Discovery and Optimization of N-Arylated Tetracyclic Dicarboximides That Target Primary Glioma Stem-like Cells.

We recently discovered a novel N-aryl tetracyclic dicarboximide MM0299 (1) with robust activity against glioma stem-like cells that potently and selectively inhibits lanosterol synthase leading to the accumulation of the toxic shunt metabolite 24(S),25-epoxycholesterol. Herein, we delineate a systematic and comprehensive SAR study that explores the structural space surrounding the N-aryl tetracyclic dicarboximide scaffold. A series of 100 analogs were synthesized and evaluated for activity against the murine glioma stem-like cell line Mut6 and for metabolic stability in mouse liver S9 fractions. This study led to several analogs with single-digit nanomolar activity in Mut6 glioblastoma cells that were metabolically stable in S9 fractions. In vivo pharmacokinetic analysis of selected analogs identified compound 52a (IC50 = 63 nM; S9 T1/2 > 240 min) which was orally available (39% plasma; 58% brain) and displayed excellent brain exposure. Chronic oral dosing of 52a during a 2-week tolerability study indicated no adverse effect on body weight nor signs of hematologic, liver, or kidney toxicity.

Asymmetric Synthesis of CIDD-0072424 via an Enantioselective Nitro-Mannich Reaction: A Central Nervous System Penetrant, Selective Small Molecule Inhibitor of Protein Kinase C Epsilon.

CIDD-0072424 is a novel small molecule developed in silico with remarkable activity for the inhibition of protein kinase C (PKC)-epsilon to treat alcohol use disorder. We developed a concise synthesis of (S)-2 that is highly enantioselective, scalable, and amenable for 3-point structure-activity relationship (SAR) studies for compound optimization. The highly enantioselective nitro-Mannich reaction was achieved through a dual-reagent catalysis system. The overall utility and the efficiency of the enantioselective route provided a scalable synthesis of both PKCε inhibitors 1 and 2.

Selective and brain-penetrant lanosterol synthase inhibitors target glioma stem-like cells by inducing 24(S),25-epoxycholesterol production.

Glioblastoma (GBM) is an aggressive adult brain cancer with few treatment options due in part to the challenges of identifying brain-penetrant drugs. Here, we investigated the mechanism of MM0299, a tetracyclic dicarboximide with anti-glioblastoma activity. MM0299 inhibits lanosterol synthase (LSS) and diverts sterol flux away from cholesterol into a "shunt" pathway that culminates in 24(S),25-epoxycholesterol (EPC). EPC synthesis following MM0299 treatment is both necessary and sufficient to block the growth of mouse and human glioma stem-like cells by depleting cellular cholesterol. MM0299 exhibits superior selectivity for LSS over other sterol biosynthetic enzymes. Critical for its application in the brain, we report an MM0299 derivative that is orally bioavailable, brain-penetrant, and induces the production of EPC in orthotopic GBM tumors but not normal mouse brain. These studies have implications for the development of an LSS inhibitor to treat GBM or other neurologic indications.

The Effect of Temperature and Moisture on Colonization of Apple Fruit and Branches by Botryosphaeria dothidea.

Botryosphaeria dothidea causes severe disease of apple trees in China. The process of conidium germination, colonization, and infection of apple fruit and branches was examined on 'Fuji' apple and the effect of temperature, surface wetness and relative humidity (RH), and host surface washates on these processes was studied in controlled environments. Initial germ tube development and hyphal growth resulted in the colonization of the host surface without forming an infection structure. Hyphae expanded radially across the host surface and, after entering lenticels, developed into a dense mycelium mass or differentiated pseudoparenchyma. Hyphae from the bottom of the pseudoparenchyma either directly penetrated the lenticel surface intercellularly through the cell layer, or formed an undifferentiated hypha that invaded the lenticel through cracks formed during the lenticel development. Conidial germination and hyphal colonization occurred at 10 to 40°C, with an optimum of approximately 28°C. Conidial germination required an RH > 95% or surface wetness but, for hyphal colonization, an RH > 90% was sufficient. Conidia germinated and formed germ tubes within 1 h under optimum conditions. However, the pathogen required a longer period at RH > 90% or surface wetness for hyphae to colonize and form pseudoparenchyma or dense mycelia on the host surface. Hyphal colonization is a crucial stage for infection of apple tissues by B. dothidea.

