The Structural Dispersity of Oligoethylene Glycol-Containing Polymer Brushes Determines Their Interfacial Properties.

Ought to their bioinert properties and facile synthesis, poly[(oligoethylene glycol)methacrylate]s (POEGMAs) have been raised as attractive alternatives to poly(ethylene glycols) (PEGs) in an array of (bio)material applications, especially when they are applied as polymer brush coatings. However, commercially available OEG-methacrylate (macro)monomers feature a broad distribution of OEG lengths, thus generating structurally polydisperse POEGMAs when polymerized through reversible deactivation radical polymerization. Here, we demonstrate that the interfacial physicochemical properties of POEGMA brushes are significantly affected by their structural dispersity, i.e., the degree of heterogeneity in the length of side OEG segments. POEGMA brushes synthesized from discrete (macro)monomers obtained through chromatographic purification of commercial mixtures show increased hydration and reduced adhesion when compared to their structurally polydisperse analogues. The observed alteration of interfacial properties is directly linked to the presence of monodisperse OEG side chains, which hamper intramolecular and intermolecular hydrophobic interactions while simultaneously promoting the association of water molecules. These phenomena provide structurally homogeneous POEGMA brushes with a more lubricious and protein repellent character with respect to their heterogeneous counterparts. More generally, in contrast to what has been assumed until now, the properties of POEGMA brushes cannot be anticipated while ruling out the effect of dispersity by (macro)monomer feeds. Simultaneously, side chain dispersity of POEGMAs emerges as a critical parameter for determining the interfacial characteristics of brushes.

Esmethadone-HCl (REL-1017): a promising rapid antidepressant.

This review article presents select recent studies that form the basis for the development of esmethadone into a potential new drug. Esmethadone is a promising member of the pharmacological class of uncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonists that have shown efficacy for major depressive disorder (MDD) and other diseases and disorders, such as Alzheimer's dementia and pseudobulbar affect. The other drugs in the novel class of NMDAR antagonists with therapeutic uses that are discussed for comparative purposes in this review are esketamine, ketamine, dextromethorphan, and memantine. We present in silico, in vitro, in vivo, and clinical data for esmethadone and other uncompetitive NMDAR antagonists that may advance our understanding of the role of these receptors in neural plasticity in health and disease. The efficacy of NMDAR antagonists as rapid antidepressants may advance our understanding of the neurobiology of MDD and other neuropsychiatric diseases and disorders.

The mode of dexamethasone decoration influences avidin-nucleic-acid-nano-assembly organ biodistribution and in vivo drug persistence.

Avidin-Nucleic-Acid-NanoASsemblies (ANANAS) possess natural tropism for the liver and, when loaded with dexamethasone, reduce clinical progression in an autoimmune hepatitis murine model. Here, we investigated the linker chemistry (hydrazide-hydrazone, Hz-Hz, or carbamate hydrazide-hydrazone, Cb-Hz bond) and length (long, 5 kDa PEG, or short, 5-6 carbons) in biotin-dexamethasone conjugates used for nanoparticle decoration through in vitro and in vivo studies. All four newly synthesized conjugates released the drug at acidic pH only. In vitro, the Hz-Hz and the PEG derivatives were less stable than the Cb-Hz and the short chain ones, respectively. Once injected in healthy mice, dexamethasone location in the PEGylated ANANAS outer layer favors liver penetration and resident macrophages uptake, while drug Hz-Hz, but not Cb-Hz, short spacing prolongs drug availability. In conclusion, the tight modulation of ANANAS decoration can significantly influence the host interaction, paving the way for the development of steroid nanoformulations suitable for different pharmacokinetic profiles.

Esmethadone (REL-1017) and Other Uncompetitive NMDAR Channel Blockers May Improve Mood Disorders via Modulation of Synaptic Kinase-Mediated Signaling.

