Design and synthesis of bioreductive prodrugs of class I histone deacetylase inhibitors and their biological evaluation in virally transfected acute myeloid leukemia cells.

Although histone deacetylase (HDAC) inhibitors show promise in treating various types of hematologic malignancies, they have some limitations, including poor pharmacokinetics and off-target side effects. Prodrug design has shown promise as an approach to improve pharmacokinetic properties and to improve target tissue specificity. In this work, several bioreductive prodrugs for class I HDACs were designed based on known selective HDAC inhibitors. The zinc-binding group of the HDAC inhibitors was masked with various nitroarylmethyl residues to make them substrates of nitroreductase (NTR). The developed prodrugs showed weak HDAC inhibitory activity compared to their parent inhibitors. The prodrugs were tested against wild-type and NTR-transfected THP1 cells. Cellular assays showed that both 2-nitroimidazole-based prodrugs 5 and 6 were best activated by the NTR and exhibited potent activity against NTR-THP1 cells. Compound 6 showed the highest cellular activity (GI50 = 77 nM) and exhibited moderate selectivity. Moreover, activation of prodrug 6 by NTR was confirmed by liquid chromatography-mass spectrometry analysis, which showed the release of the parent inhibitor after incubation with Escherichia coli NTR. Thus, compound 6 can be considered a novel prodrug selective for class I HDACs, which could be used as a good starting point for increasing selectivity and for further optimization.

Deep Proteomic Investigation of Metabolic Adaptation in Mycobacteria under Different Growth Conditions.

Understanding the complex mechanisms of mycobacterial pathophysiology and adaptive responses presents challenges that can hinder drug development. However, employing physiologically relevant conditions, such as those found in human macrophages or simulating physiological growth conditions, holds promise for more effective drug screening. A valuable tool in this pursuit is proteomics, which allows for a comprehensive analysis of adaptive responses. In our study, we focused on Mycobacterium smegmatis, a model organism closely related to the pathogenic Mycobacterium tuberculosis, to investigate the impact of various carbon sources on mycobacterial growth. To facilitate this research, we developed a cost-effective, straightforward, and high-quality pipeline for proteome analysis and compared six different carbon source conditions. Additionally, we have created an online tool to present and analyze our data, making it easily accessible to the community. This user-friendly platform allows researchers and interested parties to explore and interpret the results effectively. Our findings shed light on mycobacterial adaptive physiology and present potential targets for drug development, contributing to the fight against tuberculosis.

Increasing the Selectivity of Light-Active Antimicrobial Agents - Or How To Get a Photosensitizer to the Desired Target.

Photosensitizers combine the inherent reactivity of reactive oxygen species with the sophisticated reaction control of light. Through selective targeting, these light-active molecules have the potential to overcome certain limitations in drug discovery. Ongoing advances in the synthesis and evaluation of photosensitizer conjugates with biomolecules such as antibodies, peptides, or small-molecule drugs are leading to increasingly powerful agents for the eradication of a growing number of microbial species. This review article, therefore, summarizes challenges and opportunities in the development of selective photosensitizers and their conjugates described in recent literature. This provides adequate insight for newcomers and those interested in this field.

A Dual-Metal-Catalyzed Sequential Cascade Reaction in an Engineered Protein Cage.

Herein, we describe the creation of an artificial protein cage housing a dual-metal-tagged guest protein that catalyzes a linear, two-step sequential cascade reaction. The guest protein consists of a fusion protein of HaloTag and monomeric rhizavidin. Inside the protein capsid, we established a ruthenium-catalyzed allylcarbamate deprotection reaction followed by a gold-catalyzed ring-closing hydroamination reaction that led to indoles and phenanthridines with an overall yield of up to 66 % in aqueous solutions. Furthermore, we show that the encapsulation stabilizes the metal catalysts against deactivation by air, proteins and cell lysate.

Artificial metalloenzymes in a nutshell: the quartet for efficient catalysis.

Artificial metalloenzymes combine the inherent reactivity of transition metal catalysis with the sophisticated reaction control of natural enzymes. By providing new opportunities in bioorthogonal chemistry and biocatalysis, artificial metalloenzymes have the potential to overcome certain limitations in both drug discovery and green chemistry or related research fields. Ongoing advances in organometallic catalysis, directed evolution, and bioinformatics are enabling the design of increasingly powerful systems that outperform conventional catalysis in a growing number of cases. Therefore, this review article collects challenges and opportunities in designing artificial metalloenzymes described in recent review articles. This will provide an equitable insight for those new to and interested in the field.

