Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused of COVID-19 has posed a major global health crisis in 2019. Herein, six stilbene-coumarin derivatives were synthesized from the natural chalcone dimethylcardamonin (DMC, 5) isolated from Syzygium nervosum. Their antiviral potential against COVID-19 was evaluated by measuring the inhibition of the SARS-CoV-2 main protease (3CLpro), a biological macromolecule, using FRET-based enzymatic assay. The results demonstrated that this scaffold effectively suppressed the proteolytic activity of 3CLpro. Structure-activity relationship analysis indicated that an optimal alkyl substitution length at C-3 position of the coumarin ring comprises six carbon atoms. Among the synthesized derivatives, compound 7c exhibited the strongest inhibitory activity, with an IC50 value of 20.73 mu M, approximately four times more potent than the reference flavonoid baicalein. Furthermore, molecular dynamics simulation provided atomistic insights into the interactions between the stilbene-coumarin derivatives and 3CLpro, revealing plausible binding orientations within the active site. Complementary, Molecular Mechanics Generalized Born Surface Area (MM-GBSA) and fragment molecular orbital (FMO) calculations highlighted key residues that play critical roles in stabilizing the binding of these compounds.
2-Alkynylindolin-3-ones (pseudoindoxyl derivatives) are accessed directly from 2-arylisatogens through a CuTC-catalyzed reductive alkynylation with terminal alkynes. The transformation proceeds in a one-step cascade, combining alkyne addition at the 2-position of the isatogen nucleus with concurrent N-O bond cleavage to deliver 2-alkynylindolin-3-ones in yields of up to 89% under relatively mild thermal conditions. The method displays a broad substrate scope and operational simplicity, highlighting its value as a practical entry to this class of heterocycles. Control experiments further reveal a redox interplay of copper species, sustained by molecular oxygen, which underpins the tandem alkyne addition and N-O bond reduction events.
A mild photoinduced isomerization of isatogens to benzoxazin-4-ones is described. Upon irradiation at 370 nm, a broad range of 2-aryl- and 2-alkyl-substituted isatogens undergo efficient scaffold reorganization under catalyst-free conditions to afford benzoxazin-4-ones in good to excellent yields. The transformation tolerates diverse substitution patterns, including heteroaryl and strained alkyl groups, and is readily scalable. Mechanistic experiments support participation of an excited-state pathway, and inclusion of catalytic 2-picolinic acid provided reproducible outcomes. For a nitro-substituted substrate, a distinct reaction pathway is observed, leading to the formation of a toluene-derived adduct. These results establish isatogens as versatile photochemical precursors to benzoxazinone scaffolds under operationally simple conditions.
Background Klebsiella pneumoniae is an important pathogen of humans and animals. In the past five years, increasing reports of convergent strains that carry both virulence factors and antimicrobial resistance genes (ARGs) have raised serious public health concerns. The aim of this study is to describe the global diversity of plasmids carrying iuc3 (a key virulence factor in K pneumoniae associated with pigs and clinical isolates) from diverse settings, and their role in the emergence of convergent strains through hybridisation with plasmids carrying ARGs. Methods This population genomic analysis study was designed to describe both the global and local diversity of iuc3-carrying plasmids from diverse sources, and the co-occurrence of iuc3 with ARGs. We used all 4148 Klebsiella spp isolates from two large One-Health studies (SpARK, Italy, and OH-DART, Thailand), including 191 Klebsiella isolates from pigs, 635 from clinical isolates, 1040 from hospital and community carriage, and 2282 from other sources. Short-read sequencing of Klebsiella isolates was performed as part of the SpARK study. We sequenced Klebsiella isolates from the OH-DART (MicrobesNG, Birmingham, UK; HiSeq and NovaSeq, Illumina San Diego, CA, USA; GridION, Oxford Nanopore Technologies, Oxford, UK) and SpARK (MinION or GridION, Oxford Nanopore Technologies, Oxford, UK) studies. We also retrieved plasmid sequences carrying iuc3 from the National Centre for Biotechnology Information (NCBI). To ascertain the degree of diversity, evolutionary dynamics, and structuring across ecological and geographical axes, we detected ARGs and virulence loci, analysed clustering patterns and generated approximate maximum-likelihood phylogenetic trees. Findings We identified 48 K pneumoniae isolates with iuc3 in the SpARK data and 79 in the OH-DART data. Three (2·4%) of these 127 isolates were from clinical sources, 73 (57·5%) were from pig or pork meat. iuc3 isolates corresponded to multiple (n=47) host sequence types (STs), with ST35, ST45, ST881, ST25, and ST967 harbouring iuc3 in both datasets. We generated hybrid assemblies for 44 (SpARK) and 36 (OH-DART) isolates, plus a single iuc3 isolate from Germany. 