Atsuhiko Taniguchi

Photoinactivation allows precise spatiotemporal control of biomolecules in biological systems. Among the photoinactivation strategies, photooxygenation is advantageous, as it can be readily induced with a photosensitizer upon photoirradiation in biological environment. However, its utility is limited by poor target selectivity arising from the excessive generation and high reactivity of reactive oxygen species. Here, target-selective photoinactivation of myostatin is achieved using a target-binding ligand functionalized with a molecular rotor-based photosensitizing unit whose photooxygenation activity is enhanced upon binding to the target protein. A novel molecular rotor-based photosensitizer was developed and conjugated to a myostatin-binding peptide. The resulting functionalized peptide selectively oxygenated and inactivated myostatin under near-infrared irradiation. Treatment with the peptide followed by irradiation decreased endogenous myostatin levels in skeletal muscle, leading to increased muscle weight and muscle fiber size. This target-selective photoinactivation strategy may expand the applicability of photoresponsive approaches for the treatment of various diseases, including muscular dystrophy.

Gramicidin S (GS) is a cyclic decapeptide derived from two pentapeptides. The C‐terminal thioesterase (TE) domain of gramicidin S synthetase B (GrsB) dimerizes precursor pentapeptides and cyclizes the resulting linear decapeptide. Recently, a GS variant (GS‐SA), in which a single D‐Phe is replaced by L‐Ser(Allyl), is reported via precursor‐directed biosynthesis in a native GS producer. To understand how GrsB‐TE processes such modified precursors, its substrate specificity using synthetic linear peptides is investigated. GrsB‐TE cyclizes a substrate containing L‐Ser(Allyl) at position 6 but not at position 1. However, the enzymatically synthesized GS‐SA shows a different high‐performance liquid chromatography retention time than that of the reported GS variant. Further structural and functional analyses, including ¹ H nuclear magnetic resonance, antimicrobial assays, and circular dichroism spectroscopy, reveal that the reported GS‐SA contained D‐Ser(Allyl) rather than L‐Ser(Allyl). These findings reveal a previously unrecognized stereochemical flexibility in GrsB‐TE and support the structural revision of the reported GS variant.

Establishing an efficient method for the synthesis of disulfide bonds in peptides is an important challenge for the further development of several research fields, including peptide‐based medicinal chemistry and biochemistry. Herein, we report the development of a novel highly water‐soluble 3‐nitro‐2‐pyridine (Npy) sulfenate that efficiently forms intramolecular disulfide bonds in reduced peptides. Moreover, Npy‐sulfenate formed a disulfide bond in a thiol‐containing peptide prepared by thiol‐additive‐free native chemical ligation (NCL), as demonstrated by the one‐pot synthesis of adrenomedullin, which contains 52 amino acid residues. This study provides a new one‐pot synthetic methodology for preparing mid‐sized cyclic disulfide peptides via sulfenate‐mediated oxidation.

Chemical modification of biomolecules such as peptides and proteins is an important technology for the development of new functional molecules. Herein, a thioether‐mediated sulfenylation for tryptophan (Trp)‐selective chemical modification under mild metal‐free reaction conditions involving a weak acid/water solvent is reported. An electrophilic species generated from 4‐fluorophenyl 3‐nitro‐2‐pyridinesulfenate, Npys‐OPh(pF), in the presence of a thioether component, enables efficient sulfenylation of the Trp residue on various Trp‐containing biologically active linear and cyclic peptides. In addition, this sulfenylation to late‐stage chemical modification of a natural cyclic depsipeptide, daptomycin, is expanded and Trp‐sulfenylated derivatives, including fluorescent‐labeled derivatives, are obtained. The results indicate that the thioether‐mediated sulfenylation can be used as a robust Trp‐selective chemical modification method for peptides in a wide range of applications.

Natural macrocyclic peptides derived from microorganisms are medicinal resources that are important for the development of new therapeutic agents. Most of these molecules are biosynthesized by a nonribosomal peptide synthetase (NRPS). The thioesterase (TE) domain in NRPS is responsible for the macrocyclization of mature linear peptide thioesters in a final biosynthetic step. NRPS‐TEs can cyclize synthetic linear peptide analogs and can be utilized as biocatalysts for the preparation of natural product derivatives. Although the structures and enzymatic activities of TEs have been investigated, the substrate recognition and substrate‐TE interaction during the macrocyclization step are still unknown. To understand the TE‐mediated macrocyclization, here we report the development of a substrate‐based analog with mixed phosphonate warheads, which can react irreversibly with the Ser residue at the active site of TE. We have demonstrated that the tyrocidine A linear peptide (TLP) with a p‐nitrophenyl phosphonate (PNP) enables efficient complex formation with tyrocidine synthetase C (TycC)‐TE containing tyrocidine synthetase.