Cytotoxic effect of carbohydrate derivatives of digitoxigenin involves modulation of plasma membrane Ca2+ -ATPase.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na+ /K+ -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na+ /K+ -ATPase, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and plasma membrane Ca2+ -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na+ /K+ -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

Modulation of α1ß3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix.

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a ß-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABAA α1ß3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the ß+α- interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in ß3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [ß3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1ß3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the ß+α- interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to ß3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the ß+α- interface for the anesthetic propofol.

Infection Screening in Biofluids with Glucose Test Strips.

The four glucosyl esters were synthesized and tested for the determination of infection enzyme leukocyte esterase (LE) in human synovial (joint) fluid and urine. The esters acted as LE substrates releasing glucose in a direct proportion to the activity of LE in a sample. The freed glucose was then detected by a coupled-enzyme assay at either a nitrogen-doped carbon nanotube (N-CNT) electrode or a commercial glucose test strip. The assays at the N-CNT electrode detected LE down to 0.81 nM (25 µg L-1) and showed the fastest kinetics (2.1 × 105 M-1 s-1) for esters with the least crowded space around their carbonyl group. When used with glucose strips, the esters discerned clinically relevant levels of LE up to at least 26 nM (800 µg L-1) in the microliter-sized samples of bodily fluids. The reading of glucose strips with a potentiostat, instead of a personal glucose meter (blood glucometer), shortened the time of required sample incubation from 3 h to 5 min. Correcting the signal of incubated sample for that of original sample eliminated matrix effects and accounted for the presence of native glucose. The new esters have a potential to extend the use of glucose strips (already used by millions for diabetes monitoring) to the quantification of the severity of urinary tract and periprosthetic joint infections.

Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma.

Cancer-specific inhibitors that reflect the unique metabolic needs of cancer cells are rare. Here we describe Gboxin, a small molecule that specifically inhibits the growth of primary mouse and human glioblastoma cells but not that of mouse embryonic fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises oxygen consumption in glioblastoma cells. Gboxin relies on its positive charge to associate with mitochondrial oxidative phosphorylation complexes in a manner that is dependent on the proton gradient of the inner mitochondrial membrane, and it inhibits the activity of F0F1 ATP synthase. Gboxin-resistant cells require a functional mitochondrial permeability transition pore that regulates pH and thus impedes the accumulation of Gboxin in the mitochondrial matrix. Administration of a metabolically stable Gboxin analogue inhibits glioblastoma allografts and patient-derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin, and shows that the increased proton gradient and pH in cancer cell mitochondria is a mode of action that can be targeted in the development of antitumour reagents.

Small molecule nicotinamide N-methyltransferase inhibitor activates senescent muscle stem cells and improves regenerative capacity of aged skeletal muscle.

Aging is accompanied by progressive declines in skeletal muscle mass and strength and impaired regenerative capacity, predisposing older adults to debilitating age-related muscle deteriorations and severe morbidity. Muscle stem cells (muSCs) that proliferate, differentiate to fusion-competent myoblasts, and facilitate muscle regeneration are increasingly dysfunctional upon aging, impairing muscle recovery after injury. While regulators of muSC activity can offer novel therapeutics to improve recovery and reduce morbidity among aged adults, there are no known muSC regenerative small molecule therapeutics. We recently developed small molecule inhibitors of nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed with aging in skeletal muscles and linked to impairment of the NAD+ salvage pathway, dysregulated sirtuin 1 activity, and increased muSC senescence. We hypothesized that NNMT inhibitor (NNMTi) treatment will rescue age-related deficits in muSC activity to promote superior regeneration post-injury in aging muscle. 24-month old mice were treated with saline (control), and low and high dose NNMTi (5 and 10 mg/kg) for 1-week post-injury, or control and high dose NNMTi for 3-weeks post-injury. All mice underwent an acute muscle injury (barium chloride injection) locally to the tibialis anterior (TA) muscle, and received 5-ethynyl-2'-deoxyuridine systemically to analyze muSC activity. In vivo contractile function measurements were conducted on the injured TA muscle and tissues collected for ex-vivo analyses, including myofiber cross-sectional area (CSA) measurements to assess muscle recovery. Results revealed that muscle stem cell proliferation and subsequent fusion were elevated in NNMTi-treated mice, supporting nearly 2-fold greater CSA and shifts in fiber size distribution to greater proportions of larger sized myofibers and fewer smaller sized fibers in NNMTi-treated mice compared to controls. Prolonged NNMTi treatment post-injury further augmented myofiber regeneration evinced by increasingly larger fiber CSA. Importantly, improved muSC activity translated not only to larger myofibers after injury but also to greater contractile function, with the peak torque of the TA increased by ∼70% in NNMTi-treated mice compared to controls. Similar results were recapitulated in vitro with C2C12 myoblasts, where NNMTi treatment promoted and enhanced myoblast differentiation with supporting changes in the cellular NAD+/NADH redox states. Taken together, these results provide the first clear evidence that NNMT inhibitors constitute a viable pharmacological approach to enhance aged muscle regeneration by rescuing muSC function, supporting the development of NNMTi as novel mechanism-of-action therapeutic to improve skeletal muscle regenerative capacity and functional recovery after musculoskeletal injury in older adults.

Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice.

There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5'-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.

Novel Small-Molecule Inhibitors of Protein Kinase C Epsilon Reduce Ethanol Consumption in Mice.

BACKGROUND: Despite the high cost and widespread prevalence of alcohol use disorders, treatment options are limited, underscoring the need for new, effective medications. Previous results using protein kinase C epsilon (PKCε) knockout mice, RNA interference against PKCε, and peptide inhibitors of PKCε predict that small-molecule inhibitors of PKCε should reduce alcohol consumption in humans. METHODS: We designed a new class of PKCε inhibitors based on the Rho-associated protein kinase (ROCK) inhibitor Y-27632. In vitro kinase and binding assays were used to identify the most potent compounds. Their effects on ethanol-stimulated synaptic transmission; ethanol, sucrose, and quinine consumption; ethanol-induced loss of righting; and ethanol clearance were studied in mice. RESULTS: We identified two compounds that inhibited PKCε with Ki <20 nM, showed selectivity for PKCε over other kinases, crossed the blood-brain barrier, achieved effective concentrations in mouse brain, prevented ethanol-stimulated gamma-aminobutyric acid release in the central amygdala, and reduced ethanol consumption when administered intraperitoneally at 40 mg/kg in wild-type but not in Prkce-/- mice. One compound also reduced sucrose and saccharin consumption, while the other was selective for ethanol. Both transiently impaired locomotion through an off-target effect that did not interfere with their ability to reduce ethanol intake. One compound prolonged recovery from ethanol-induced loss of righting but this was also due to an off-target effect since it was present in Prkce-/- mice. Neither altered ethanol clearance. CONCLUSIONS: These results identify lead compounds for development of PKCε inhibitors that reduce alcohol consumption.

Structure-Activity Relationship for Small Molecule Inhibitors of Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) is a fundamental cytosolic biotransforming enzyme that catalyzes the N-methylation of endogenous and exogenous xenobiotics. We have identified small molecule inhibitors of NNMT with >1000-fold range of activity and developed comprehensive structure-activity relationships (SARs) for NNMT inhibitors. Screening of N-methylated quinolinium, isoquinolinium, pyrididium, and benzimidazolium/benzothiazolium analogues resulted in the identification of quinoliniums as a promising scaffold with very low micromolar (IC50 ∼ 1 µM) NNMT inhibition. Computer-based docking of inhibitors to the NNMT substrate (nicotinamide)-binding site produced a robust correlation between ligand-enzyme interaction docking scores and experimentally calculated IC50 values. Predicted binding orientation of the quinolinium analogues revealed selective binding to the NNMT substrate-binding site residues and essential chemical features driving protein-ligand intermolecular interactions and NNMT inhibition. The development of this new series of small molecule NNMT inhibitors direct the future design of lead drug-like inhibitors to treat several metabolic and chronic disease conditions characterized by abnormal NNMT activity.

Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity.

