A multi-enzyme cascade for the cell-free, one-pot synthesis of CDP-glycerol has been developed. Through a design-of-experiments approach, the yield of the cascade was increased from 10 to 89 % with respect to cytidine as a substrate. The final product titer after a batch time of 24 h was 31.2 mM CDP-glycerol.

Abstract

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in Escherichia coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is regenerated in situ from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at the laboratory scale, and the products were analyzed by high-performance anion-exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis had been confirmed, a design-of-experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl₂ concentration). Overall, a substrate conversion of 89 % was achieved with respect to the substrate cytidine.

The receptor activated by GLP-1 is a target for drugs that treat type 2 diabetes and obesity. Since glycine has a low α-helix propensity, and Gly22 of GLP-1 does not contact the receptor, we replaced Gly22 with residues that have a higher or lower helix propensity. Most substitutions had little effect on receptor affinity or activation and raised the possibility that the receptor-bound state of agonist peptides encompasses a broader conformational envelope.

Abstract

Agonists of the glucagon-like peptide-1 receptor (GLP-1R) are used to treat diabetes and obesity. Cryo-EM structures indicate that GLP-1 is completely α-helical when bound to the GLP-1R. The mature form of this hormone, GLP-1(7-36), contains a glycine residue near the center (Gly22). Since glycine has the second-lowest α-helix propensity among the proteinogenic α-amino acid residues, and Gly22 does not appear to make direct contact with the receptor, we were motivated to explore the impact on agonist activity of altering the α-helix propensity at this position. We examined GLP-1 analogues in which Gly22 was replaced with L-Ala, D-Ala, or β-amino acid residues with varying helix propensities. The results suggest that the receptor is reasonably tolerant of variations in helix propensity, and that the functional receptor-agonist complex may comprise a conformational spectrum rather than a single fixed structure.

A bifunctional HaloPROTAC ligand was developed to recruit the CRBN E3 ubiquitin ligase to degrade target proteins fused with a HaloTag in the cell. The CRBN-recruiting HaloPROTAC can expand the use of the HaloPROTAC system for screening the degradation targets for therapeutic discovery.

Abstract

Target validation is key to the development of protein degrading molecules such as proteolysis-targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin-proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3-catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN-pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP-HaloTag fusion with higher activities than VHL-engaging HaloPROTACs at sub-micromolar concentrations of the compound. The CRBN-engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.

Car crashes on highways require responses like rescuing victims, deviating traffic, towing away damaged vehicles, and sometimes even blocking a road. Similarly, during translation, ribosomes can collide into each other with potentially dangerous outcomes for the cell. In response to ribosome collisions, cells mount a multi-pronged response, activating several molecular ′squads′, symbolized by the Highway Police badge. These include degradation pathways for faulty mRNAs, peptide products and sometimes ribosomal constituents, regulation of translation, and stress as well as immune responses. More information can be found in the Review by M.-L. Winz et al.

We have synthesized perfluoroalkyl-containing amino acids (R_F-AA) and demonstrated that tripeptides composed of the R_F-AA have high cellular uptake efficiency. Furthermore, the absolute configuration of R_F-AA affected the uptake efficiency due to the change in its ability to form nanoparticles. We anticipate that these findings provide the foundation for the development of efficient cell-penetrating peptide (CPP). The cover picture shows tripeptides with perfluoroalkyl groups labeled with hydrophilic dyes that form particles in water and are efficiently taken up into cells. More information can be found in the Research Article by K. Aikawa et al.

Shrooms going terpenoid: The sesquiterpene synthase CubA of the “magic mushroom” Psilocybe cubensis was identified as a multi-functional enzyme producing cubebol, β-copaene, and various other mono-, bi-, and tricyclic sesquiterpenes or -terpenoids. As related enzymes are encoded in numerous Psilocybe species, our results provide further insight into the true metabolic diversity of this genus.

Abstract

Psilocybe “magic mushrooms” are chemically well understood for their psychotropic tryptamines. However, the diversity of their other specialized metabolites, in particular terpenoids, has largely remained an open question. Yet, knowledge on the natural product background is critical to understand if other compounds modulate the psychotropic pharmacological effects. CubA, the single clade II sesquiterpene synthase of P. cubensis, was heterologously produced in Escherichia coli and characterized in vitro, complemented by in vivo product formation assays in Aspergillus niger as a heterologous host. Extensive GC-MS analyses proved a function as multi-product synthase and, depending on the reaction conditions, cubebol, β-copaene, δ-cadinene, and germacrene D were detected as the major products of CubA. In addition, mature P. cubensis carpophores were analysed chromatographically which led to the detection of β-copaene and δ-cadinene. Enzymes closely related to CubA are encoded in the genomes of various Psilocybe species. Therefore, our results provide insight into the metabolic capacity of the entire genus.

