Small‐Molecule Inhibitors of Store‐Operated Calcium Entry

Molecules that inhibit store‐operated calcium entry (SOCE) are potentially useful immunomodulating agents. The identification of proteins involved in this pathway may further enable the identification of selective inhibitors. Herein we document some examples of the small‐molecule inhibitors of SOCE that have been reported to date. We also describe methods that were used to characterize the mechanism of action of these inhibitors.

     

Characterization of a Cyclic Nucleotide‐Activated K+ Channel and its Lipid Environment by Using Solid‐State NMR Spectroscopy

Voltage‐gated ion channels are large tetrameric multidomain membrane proteins that play crucial roles in various cellular transduction pathways. Because of their large size and domain‐related mobility, structural characterization has proved challenging. We analyzed high‐resolution solid‐state NMR data on different isotope‐labeled protein constructs of a bacterial cyclic nucleotide‐activated K⁺ channel (MlCNG) in lipid bilayers. We could identify the different subdomains of the 4×355 residue protein, such as the voltage‐sensing domain and the cyclic nucleotide binding domain. Comparison to ssNMR data obtained on isotope‐labeled cell membranes suggests a tight association of negatively charged lipids to the channel. We detected spectroscopic polymorphism that extends beyond the ligand binding site, and the corresponding protein segments have been associated with mutant channel types in eukaryotic systems. These findings illustrate the potential of ssNMR for structural investigations on large membrane‐embedded proteins, even in the presence of local disorder.     

Tricycloundecane Derivatives as Potential N‐Methyl‐D‐aspartate (NMDA) Receptor and Voltage‐Gated Calcium Channel Modulators

Neurodegenerative disorders are debilitating conditions characterised by progressive dysfunction and death of neuronal cells. Amidst the proposed mechanisms of neurodegeneration, the effects of excitotoxicity via N‐methyl‐D ‐aspartate (NMDA) receptor stimulation and activation of voltage‐gated calcium channels (VGCC) on neuronal cells are prominent. This has led to the development of polycyclic cage molecules such as NGP1‐01, which exhibit neuroprotective properties through NMDA receptor and VGCC modulation. The medicinal potential of structurally related tricycloundecanes that are open‐cage or rearranged polycyclic moieties has not been explored. This study is therefore focused on the synthesis of a series of novel tricycloundecane derivatives and their ability to inhibit NMDA receptors and VGCC. Significant NMDA receptor inhibition was observed for tricyclo[6.2.1.0^2,7]undec‐9‐ene‐3,6‐dione ( 4 , 78 %) and 6‐hydroxytricyclo[6.2.1.0^2,7]undec‐9‐en‐3‐one ( 5 , >95 %) at a concentration of 100 μM . The highest inhibitory activity was observed for 6‐(benzylimino)tricyclo[6.2.1.0^2,7]undec‐9‐en‐3‐one ( 9 , >95 %), which is in the same range as the inhibitory activity of MK‐801 (dizocilpine). In the VGCC inhibition assay, 6‐(benzylamino)tricyclo[6.2.1.0^2,7]undeca‐4,9‐dien‐3‐one ( 8 , 34 %), 9 (38 %) and 2‐(benzylamino)‐3,6‐epoxytricyclo[6.2.1.0^5,10]undecan‐9‐ol ( 12 , 40 %) showed statistically significant (p<0.05) VGCC inhibition.     

Synthetic and Biological Studies of Sesquiterpene Polygodial: Activity of 9‐Epipolygodial against Drug‐Resistant Cancer Cells

Polygodial, a terpenoid dialdehyde isolated from Polygonum hydropiper L., is a known agonist of the transient receptor potential vanilloid 1 (TRPV1). In this investigation a series of polygodial analogues were prepared and investigated for TRPV1‐agonist and anticancer activities. These experiments led to the identification of 9‐epipolygodial, which has antiproliferative potency significantly exceeding that of polygodial. 9‐Epipolygodial was found to maintain potency against apoptosis‐resistant cancer cells as well as those displaying the multidrug‐resistant (MDR) phenotype. In addition, the chemical feasibility for the previously proposed mechanism of action of polygodial, involving the formation of a Paal–Knorr pyrrole with a lysine residue on the target protein, was demonstrated by the synthesis of a stable polygodial pyrrole derivative. These studies reveal rich chemical and biological properties associated with polygodial and its direct derivatives. These compounds should inspire further work in this area aimed at the development of new pharmacological agents, or the exploration of novel mechanisms of covalent modification of biological molecules with natural products.     

Structurally Minimized μ‐Conotoxin Analogues as Sodium Channel Blockers: Implications for Designing Conopeptide‐Based Therapeutics

Transforming the neuroactive toxins of cone snails into small‐size compounds poses a challenge due to the presence of multiple disulfide bridges. Herein we describe our successful efforts in minimizing the size of μ‐conotoxin while retaining its biological activity.

