ACS Chemical Biology最新文献

Cross-link containing products from ribosomally synthesized and post-translationally modified peptides (RiPPs) are generated by radical SAM enzymes (rSAM). Here, we bioinformatically expanded rSAM enzymes based on the known families StrB, NxxcB, WgkB, RrrB, TqqB and GggB. Through in vivo functional studies in E. coli, the newly identified enzyme WprB from Xenorhabdus sp. psl was found to catalyze formation of a cross-link between Trp-C5 and Arg-Cγ at three WPR motifs on the precursor peptide WprA. This represents the first report of this type of cross-link by rSAM enzymes.

Proteasomes catalyze protein degradation in cells and play an integral role in cellular homeostasis. Its activity decreases with age alongside the load of defective proteins, resulting from mutations or oxidative stress-induced damage. Such proteins are prone to aggregation and, if not efficiently degraded, can form toxic oligomers and amyloid plaques. Developing an effective way to activate the proteasome could prevent such pathologies. Designing activators is not easy because they do not bind in the active site, which is well-defined and highly conserved, but away from it. The structures of proteasome complexes with natural activators can help here, but these are large proteins, some even multimeric, whose activity is difficult to replace with a small-molecule compound. Nevertheless, the use of fragments of such proteins makes it possible to accumulate knowledge about the relevance of various structural elements for efficient and selective activation. Here, we presented peptidic activators of the 20S proteasome, which were designed based on both the C-terminal sequence of the yeast proteasome activator, Blm10 protein, and the interactions predicted by molecular modeling. These Blm analogs were able to stimulate human 20S proteasome to more efficiently degrade both small fluorogenic substrates and proteins. The best activators also demonstrated their efficacy in cell lysates. X-ray crystallography indicated that an effective modulator can bind to several sites on the surface of the proteasome without causing permanent structural changes in its immediate vicinity but affecting the active sites.

Ligand-conjugated small interfering RNAs (siRNAs) have emerged as a powerful approach to developing nucleic acid-based medicines. To achieve efficient mRNA knockdown, it is important to select targeting receptors with high expression and ligands that exhibit rapid internalization. However, the key characteristics of ligand-receptor sets involved in the postinternalization process remain largely unclear. In this study, we investigated the effect of ligand-receptor binding dissociation under low pH conditions, known as a postendocytic environment. Specifically, we chemically synthesized several modified epidermal growth factor (EGF) ligands that showed a variety of binding activities to the EGF receptor (EGFR) at low pH. Among these modified ligands, the siRNA conjugate with chemically synthesized EGF H10Y/H16Y, which is a less pH-responsive variant, exhibited reduced internalization and mRNA knockdown activity at high concentrations in EGFR-expressing cells. Additionally, we explored the use of antibody-related molecules (anti-EGFR IgG and Fab) as targeting moieties for siRNA conjugates. The anti-EGFR Fab-siRNA, which showed dissociation of EGF under low pH conditions, demonstrated stronger internalization and mRNA knockdown activity compared to the anti-EGFR IgG-siRNA, which strongly binds EGF at low pH. These data emphasize the importance of intracellular ligand-receptor dissociation and provide insights for future advancements in the field.

We report silyl-lipid derivatives of N-acyl l-homoserine lactones (AHLs) that have nanomolar activities in LuxR-type quorum sensing receptors in Gram-negative bacterial pathogens. A collection of silyl-lipid AHLs were designed and synthesized to represent three general structural classes based on native AHL signals and synthetic LuxR-type receptor modulators. The synthetic routes feature straightforward hydrosilylation and aryl silylation reactions to access silyl-lipid groups that are not readily accessible in analogous all-carbon chemistry. Of the 17 compounds evaluated, eight silyl-lipid AHLs were identified with either nanomolar agonistic or submicromolar antagonistic activities in the LasR receptor from the common pathogen Pseudomonas aeruginosa using E. coli reporter gene assays. Several silyl-lipid AHL agonists retained high activities in LasR in a native P. aeruginosa reporter system and also were active in another related LuxR-type receptor, EsaR from Pantoea stewartii. Light scattering and computational experiments indicate that the silyl-lipid group can alter the aggregation capabilities and lipophilicities of AHLs relative to native all-carbon tails, engendering larger aggregate formation in water and higher lipophilicities on average. These properties, along with their strong activity profiles in LuxR-type receptors, suggest silyl-lipid AHLs could provide value as chemical probes to study the mechanisms of quorum sensing in Gram-negative bacteria and the roles of signal lipophilicity in this chemical communication process.

Acyclic and cyclic N-acyl O-aminophenol prodrugs of duocarmycin analogues were reported as members of a unique class of reductively cleaved prodrugs that map seamlessly onto the duocarmycin family of natural products. Although these prodrugs were explored with the expectations that they may be cleaved selectively within hypoxic tumor environments that have intrinsically higher concentrations of reducing nucleophiles, the remarkable stability of some such prodrugs suggests another mechanism of free drug release is operative. The prototype of such chemically unreactive N-acyl O-aminophenol prodrugs is 1, which proved remarkably efficacious in vivo in vertebrate tumor models; was found to lack the toxicity that is characteristic of traditional chemotherapeutic drugs as well as the free drugs in the class (e.g., myelosuppression); and displayed a preferential site (intracellular), a slow and sustained rate, and a potentially unique mechanism of free drug release. Herein, we detail studies that provide insights into this stereoselective mechanism of free drug release. Combined, the results of the studies are consistent with an exclusive protein-mediated (enantio)selective activation and free drug release from prodrug 1 by N-O bond cleavage preferentially in cancer cell lines versus cultured normal human cell lines effected by a cytosolic cysteine-based enzyme and suggest that the activating protein is one that is selectively expressed, upregulated, or preferentially activated in cancer cell lines, potentially constituting a new oncology targeted precision therapy.

