Discovery of 5-(Pyrimidin-2-ylamino)-1H-indole-2-carboxamide Derivatives as Nur77 Modulators with Selective and Potent Activity Against Triple-Negative Breast Cancer.

The orphan nuclear receptor Nur77 has been validated as a potential drug target for treating breast cancer. Therefore, focusing on the SAR study of the lead 8b (KDSPR(Nur77) = 354 nM), we found the active compound ja which exhibited improved Nur77-binding capability (KDSPR(Nur77) = 91 nM) and excellent antiproliferative activities against breast cancer cell lines. Interestingly, ja acted as a potent and selective Nur77 antagonist, displaying good potency against triple-negative breast cancer (TNBC) cell lines but did not inhibit human normal breast cancer cell line MCF-10A (SI > 20). Exceptionally, ja Nur77-dependently caused mitochondria dysfunction and induced the caspase-dependent apoptosis by mediating the TP53 phosphorylation pathway. Moreover, ja significantly suppressed MDA-MB-231 xenograft tumor growth but had no apparent side effects in mice and zebrafish. Overall, ja demonstrated to be the first Nur77 modulator mediating the TP53 phosphorylation pathway that has the potential as a novel anticancer agent for TNBC.

Virus-like particles nanoreactors: from catalysis towards bio-applications.

Virus-like particles (VLPs) are self-assembled supramolecular structures found in nature, often used for compartmentalization. Exploiting their inherent properties, including precise nanoscale structures, monodispersity, and high stability, these architectures have been widely used as nanocarriers to protect or enrich catalysts, facilitating catalytic reactions and avoiding interference from the bulk solutions. In this review, we summarize the current progress of virus-like particles (VLPs)-based nanoreactors. First, we briefly introduce the physicochemical properties of the most commonly used virus particles to understand their roles in catalytic reactions beyond the confined space. Next, we summarize the self-assembly of nanoreactors forming higher-order hierarchical structures, highlighting the emerging field of nanoreactors as artificial organelles and their potential biomedical applications. Finally, we discuss the current findings and future perspectives of VLPs-based nanoreactors.

Ultrasound meets the cell membrane: for enhanced endocytosis and drug delivery.

Endocytosis plays a crucial role in drug delivery for precision therapy. As a non-invasive and spatiotemporal-controllable stimulus, ultrasound (US) has been utilized for improving drug delivery efficiency due to its ability to enhance cell membrane permeability. When US meets the cell membrane, the well-known cavitation effect generated by US can cause various biophysical effects, facilitating the delivery of various cargoes, especially nanocarriers. The comprehension of recent progress in the biophysical mechanism governing the interaction between ultrasound and cell membranes holds significant implications for the broader scientific community, particularly in drug delivery and nanomedicine. This review will summarize the latest research results on the biological effects and mechanisms of US-enhanced cellular endocytosis. Moreover, the latest achievements in US-related biomedical applications will be discussed. Finally, challenges and opportunities of US-enhanced endocytosis for biomedical applications will be provided.

Alterations in the Gut Microbiome of Individuals With Tuberculosis of Different Disease States.

Objective: There is evidence that the gut microbiota play a regulatory role in the occurrence and progression of tuberculosis. The purpose of the current study was to explore the alterations in gut microbiome under different tuberculosis disease stages in the Uyghur population, clarify the composition of microbial taxonomy, search for microbial biomarkers and provide innovative ideas for individual immune prevention and for control strategies. Design: A case-control study of Uyghur individuals was performed using 56 cases of pulmonary tuberculosis (PTB), 36 cases of latent tuberculosis infection (LTBI) and 50 healthy controls (HC), from which stool samples were collected for 16S rRNA gene sequencing. Results: The results showed that the alpha diversity indexes of the PTB group were lower than those of the other two groups (P <0.001), while only observed species were different between LTBI and HC (P <0.05). Beta diversity showed differences among the three groups (P = 0.001). At the genus level, the relative abundance of Bifidobacterium and Bacteroides increased, while Roseburia and Faecalibacterium decreased in the PTB group, when compared with the other two groups, but the changes between the LTBI and HC groups were not significant. The classifier in the test set showed that the ability of the combined genus to distinguish between each two groups was 81.73, 87.26, and 86.88%, respectively, and the validation efficiency was higher than that of a single screened genus. Conclusion: The gut microbiota of PTB patients was significantly disordered compared with LTBI and HC, while the changes of LTBI and HC were not significant. In the future, gut microbiota could be used as a non-invasive biomarker to assess disease activity.