     

Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of KATP Channels

4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides with various substituents in positions 3, 5, and 7 were synthesized and tested as K_ATP channel agonists in artificial cell systems (CHO cells transfected with SUR1/Kir6.2, and HEK 293 transfected with SUR2B/Kir6.1) as model systems for insulin‐secreting pancreatic β‐cells and for smooth muscle cells, respectively. The effects of agonists were tested in intact cells using DiBAC4(3) [bis‐(1,3‐dibarbituric acid)trimethine oxanol] as a membrane potential dye, and the results compared with their binding affinity for the SUR2B‐type K_ATP channels using the radioligand [³H]P1075. Compounds with cycloalkyl and (cycloalkyl)methyl side chains in position 3 had higher affinities towards the SUR2B/Kir6.1 receptor compared with the parent compound diazoxide ( 1 a ). Compounds with bulky, nonpolar residues in position 3 exhibited remarkable selectivity for SUR2B‐type K_ATP channels. The compound substituted with a bulky (1‐adamantyl)methyl residue exhibited micromolar affinity and activity on SUR2B‐type K_ATP channels without being able to activate the SUR1‐type K_ATP channels.     

Bis‐(1,2,3,4‐tetrahydroisoquinolinium): A Chiral Scaffold for Developing High‐Affinity Ligands for SK Channels

N‐Methyl‐bis‐(1,2,3,4‐tetrahydroisoquinolinium) analogues derived from AG525 (1,1′‐(propane‐1,3‐diyl)‐bis‐(6,7‐dimethoxy‐2‐methyl‐1,2,3,4‐tetrahydroisoquinoline)) stereoisomers and tetrandrine, a rigid bis‐(1,2,3,4‐tetrahydroisoquinoline) analogue with an S,S configuration, were synthesized and tested for their affinity for small‐conductance calcium‐activated potassium channel (SK/K_Ca2) subtypes using radioligand binding assays. A significant increase in affinity was observed for the quaternized analogues over the parent 1,2,3,4‐tetrahydroisoquinoline compounds. Interestingly, the impact of stereochemistry was not the same in the two groups of compounds. For quaternized analogues, affinities of S,S and R,R isomers for SK2 and SK3 channels were similar and in both cases higher than that of the meso derivative. Among the bis‐tetrahydroisoquinoline compounds, the S,S isomers exhibited high affinity, while the R,R and meso isomers had similarly lower affinities. Furthermore, the SK2/SK3 selectivity ratio was slightly increased for quaternized analogues. Bis‐(1,2,3,4‐tetrahydroisoquinolinium) represents a new scaffold for the development of high‐affinity ligands for SK channel subtypes.     

Photoactivation of an Acid‐Sensitive Ion Channel Associated with Vision and Pain

We describe the reversible photoactivation of the acid sensitive ligand‐gated ion channel ASIC2a, a mammalian channel found throughout the central and peripheral nervous systems that is associated with vision and pain. We also show the activation of GLIC, an acid‐sensitive prokaryotic homologue of the nicotinic acetylcholine receptor. Photoactivation was achieved by using visible light irradiation of a newly synthesized water‐soluble merocyanine photoacid, 1 , which was designed to remove adverse channel blocking effects of a related system. Activation of ASIC2a and GLIC occurs reversibly, in a benign manner, and only upon irradiation. Further studies using transient absorption spectroscopy showed that protonation of a colorimetric base occurred rapidly (ca. 100 μs) after excitation of 1 . These results demonstrate that irradiation of 1 can induce rapid, local pH changes that can be used to investigate both biological and chemical proton transfer reactions.     

New Disaccharide‐Based Ether Lipids as SK3 Ion Channel Inhibitors

The SK3 potassium channel is involved in the development of bone metastasis and in the settlement of cancer cells in Ca²⁺‐rich environments. Ohmline, which is a lactose‐based glycero‐ether lipid, is a lead compound that decreases SK3 channel activity and consequently limits the migration of SK3‐expressing cells. Herein we report the synthesis of three new ohmline analogues in which the connection of the disaccharide moieties (1→6 versus 1→4) and the stereochemistry of the glycosyl linkage was studied. Compound 2 [3‐(hexadecyloxy)‐2‐methoxypropyl‐6‐O‐α‐d ‐glucopyranosyl‐β‐d ‐galactopyranoside], which possesses an α‐glucopyranosyl‐(1→6)‐β‐galactopyranosyl moiety, was found to decrease SK3 current amplitude (70 % inhibition at 10 μm ), displace SK3 protein outside caveolae, and decrease constitutive Ca²⁺ entry (50 % inhibition at 300 nm ) and SK3‐dependent cell migration (30 % at 300 nm ) at a level close to that of the benchmark compound ohmline. Compound 2 , which decreases the activity of SK3 channel (but not SK2 channel), is a new drug candidate to reduce cancer cell migration and to prevent bone metastasis.     

