids" was introduced decades ago to describe peptide analogues that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo(N-substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible, and ensuring a defined shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose oligo(N-substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies, and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. This new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as a scaffold for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular recognition.

Quantum-tunneling-based DNA sequencing is a single molecular technology that has great potential for achieving facile and high-throughput DNA sequencing. In principle, the sequence of DNA could be read out by the time trace of the tunnel current that can be changed according to molecular conductance of nucleobases passing through nanosized gap electrodes. However, efficient base-calling of four genetic alphabets has been seriously impeded due to the similarity of molecular conductance among canonical nucleotides. In this article, we demonstrate that replacement of canonical 2'-deoxyadenosine (dA) with a highly conductive dA analogue, 7-deaza dA, could expand the difference of molecular conductance between four genetic alphabets. Additionally, systematic evaluation of molecular conductance using a series of dA and dG analogues revealed that molecular conductance of the nucleotide is highly dependent on the HOMO level. Thus, the present study demonstrating that signal characteristics of the nucleotide can be modulated based on the HOMO level provides a widely applicable chemical approach and insight for facilitation of single molecular sensing as well as DNA sequencing based on quantum tunneling.

Serine hydroxymethyltransferase (SHMT) is an enzyme that catalyzes the reaction that converts serine to glycine. It plays an important role in one-carbon metabolism. Recently, SHMT has been shown to be associated with various diseases. Therefore, SHMT has attracted attention as a biomarker and drug target. However, the development of molecular probes responsive to SHMT has not yet been realized. This is because SHMT catalyzes an essential yet simple reaction; thus, the substrates that can be accepted into the active site of SHMT are limited. Here, we focus on the SHMT-catalyzed retro-aldol reaction rather than the canonical serine–glycine conversion and succeed in developing fluorescent and 19F NMR molecular probes. Taking advantage of the facile and direct detection of SHMT, the developed fluorescent probe is used in the high-throughput screening for human SHMT inhibitors, and two hit compounds are obtained., The enzyme serine hydroxymethyltransferase (SHMT) has been implicated in several diseases, however is hard to investigate. Here, the authors used a design strategy based on the retro-aldol-type reaction catalyzed by SHMT to develop SHMT-responsive fluorescence and 19F NMR molecular probes.

Cell culture media are often supplemented with recombinant growth factors and cytokines to reproduce biological conditions in vitro. Basic fibroblast growth factor (bFGF) has been widely used to support the pluripotency and self-renewal activity of human induced pluripotent stem cells (hiPSCs). We had previously developed a synthetic surrogate for bFGF on the basis of a DNA aptamer that binds to one of the FGF receptors. Since DNA aptamers have advantages over recombinant proteins in terms of thermal stability and production cost, replacing recombinant growth factors in cell culture media with DNA aptamers would be of great interest. Herein, we describe our protocol for feeder-free hiPSC culture using a DNA aptamer-based mimic of bFGF.

A quantum sequencer offers a scalable electrical platform for single-molecule analysis of genomic events. A thymidine (dT) analog exhibiting uniquely high single-molecule conductance is a key element in capturing DNA synthesis dynamics by serving as a decodable marker for enzymatic labeling of nascent strands. However, the current design strategies of dT analogs that focus on their molecular orbital energy levels require bulky chemical modifications to extend the π-conjugation, which hinders polymerase recognition. We report herein a polymerase-compatible dT analog that is highly identifiable in quantum sequencing. An ethynyl group is introduced as a small gold-binding motif to differentiate the nucleobase–gold electronic coupling, which has been an overlooked factor in modifying nucleobase conductance. The resulting C5-ethynyl-2′-deoxyuridine exhibits characteristic signal profiles that allowed its correct identification at a 93% rate while maintaining polymerase compatibility. This study would expand the applicability of quantum sequencing by demonstrating a robust nucleoside marker with high identifiability., Introduction of an aurophilic ethynyl group demonstrates a highly conductive dT analog accurately identifiable by quantum sequencing.