53 (65·4%) of these isolates were from pigs, three (3·7%) from clinical sources, and 25 (30·9%) from other sources. There were an additional 48 iuc3 positive isolates from our collections for which only short read data was available. A single iuc3-positive Klebsiella oxytoca isolate from a pig farm was detected in the SpARK data, which was also sequenced. We identified 330 iuc3-positive isolates and 58 iuc3-carrying plasmid assemblies from NCBI, of which 83 (21·4%) were from clinical sources, 120 from pigs (30·9%), and 185 (47·7%) from other sources or of unknown provenance. These isolates were from K pneumoniae except two isolates of Klebsiella quasipneumoniae subsp similipneumoniae and one of Enterobacter hormaechei. The combined dataset of 517 iuc3 plasmids ranged in size from 110 375 bp to 365 580 bp and mostly corresponded to multiple IncFIB(K) and IncFII replicon types. We found seven convergent K pneumoniae plasmids in the Thai data: six from fresh markets and one from a neighbouring hospital. These plasmids emerged through the hybridisation of cocirculating iuc3 plasmids and plasmids encoding extended-spectrum β-lactamases (ESBLs), although none of these seven plasmids carried genes encoding carbapenemases. We also identified putative cocirculating parental plasmids carrying iuc3 and ESBL-encoding genes. Clustering and phylogenetic analysis resolved the iuc3 plasmid sequences into three groups, which were consistent using both complete plasmid sequences (n=139) and short-read data (n=517). In the complete plasmid sequence data, 66 strains contained group 1 plasmids, 38 strains contained group 2 plasmids, and 35 strains contained group 3 plasmids. Group 3 plasmids are mostly carried by isolates circulating in hospitals throughout Asia, with occasional examples in Europe and elsewhere, and carry multiple ARGs and potential virulence factors. By contrast, group 1 plasmids are commonly carried by porcine isolates in Europe, and group 2 are a heterogeneous mixture of geographical and ecological sources. Interpretation Plasmid hybridisation occurs frequently outside of the health-care environment and can lead to the convergence of resistance and virulence traits. Generating complete plasmid sequences from regional population-scale samples facilitates the identification of convergent plasmids and their putative parental plasmids. Three robust groups of iuc3 plasmids were resolved, which show both epidemiological and geographical differences; one of these groups was associated with clinical isolates in Asia and warrants targeted plasmid surveillance. Funding UKRI, JPIAMR, Evolution Education Trust, and a Schlumberger Foundation Fellowship.
Agrobacterium tumefaciens, a soil bacterium, was used as a model organism to study the mechanisms of triclosan (TCS) resistance in environmental bacteria. Adaptive laboratory evolution tests were performed to select TCS-resistant strains by challenging a wild-type (WT) strain with increasing concentrations of TCS (8, 12, 16, and 20 µg/mL). Two high-dose-resistant strains, HDR-12a and HDR-20a, were isolated and used for detailed examination. In comparison to the minimum inhibitory concentration of the WT strain (10 µg/mL), HDR-12a (20 µg/mL), and HDR-20a (32 µg/mL) showed increased resistance to TCS. Whole-genome sequencing and transcriptomic analysis performed to identify mechanisms underlying the different degrees of TCS resistance among the two evolved A. tumefaciens strains revealed a nucleotide base change (missense mutation, Asn157Thr) in the transcriptional repressor triR gene as the key mechanism of TCS resistance in HDR-20a. This change reduced the DNA-binding ability of TriR, causing overexpression of the triABC operon that encodes the TCS-specific efflux pump. In contrast, HDR-12a had no mutation in the triR gene. HDR-12a exhibited transcriptomic changes in several genes involved in ATP-binding cassette (ABC) transporters and in the metabolism of sulfur, fatty acids, and carbohydrates. However, it remains unclear whether these transcriptomic changes are directly responsible for TCS resistance in HDR-12a. Both the TCS-adapted strains also showed increased resistance to chloramphenicol and erythromycin. Overall, these results demonstrate that TCS pollution in environmental hotspots can select for adaptive and cross-resistant bacteria.IMPORTANCETCS is widely used as a preservative and disinfectant in many personal healthcare products. TCS is subsequently released into aquatic and terrestrial environments. The emergence and spread of multidrug-resistant pathogens from the use of antimicrobials like TCS and the misuse of antibiotic drugs now pose a serious global public health threat. Understanding how resistance develops has implications for preventing the emergence of antimicrobial resistance. The adapted TCS-resistant strains showed cross-resistance to chloramphenicol and erythromycin. This study provides insight into how environmental exposure to triclosan can drive adaptive and cross-resistance mechanisms in a soil bacterium, highlighting its relevance to environmental antimicrobial resistance and public health risk.
