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.

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.

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.

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.

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.

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.

Discovery of a follistatin-derived myostatin inhibitory peptide.

Follistatin is well known as an inhibitor of transforming growth factor (TGF)-ß superfamily ligands including myostatin and activin A. Myostatin, a negative regulator of muscle growth, is a promising target with which to treat muscle atrophic diseases. Here, we focused on the N-terminal domain (ND) of follistatin (Fst) that interacts with the type I receptor binding site of myostatin. Through bioassay of synthetic ND-derived fragment peptides, we identified DF-3, a new myostatin inhibitory 14-mer peptide which effectively inhibits myostatin, but fails to inhibit activin A or TGF-ß1, in an in vitro luciferase reporter assay. Injected intramuscularly, DF-3 significantly increases skeletal muscle mass in mice and consequently, it can serve as a platform for development of muscle enhancement based on myostatin inhibition.

Inactivation of myostatin by photo-oxygenation using catalyst-functionalized peptides.

Inhibition of myostatin is an attractive treatment for muscular dystrophy and other amyotrophic diseases. A myostatin-binding peptide was functionalized by equipped with an on/off switchable photo-oxygenation catalyst. This peptide induces a selective oxygenation of myostatin under near-infrared light, resulting in inactivation of myostatin. This peptide shows several orders of magnitude greater inhibitory effect than the original peptide.

Discovery of a Human Neuromedin U Receptor 1-Selective Hexapeptide Agonist with Enhanced Serum Stability.

Neuromedin U (NMU) activates two NMU receptors (NMUR1 and NMUR2) and is a useful antiobesity drug lead. We report discovery of a hexapeptide agonist, 2-thienylacetyl-Trp1-Phe(4-F)2-Arg3-Pro4-Arg5-Asn6-NH2 (4). However, the NMUR1 selectivity and serum stability of this agonist were unsatisfactory. Through a structure-activity relationship study focused on residue 2 of agonist 4, serum stability, and pharmacokinetic properties, we report here the discovery of a novel NMUR1 selective hexapeptide agonist 7b that suppresses body weight gain in mice.

Switchable photooxygenation catalysts that sense higher-order amyloid structures.

Proteins can misfold into amyloid structures that are associated with diseases; however, the same proteins often have important biological roles. To degrade selectively the amyloid form without affecting the fraction of functional protein is, therefore, an attractive goal. Here we report target-state-dependent photooxygenation catalysts that are active only when bound to the cross-ß-sheet structure that is characteristic of pathogenic aggregated amyloid proteins. We show these catalysts can selectively oxygenate the amyloid form of amyloid ß-protein (Aß) 1-42 in the presence of non-amyloid off-target substrates. Furthermore, photooxygenation with a catalyst that bears an Aß-binding peptide attenuated the Aß pathogenicity in the presence of cells. We also show that selective photooxygenation is generally applicable to other amyloidogenic proteins (amylin, insulin, ß2-microglobulin, transthyretin and α-synuclein) and does not affect the physiologically functional non-aggregate states of these proteins. This is the first report of an artificial catalyst that can be selectively and reversibly turned on and off depending on the structure and aggregation state of the substrate protein.

Rational design and identification of a non-peptidic aggregation inhibitor of amyloid-ß based on a pharmacophore motif obtained from cyclo[-Lys-Leu-Val-Phe-Phe-].

Inhibition of pathogenic protein aggregation may be an important and straightforward therapeutic strategy for curing amyloid diseases. Small-molecule aggregation inhibitors of Alzheimer's amyloid-ß (Aß) are extremely scarce, however, and are mainly restricted to dye- and polyphenol-type compounds that lack drug-likeness. Based on the structure-activity relationship of cyclic Aß16-20 (cyclo-[KLVFF]), we identified unique pharmacophore motifs comprising side-chains of Leu(2), Val(3), Phe(4), and Phe(5) residues without involvement of the backbone amide bonds to inhibit Aß aggregation. This finding allowed us to design non-peptidic, small-molecule aggregation inhibitors that possess potent activity. These molecules are the first successful non-peptidic, small-molecule aggregation inhibitors of amyloids based on rational molecular design.

Attenuation of the aggregation and neurotoxicity of amyloid-ß peptides by catalytic photooxygenation.

Alzheimer's disease (AD), a progressive severe neurodegenerative disorder, is currently incurable, despite intensive efforts worldwide. Herein, we demonstrate that catalytic oxygenation of amyloid-ß peptides (Aß) might be an effective approach to treat AD. Aß1-42 was oxygenated under physiologically-relevant conditions (pH 7.4, 37 °C) using a riboflavin catalyst and visible light irradiation, with modifications at the Tyr(10) , His(13) , His(14) , and Met(35) residues. The oxygenated Aß1-42 exhibited considerably lower aggregation potency and neurotoxicity compared with native Aß. Photooxygenation of Aß can be performed even in the presence of cells, by using a selective flavin catalyst attached to an Aß-binding peptide; the Aß cytotoxicity was attenuated in this case as well. Furthermore, oxygenated Aß1-42 inhibited the aggregation and cytotoxicity of native Aß.