A natural deep eutectic solvent, CAGE, derived from choline and geranic acid, perturb the metabolome, proteome, and transcriptome of human cell line. Specifically, CAGE upregulated 4-hydroxyphenyllactic acid and indole-3-lactic acid to activate the aryl hydrocarbon receptor, leading to the expression of IL1A, IL1B, CYP1A1, CYP1B1, ALDH31, NQO1. CAGE also attenuate the intracellular levels of glutamine.

Abstract

Natural deep eutectic solvents (NADESs) are emerging sustainable alternatives to conventional organic solvents. Beyond their role as laboratory solvents, NADESs are increasingly explored in drug delivery and as therapeutics. Their increasing applications notwithstanding, our understanding of how they interact with biomolecules at multiple levels - metabolome, proteome, and transcriptome - within human cell remain poor. Here, we deploy integrated metabolomics, proteomics, and transcriptomics to probe how NADESs perturb the molecular landscape of human cells. In a human cell line model, we found that an archetypal NADES derived from choline and geranic acid (CAGE) significantly altered the metabolome, proteome, and transcriptome. CAGE upregulated indole-3-lactic acid and 4-hydroxyphenyllactic acid levels, resulting in ligand-independent activation of aryl hydrocarbon receptor to signal the transcription of genes with implications for inflammation, immunomodulation, cell development, and chemical detoxification. Further, treating the cell line with CAGE downregulated glutamine biosynthesis, a nutrient rapidly proliferating cancer cells require. CAGE's ability to attenuate glutamine levels is potentially relevant for cancer treatment. These findings suggest that NADESs, even when derived from natural components like choline, can indirectly modulate cell biology at multiple levels, expanding their applications beyond chemistry to biomedicine and biotechnology.

In a screening of over 1000 FKBP-inhibitors [4.3.1]-bicyclic sulfonamides turned out to be the preferred binding scaffold for LpMip, a virulence factor of Legionella pneumophila. Although selected [4.3.1]-bicyclic sulfonamides showed anti-infective properties, LpMip was ruled out as sole target, with the results suggesting another FKBP is responsible for the observed effects.

Abstract

Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.

A novel drug-delivery system with a dual response to GSH and enzymes is based on the newly designed PEG-functionalized pillararene for efficient and rapid drug release, and cancer therapy.

Abstract

The construction of a smart drug-delivery system based on amphiphilic pillararenes with multiple responsiveness properties has become an important way to improve the efficacy of tumor chemotherapy. Here, a new PEG-functionalized pillararene (EtP5-SS-PEG) containing disulfide and amido bonds was designed and synthesized, which has been used to construct a novel supramolecular nanocarrier through a host-guest interaction with a perylene diimide derivative (PDI-2NH₄) and their supramolecular self-assembly. This nanocarrier showed good drug loading capability, and dual stimulus responsiveness to enzyme and GSH (glutathione). After loading of doxorubicin (DOX), the prepared nanodrugs displayed efficient DOX release and outstanding cancer theranostics ability.

Garcinolic acid is a topologically complex natural product derived from Garcinia hanburyi. It engages the KIX domain in the master coactivator CBP/p300 selectively over other closely related motifs and in doing so disrupts CBP/p300 KIX protein-protein interactions. In the cellular context this enables downregulation of transcriptional circuits essential for cMyb-dependent leukemias.

Abstract

Natural products are often uniquely suited to modulate protein-protein interactions (PPIs) due to their architectural and functional group complexity relative to synthetic molecules. Here we demonstrate that the natural product garcinolic acid allosterically blocks the CBP/p300 KIX PPI network and displays excellent selectivity over related GACKIX motifs. It does so via a strong interaction (K _D 1 μM) with a non-canonical binding site containing a structurally dynamic loop in CBP/p300 KIX. Garcinolic acid engages full-length CBP in the context of the proteome and in doing so effectively inhibits KIX-dependent transcription in a leukemia model. As the most potent small-molecule KIX inhibitor yet reported, garcinolic acid represents an important step forward in the therapeutic targeting of CBP/p300.