Inside a Shell-Organometallic Catalysis Inside Encapsulin Nanoreactors.

Compartmentalization of chemical reactions inside cells are a fundamental requirement for life. Encapsulins are self-assembling protein-based nanocompartments from the prokaryotic repertoire that present a highly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single-molecule Förster resonance energy transfer and 3D-MINFLUX analysis, we analyze fluorescently labeled encapsulins on a single-molecule basis. Furthermore, by equipping these capsules with a synthetic ruthenium catalyst via covalent attachment to a non-native host protein, we are able to perform in vitro catalysis and go on to show that engineered encapsulins can be used as hosts for transition metal catalysis inside living cells in confined space.

Pyridinium Modified Anthracenes and Their Endoperoxides Provide a Tunable Scaffold with Activity against Gram-Positive and Gram-Negative Bacteria.

Due to the emergence of multidrug resistant bacteria, the development of new antibiotics is required. We introduce here asymmetrically modified positively charged bis(methylpyridinium) anthracenes as a novel tunable scaffold, in which the two positive charges can be placed at a defined distance and angle. Our structure-activity relationship reveals that coupling the methylpyridiniums with alkynyl linkers to the central anthracene unit yields antibacterial compounds against a wide range of bacteria, including Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis. Also, different mycobacteria, such as Mycobacterium smegmatis and Mycobacterium tuberculosis, are efficiently targeted by these compounds. The antibacterial activity depends on the number of alkynyl linkers and consequently also on the distance of the positive charges in the rigid anthracene scaffold. Additionally, the formation of an anthracene endoperoxide further increases the antibacterial activity, likely due to the release of toxic singlet oxygen that converts the endoperoxide back to the antibacterial anthracene scaffold with half-lives of several hours.

The Two-Component Locus MSMEG_0244/0246 Together With MSMEG_0243 Affects Biofilm Assembly in M. smegmatis Correlating With Changes in Phosphatidylinositol Mannosides Acylation.

Ferric and ferrous iron is an essential transition metal for growth of many bacterial species including mycobacteria. The genomic region msmeg_0234 to msmeg_0252 from Mycobacterium smegmatis is putatively involved in iron/heme metabolism. We investigate the genes encoding the presumed two component system MSMEG_0244/MSMEG_0246, the neighboring gene msmeg_0243 and their involvement in this process. We show that purified MSMEG_0243 indeed is a heme binding protein. Deletion of msmeg_0243/msmeg_0244/msmeg_0246 in Mycobacterium smegmatis leads to a defect in biofilm formation and colony growth on solid agar, however, this phenotype is independent of the supplied iron source. Further, analysis of the corresponding mutant and its lipids reveals that changes in morphology and biofilm formation correlate with altered acylation patterns of phosphatidylinositol mannosides (PIMs). We provide the first evidence that msmeg_0244/msmeg_0246 work in concert in cellular lipid homeostasis, especially in the maintenance of PIMs, with the heme-binding protein MSMEG_0243 as potential partner.

Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis.

The complex phosphorylation pattern of natural and modified pentaphosphorylated magic spot nucleotides is generated in a highly efficient way. A cyclic pyrophosphoryl phosphoramidite (cPyPA) reagent is used to introduce four phosphates on nucleosides regioselectively in a one-flask key transformation. The obtained magic spot nucleotides are used to develop a capillary electrophoresis UV detection method, enabling nucleotide assignment in complex bacterial extracts.

Understanding the mechanism of action of pyrrolo[3,2-b]quinoxaline-derivatives as kinase inhibitors.

The X-ray structure of the catalytic domain of the EphA3 tyrosine kinase in complex with a previously reported type II inhibitor was used to design two novel quinoxaline derivatives, inspired by kinase inhibitors that have reached clinical development. These two new compounds were characterized by an array of cell-based assays and gene expression profiling experiments. A global chemical proteomics approach was used to generate the drug-protein interaction profile, which suggested suitable therapeutic indications. Both inhibitors, studied in the context of angiogenesis and in vivo in a relevant lymphoma model, showed high efficacy in the control of tumor size.

