The antibiotic darobactin mimics a ß-strand to inhibit outer membrane insertase.

Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis1-3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets4,5. The natural compound darobactin targets the bacterial insertase BamA6-the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins7-13. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid ß-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.

A new antibiotic selectively kills Gram-negative pathogens.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.

A small-molecule degron with a phenylpropionic acid scaffold.

Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.

The effect of saraglitazar on TGF-ß-induced smad3 phosphorylation and expression of genes related to liver fibrosis in LX2 cell line.

BACKGROUND AND PURPOSE: Liver fibrosis is a reversible liver injury that occurs as a result of many chronic inflammatory diseases and can lead to cirrhosis, which is irreversible and fatal. So, we studied the anti-fibrotic effects of saroglitazar on LX-2 cell lines, as a dual PPARα/γ agonist. METHODS: Cells, after 80% confluence, were treated with TGF-ß (2 ng/mL) for 24 h. Then cells were treated with saroglitazar at different doses (2.5, 5, 10 µM) for 24 h. After same incubation, the cells of control group, TGF-ß group, and TGF-ß + saroglitazar group were harvested for RNA and protein extraction to determine the effects of saroglitazar. RT-PCR and western blot methods were used to express genes related to fibrosis. RESULTS: Our results show that the relative expression of α-SMA, collagen1α, N-cadherin, NOX (1, 2, and 4), and phosphorylated Smad3 protein was significantly higher in TGF-ß-treated cells compared with the normal group, and E-cadherin expression was decreased in TGF-ß-treated cells. After TGF-ß-treated cells were exposed to saroglitazar, the expression of these genes was significantly reversed (P < 0.05). CONCLUSIONS: Our results clearly show the short-term inhibitory role of saroglitazar in the expression of fibrotic factors using the TGF-ß/Smad signaling pathway. These results suggest that saroglitazar can be considered as a suitable therapeutic strategy for fibrotic patients. Although more studies are needed.

Syringin from Tinospora crispa downregulates pro-inflammatory mediator production through MyD88-dependent pathways in lipopolysaccharide (LPS)-induced U937 macrophages.

BACKGROUND: Syringin, a phenylpropanoid glycoside, has exhibited numerous biological properties including inhibitory activities against various immune and inflammatory disorders. In this study, syringin isolated from Tinospora crispa was evaluated for its ability to down-regulate activated nuclear factor-kappa B (NF-κB), phosphoinositide-3-kinase-Akt (PI3K-Akt) and mitogen-activated protein kinases (MAPKs) signal transducing networks in U937 macrophages activated by lipopolysaccharide. METHODS: The attenuating effects of syringin on the productions of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α), and the expressions of signaling molecules of the signaling pathways were investigated by using ELISA, Western blot, and qRT-PCR. RESULTS: Syringin downregulated the NF-κB, MAPKs, and PI3K-Akt signal networks by significantly reducing PGE2 production in the macrophages via suppression of COX-2 gene and protein expression levels. It also reduced TNF-α and IL-1ß secretion and their mRNA expression, suppressed phosphorylation of NF-κB (p65), IKKα/ß, and IκBα, and restored ability of IκBα to degrade. Syringin dose-dependently attenuated Akt, p38 MAPKs, JNK, and ERK phosphorylation. Also, the expression of corresponding upstream signaling molecules toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) were down-regulated in response to syringin treatment. CONCLUSION: The suppressive effect of syringin on the inflammatory signaling molecules in MyD88-dependent pathways suggested it's potential as a drug candidate for development into an agent for treatment of various immune-mediated inflammatory disorders.

High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function.

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the ß-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF's partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

Mechanism of multiple resistance to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon in Avena fatua L. from China.

