Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 66
Filter
Add more filters










Publication year range
1.
Bioorg Med Chem Lett ; 98: 129588, 2024 Jan 15.
Article in English | MEDLINE | ID: mdl-38086467

ABSTRACT

In vitro screening of a focused library of compounds containing an electrophilic warhead identified N-chloroacetyl-bis(trifluoromethyl)aniline derivative 15 as a potent inhibitor of BMAL1-CLOCK heterodimer binding to an E-box DNA fragment. Kinetic analysis of thiol-reactivity demonstrated that iodoacetamide and structurally related 20 are significantly more reactive than or equally reactive as 15, respectively, whereas none inhibited BMAL1-CLOCK interaction with the E-box DNA fragment. These results suggest that 15 binds and reacts with a specific nucleophilic residue. This low-molecular-weight compound may serve as a useful lead for further development of BMAL1-CLOCK inhibitors.


Subject(s)
Aniline Compounds , Circadian Clocks , ARNTL Transcription Factors/antagonists & inhibitors , ARNTL Transcription Factors/metabolism , Circadian Clocks/drug effects , Circadian Clocks/genetics , Circadian Rhythm/drug effects , DNA/metabolism , Kinetics , Aniline Compounds/chemistry
2.
Chem Commun (Camb) ; 59(72): 10809-10812, 2023 Sep 07.
Article in English | MEDLINE | ID: mdl-37599611

ABSTRACT

Xanthine derivatives were identified as inhibitors of the N6-methyladenosine (m6A) demethylase activity of fat-mass-and-obesity-associated protein (FTO) by activity-based high-throughput screening using the m6A-sensitive ribonuclease MazF. Pentoxifylline exhibited L-ascorbic acid concentration-dependent inhibitory activity against FTO, an unprecedented mode of inhibition, indicating that L-ascorbic acid is a promising key for designing FTO-specific inhibitors.


Subject(s)
Alkaloids , Ascorbic Acid/pharmacology , High-Throughput Screening Assays , Ribonucleases , Xanthines/pharmacology
3.
Chemistry ; 29(29): e202300129, 2023 May 22.
Article in English | MEDLINE | ID: mdl-36878866

ABSTRACT

Spatiotemporal structural alterations in cellular membranes are the hallmark of many vital processes. In these cellular events, the induction of local changes in membrane curvature often plays a pivotal role. Many amphiphilic peptides are able to modulate membrane curvature, but there is little information on specific structural factors that direct the curvature change. Epsin-1 is a representative protein thought to initiate invagination of the plasma membrane upon clathrin-coated vesicles formation. Its N-terminal helical segment (EpN18) plays a key role in inducing positive membrane curvature. This study aimed to elucidate the essential structural features of EpN18 in order to better understand general curvature-inducing mechanisms, and to design effective tools for rationally controlling membrane curvature. Structural dissection of peptides derived from EpN18 revealed the decisive contribution of hydrophobic residues to (i) enhancing membrane interactions, (ii) helix structuring, (iii) inducing positive membrane curvature, and (iv) loosening lipid packing. The strongest effect was obtained by substitution with leucine residues, as this EpN18 analog showed a marked ability to promote the influx of octa-arginine cell-penetrating peptides into living cells.


Subject(s)
Adaptor Proteins, Vesicular Transport , Peptides , Peptides/chemistry , Adaptor Proteins, Vesicular Transport/analysis , Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Cell Membrane/metabolism
4.
J Biol Inorg Chem ; 28(3): 249-261, 2023 04.
Article in English | MEDLINE | ID: mdl-36749405

ABSTRACT

Zinc finger proteins are abundant in the human proteome and are responsible for a variety of functions. The domains that constitute zinc finger proteins are compact spherical structures, each comprising approximately 30 amino acid residues, but they also have precise molecular factor functions: zinc binding and DNA recognition. Due to the biological importance of zinc finger proteins and their unique structural and functional properties, many artificial zinc finger proteins have been created and are expected to improve their functions and biological applications. In this study, we review previous studies on the redesign and application of artificial zinc finger proteins, focusing on the experimental results obtained by our research group. In addition, we systematically review various design strategies used to construct artificial zinc finger proteins and discuss in detail their potential biological applications, including gene editing. This review will provide relevant information to researchers involved or interested in the field of artificial zinc finger proteins as a potential new treatment for various diseases.