Trehalose Conjugation Enhances Toxicity of Photosensitizers against Mycobacteria.

Trehalose is a natural glucose-derived disaccharide found in the cell wall of mycobacteria. It enters the mycobacterial cell through a highly specific trehalose transporter system. Subsequently, trehalose is equipped with mycolic acid species and is incorporated into the cell wall as trehalose monomycolate or dimycolate. Here, we investigate the phototoxicity of several photosensitizer trehalose conjugates and take advantage of the promiscuity of the extracellular Ag85 complex, which catalyzes the attachment of mycolic acids to trehalose and its analogues. We find that processing by Ag85 enriches and tethers photosensitizer trehalose conjugates directly into the mycomembrane. Irradiation of the conjugates triggers singlet oxygen formation, killing mycobacterial cells more efficiently, as compared to photosensitizers without trehalose conjugation. The conjugates are potent antimycobacterial agents that are, per se, affected neither by permeability issues nor by detoxification mechanisms via drug efflux. They could serve as interesting scaffolds for photodynamic therapy of mycobacterial infections.

Magic spot nucleotides: tunable target-specific chemoenzymatic synthesis.

A tunable chemoenzymatic strategy provides access to the entire class of magic spot nucleotides and modified analogues. The approach combines chemoselective bisphosphorylations using phosphoramidites with regioselective ribonuclease T2 cyclo-phosphate hydrolysis, leading to flexible and simple gram-scale operations.

Detection and Characterization of a Mycobacterial L-Arabinofuranose ABC Transporter Identified with a Rapid Lipoproteomics Protocol.

Nutrient uptake is essential for survival of organisms, and carbohydrates serve as a crucial carbon and energy source for most microorganisms. Given the importance of mycobacteria as human pathogens a detailed knowledge of carbohydrate uptake transporters is highly desirable, but currently available information is severely limited and mainly based on in silico analyses. Moreover, there is only very little data available on the in vitro characterization of carbohydrate transporters from mycobacterial species. To overcome these significant limitations there is a strong demand for innovative approaches to experimentally match substrates to ATP-binding cassette (ABC) transporters in a straightforward manner. Our study focuses on the model organism Mycobacterium smegmatis and identifies a mycobacterial ABC transport system based on a rapid label-free mass spectrometry lipoproteomics assay with broad applicability. Further validation and X-ray structure analyses reveal a highly selective mycobacterial L-arabinose uptake system.

New WS9326A Derivatives and One New Annimycin Derivative with Antimalarial Activity are Produced by Streptomyces asterosporus DSM 41452 and Its Mutant.

In this study, we report that Streptomyces asterosporus DSM 41452 is a producer of new molecules related to the nonribosomal cyclodepsipeptide WS9326A and the polyketide annimycin. S. asterosporus DSM 41452 is shown to produce six cyclodepsipeptides and peptides, WS9326A to G. Notably, the compounds WS9326F and WS9326G have not been described before. The genome of S. asterosporus DSM 41452 was sequenced, and a putative WS9326A gene cluster was identified. Gene-deletion experiments confirmed that this cluster was responsible for the biosynthesis of WS9326A to G. Additionally, a gene-deletion experiment demonstrated that sas16 encoding a cytochrome P450 monooxygenase was involved in the synthesis of the novel (E)-2,3-dehydrotyrosine residue found in WS9326A and its derivatives. An insertion mutation within the putative annimycin gene cluster led to the production of a new annimycin derivative, annimycin B, which exhibited modest inhibitory activity against Plasmodium falciparum.

Wide Distribution of Foxicin Biosynthetic Gene Clusters in Streptomyces Strains - An Unusual Secondary Metabolite with Various Properties.

Streptomyces diastatochromogenes Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the pokOIV oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A-D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of S. diastatochromogenes. The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A foxBII gene-inactivated mutant of S. diastatochromogenes Tü6028 ΔpokOIV is unable to produce foxicins. Homologous fox biosynthetic gene clusters were found in more than 20 additional Streptomyces strains, overall in about 2.6% of all sequenced Streptomyces genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the Escherichia coli aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in Streptomyces strains.