Detection of Active SARS-CoV-2 3CL Protease in Infected Cells Using Activity-Based Probes with a 2,6-Dichlorobenzoyloxymethyl Ketone Reactive Warhead.

The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe 2) or 2,6-dichlorobenzoyloxymethyl ketone (probe 3) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe 3 displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe 3 for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe 3, suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe 3 will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.

Peptide mixed phosphonates for covalent complex formation with thioesterases in nonribosomal peptide synthetases.

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.

Development of Conformationally Restricted Negamycin Derivatives for Potent Readthrough Activity.

(+)-Negamycin, which is a dipeptide-like antibiotic containing a hydrazide structure, exhibits readthrough activity, resulting in the restoration of dystrophin in the mdx mouse model of Duchenne muscular dystrophy (DMD). In our previous structure-activity relationship study of negamycin, we found that its natural analogue 3-epi-deoxynegamycin (TCP-107), without antimicrobial activity, showed a higher readthrough activity than negamycin. In this study, we designed and synthesized cyclopropane-based conformationally restricted derivatives of TCP-107 and evaluated their readthrough activity in the cell-based reporter assay against a TGA-type mutation derived from DMD. As a result, a down-cis isomer, TCP-304, showed significant readthrough activity among the four isomers. Moreover, TCP-306, a derivative acylated by l-α-aminoundecanoic acid, possessed approximately 3 times higher activity than TCP-304. These down-cis derivatives showed dose-dependent readthrough activity and were effective for not only TGA but also TAG mutations. These results suggest that the conformational restriction of negamycin derivatives by the introduction of the cyclopropane ring is effective for an exhibition of potent readthrough activity.

X-Ray Conformation and Structure-Activity Relationships of MA026, a Reversible Tight Junction Opener.

MA026, a cyclic lipodepsipeptide, opens the tight junction (TJ) probably via binding to claudin-1. We reported that (1) TJ-opening activity is dependent on the amino acid sequence order at Glu10-Leu11; (2) an epimer at the C3 position of the N-terminal acyl tail decreased the TJ-opening activity; and (3) the epimers D-Leu1/L-Gln6 and L-Leu1/D-Gln6 showed more potent TJ-opening activity than natural MA026, although no systematic structure-activity relationship (SAR) study was conducted. Here, we report the three-dimensional structure and systematic SAR study of MA026. X-Ray crystallography and circular dichroism analysis of MA026 revealed that MA026 forms a left-handed α-helical structure, and hydrophobic amino acids are clustered on one side. Furthermore, the SAR results clearly showed that the hydrophobic region of MA026 is important for TJ-opening activity. These results suggest that MA026 interacts with claudin-1 via the hydrophobic cluster region and provide novel structural insights toward the development of a TJ opener targeting claudin-1.

Establishment of One-Pot Disulfide-Driven Cyclic Peptide Synthesis with a 3-Nitro-2-pyridinesulfenate.

We have developed a new one-pot disulfide-driven cyclic peptide synthesis. The entire process is carried out in the solid phase, thus eliminating complicated work up procedures to remove by-products and unreacted reagents and enabling production of high-purity cyclic disulfide peptides by simple cleavage of a peptidyl resin. The one-pot synthesis of oxytocin was accomplished in this way with an isolated yield of 28% over 13 steps. These include peptide chain elongation from an initial resin, sulfenylation of the protected side chain of a cysteine (Cys) residue, disulfide ligation between thiols in an additional peptide fragment and a 3-nitro-2-pyridinesulfenyl-protected cysteine (Cys(Npys))-containing peptide resin, subsequent intramolecular amide bond formation of the disulfide-connected fragments by an Ag+-promoted thioester method, followed by deprotection and HPLC purification.

Increasing Skeletal Muscle Mass in Mice by Non-Invasive Intramuscular Delivery of Myostatin Inhibitory Peptide by Iontophoresis.

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.

Inactivation of myostatin by photooxygenation using functionalized d-peptides.

Inhibition of myostatin is an attractive strategy for the treatment of muscular atrophic diseases such as muscular dystrophy. For the efficient inhibition of myostatin, functionalized peptides were developed by the conjugation of a 16-mer myostatin-binding d-peptide with a photooxygenation catalyst. These peptides induced myostatin-selective photooxygenation and inactivation under near-infrared irradiation, and were associated with little cytotoxicity or phototoxicity. The peptides are resistant to enzymatic digestion due to their d-peptide chains. These properties could contribute to the in vivo use of photooxygenation-based inactivation strategies targeting myostatin.

Development of Myostatin Inhibitory d-Peptides to Enhance the Potency, Increasing Skeletal Muscle Mass in Mice.

