A pentameric TRPV3 channel with a dilated pore.

Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets1-5. More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers4. Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification6,7. Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research.

Talarodrides A-F, Nonadrides from the Antarctic Sponge-Derived Fungus Talaromyces sp. HDN1820200.

Six new nonadride derivatives, named talarodrides A-F (1-6), were isolated from the Antarctic sponge-derived fungus Talaromyces sp. HDN1820200. All structures including the absolute configurations were deduced by extensive spectroscopic analysis and computational ECD calculations. Compounds 1-4 share a rare caged bicyclo[4.3.1]-deca-1,6-diene with a bridgehead olefin and maleic anhydride core skeleton, while compounds 5 and 6 possess the first case of a naturally occurring 5/7/6 methanocyclonona[c]furan skeleton. Talarodride A (1) and talarodride B (2) showed selective inhibitory effects against Proteus mirabilis and Vibrio parahemolyticus with MICs of 3.13-12.5 µM.

Water-Insensitive Synthesis of Poly-ß-Peptides with Defined Architecture.

Biocompatible and proteolysis-resistant poly-ß-peptides have broad applications and are dominantly synthesized via the harsh and water-sensitive ring-opening polymerization of ß-lactams in a glovebox or using a Schlenk line, catalyzed by the strong base LiN(SiMe3 )2 . We have developed a controllable and water-insensitive ring-opening polymerization of ß-amino acid N-thiocarboxyanhydrides (ß-NTAs) that can be operated in open vessels to prepare poly-ß-peptides in high yields, with diverse functional groups, variable chain length, narrow dispersity and defined architecture. These merits imply wide applications of ß-NTA polymerization and resulting poly-ß-peptides, which is validated by the finding of a HDP-mimicking poly-ß-peptide with potent antimicrobial activities. The living ß-NTA polymerization enables the controllable synthesis of random, block copolymers and easy tuning of both terminal groups of polypeptides, which facilitated the unravelling of the antibacterial mechanism using the fluorophore-labelled poly-ß-peptide.

The effects of licogliflozin, a dual SGLT1/2 inhibitor, on body weight in obese patients with or without diabetes.

BACKGROUND: There is an unmet need for a safer and more effective treatment for obesity. This study assessed the effects of licogliflozin, a dual inhibitor of sodium-glucose co-transporter (SGLT) 1/2, on body weight, metabolic parameters and incretin hormones in patients with type 2 diabetes mellitus (T2DM) and/or obesity. METHODS: Patients with obesity (BMI, 35-50 kg/m2 ) were enrolled into a 12-week study (N = 88; licogliflozin 150 mg q.d.). Patients with T2DM were enrolled into a second, two-part study, comprising a single-dose cross-over study (N = 12; 2.5 - 300 mg) and a 14-day dosing study (N = 30; 15 mg q.d). Primary endpoints included effects on body weight, effects on glucose, safety and tolerability. Secondary endpoints included urinary glucose excretion (UGE24 ) and pharmacokinetics, while exploratory endpoints assessed the effects on incretin hormones (total GLP-1, PYY3-36 , and GIP), insulin and glucagon. RESULTS: Treatment with licogliflozin 150 mg q.d. for 12 weeks in patients with obesity significantly reduced body weight by 5.7% vs placebo (P < 0.001) and improved metabolic parameters such as significantly reduced postprandial glucose excursion (21%; P < 0.001), reduced insulin levels (80%; P < 0.001) and increased glucagon (59%; P < 0.001). In patients with T2DM, a single dose of licogliflozin 300 mg in the morning prior to an oral glucose tolerance test (OGTT) remarkably reduced glucose excursion by 93% (P < 0.001; incremental AUC0-4h ) and suppressed insulin by 90% (P < 0.01; incremental AUC0-4h ). Treatment with licogliflozin 15 mg q.d. for 14 days reduced 24-hour average glucose levels by 26% (41 mg/dL; P < 0.001) and increased UGE24 to 100 g (P < 0.001) in patients with T2DM. In addition, this treatment regimen significantly increased total GLP-1 by 54% (P < 0.001) and PYY3-36 by 67% (P < 0.05) post OGTT vs placebo, while significantly reducing GIP levels by 53% (P < 0.001). Treatment with licogliflozin was generally safe and well tolerated. Diarrhea (increased numbers of loose stool) was the most common adverse event in all studies (90% with licogliflozin vs 25% with placebo in the 12-week study), while a lower incidence of flatulence, abdominal pain and abdominal distension (25%-43% with licogliflozin vs 9%-11% with placebo in the 12-week study) were among the other gastrointestinal events reported. CONCLUSION: Licogliflozin treatment (1-84 days) leads to significant weight loss and favourable changes in a variety of metabolic parameters and incretin hormones. Dual inhibition of SGLT1/2 with licogliflozin in the gut and kidneys is an attractive strategy for treating obesity and diabetes.

