Dronedarone Enhances the Antibacterial Activity of Polymyxin B and Inhibits the Quorum Sensing System by Interacting with LuxS.

Quorum sensing (QS) is considered an appealing target for interference with bacterial infections. ß-Adrenergic blockers are promising anti-QS agents but do not have antibacterial activity. We assessed the potential ability of adrenergic receptor inhibitors to enhance the antibacterial activity of polymyxin B (PB) against Klebsiella pneumoniae and determined that dronedarone has the most potent activity both in vitro and in vivo. We found that dronedarone increases the thermal stability of LuxS, decreases the production of AI-2, and affects the biofilm formation of K. pneumoniae. We also identified the direct binding of dronedarone to LuxS. However, the mechanism by which dronedarone enhances the antibacterial activity of PB has not been elucidated and is worthy of further exploration. Our study provides a basis for the future development of drug combination regimens.

The enhancement effect of small molecule Lyb24 reveals AzoR as a novel target of polymyxin B.

Given the important role of polymyxin B (PB) in the treatment of drug-resistant Gram-negative bacterial infections, the emergence of PB resistance poses a serious threat to public health. Adjuvant development is a supplementary strategy that can compensate for the lack of novel antibiotics by protecting PB. In this study, we found a small molecule named Lyb24 that showed weak antibacterial activity (minimum inhibitory concentration ≥ 10 µg/ml) but potentiated and revitalized the efficacy of PB against Gram-negative pathogens, including mcr-1- and mgrB-deletion-mediated PB-resistant strains. Our results showed that Lyb24 inhibits the translational levels of genes associated with the modification of lipid A. In addition, Lyb24 increases the permeability, disrupts the integrity and induces the depolarization of the membrane. We further found that both Lyb24 and PB could directly bind to AzoR and inhibit its activity. Structural analysis showed that Lyb24 binds to the isoalloxazine ring of flavin mononucleotide (FMN) through pi-pi stacking and loop η4 of AzoR. A pneumonia model was used to confirm that the activity against clinical PB-resistant Klebsiella pneumoniae was enhanced due to Lyb24 on PB. In conclusion, we provide a potential therapeutic regimen by combining Lyb24 and PB to treat Gram-negative-resistant bacterial infections. Our findings not only explain the synergistic effect of Lyb24, but also expand our knowledge on the mechanism of action of PB.

Pyrimirhodomyrtone inhibits Staphylococcus aureus by affecting the activity of NagA.

The epidemic of methicillin-resistant Staphylococcus aureus (MRSA) infections has created a critical health threat. The drug resistance of MRSA makes the development of drugs with new modes of action particularly urgent. In this study, we found that a natural product derivative pyrimirhodomyrtone (PRM) exerted antibacterial activity against S. aureus, including MRSA, both in vitro and in vivo. Genetic and biochemical studies revealed the interaction between PRM and N-acetylglucosamine-6-phosphate deacetylase (NagA) and the inhibitory effect of PRM on its deacetylation activity. We also found that PRM causes depolarization and destroys the integrity of the cell membrane. The elucidation of the antibacterial mechanism will inspire the subsequent development of new anti-MRSA drugs based on PRM.

A cyclic adenosine monophosphate response element-binding protein inhibitor enhances the antibacterial activity of polymyxin B by inhibiting the ATP hydrolyzation activity of CrrB.

The emergence of polymyxin B (PB) resistant Gram-negative bacteria poses an important clinical and public health threat. Antibiotic adjuvants development is a complementary strategy that fills the gap in new antibiotics. Here, we described the discovery of the enhancement capacity of compound 666-15, previously identified as an inhibitor of cyclic adenosine monophosphate response element-binding protein (CREB), on the activity of PB against Klebsiella pneumoniae in vitro and in vivo. Mechanistic studies showed that this compound reduced the transcription and translation levels of genes related to lipid A modification in the presence of PB. We also identified that 666-15 reduces the ATP hydrolyzation activity of CrrB, and P151L mutation mediates the resistance of bacteria to the enhancement of 666-15. Our results demonstrated the potential of 666-15 in clinical application and support the further development of a PB synergist based on this compound.

A discovery of clinically approved Panlongqi Tablet for repositioning to treat osteoarthritis by inhibiting PI3K/AKT activation.