Myostatin is a key negative regulator of skeletal muscle growth, and myostatin inhibitors are attractive tools for the treatment of muscular atrophy. Previously, we reported a series of 14-29-mer peptide myostatin inhibitors, including a potent derivative, MIPE-1686, a 16-mer N-terminal-free l-peptide with three unnatural amino acids and a propensity to form ß-sheets. However, the in vivo biological stability of MIPE-1686 is a concern for its development as a drug. In the present study, to develop a more stable myostatin inhibitory d-peptide (MID), we synthesized various retro-inverso versions of a 16-mer peptide. Among these, an arginine-containing derivative, MID-35, shows a potent and equivalent in vitro myostatin inhibitory activity equivalent to that of MIPE-1686 and considerable stability against biodegradation. The in vivo potency of MID-35 to increase the tibialis anterior muscle mass in mice is significantly enhanced over that of MIPE-1686, and MID-35 can serve as a new entity for the prolonged inactivation of myostatin in skeletal muscle.

3CL Protease Inhibitors with an Electrophilic Arylketone Moiety as Anti-SARS-CoV-2 Agents.

The novel coronavirus, SARS-CoV-2, has been identified as the causative agent for the current coronavirus disease (COVID-19) pandemic. 3CL protease (3CLpro) plays a pivotal role in the processing of viral polyproteins. We report peptidomimetic compounds with a unique benzothiazolyl ketone as a warhead group, which display potent activity against SARS-CoV-2 3CLpro. The most potent inhibitor YH-53 can strongly block the SARS-CoV-2 replication. X-ray structural analysis revealed that YH-53 establishes multiple hydrogen bond interactions with backbone amino acids and a covalent bond with the active site of 3CLpro. Further results from computational and experimental studies, including an in vitro absorption, distribution, metabolism, and excretion profile, in vivo pharmacokinetics, and metabolic analysis of YH-53 suggest that it has a high potential as a lead candidate to compete with COVID-19.

Strategic structure-activity relationship study on a follistatin-derived myostatin inhibitory peptide.

Myostatin, a negative regulator of muscle mass is a promising target for the treatment of muscle atrophic diseases. The novel myostatin inhibitory peptide, DF-3 is derived from the N-terminal α-helical domain of follistatin, which is an endogenous inhibitor of myostatin and other TGF-ß family members. It has been suggested that the optimization of hydrophobic residues is important to enhance the myostatin inhibition. This study describes a structure-activity relationship study focused on hydrophobic residues of DF-3 and designed to obtain a more potent peptide. A methionine residue in DF-3, which is susceptible to oxidation, was successfully converted to homophenylalanine in DF-100, and a new derivative DF-100, with four amino acid substitutions in DF-3 shows twice the potent inhibitory ability as DF-3. This report provides a new platform of a 14-mer peptide muscle enhancer.

Proposal for the binding mode of the 23-mer inhibitory peptide to myostatin.

Inhibition of myostatin is a promising strategy for the treatment of amyotrophic disorders. Previously, we identified a minimum 23-mer peptide spanning positions 21-43 of a mouse myostatin precursor-derived prodomain and identified the nine key residues for effective myostatin inhibition through Ala scanning. We also reported the 23-mer peptides that show the propensity to form an α-helical structure around positions 32-36. Here, based on these findings, we conducted a docking simulation of a peptide-myostatin interaction. The results showed that by α-helix restraint docking of the 30-41 main chain, we obtained a proposed binding mode in which all nine of the key residues interact with myostatin. By analyzing the binding mode of four proposed docking models, we identified six of the myostatin residues that play an important role in the interaction with the peptide. This result provides a valuable insight into the relationship between myostatin and peptide interaction sites and may help in the design of future inhibitors.

Synthesis and biological evaluation of a monocyclic Fc-binding antibody-recruiting molecule for cancer immunotherapy.

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.

Photo-oxygenation by a biocompatible catalyst reduces amyloid-ß levels in Alzheimer's disease mice.

Amyloid formation and the deposition of the amyloid-ß peptide are hallmarks of Alzheimer's disease pathogenesis. Immunotherapies using anti-amyloid-ß antibodies have been highlighted as a promising approach for the prevention and treatment of Alzheimer's disease by enhancing microglial clearance of amyloid-ß peptide. However, the efficiency of antibody delivery into the brain is limited, and therefore an alternative strategy to facilitate the clearance of brain amyloid is needed. We previously developed an artificial photo-oxygenation system using a low molecular weight catalytic compound. The photocatalyst specifically attached oxygen atoms to amyloids upon irradiation with light, and successfully reduced the neurotoxicity of aggregated amyloid-ß via inhibition of amyloid formation. However, the therapeutic effect and mode of actions of the photo-oxygenation system in vivo remained unclear. In this study, we demonstrate that photo-oxygenation facilitates the clearance of aggregated amyloid-ß from the brains of living Alzheimer's disease model mice, and enhances the microglial degradation of amyloid-ß peptide. These results suggest that photo-oxygenation may represent a novel anti-amyloid-ß strategy in Alzheimer's disease, which is compatible with immunotherapy.

Development of Methods for Convergent Synthesis of Chloroalkene Dipeptide Isosteres and Its Application.

Improved methods of convergent synthesis for peptidomimetic utilizing a chloroalkene dipeptide isostere (CADI) are reported. In this synthesis, Fmoc- or Boc-protected carboxylic acids can be produced from N- and C-terminal analogues corresponding to each amino acid starting material via an Evans syn aldol reaction, followed by a [3.3] sigmatropic rearrangement utilizing the Ichikawa allylcyanate rearrangement reaction. With this strategy, an Fmoc-protected CADI can be directly applied for solid-phase peptide synthesis. Using this approach, we have also identified the CADI-containing cyclo[-Lys-Leu-Val-Phe-Phe-] peptidomimetic, which is a superior inhibitor of amyloid-ß aggregation than the parent peptide.

