Exploring the Localization of Siderophore-Mediated Cargo Delivery in Gram-Negative Bacteria Using 3-Hydroxypyridin-4(1H)-one-Fluorescein Probes.

The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1H)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate 20 in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1H)-one-MG conjugate 26 was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of Escherichia coli and Pseudomonas aeruginosa PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1H)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1H)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1H)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1H)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1H)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1H)-ones as siderophores.

Cajaninstilbene acid derivatives conjugated with siderophores of 3-hydroxypyridin-4(1H)-ones as novel antibacterial agents against Gram-negative bacteria based on the Trojan horse strategy.

The low permeability of the outer membrane of Gram-negative bacteria is a serious obstacle to the development of new antibiotics against them. Conjugation of antibiotic with siderophore based on the "Trojan horse strategy" is a promising strategy to overcome the outer membrane obstacle. In this study, series of antibacterial agents were designed and synthesized by conjugating the 3-hydroxypyridin-4(1H)-one based siderophores with cajaninstilbene acid (CSA) derivative 4 which shows good activity against Gram-positive bacteria by targeting their cell membranes but is ineffective against Gram-negative bacteria. Compared to the inactive parent compound 4, the conjugates 45c or 45d exhibits significant improvement in activity against Gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae and especially P. aeruginosa (minimum inhibitory concentrations, MICs = 7.8-31.25 µM). The antibacterial activity of the conjugates is attributed to the CSA derivative moiety, and the action mechanism is by disruption of bacterial cell membranes. Further studies on the uptake mechanisms showed that the bacterial siderophore-dependent iron transport system was involved in the uptake of the conjugates. In addition, the conjugates 45c and 45d showed a lower cytotoxic effects in vivo and in vitro and a positive therapeutic effect in the treatment of C. elegans infected by P. aeruginosa. Overall, our work describes a new class and a promising 3-hydroxypyridin-4(1H)-one-CSA derivative conjugates for further development as antibacterial agents against Gram-negative bacteria.

3-Hydroxypyridin-4(1H)-one Derivatives as pqs Quorum Sensing Inhibitors Attenuate Virulence and Reduce Antibiotic Resistance in Pseudomonas aeruginosa.

The development of quorum sensing inhibitors capable of decreasing the production of virulence factors is an effective strategy to overcome resistance in Pseudomonas aeruginosa due to the less selective pressure exerted on bacteria. In this study, a series of 3-hydroxypyridin-4(1H)-one derivatives bearing a 4-aminomethyl-1,2,3-triazole linker were designed and synthesized as antivirulence agents against P. aeruginosa. The most potent derivative 16e was identified as a selective inhibitor of the pqs system (IC50 = 3.7 µM) and its related virulence factor pyocyanin (IC50 = 2.7 µM). In addition, 16e exhibited moderate biofilm inhibition and significant inhibition of P. aeruginosa motility phenotypes with low cytotoxicity. Compound 16e showed an obvious antibacterial synergistic effect in combination with antibiotics such as ciprofloxacin and tobramycin in in vitro and in vivo Caenorhabditis elegans infection models. Overall, the excellent antivirulence properties of compound 16e make it a potential antibiotic adjuvant for the treatment of P. aeruginosa infections that may be advanced into preclinical development in the future.

Design, synthesis and biological evaluation of novel 3-hydroxypyridin-4(1H)-ones based hybrids as Pseudomonas aeruginosa biofilm inhibitors.

Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative pathogenic bacterium, often causative drug-resistance related human infections, given its great capacity to form bioflm. It uses three major quorum sensing (QS) systems, las, rhl, and pqs, to regulate the expression of genes related to virulence and biofilm formation. Consequently, strategies for inhibiting QS have garnered considerable attention as antimicrobial therapies. In this study, we designed and synthesized several 3-hydroxypyridin-4(1H)-one hybrids and assessed their potential as the inhibitors of P. aeruginosa biofilm formation. The most active compound identified was 12h; it exhibited satisfactory biofilm inhibitory activity (IC50: 10.59 ± 1.17 µM). Mechanistic studies revealed that 12h significantly inhibited the fluorescence of the PAO1-lasB-gfp and PAO1-pqsA-gfp fluorescent reporter strains and the production of Las-regulated (elastase) and Pqs-regulated (pyocyanin) virulence factors. These findings indicate that 12h inhibited biofilm formation by suppressing the expression of lasB and pqsA, thereby inactivating the las and pqs pathways. Furthermore, 12h improved the antibiotic susceptibility of P. aeruginosa and reduced the acute virulence of this bacterium in the African green monkey kidney cell line Vero. In conclusion, 3-hydroxypyridin-4(1H)-one hybrids, such as 12h, represent a promising class of antibacterial agents against P. aeruginosa.