Rational Design,Synthesis, and Biological Evaluation of Lactam‐Bridged Gramicidin A Analogues: Discovery of a Low‐Hemolytic Antibacterial Peptide

A linear peptide, gramicidin A (GA), folds into a β^6.3‐helix, functions as an ion channel in the cell membrane, and exerts antibacterial activity. Herein we describe the rational design, synthesis, and biological evaluation of lactam‐bridged GA analogues. The GA analogue with a 27‐membered macrolactam was found to adopt a stable β^6.3‐helical conformation and exhibits higher ion‐exchange activity than GA. Furthermore, this GA analogue retains the potent antibiotic activity of GA, but its hemolytic activity and toxicity toward mammalian cells are significantly lower than those of GA. This study thus dissociates the antibacterial and hemolytic/cytotoxic activities of GA, and charts a rational path forward for the development of new ion‐channel‐based antibiotics.     

Do the short‐wave cones signal blueness?

A simple correspondence between S‐cone response and the sensation of blueness has long been questioned. We measured color appearance using a series of S‐cone isolating stimuli and a paired comparison paradigm to identify a direct correspondence between S‐cone response and perceived hue strength. The results revealed a non‐monotonic reversal in perceived blueness at the unique‐blue line: The S‐cone increment initially increased blueness, but as S‐cone excitation grew past the pure‐blue line, blueness decreased and redness increased. It is difficult to account for this pattern of results by supposing that the sensation of blueness is mediated by a conventional linear channel that extracts (S+M)/L: If the ratio L:M is held constant, the output of such a channel should always increase with increasing S, and so inhibition from a second channel would be needed to account for our result. We also consider another category of explanation: The appearance of a given chromaticity may depend on its position relative to the fundamental axis of daylight locus, which serves as an internalized reference. In an account of the latter kind, there is a systematic mapping between chromaticities and sensations, but there does not need to be a distinct neural signal that represents “blueness.” © 2014 Wiley Periodicals, Inc. Col Res Appl, 40, 323–328, 2015     

Expanding the Scope of Protein Trans‐Splicing to Fragment Ligation of an Integral Membrane Protein: Towards Modulation of Porin‐Based Ion Channels by Chemical Modification

It's raining, it's porin : Fragment ligation of OmpF ion channels was achieved by using the split Psp‐GBD Pol intein; this allowed reconstitution of active trimeric porin. In combination with cysteine modification at an internal position, the porin's conductance properties were altered.

     

Overview of the Experimental Trends in Water‐Assisted Injection Molding

In recent years, a new and promising polymer processing technology known as water‐assisted injection molding (WAIM) has attracted attention not only for academic reasons but also for its industrial applications. WAIM technology provides a new way to fabricate hollow or other complicated products due to its faster cycling time and light weight. This paper aims to give an overview of the basic principles and applications of WAIM as well as the current research status in academia. The origin and development of WAIM technology are first described and then, their advantages and applications are given. This review focuses on the experimental trends of WAIM such as computer simulation, the effect of processing parameters on the WAIM samples, and the morphology as well as related WAIM–molded composites and polymer blends' work.     

Isocyanides as Influenza A Virus Subtype H5N1 Wild‐Type M2 Channel Inhibitors

Basic bulky amines such as amantadine are well‐characterized M2 channel blockers, useful for treating influenza. Herein we report our surprising findings that charge‐neutral, bulky isocyanides exhibit activities similar to—or even higher than—that of amantadine. We also demonstrate that these isocyanides have potent growth inhibitory activity against the H5N1 virus. The ?NH₂ to ?N≡C group replacement within current anti‐influenza drugs was found to give compounds with high activities at low‐micromolar concentrations. For example, a tenfold improvement in potency was observed for 1‐isocyanoadamantane ( 27 ), with an EC₅₀ value of 0.487 μm against amantadine‐sensitive H5N1 virus as determined by both MTT and plaque‐reduction assays, without showing cytotoxicity. Furthermore, the isocyanide analogues synthesized in this study did not inhibit the V27A or S31N mutant M2 ion channels, according to electrophysiology experiments, and did not exhibit activity against amantadine‐resistant virus strains.     

Probing the Role of Backbone Hydrogen Bonding in a Critical β Sheet of the Extracellular Domain of a Cys‐Loop Receptor

Probing the sheet : The network of hydrogen bonds formed in the outer β sheet of the nicotinic acetylcholine receptor (nAChR; see figure) is fairly robust and tolerates single amide‐to‐ester mutations throughout. However, eliminating two proximal hydrogen bonds completely destroys receptor function; this adds further support to gating models that ascribe important roles to these β strands of the nAChR extracellular domain.

     

Bifurcating channels supplying “numbered-up” microreactors

This study is intended to be the starting point for theory of branching channels that supply reactants into chemical microreactors positioned on the surface of a microchip. The channels are assumed to be made by isotropic etching - and therefore of the same depths everywhere, irrespective of their cross-section area. When required to handle larger flows, the channels are therefore wide and thus occupy a substantial proportion of the available surface area. Because of the intended starting-point character, this discussion of the branchings assumes the simplest, isokinetic bifurcation. The branching pattern may resemble the self-similar pattern of fractal objects, but the constant depth makes the fractal theory inadequate. The evaluation of the chip surface areas occupied by the three basic components of the system - the reactors, the sealing strips, and the channels - uses a two-parametric model, with widths of the channels and sealing strips as the independent variables.