This article presents a mechanism of action hypothesis to explain the rapid antidepressant effects of esmethadone (REL-1017) and other uncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonists and presents a corresponding mechanism of disease hypothesis for major depressive disorder (MDD). Esmethadone and other uncompetitive NMDAR antagonists may restore physiological neural plasticity in animal models of depressive-like behavior and in patients with MDD via preferential tonic block of pathologically hyperactive GluN2D subtypes. Tonic Ca2+ currents via GluN2D subtypes regulate the homeostatic availability of synaptic proteins. MDD and depressive behaviors may be determined by reduced homeostatic availability of synaptic proteins, due to upregulated tonic Ca2+ currents through GluN2D subtypes. The preferential activity of low-potency NMDAR antagonists for GluN2D subtypes may explain their rapid antidepressant effects in the absence of dissociative side effects.

Dispersity within Brushes Plays a Major Role in Determining Their Interfacial Properties: The Case of Oligoxazoline-Based Graft Polymers.

Many synthetic polymers used to form polymer-brush films feature a main backbone with functional, oligomeric side chains. While the structure of such graft polymers mimics biomacromolecules to an extent, it lacks the monodispersity and structural purity present in nature. Here we demonstrate that side-chain heterogeneity within graft polymers significantly influences hydration and the occurrence of hydrophobic interactions in the subsequently formed brushes and consequently impacts fundamental interfacial properties. This is demonstrated for the case of poly(methacrylate)s (PMAs) presenting oligomeric side chains of different length (n) and dispersity. A precise tuning of brush structure was achieved by first synthesizing oligo(2-ethyl-2-oxazoline) methacrylates (OEOXMAs) by cationic ring-opening polymerization (CROP), subsequently purifying them into discrete macromonomers with distinct values of n by column chromatography, and finally obtaining poly[oligo(2-ethyl-2-oxazoline) methacrylate]s (POEOXMAs) by reversible addition-fragmentation chain-transfer (RAFT) polymerization. Assembly of POEOXMA on Au surfaces yielded graft polymer brushes with different side-chain dispersities and lengths, whose properties were thoroughly investigated by a combination of variable angle spectroscopic ellipsometry (VASE), quartz crystal microbalance with dissipation (QCMD), and atomic force microscopy (AFM) methods. Side-chain dispersity, or dispersity within brushes, leads to assemblies that are more hydrated, less adhesive, and more lubricious and biopassive compared to analogous films obtained from graft polymers characterized by a homogeneous structure.

Synthesis and cellular effects of a mitochondria-targeted inhibitor of the two-pore potassium channel TASK-3.

The two-pore potassium channel TASK-3 has been shown to localize to both the plasma membrane and the mitochondrial inner membrane. TASK-3 is highly expressed in melanoma and breast cancer cells and has been proposed to promote tumor formation. Here we investigated whether pharmacological modulation of TASK-3, and specifically of mitochondrial TASK-3 (mitoTASK-3), had any effect on cancer cell survival and mitochondrial physiology. A novel, mitochondriotropic version of the specific TASK-3 inhibitor IN-THPP has been synthesized by addition of a positively charged triphenylphosphonium moiety. While IN-THPP was unable to induce apoptosis, mitoIN-THPP decreased survival of breast cancer cells and efficiently killed melanoma lines, which we show to express mitoTASK-3. Cell death was accompanied by mitochondrial membrane depolarization and fragmentation of the mitochondrial network, suggesting a role of the channel in the maintenance of the correct function of this organelle. In accordance, cells treated with mitoIN-THPP became rapidly depleted of mitochondrial ATP which resulted in activation of the AMP-dependent kinase AMPK. Importantly, cell survival was not affected in mouse embryonic fibroblasts and the effect of mitoIN-THPP was less pronounced in human melanoma cells stably knocked down for TASK-3 expression, indicating a certain degree of selectivity of the drug both for pathological cells and for the channel. In addition, mitoIN-THPP inhibited cancer cell migration to a higher extent than IN-THPP in two melanoma cell lines. In summary, our results point to the importance of mitoTASK-3 for melanoma cell survival and migration.

