Cell surface glycolipids are implicated in the formation of lipid rafts and membrane microdomains, where they interact with protein receptors to mediate a variety of cellular processes such as ...cell-cell recognition, cell adhesion, and membrane signaling. Studies of glycolipid function at the local membrane structures have not been straightforward to date, because the locally clustered structures are labile and their protein binding affinities tend to be weak. While specific glycolipid-binding proteins have been employed as molecular probes for detecting lipid rafts, small peptides may be more suitable for probing glycolipids at the cell surface due to their small size as well as their ease of synthetic preparation and functionalization. Here we report an application of the retro-inverso approach as a rapid method to obtain novel glycolipid-binding D-peptide sequences. We have prepared analogues of two known GM1-binding peptides by replacing L-amino acids with D-amino acids, followed by inverting the sequences and characterized their conformational propensity and glycolipid binding properties. Circular dichroism (CD) spectroscopic analysis indicated that all the peptide sequences interacted with GM1 under a micellar condition. We found, by a microplate-based competitive glycolipid binding assay, that one of the retroinverso D-peptide analogues, peptide 3, also binds GM1 as the parent L-peptide 1. These results suggested that in this glycolipid-peptide interaction, the positioning of the side chain functionalities of the peptide is important, while the peptide backbone polarity is not. Glycolipid binding retroinverso D-peptides should be useful for the design of new peptide-based probes for investigating the biological role of cell surface glycolipids.
Here we explored the reactivity of a set of multivalent electrophiles cofunctionalized with a carbohydrate ligand on gold nanoparticles to achieve efficient affinity labeling for target protein ...analysis. Evaluation of the reactivity and selectivity of the electrophiles against three different cognate binding proteins identified arylsulfonyl fluoride as the most efficient protein‐reactive group in this study. We demonstrated that multivalent arylsulfonyl fluoride probe 4 at 50 nm concentration achieved selective affinity labeling and enrichment of a model protein PNA in cell lysate, which was more effective than photoaffinity probe 1 with arylazide group. Labeling site analysis by LC–MS/MS revealed that the nanoparticle‐immobilized arylsulfonyl fluoride group can target multiple amino acid residues around the ligand binding site of the target proteins. Our study highlights the utility of arylsulfonyl fluoride as a highly effective multivalent affinity label suitable for covalently capturing unknown target proteins.
Chemical probes were prepared by multivalently presenting electrophilic groups and ligands on gold nanoparticles (NPs), and their reactivities as novel affinity labeling probes were explored. An arylsulfonyl fluoride probe at a nm concentration achieved an efficient and selective labeling of model proteins in cell lysate. The NP‐immobilized arylsulfonyl fluoride groups can target multiple amino acid residues around the ligand binding site.
The Golgi apparatus plays an important role in maintaining cell homeostasis by serving as a biosynthetic center for glycans, lipids and post-translationally modified proteins and as a sorting center ...for vesicular transport of proteins to specific destinations. Moreover, it provides a signaling hub that facilitates not only membrane trafficking processes but also cellular response pathways to various types of stresses. Altered signaling at the Golgi apparatus has emerged as a key regulator of tumor growth and survival. Among the small molecules that can specifically perturb or modulate Golgi proteins and organization, natural products with anticancer property have been identified as powerful chemical probes in deciphering Golgi-related pathways and, in particular, recently described Golgi stress response pathways. In this review, we highlight a set of Golgi-targeting natural products that enabled the characterization of the Golgi-mediated signaling events leading to cancer cell death and discuss the potential for selectively exploiting these pathways for the development of novel chemotherapeutic agents.
We have designed and synthesized six different multivalent electrophiles as carbohydrate affinity labeling probes. Evaluation of the reactivity of the electrophiles against peanut agglutinin (PNA) ...and Ricinus communis agglutinin (RCA) showed that p- and m-aryl sulfonyl fluoride are effective protein reactive groups that label carbohydrate binding lectins in a ligand-dependent fashion at a nanomolar probe concentration. Analysis of the selectivity of affinity labeling in the presence of excess BSA as a nonspecific protein indicated that m-arylsulfonyl fluoride is a more selective protein-reactive group, albeit with attenuated reactivity. Further analysis showed that the labeling efficiency of the multivalent electrophilic probes can be improved by employing reaction conditions involving 25 °C instead of typically employed 4 °C. Both isomers of arylsulfonyl fluoride groups together represent promising affinity labels for target identification studies that could serve as more efficient alternatives to photoreactive groups.
Chemical probe-based approaches have proven powerful in recent years in the target identification studies of natural products. OSW-1 is a saponin class of natural products with highly potent and ...selective cytotoxicity against various cancer cell lines. Understanding its mechanism of action is important for the development of anticancer drugs with potentially novel target pathways. This account reviews recent progress in the development of OSW-1 derived probes for exploring the mechanism of its action. The key to the probe development is a judicious choice of functionalization sites and a selective functionalization strategy. The types of probes include fluorescent probes for cellular imaging analysis and affinity probes for target identification analysis.