Avena fatua L. is one of the most damaging and malignant weeds in wheat fields in China. Fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon, which belong to Acetyl-CoA carboxylase- (ACCase), acetolactate synthase- (ALS), and photosystem II- (PS II) inhibitors, respectively, are commonly used in wheat fields and have a long history of use on A. fatua. An A. fatua population (R) resistant to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon was collected from a wheat field in 2020. This study explored the mechanisms of target site resistance (TSR) and non-target site resistance (NTSR) in the multi-resistant A. fatua. Whole-plant bioassays showed that the R population had evolved high resistance to fenoxaprop-P-ethyl and moderate resistance to mesosulfuron-methyl and isoproturon. However, no mutations were detected in the ACCase, ALS, or psbA genes in the R population. In addition, the ACCase and ALS gene expression levels in the R group were significantly higher than those in the susceptible population (S) after treatment with fenoxaprop-P-ethyl or mesosulfuron-methyl. In vitro ACCase and ALS activity assays showed that ACCase and ALS from the R population were insensitive to fenoxaprop and mesosulfuron-methyl, respectively, with resistance indices 6.12-fold and 17.46-fold higher than those of the S population. Furthermore, pretreatment with P450 inhibitors significantly (P < 0.05) reversed the multi-resistant A. fatua's resistance to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon. Sethoxydim, flucarbazone­sodium, chlortoluron, and cypyrafluone were effective in controlling multi-resistance A. fatua. Therefore, the overexpression of ACCase and ALS to synthesize sufficient herbicide-targeting proteins, along with P450-mediated metabolism, conferred resistance to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon in the R population.

New Phenylpropanoids and Disulphides Dimers from Ferula Kuhistanica.

Five undescribed compounds, including three phenylpropanoid derivatives, 4'-methoxycinnamyl isobutyrate (1), 4'-methoxycinnamyl-2"-methyl butyrate (2) and (2Z)-3',4'-dimethoxycinnamyl isovalerate (3) and two disulphides dimers, kuhistanicasulphide A (7) and kuhistanicasulphide B (8) together with five known ones, including three phenylpropanoids (4-6) and two disulphides (9-10), were isolated from the roots of Ferula kuhistanica Korovin. Their structures were elucidated on the basis of spectroscopic analysis, including IR, UV, HRESIMS, NMR and quantum 13C NMR DP4+ probability. Anti-inflammatory and cytotoxic (Hela, A549 and HT-29 cell lines) activities of the obtained compounds was tested, which compounds 4 and 5 demonstrated good anti-inflammatory with IC50 values of 25.41±2.30 µM and 31.70±3.82 µM, respectively.

Nanoparticles Encapsulated γ-Oryzanol as a Natural Prodrug of Ferulic Acid for the Treatment of Oxidative Liver Damage.

Antioxidants are promising therapeutics for treating oxidative stress-mediated liver diseases. Previously, we studied a potent natural antioxidant, ferulic acid, and developed a liposomal formulation of ferulic acid (ferulic-lipo) to improve its solubility. Ferulic-lipo significantly attenuated oxidative damage in the liver by inhibiting reactive oxygenase species (ROS). However, antioxidative liposomes must be less reactive with ROS prior to reaching the target sites to effectively neutralize existing ROS. But ferulic-lipo tends to be oxidized before reaching the liver. Besides, γ-oryzanol has been reported to decompose into ferulic acid in vivo; accordingly, we hypothesized that γ-oryzanol could be employed as a natural prodrug of ferulic acid to improve stability and antioxidative effectiveness. Therefore, in this study, we prepared a liposomal formulation of γ-oryzanol (γ-ory-lipo) and investigated its therapeutic effects in a CCl4-induced rat model of liver injury. We found that γ-ory-lipo has a higher chemical stability than does free γ-oryzanol. Although the antioxidative effect of γ-ory-lipo was lower than that of ferulic-lipo, pretreatment of the HepG2 cells with γ-ory-lipo improved the viability of CCl4-treated cells to a similar level as treatment with ferulic-lipo. γ-Oryzanol was shown to be converted into ferulic acid in vitro and in vivo. Furthermore, intravenous administration of γ-ory-lipo exhibited a similar effectiveness as ferulic-lipo against CCl4-induced hepatotoxicity, which should be the due to the conversion of γ-oryzanol into ferulic acid. These findings demonstrated that γ-ory-lipo could be a good natural prodrug of ferulic acid for eradicating its stability problem.

Evaluation of syringin's neuroprotective effect in a model of neonatal hypoxic-ischemic brain injury.

