We present a series of bifunctional photoaffinity CB₂R selective probes based on the 5-fluoropyridin-2-yl-benzyl-imidazoleidine-2,4-dione derivative LEI-102, with both inverse agonist and partial agonist behaviour in vitro. These photoaffinity probes have improved affinity and potency compared to previously published LEI-121.

Abstract

The cannabinoid receptor type 2 (CB₂R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI-121 that stabilized the inactive conformation of the CB₂R. Here, we report the structure-based design of a novel bifunctional probe that captures the active conformation of the CB₂R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click-chemistry with fluorophore-azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function.

NP-TBZ is developed by covalently linking o-nitrobenzyl (NP) with thiabendazole (TBZ). Compound NP-TBZ can be controlled to release TBZ in dependent to light, which resulted in controllable in-vivo activity against wheat scab. It is promising for spatiotemporal controlled-release of TBZ in factual applications, posing a theoretical guidance in development of controlled-release pesticides.

Abstract

Pesticides are essential in agricultural development. Controlled-release pesticides have attracted great attentions. Base on a principle of spatiotemporal selectivity, we extended the photoremovable protective group (PRPG) into agrochemical agents to achieve controllable release of active ingredients. Herein, we obtained NP-TBZ by covalently linking o-nitrobenzyl (NP) with thiabendazole (TBZ). Compound NP-TBZ can be controlled to release TBZ in dependent to light. The irradiated and unirradiated NP-TBZ showed significant differences on fungicidal activities both in vitro and in vivo. In addition, the irradiated NP-TBZ displayed similar antifungal activities to the directly-used TBZ, indicating a factual applicability in controllable release of TBZ. Furthermore, we explored the action mode and microcosmic variations by SEM analysis, and demonstrated that the irradiated NP-TBZ retained a same action mode with TBZ against mycelia growth.

A convergent synthesis was used to access both enantiomers of the polyether antibiotic routiennocin, and a diastereomeric variant, to probe the biological consequences of altering the naturally configured stereochemistry. Testing these compounds against a broad panel of bacteria and mammalian cells revealed independence of absolute stereochemistry in all tested cells.

Abstract

Carboxylic polyether ionophores (CPIs) are among the most prevalent agricultural antibiotics (notably in the US) and these compounds have been in use for decades. The potential to reposition CPIs beyond veterinary use, e. g. through chemical modifications to enhance their selectivity window, is an exciting challenge and opportunity, considering their general resilience towards resistance development. Given the very large societal impact of these somewhat controversial compounds, it is surprising that many aspects of their mechanisms and activities in cells remain unclear. Here, we report comparative biological activities of the CPI routiennocin and two stereoisomers, including its enantiomer. We used an efficient convergent synthesis strategy to access the compounds and conducted a broad survey of antibacterial activities against planktonic cells and biofilms as well as the compounds’ effects on mammalian cells, the latter assessed both via standard cell viability assays and broad morphological profiling. Interestingly, similar bioactivity of the enantiomeric pair was observed across all assays, strongly suggesting that chiral interactions do not play a decisive role in the mode of action. Overall, our findings are consistent with a mechanistic model involving highly dynamic behaviour of CPIs in biological membranes.

Fus-SMO, a chimera of StyA and StyB in styrene monooxygenase, has a trimeric structure with one bound flavin. In silico modelling indicated a well-distanced arrangement due to the flexible linker. Pre-steady-state kinetics highlighted the intricacies of FAD_ox reduction and aerobic FADH₂ oxidation. NADH consumption vs. styrene epoxidation revealed quantitative coupling efficiency. These findings advance our understanding of FADH₂ transfer mechanisms in SMO and underscore protein fusion‘s role in enhancing biocatalysis.

