Insulin-regulated aminopeptidase (IRAP) is an important biomarker for various diseases, including cancer. However, its selective detection remains challenging due to competing activity from closely related enzymes such as aminopeptidase N (APN). Dynamic nuclear polarization-enhanced magnetic resonance imaging (DNP-MRI) enables noninvasive detection of enzymatic activity in vivo and represents a powerful platform for metabolic imaging. Although a previously reported IRAP-responsive DNP-MRI probe, Leu-[1- 13 C]Gly- d 2 -NMe 2 , allows detection of metabolic activity in vivo, its susceptibility to APN-mediated cleavage limits its selectivity for IRAP. Here, we report the development of a hyperpolarized molecular probe, Ala( t Bu)-[1- 13 C]Gly- d 2 -NMe 2 , through minimal structural modification of the previous probe. Replacement of the N-terminal leucine residue with a bulky Ala( t Bu) moiety markedly suppressed APN-mediated cleavage, reducing the catalytic efficiency toward APN by approximately 130-fold, while maintaining comparable catalytic efficiency toward IRAP. Furthermore, in vivo DNP-MRI experiments demonstrated successful metabolic imaging of Ala( t Bu)-[1- 13 C]Gly- d 2 -NMe 2 in MCF-7 tumor-bearing mice.

To establish design principles for membrane-permeable cyclic peptides and thereby enable drug discovery targeting intracellular proteins, it is essential to develop technologies capable of generating large-scale structure–permeability datasets across broad chemical space. However, conventional mass spectrometry-based sequence identification methods for cyclic peptides suffer from low identification rates due to the complexity of fragment analysis and have difficulty distinguishing stereoisomers. These limitations have posed a significant barrier to acquiring large-scale membrane permeability data across broad chemical space. In this study, we addressed these challenges by employing DNA-encoded library (DEL) technology, thereby establishing a platform for evaluating the membrane permeability of cyclic peptides across broad chemical space. We constructed a hexamer cyclic peptide library with a theoretical diversity of 10,240 members and evaluated the membrane permeability of approximately 100 peptides using the parallel artificial membrane permeability assay (PAMPA). Whereas a conventional MS/MS-based method yielded a sequence assignment rate of less than 10% and low data acquisition efficiency, our approach achieved a high sequence assignment rate of approximately 90% and a data acquisition efficiency of approximately 80%. Furthermore, the accuracy of sequence assignment reached 100% in the validated set, enabling the acquisition of highly reliable data compared with conventional approaches. These results demonstrate that our method provides a powerful and high-throughput platform for reliably evaluating the membrane permeability of cyclic peptide libraries across broad chemical space.

Polymer conjugation is a common strategy to improve the pharmacokinetics of aptamers, yet its effects on aptamer properties are incompletely understood. Poly(ethylene glycol) (PEG) is the most widely used polymer for this purpose, but concerns about anti-PEG immune responses have prompted interest in alternative polymers. We previously reported that conjugation with the zwitterionic polymer poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) significantly prolongs the circulation time of a DNA aptamer while avoiding anti-PEG antibody recognition. In this study, we evaluated the physicochemical and functional consequences of PMPC conjugation of aptamers. Biophysical analyses suggested that the secondary structure and target-binding affinity of the aptamer were preserved, while functional consequences upon PMPC conjugation varied with the targets. The activity of a membrane receptor-targeting aptamer partially decreased, likely due to spatial constraints around the cell membrane, while RB005, targeting soluble activated coagulation factor IX, retained its full activity. In addition, PMPC conjugation significantly prolonged the in vivo plasma retention of RB005. By elucidating the effects of PMPC on aptamer properties and introducing another example that further supports the general applicability of PMPC conjugation in enhancing aptamer pharmacokinetics, these findings support PMPC as a promising alternative to PEG.

The fibroblast growth factor (FGF) family exhibits distinct yet overlapping specificities toward seven fibroblast growth factor receptor (FGFR) isoforms, forming highly complex signaling networks through broad binding and activation profiles. FGF2 plays important physiological roles and has considerable therapeutic potential; however, its pleiotropic and non-selective activation of FGFRs can lead to adverse effects. Therefore, molecular engineering of FGF ligands with controlled receptor isoform specificity is required. Here, we engineered FGF2 variants with altered FGFR isoform specificity using mRNA display-based deep mutational scanning (DMS), thereby enabling systematic evaluation of residues within the FGFR-binding interface that contribute to FGFR isoform specificity despite limited structural information. We demonstrate for the first time that FGFs with altered FGFR isoform specificity can be generated. Notably, FGFR isoform specificity proved malleable and could be reprogrammed by single-amino acid substitutions. These findings reveal that receptor selectivity in FGF2 is tunable and provide a framework for rationally designing FGF ligands with tailored FGFR isoform specificity, advancing the understanding and control of complex FGF/FGFR signaling networks.