SepF supports the recruitment of the DNA translocase SftA to the Z-ring.

In many bacteria, cell division begins before the sister chromosomes are fully segregated. Specific DNA translocases ensure that the chromosome is removed from the closing septum, such as the transmembrane protein FtsK in Escherichia coli. Bacillus subtilis contains two FtsK homologues, SpoIIIE and SftA. SftA is active during vegetative growth whereas SpoIIIE is primarily active during sporulation and pumps the chromosome into the spore compartment. FtsK and SpoIIIE contain several transmembrane helices, however, SftA is assumed to be a cytoplasmic protein. It is unknown how SftA is recruited to the cell division site. Here we show that SftA is a peripheral membrane protein, containing an N-terminal amphipathic helix that reversibly anchors the protein to the cell membrane. Using a yeast two-hybrid screen we found that SftA interacts with the conserved cell division protein SepF. Based on extensive genetic analyses and previous data we propose that the septal localization of SftA depends on either SepF or the cell division protein FtsA. Since SftA seems to interfere with the activity of SepF, and since inactivation of SepF mitigates the sensitivity of a ∆sftA mutant for ciprofloxacin, we speculate that SftA might delay septum synthesis when chromosomal DNA is in the vicinity.

Control of septum thickness by the curvature of SepF polymers.

Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and/or SepF. We have isolated SepF homologs from different bacterial species and found that they all polymerize into large protein rings with diameters varying from 19 to 44 nm. Interestingly, these values correlated well with the thickness of their septa. To test whether ring diameter determines septal thickness, we tried to construct different SepF chimeras with the purpose to manipulate the diameter of the SepF protein ring. This was indeed possible and confirmed that the conserved core domain of SepF regulates ring diameter. Importantly, when SepF chimeras with different diameters were expressed in the bacterial host Bacillus subtilis, the thickness of its septa changed accordingly. These results strongly support a model in which septal thickness is controlled by curved molecular clamps formed by SepF polymers attached to the leading edge of nascent septa. This also implies that the intrinsic shape of a protein polymer can function as a mold to shape the cell wall.

Theaflavin-3,3´-digallate increases the antibacterial activity of ß-lactam antibiotics by inhibiting metallo-ß-lactamase activity.

Metallo-ß-lactamases (MBLs) are some of the best known ß-lactamases produced by common Gram-positive and Gram-negative pathogens and are crucial factors in the rise of bacterial resistance against ß-lactam antibiotics. Although many types of ß-lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time-kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand-residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin-3,3´-digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time-kill assays, we observed a synergistic effect of TFDG with ß-lactam antibiotics against methicillin-resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with ß-lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of ß-lactam antibiotics against pathogens in vitro and in vivo.

Aloe-emodin Attenuates Staphylococcus aureus Pathogenicity by Interfering With the Oligomerization of α-Toxin.

α-toxin, an essential virulence factor secreted by Staphylococcus aureus (S. aureus), is a critical exotoxin in multiple infections. In this study, we found that aloe-emodin (AE), a natural compound lacking anti-S. aureus activity, could inhibit the hemolytic activity of α-toxin. Oligomerization assays, molecular dynamics simulations, and fluorescence-quenching analyses were used to determine the mechanism of this inhibition. The oligomerization of α-toxin was restricted by the engagement of AE with K110, T112, and M113 of the toxin, which eventually resulted in inhibition of the hemolytic activity. Lactate dehydrogenase and live/dead assays demonstrated that AE decreased the injury of human lung epithelial cells (A549) and mouse lung macrophages (MH-S) mediated by S. aureus. Furthermore, treatment with AE showed robust protective effects in mice infected by S. aureus. These findings suggest that AE effectively inhibited the pore-forming activity of α-toxin and showed a protective effect against S. aureus virulence in vitro and in vivo, which may provide a new strategy and new antibacterial agent for clinical treatment of S. aureus infections.

