A quest for novel antimicrobial targets: Inhibition of Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) by synthetic analogs of aminoacyl-adenosine in vitro and live bacteria.

The Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) has been proposed as a novel antibacterial drug target due to its indispensability in prominent human pathogens. While several inhibitors with in vitro activity have been identified, none have been demonstrated to have potent activity against live bacteria. In this work, seven non-hydrolyzable transition state mimics of GatCAB were synthesized and tested as the transamidase inhibitors against GatCAB from the human pathogen Helicobacter pylori. Notably, the methyl sulfone analog of glutamyl-adenosine significantly reduced GatCAB's transamination rate. Additionally, four lipid-conjugates of these mimics displayed antibacterial activity against Bacillus subtilis, likely due to enhanced cell permeability. Inhibitory activity against GatCAB in live bacteria was confirmed using a sensitive gain-of-function dual luciferase reporter in Mycobacterium bovis-BCG. Only the lipid-conjugated methyl sulfone analog exhibited a significant increase in mistranslation rate, highlighting its cell permeability and inhibitory potential. This study provides insights for developing urgently needed novel antibacterial agents amidst emerging antimicrobial drug resistance.

Design, Synthesis, and Characterization of Novel Styryl Dyes as Fluorescent Probes for Tau Aggregate Detection in Vitro and in Cells.

A series of novel styryl dye derivatives incorporating indolium and quinolinium core structures were successfully synthesized to explore their interacting and binding capabilities with tau aggregates in vitro and in cells. The synthesized dyes exhibited enhanced fluorescence emission in viscous environments due to the rotatable bond confinement in the core structure. Dye 4, containing a quinolinium moeity and featuring two cationic sites, demonstrated a 28-fold increase in fluorescence emission upon binding to tau aggregates. This dye could also stain tau aggregates in living cells, confirmed by cell imaging using confocal fluorescence microscopy. A molecular docking study was conducted to provide additional visualization and support for binding interactions. This work offers novel and non-cytotoxic fluorescent probes with desirable photophysical properties, which could potentially be used for studying tau aggregates in living cells, prompting further development of new fluorescent probes for early Alzheimer's disease detection.

Anthraquinones from the roots of Morinda scabrida Craib exhibit antiproliferative activity against A549 lung cancer cells and antitubulin polymerization.

Six anthraquinones were isolated from Morinda scabrida Craib, an unexplored species of Morinda found in the tropical forest of Thailand. All six anthraquinones showed cytotoxicity against A549 lung cancer cells, with the most active compound, nordamnacanthal (MS01), exhibiting the IC50 value of 16.3 ± 2.5 µM. The cytotoxic effect was dose-dependent and led to cell morphological changes characteristic of apoptosis. In addition, flow cytometric analysis showed dose-dependent apoptosis induction and the G2/M phase cell cycle arrest, which was in agreement with the tubulin polymerization inhibitory activity of MS01. Molecular docking analysis illustrated the binding between MS01 and the α/ß-tubulin heterodimer at the colchicine binding site, and UV-visible absorption spectroscopy revealed the DNA binding capacity of MS01.

Quantitative analysis of steric effects on the regioselectivity of the Larock heteroannulation reaction.

Alkylphenylacetylene derivatives were synthesized and used as reactants in the Larock heteroannulation reaction to investigate the steric influence on regioselectivity. Large alkyl groups preferentially yielded 2-alkyl-3-phenylindole products, while smaller alkyl groups provided 3-alkyl-2-phenylindole as major products. The logarithm of regioisomeric product ratios exhibited good correlations with various steric parameters. Notably, the Charton values provided the best correlation when excluding the cyclopropyl group. In addition, the Boltzmann-weighted Sterimol parameter (wSterimol) was utilized to generate a good predictive model, indicating the B1 wSterimol as the significant regiochemical determining parameter with no obvious deviation for the cyclopropyl group. Relative atomic distances within the DFT-optimized transition state structures revealed good correlations with the logarithm of regioisomeric ratios. Furthermore, the cyclopropyl adsorption complex indicated electronic contribution, explaining the peculiar behavior of this substituent in the experimental observation.

Absolute configuration of azaphilones from Monascus kaoliang KB9 and solvent effects on their keto and enol forms.