Synthesis of Canthardin Sulfanilamides and Their Acid Anhydride Analogues via a Ring-Opening Reaction of Activated Aziridines and Their Associated Pharmacological Effects.

The cantharidinimide derivatives, 5a-h, including sulfanilamides containing pyrimidyl, pyrazinyl, hydrogen, thiazolyl, and oxazolyl groups were synthesized. Modification of cantharidinimide by means of the reaction of activated aziridine ring opening led to the discovery of a novel class of antitumor compounds. The analogues 10i-k, 11l-n, 12o-p, and 16q-s were obtained from treating cantharidinimide 6 and analogues (7, 8, and 13) with activated aziridines, which produced a series of ring-opened products including normal and abnormal types. Some of these compounds showed cytotoxic effects in vitro against HL-60, Hep3B, MCF7, and MDA-MB-231 cancer cells. The most potent cytostatic compound, N-cantharidinimido-sulfamethazine (5a), exhibited anti-HL-60 and anti-Hep3B cell activities. Two compounds 5g and 5h displayed slight effects on the Hep3B cell line, while the other compounds produced no response in these four cell lines.

Poly(sebacic anhydride) nanocapsules as carriers: effects of preparation parameters on properties and release of doxorubicin.

Poly(sebacic anhydride) (PSA) is a promising polymer for the production of drug delivery vehicles. The aim of this work is to study the effect of preparation parameters on the quality of the nanoparticles. In this study, doxorubicin (DOX)-loaded PSA nanocapsules were prepared by an emulsion method. Effects of factors such as type of organic solvent, co-solute (surfactant) and its concentration on drug-loading efficiency, particle size and size distribution, morphology and release profile were examined to gain insight in the preparation and stability of nanostructures. Particles with sizes in the range of 218-1198 nm were prepared. The smallest particles with a narrow size distribution were prepared by using polyvinyl alcohol as a co-solute and dichloromethane as a solvent. Efficiency and intracellular release of doxorubicin from the formulated particles were studied on MDA-MB-231 cells. It was observed that DOX-loaded PSA particles can diffuse into the cells and intracellular antitumour activity is directly related to the released amount of drug from the PSA nanocapsules.

Sensitivity of Neurospora crassa to a marine-derived Aspergillus tubingensis anhydride exhibiting antifungal activity that is mediated by the MAS1 protein.

The fungus Aspergillus tubingensis (strain OY907) was isolated from the Mediterranean marine sponge Ircinia variabilis. Extracellular extracts produced by this strain were found to inhibit the growth of several fungi. Among the secreted extract components, a novel anhydride metabolite, tubingenoic anhydride A (1) as well as the known 2-carboxymethyl-3-hexylmaleic acid anhydride, asperic acid, and campyrone A and C were purified and their structure elucidated. Compound 1 and 2-carboxymethyl-3-hexylmaleic acid anhydride inhibited Neurospora crassa growth (MIC = 330 and 207 µM, respectively) and affected hyphal morphology. We produced a N. crassa mutant exhibiting tolerance to 1 and found that a yet-uncharacterized gene, designated mas-1, whose product is a cytosolic protein, confers sensitivity to this compound. The ∆mas-1 strain showed increased tolerance to sublethal concentrations of the chitin synthase inhibitor polyoxin D, when compared to the wild type. In addition, the expression of chitin synthase genes was highly elevated in the ∆mas-1 strain, suggesting the gene product is involved in cell wall biosynthesis and the novel anhydride interferes with its function.

Acidity-activated aggregation and accumulation of self-complementary zwitterionic peptide-decorated gold nanoparticles for photothermal biofilm eradication.