BACKGROUND: Panlongqi Tablet (PLQT) is a Chinese patent drug composed of 29 kinds of traditional Chinese medicines. Clinical practice has shown that PLQT can relieve osteoarthritis-caused joint pain, but its effects and mechanisms in other pathological links of osteoarthritis have not been characterized. PURPOSE: The purpose of this study is to reposition the pharmacodynamic effects of PLQT through network pharmacology analysis combined with experimental validation, and also to preliminarily explore its possible mechanism. METHODS: On the basis of integrating the relevant targets of PLQT in multiple drug databases and osteoarthritis-related targets in the disease database, an interaction network of related genes was constructed. The hub candidate targets of PLQT in the treatment of osteoarthritis were determined by calculating the main network topological characteristics, The specific functions and pathways of these targets acting on osteoarthritis were modularly analyzed. In addition, the modified Hulth-induced rat model of osteoarthritis and IL-1ß-induced in vitro model of osteoarthritis were established to further validate the potential efficacy and possible mechanism of PLQT. RESULTS: A total of 138 key targets related to osteoarthritis were selected based on topological parameters, and their biological functions were mainly enriched in four over-expressed modules of cartilage degeneration, inflammatory response, immune response, and subchondral bone metabolism. The hub candidate targets had the highest enrichment degree in the TLR4-RAC1-PIK3CA-Akt-NFκB signaling axis of the PI3K/Akt signaling pathway. In vivo results showed that PLQT treatment significantly inhibited the degeneration of proteoglycan and collagen in the cartilage of osteoarthritis rats, suppressed chondrocyte apoptosis, and reduced the Mankin score of joints. Moreover, PLQT alleviated synovial inflammation, reduced the Krenn score of synovium, inhibited the formation of osteophytes in osteoarthritis rats, reduced the bone mineral density (BMD), fractional bone volume (BV/TV), and trabecular thickness (Tb.Th.), as well as increased the trabecular separation (Tb.Sp.) of subchondral bone and the thickness of the subchondral bone plate (SBP.Th.). PLQT suppressed the expressions of TLR4, RAC1, PIK3CA, p-Akt, MMP-13, and ADAMTS-5 in the cartilage, and inhibited the expression of NFκB p65 in the chondrogenic nucleus. Meanwhile, as downstream effector factors of the predictive pathways, the levels of serum interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2) were decreased after PLQT treatment. In vitro results also showed that PLQT could inhibit the expression of key proteins and downstream effector factors of the signaling axis, and this inhibition disappeared when pathway agonists were added. CONCLUSION: PLQT exerted pharmacological effects on the key pathological links of osteoarthritis including chondrocyte apoptosis, extracellular matrix degradation, inflammation, and subchondral bone metabolism by inhibiting the TLR4-RAC1-PIK3CA-Akt-NFκB axis-related proteins.

Disulfiram Enhances the Activity of Polymyxin B Against Klebsiella pneumoniae by Inhibiting Lipid A Modification.

BACKGROUND: The use of antibiotic adjuvants is a complementary strategy to the development of new antibiotics. The essential role of the ArnA dehydrogenase domain (ArnA_DH) in the addition of 4-amino-L-arabinose (L-Ara4N) to lipid A makes it a potential target in polymyxin adjuvant design. PURPOSE: This study aimed to identify a dehydrogenase inhibitor that enhances the antibacterial effect of polymyxin B (PB) and to further understand the mechanism of this drug combination. METHODS: A susceptible K. pneumoniae strain, ATCC13883, was used to screen a dehydrogenase inhibitor library based on 3-(4,5)-dimethylthiazol(-z-y1)-2,5-diphenyltetrazolium bromide (MTT) and chequerboard assays. The protein- and cell-based effects of disulfiram (DSF) on ArnA activity were assessed, and the transcription levels of genes in the arn operon were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Lipid A was isolated, and a structural analysis was performed. The cell wall function was evaluated through membrane integrity and bacterial viability assays. The in vivo antibacterial activity was evaluated using a mouse pulmonary infection model. RESULTS: We screened a dehydrogenase inhibitor library and found that the anti-alcoholism drug DSF significantly enhanced the antibacterial activity of PB in vitro and in vivo. The protein-based enzyme activity assay showed that DSF exerted no direct effect on the dehydrogenase activity of ArnA. Treatment with the combination of DSF and PB but not with PB alone decreased both the transcription level of genes in the arn operon and the modification level of lipid A. DSF also strengthened the disruption of the cell membrane integrity of PB. Moreover, the enhanced PB antibacterial activity was effective against clinical PB-resistant strains. CONCLUSION: We identified a new drug combination that can be used to reduce the necessary dosage of PB and overcome PB resistance, and this drug combination has good prospects for clinical application.