Subject(s)
DNA , Zinc Fingers , Humans , DNA/chemistry
5.
Acta Histochem Cytochem ; 55(5): 169-184, 2022 Oct 28.
Article in English | MEDLINE | ID: mdl-36405550

ABSTRACT

We investigated the role of cancer stem cells (CSCs) in a population of triple-negative breast cancer (TNBC) cells that are resistant to apoptosis. A human breast cancer cell population capable of inducing p53 expression with doxycycline (Dox) was created and used as an untreated control (UT). After the addition of Dox to UT for 5 days, the cell population reconstituted with cells showing resistance to apoptosis was named RE. Fluorescence-activated cell sorting (FACS) and immunostaining revealed that after the addition of Dox, the ratio of cells in the S and G2/M phases decreased in UT as apoptosis proceeded, but did not markedly change in apoptosis-resistant RE. CSC-like cells in RE exhibited a cell morphology with a larger ratio of the major/minor axis than UT. FACS showed that RE had a higher proportion of CSC-like cells and contained more CD44+CD24- mesenchymal CSCs than ALDH1A3+ epithelial-like CSCs. In a Matrigel invasion assay, UT was more likely to form a three-dimensional cell population, whereas RE exhibited a planar population, higher migration ability, and the up-regulated expression of epithelial-mesenchymal transition-related genes. These results provide insights into the mechanisms by which TNBC cells acquire treatment resistance at the time of recurrence.

6.
RSC Chem Biol ; 3(8): 1076-1084, 2022 Aug 03.
Article in English | MEDLINE | ID: mdl-35975000

ABSTRACT

We investigated the cell penetration of Sp1 zinc finger proteins (Sp1 ZF) and the mechanism via which the total cationic charge and distribution of cationic residues on the protein surface affect intracellular trafficking. Sp1 ZFs showed intrinsic cell membrane permeability. The intracellular transfer of Sp1 ZFs other than 1F3 was dependent on the total cationic charge. Investigation of the effect of cationic residue distribution on intracellular membrane permeability revealed that the cellular uptake of unfolded Zn2+-non-coordinating Ala mutants was lower than that of the wild type. Therefore, the total cationic charge and distribution of cationic residues on the protein played crucial roles in intracellular translocation. Mutational studies revealed that the two-dimensional cation cluster on the protein surface significantly improved their cellular uptake. This study will contribute to the design of artificial cargoes that can efficiently transport target substances into cells.

7.
Bioorg Med Chem Lett ; 72: 128875, 2022 09 15.
Article in English | MEDLINE | ID: mdl-35798239

ABSTRACT

Intracellular delivery of biomacromolecules is challenging as these molecules are taken up by cells and encapsulated into vesicular compartments called endosomes, and the fraction of molecules that are translocated to the cytosol are particularly important to obtain desired biological responses. This study aimed to estimate the cytosolic concentrations of intracellularly delivered peptides and proteins to aid the design of novel and effective biopharmaceutical delivery systems. To this end, we employed the split NanoLuc luciferase system, using the 11-residue HiBiT peptide segment as a probe for the delivered molecules in cells expressing the complementary LgBiT protein segment. The efficacy in cytosolic HiBiT delivery was determined by measuring the resultant luciferase activity when the HiBiT segment delivered into the cytosol forms a complex with LgBiT. Mean cytosolic HiBiT concentration was calculated using cell number and cell volume analysis. L17E and HAad peptides, developed in our laboratory for intracellular protein delivery, yielded approximately 6-fold cellular HiBiT concentrations than that obtained in their absence.


Subject(s)
Endosomes , Peptides , Cations/metabolism , Cytosol/metabolism , Endosomes/metabolism , Luciferases/metabolism , Peptides/chemistry
8.
Chem Asian J ; 17(16): e202200367, 2022 Aug 15.
Article in English | MEDLINE | ID: mdl-35750635

ABSTRACT

N6 -Methyladenosine (m6 A) is the most common internal RNA modification in the consensus sequence of 5'-RRACH-3'. The methyl mark is added by writer proteins (METTL3/METTL14 metyltransferase complex) and removed by eraser proteins (m6 A demethylases; FTO and ALKBH5). Recognition of this methyl mark by m6 A reader proteins leads to changes in RNA metabolism. How the writer and eraser proteins determine their targets is not well-understood, despite the importance of this information in understanding the regulatory mechanisms and physiological roles of m6 A. However, approaches for targeted manipulation of the methylation state at specific sites are being developed. In this review, I summarize the recent findings on the mechanisms of target identification of m6 A regulatory proteins, as well as recent approaches for targeted m6 A modifications.