Abstract

The styrene monooxygenase, a two-component enzymatic system for styrene epoxidation, was characterised through the study of Fus-SMO – a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH₂ from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus-SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well-distanced arrangement (45–50 Å) facilitated by the flexible linker‘s loopy structure. Pre-steady-state kinetics elucidated the FAD_ox reduction intricacies (k_red=110 s⁻¹ for bound FAD_ox), identifying free FAD_ox binding as the rate-determining step. The aerobic oxidation of FADH₂ (k_ox=90 s⁻¹) and subsequent decomposition to FAD_ox and H₂O₂ demonstrated StyA′s protective effect on the bound hydroperoxoflavin (k_dec=0.2 s⁻¹) compared to free cofactor (k_dec=1.8 s⁻¹). At varied styrene concentrations, k_ox for FADH₂ ranged from 80 to 120 s⁻¹. Studies on NADH consumption vs. styrene epoxidation revealed Fus-SMO′s ability to achieve quantitative coupling efficiency in solution, surpassing natural two-component SMOs. The results suggest that Fus-SMO exhibits enhanced FADH₂ channelling between subunits. This work contributes to comprehending FADH₂ transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.

The human enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 (HsDNPH1) is a promising target for inhibition towards anticancer drug development. Crystal structures, site-directed mutagenesis, and kinetic analysis, including pH-rate profiles and solvent deuterium isotope effects, help quantify contributions from conserved residues Tyr24 and Asp80 to HsDNPH1-catalysed hydrolysis of 5-hydroxymethyl-2′-deoxyuridine 5′-phosphate. These include nucleophile and substrate positioning and general-acid catalysis.

Abstract

The human enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 (HsDNPH1) catalyses the hydrolysis of 5-hydroxymethyl-2′-deoxyuridine 5′-phosphate to generate 5-hydroxymethyluracil and 2-deoxyribose-5-phosphate via a covalent 5-phospho-2-deoxyribosylated enzyme intermediate. HsDNPH1 is a promising target for inhibitor development towards anticancer drugs. Here, site-directed mutagenesis of conserved active-site residues, followed by HPLC analysis of the reaction and steady-state kinetics are employed to reveal the importance of each of these residues in catalysis, and the reaction pH-dependence is perturbed by each mutation. Solvent deuterium isotope effects indicate no rate-limiting proton transfers. Crystal structures of D80N-HsDNPH1 in unliganded and substrate-bound states, and of unliganded D80A- and Y24F-HsDNPH1 offer atomic level insights into substrate binding and catalysis. The results reveal a network of hydrogen bonds involving the substrate and the E104-Y24-D80 catalytic triad and are consistent with a proposed mechanism whereby D80 is important for substrate positioning, for helping modulate E104 nucleophilicity, and as the general acid in the first half-reaction. Y24 positions E104 for catalysis and prevents a catalytically disruptive close contact between E104 and D80.

Bacteria can use DNA origami nanostructures as a nutrient source, leading to increased population growth. This process depends not only on the competence signal and uptake mechanisms of each species, but also on DNA origami shape and superstructure. It should thus be considered in the design and development of antimicrobial DNA origami nanostructures.

Abstract

DNA origami nanostructures are a powerful tool in biomedicine and can be used to combat drug-resistant bacterial infections. However, the effect of unmodified DNA origami nanostructures on bacteria is yet to be elucidated. With the aim to obtain a better understanding of this phenomenon, the effect of three DNA origami shapes, i.e., DNA origami triangles, six-helix bundles (6HBs), and 24-helix bundles (24HBs), on the growth of Gram-negative Escherichia coli and Gram-positive Bacillus subtilis is investigated. The results reveal that while triangles and 24HBs can be used as a source of nutrients by E. coli and thereby promote population growth, their effect is much smaller than that of genomic single- and double-stranded DNA. However, no effect on E. coli population growth is observed for the 6HBs. On the other hand, B. subtilis does not show any significant changes in population growth when cultured with the different DNA origami shapes or genomic DNA. The detailed effect of DNA origami nanostructures on bacterial growth thus depends on the competence signals and uptake mechanism of each bacterial species, as well as the DNA origami shape. This should be considered in the development of antimicrobial DNA origami nanostructures.

The organocatalyzed reaction between thiazolidine-3-carboxylates and aryl azides resulted in the metal-free synthesis of novel 1,2,3-triazolyl-thiazolidine hybrids in good yields. Spectroscopic methods (absorption and emission), viscosimetry, and molecular docking were employed to assess the interactions between the synthesized compounds and DNA, revealing promising results for the development of novel therapeutic agents targeting DNA-related processes.

