PTPs: Degrading the Undruggable Barrios, Amy M
Journal of medicinal chemistry,
07/2020, Letnik:
63, Številka:
14
Journal Article
Recenzirano
By using a proteolysis-targeting chimera (PROTAC) strategy that couples an allosteric, reversible PTP inhibitor with an E3 ligase targeting ligand through a well-designed linker, the first molecule ...to successfully inhibit PTP activity through degradation has been developed.
The ability to visualize enzyme activity in a cell, tissue, or living organism can greatly enhance our understanding of the biological roles of that enzyme. While many aspects of cellular signaling ...are controlled by reversible protein phosphorylation, our understanding of the biological roles of the protein phosphatases involved is limited. Here, we provide an overview of progress toward the development of fluorescent probes that can be used to visualize the activity of protein phosphatases. Significant advances include the development of probes with visible and near-infrared (near-IR) excitation and emission profiles, which provides greater tissue and whole-animal imaging capabilities. In addition, the development of peptide-based probes has provided some selectivity for a phosphatase of interest. Key challenges involve the difficulty of achieving sufficient selectivity for an individual member of a phosphatase enzyme family and the necessity of fully validating the best probes before they can be adopted widely.
Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was ...investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4–12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.
Buzzing with activity: A hydrogen sulfide selective fluorogenic probe, 7‐azido‐4‐methylcoumarin (AzMC), serves as a highly sensitive assay for cystathionine β‐synthase activity, and is suitable for ...the high‐throughput discovery of novel enzyme inhibitors.
Buzzing with activity: A hydrogen sulfide selective fluorogenic probe, 7-azido-4-methylcoumarin (AzMC), serves as a highly sensitive assay for cystathionine beta-synthase activity, and is suitable ...for the high-throughput discovery of novel enzyme inhibitors.
Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to ...protozoan infections worldwide(1), resulting in ~70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole(2), which has adverse effects(3), and potential resistance of E. histolytica to the drug is an increasing concern(4,5). To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Land application of swine manure slurry is a common practice to supplement nutrients to soil for crop production. This practice can introduce antibiotic residues and antibiotic resistance genes ...(ARGs) into the environment. Field testing is critical in identifying manure management practices effective in minimizing the environmental impacts of manure-borne antibiotic and ARGs. The objective of this study was to determine how the timing of swine manure application relative to rainfall events impacts the fate and transport of antibiotics and ARGs in surface runoff and manure-amended soil. Swine manure slurry was either broadcast or injected on test plots in the field. A set of three 30-min simulated rainfall events, 24 h apart, were initiated on manured plots 1 day, 1 week, 2 weeks, or 3 weeks after the manure application. Results showed that an interval longer than 2 weeks between application and rainfall often significantly reduced the levels of antibiotics and ARGs tested in runoff with the exception of tet(X). For soil samples from broadcast plots, concentrations of two of the three antibiotics tested (lincomycin and tiamulin) decreased substantially in the first two weeks after manure application. In contrast, concentrations of most of the ARGs tested (tet(Q), tet(X), and erm(A)) in soil did not change significantly during the test period. Information obtained from the study can be beneficial in designing manure management practices and estimating the environmental loading of antibiotics and ARGs resulting from manure application.
Display omitted
•Following manure application two weeks are needed to yield low ARG levels in runoff.•Two weeks are also needed to yield low antibiotic levels in runoff.•ARG levels in soil hardly changed in the four weeks following manure application.•Antibiotic levels in soil dropped in the first two weeks after manure application.
Post-translational modifications (PTMs) are invaluable regulatory tools for the control of catalytic functionality, protein-protein interactions, and signaling pathways. Historically, the study of ...phosphorylation as a PTM has been focused on serine, threonine, and tyrosine residues. In contrast, the significance of mammalian histidine phosphorylation remains largely unexplored. This gap in knowledge regarding the molecular basis for histidine phosphorylation as a regulatory agent exists in part because of the relative instability of phosphorylated histidine as compared with phosphorylated serine, threonine and tyrosine. However, the unique metal binding abilities of histidine make it one of the most common metal coordinating ligands in nature, and it is interesting to consider how phosphorylation would change the metal coordinating ability of histidine, and consequently, the properties of the phosphorylated metalloprotein. In this review, we examine eleven metalloproteins that have been shown to undergo reversible histidine phosphorylation at or near their metal binding sites. These proteins are described with respect to their biological activity and structure, with a particular emphasis on how phosphohistidine may tune the primary coordination sphere and protein conformation. Furthermore, several common methods, challenges, and limitations of studying sensitive, high affinity metalloproteins are discussed.
Display omitted
•Histidine phosphorylation is a key posttranslational modification in biology.•Histidine phosphorylation has been found in several metalloprotein metal-binding sites.•Possible impacts of histidine phosphorylation in metalloproteins are discussed.
Cyclic peptides hold great potential as therapeutic agents and research tools, but their broad application has been limited by poor membrane permeability. Here, we report a potentially general ...approach for intracellular delivery of cyclic peptides. Short peptide motifs rich in arginine and hydrophobic residues (e.g., FΦRRRR, where Φ is l-2-naphthylalanine), when embedded into small- to medium-sized cyclic peptides (7–13 amino acids), bound to the plasma membrane of mammalian cultured cells and were subsequently internalized by the cells. Confocal microscopy and a newly developed peptide internalization assay demonstrated that cyclic peptides containing these transporter motifs were translocated into the cytoplasm and nucleus at efficiencies 2–5-fold higher than that of nonaarginine (R9). Furthermore, incorporation of the FΦRRRR motif into a cyclic peptide containing a phosphocoumaryl aminopropionic acid (pCAP) residue generated a cell permeable, fluorogenic probe for detecting intracellular protein tyrosine phosphatase activities.
Although the importance of protein histidine phosphorylation in mammals has been a subject of increasing interest, few chemical probes are available for monitoring and manipulating PHP activity. ...Here, we present an optimized and validated protocol for assaying the activity of PHPT1 using the fluorogenic substrate DiFMUP. The kinetic parameters of our optimized assay are significantly improved as compared with other PHPT1 assays in the literature, with a k cat of 0.39 ± 0.02 s–1, a K m of 220 ± 30 μM, and a k cat/K m of 1800 ± 200 M–1 s–1. In addition, the assay is significantly more sensitive as a result of using a fluorescent probe, requiring only 109 nM enzyme as compared with 2.4 μM as required by previously published assays. In the process of assay optimization, we discovered that PHPT1 is sensitive to a reducing environment and inhibited by transition-metal ions, with one apparent Cu(II) binding site with IC50 value of 500 ± 20 μM and two apparent Zn(II) binding sites with IC50 values of 25 ± 1 and 490 ± 20 μM.
PDF