Subject(s)
Adenosine , RNA , Methylation , RNA/metabolism
10.
Sci Rep ; 12(1): 6322, 2022 04 15.
Article in English | MEDLINE | ID: mdl-35428847

ABSTRACT

Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools.


Subject(s)
Carcinoma, Squamous Cell , Epidermal Growth Factor , Ion Channels , Pyrazines , Thiadiazoles , Biological Transport , Calcium/metabolism , Cell Line, Tumor , Epidermal Growth Factor/pharmacology , Humans , Ion Channels/agonists , Ion Channels/metabolism , Pinocytosis/drug effects
11.
Bioorg Med Chem ; 61: 116728, 2022 05 01.
Article in English | MEDLINE | ID: mdl-35395514

ABSTRACT

We have developed a series of attenuated cationic amphiphilic lytic (ACAL) peptides that can efficiently bring immunoglobulin G (IgG) and other functional proteins into cells. Delivery is generally achieved through the coadministration of ACAL peptides with cargo proteins. However, conjugation of ACAL peptides with cargos may be a promising approach for in vivo application to link in vivo outcomes of ACAL peptides and cargos. This study describes the creation of a new cell-permeable ACAL peptide, L17ER4. L17E is an optimized prototype of ACAL peptides previously developed in our laboratory for efficient delivery of IgGs into cells. Delivery was improved by functionalizing L17E with a tetra-arginine (R4) tag. Compared to the use of R8, a representative cell-penetrating peptide with high intracellular delivery efficacy, conjugation with L17ER4 afforded approximately four-fold higher cellular uptake of model small-molecule cargos (fluorescein isothiocyanate and HiBiT peptide). L17ER4 was also able to deliver proteins to cells. Fused with L17ER4, Cre recombinase was delivered into cells. Intracerebroventricular injection of Cre-L17ER4 into green red reporter mice, R26GRR, led to significant in vivo gene recombination in ependymal cells, suggesting that L17ER4 may be used as a cell-penetrating peptide for delivering protein therapeutics into cells in vivo.


Subject(s)
Cell-Penetrating Peptides , Animals , Cations , Cell-Penetrating Peptides/chemistry , Mice
12.
Bioconjug Chem ; 33(2): 311-320, 2022 02 16.
Article in English | MEDLINE | ID: mdl-35049280

ABSTRACT

Nanocarriers that deliver functional proteins to cell interiors are an attractive platform for the intracellular delivery of intact proteins without further modification, with in vivo compatibility. Development of efficient methods for cargo protein encapsulation and release in recipient cell cytosol is needed. Herein, we assess the feasibility of the abovementioned requirements using a protein nanocage (artificial nanocage) without compromising the structure and functions of the original protein and allowing for design flexibility of the surfaces and interiors. The protein nanocage formed via the self-assembly of the ß-annulus peptide (24-amino acid peptide) in water was used as a model framework. The nitrilotriacetic acid moiety was displayed on the nanocage lumen for effective encapsulation of hexahistidine-tagged proteins in the presence of Ni2+, and the amphiphilic cationic lytic peptide HAad was displayed on a nanocage surface to attain cell permeability. Successful intracellular delivery of cargo proteins and targeting of cytosolic proteins by a nanobody were achieved, indicating the validity of the approach employed in this study.


Subject(s)
Peptides , Proteins , Cytosol/metabolism , Nitrilotriacetic Acid , Peptides/chemistry , Proteins/chemistry
13.
Mol Pharm ; 19(2): 558-567, 2022 02 07.
Article in English | MEDLINE | ID: mdl-34958576

ABSTRACT

Stapled peptides are a promising class of conformationally restricted peptides for modulating protein-protein interactions (PPIs). However, the low membrane permeability of these peptides is an obstacle to their therapeutic applications. It is common that only a few hydrophobic amino acid residues are mandatory for stapled peptides to bind to their target proteins. Hoping to create a novel class of membrane-permeable PPI inhibitors, the phenylalanine, tryptophan, and leucine residues that play a critical role in inhibiting the p53-HDM2 interaction were grafted into the framework of CADY2─a cell-penetrating peptide (CPP) having a helical propensity. Two analogues (CADY-3FWL and CADY-10FWL) induced apoptotic cell death but lacked the intended HDM2 interaction. Pull-down experiments followed by proteomic analysis led to the elucidation of nesprin-2 as a candidate binding target. Nesprin-2 is considered to play a role in the nuclear translocation of ß-catenin upon activation of the Wnt signaling pathway, which leads to the expression of antiapoptosis proteins and cell survival. Cells treated with the two analogues showed decreased nuclear localization of ß-catenin and reduced mRNA expression of related antiapoptotic proteins. These data suggest inhibition of ß-catenin nuclear translocation as a possible mode of action of the described cell-penetrating stapled peptides.