Small-Molecule Modulators of Mitochondrial Channels as Chemotherapeutic Agents.

Ion channels residing in the inner (IMM) and outer (OMM) mitochondrial membranes are emerging as noteworthy pharmacological targets in oncology. While these aspects have not been investigated for all of them, a role in cancer growth and/or metastasis and/or drug resistance has been shown at least for the IMM-residing Ca2+ uniporter complex and K+- selective mtKV1.3, mtIKCa, mtSKCa and mtTASK-3, and for the OMM Voltage-Dependent Anion Channel (mitochondrial porin). A special case is that of the Mitochondrial Permeability Transition Pore, a large pore which forms in the IMM of severely stressed cells, and which may be exploited to precipitate the death of cancerous cells. Here we briefly discuss the oncological relevance of mitochondria and their channels, and summarize the methods that can be adopted to selectively target these intracellular organelles. We then present an updated list of known mitochondrial channels, and review the pharmacology of those with proven relevance for cancer.

A Synthetic Fluorescent Mitochondria-Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells.

Ca2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca2+ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca2+ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca2+ indicator fura-2. Mt-fura-2 binds Ca2+ with a dissociation constant of ≈1.5 µm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca2+ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.

Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals.

Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins. They are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol where their substrates, the three SNARE proteins, are localised. L translocation is accompanied by unfolding, and it has to be reduced and reacquire the native fold to exert its neurotoxicity. The Thioredoxin reductase-Thioredoxin system is responsible for the reduction, but it is unknown whether the refolding of L is spontaneous or aided by host chaperones. Here we report that geldanamycin, a specific inhibitor of heat shock protein 90, hampers the refolding of L after membrane translocation and completely prevents the cleavage of SNAREs. We also found that geldanamycin strongly synergises with PX-12, an inhibitor of thioredoxin, suggesting that the processes of L chain refolding and interchain disulfide reduction are strictly coupled. Indeed we found that the heat shock protein 90 and the Thioredoxin reductase-Thioredoxin system physically interact on synaptic vesicle where they orchestrate a chaperone-redox machinery which is exploited by clostridial neurotoxins to deliver their catalytic part into the cytosol.

A Photo-Crosslinkable Biotin Derivative of the Phosphoantigen (E)-4-Hydroxy-3-Methylbut-2-Enyl Diphosphate (HMBPP) Activates Vγ9Vδ2 T Cells and Binds to the HMBPP Site of BTN3A1.

Vγ9Vδ2 T cells play an important role in the cross talk of the innate and adaptive immune system. For their activation by phosphoantigens (PAgs), both cell surface receptors, the eponymous Vγ9Vδ2 T cell antigen receptors (Vγ9Vδ2 TCRs) on Vγ9Vδ2 T cells and butyrophilin 3A1 (BTN3A1) on the phosphoantigen-"presenting" cell, are mandatory. To find yet undetected but further contributing proteins, a biotinylated, photo-crosslinkable benzophenone probe BioBP-HMBPP (2) was synthesized from a known allyl alcohol in nine steps and overall 16 % yield. 2 is based on the picomolar PAg (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 1). Laser irradiation of 2 at 308 nm initiated the photo-crosslinking reaction with proteins. When the B30.2 domain of BTN3A1, which contains a positively charged PAg-binding pocket, was exposed to increasing amounts of HMBPP (1), labeling by BioBP-HMBPP (2) was reduced significantly. Because BSA labeling was not impaired, 2 clearly binds to the same site as natural ligand 1. Thus, BioBP-HMBPP (2) is a suitable tool to identify co-ligands or receptors involved in PAg-mediated T cell activation.

Targeting the Potassium Channel Kv1.3 Kills Glioblastoma Cells.

