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Neuromedin U (NMU) activates two types of receptors (NMUR1 and NMUR2), and the former is mainly expressed in the peripheral tissues, including the intestinal tract and lung tissues. Since NMUR1 contributes to the promotion of type 2 inflammation in these tissues, it is a potential target to suppress inflammatory responses. However, promising antagonist candidates for human NMUR1 have not yet been developed. Here we successfully identified pentapeptide antagonist 9a through a structure-activity relationship study based on hexapeptide lead 1. Its antagonistic activity against human NMUR1 was 10 times greater than that against NMUR2. This is a breakthrough in the development of NMUR1-selective antagonists. Although 9a was relatively stable in the plasma, the C-terminal amide was rapidly degraded to the carboxylic acid by the serum endopeptidase thrombin, which acted as an amidase. This basic information would aid in sample handling in future biological evaluations.
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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.
Assuntos
Proteases 3C de Coronavírus , SARS-CoV-2 , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/metabolismo , Chlorocebus aethiops , Animais , Células COS , Humanos , Cetonas/química , Cetonas/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Sondas Moleculares/químicaRESUMO
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.
Assuntos
Peptídeos , Tirocidina , Peptídeo Sintases/química , Tirocidina/químicaRESUMO
(+)-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.
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To overcome the decline in the number of students advancing to doctor of philosophy (PhD) courses in graduate schools in Japan, the government of Japan launched a new five-year program in 2021: Support for Pioneering Research Initiated by the Next Generation (SPRING). SPRING is overseen by the Japan Science and Technology Agency (JST) as an integrated program that provides outstanding PhD course students financial support in an amount equivalent to their living expenses and research expenses, as well as career development and human development courses and support. The proposed advanced doctoral program for medical innovators at Tokyo University of Pharmacy and Life Sciences was selected for SPRING support under the theme of "Beef Up Toyaku talents TO go BEyond the borders" (BUTTOBE). This report describes the purpose and current activities of the BUTTOBE program.
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Disciplinas das Ciências Biológicas , Educação de Pós-Graduação em Farmácia , Animais , Bovinos , Humanos , Tóquio , Educação de Pós-Graduação , CurrículoRESUMO
Bovine leukemia virus (BLV) causes enzootic bovine leukosis, a fatal cattle disease that leads to significant economic losses in the livestock industry. Currently, no effective BLV countermeasures exist, except testing and culling. In this study, we developed a high-throughput fluorogenic assay to evaluate the inhibitory activity of various compounds on BLV protease, an essential enzyme for viral replication. The developed assay method was used to screen a chemical library, and mitorubrinic acid was identified as a BLV protease inhibitor that exhibited stronger inhibitory activity than amprenavir. Additionally, the anti-BLV activity of both compounds was evaluated using a cell-based assay, and mitorubrinic acid was found to exhibit inhibitory activity without cytotoxicity. This study presents the first report of a natural inhibitor of BLV protease-mitorubrinic acid-a potential candidate for the development of anti-BLV drugs. The developed method can be used for high-throughput screening of large-scale chemical libraries.
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Vírus da Leucemia Bovina , Peptídeo Hidrolases , Animais , Bovinos , Vírus da Leucemia Bovina/química , Replicação ViralRESUMO
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.
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Junções Íntimas , Sequência de Aminoácidos , Claudina-1/metabolismo , Relação Estrutura-Atividade , Junções Íntimas/metabolismo , Raios XRESUMO
Crush syndrome induced by skeletal muscle compression causes fatal rhabdomyolysis-induced acute kidney injury (RIAKI) that requires intensive care, including hemodialysis. However, access to crucial medical supplies is highly limited while treating earthquake victims trapped under fallen buildings, lowering their chances of survival. Developing a compact, portable, and simple treatment method for RIAKI remains an important challenge. Based on our previous finding that RIAKI depends on leukocyte extracellular traps (ETs), we aimed to develop a novel medium-molecular-weight peptide to provide clinical treatment of Crush syndrome. We conducted a structure-activity relationship study to develop a new therapeutic peptide. Using human peripheral polymorphonuclear neutrophils, we identified a 12-amino acid peptide sequence (FK-12) that strongly inhibited neutrophil extracellular trap (NET) release in vitro and further modified it by alanine scanning to construct multiple peptide analogs that were screened for their NET inhibition ability. The clinical applicability and renal-protective effects of these analogs were evaluated in vivo using the rhabdomyolysis-induced AKI mouse model. One candidate drug [M10Hse(Me)], wherein the sulfur of Met10 is substituted by oxygen, exhibited excellent renal-protective effects and completely inhibited fatality in the RIAKI mouse model. Furthermore, we observed that both therapeutic and prophylactic administration of M10Hse(Me) markedly protected the renal function during the acute and chronic phases of RIAKI. In conclusion, we developed a novel medium-molecular-weight peptide that could potentially treat patients with rhabdomyolysis and protect their renal function, thereby increasing the survival rate of victims affected by Crush syndrome.