Chalcone Attenuates Staphylococcus aureus Virulence by Targeting Sortase A and Alpha-Hemolysin.

Staphylococcus aureus (S.aureus) resistance, considered a dilemma for the clinical treatment of this bacterial infection, is becoming increasingly intractable. Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections. Sortase A (SrtA), an enzyme responsible for anchoring virulence-related surface proteins, and alpha-hemolysin (Hla), a pore-forming cytotoxin, have aroused great scientific interest, as they have been regarded as targets for promising agents against S. aureus infection. In this study, we discovered that chalcone, a natural small compound with little anti-S. aureus activity, could significantly inhibit SrtA activity with an IC50 of 53.15 µM and Hla hemolysis activity with an IC50 of 17.63 µM using a fluorescence resonance energy transfer (FRET) assay and a hemolysis assay, respectively. In addition, chalcone was proven to reduce protein A (SpA) display in intact bacteria, binding to fibronectin, formation of biofilm and S. aureus invasion. Chalcone could down-regulate the transcriptional levels of the hla gene and the agrA gene, thus leading to a reduction in the expression of Hla and significant protection against Hla-mediated A549 cell injury; more importantly, chalcone could also reduce mortality in infected mice. Additionally, molecular dynamics simulations and mutagenesis assays were used to identify the mechanism of chalcone against SrtA, which implied that the inhibitory activity lies in the bond between chalcone and SrtA residues Val168, Ile182, and Arg197. Taken together, the in vivo and in vitro experiments suggest that chalcone is a potential novel therapeutic compound for S. aureus infection via targeting SrtA and Hla.

Lysionotin attenuates Staphylococcus aureus pathogenicity by inhibiting α-toxin expression.

α-Toxin, one of the best known pore-forming proteins produced by Staphylococcus aureus (S. aureus), is a critical virulence factor in multiple infections. The necessity of α-toxin for S. aureus pathogenicity suggests that this toxin is an important target for the development of a potential treatment strategy. In this study, we showed that lysionotin, a natural compound, can inhibit the hemolytic activity of culture supernatants by S. aureus by reducing α-toxin expression. Using real-time PCR analysis, we showed that transcription of hla (the gene encoding α-toxin) and agr (the locus regulating hla) was significantly inhibited by lysionotin. Lactate dehydrogenase and live/dead assays indicated that lysionotin effectively protected human alveolar epithelial cells against S. aureus, and in vivo studies also demonstrated that lysionotin can protect mice from pneumonia caused by S. aureus. These findings suggest that lysionotin is an efficient inhibitor of α-toxin expression and shows significant protection against S. aureus in vitro and in vivo. This study supports a potential strategy for the treatment of S. aureus infection by inhibiting the expression of virulence factors and indicates that lysionotin may be a potential treatment for S. aureus pneumonia.

Curcumin Promotes the Clearance of Listeria monocytogenes both In Vitro and In Vivo by Reducing Listeriolysin O Oligomers.

The pore-forming toxin listeriolysin O (LLO), an essential virulence factor that is secreted by Listeria monocytogenes (L. monocytogenes), is responsible for bacterial breaching at the phagosomal membranes and subsequent release into the cytoplasm; it cannot be recognized by the host immune system. The vital role that LLO plays in bacterial pathogenicity and evading host immune clearance makes this virulence a promising target for addressing L. monocytogenes infection. In this study, we hypothesized that curcumin, a polyphenol derived from turmeric that could effectively inhibit LLO pore-forming activity, might be useful in the prevention or treatment of L. monocytogenes infection. Thus, the in vitro protective effects of curcumin against L. monocytogenes infection by targeting LLO were assessed via hemolytic activity assays, cytotoxicity tests, intracellular growth assays, and confocal microscopy. Our results revealed that treating infected macrophages with curcumin can lead to a decrease in LLO-mediated bacteria phagosomal escape and limit the intracellular growth of L. monocytogenes. Moreover, results from animal experiments show that this natural compound effectively increases protection against bacterial infection and helps the host to clear the invading pathogen completely from an animal model, establishing it as a potent antagonist of L. monocytogenes. The results from our molecular modeling and mutational analysis demonstrated that curcumin directly engages with domains 2 and 4 of LLO, thereby decreasing the hemolytic activity of LLO by influencing its oligomerization. Taken together, these results suggest that, as an antitoxin agent, curcumin can be further developed into a novel therapy against L. monocytogenes infections by targeting LLO.