Self-assembly pathways of E22Δ-type amyloid ß peptide mutants generated from non-aggregative O-acyl isopeptide precursors.

The recently identified E22Δ-type amyloid ß peptide (Aß) mutants are reported to favor oligomerization over fibrillization and to exhibit more-potent synaptotoxicity than does wild-type (WT) Aß. Aß(E22Δ) mutants can thus be expected to serve as tools for clarifying the impact of Aß oligomers in Alzheimer's disease (or Alzheimer's-type dementia). However, the biochemical and biophysical properties of Aß(E22Δ) have not been conclusively determined. Here, we evaluated the self-assembly pathways of Aß(E22Δ) mutants generated from water-soluble, non-aggregative O-acyl isopeptide precursors. Circular dichroism spectroscopy, Western blot analysis, and thioflavin-T fluorescence intensity and cellular toxicity assays suggest that the self-assembly pathways of Aß(E22Δ) differed from those of Aß(WT). Aß1-40(E22Δ) underwent a rapid random coil→ß-sheet conformational change in its monomeric or low-molecular-weight oligomeric states, whereas Aß1-40(WT) self-assembled gradually without losing its propensity to form random coil structures. The Aß1-42(E22Δ) monomer formed ß-sheet-rich oligomers more rapidly than did Aß1-42(WT). Additionally, the Aß1-42(E22Δ) oligomers appear to differ from Aß1-42(WT) oligomers in size, shape, or both. These results should provide new insights into the functions of Aß(E22Δ) mutants.

'Click peptide' using production of monomer Aß from the O-acyl isopeptide: application to assay system of aggregation inhibitors and cellular cytotoxicity.

The O-acyl isopeptide of Aß1-42 (1), possessing an ester bond at the Gly(25)-Ser(26) sequence, is a water-soluble and non-aggregative precursor molecule and is capable of production of monomer Aß1-42. The SDS-PAGE result showed that the Aß1-42, produced from 1, adopted monomeric state at first and then self-assembled to oligomer. The oligomeric state was stabilized by nordihydroguaiaretic acid. The Thioflavin-T (ThT) fluorescence intensity derived from Aß1-42 (generated from 1) was suppressed by various aggregation inhibitors. Finally, 1 could generate Aß1-42 via the O-to-N acyl migration under cellular medium conditions and the produced Aß1-42 exhibited cytotoxicity against PC12 cells. These results suggest that the click peptide system, which enables us to predominantly produce monomer Aß1-42 under physiological conditions, would be adoptable to various biochemical and biophysical experiments including cellular system to investigate the functions of Aß1-42.

[Development of click peptide: stimuli-responsive precursor producing Alzheimer's disease-related amyloid beta peptide].

In a pathological mechanism of Alzheimer's disease (AD), amyloid beta peptide (Abeta) 1-42 plays a crucial role. However, the detailed pathological mechanism remains unclear. This elucidation is hampered by handling difficulties of Abeta1-42 due to its poor water-solubility and uncontrollable aggregation. These properties prevent reproducing neurotoxicity-related assembly events of Abeta1-42 in the experiments, leading to discrepant study outcomes. Namely, such properties of Abeta1-42 are serious obstacles to establish an experiment system that clarifies the pathological mechanism of Abeta1-42 in AD. To solve these problems, we developed "click peptide" of Abeta1-42 based on the "Omicron-acyl isopeptide method". The click peptide, which contains an Omicron-acyl instead of N-acyl residue at Gly(25)-Ser(26) of Abeta1-42, is converted to Abeta1-42 via an Omicron-to-N intramolecular acyl migration upon being triggered by pH-change (pH-click) or photo-irradiation (photo-click). The click peptide was 100-fold more water-soluble than Abeta1-42 and clearly adopted a monomeric random coil structure due to the Omicron-acyl moiety in the peptide backbone. The click peptide was quickly converted to monomer Abeta1-42 with a random coil structure under physiological conditions upon an action (click). The obtained Abeta1-42 underwent both self-assembly and conformational changes with time. Because the in situ production of intact Abeta1-42 from the water-soluble and non-aggregative precursor could overcome the handling problems of Abeta1-42, this click peptide strategy would provide a reliable experiment system to investigate the pathological functions of Abeta1-42 in AD.