Yaku’amide B (1b) is a structurally unique tetradecapeptide bearing four β,β-dialkylated α,β-unsaturated amino acid residues. Growth-inhibitory profile of 1b against a panel of 39 human cancer cell ...lines is distinct from those of clinically used anticancer drugs, suggesting a novel mechanism of action. We achieved total syntheses of chemical probes based on 1b and elucidated the cellular target and mode of action of 1b. Fluorescent (3, 4) and biotinylated (5, 6) derivatives of 1b were prepared for cell imaging studies and pull-down assays, respectively. In addition, the unnatural enantiomer of 1b ( ent-1b) and its fluorescent probe ( ent-3) were synthesized for control experiments. Subcellular localization analysis using 3 and 4 showed that 1b selectively accumulates in the mitochondria of MCF-7 human breast cancer cells. Pull-down assays with 6 revealed FoF1-ATP synthase as the major target protein of 1b. Consistent with these findings, biochemical activity assays showed that 1b inhibits ATP production catalyzed by mitochondrial FoF1-ATP synthase. Remarkably, 1b was also found capable of enhancing the ATP hydrolytic activity of FoF1-ATP synthase. On the other hand, ent-1b inhibits ATP synthesis more weakly than does 1b and does not affect ATP hydrolysis, suggesting the stereospecific requirement for the characteristic multimodal functions of 1b. These findings corroborate that 1b causes growth arrest in MCF-7 cells by inhibiting ATP production and enhancing ATP hydrolysis, thereby depleting the cellular ATP pool. This study provides, for the first time, a structural basis for the design and development of anticancer agents exploiting the novel mode of action of 1b.
OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed ...to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.
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•OSW-1 has strong hemolytic and liposomal membrane activity owing to interactions with cholesterol in the membrane.•Cholesterol recognition by OSW-1 is essential for the subsequent membrane permeabilization.•OSW-1 induces membrane permeabilization via pore formation.
A octanuclear heterometallic complex of {Rh2(O2CCH3)4(OH2)}{Rh2(O2CCH3)4(O2CCF3)}{Pt2(acam)2(bpy)2}2(PF6)3 (2), which is aligned as Rh–Rh–Pt–Pt–Pt–Pt–Rh–Rh with metal–metal bonds, is synthesized and ...characterized. The metal–metal to ligand charge transfer around 2.0 eV, which is characteristic for dinuclear platinum complexes with π conjugated ligands, is admixed with three characteristic bands attributed to heterometallic Pt–Rh bonds.
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•1D octanuclear complex with bpy aligned as Rh–Rh–Pt–Pt–Pt–Pt–Rh–Rh is prepared.•The heterometallic octanuclear complex shows MMCLT.•The absorption for MMCLT and the transition between the σ-type orbitals are coupled.
In this study, {Rh2(O2CCH3)4(OH2)}{Rh2(O2CCH3)4(O2CCF3)}{Pt2(acam)2(bpy)2}2(PF6)3 (2, acam = acetamidate, bpy = 2,2ʹ-bipyridine), a one-dimensional (1D) octanuclear complex aligned as Rh–Rh–Pt–Pt–Pt–Pt–Rh–Rh, was successfully synthesized and characterized. Compound 2 contained a tetranuclear platinum complex where each Pt atom was coordinated with bpy as the co-ligand, exhibiting metal–metal to ligand charge transfer (MMLCT) around 2.0 eV. It admixed with three characteristic bands attributed to heterometallic Pt–Rh bonds in the range of 1.3–2.5 eV. This success paves the way for the development of further novel heterometallic one-dimensional chains containing co-ligands that affect the σ-type orbitals of metal–metal bonds.
An ion-channel-forming natural peptide, gramicidin A (1), exhibits potent antimicrobial activity against Gram-positive bacteria, although medical applications are limited to topical use due to its ...mammalian cytotoxicity. We recently reported that the artificial macrocyclic analogue 2 provides a promising starting point for developing new ion-channel-based systemic antibacterial agents because of its low mammalian cytotoxicity compared to that of the parent 1. To dissect the molecular factors involved in the species selectivity of 2, we evaluated the ion transport activities, phospholipid affinities, and conformational properties of 1 and 2 using various compositions of phospholipids. A combination of lipid dot blot assays and circular dichroism (CD) analysis with H+/Na+ exchange assays revealed that the higher H+/Na+ exchange activity of 2 than that of 1 in liposomes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) is attributable to its higher affinity towards the phospholipids than that of 1. Notably, we also discovered that 2 showed weaker H+/Na+ exchange activity in liposomes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE). CD analysis of 2 in liposomes indicated that the weak H+/Na+ exchange activity is induced by disturbance of the ion-conducting β6.3-helical conformation in the POPE-containing lipid bilayer. These results suggest that the POPE-induced attenuation of the ion-conducting activity of 2 contributes to the alleviation of undesirable mammalian cytotoxicity of 2 compared to that of 1.
Multivalent carbohydrate photoaffinity probes were developed based on gold nanoparticles (AuNPs) to provide a streamlined approach toward identification of carbohydrate-binding proteins. By using ...AuNPs as scaffolds, a carbohydrate ligand and a photoreactive group could be readily assembled on a probe in a modular fashion, which greatly accelerated the process of optimizing the probe design. The novel AuNP-based probes serve dual functions by facilitating photoaffinity labeling and by directly enriching the crosslinked proteins by centrifugation. We demonstrated that their ability to enhance the affinity and to stringently remove nonspecific proteins allowed selective photoaffinity labeling and isolation of a low affinity carbohydrate-binding protein in cell lysate.
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