Monascus fermented rice, also known as red yeast rice, exhibits a broad spectrum of biological activities due to its chemical constituents, such as monacolins and azaphilone pigments. Here, we cultured Monascus kaoliang KB9 in a liquid malt medium instead of on rice as a carbon source. Eleven known compounds (1-11) containing azaphilones and their early intermediate were isolated and identified. However, this was the first time that angular tricyclic azaphilones, monasfluols A (4) and B (7), acetyl-monasfluol A (5) and monasfluore A (6), were isolated from this species. Interestingly, all isolated tricyclic azaphilones existed exclusively in enol form in CD3OD, as evidenced by NMR spectroscopy. The absolute configuration of compounds 4-7 was also first experimentally identified based on ECD spectroscopy combined with conformational analyses using computational techniques. The assigned stereochemistry of Monascus azaphilones in this work provides essential structural information that will benefit future biological and pharmaceutical investigations.

Titanium Complexes of Salicylbenzoxazole and Salicylbenzothiazole Ligands for the Ring-Opening Polymerization of ε-Caprolactone and Substituted ε-Caprolactones and Their Copolymerizations.

Two series of titanium complexes, including salicylbenzoxazole titanium complexes (1-4) and salicylbenzothiazole titanium complexes (5-8), were successfully synthesized and characterized by NMR spectroscopy, elemental analysis, and X-ray diffraction crystallography (for 2 and 5). The 1H NMR spectra of complexes 7 and 8 reveal fluxional behavior in solution at room temperature, and the activation parameters were determined by lineshape analysis of variable-temperature (VT) NMR spectra in toluene-d8: for 7, ΔH⧧ = 73.0 ± 1.8 kJ mol-1, ΔS⧧ = 22.1 ± 5.5 J mol-1 K-1; for 8, ΔH⧧ = 73.7 ± 1.2 kJ mol-1, ΔS⧧ = 20.3 ± 3.8 J mol-1 K-1. The positive values of ΔS⧧ suggested that the isomerization occurred via a dissociative mechanism. All complexes were active initiators for the ring-opening polymerization of ε-caprolactone (ε-CL) and three substituted ε-CLs: γ-methyl-ε-caprolactone (γMeCL), γ-ethyl-ε-caprolactone (γEtCL), and γ-phenyl-ε-caprolactone (γPhCL). Of all complexes, complex 5 was found to be the most active initiator in this study. The copolymerizations between ε-CL and three substituted ε-CLs produced completely random copolymers. The polymerization was proposed to proceed via a dissociative coordination-insertion mechanism. The catalytic activity of the salicylbenzoxazole titanium complex was lower than that of its closely related salicylbenzothiazole titanium congener. Additionally, DFT calculations unveiled that the ligand decoordination step and the less steric congestion at the titanium center in the salicylbenzothiazole titanium complexes were the key factors in enhancing the catalytic rate.

Biphasic Dose-Dependent G0/G1 and G2/M Cell-Cycle Arrest by Synthetic 2,3-Arylpyridylindole Derivatives in A549 Lung Cancer Cells.

A collection of 2,3-arylpyridylindole derivatives were synthesized via the Larock heteroannulation and evaluated for their in vitro cytotoxic activity against A549 human lung cancer cells. Two derivatives expressed good cytotoxicity with IC50 values of 1.18±0.25 µM and 0.87±0.10 µM and inhibited tubulin polymerization in vitro, with molecular docking studies suggesting the binding modes of the compounds in the colchicine binding site. Both derivatives have biphasic cell cycle arrest effects depending on their concentrations. At a lower concentration (0.5 µM), the two compounds induced G0/G1 cell cycle arrest by activating the JNK/p53/p21 pathway. At a higher concentration (2.0 µM), the two derivatives arrested the cell cycle at the G2/M phase via Akt signaling and inhibition of tubulin polymerization. Additional cytotoxic mechanisms of the two compounds involved the decreased expression of Bcl-2 and Mcl-1 antiapoptotic proteins through inhibition of the STAT3 and Akt signaling pathways.

Dissecting the Electronic Contribution to the Regioselectivity of the Larock Heteroannulation Reaction in the Oxidative Addition and Carbopalladation Steps.