3-Hydroxy-pyridin-4(1H)-ones as siderophores mediated delivery of isobavachalcone enhances antibacterial activity against pathogenic Pseudomonas aeruginosa.

The natural prenylated chalcone isobavachalcone (IBC) shows good antibacterial activity against Gram-positive bacteria but is ineffective against Gram-negative bacteria, most likely due to the outer membrane barrier of Gram-negative bacteria. The Trojan horse strategy has been shown to be an effective strategy to overcome the reduction in the permeability of the outer membrane of Gram-negative bacteria. In this study, eight different 3-hydroxy-pyridin-4(1H)-one-isobavachalcone conjugates were designed and synthesized based on the siderophore Trojan horse strategy. The conjugates exhibited 8- to 32-fold lower minimum inhibitory concentrations (MICs) and 32- to 177-fold lower half-inhibitory concentrations (IC50s) against Pseudomonas aeruginosa PAO1 as well as clinical multidrug-resistant (MDR) strains compared to the parent IBC under iron limitation. Further studies showed that the antibacterial activity of the conjugates was regulated by the bacterial iron uptake pathway under different iron concentration conditions. Studies on the antibacterial mechanism of conjugate 1b showed that it exerts antibacterial activity by disrupting cytoplasmic membrane integrity and inhibiting cell metabolism. Finally, conjugate 1b showed a lower cytotoxic effects on Vero cells than IBC and a positive therapeutic effect in the treatment of bacterial infections caused by Gram-negative bacteria PAO1. Overall, this work demonstrates that IBC can be delivered to Gram-negative bacteria when combined with 3-hydroxy-pyridin-4(1H)-ones as siderophores and provides a scientific basis for the development of effective antibacterial agents against Gram-negative bacteria.

The impact of BRAF targeting agents in advanced anaplastic thyroid cancer: a multi-institutional retrospective study in Taiwan.

Anaplastic thyroid cancer (ATC) is a rare but lethal thyroid cancer. Dabrafenib and trametinib has been the standard treatment for the patients with BRAF mutation based on phase II study. This study aimed to exam the impact of dabrafenib and trametinib in ATC patients. ATC patients treated in three institutes in Taiwan were retrospectively reviewed. The clinical features, BRAF status, and survivals were collected. Multivariate analysis was performed to determine the independent prognostic factors. A total of 44 ATC patients were enrolled in current study. Twelve (50%) out of 24 detected patients had BRAF V600E mutation and eleven received dabrafenib and trametinib treatment. Patients treated with dabrafenib and trametinib had longer overall survival (OS) than the patients without treatment with dabrafenib and trametinib (median OS: 10.4 months vs. 3.3 months, P=0.05). The objective response rate was 81.8% and progress-free survival was 7.4 months. Multivariate analysis identified prior surgery, treatment with dabrafenib and trametinib and metastasis to lung, brain, and bone were significant prognostic factors for OS. The benefit of prior surgery was significant in patients receiving dabrafenib and trametinib (P=0.017) rather than those without dabrafenib and trametinib (P=0.067). The current study provides the real-world evidence that targeted therapy with dabrafenib and trametinib was effective and significantly improved the OS for ATC patients. The role of prior surgery became important in the era of targeted therapy. Future studies should focus on resistance mechanisms and combination with immunotherapy for ATC patients.

Invasion and Propagation of White Spot Syndrome Virus: Hijacking of the Cytoskeleton, Intracellular Transport Machinery, and Nuclear Import Transporters.

