Shinsuke Sando

MRNA display-based DMS systematically explores FGF2/FGFR interaction interfaces and evaluates residues contributing to FGFR isoform specificity, enabling engineering of variants that shift broad FGFR binding toward isoform-selective binding.

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 C α‐substituents, we isolated the backbone steric effects independently of amide hydrogen removal. N ‐Alkyl peptides bearing an N / C α‐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.

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 C α‐substituents, we isolated the backbone steric effects independently of amide hydrogen removal. N ‐Alkyl peptides bearing an N / C α‐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.

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.

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.

Polymer conjugation is a common strategy to improve the pharmacokinetics of aptamers, yet its effects on aptamer properties are incompletely understood.

Fibroblast growth factor 2 (FGF2)regulates signal transduction by forming complexes with its receptors, FGF receptors (FGFRs), and heparan sulfate (HS), playing a crucial role in biological systems. Although HS has been suggested to modulate FGF/FGFR signaling as a coreceptor, multiple hypotheses exist regarding how HS affects FGF/FGFR signaling and the mechanism remains unclear. Herein, to highlight the role of FGF2/HS interaction in FGF2/FGFR1 signaling, FGF2 mutants with reduced HS‐binding affinity are rationally designed through in silico analysis. These FGF2 mutants exhibit reduced HS affinity by more than two orders of magnitude while maintaining binding affinity to FGFR1. In addition, these mutants retain their thermal stability. Cellular assays using the FGF2 mutant suggest that, contrary to previous reports, the contribution of the FGF2/HS interaction in FGF2/FGFR1 signaling may be limited. The mutant FGFs that specifically alter the interaction with HS, achieved in this study, would contribute to an understanding of the role of FGF/HS interaction in FGF/FGFR signaling.

We report the construction of a polyunsaturated fatty acid (PUFA) library by the solid-phase synthesis, the investigation of agonist activities for free fatty acid receptors 1 and 4, and the evaluation of anti-inflammatory effect.