Cancer cells sustain glycolysis despite oxygen availability, creating an acidic microenvironment via proton and lactate export, but how they survive acid stress is unclear. We show that severe acidification (pH 5.6) induces necroptosis, whereas moderate acidity (pH 6.8) prevents death and enables anchorage-independent survival and tumor initiation. RNA sequencing of suspended cells at pH 6.8 revealed activation of respiratory chain complex and complement pathways, consistent with adaptation to this pH. A genome-wide CRISPR-Cas9 knockout screen in PANC1 cells under chronic acidity identified FAM129C as a regulator of acid tolerance and survival. In xenografts, FAM129C overexpression reduced PIGR expression, implicating this axis in tumor growth and immune infiltration. Anti-PD-L1 plus a complement inhibitor showed synergistic anti-tumor activity in PIGR-overexpressing tumors. Thus, acidic stress engages a pathway that allows cancer cells to evade necroptosis and promote tumor plasticity, providing potential avenues for therapeutic intervention targeting pH-dependent cell-death pathways.

N-Alkyl peptides have emerged as promising drug modalities, yet the structural determinants governing passive membrane permeability beyond amide hydrogen removal remain poorly understood. Here, we show that sterically constrained N-alkyl peptide backbones, generated by dual substitution at the amide nitrogen and the α-carbon, play a critical role in promoting passive membrane permeability. By directly comparing N-alkyl peptides with oligo(N-alkyl glycines) lacking Cα-substituents, we isolated the backbone steric effects independently of amide hydrogen removal. N-Alkyl peptides bearing an N/Cα-dually substituted backbone architecture consistently exhibited enhanced permeability across a broad range of lipophilicity and diverse sequences. Molecular dynamics simulations revealed two cooperative mechanisms: conformational restriction that favors less hydrated states and steric limitation of backbone hydration by β-carbons during membrane permeation. Together, these findings uncover a previously unrecognized structural basis for the high passive membrane permeability of N-alkyl peptides.

DNA-Encoded Library (DEL) enables the efficient discovery of bioactive ligands. However, conventional DELs utilize unprotected DNA barcodes, which are susceptible to damage during library construction due to chemical reactions. The instability of DNA barcodes limits the applicable reaction conditions and building blocks, and restricts the number of synthesis cycles to approximately 2-4. As a result, the application of DELs has been mainly confined to small molecules, making it challenging to extend the approach to medium-sized molecules such as peptides. In this study, we report a chemically stable DNA barcode that addresses this issue. We focused on the Recording-by-Synthesis method, which chemically constructs DNA barcodes, and developed the N -acetylpyrrolidin-2-ylmethyl (NAPM) protective group to overcome one of its challenges, the fragility of protected phosphodiester group. The NAPM protective group is stable under conditions used for the solid-phase peptide synthesis, while allowing for deprotection under neutral to weakly basic heating conditions. By integrating the NAPM protective group in short DNA barcode technology, we established a new Recording-by-Synthesis approach that enables ligand synthesis under standard organic solvent-based reaction conditions. Using this strategy, we successfully achieved 10 cycles of alternating synthesis of DNA barcode and peptide, even incorporating bulky N -methyl amino acids.

Aminopeptidases (APs) in the renin-angiotensin system (RAS) and their activity balance play crucial roles in regulating vascular functions. Multiplexed analysis of RAS-related AP activities is useful for diagnosing diseases including cancer. Dynamic nuclear polarization-coupled magnetic resonance imaging (DNP-MRI) enables the simultaneous detection of multiple enzymatic activities in vivo. However, developing practical DNP-MRI probes, especially for multiplexed detection, remains challenging. Here, we report the design of DNP-MRI probes for the in vivo multiplexed analysis of AP activities. By integrating quantum mechanical calculations, organic synthesis, and physicochemical and biochemical evaluations, we developed a series of AP-responsive DNP-MRI probes with high enzymatic reactivities and distinguishable chemical shifts. Using these probes, we successfully detected and visualized multiple AP activities in vivo. Furthermore, we performed in vivo multiplexed analysis of RAS-related AP activities in tumor-bearing mice, demonstrating the potential of this approach for monitoring the efficacy of antiangiogenic cancer therapy and for the accurate discrimination of tumor types.