Drug-resistant biofilm infection is an extremely serious clinical problem, that easily leads to failure of antibiotic treatment. Although gold nanoparticles (AuNPs) as photothermal agents have been widely used in biofilm eradication, there are still challenges to be addressed, such as insignificantly redshifted absorption and slow assembly process of aggregated AuNPs. Herein, we developed an acidity-activated dispersion-to-aggregation transition to enhance the accumulation of self-complementary zwitterionic peptide-decorated AuNPs for photothermal eradication of drug-resistant biofilm infections. AuNPs were decorated with self-complementary zwitterionic peptides (ZP1 and ZP2) coupled with pH-sensitive anhydride (DMA) and pH-insensitive anhydride (SA), respectively. ZP2-decorated AuNPs with DMA modification (AuNP@ZP2(DMA)) exhibited prolonged blood circulation and enhanced accumulation in acidic biofilm microenvironment. Moreover, the electrostatic attraction between self-complementary ligands drove AuNPs to form closely packed aggregates with strong near-infrared absorption, leading to in vivo photoacoustic imaging ability and photothermal effect against drug-resistant bacteria and fungus, as well as microbial biofilms. AuNP@ZP2(DMA) with longer charge domains and a polyethylene glycol oligomer spacer showed greater photothermal antimicrobial and biofilm resistance in vitro and in vivo. This study develops an innovative acidity-activated AuNP photothermal agent, which provides an effective approach for treatment of biofilm infections.

Biodegradable ferulic acid-containing poly(anhydride-ester): degradation products with controlled release and sustained antioxidant activity.

Ferulic acid (FA) is an antioxidant and photoprotective agent used in biomedical and cosmetic formulations to prevent skin cancer and senescence. Although FA exhibits numerous health benefits, physicochemical instability leading to decomposition hinders its efficacy. To minimize inherent decomposition, a FA-containing biodegradable polymer was prepared via solution polymerization to chemically incorporate FA into a poly(anhydride-ester). The polymer was characterized using nuclear magnetic resonance and infrared spectroscopies. The molecular weight and thermal properties were also determined. In vitro studies demonstrated that the polymer was hydrolytically degradable, thus providing controlled release of the chemically incorporated bioactive with no detectable decomposition. The polymer degradation products were found to exhibit antioxidant and antibacterial activity comparable to that of free FA, and in vitro cell viability studies demonstrated that the polymer is noncytotoxic toward fibroblasts. This renders the polymer a potential candidate for use as a controlled release system for skin care formulations.

Cantharidin and its anhydride-modified derivatives: relation of structure to insecticidal activity.

Cantharidin is a natural compound of novel structure with ideal insecticidal activity. However, the relationship of structure to insecticidal activity of cantharidin and its derivatives has not been ever clarified. To explore what determines the insecticidal activity structurally of cantharidin-related compounds, two series target compounds 6 and 7 were synthesized by replacing the anhydride ring of norcantharidin with an aromatic amine or fatty amine with different electron density, respectively. The structures of these compounds were characterized by 1H NMR, 13C NMR and HRMS-ESI. A bioassay showed that compounds 6 (a-m) lacked any larvicidal activity against Plutella xylostella; whereas their ring-opened partners 7 (a-m) provided a variety of larvicidal activities against P. xylostella, and compound 7f indicated the highest larvicidal activity with LC(50) value of 0.43 mM. The present work demonstrated that the form of the compound (cyclic or ring-opened) or their ability to hydrolyze facilely was the key to determine whether it exhibits larvicidal activity. Moreover, it revealed that the improvement of insecticidal activity required a reasonable combination of both aliphatic amide and aromatic amide moieties, and the type of substituent Y on the aniline ring was critical.

Licogliflozin for nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled, phase 2a study.

Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease that may advance to fibrosis and lead to mortality; however, no pharmacotherapy is currently available. We tested the hypothesis that inhibition of both the sodium-glucose cotransporters 1 and 2 with licogliflozin would lead to improvement in NASH. A total of 107 patients with phenotypic or histologic NASH were randomized (1:2:2) to receive oral administration of either placebo (n = 21), licogliflozin 30 mg (n = 43) or 150 mg (n = 43) once daily for 12 weeks. Licogliflozin 150 mg showed a significant 32% (80% confidence interval (CI): 21-43%; P = 0.002) placebo-adjusted reduction in serum alanine aminotransferase after 12 weeks of treatment, the primary endpoint of the study. However, the 30 mg dose of licogliflozin did not meet the primary endpoint (placebo-adjusted reduction 21% (80% CI: 7-32%; P = 0.061)). Diarrhea occurred in 77% (33 of 43), 49% (21 of 43) and 43% (9 of 21) of patients treated with licogliflozin 150 mg, 30 mg and placebo, respectively, which was mostly mild in severity. No other major safety concerns were identified. Treatment with 150 mg licogliflozin led to reductions in serum alanine aminotransferase in patients with NASH. Studies of longer duration and in combination with drugs that have different mechanisms of action are needed to validate these findings and to define a role of licogliflozin as a therapeutic option for NASH. ClinicalTrials.gov identifier: NCT03205150.