Substituted 2-iodoaniline derivatives were prepared and utilized as reactants, along with asymmetric diarylacetylenes, to synthesize a series of 6-substituted-2,3-diarylindole derivatives via the Larock heteroannulation reaction. Electron-donating substituents on the 2-iodoaniline derivatives retarded the reaction, while electron-withdrawing substituents provided a complete conversion to the indole products. In addition, the electronic properties of the substituted 2-iodoaniline reactants displayed no influence toward regioselectivity. On the contrary, the electronic effect from unsymmetrical diarylacetylenes significantly influenced the regiochemical outcome of the reaction. Density functional theory calculations of the oxidative addition and carbopalladation steps revealed the electronic influences of the substituted 2-iodoaniline derivatives toward the overall rate of the reaction. In contrast, the electronic properties of the asymmetric diarylacetylene remained the critical product-determining factor of regioselectivity.

Titanium complexes of pyrrolylaldiminate ligands and their exploitation for the ring-opening polymerization of cyclic esters.

A series of six-coordinate titanium complexes 1-6 supported by pyrrolylaldiminate ligands were prepared via the reaction of 2 equivalents of ligands and Ti(OiPr)4 in toluene at 70 °C. The X-ray structure of 2 revealed that the two ligands were κ2-coordinated to the titanium center with the two pyrrole nitrogen atoms in trans positions and the two imine nitrogen atoms in cis positions. All complexes were active initiators for the ring-opening polymerization (ROP) of rac-lactide (rac-LA), ε-caprolactone (ε-CL), and three substituted ε-caprolactones (γ-methyl-ε-caprolactone (γMeCL), γ-ethyl-ε-caprolactone (γEtCL), and γ-phenyl-ε-caprolactone (γPhCL)). Polymerizations of all monomers were well controlled, affording predetermined molar masses and narrow dispersity values. Complex 5 exhibited the highest polymerization activities with rac-LA and ε-CL and its performance was comparable to other highly active six-coordinate titanium complexes reported thus far. Kinetic results revealed a first-order dependency on the monomer concentration, and the rate of polymerization was greatly influenced by the substituent on the imine nitrogen. End-group analysis of the isolated PLA and PCL suggested a coordination-insertion mechanism.

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity.

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.

A synthetic 2,3-diarylindole induces microtubule destabilization and G2/M cell cycle arrest in lung cancer cells.

The anticancer potential of a synthetic 2,3-diarylindole (PCNT13) has been demonstrated in A549 lung cancer cells by inducing both apoptosis and autophagic cell death. In this report, we designed to connect a fluorophore to the compound via a hydrophilic linker for monitoring intracellular localization. The best position for linker attachment was identified from cytotoxicity and effect on cell morphology of newly synthesized PCNT13 derivatives bearing hydrophilic linker. Cytotoxicity and effect on cell morphology related to the parental compound were used to identify the optimum position for linker attachment in the PCNT13 chemical structure. The fluorophore-PCNT13 conjugate was found to localize in the cytoplasm. Microtubules were found to be one of the cytosolic target proteins of PCNT13, as the compound could inhibit tubulin polymerization in vitro. A molecular docking study revealed that PCNT13 binds at the colchicine binding site on the α/ß-tubulin heterodimer. The effect of PCNT13 on microtubule dynamics caused cell cycle arrest in the G2/M phase as analyzed by flow cytometric analysis.

Cloning, overexpression, and purification of a gene of unknown function of prophage loci from 'Candidatus Liberibacter asiaticus,' the destructive bacterial pathogen of huanglongbing disease in citrus plants.

