Time-lapse confocal imaging-induced calcium ion discharge from the cumulus-oocyte complex at the time of cattle oocyte activation.

Oocyte activation, the dynamic transformation of an oocyte into an embryo, is largely driven by Ca2+ oscillations that vary in duration and amplitude across species. Previous studies have analysed intraoocyte Ca2+ oscillations in the absence of the oocyte's supporting cumulus cells. Therefore, it is unknown whether cumulus cells also produce an ionic signal that reflects fertilisation success. Time-lapse confocal microscopy and image analysis on abattoir-derived cattle cumulus-oocyte complexes coincubated with spermatozoa revealed a distinct discharge of fluorescence from the cumulus vestment. This study demonstrated that this Ca2+ fluorescence discharge was an artefact induced by the imaging procedure independently of oocyte activation success. The fluorescence discharge was a direct result of cumulus cell membrane integrity loss, and future studies should consider the long-term effect of fluorescent labels on cells in time-lapse imaging. However, this study also demonstrated that the distinctive pattern of a coordinated fluorescence discharge was associated with both the presence of spermatozoa and subsequent embryo development to the morula stage, which was affected by Ca2+ chelation and a reduction in the active efflux of the fluorophore. This indicates that the cumulus vestment may have a relationship with oocyte activation at and beyond fertilisation that requires further investigation.

Surface Functionalization of Exposed Core Glass Optical Fiber for Metal Ion Sensing.

One of the biggest challenges associated with exposed core glass optical fiber-based sensing is the availability of techniques that can be used to generate reproducible, homogeneous and stable surface coating. We report a one step, solvent free method for surface functionalization of exposed core glass optical fiber that allows achieving binding of fluorophore of choice for metal ion sensing. The plasma polymerization-based method yielded a homogeneous, reproducible and stable coating, enabling high sensitivity aluminium ion sensing. The sensing platform reported in this manuscript is versatile and can be used to bind different sensing molecules opening new avenues for optical fiber-based sensing.

Development of a Photoswitchable Lithium-Sensitive Probe to Analyze Nonselective Cation Channel Activity in Migrating Cancer Cells.

This is the first work to use a newly designed Li+-selective photoswitchable probe Sabrina Heng Lithium (SHL) in living colon cancer cells to noninvasively monitor cation channel activity in real time by the appearance of lithium hot spots detected by confocal microscopy. Punctate Li+ hot spots are clustered in the lamellipodial leading edges of HT29 human colon cancer cells and are colocalized with aquaporin-1 (AQP1) channels. AQP1 is a dual water and cyclic-nucleotide-gated cation channel located in lamellipodia and is essential for rapid cell migration in a subset of aggressive cancers. Both the Li+ hot spots and cell migration are blocked in HT29 cells by the AQP1 ion channel antagonist AqB011. In contrast, Li+ hot spots are not evident in a poorly migrating colon cancer cell line, SW620, which lacks comparable membrane expression of AQP1. Knockdown of AQP1 by RNA interference in HT29 cells significantly impairs Li+ hot spot activity. The SHL probe loaded in living cells shows signature chemical properties of ionic selectivity and reversibility. Dynamic properties of the Li+ hot spots, turning on and off, are confirmed by time-lapse imaging. SHL is a powerful tool for evaluating cation channel function in living cells in real time, with particular promise for studies of motile cells or interlinked networks not easily analyzed by electrophysiological methods. The ability to reset SHL by photoswitching allows monitoring of dynamic signals over time. Future applications of the Li+ probe could include high-throughput optical screening for discovering new classes of channels, or finding new pharmacological modulators for nonselective cation channels.

Spiropyran-Based Nanocarrier: A New Zn2+ -Responsive Delivery System with Real-Time Intracellular Sensing Capabilities.

A new spiropyran-based stimuli-responsive delivery system is fabricated. It encapsulates and then releases an extraneous compound in response to elevated levels of Zn2+ , a critical factor in cell apoptosis. A C12 -alkyl substituent on the spiropyran promotes self-assembly into a micelle-like nanocarrier in aqueous media, with nanoprecipitation and encapsulation of added payload. Zn2+ binding occurs to an appended bis(2-pyridylmethyl)amine group at biologically relevant micromolar concentration. This leads to switching of the spiropyran (SP) isomer to the strongly fluorescent ring opened merocyanine-Zn2+ (MC-Zn2+ ) complex, with associated expansion of the nanocarriers to release the encapsulated payload. Payload release is demonstrated in solution and in HEK293 cells by encapsulation of a blue fluorophore, 7-hydroxycoumarin, and monitoring its release using fluorescence spectroscopy and microscopy. Furthermore, the use of the nanocarriers to deliver a caspase inhibitor, Azure B, into apoptotic cells in response to an elevated Zn2+ concentration is demonstrated. This then inhibits intracellular caspase activity, as evidenced by confocal microscopy and in real-time by time-lapsed microscopy. Finally, the nanocarriers are shown to release an encapsulated proteasome inhibitor (5) in Zn2+ -treated breast carcinoma cell line models. This then inhibits intracellular proteasome and induces cytotoxicity to the carcinoma cells.

A Liposomal Platform for Sensing of Extracellular Analytes Near Cells.