Assuntos
Injúria Renal Aguda , Síndrome de Esmagamento , Armadilhas Extracelulares , Rabdomiólise , Animais , Camundongos , Humanos , Síndrome de Esmagamento/complicações , Síndrome de Esmagamento/tratamento farmacológico , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/tratamento farmacológico , Rabdomiólise/complicações , Rabdomiólise/tratamento farmacológico , Leucócitos , Peptídeos/farmacologia , Peptídeos/uso terapêuticoRESUMO
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.
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Cisteína , Peptídeos Cíclicos , Cisteína/química , Dissulfetos , Peptídeos/química , Compostos de Sulfidrila/químicaRESUMO
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.
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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.
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Cancer cachexia is a multifactorial disease that causes continuous skeletal muscle wasting. Thereby, it seems to be a key determinant of cancer-related death. Although anamorelin, a ghrelin receptor agonist, has been approved in Japan for the treatment of cachexia, few medical treatments for cancer cachexia are currently available. Myostatin (MSTN)/growth differentiation factor 8, which belongs to the transforming growth factor-ß family, is a negative regulator of skeletal muscle mass, and inhibition of MSTN signaling is expected to be a therapeutic target for muscle-wasting diseases. Indeed, we have reported that peptide-2, an MSTN-inhibiting peptide from the MSTN prodomain, alleviates muscle wasting due to cancer cachexia. Herein, we evaluated the therapeutic benefit of myostatin inhibitory D-peptide-35 (MID-35), whose stability and activity were more improved than those of peptide-2 in cancer cachexia model mice. The biologic effects of MID-35 were better than those of peptide-2. Intramuscular administration of MID-35 effectively alleviated skeletal muscle atrophy in cachexia model mice, and the combination therapy of MID-35 with anamorelin increased food intake and maximized grip strength, resulting in longer survival. Our results suggest that this combination might be a novel therapeutic tool to suppress muscle wasting in cancer cachexia.
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Produtos Biológicos , Neoplasias , Animais , Produtos Biológicos/farmacologia , Caquexia/tratamento farmacológico , Caquexia/etiologia , Caquexia/patologia , Modelos Animais de Doenças , Hidrazinas , Camundongos , Músculo Esquelético , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Miostatina , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Oligopeptídeos , Peptídeos/farmacologia , Receptores de Grelina/uso terapêutico , Fatores de Crescimento Transformadores/farmacologia , Fatores de Crescimento Transformadores/uso terapêuticoRESUMO
Translational readthrough-inducing agents have been developed for the treatment of nonsense mutations in hereditary diseases. The clinical effectiveness of readthrough agents has been reported, although newly developed agents are still desired because of their toxicities or limited clinical effectiveness. Recently, novel negamycin-derived readthrough agents without antimicrobial activity have been developed. Our aim was to evaluate the activities of these readthrough agents by monitoring the production of large myelin protein zero (L-MPZ), the programmed translational readthrough isoform of myelin protein zero (P0, MPZ) mRNA, and to clarify the influence of these agents on the sciatic nerve in vivo. First, we examined the readthrough activities of novel negamycin-derived agents using cell-free and cell culture systems using plasmids encoding human MPZ (hP0) cDNA. Three of the negamycin derivatives, TCP-112, TCP-169, and TCP-1109, suppressed the canonical stop codon to induce readthrough. Direct injection of TCP-1109, which showed higher readthrough activity for Mpz in mouse sciatic nerves, exhibited a 1.3-fold increase in the L-MPZ/P0 ratio compared to that with the vehicle control on western blotting. The nerve conduction velocity and beam walk test showed abnormalities in the classical readthrough agent G418-treated group, but not in the TCP-1109-treated group. Immunofluorescence analysis showed that TCP-1109 caused less damage to the sciatic nerve than G418. In the semi-thin sections, a lower g-ratio and more tomacula-like structures were observed in TCP-1109-treated nerves. Thus, the present results indicate that negamycin-derived readthrough agents enhance programmed translational readthrough, and the management of readthrough activities using canonical stop codons may be important.
Assuntos
Proteína P0 da Mielina , Biossíntese de Proteínas , Animais , Códon de Terminação , Camundongos , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Sistema Nervoso Periférico/metabolismo , RNA Mensageiro/metabolismoRESUMO
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.
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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.
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Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Cetonas/farmacologia , Peptidomiméticos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/química , COVID-19/metabolismo , Chlorocebus aethiops , Proteases 3C de Coronavírus/isolamento & purificação , Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Humanos , Cetonas/química , Masculino , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Molecular , Peptidomiméticos/síntese química , Peptidomiméticos/química , Ratos , Ratos Wistar , SARS-CoV-2/enzimologia , Células Vero , Tratamento Farmacológico da COVID-19RESUMO
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.
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Folistatina/química , Miostatina/antagonistas & inibidores , Peptídeos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Miostatina/metabolismo , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
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.
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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.
Assuntos
Miostatina/antagonistas & inibidores , Peptídeos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
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.
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Dipeptídeos , Peptidomiméticos , Aminoácidos , Peptídeos , Técnicas de Síntese em Fase SólidaRESUMO
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 =8.19â 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 =24.3â nM) than that of 15-IgBP (Kd =267â 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.