Citrus Huanglongbing (HLB) or citrus greening is one of the most destructive diseases affecting citrus industry worldwide. The causal agent in Asia is a phloem-limited, Gram-negative bacterium, 'Candidatus Liberibacter asiaticus' (CLas). Within the genome of CLas lies prophage regions, classified as Type-A, B, C, and D. In particular, Type-D has been indicated to correlate with the blotchy-mottle symptoms of citrus trees. Here we reported the cloning, overexpression, and purification of the ORF1, an open reading frame from the partial Type-D region of CLas obtained from an infected lime tree (Citrus aurantifolia Swingle). Overexpression of the ORF1 was toxic to the E. coli BL21(DE3), and the transient expression of ORF1 in Arabidopsis seedlings by Agrobacterium-mediated transformation exhibited rapid and total chlorosis of the seedlings within two days post-transformation. The native-PAGE of the purified protein showed multiple bands, indicative of various conformations in solution. The ESI-TOF mass spectrum confirmed the molecular weight of the purified ORF1 to be 15,364.3150 Da, corresponding to the [M+1]+ of the ORF1 without an N-terminal methionine. The protein predominantly consisted of α-helix as evidenced by circular dichroism (CD), and the transition toward random coil structure upon heating was reversible. The template-based modeling (I-TASSER) of the ORF1 indicated eight α-helices connected through variable loops. The simulated CD spectrum, generated from the atomic coordinates of the I-TASSER model, was notably similar to the experimental spectrum. Our report offers the basis for understanding the contributions of genes within Type-D prophage region toward the disease pathogenicity of citrus HLB.

Aluminium complexes containing salicylbenzothiazole ligands and their application in the ring-opening polymerisation of rac-lactide and ε-caprolactone.

Two series of aluminium complexes bearing salicylbenzothiazole ligands, namely four-coordinate aluminium complexes (1a-7a) and five-coordinate aluminium complexes (1b-7b), were synthesized and characterized by NMR spectroscopy, elemental analysis and X-ray diffraction crystallography (for 5a and 1b). Their application in the ring-opening polymerisation of rac-lactide and ε-caprolactone was studied with the aim of drawing comparisons to closely related aluminium salicylbenzoxazole complexes investigated previously. In the presence of benzyl alcohol, all complexes were active initiators and polymerisations were all well controlled and living. Kinetic studies revealed first-order kinetics in the monomer. In contrast, the catalytic activity of aluminium salicylbenzothiazole complexes was lower than that of aluminium salicylbenzoxazole counterparts. Detailed DFT calculations were performed and indicated that the observed lower catalytic activity of aluminium salicylbenzothiazole complexes agreed well with the observed higher Gibbs free energy at the ring-opening transition state.

Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori.

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAsp and tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Šresolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAsp and tRNAAsn. It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp (34GUC36) and tRNAAsn (34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

Aluminum complexes containing salicylbenzoxazole ligands and their application in the ring-opening polymerization of rac-lactide and ε-caprolactone.

Two series of four-coordinate aluminum () and five-coordinate aluminum () complexes were successfully synthesized via the reactions between the corresponding salicylbenzoxazole ligands and 1 or 0.5 equivalents of AlMe3, respectively. The synthesized aluminum complexes were characterized by (1)H and (13)C NMR spectroscopy and elemental analysis. The solid-state structures of complexes and were determined using single crystal X-ray diffraction. Upon addition of 1 equivalent of benzyl alcohol, all complexes were efficient initiators for the ring-opening polymerization (ROP) of rac-lactide (rac-LA) and ε-caprolactone (ε-CL). The polymerizations were living with a good control over molecular weights and molecular weight distributions. Under immortal polymerization conditions, all four-coordinate aluminum complexes () exhibited a living polymerization with the obtained molecular weights proportional to the ratio of monomer/benzyl alcohol and the PDIs were narrow. Kinetic studies revealed that both rac-LA and ε-CL polymerizations mediated by all complexes were first-order in monomers. The effects of ligand structure and coordination geometry on the catalytic activity and stereoselectivity were discussed. A good isoselectivity control was achieved for the polymerizations mediated by complexes (Pm = 0.75), (Pm = 0.74), and (Pm = 0.74).

A synthetic 2,3-diarylindole induces cell death via apoptosis and autophagy in A549 lung cancer cells.

A series of 2,3-diarylindoles were synthesized via the Larock heteroannulation, and evaluated for their anticancer activity against A549 lung cancer cells. The most potent compound, PCNT13 with IC50=5.17 µM, caused the induction of two modes of programmed cell death, apoptosis and autophagy.

Improvement of chloramphenicol production in Streptomyces venezuelae ATCC 10712 by overexpression of the aroB and aroK genes catalysing steps in the shikimate pathway.