The pathogenesis of white spot syndrome virus (WSSV) is largely unclear. In this study, we found that actin nucleation and clathrin-mediated endocytosis were recruited for internalization of WSSV into crayfish hematopoietic tissue (Hpt) cells. This internalization was followed by intracellular transport of the invading virions via endocytic vesicles and endosomes. After envelope fusion within endosomes, the penetrated nucleocapsids were transported along microtubules toward the periphery of the nuclear pores. Furthermore, the nuclear transporter CqImportin α1/ß1, via binding of ARM repeat domain within CqImportin α1 to the nuclear localization sequences (NLSs) of viral cargoes and binding of CqImportin ß1 to the nucleoporins CqNup35/62 with the action of CqRan for docking to nuclear pores, was hijacked for both targeting of the incoming nucleocapsids toward the nuclear pores and import of the expressed viral structural proteins containing NLS into the cell nucleus. Intriguingly, dysfunction of CqImportin α1/ß1 resulted in significant accumulation of incoming nucleocapsids on the periphery of the Hpt cell nucleus, leading to substantially decreased introduction of the viral genome into the nucleus and remarkably reduced nuclear import of expressed viral structural proteins with NLS; both of these effects were accompanied by significantly inhibited viral propagation. Accordingly, the survival rate of crayfish post-WSSV challenge was significantly increased after dysfunction of CqImportin α1/ß1, also showing significantly reduced viral propagation, and was induced either by gene silencing or by pharmacological blockade via dietary administration of ivermectin per os. Collectively, our findings improve our understanding of WSSV pathogenesis and support future antiviral designing against WSSV. IMPORTANCE As one of the largest animal DNA viruses, white spot syndrome virus (WSSV) has been causing severe economical loss in aquaculture due to the limited knowledge on WSSV pathogenesis for an antiviral strategy. We demonstrate that the actin cytoskeleton, endocytic vesicles, endosomes, and microtubules are hijacked for WSSV invasion; importantly, the nuclear transporter CqImportin α1/ß1 together with CqRan were recruited, via binding of CqImportin ß1 to the nucleoporins CqNup35/62, for both the nuclear pore targeting of the incoming nucleocapsids and the nuclear import of expressed viral structural proteins containing the nuclear localization sequences (NLSs). This is the first report that NLSs from both viral structure proteins and host factor are elaborately recruited together to facilitate WSSV infection. Our findings provide a novel explanation for WSSV pathogenesis involving systemic hijacking of host factors, which can be used for antiviral targeting against WSSV disease, such as the blockade of CqImportin α1/ß1 with ivermectin.

[Effect of Ferula ferulaeoides on growth and apoptosis of human gastric cancer MGC-803 transplantation tumor in nude mice].

In this paper,the effects of active fractions of Ferula ferulaeoides on the growth and apoptosis of human gastric cancer cell MGC-803 transplantation tumor were systematically studied. The subcutaneous ectopic transplantation tumor model was established in human gastric cancer MGC-803 nude mice by cell suspension implantation method. The anti-tumor rate and organ index were used to evaluate the anti-tumor effect of the active fractions of F. ferulaeoides on the tumor-bearing nude mice. HE staining,TUNEL staining,RT-PCR,Western-blot and ELISA were used for pathological examination,apoptosis observation,and detection of apoptosis-related genes,proteins and cytokines expression. The results showed that as compared with the model group,the low,medium and high doses of the active fraction of F. ferulaeoides had inhibitory effects on xenografts in nude mice,respectively,in a dose-dependent manner; the apoptotic ratio was increased with the increase of drug concentration. As compared with the model group,F. ferulaeoides could down-regulate the expression of survivin mRNA in nude mice,and the protein expression levels of Bax,Bcl-2,caspase-3 and caspase-9 in tumor tissues of nude mice could be increased to different degrees in F. ferulaeoides groups. The contents of IL-10 and TGF-ß1 in plasma of nude mice were decreased in high dose group of F. ferulaeoides active fractions. The results indicated that F. ferulaeoides can significantly inhibit the growth of human gastric cancer MGC-803 subcutaneously transplanted tumor,and its mechanism may be related with down-regulating the expression of survivin mRNA,and up-regulating the expression of apoptosis-related proteins Bax,caspase-3 and caspase-9.

PI3K inhibitor provides durable response in metastatic metaplastic carcinoma of the breast: A hidden gem in the BELLE-4 study.

PURPOSE: Metaplastic carcinoma of the breast (MCB) is a rare cancer characterized by the histologic presence of two or more histological cell types originating from epithelial and mesenchymal stem cells. Patients with metastatic MCB have a low response rate to conventional chemotherapy and poor survival. Optimal treatment strategies for metastatic MCB are urgently needed. METHODS: We retrospectively reviewed a patient who had enrolled in the phase II/III seamless study, BELLE-4 (NCT01572727). The patient's response to the study drug assessed by an investigator per protocol and clinical course were examined and compared with those of the main cohorts in the BELLE-4 study. RESULTS: Our patient exhibited metastatic MCB and received systemic chemotherapy, paclitaxel (70 mg/m2/week) and buparlisib (80 mg/day), a pan-class I phosphatidylinositol-3 kinase (PI3K) inhibitor. The optimal response was a confirmed partial response for 17 months in total. During the compassionated use program period, the tumor regrew when buparlisib was stop because of toxicity, and responded to the treatment again after resumed the buparlisib treatment. The overall survival of the patient after the development of metastatic MCB was 42 months. She experienced grade 3 hyperglycemia similar to that observed in the main cohort. CONCLUSION: Buparlisib plus weekly paclitaxel might be a new treatment option for patients with metastatic MCB harboring a PIK3CA mutation. Additional prospective studies for investigating the efficacy of the proposed combination are warranted.