Monoclonal antibodies (mAb) produced in 1,4-mannosyl-glycoprotein 4-N-acetylglucosaminyltransferase (MGAT3) overexpressing cell lines have superior in vitro and in vivo activities. The N-glycan of the Fc-region of these mAbs have increased levels of bisecting N-acetylglucosamine (GlcNAc) and reduced core-fucosylation. Although a reduction in core-fucosylation will improve FcγRIIIa binding and antibody-dependent cellular cytotoxicity (ADCC) activity, the influence of bisecting GlcNAc on these activities has been difficult to probe. Here, we describe the preparation of a unique series of homogeneous glycoforms of trastuzumab (Herceptin) with and without core-fucose and with and without bisecting GlcNAc and examine binding to a comprehensive panel of Fcγ receptors. The glycoforms of trastuzumab were prepared by treatment with wild-type Endo-S2 to cleave the chitobiose core of the N-glycan to leave GlcNAc-Fuc that was exposed to an α-fucosidase to provide trastuzumab-GlcNAc. Glycan oxazolines with and without bisecting GlcNAc were prepared by enzymatic remodeling of a sialoglycopeptide isolated from egg yolk powder, which were employed in transglycosylations with trastuzumab-GlcNAc and trastuzumab-GlcNAc-Fuc catalyzed by Endo-S2 D184M resulting in well-defined glycoforms. As expected, core-fucosylation had a major effect on FcγRIIIa binding, which was not influenced by the presence of bisecting GlcNAc. It was found that an A2-glycan (GlcNAc₂Man₃GlcNAc₂) modified by bisecting GlcNAc cannot be core-fucosylated by FUT8. Thus, bisecting GlcNAc has only an indirect influence on FcγRIIIa binding and subsequent ADCC activity by inhibiting core-fucosylation. The results described here provide an understanding of the properties of therapeutic monoclonal antibodies.

Cellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative to protein-based RNA labeling approaches. Aptamer-based systems are genetically encodable and cover the entire visible spectrum. However, the inherently temporary nature of the noncovalent aptamer–fluorogen interaction limits the utility of these systems in that imaging does not withstand dye washout, and dye dissociation can compromise RNA tracking. We propose that these limitations can be averted through covalent RNA labeling. Here, we describe a photoaffinity approach in which the aptamer ligand is functionalized with a photoactivatable diazirine reactive group such that irradiation with UV light results in covalent attachment to the RNA of interest. In addition to the robustness of the covalent linkage, this approach benefits from the ability to achieve spatiotemporal control over RNA labeling. To demonstrate this approach, we incorporated a photoaffinity linker into malachite green and fused a single copy of the malachite green aptamer to a Cajal body-associated small nuclear RNA of interest as well as a cytoplasmic mRNA. We observed improved sensitivity for live cell imaging of the target RNA upon UV irradiation and demonstrated visualization of RNA dynamics over a time scale of minutes. The covalent attachment uniquely enables these time-resolved experiments, whereas in noncovalent approaches, the dye molecule can be transferred between different RNA molecules, compromising tracking. We envision future applications of this method for a wide range of investigations into the cellular localization, dynamics, and protein-binding properties of cellular RNAs.

Flavonoids such as sterubin and fisetin─and derivatives thereof─show strong neuroprotective effects in vitro as well as in vivo, combined with negligible toxicity and can therefore be considered novel treatment options for neurodegenerative diseases such as Alzheimer’s disease. However, their subcellular locations responsible for neuroprotection and exact modes of action still remain unclear. Here, we present chemical probes based on both flavonoids sterubin and fisetin that were utilized in fluorescence microscopy and click-correlative light and electron microscopy to detect and visualize the localization of specific intracellular targets. We successfully adapted the workflow of correlative light and electron microscopy to a click-chemistry-based approach in a murine hippocampal cell line (HT22) on ultrathin resin sections making visualization of a small molecule for the first time possible in this setup. Utilizing this newly adapted technique, we could demonstrate that sterubin and fisetin show specific enrichment in the endoplasmic reticulum.

We report silyl-lipid derivatives of N-acyl l-homoserine lactones (AHLs) that have nanomolar activities in LuxR-type quorum sensing receptors in Gram-negative bacterial pathogens. A collection of silyl-lipid AHLs were designed and synthesized to represent three general structural classes based on native AHL signals and synthetic LuxR-type receptor modulators. The synthetic routes feature straightforward hydrosilylation and aryl silylation reactions to access silyl-lipid groups that are not readily accessible in analogous all-carbon chemistry. Of the 17 compounds evaluated, eight silyl-lipid AHLs were identified with either nanomolar agonistic or submicromolar antagonistic activities in the LasR receptor from the common pathogen Pseudomonas aeruginosa using E. coli reporter gene assays. Several silyl-lipid AHL agonists retained high activities in LasR in a native P. aeruginosa reporter system and also were active in another related LuxR-type receptor, EsaR from Pantoea stewartii. Light scattering and computational experiments indicate that the silyl-lipid group can alter the aggregation capabilities and lipophilicities of AHLs relative to native all-carbon tails, engendering larger aggregate formation in water and higher lipophilicities on average. These properties, along with their strong activity profiles in LuxR-type receptors, suggest silyl-lipid AHLs could provide value as chemical probes to study the mechanisms of quorum sensing in Gram-negative bacteria and the roles of signal lipophilicity in this chemical communication process.