Background/aim: A significant cause of mortality and morbidity in the neonatal era is hypoxic-ischemic encephalopathy (HIE). This study examined the histopathological analysis and neuroprotective impact of syringin (SYR) in an experimental HIE rat model. Material and methods: On the 7th postnatal day, 24 Wistar albino rats were evaluated in 3 groups using the HIE model under gas anesthesia. In the experiment, Group A received 10 mg/kg SYR plus dimethyl sulfoxide (DMSO), Group B received DMSO only, and Group C served as a sham group. Immunohistochemical techniques were used to assess apoptotic cell measurement and proinflammatory cytokines (TNF-α and IL-1ß primary antibodies). Results: Rats suffering from hypoxic-ischemic brain damage had their apoptosis assessed. The SYR and sham groups had statistically fewer cells undergoing apoptosis (p < 0.001). There was no difference between the groups in terms of IL-1ß and TNF-α during immunohistochemical staining. Neuronal degeneration was significantly lower in the histological evaluation of the hippocampus in the SYR group (p = 0.01). A statistically significant difference (p = 0.01) was observed between the SYR and the control groups regarding pericellular and perivascular edema. Conclusion: SYR reduced apoptosis, perivascular and pericellular edema, and neuronal degeneration in rat cerebral tissue. These results raise the possibility that SYR may have a neuroprotective effect on the harm brought on by HIE. This is the first investigation of SYR's function within the HIE paradigm.

Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering.

Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects.

Proof-of-concept study to evaluate the safety and efficacy of saroglitazar in patients with primary biliary cholangitis.

BACKGROUND & AIM: Saroglitazar is a novel peroxisome proliferator-activated receptor (PPAR) agonist with dual agonistic properties (α/γ). Due to a strong mechanistic rationale, we aimed to test the safety and efficacy of saroglitazar in patients with primary biliary cholangitis (PBC) who were either ursodeoxycholic acid (UDCA) resistant or intolerant. METHODS: In this double-blind, phase II proof-of-concept trial, 37 patients with PBC were randomized to saroglitazar 4 mg (n = 13), saroglitazar 2 mg (n = 14), or placebo (n = 10) daily for 16 weeks. The primary efficacy endpoint was the reduction in alkaline phosphatase (ALP) level at Week 16. RESULTS: A significant reduction of mean ALP levels was observed at Week 16 relative to baseline in both the saroglitazar 4 mg (least-squares [LS] mean =-163.3 U/L, SE = 25.1, p <0.001) and 2 mg (LS mean =-155.8 U/L, SE = 24.4, p <0.001) groups, compared with placebo (LS mean =-21.1 U/L, SE = 28.9). Treatment with saroglitazar resulted in a rapid reduction of ALP concentration at Week 4 that was sustained through the study duration. At least 1 treatment-emergent adverse event occurred in 11 (84.6%) patients in the saroglitazar 4 mg group, in 12 (85.7%) patients in the 2 mg group and in 8 (80%) patients in the placebo group. Study drug was discontinued in 4 patients (3 patients in the 4 mg group and 1 patient in the 2 mg group) due to aminotransferase increases that promptly returned to baseline values after drug discontinuation. CONCLUSIONS: Saroglitazar at 2 mg and 4 mg daily was tolerated and resulted in rapid and sustained improvements in ALP. Further studies are underway at a daily dose of 2 mg and 1 mg due to the higher incidence of elevated liver enzymes observed with the 4 mg dose. CLINICALTRIALS. GOV IDENTIFIER: NCT03112681 LAY SUMMARY: Saroglitazar resulted in a rapid and sustained improvement in alkaline phosphatase levels in patients with primary biliary cholangitis. The mean percentage reductions in alkaline phosphatase levels were 49% and 51% in the saroglitazar 4 mg and 2 mg groups compared to 3% in the placebo group.

Syringin exerts anti-breast cancer effects through PI3K-AKT and EGFR-RAS-RAF pathways.

BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors with the highest mortality in the world. Modern pharmacological studies have shown that Syringin has an inhibitory effect on many tumors, but its anti-BC efficacy and mechanism are still unclear. METHODS: First, Syringin was isolated from Acanthopanax senticosus (Rupr. & Maxim.) Harms (ASH) by systematic solvent extraction and silica gel chromatography column. The plant name is composed of genus epithet, species additive words and the persons' name who give its name. Then, the hub targets of Syringin against BC were revealed by bioinformatics. To provide a more experimental basis for later research, the hub genes which could be candidate biomarkers of BC and a ceRNA network related to them were obtained. And the potential mechanism of Syringin against BC was proved in vitro experiments. RESULTS: Syringin was obtained by liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and high-performance liquid chromatography (HPLC). Bioinformatics results showed that MAP2K1, PIK3CA, HRAS, EGFR, Caspase3, and PTGS2 were the hub targets of Syringin against BC. And PIK3CA and HRAS were related to the survival and prognosis of BC patients, the PIK3CA-hsa-mir-139-5p-LINC01278 and PIK3CA-hsa-mir-375 pathways might be closely related to the mechanism of Syringin against BC. In vitro experiments confirmed that Syringin inhibited the proliferation and migration and promoted apoptosis of BC cells through the above hub targets. CONCLUSIONS: Syringin against BC via PI3K-AKT-PTGS2 and EGFR-RAS-RAF-MEK-ERK pathways, and PIK3CA and HRAS are hub genes for adjuvant treatment of BC.

Gamma-Oryzanol-Rich Fraction from Purple Rice Extract Attenuates Lipopolysaccharide-Stimulated Inflammatory Responses, Migration and VEGFA Production in SW480 Cells via Modulation of TLR4 and NF-κB Pathways.

Inflammatory response facilitating colorectal cancer (CRC) progression is a serious event following operative infection, which can occur in CRC patients. This event is mainly mediated by bacterial lipopolysaccharide (LPS), via a toll like receptor 4 (TLR4) and NF-κB. Hexane soluble fraction (HSF) from purple rice extract (PRE) has been identified as a γ-oryzanol (OR)-rich fraction. Recently, HSF possessed inhibitory effect of LPS-stimulated metastasis of human colon cancer SW480 cells, however the related mechanism was unknown. Thus, this study aimed to investigate the effect of HSF on inflammatory response-associated cancer progression of LPS-stimulated SW480 cells. The various inflammatory mediators, vascular endothelial growth factor-A (VEGFA) and related pathways were evaluated by Western blot and ELISA. Furthermore, cell migration was also determined by migration assays. Of all, HSF seemed to be stronger than OR to attenuate the responsiveness of LPS on various inflammatory mediators, which was related to an obvious reduction of cancer cell migration as well as indistinct disruption on VEGFA production in SW480 cells, via downregulation of TLR4 and NF-κB. Therefore, OR-rich fraction from PRE, against the subsequent inflammatory response and CRC progression following surgery, which could be combined with conventional treatments to increase the survival rate.

Predicting antimicrobial mechanism-of-action from transcriptomes: A generalizable explainable artificial intelligence approach.

To better combat the expansion of antibiotic resistance in pathogens, new compounds, particularly those with novel mechanisms-of-action [MOA], represent a major research priority in biomedical science. However, rediscovery of known antibiotics demonstrates a need for approaches that accurately identify potential novelty with higher throughput and reduced labor. Here we describe an explainable artificial intelligence classification methodology that emphasizes prediction performance and human interpretability by using a Hierarchical Ensemble of Classifiers model optimized with a novel feature selection algorithm called Clairvoyance; collectively referred to as a CoHEC model. We evaluated our methods using whole transcriptome responses from Escherichia coli challenged with 41 known antibiotics and 9 crude extracts while depositing 122 transcriptomes unique to this study. Our CoHEC model can properly predict the primary MOA of previously unobserved compounds in both purified forms and crude extracts at an accuracy above 99%, while also correctly identifying darobactin, a newly discovered antibiotic, as having a novel MOA. In addition, we deploy our methods on a recent E. coli transcriptomics dataset from a different strain and a Mycobacterium smegmatis metabolomics timeseries dataset showcasing exceptionally high performance; improving upon the performance metrics of the original publications. We not only provide insight into the biological interpretation of our model but also that the concept of MOA is a non-discrete heuristic with diverse effects for different compounds within the same MOA, suggesting substantial antibiotic diversity awaiting discovery within existing MOA.