The development of inhibitors of intracellular protein–protein interactions (PPIs) is of great significance for drug discovery, but the generation of a cell-permeable molecule with high affinity to protein is challenging. Oligo(N-substituted glycines) (oligo-NSGs), referred to as peptoids, are attractive as potential intracellular PPI inhibitors owing to their high membrane permeability. However, their intrinsically flexible backbones make the rational design of inhibitors difficult. Here, we propose a peptoid-based rational approach to develop cell-permeable PPI inhibitors using oligo(N-substituted alanines) (oligo-NSAs). The rigid structures of oligo-NSAs enable independent optimization of each N-substituent to improve binding affinity and membrane permeability, while preserving the backbone shape. A molecule with optimized N-substituents inhibited a target PPI in cells, which demonstrated the utility of oligo-NSA as a reprogrammable template to develop intracellular PPI inhibitors., A peptoid-based modular approach using oligo(N-substituted alanine) as a reprogrammable template enables independent optimization of N-substituents and facile development of cell-permeable inhibitors of protein–protein interactions.

Summary Bile acids are metabolites of cholesterol that facilitate lipid digestion and absorption in the small bowel. Bile acids work as agonists of receptors to regulate their own metabolism. Bile acids also regulate other biological systems such as sugar metabolism, intestinal multidrug resistance, and adaptive immunity. However, numerous physiological roles of bile acids remain undetermined. In this study, we solved the crystal structure of human serine hydroxymethyltransferase (hSHMT) in complex with an endogenous secondary bile acid glycine conjugate. The specific interaction between hSHMT and the ligand was demonstrated using mutational analyses, biophysical measurements, and structure-activity relationship studies, suggesting that secondary bile acid conjugates may act as modulators of SHMT activity., Graphical abstract, Highlights • The crystal structures of hSHMT in complex with secondary bile acid glycine conjugate • Specific interactions between hSHMT and secondary bile acid conjugate were validated • Biological role of bile acids as modulators for one-carbon metabolism is suggested, Molecular Interaction; Structural Biology; Metabolic Engineering

Cell culture media are often supplemented with recombinant growth factors and cytokines to reproduce biological conditions in vitro. Basic fibroblast growth factor (bFGF) has been widely used to support the pluripotency and self-renewal activity of human induced pluripotent stem cells (hiPSCs). We had previously developed a synthetic surrogate for bFGF on the basis of a DNA aptamer that binds to one of the FGF receptors. Since DNA aptamers have advantages over recombinant proteins in terms of thermal stability and production cost, replacing recombinant growth factors in cell culture media with DNA aptamers would be of great interest. Herein, we describe our protocol for feeder-free hiPSC culture using a DNA aptamer-based mimic of bFGF.

Growth factor receptors are activated through dimerization by the binding of their ligands and play pivotal roles in normal cell function. However, in cancer cells, the overexpression of receptors often causes the formation of unliganded receptor dimers, which can be activated in a ligand-independent manner. Thus, the unliganded receptor dimer is a promising target to inhibit aberrant signaling in cancer. Here, we report an aptamer that inhibits ligand-independent receptor activation via preventing the formation of unliganded receptor dimer. By biasing the receptor monomer–dimer equilibrium to the monomer, this aptamer inhibited aberrant cell signaling caused by the unliganded receptor dimer. This work presents a new possibility of oligonucleotide-based therapeutics for cancer.

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the on-rate of X dimerization. Our findings will impact on a strategy for kinetic regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules.

Cyclic peptides are attractive molecules as inhibitors with high affinity and selectivity against intracellular protein-protein interactions (PPIs). On the other hand, cyclic peptides generally have low passive cell-membrane permeability, which makes it difficult to discover cyclic peptides that efficiently permeate into cells and inhibit intracellular PPIs. Here, we show that backbone amide-to-ester substitutions are useful for improving membrane permeability of peptides. Permeability in a series of model dipeptides increased upon amide-to-ester substitution. Amide-to-ester substitutions increased permeability in the same manner as amide-to-N-methyl amide substitutions, which are conventionally used for increasing permeability. Furthermore, amide-to-ester substitutions of exposed amides of a cyclic peptide successfully improved permeability. Conformational studies of the cyclic peptides using NMR and molecular mechanics calculations revealed that an amide-to-ester substitution of an exposed amide bond did not affect its low-energy conformation in CDCl3, in contrast with an N-methyl amide substitution. We envision that amide-to-ester substitution will be a potentially useful strategy for rational design of bioactive peptides with high membrane permeability.

Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. γ-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP γ-glutamyl-[1-13C]glycine and acquiring dynamic 13C data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of γ-glutamyl-[1-13C]glycine and its product [1-13C]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in γ-glutamyl-[1-13C]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1-13C]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1-13C]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP γ-glutamyl-[1-13C]glycine to monitor GGT expression in the brain and thus to detect glioblastoma.