Subject(s)
Cell-Penetrating Peptides , Amino Acids , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Hydrophobic and Hydrophilic Interactions , Proteomics , Wnt Signaling Pathway
14.
Nucleic Acids Res ; 50(1): 449-457, 2022 01 11.
Article in English | MEDLINE | ID: mdl-34908152

ABSTRACT

N6-methyladenosine (m6A) is an important epitranscriptomic chemical modification that is mainly catalyzed by the METTL3/METTL14 RNA methyltransferase heterodimer. Although m6A is found at the consensus sequence of 5'-DRACH-3' in various transcripts, the mechanism by which METTL3/METTL14 determines its target is unclear. This study aimed to clarify the RNA binding property of METTL3/METTL14. We found that the methyltransferase heterodimer itself has a binding preference for RNA G-quadruplex (rG4) structures, which are non-canonical four-stranded structures formed by G-rich sequences, via the METTL14 RGG repeats. Additionally, the methyltransferase heterodimer selectively methylated adenosines close to the rG4 sequences. These results suggest a possible process for direct recruitment of METTL3/METTL14 to specific methylation sites, especially near the G4-forming regions. This study is the first to report the RNA binding preference of the m6A writer complex for the rG4 structure and provides insights into the role of rG4 in epitranscriptomic regulation.


Subject(s)
Adenosine/analogs & derivatives , G-Quadruplexes , Methyltransferases/metabolism , RNA/metabolism , Adenosine/metabolism , Humans
15.
Sci Rep ; 11(1): 19896, 2021 10 06.
Article in English | MEDLINE | ID: mdl-34615928

ABSTRACT

Although proteins have attractive features as biopharmaceuticals, the difficulty in delivering them into the cell interior limits their applicability. Lipid nanoparticles (LNPs) are a promising class of delivery vehicles. When designing a protein delivery system based on LNPs, the major challenges include: (i) formulation of LNPs with defined particle sizes and dispersity, (ii) efficient encapsulation of cargo proteins into LNPs, and (iii) effective cellular uptake and endosomal release into the cytosol. Dioleoylglycerophosphate-diethylenediamine (DOP-DEDA) is a pH-responsive, charge-reversible lipid. The aim of this study was to evaluate the applicability of DOP-DEDA-based LNPs for intracellular protein delivery. Considering the importance of electrostatic interactions in protein encapsulation into LNPs, a negatively charged green fluorescent protein (GFP) analog was successfully encapsulated into DOP-DEDA-based LNPs to yield diameters and polydispersity index of < 200 nm and < 0.2, respectively. Moreover, ~ 80% of the cargo proteins was encapsulated into the LNPs. Cytosolic distribution of fluorescent signals of the protein was observed for up to ~ 90% cells treated with the LNPs, indicating the facilitated endocytic uptake and endosomal escape of the cargo attained using the LNP system.


Subject(s)
Drug Carriers , Hydrogen-Ion Concentration , Liposomes , Nanoparticles , Proteins/administration & dosage , Chemical Phenomena , Cytosol/metabolism , Drug Delivery Systems , Lipids/chemistry , Liposomes/chemistry , Nanoparticles/chemistry , Proteins/chemistry , Recombinant Proteins/administration & dosage , Static Electricity
16.
Angew Chem Int Ed Engl ; 60(36): 19804-19812, 2021 09 01.
Article in English | MEDLINE | ID: mdl-34114295

ABSTRACT

Fc region binding peptide conjugated with attenuated cationic amphiphilic lytic peptide L17E trimer [FcB(L17E)3 ] was designed for immunoglobulin G (IgG) delivery into cells. Particle-like liquid droplets were generated by mixing Alexa Fluor 488 labeled IgG (Alexa488-IgG) with FcB(L17E)3 . Droplet contact with the cellular membrane led to spontaneous influx and distribution of Alexa488-IgG throughout cells in serum containing medium. Involvement of cellular machinery accompanied by actin polymerization and membrane ruffling was suggested for the translocation. Alexa488-IgG negative charges were crucial in liquid droplet formation with positively charged FcB(L17E)3 . Binding of IgG to FcB(L17E)3 may not be necessary. Successful intracellular delivery of Alexa Fluor 594-labeled anti-nuclear pore complex antibody and anti-mCherry-nanobody tagged with supernegatively charged green fluorescence protein allowed binding to cellular targets in the presence of FcB(L17E)3 .