BACKGROUND/AIMS: Glioblastoma (GBM) is one of the most aggressive cancers, counting for a high number of the newly diagnosed patients with central nervous system (CNS) cancers in the United States and Europe. Major features of GBM include aggressive and invasive growth as well as a high resistance to treatment. Kv1.3, a potassium channel of the shaker family, is expressed in the inner mitochondrial membrane of many cancer cells. Inhibition of mitochondrial Kv1.3 was shown to induce apoptosis in several tumor cells at doses that were not lethal for normal cells. METHODS: We investigated the expression of Kv1.3 in different glioma cell lines by immunocytochemistry, western blotting and electron microscopy and analyzed the effect of newly synthesized, mitochondria-targeted, Kv1.3 inhibitors on the induction of cell death in these cells. Finally, we performed in vivo studies on glioma bearing mice. RESULTS: Here, we report that Kv1.3 is expressed in mitochondria of human and murine GL261, A172 and LN308 glioma cells. Treatment with the novel Kv1.3 inhibitors PAPTP or PCARBTP as well as with clofazimine induced massive cell death in glioma cells, while Psora-4 and PAP-1 were almost without effect. However, in vivo experiments revealed that the drugs had no effect on orthotopic brain tumors in vivo. CONCLUSION: These data serve as proof of principle that Kv1.3 inhibitors kills GBM cells, but drugs that act in vivo against glioblastoma must be developed to translate these findings in vivo.

Potential anti-cancer activity of 7-O-pentyl quercetin: Efficient, membrane-targeted kinase inhibition and pro-oxidant effect.

Quercetin is a redox-active plant-derived flavonoid with potential anticancer effects, stemming largely from its interaction with a number of proteins, and in particular from inhibition of pro-life kinases. To improve efficacy, we reasoned that a local increase in concentration of the compound at the level of cell membranes would result in a more efficient interaction with membrane-associated signaling kinases. We report here the synthesis of all five isomeric quercetin derivatives in which an n-pentyl group was linked via an ether bond to each hydroxyl of the flavonoid kernel. This strategy proved effective in directing quercetin to cellular membranes, and revealed a remarkable dependence of the derivatives' bioactivity on the specific site of functionalization. The isomer bearing the pentyl group in position 7, Q-7P, turned out to be the most effective and promising derivative, selectively inducing apoptosis in tumoral and fast-growing cells, while sparing slow-growing, non-tumoral ones. Cytotoxicity for tumoral cells was strongly enhanced compared to quercetin itself. Q-7P induced massive ROS production, which however accounted only partially for cell death. Alterations in the levels of various signaling phospho-proteins were observed in a proteomics screen. An important contribution seems to come from inhibition of the PI3K/Akt pathway. This work opens new perspectives in developing membrane-associating, polyphenol-based anticancer agents.

Cytotoxicity of mitochondria-targeted resveratrol derivatives: interactions with respiratory chain complexes and ATP synthase.

We recently reported that mitochondria-targeted derivatives of resveratrol are cytotoxic in vitro, selectively inducing mostly necrotic death of fast-growing and tumoral cells when supplied in the low µM range (N. Sassi et al., Curr. Pharm. Des. 2014). Cytotoxicity is due to H2O2 produced upon accumulation of the compounds into mitochondria. We investigate here the mechanisms underlying ROS generation and mitochondrial depolarization caused by these agents. We find that they interact with the respiratory chain, especially complexes I and III, causing superoxide production. "Capping" free hydroxyls with acetyl or methyl groups increases their effectiveness as respiratory chain inhibitors, promoters of ROS generation and cytotoxic agents. Exposure to the compounds also induces an increase in the occurrence of short transient [Ca(2+)] "spikes" in the cells. This increase is unrelated to ROS production, and it is not the cause of cell death. These molecules furthermore inhibit the F0F1 ATPase. When added to oligomycin-treated cells, the acetylated/methylated ones cause a recovery of the cellular oxygen consumption rates depressed by oligomycin. Since a protonophoric futile cycle which might account for the uncoupling effect is impossible, we speculate that the compounds may cause the transformation of the ATP synthase and/or respiratory chain complex(es) into a conduit for uncoupled proton translocation. Only in the presence of excess oligomycin the most effective derivatives appear to induce the mitochondrial permeability transition (MPT) within the cells. This may be considered to provide circumstantial support for the idea that the ATP synthase is the molecular substrate for the MPT pore.