“Peptoids” was proposed, over decades ago, as a term describing analogs of peptides that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo-(N-substituted glycines) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible and is difficult to achieve a defined shape in water. This conformational flexibility is severely limiting biological application of oligo-NSG. Here, we propose oligo-(N-substituted alanines) (oligo-NSA) as a new peptoid that forms a defined shape in water. A 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. The new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as an ideal scaffold for displaying functional groups in well-defined three-dimensional space, which leads to effective biomolecular recognition.

Enzymes play a crucial role in regulating physiological functions, and abnormal enzyme activity is associated with various pathological conditions. Precise imaging of enzyme activity in tissues, providing detailed spatial and quantitative information, advances our understanding of physiological and pathological processes. Despite their importance, there is still a lack of methods for high-resolution 3D imaging of enzyme activity across entire tissues. In this research, we report a methodology for high-resolution, whole-organ 3D mapping of enzyme activity, which combines tissue clearing with an activity-based covalent chemical probe. Focusing on aminopeptidase N (APN) as a representative target of peptidase, we developed ANA-o-BODIPY, an activity-based covalent fluorescent probe compatible with tissue clearing for imaging APN activity. Upon activation by APN, ANA-o-BODIPY produces a reactive intermediate, aza-quinone methide, which covalently binds to proximal proteins. This covalent probe is successfully utilized to record the location of APN activity during the tissue-clearing process. By combining the probe with tissue clearing, we have achieved high-resolution 3D mapping of APN activity across whole organs for the first time. Moreover, this advancement allowed us to visualize the heterogeneity of APN activity in individual tubular structures and to uncover the inhibitory effects of different APN inhibitors.

Liquid-crystalline (LC) biomaterials are garnering attention as promising materials due to their ordered nanostructures and dynamic properties. The introduction of biobased molecular moieties into LC materials may be useful to develop highly functional biomaterials. The integration of biodegradable polymers with naturally occurring molecules is favored for bioadaptive LC materials. Poly(L,L-lactide) (PLLA), has been extensively studied for biomedical applications, from drug delivery to tissue engineering due to its in vivo degradability and compatibility with tissues. However, there is a demand for PLLA-based materials to possess additional bioactive functionalities, such as improved interactions with cells. Here, we present the synthesis and self-assembled properties of A-B-A triblock copolymers incorporating PLLA segments (B) and mesogenic dendrons with cholesteryl moieties (A) at their peripheral parts. Mesogenic copolymers exhibit thermotropic LC properties attributed to their self-assembled lamellar nanostructures. The presence of peripheral mesogenic dendrons was observed to disrupt the crystallization of PLLA, thereby preserving the dynamic LC properties unhindered by PLLA crystals. Additionally, the surface wettability of mesogen-functionalized PLLA demonstrated greater hydrophobicity compared to pristine PLLA, owing to the incorporation of hydrophobic cholesterol moieties. Despite the common notion that hydrophobic surfaces are less bioactive, our results show significantly enhanced cell adhesion and proliferation on mesogen-functionalized PLLA surfaces in comparison to PLLA alone. The cholesterol trident dendrons conjugating the central PLLA segment appear to significantly promote the cellular surfaces. This bioactivity can be attributed to the soft and dynamic properties of mesogen-functionalized PLLA based on the LC nanostructure. Microstructural analysis in the hydrated state revealed a macroscopically smooth surface with nanometer-scale roughness for mesogen-functionalized PLLA, and the LC nanostructure demonstrated responsiveness to hydration. This research sheds light on the potential of LC biomaterials for enhanced biomedical applications.

Introducing substituents on backbone amide nitrogens of peptides is an attractive strategy to improve the pharmacological properties of peptides. Here, we describe a solid-phase synthetic method for peptides with various substituents on backbone amide nitrogens. The method utilizes the procedures for introducing substituents on resin, which facilitates introducing diverse functional groups on backbone amide nitrogens.

In this study, a passively membrane-permeable short peptide inhibitor targeting the measles virus fusion protein (MeV-F) is reported. Measles virus (MeV) is highly contagious, yet no approved antiviral drugs are currently available. MeV-F plays a crucial role in viral infection, making it an attractive target for drug development. The fusion inhibitor peptide (FIP) is a well-known short peptide that binds to MeV-F and prevents its structural rearrangement. However, improving both inhibitory activity and passive membrane permeability is essential for developing orally available MeV-F inhibitors. Herein, FIP derivatives are explored through hydrogen-to-fluorine substitution and a derivative with enhanced inhibitory activity (IC50 = 90 nM) and passive membrane permeability (Pe = 1.4 × 10-6 cm s-1) was identified. This study highlights the potential of the long-studied fusion inhibitor peptide as a promising lead compound for the development of orally available drugs against measles infection.