Epigallocatechin gallate inhibits Streptococcus pneumoniae virulence by simultaneously targeting pneumolysin and sortase A.

Streptococcus pneumoniae (pneumococcus), the causative agent of several human diseases, possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. Pneumolysin (PLY), an important virulence factor, is a member of the cholesterol-dependent cytolysin family and has cytolytic activity. Sortase A (SrtA), another crucial pneumococcal virulence determinate, contributes greatly to the anchoring of many virulence-associated surface proteins to the cell wall. In this study, epigallocatechin gallate (EGCG), a natural compound with little known antipneumococcal activity, was shown to directly inhibit PLY-mediated haemolysis and cytolysis by blocking the oligomerization of PLY and simultaneously reduce the peptidase activity of SrtA. The biofilm formation, production of neuraminidase A (NanA, the pneumococcal surface protein anchored by SrtA), and bacterial adhesion to human epithelial cells (Hep2) were inhibited effectively when S. pneumoniae D39 was cocultured with EGCG. The results from molecular dynamics simulations and mutational analysis confirmed the interaction of EGCG with PLY and SrtA, and EGCG binds to Glu277, Tyr358, and Arg359 in PLY and Thr169, Lys171, and Phe239 in SrtA. In vivo studies further demonstrated that EGCG protected mice against S. pneumoniae pneumonia. Our results imply that EGCG is an effective inhibitor of both PLY and SrtA and that an antivirulence strategy that directly targets PLY and SrtA using EGCG is a promising therapeutic option for S. pneumoniae pneumonia.

Phloretin Attenuates Listeria monocytogenes Virulence Both In vitro and In vivo by Simultaneously Targeting Listeriolysin O and Sortase A.

The critical roles of sortase A (SrtA) and listeriolysin O (LLO) in Listeria monocytogenes pathogenicity render these two virulence factors as ideal targets for the development of anti-virulence agents against L. monocytogenes infection. Additionally, the structures of SrtA and LLO are highly conserved among the members of sortase enzyme family and cholesterol dependent toxin family. Here, phloretin, a natural polyphenolic compound derived from apples and pears that has little anti-L. monocytogenes activity, was identified to simultaneously inhibit LLO expression and neutralize SrtA catalytic activity. Phloretin neutralized SrtA activity by causing a conformational change in the protein's active pocket, which prevented engagement with its substrate. Treatment with phloretin simultaneously reduced L. monocytogenes invasion into host cells and blocked the escape of vacuole-entrapped L. monocytogenes into cytoplasm. Further, L. monocytogenes-infected mice that received phloretin showed lower mortality, decreased bacterial burden and reduced pathological injury. Our results demonstrate that phloretin is a promising anti-infective therapeutic for infections caused by L. monocytogenes due to its simultaneous targeting of SrtA and LLO, which may result in fewer side effects than those caused by other antibiotics.

Inhibition of listeriolysin O oligomerization by lutein prevents Listeria monocytogenes infection.

The foodborne pathogenic bacterial species Listeria monocytogenes (L. monocytogenes) has caused incalculable damages to public health, and its successful infection requires various virulence factors, including Listeriolysin O (LLO). By forming pores in phagosomal membranes and even in some organelles, LLO plays an indispensable role in the ability of L. monocytogenes to escape from host immune attacks. Because of its critical role, LLO offers an appropriate therapeutic target against L. monocytogenes infection. Here, lutein, a natural small molecule existing widely in fruits and vegetables, is demonstrated as an effective inhibitor of LLO that works by blocking its oligomerization during invasion without showing significant bacteriostatic activity. Further assays applying lutein in cell culture models of invasion and in animal models showed that lutein could effectively inhibit L. monocytogenes infection. Overall, our results indicate that lutein may represent a promising and novel therapeutic agent against L. monocytogenes infection.