Abstract

An organocatalytic [3+2] cycloaddition reaction between thiazolidine-containing β-ketoester 1 and aryl azides 2 was employed to synthesize new 1,2,3-triazolyl-thiazolidine hybrids 3. In this metal-free approach, twelve compounds were isolated in yields ranging from 23 % to 96 % by using diethylamine (10 mol%) and DMSO at 75 °C for 24 hours. DNA-binding assays were conducted through absorption, emission spectroscopy and viscosimetry analysis, to evaluate the interaction capacity of the studied derivatives with nucleic acids. All the synthesized compounds were evaluated for their interactions with a specific group of compounds containing the pharmacophoric groups triazole and thiazolidine through a molecular docking speculative study, aimed at identifying the interaction profile of these compounds with DNA. The obtained results suggest that 1,2,3-triazolyl-thiazolidine hybrids could be a promising approach in the development of novel therapeutic agents targeting DNA-related processes.

Antimicrobial resistance is an urgent global public health problem that has made the search for new antibiotics essential. Ribosomally synthesized and post-translationally modified peptides are a promising new class of antibiotics and in this work, we report site-selective modification of their dehydroamino acids by β-amination in order to increase water solubility: the singly modified thiopeptide Thiostrepton showed an increase up to 35-fold and minimum inhibitory concentration tests demonstrated that the antimicrobial activity was still good, albeit lower than the natural peptide.

Abstract

We report the efficient and site selective modification of non-canonical dehydroamino acids in ribosomally synthesized and post-transationally modified peptides (RiPPs) by β-amination. The singly modified thiopeptide Thiostrepton showed an up to 35-fold increase in water solubility, and minimum inhibitory concentration (MIC) assays showed that antimicrobial activity remained good, albeit lower than the unmodified peptide. Also the lanthipeptide nisin could be modified using this method.

A new (R)-selective amine transaminase (R-ATA) from Mycobacterium sp. ACS1612 was identified via in silico prediction combined with genome and protein database information. The newly identified and characterized R-ATA displayed a broad substrate spectrum and strong tolerance to organic solvents. Moreover, the synthetic applicability of M16AT was demonstrated by the asymmetric synthesis of (R)-fendiline.

Abstract

Biocatalysis has emerged as a powerful alternative to traditional chemical methods, especially for asymmetric synthesis. As biocatalysts usually exhibit excellent chemical, regio- and enantioselectivity, they facilitate and simplify many chemical processes for the production of a broad range of products. Here, a new biocatalyst called, R-selective amine transaminases (R-ATAs), was obtained from Mycobacterium sp. ACS1612 (M16AT) using in–silico prediction combined with a genome and protein database. A two–step simple purification process could yield a high concentration of pure enzyme, suggesting that industrial application would be inexpensive. Additionally, the newly identified and characterized R-ATAs displayed a broad substrate spectrum and strong tolerance to organic solvents. Moreover, the synthetic applicability of M16AT has been demonstrated by the asymmetric synthesis of (R)-fendiline from of (R)-1-phenylethan-1-amine.

Characterization of the aromatic ammonia-lyase from Loktanella atrilutea (LaAAL) revealed reduced activity towards canonical AAL substrates: L-Phe, L-Tyr, and L-His, contrasted by its pronounced efficiency towards 3,4-dimethoxy-L-phenylalanine. Assessing optimal conditions, LaAAL exhibited maximal activity at pH 9.5 in the ammonia elimination reaction route, distinct from the typical pH ranges of most PALs and TALs. Within the exploration of the ammonia source for the opposite, synthetically valuable ammonia addition reaction, the stability of LaAAL, exhibited a positive correlation with the ammonia concentration, with the highest stability in 4 M ammonium carbamate of unadjusted pH of ~9.5. While the enzyme activity increased with rising temperatures yet, the highest operational stability and highest stationary conversions of LaAAL were observed at 30 °C. The substrate scope analysis highlighted the catalytic adaptability of LaAAL in the hydroamination of diverse cinnamic acids, especially of meta-substituted and di-/multi-substituted analogues, with structural modelling exposing steric clashes between the substrates’ ortho-substituents and catalytic site residues. LaAAL showed a predilection for ammonia elimination, while classifying as a tyrosine ammonia-lyase (TAL) among the natural AAL classes. However, its distinctive attributes, such as genomic context, unique substrate specificity and catalytic fingerprint, suggest a potential natural role beyond those of known AAL classes.