Photothermal Hydrogel Encapsulating Intelligently Bacteria-Capturing Bio-MOF for Infectious Wound Healing.

Chronic wounds are characterized by long-term inflammation and persistent infection, which make them difficult to heal. Therefore, an urgent desire is to develop a multifunctional wound dressing that can prevent wound infection and promote wound healing by creating a favorable microenvironment. In this study, a curcumin-based metal-organic framework (QCSMOF-Van), loaded with vancomycin and coated with quaternary ammonium salt chitosan (QCS), was prepared. Multifunctional composite hydrogels were conveniently synthesized by combining methacrylic anhydride modified gelatin and methacrylic anhydride modified oxidized sodium alginate with QCSMOF-Van through radical polymerization and Schiff base reaction. It is important to note that the QCSMOF-Van could capture bacteria through the positive charges on the surface of QCS. In this process, due to the synergistic effect of broad-spectrum antibacterial Zn2+ and vancomycin, the metabolism of bacteria was well inhibited, and the efficient capturing and rapid killing of bacteria were achieved. The QCSMOF-Van hydrogels could precisely regulate the balance of M1/M2 phenotypes of macrophages, thereby promoting the regeneration of nerves and blood vessels, which promotes the rapid healing of chronic wounds. This advanced cascade management strategy for tissue regeneration highlights the potential of multifunctional composite hydrogels in chronic wound dressings.

The effects of 2-aminoethoxydiphenyl borate and diphenylboronic anhydride on gap junctions composed of Connexin43 in TM4 sertoli cells.

2-Aminoethoxydiphenyl borate (2-APB) has recently been demonstrated to inhibit gap junction (GJ) channels, whereas the underlying mechanisms are still unknown. Using mouse TM4 Sertoli cell which expresses connexin43 (Cx43), we explored the effects of 2-APB and its analogues on dye-coupling through junctional channels formed by Cx43 and on expression of Cx43. Exposure of the cells to 2-APB (1-50 µM) and one of its analogues diphenylboronic anhydride (DPBA) (1-30 µM) for 4 h leads to a significant decrease in dye coupling of GJ in a concentration-dependent manner. The inhibitory effects of 2-APB and DPBA are reversible since decreased GJ coupling resumes after the two compounds are washed out. The disfunction of GJ induced by 2-APB and DPBA is associated with a decrease in total amount of Cx43 protein and number of GJs on the cell membrane. 2-APB and DPBA do not alter Cx43 phosphorylation state and the level of Cx43 mRNA expression. The loss of Cx43 protein is prevented by either lysosomal or proteasomal inhibitor, suggesting that the decrease in Cx43 results from a 2-APB or DPBA-enhanced degradation of Cx43. The present results indicate that 2-APB and DPBA inhibit GJ communication through decreasing Cx43 expression in TM4 cells.

Amino acid-based H2S donors: N-thiocarboxyanhydrides that release H2S with innocuous byproducts.

A library of N-thiocarboxyanhydrides (NTAs) derived from natural amino acids with benign byproducts and controlled H2S-release kinetics is reported. Minimal acute in vitro toxicity was observed in multiple cell lines, while longer-term toxicity in cancer cells was observed, with slow-releasing donors exhibiting the greatest cytotoxic effects.

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride. A novel and highly potent anti-inflammatory and cytoprotective agent.

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride (CDDO anhydride) has been synthesized, which is the first example of an oleanane triterpenoid anhydride. CDDO anhydride shows potency similar to or higher than the corresponding acid (CDDO) in various in vitro and in vivo assays related to inflammation and carcinogenesis. Notably, preliminary phamacokinetics studies show that CDDO anhydride levels are higher than CDDO levels in mouse tissues and blood. Further evaluation of CDDO anhydride is in progress.

Study on the secondary metabolites of grasshopper-derived fungi Arthrinium sp. NF2410.

Two new 2-carboxymethyl-3-hexyl-maleic anhydride derivatives, arthrianhydride A (1) and B (2), along with three known compounds 3-5, were isolated from the fermentation broth of a grasshopper-associated fungus Arthrinium sp. NF2410. The structures of new compounds 1 and 2 were determined based on the analysis of the HR-ESI-MS and NMR spectroscopic data. Furthermore, compounds 1 and 2 were evaluated on inhibitory activity against the enzyme SHP2 and both of them showed moderate inhibitory activity against SHP2.