Streptomyces venezuelae ATCC 10712 produces chloramphenicol in small amounts. To enhance chloramphenicol production, two genes, aroB and aroK, encoding rate-limiting enzymes of the shikimate pathway were overexpressed using the expression vector pIJ86 under the control of the strong constitutive ermE* promoter. The recombinant strains, S. venezuelae/pIJ86-aroB and S. venezuelae/pIJ86-aroK, produced 2.5- and 4.3-fold greater amounts respectively of chloramphenicol than wild type at early stationary phase of growth. High transcriptional levels of aroB and aroK genes were detected at the early exponential growth of both recombinant strains and consistent with the enhanced expression of pabB gene encoding an early enzyme in chloramphenicol biosynthesis. The results suggested that the increment of carbon flux was directed towards intermediates in the shikimate pathway required for the production of chorismic acid, and consequently resulted in the enhancement of chloramphenicol production. This work is the first report of a convenient genetic approach to manipulate primary metabolite genes in S. venezuelae in order to increase chloramphenicol production.

Facile and Divergent Synthesis of Lamellarins and Lactam-Containing Derivatives with Improved Drug Likeness and Biological Activities.

With the goal to improve the aqueous solubility of lamellarins, the lactone ring in their skeleton was replaced with a lactam moiety in azalamellarins. However, the reported synthetic route produced such derivatives in very low yields. Hence, this study focused on developing an efficient simplified total synthetic scheme that could furnish both azalamellarins and the parent lamellarins from the same pyrrole ester intermediates. Subsequent comparative profiling revealed that the introduced lactone-to-lactam replacement rendered these molecules less lipophilic, whereas their cancer cytotoxicity remained equipotent to that of the parent compounds. Interestingly, their inhibitory activity was significantly enhanced towards the multifaceted GSK-3ß enzyme. Our results clearly demonstrate the therapeutic potential of this promising class of marine-derived natural products and justify their further development, especially into anticancer agents.

Regioselectivity of Larock heteroannulation: a contribution from electronic properties of diarylacetylenes.

A series of 2,3-diarylindoles were synthesized from 2-iodoaniline and unsymmetrical diarylacetylenes using the Larock heteroannulation. Diarylacetylenes bearing electron-withdrawing substituents lead to 2,3-diarylindoles with substituted phenyl moieties at the 2-position as major products, while those with electron-donating groups preferably yield indole products with substituted phenyl moieties at the 3-position. The regioisomeric product ratios exhibit a clear correlation with Hammett σ(p) values. DFT calculations reveal the origin of this effect, displaying smaller activation energy barriers for those pathways leading to the major regioisomer.

Overproduction of the N-terminal anticodon-binding domain of the non-discriminating aspartyl-tRNA synthetase from Helicobacter pylori for crystallization and NMR measurements.

Aminoacyl-tRNA synthetases (aaRSs) covalently attach an amino acid to its cognate tRNA isoacceptors through an ester bond. The standard set of 20 amino acids implies 20 aaRSs for each pair of amino acid/tRNA isoacceptors. However, the genomes of all archaea and some bacteria do not encode for a complete set of 20 aaRSs. For the human pathogenic bacterium Helicobacter pylori, a gene encoding asparaginyl-tRNA synthetase (AsnRS) is absent whilst an aspartyl-tRNA synthetase (AspRS) aminoacylates both tRNA(Asp) and tRNA(Asn) with aspartate. The structural and functional basis for this non-discriminatory behavior is not well understood. Here we report the over-production of the N-terminal anticodon-binding domain of H. pylori ND-AspRS using Escherichia coli BL21(DE3) host cells. Prolonged expression of this protein resulted in a toxic phenotype, limiting the expression period to just 30min. Purified protein was monomeric in solution by gel filtration chromatography and stable up to 42°C as observed in temperature-dependent dynamic light scattering measurements. Circular dichroism indicated a mixture of α-helix and ß-sheet secondary structure at 20°C and predominantly ß-sheet at 70°C. Optimized crystallization conditions at pH 5.6 with PEG 4000 as a co-precipitant produced well-formed crystals and (1)H NMR spectrum showed a well dispersed chemical shift envelope characteristic of a folded protein.