Sarcopenia is a major public health issue that affects older adults. Myostatin inhibitory-D-peptide-35 (MID-35) can increase skeletal muscle and is a candidate therapeutic agent, but a non-invasive and accessible technology for the intramuscular delivery of MID-35 is required. Recently, we succeeded in the intradermal delivery of various macromolecules, such as siRNA and antibodies, by iontophoresis (ItP), a non-invasive transdermal drug delivery technology that uses weak electricity. Thus, we expected that ItP could deliver MID-35 non-invasively from the skin surface to skeletal muscle. In the present study, ItP was performed with a fluorescently labeled peptide on mouse hind leg skin. Fluorescent signal was observed in both skin and skeletal muscle. This result suggested that the peptide was effectively delivered to skeletal muscle from skin surface by ItP. Then, the effect of MID-35/ItP on skeletal muscle mass was evaluated. The skeletal muscle mass increased 1.25 times with ItP of MID-35. In addition, the percentage of new and mature muscle fibers tended to increase, and ItP delivery of MID-35 showed a tendency to induce alterations in the levels of mRNA of genes downstream of myostatin. In conclusion, ItP of myostatin inhibitory peptide is a potentially useful strategy for treating sarcopenia.

Myostatin-binding d-peptides functionalized with photocatalyst efficiently inactivate myostatin by photooxygenation and show no phototoxicity and high resistance to proteolytic enzymes, leading to the treatment of muscular atrophic diseases.

A revised structure of natural 14‐mer cyclic depsipeptide MA026, isolated from Pseudomonas sp. RtlB026 in 2002 was established by physicochemical analysis with HPLC, MS/MS, and NMR and confirmed by total solid‐phase synthesis. The revised structure differs from that previously reported in that two amino acid residues, assigned in error, have been replaced. Synthesized MA026 with the revised structure showed a tight junction (TJ) opening activity like that of the natural one in a cell‐based TJ opening assay. Bioinformatic analysis of the putative MA026 biosynthetic gene cluster (BGC) of RtIB026 demonstrated that the stereochemistry of each amino acid residue in the revised structure can be reasonably explained. Phylogenetic analysis with xantholysin BGC indicates an exceptionally high homology (ca. 90 %) between xantholysin and MA026. The TJ opening activity of MA026 when binding to claudin‐1 is a key to new avenues for transdermal administration of large hydrophilic biologics.

A revised structure of natural 14‐mer cyclic depsipeptide MA026, isolated from Pseudomonas sp. RtlB026 in 2002 was established by physicochemical analysis with HPLC, MS/MS, and NMR and confirmed by total solid‐phase synthesis. The revised structure differs from that previously reported in that two amino acid residues, assigned in error, have been replaced. Synthesized MA026 with the revised structure showed a tight junction (TJ) opening activity like that of the natural one in a cell‐based TJ opening assay. Bioinformatic analysis of the putative MA026 biosynthetic gene cluster (BGC) of RtIB026 demonstrated that the stereochemistry of each amino acid residue in the revised structure can be reasonably explained. Phylogenetic analysis with xantholysin BGC indicates an exceptionally high homology (ca. 90 %) between xantholysin and MA026. The TJ opening activity of MA026 when binding to claudin‐1 is a key to new avenues for transdermal administration of large hydrophilic biologics.

Antibody-recruiting molecules (ARMs) are bispecific molecules composed of an antibody-binding motif and a target-binding motif that redirect endogenous antibodies to target cells to elicit immune responses. To enhance the translational potential of ARMs, it is crucial to design antibody/target-binding motifs that have strong affinity and are easy to synthesize. Here, we synthesized a novel Fc-binding ARM (Fc-ARM) that targets folate receptor (FR)-positive cancer cells, Reo-3, using a recently developed monocyclic peptide 15-Lys8Leu, which binds strongly to the Fc region of an antibody. Reo-3 bound to the Fc region of the antibody with a K_d of 5.8 nM, and recruited a clinically used antibody mixture to attack FR-positive IGROV-1 cells as efficiently as Fc-ARM2, in which a bicyclic Fc-binding peptide was used. These results indicate that 15-Lys8Leu, which can be synthesized readily, is suitable for various applications including the development of Fc-ARMs.