Subject(s)
Cytosol/metabolism , Immunoglobulin G/metabolism , Peptides/metabolism , Surface-Active Agents/metabolism , Cations/chemistry , Cations/metabolism , Cytosol/chemistry , Humans , Immunoglobulin G/chemistry , Molecular Structure , Particle Size , Peptides/chemistry , Surface-Active Agents/chemistry
17.
Bioorg Med Chem Lett ; 40: 127925, 2021 05 15.
Article in English | MEDLINE | ID: mdl-33705909

ABSTRACT

Our research group has been studying the design of intracellular delivery peptides based on cationic lytic peptides. By placing negatively charged amino acids on potentially hydrophobic faces of the peptides, membrane lytic activity is attenuated on the cell surface, whereas it recovers in endosomes, enabling cytosolic delivery of proteins including antibodies. These lytic peptides generally contain multiple lysines, facilitating cell surface interaction and membrane perturbation. This study evaluated the effect of lysine-to-homoarginine substitution using HAad as a model delivery peptide. The resulting peptide had a comparable or better delivery efficacy for Cre recombinase, antibodies, and the Cas9/sgRNA complex with one-quarter of the concentration of HAad, implying that a subtle structural difference can affect delivery activity.


Subject(s)
Drug Carriers/chemistry , Endosomes/metabolism , Homoarginine/chemistry , Intracellular Membranes/metabolism , Peptides/chemistry , Amino Acid Sequence , CRISPR-Associated Protein 9/pharmacology , Dextrans/chemistry , Drug Carriers/toxicity , Drug Liberation , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Immunoglobulin G/pharmacology , Integrases/pharmacology , Liposomes/chemistry , Peptides/toxicity , RNA, Guide, Kinetoplastida/pharmacology , Sulfonic Acids/chemistry
18.
Angew Chem Int Ed Engl ; 60(21): 11928-11936, 2021 05 17.
Article in English | MEDLINE | ID: mdl-33629482

ABSTRACT

Macropinocytosis is a ubiquitous cellular uptake mechanism of peptide-based intracellular delivery. This entry pathway shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. In this work, we obtained the 8-residue analogue P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A and the membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins.


Subject(s)
Peptides/metabolism , Pinocytosis/drug effects , Protein Transport/drug effects , Amino Acid Sequence , Cysteine/chemistry , Cysteine/metabolism , Disulfides/metabolism , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Integrases/metabolism , Peptides/chemistry
19.
Chem Commun (Camb) ; 56(76): 11203-11206, 2020 Sep 24.
Article in English | MEDLINE | ID: mdl-32969426

ABSTRACT

Essential components of the human circadian clock, BMAL1 and CLOCK, which are intrinsically disordered transcription factors, were expressed and subjected to a fluorescent in vitro binding assay using an E-box DNA fragment. Screening of a chemical library identified 5,8-quinoxalinedione (1), which was found to inhibit binding of the heterodimer BMAL1/CLOCK to E-box at low micromolar concentrations.


Subject(s)
ARNTL Transcription Factors/antagonists & inhibitors , CLOCK Proteins/antagonists & inhibitors , Circadian Clocks , DNA/metabolism , Intrinsically Disordered Proteins/antagonists & inhibitors , Quinoxalines/pharmacology , ARNTL Transcription Factors/chemistry , ARNTL Transcription Factors/metabolism , CLOCK Proteins/chemistry , CLOCK Proteins/metabolism , DNA/chemistry , Dose-Response Relationship, Drug , Humans , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/metabolism , Molecular Structure , Protein Binding/drug effects
20.
Angew Chem Int Ed Engl ; 59(45): 19990-19998, 2020 11 02.
Article in English | MEDLINE | ID: mdl-32557993

ABSTRACT

Endocytic pathways are practical routes for the intracellular delivery of biomacromolecules. Along with this, effective strategies for endosomal cargo release into the cytosol are desired to achieve successful delivery. Focusing on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes, we designed the lipid-sensitive and pH-responsive endosome-lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues, which serve as a safety catch for preferential permeabilization of endosomal membranes over cell membranes, and His-to-Ala substitutions enhance the endosomolytic activity. The ability of HAad to destabilize endosomal membranes was supported by model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells. Cerebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in a mouse model, thus demonstrating potential applicability of HAad in vivo.


Subject(s)
Endosomes/metabolism , Peptides/metabolism , Cell Membrane/metabolism , Endocytosis , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions
SELECTION OF CITATIONS
SEARCH DETAIL
...