Overcoming poor in vivo pharmacokinetics is a critical challenge in developing therapeutic aptamers, and conjugation to poly(ethylene glycol) (PEG) is a well-established technique for aptamers to prolong blood circulation. However, the existence of antibodies that specifically recognize PEG and their adverse effects on in vivo behaviors have been increasingly reported, highlighting the necessity of alternative modification strategies for aptamers. To address this issue, we focused on a zwitterionic polymer, particularly poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), as a PEG alternative to modify DNA aptamers. We conjugated PMPC to a DNA aptamer targeting IFN-gamma and investigated the properties of the PMPC-conjugated DNA aptamer as a therapeutic agent. PMPC modification did not affect the neutralizing activity of the aptamer. PMPC demonstrated lower reactivity against anti-PEG antibodies than PEG-like aptamer modifiers previously reported to exhibit low reactivity against PEG antibodies. In addition, PMPC extended the blood circulation time of the aptamer as long as or longer than PEG with a similar molecular size. In the LPS-induced inflammation animal model, the survival rate after treatment with the PMPC-aptamer conjugate was significantly superior to that with unmodified aptamer. These results indicate that PMPC has potential as an aptamer or other nucleic acid drug modifier to replace or be compatible with PEG.

Fusion inhibitor peptide (FIP), a short peptide known as a measles virus (MeV) infection inhibitor, inhibits membrane fusion between the viral envelope of MeV and the host cell membrane. Therefore, FIP is potentially useful as a drug candidate for treating MeV infection, but improvement of inhibitory activity is desirable. In this study, we conducted a structure-activity relationship study of FIP and, based on the result and the previously reported crystal structure of the complex, we designed FIP derivatives. From a series of derivatives, we discovered an FIP derivative with a strong inhibitory activity (IC50 = 210 nM) derived from the enhanced binding affinity (KD = 6.6 nM) to the MeV fusion protein.

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.

Receptor tyrosine kinases (RTKs) play a pivotal role in cell signaling through their activation via dimerization. Recent studies have demonstrated the importance of the temporal dynamics of RTK activity and downstream signals, such as ERK, in determining the cell fate. To better understand these dynamics, it is essential to develop methods capable of controlling the RTK activity with high temporal resolution. However, techniques for precisely modulating the activity of endogenous RTKs without requiring genetic modification remain insufficiently established. In this study, we developed a DNA aptamer agonist, Met-azo-aptamer, which enables reversible optical control of the activity of the c-Met receptor, a member of the RTK family. This was achieved by incorporating azobenzene, a photoisomerizable molecule, into a DNA aptamer that binds to c-Met. This design allows light-induced switching between the active and inactive structures of the aptamer. When the aptamer was applied to HeLa cells and exposed to ultraviolet or blue light, phosphorylation signals within the cells were activated in response to the light patterns. Furthermore, by variation of the light patterns, the Met-azo-aptamer successfully controlled the timing, amplitude, and duration of downstream ERK activation. The Met-azo-aptamer developed in this study offers a high-resolution method for investigating the relationship between RTK activation patterns and cell function or fate.

Aminopeptidase N is a key biomarker associated with various diseases, including cancers and cardiovascular diseases. Nuclear magnetic resonance imaging coupled with dynamic nuclear polarization represents a powerful technique for in vivo detection of enzymatic activity. We previously developed Ala-[1-13C]Gly-d2-NMe2 as a dynamic nuclear polarization-coupled magnetic resonance imaging molecular probe capable of visualizing and mapping aminopeptidase N activity in vivo. However, the broader application of this probe to disease animal models is hindered by significant signal attenuation during sample transfer through low magnetic fields, such as the Earth's magnetic field, primarily due to scalar relaxation via the 13C-14N bond. Here, we present Ala-[1-13C]Gly-d2-15NMe2 as an alternative, incorporating a 15N nucleus in place of 14N. This modification produces markedly enhanced magnetic resonance signal intensity without the need for stringent sample handling to avoid exposure to low magnetic fields. Consequently, this developed probe can potentially offer significant advantages for cellular studies and applications in complex organisms, where elaborate preinjection procedures are often required.