Verdinexor, a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis.

Esophageal carcinoma (EC) ranks sixth among cancers in mortality worldwide and effective drugs to reduce EC incidence and mortality are lacking. To explore potential anti-esophageal cancer drugs, we conducted drug screening and discovered that verdinexor, a selective inhibitor of nuclear exportin 1 (XPO1/CRM1), has anti-esophageal cancer effects both in vivo and in vitro. However, the mechanism and role of verdinexor in esophageal cancer remain unknown. In the present study, we observed that verdinexor inhibited the proliferation and migration of EC cells in vitro and suppressed tumor growth in vivo. Additionally, we found that verdinexor induced cleavage of PARP and downregulated XPO1, c-Myc, and FOSL1 expression. RNA-sequence analysis and protein-protein interaction (PPI) analysis revealed that verdinexor regulated the XPO1/c-Myc/FOSL1 axis. The results of immunoprecipitation and proximity ligation assays confirmed that verdinexor disrupted the interaction between XPO1 and c-Myc. Overexpression of c-Myc rescued the inhibition of cell proliferation and cell migration caused by verdinexor. Overexpressed FOSL1 restored the inhibited migration by verdinexor. Taken together, verdinexor inhibited cell proliferation and migration of esophageal cancer via XPO1/c-Myc/FOSL1 axis. Our findings provide a new option for the development of anti-esophageal cancer drugs.

A small molecule interacts with pMAC-derived hydroperoxide reductase and enhances the activity of aminoglycosides.

The threat of antimicrobial resistance calls for more efforts in basic science, drug discovery, and clinical development, particularly gram-negative carbapenem-resistant pathogens. We sought to identify novel antibacterial agents against Acinetobacter baumannii ATCC19606 using whole cell-based screening. A small molecule named 6D1 with the chemical structure of 6-fluorobenzo[d]isothiazol-3(2H)-one was identified and exhibited activity against A. baumannii ATCC19606 strain (minimal inhibitory concentration, MIC = 1 mg l-1). The mutation in the plasmid-derived ohrB gene that encodes a peroxidase was identified in spontaneously resistant mutants. Treatment of the bacteria with 6D1 resulted in increased sensitivity to peroxide, such as tert-butyl hydroperoxide. The binding of 6D1 and OhrB was confirmed by surface plasmon resonance. Interestingly, the MIC of kanamycin and gentamicin against spontaneously resistant mutants decreased. Finally, we identified the effect of 6D1 on enhancing the antibacterial activity of kanamycin and gentamicin, including against New Delhi metallo-ß-lactamase (NDM-1)-producing carbapenem-resistant Klebsiella pneumoniae, but not in strains carrying aminoglycosides resistance genes. In this study, we identified a small molecule that suppresses the growth of A. baumannii, interacts with hydroperoxide reductase from A. baumannii ATCC19606 plasmid pMAC, and enhances the antibacterial activity of kanamycin and gentamicin. We propose that peroxidase may be potentially used as a target for aminoglycosides adjuvant development.

Rapid Identification of KL49 Acinetobacter baumannii Associated with Clinical Mortality.