Structural Revision of Natural Cyclic Depsipeptide MA026 Established by Total Synthesis and Biosynthetic Gene Cluster Analysis.

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.

Development of a High-Affinity Antibody-Binding Peptide for Site-Specific Modification.

Immunoglobulin G (IgG)-binding peptides such as 15-IgBP are convenient tools for the site-specific modification of antibodies and the preparation of homogeneous antibody-drug conjugates. A peptide such as 15-IgBP can be selectively crosslinked to the fragment crystallizable region of human IgG in an affinity-dependent manner via the ϵ-amino group of Lys8. Previously, we found that the peptide 15-Lys8Leu has a high affinity (Kd =8.19 nM) due to the presence of the γ-dimethyl group in Leu8. The primary amino group required for the crosslinking to the antibodies has, however, been lost. Here, we report the design and synthesis of a novel unnatural amino acid, 4-(2-aminoethylcarbamoyl)leucine (Aecl), which possesses both the γ-dimethyl fragment and a primary amino group. A peptide containing Aecl8 (15-Lys8Aecl) was synthesized and showed a binding affinity ten times higher (Kd =24.3 nM) than that of 15-IgBP (Kd =267 nM). Fluorescein isothiocyanate (FITC)-labeled 15-Lys8Aecl with an N-hydroxy succinimide ester at the side chain of Aecl8 (FITC-15-Lys8Aecl(OSu)) successfully labeled an antibody (trastuzumab, Herceptin® ) with the fluorophore. This peptide scaffold has both strong binding affinity and crosslinking capability, and could be a useful tool for the selective chemical modification of antibodies with molecules of interest such as drugs.

Development of functionalized peptides for efficient inhibition of myostatin by selective photooxygenation.

For the inhibition of myostatin, which is an attractive strategy for the treatment of muscle atrophic disorders including muscular dystrophy, myostatin-binding peptides were synthesized with an on/off-switchable photooxygenation catalyst at different positions on the peptide chain. These functionalized peptides oxygenated and inactivated myostatin upon irradiation with near-infrared light. Among the peptides tested, a peptide (5) with the catalyst moiety at the 16 position induced myostatin-selective photooxygenation, and efficiently inhibited myostatin. These peptides exhibited low phototoxicity. Such functionalized peptides would provide a precedented strategy for myostatin-targeting therapy, in which myostatin is irreversibly and catalytically inactivated by photooxygenation.

Use of solid-supported 4-fluorophenyl 3-nitro-2-pyridinesulfenate in the construction of disulfide-linked hybrid molecules.

To construct disulfide-linked hybrid molecules systematically and efficiently, we established a more practical solid-phase disulfide ligation (SPDSL) system with enhanced utility. The group Npys-OPh(pF) shows reactivity similar to that of Npys-Cl, but it is more stable. An efficient synthesis of the cyclic peptide oxytocin and a peptide-sugar conjugate was accomplished as models. These results indicate that the Npys-OPh(pF) resin functions as a common synthetic platform in SPDSL.

Design and synthesis of peptidic partial agonists of human neuromedin U receptor 1 with enhanced serum stability.

Neuromedin U (NMU) activates two receptors (NMUR1 and NMUR2) and is a promising candidate for development of drugs to combat obesity. Previously, we obtained hexapeptides as selective full NMUR agonists. Development of a partial agonist which mildly activates receptors is an effective strategy which lead to an understanding of the functions of NMU receptors. In 2014, we reported hexapeptide 3 (CPN-124) as an NMUR1-selective partial agonist but its selectivity and serum stability were unsatisfactory. Herein, we report the development of a hexapeptide-type partial agonist (8, CPN-223) based on a peptide (3) but with higher NMUR1-selectivity and enhanced serum stability. A structure-activity relationship study of synthetic pentapeptide derivatives suggested that a hexapeptide is a minimum structure consistent with both good NMUR1-selective agonistic activity and serum stability.

Enzymatic Stability of Myostatin Inhibitory 16-mer Peptides.

Inhibition of myostatin is a promising strategy for treatment of muscle atrophic disorders. A 16-mer myostatin inhibitory linear peptide, MIPE-1686, administered intramuscularly, significantly increases muscle mass and hindlimb grip strength in Duchenne muscular dystrophic model mice. In this paper, we describe our examination of the enzymatic stabilities of this peptide with recombinant human proteases, aminopeptidase N, chymotrypsin C, and trypsin 3. MIPE-1686 was found to be stable in the presence of these enzymes, in contrast to a peptide (1), from which MIPE-1686 was developed. Modification of the peptides at a position distant from the protease cleavage site altered their enzymatic stability. These results suggest the possibility that the stability to proteases of 16-mer myostatin inhibitory peptides is associated with an increase in their known ß-sheet formation properties. This study suggests that MIPE-1686 has a potential to serve as a long-lasting agent in vivo.