Synthesis and Evaluation as Prodrugs of Hydrophilic Carbamate Ester Analogues of Resveratrol.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is an unfulfilled promise for health care: its exploitation is hindered by rapid conjugative metabolism in enterocytes and hepatocytes; low water solubility is a serious practical problem. To advantageously modify the physicochemical properties of the compound we have developed prodrugs in which all or part of the hydroxyl groups are linked via an N-monosubstituted carbamate ester bond to promoieties derived from glycerol or galactose, conferring higher water solubility. Kinetic studies of hydrolysis in aqueous solutions and in blood indicated that regeneration of resveratrol takes place in an appropriate time frame for delivery via oral administration. Despite their hydrophilicity some of the synthesized compounds were absorbed in the gastrointestinal tract of rats. In these cases the species found in blood after administration of a bolus consisted mainly of partially deprotected resveratrol derivatives and of the products of their glucuronidation, thus providing proof-of-principle evidence of behavior as prodrugs. The soluble compounds largely reached the lower intestinal tract. Upon administration of resveratrol, the major species found in this region was dihydroresveratrol, produced by enzymes of the intestinal flora. In experiments with a fully protected (trisubstituted) deoxygalactose containing prodrug, the major species were the prodrug itself and partially deprotected derivatives, along with small amounts of dihydroresveratrol. We conclude that the N-monosubstituted carbamate moiety is suitable for use in prodrugs of polyphenols.

N-Monosubstituted Methoxy-oligo(ethylene glycol) Carbamate Ester Prodrugs of Resveratrol.

Resveratrol is a natural polyphenol with many interesting biological activities. Its pharmacological exploitation in vivo is, however, hindered by its rapid elimination via phase II conjugative metabolism at the intestinal and, most importantly, hepatic levels. One approach to bypass this problem relies on prodrugs. We report here the synthesis, characterization, hydrolysis, and in vivo pharmacokinetic behavior of resveratrol prodrugs in which the OH groups are engaged in an N-monosubstituted carbamate ester linkage. As promoiety, methoxy-oligo(ethylene glycol) groups (m-OEG) (CH3-[OCH2CH2]n-) of defined chain length (n = 3, 4, 6) were used. These are expected to modulate the chemico-physical properties of the resulting derivatives, much like longer poly(ethylene glycol) (PEG) chains, while retaining a relatively low MW and, thus, a favorable drug loading capacity. Intragastric administration to rats resulted in the appearance in the bloodstream of the prodrug and of the products of its partial hydrolysis, confirming protection from first-pass metabolism during absorption.

New water-soluble carbamate ester derivatives of resveratrol.

Low bioavailability severely hinders exploitation of the biomedical potential of resveratrol. Extensive phase-II metabolism and poor water solubility contribute to lowering the concentrations of resveratrol in the bloodstream after oral administration. Prodrugs may provide a solution-protection of the phenolic functions hinders conjugative metabolism and can be exploited to modulate the physicochemical properties of the compound. We report here the synthesis and characterization of carbamate ester derivatives of resveratrol bearing on each nitrogen atom a methyl group and either a methoxy-poly(ethylene glycol)-350 (mPEG-350) or a butyl-glucosyl promoiety conferring high water solubility. Ex vivo absorption studies revealed that the butyl-glucosyl conjugate, unlike the mPEG-350 one, is able to permeate the intestinal wall. In vivo pharmacokinetics confirmed absorption after oral administration and showed that no hydrolysis of the carbamate groups takes place. Thus, sugar groups can be attached to resveratrol to obtain soluble derivatives maintaining to some degree the ability to permeate biomembranes, perhaps by facilitated or active transport.