Nicotinamide N-methyltransferase (NNMT) is an important biotransforming enzyme that catalyzes the transfer of a labile methyl group from the ubiquitous cofactor S-5'-adenosyl-l-methionine (SAM) to endogenous and exogenous small molecules to form methylated end products. NNMT has been implicated in a number of chronic disease conditions, including metabolic disorders, cardiovascular disease, cancer, osteoarthritis, kidney disease, and Parkinson's disease. We have developed a novel noncoupled fluorescence-based methyltransferase assay that allows direct ultrasensitive real-time detection of the NNMT reaction product 1-methylquinolinium. This is the first assay reported to date to utilize fluorescence spectroscopy to directly monitor NNMT product formation and activity in real time. This assay provided accurate kinetic data that allowed detailed comparative analysis of the NNMT reaction mechanism and kinetic parameters. A reaction model based on a random bireactant mechanism produced global curve fits that were most consistent with steady-state initial velocity data collected across an array of substrate concentrations. On the basis of the reaction mechanism, each substrate could independently bind to the NNMT apoenzyme; however, both substrates bound to the complementary binary complexes with an affinity ∼20-fold stronger compared to their binding to the apoenzyme. This reaction mechanism implies either substrate-induced conformational changes or bireactant intermolecular interactions may stabilize the binding of the substrate to the binary complex and formation of the ternary complex. Importantly, this assay could rapidly generate concentration response curves for known NNMT inhibitors, suggesting its applicability for high-throughput screening of chemical libraries to identify novel NNMT inhibitors. Furthermore, our novel assay potentially offers a robust detection technology for use in SAM substrate competition assays for the discovery and development of SAM-dependent methyltransferase inhibitors.

Recent advances in acetylcholinesterase Inhibitors and Reactivators: an update on the patent literature (2012-2015).

INTRODUCTION: Acetylcholinesterase (AChE) is the major enzyme that hydrolyzes acetylcholine, a key neurotransmitter for synaptic transmission, into acetic acid and choline. Mild inhibition of AChE has been shown to have therapeutic relevance in Alzheimer's disease (AD), myasthenia gravis, and glaucoma among others. In contrast, strong inhibition of AChE can lead to cholinergic poisoning. To combat this, AChE reactivators have to be developed to remove the offending AChE inhibitor, restoring acetylcholine levels to normal. Areas covered: This article covers recent advances in the development of acetylcholinesterase modulators, including both inhibitors of acetylcholinesterase for the efforts in development of new chemical entities for treatment of AD, as well as re-activators for resurrection of organophosphate bound acetylcholinesterase. Expert opinion: Over the past three years, research efforts have continued to identify novel small molecules as AChE inhibitors for both CNS and peripheral diseases. The more recent patent activity has focused on three AChE ligand design areas: derivatives of known AChE ligands, natural product based scaffolds and multifunctional ligands, all of which have produced some unique chemical matter with AChE inhibition activities in the mid picomolar to low micromolar ranges. New AChE inhibitors with polypharmacology or dual inhibitory activity have also emerged as highlighted by new AChE inhibitors with dual activity at L-type calcium channels, GSK-3, BACE1 and H3, although most only show low micromolar activity, thus further research is warranted. New small molecule reactivators of organophosphate-inhibited AChE have also been disclosed, which focused on the design of neutral ligands with improved pharmaceutical properties and blood-brain barrier (BBB) penetration. Gratifyingly, some research in this area is moving away from the traditional quaternary pyridinium oximes AChE reactivators, while still employing the necessary reactivation group (oximes). However, selectivity over inhibition of native AChE enzyme, effectiveness of reactivation, broad-spectrum reactivation against multiple organophosphates and reactivation of aged-enzyme continue to be hurdles for this area of research.

Population-representative Incidence of Acute-On-Chronic Liver Failure: A Prospective Cross-Sectional Study.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a major cause of hepatic death in the world, but no population-based studies have evaluated the incidence of ACLF. This study was conducted to determine the incidence and short-term outcomes of ACLF in a region of Eastern China. METHODS: In this prospective cross-sectional study, we collected data from public hospitals in Nantong city between January 1, 2005, and December 31, 2014. All hospitals with admission potential for ACLF patients were included. The primary outcome was ACLF defined as severe jaundice and coagulopathy with underlying chronic liver disease, according to diagnostic and laboratory criteria suggested by Chinese Society for Hepatology (CSH). RESULTS: During the 10-year period, a consecutive sample of 1934 ACLF patients was included in this study. The overall ACLF incidence rate over the 10-year period was 2.53 (95% confidence interval, 2.16-2.91) per 100,000 population per year, decreasing from 3.35 in 2005 to 2.06 in 2014. Chronic hepatitis B virus (HBV) infection was the leading cause of chronic liver disease and HBV reactivation was the most common cause of acute hepatic event. The 28-day mortality for the ACLF patients had a clear decline during the study period, form 50.39% in 2005 to 35.44% in 2014. CONCLUSIONS: In the Eastern China population, the incidence of ACLF is decreasing and the prognosis improving. Short-term mortality was associated with the presence of cirrhosis and growing age. While ACLF remains a life-threatening disorder, our findings suggest that nationwide and long-term cohorts should be conducted for the natural history of ACLF.