Functionalization of PEO layer formed on Ti-15Mo for biomedical application.

In the present work, deposition of poly(sebacic anhydride) PSBA loaded by amoxicillin, cefazolin, or vancomycin on a previously anodized Ti-15Mo surface is presented. PSBA loaded by the drug was deposited so as not to lose the functionality of the porous oxide layer microstructure. The morphology was evaluated using scanning electron microscopy, surface roughness, and wettability. The drug concentration was evaluated using high-performance liquid chromatography. It was determined that the drugs were loaded into coatings in the range of 35.2-122.87 µg/cm2 of Ti sample. The drugs released more than 16% after 0.5 hr of the hybrid coating immersion in artificial saliva. After 3 days, the PSBA coatings were degraded by 51.3 mol %, and after 7 days by 77.8 mol %, which makes it possible to load the material by different, biologically active substances. An antimicrobial investigation of Staphylococcus aureus (DSM 24167) and Staphylococcus epidermidis (ATCC 700296) confirmed the activity of the hybrid layers against the pathogens. Hybrid layer with vancomycin best inhibits the adhesion of the bacteria, whereas coatings with amoxicillin and cefazolin showed a much better bactericidal activity. In this article, the difference in the obtained results is discussed, as well as the possibility of the application of this functional material in biomedicine.

(-)-ß-Homoarginine anhydride, a new antioxidant and tyrosinase inhibitor, and further active components from Trichosanthes truncata.

One new amino acid derivative, (-)-ß-homoarginine anhydride 1, as well as nine known compounds were isolated from Trichosanthes truncata. The structures of the isolates were elucidated by spectroscopic methods. Among them, compounds 5 and 11 could notably dose-dependently inhibit ROS productions in HaCaT keratinocyte cells without cytotoxicity in the concentration range of 0.2-20 µM. In cell-free mushroom tyrosinase assay, compounds 1-5, 10 and 11 had more potential anti-tyrosinase activities with IC50 values of 106.9-255.6 µM than arbutin that were similar to predicted values of binding affinity calculated by molecule docking. The most active 2 had hydrogen bonds (Ser77, Glu309, Phe454) and electrostatic charges (Glu309, Glu248) interactions with mushroom tyrosinase, respectively. Our data manifested that T. truncata and its components are potentially to be developed as anti-aging and whitening agents for skin disorders.

Licogliflozin, a Novel SGLT1 and 2 Inhibitor: Body Weight Effects in a Randomized Trial in Adults with Overweight or Obesity.

OBJECTIVE: The aim of this study was to explore the dose response of licogliflozin, a dual inhibitor of sodium/glucose cotransporter 1 (SGLT1) and 2 (SGLT2), by evaluating change in body weight in adults with overweight or obesity. METHODS: This dose-response analysis evaluated change in body weight following 24 weeks with four once-daily and twice-daily licogliflozin doses (2.5-150 mg) versus placebo (primary end point). A further 24-week analysis evaluated the efficacy and safety of two once-daily licogliflozin doses in maintaining initial weight reduction. RESULTS: Licogliflozin once daily or twice daily produced a significant dose-response signal for weight loss versus placebo (P < 0.0001). However, mean adjusted percent changes in body weight after 24 weeks were modest, ranging from -0.45% to -3.83% (in the 50 mg twice daily group [95% CI: -5.26% to -2.48%]; n = 75). Responder analysis of ≥ 5% weight loss at week 24 revealed significant differences versus placebo, which were most pronounced with highest doses of 50 mg twice daily (45.3%) and 150 mg once daily (42.9%) (both P < 0.01). While weight loss was greater at higher doses, gastrointestinal adverse events were also more frequent. The 50-mg once-daily dose had perhaps the best balance between efficacy and tolerability. CONCLUSIONS: Licogliflozin produced significant reductions in body weight versus placebo. However, the magnitude of weight reduction was modest.

Lipase-catalyzed ring-opening polymerization of the O-carboxylic anhydride derived from lactic acid.

Both lipase PS and Novozym 435 promote the ring-opening polymerization of lacOCA, the O-carboxylic anhydride derived from lactic acid. Accordingly, PLA of relatively high molecular weights (M(n) up to 38400 g/mol) and low polydispersities (M(w)/M(n) < 1.4) are obtained in high yields within a few hours at 80 degrees C. Slight preference for l-lacOCA over d-lacOCA is observed, and with Novozym 435, the molecular weight of the obtained PLA can be controlled by varying the lipase loading.