PTEN protects kidney against acute kidney injury by alleviating apoptosis and promoting autophagy via regulating HIF1-α and mTOR through PI3K/Akt pathway.

Phosphatase and tensin homolog (PTEN) deleted on human chromosome 10 is a tumor suppressor with bispecific phosphatase activity, which is often involved in the study of energy metabolism and tumorigenesis. PTEN is recently reported to participate in the process of acute injury. However, the mechanism of PTEN in Ischemia-Reperfusion Injury (IRI) has not yet been clearly elucidated. In this study, mice with bilateral renal artery ischemia-reperfusion and HK-2 cells with hypoxia/reoxygenation (H/R) were used as acute kidney injury models. We demonstrated that PTEN was downregulated in IRI-induced kidney as well as in H/R-induced HK-2 cells. By silencing and overexpressing PTEN with si-PTEN RNA and PHBLV-CMV-PTEN-flag lentivirus before H/R, we found that PTEN protected HK-2 cells against H/R-induced injury reflected by the change in cell activity and the release of LDH. Furthermore, we inhibited HIF1-α with PX-478 and inactivated mTOR with Rapamycin before the silence of PTEN in H/R model. Our data indicated that the renoprotective effect of PTEN worked via PI3K/Akt/mTOR pathway and PI3K/Akt/HIF1-α pathway, hence alleviating apoptosis and improving autophagy respectively. Our findings provide valuable insights into the molecular mechanism underlying renoprotection of PTEN on autophagy and apoptosis induced by renal IRI, which offers a novel therapeutic target for the treatment of AKI.

Isolation, Characterization and In Silico Studies of Secondary Metabolites from the Whole Plant of Polygala inexpectata Pesmen & Erik.

Polygala species are frequently used worldwide in the treatment of various diseases, such as inflammatory and autoimmune disorders as well as metabolic and neurodegenerative diseases, due to the large number of secondary metabolites they contain. The present study was performed on Polygala inexpectata, which is a narrow endemic species for the flora of Turkey, and resulted in the isolation of nine known compounds, 6,3'-disinapoyl-sucrose (1), 6-O-sinapoyl,3'-O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2), 3'-O-(O-methyl-feruloyl)-sucrose (3), 3'-O-(sinapoyl)-sucrose (4), 3'-O-trimethoxy-cinnamoyl-sucrose (glomeratose) (5), 3'-O-feruloyl-sucrose (sibiricose A5) (6), sinapyl alcohol 4-O-glucoside (syringin or eleutheroside B) (7), liriodendrin (8), and 7,4'-di-O-methylquercetin-3-O-ß-rutinoside (ombuin 3-O-rutinoside or ombuoside) (9). The structures of the compounds were determined by the spectroscopic methods including 1D-NMR (1H NMR, 13C NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC), and HRMS. The isolated compounds were shown in an in silico setting to be accommodated well within the inhibitor-binding pockets of myeloperoxidase and inducible nitric oxide synthase and anchored mainly through hydrogen-bonding interactions and π-effects. It is therefore plausible to suggest that the previously established anti-inflammatory properties of some Polygala-derived phytochemicals may be due, in part, to the modulation of pro-inflammatory enzyme activities.

Inhibition of hypoxia-inducible factor-1α alleviates acinar cell necrosis in a mouse model of acute pancreatitis.