OBJECTIVE: We aimed to establish a tool for rapid identification of KL49 Acinetobacter baumannii. METHODS: Based on the capsular polysaccharide (CPS) synthesis genes database, we investigated the distribution of K locus type 49 (KL49) genes in other KL types and established a rapid identification method for KL49. We collected 61 clinical carbapenem-resistant A. baumannii (CRAB) strains, identified KL49 by gtr100 detection, and used whole genome sequencing (WGS) for verification. A mouse pneumonia model was used to confirm the hypervirulence phenotype. We tested the presence of gtr100 gene in 165 CRAB strains from three provinces in China and evaluated the correlation of gtr100 carrying CRAB infection with mortality. RESULTS: The gtr100 gene is the CPS synthesis gene found only in KL49. We screened out nine WGS-validated KL49 strains from 61 CRAB clinical strains using polymerase chain reaction (PCR) to detect the gtr100 gene. The survival rates of KL49 strains were significantly lower than nonKL49 strains in a mouse pneumonia model. The survival rates of LAC-4 gtr100 knockout strain decreased significantly. Analysis of phylogenetics showed the worldwide spread of KL49 A. baumannii. Infection of gtr100 carrying CRAB is an independent risk for mortality (OR, 10.76; 95%CI: 3.08-37.55; p<0.001). CONCLUSION: The hypervirulence phenotype of KL49 CRAB and the association with mortality highlight the urgent need for implementing control measures. The rapid identification assay has the potential to facilitate early medical intervention and worldwide surveillance.

Liuweidihuang Pill Alleviates Inflammation of the Testis via AMPK/SIRT1/NF-κB Pathway in Aging Rats.

Liuweidihuang Pill (LP) is a traditional Chinese herbal formula that is often used in clinical practice to treat kidney deficiency syndrome. The present study investigated the antiaging effects of LP in a D-galactose- (D-Gal-) induced subacute aging rat model. The study also attempted to explore whether anti-inflammatory mechanisms that underpin the antiaging effects are mediated by the AMPK/SIRT1/NF-κB signaling pathway. Rats were subcutaneously injected with D-Gal at a dosage of 100 mg/kg/d for 8 weeks. Upon successful induction of aging in the rats, the animal was administered LP at 0.9 g/kg/d by gavage for 4 weeks. Proteins of the testis were subsequently examined by western blot analysis, and associated locations in the testicular tissue were determined by immunohistochemistry. We observed that LP exerted antiaging effects in aging rats following the activation of AMPK/SIRT1. It was also observed that LP inhibited the expression of NF-κB, thereby further attenuating inflammation of the testis. Therefore, LP can alleviate inflammation of the testis via the AMPK/SIRT1/NF-κB pathway in aging rats.

A new periplogenin cardenolide from the seeds of Antiaris toxicaria.

A new periplogenin cardenolide, periplogulcoside (1), together with three known cardenolides, was isolated from the seeds of Antiaris toxicaria. The structure of the new compound was characterized as periplogenin-3-O-ß-D-glucopyranosyl-(1 → 4)-ß-D-glucopyranoside (1) by spectroscopic methods including 1D and 2D NMR, HR-TOF-MS, and CD spectrometry, and the known compounds were identified by comparison of their NMR and HR-TOF-MS data with those reported in the literature. Compound 1 showed significant cytotoxicity against Hela and HepG-2 cell lines.

[Effects of multi-glycosides of tripterygium wilfordii on histological structures and c-kit expression in testes of pubertal rats].

OBJECTIVE: To study the short- and long-term effects of multi-glycosides of tripterygium wilfordii (GTWon) the histological structures of testes in pubertal rats and possible mechanisms. METHODS: Forty-eight 5-week-old male Sprague-Dawley rats were randomly intragastrically administered with low-does GTW(6 g/kg daily)and high-does GTW (12 mg/kg daily) or 1% sodium carboxymethylcellulose (6 mL/kg, control group) for four weeks. The testes were sampled for detecting histological structures and c-kit expression by immunohistochemistry 24 hrs and four weeks after drug discontinuance. RESULTS: The number of spermatogenic cells and the expression of c-kit in testes were reduced in the two GTW treatment groups 24 hrs and 4 weeks after drug discontinuance compared with those in the control group(P<0.05). Four weeks after drug discontinuance atrophy and interstitial edema of seminiferous epitheliumin in testes were observed, and the testis weight and the expression of c-kit in testes were reduced in the high-does GTW group compared with those in the control group (P<0.01). There were no significant differences in the parameters observed between the low-dose GTW and the control group 4 weeks after drug discontinuance. CONCLUSIONS: GTW has adverse effects on testes in a dose-dependent manner in puberty rats. Low-dose GTW may cause reversible short-term injuries to testis tissues. The damage of the interstitial tissue of testes induced by high-dose GTW may be one of the causes of long-term injuries of testes.