Sphingosine is involved in PAPTP-induced death of pancreas cancer cells by interfering with mitochondrial functions.

Pancreas ductal adenocarcinoma belongs to the most common cancers, but also to the tumors with the poorest prognosis. Here, we pharmacologically targeted a mitochondrial potassium channel, namely mitochondrial Kv1.3, and investigated the role of sphingolipids and mutated Kirsten Rat Sarcoma Virus (KRAS) in Kv1.3-mediated cell death. We demonstrate that inhibition of Kv1.3 using the Kv1.3-inhibitor PAPTP results in an increase of sphingosine and superoxide in membranes and/or membranes associated with mitochondria, which is enhanced by KRAS mutation. The effect of PAPTP on sphingosine and mitochondrial superoxide formation as well as cell death is prevented by sh-RNA-mediated downregulation of Kv1.3. Induction of sphingosine in human pancreas cancer cells by PAPTP is mediated by activation of sphingosine-1-phosphate phosphatase and prevented by an inhibitor of sphingosine-1-phosphate phosphatase. A rapid depolarization of isolated mitochondria is triggered by binding of sphingosine to cardiolipin, which is neutralized by addition of exogenous cardiolipin. The significance of these findings is indicated by treatment of mutated KRAS-harboring metastasized pancreas cancer with PAPTP in combination with ABC294640, a blocker of sphingosine kinases. This treatment results in increased formation of sphingosine and death of pancreas cancer cells in vitro and, most importantly, prolongs in vivo survival of mice challenged with metastatic pancreas cancer. KEY MESSAGES: Pancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis. The mitochondrial Kv1.3 ion channel blocker induced mitochondrial sphingosine. Sphingosine binds to cardiolipin thereby mediating mitochondrial depolarization. Sphingosine is formed by a PAPTP-mediated activation of S1P-Phosphatase. Inhibition of sphingosine-consumption amplifies PAPTP effects on PDAC in vivo.

Effect of Time From Onset of Major Depressive Disorder on the Therapeutic Response to Esmethadone (REL-1017).

Objective: The relationship between the duration of major depressive disorder (MDD) and therapeutic response to standard antidepressant treatment (SAT) is unknown. N-methyl-D-aspartate receptor uncompetitive antagonists are emerging drugs for MDD. We investigated whether the antidepressant effect of esmethadone (REL-1017) could be related to the duration of depression.Methods: We analyzed data from a Phase 2a study of adjunctive treatment with esmethadone in MDD patients (DSM-5) with inadequate response to ongoing SAT (May 2018-August 2019). Patients were randomized to treatment with esmethadone 25 mg, esmethadone 50 mg, or placebo for 7 days, followed by an observation period (Days 7-14). Duration of depression was derived from 2 measures: (1) time from onset (TFO), calculated as the difference in years between age at trial enrollment and age at the onset of the first major depressive episode (MDE), and (2) TFO index, calculated by computing the years of illness duration (number of years from the beginning of MDD), divided by age and multiplied by 100. First, bivariate correlations between TFO and change from baseline (CFB) were calculated by Spearman ρ. Linear mixed-model analyses were also conducted.Results: A total of 62 patients participated in the trial. The median values of time from MDD onset for the 62 patients were 11 years (absolute value) and 22% (percentage of life-years). Duration of depression was significantly correlated with Montgomery-Asberg Depression Rating Scale (MADRS) CFB on Day 14, even when controlling for the effect of current depression severity (MADRS baseline). In the linear mixed-model analyses, we found a significant effect of duration on reduction in MADRS score from T0 to subsequent assessments (P < .05). Number of previous MDEs and effect of esmethadone 50 mg when compared to 25 mg were not significant.Conclusion: Esmethadone 25 and 50 mg were more effective in reducing MADRS scores in patients with shorter time from first MDE onset.Trial Registration: ClinicalTrials.gov identifier: NCT03051256.