Hypoxia-inducible factor-1α (Hif1α) is activated in hypoxia and is closely related to oxidative stress, immunity and cell metabolism. Recently, it is reported that Hif1α is involved in atherosclerosis, ischemia-reperfusion (I/R) injury, alcoholic liver disease and pancreatic tumors. In this study, we found that Hif1 signal pathway is significantly changed in pancreas of acute pancreatitis (AP) mice. Meanwhile, we verified that the high expression of Hif1α injured pancreatic tissues of cerulean-induced AP mice, which prompting that Hif1α participated in the progress of histopathology on AP. We applied a Hif1α inhibitor PX478 and observed that it could alleviate histological injury of pancreas as well as the levels of serum amylase, lipase and proinflammatory cytokine in the murine model of AP induced by caerulein. In addition, PX478 could reduce the formation of necrosome (RIP3 and p-MLKL) and the generation of reactive oxygen species (ROS) in AP mice. Correspondingly, we further confirmed the effectiveness of PX478 in vitro and found that inhibiting Hif1α could mitigated the necrosis of pancreatic acinar cells via reducing the RIP3 and p-MLKL expression and the ROS production. In conclusion, inhibiting Hif1α could protect against acinar cells necrosis in AP, which may provide a new target for the prevention and treatment of AP clinically.

Downregulation of the αvß6 Integrin via RGD Engagement Is Affinity and Time Dependent.

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (αvß6) has been identified as playing a key role in the activation of transforming growth factor-ß (TGFß) that is hypothesized to be pivotal in the development of fibrosis and other diseases. In this study, αvß6 small molecule inhibitors were characterized in a range of in vitro systems to determine affinity, kinetics, and duration of TGFß inhibition. High αvß6 binding affinity was shown to be correlated with slow dissociation kinetics. Compound 1 (high αvß6 affinity, slow dissociation) and SC-68448 (low αvß6 affinity, fast dissociation) induced concentration- and time-dependent internalization of αvß6 in normal human bronchial epithelial (NHBE) cells. After washout, the αvß6 cell surface repopulation was faster for SC-68448 compared with compound 1 In addition, αvß6-dependent release of active TGFß from NHBE cells was inhibited by compound 1 and SC-68448. After washout of SC-68448, release of active TGFß was restored, whereas after washout of compound 1 the inhibition of TGFß activation was maintained and only reversible in the presence of a lysosomal inhibitor (chloroquine). However, SC-68448 was able to reduce total levels of αvß6 in NHBE cells if present continuously. These observations suggest αvß6 can be degraded after high affinity RGD binding that sorts the integrin for lysosomal degradation after internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the αvß6 integrin can also be downregulated after sustained engagement of the RGD binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation. SIGNIFICANCE STATEMENT: The fate of RGD integrin after ligand binding has not been widely investigated. Using the αvß6 integrin as a case study, we have demonstrated that RGD-induced downregulation of αvß6 is both affinity and time dependent. High affinity ligands induced downregulation via lysosomal degradation, likely due to slow dissociation, whereas sustained low affinity ligand engagement was only able to decrease αvß6 expression over longer periods of time. Our study provides a potential unique mechanism for obtaining duration of action for drugs targeting integrins.

Characterization of Negative Allosteric Modulators of the Calcium-Sensing Receptor for Repurposing as a Treatment of Asthma.

Asthma is still an incurable disease, and there is a recognized need for novel small-molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, which were originally developed for oral therapy for osteoporosis and previously tested in the clinic as a novel, single, and comprehensive topical antiasthma therapy. We address the hypotheses, using murine asthma surrogates, that topically delivered CaSR NAMs 1) abolish AHR; 2) are unlikely to cause unwanted systemic effects; 3) are suitable for topical application; and 4) inhibit airway inflammation to the same degree as the current standard of care, inhaled corticosteroids, and, furthermore, inhibit airway remodeling. All four CaSR NAMs inhibited poly-L-arginine-induced AHR in naïve mice and suppressed both AHR and airway inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate, or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, pharmacokinetics/pharmacodynamics, formulation, and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe, and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation, and abrogating some features of airway remodeling. SIGNIFICANCE STATEMENT: Calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs) reduce airway smooth muscle hyperresponsiveness, reverse airway inflammation as efficiently as topical corticosteroids, and suppress airway remodeling in asthma surrogates. CaSR NAMs, which were initially developed for oral therapy of osteoporosis proved inefficacious for this indication despite being safe and well tolerated. Here we show that structurally unrelated CaSR NAMs are suitable for inhaled delivery and represent a one-stop, steroid-free approach to asthma control and prophylaxis.