Digitoxin analogues with improved anticytomegalovirus activity.

Cardiac glycosides are potent inhibitors of cancer cell growth and possess antiviral activities at nanomolar concentrations. In this study we evaluated the anticytomegalovirus (CMV) activity of digitoxin and several of its analogues. We show that sugar type and sugar length attached to the steroid core structure affects its anticytomegalovirus activity. Structure-activity relationship (SAR) studies identified the l-sugar containing cardiac glycosides as having improved anti-CMV activity and may lead to better understanding of how these compounds inhibit CMV replication.

Merremoside D: de novo synthesis of the purported structure, NMR analysis, and comparison of spectral data.

The first synthesis of the purported structure of Merremoside D has been achieved in 22 longest linear steps. The de novo asymmetric synthesis relied on the use of asymmetric catalysis to selectively install all 21 stereocenters in the final compounds from commercially available achiral starting materials. Adiabatic gradient 2D NMR techniques (gHSQCAD, gHMBCAD, gH2BCAD, gHSQCTOXYAD, ROESYAD) were used to completely assign the structure of synthetic Merremoside D. Comparison of our assignments with the limited NMR data reported for natural Merremoside D allows for the tentative confirmation of its structure.

Monosaccharide digitoxin derivative sensitize human non-small cell lung cancer cells to anoikis through Mcl-1 proteasomal degradation.

Advanced stage cancers acquire anoikis resistance which provides metastatic potential to invade and form tumors at distant sites. Suppression of anoikis resistance by novel molecular therapies would greatly benefit treatment strategies for metastatic cancers. Recently, digitoxin and several of its novel synthetic derivatives, such as α-l-rhamnose monosaccharide derivative (D6-MA), have been synthesized and studied for their profound anticancer activity in various cancer cell lines. In this study, we investigated the anoikis sensitizing effect of D6-MA compared with digitoxin to identify their anti-metastatic mechanism of action. D6-MA sensitized NSCLC H460 cells to detachment-induced apoptosis with significantly greater cytotoxicity (IC50=11.9 nM) than digitoxin (IC50=90.7 nM) by activating caspase-9. Screening of the Bcl-2 protein family revealed that degradation of anti-apoptotic Mcl-1 protein is a favorable target. Mcl-1 over-expression and knockdown studies in D6-MA and digitoxin exposed cells resulted in rescue and enhancement, respectively, indicating a facilitative role for decreased Mcl-1 expression in NSCLC anoikis. Transfection with mutant Mcl-1S159 attenuated detachment-induced cell death and correlated with a remaining of Mcl-1 level. Furthermore, D6-MA suppressed Mcl-1 expression via ubiquitin proteasomal degradation that is dependent on activation of glycogen synthase kinase (GSK)-3ß signaling. In addition, D6-MA also targeted Mcl-1 degradation causing an increased anoikis in A549 lung cancer cells. Anoikis sensitizing effect on normal small airway epithelial cells was not observed indicating the specificity of D6-MA and digitoxin for NSCLC. These results identify a novel cardiac glycoside (CG) sensitizing anoikis mechanism and provide a promising anti-metastatic target for lung cancer therapy.

De novo asymmetric synthesis of oligo-rhamno di- and tri-saccharides related to the anthrax tetrasaccharide.

An asymmetric synthesis of the di- and trisaccharide portion of the naturally occurring anthrax tetrasaccharide from acetylfuran has been developed. The construction of the di- and trisaccharide subunits is based upon our previously disclosed route to anthrax tetrasaccharide. The approach uses iterative diastereoselective palladium-catalyzed glycosylations, Luche reductions, diastereoselective dihydroxylations, and regioselective protections for the assembly of the rhamno- di- and tri-saccharide. The route was also modified for the preparation of the mixed D-/L-disaccharide analogue.