Cell-permeable fluorescent chemosensors (calcein, monochlorobimane, and a recently reported spiropyran-based sensor SP2) have been incorporated into yeast total lipid extract-based liposomes to suppress inherent cell permeability to allow the detection of extracellular Ca2+, GSH, and Zn2+, respectively. The repurposed sensors have enhanced aqueous solubility and the ability to quantitatively measure biologically relevant concentrations of Ca2+ (0.25 mM⁻1 mM), Zn2+ (6.25 µM⁻50 µM), and GSH (0.25 mM⁻1 mM) by fluorescence in aqueous media. In addition, the liposomal sensors are nontoxic to HEK293 cells and have the ability to detect exogenously added Zn2+ (1 mM), Ca2+ (1 mM), or GSH (1 mM) near cells without internalisation. This new sensing platform provides a means to repurpose a range of intracellular fluorescent sensors to specifically detect extracellular analytes, while also improving biocompatibility for overall enhanced use in a wide range of biomedical applications.

Control of Molecular Recognition via Modulation of the Nanoenvironment.

Many biological processes are driven by the interaction of a host with a guest molecule. We show such interactions can be modulated by carefully defining the local molecular environment to give a specific chemical outcome. Particularly, the selectivity of a host toward two different ions (Ca2+ and Al3+) is defined by it being in solution or the physisorbed state. In solution, the host displays greater selectivity toward Ca2+. When physisorbed, the selectivity profile of the host is reversed with enhanced binding of Al3+. This demonstrates a single host molecule can be tailored to selectively bind multiple guests by altering its nanoenvironment.

A Rationally Designed Reversible 'Turn-Off' Sensor for Glutathione.

γ-Glutamyl-cysteinyl-glycine (GSH) plays a critical role in maintaining redox homeostasis in biological systems and a decrease in its cellular levels is associated with diseases. Existing fluorescence-based chemosensors for GSH acts as irreversible reaction-based probes that exhibit a maximum fluorescence ('turn-on') once the reaction is complete, regardless of the actual concentration of GSH. A reversible, reaction-based 'turn-off' probe ( 1 ) is reported here to sense the decreasing levels of GSH, a situation known to occur at the onset of various diseases. The more fluorescent merocyanine (MC) isomer of 1 exists in aqueous solution and this reacts with GSH to induce formation of the ring-closed spiropyran (SP) isomer, with a measurable decrease in absorbance and fluorescence ('turn-off'). Sensor 1 has good aqueous solubility and shows an excellent selectivity for GSH over other biologically relevant metal ions and aminothiol analytes. The sensor permeates HEK 293 cells and an increase in fluorescence is observed on adding buthionine sulfoximine, an inhibitor of GSH synthesis.

Rationally Designed Probe for Reversible Sensing of Zinc and Application in Cells.

Biologically compatible fluorescent ion sensors, particularly those that are reversible, represent a key tool for answering a range of fundamental biological questions. We report a rationally designed probe with a 6'-fluoro spiropyran scaffold (5) for the reversible sensing of zinc (Zn2+) in cells. The 6'-fluoro substituent overcomes several limitations normally associated with spiropyran-based sensors to provide an improved signal-to-background ratio and faster photoswitching times in aqueous solution. In vitro studies were performed with 5 and the 6'-nitro analogues (6) in HEK 293 and endothelial cells. The new spiropyran (5) can detect exogenous Zn2+ inside both cell types and without affecting the proliferation of endothelial cells. Studies were also performed on dying HEK 293 cells, with results demonstrating the ability of the key compound to detect endogenous Zn2+ efflux from cells undergoing apoptosis. Biocompatibility and photoswitching of 5 were demonstrated within endothelial cells but not with 6, suggesting the future applicability of sensor 5 to study intracellular Zn2+ efflux in these systems.

Microstructured Optical Fiber-based Biosensors: Reversible and Nanoliter-Scale Measurement of Zinc Ions.

Sensing platforms that allow rapid and efficient detection of metal ions would have applications in disease diagnosis and study, as well as environmental sensing. Here, we report the first microstructured optical fiber-based biosensor for the reversible and nanoliter-scale measurement of metal ions. Specifically, a photoswitchable spiropyran Zn(2+) sensor is incorporated within the microenvironment of a liposome attached to microstructured optical fibers (exposed-core and suspended-core microstructured optical fibers). Both fiber-based platforms retains high selectivity of ion binding associated with a small molecule sensor, while also allowing nanoliter volume sampling and on/off switching. We have demonstrated that multiple measurements can be made on a single sample without the need to change the sensor. The ability of the new sensing platform to sense Zn(2+) in pleural lavage and nasopharynx of mice was compared to that of established ion sensing methodologies such as inductively coupled plasma mass spectrometry (ICP-MS) and a commercially available fluorophore (Fluozin-3), where the optical-fiber-based sensor provides a significant advantage in that it allows the use of nanoliter (nL) sampling when compared to ICP-MS (mL) and FluoZin-3 (µL). This work paves the way to a generic approach for developing surface-based ion sensors using a range of sensor molecules, which can be attached to a surface without the need for its chemical modification and presents an opportunity for the development of new and highly specific ion sensors for real time sensing applications.

Photoregulation of α-Chymotrypsin Activity by Spiropyran-Based Inhibitors in Solution and Attached to an Optical Fiber.

Here the synthesis and characterization of a new class of spiropyran-based protease inhibitor is reported that can be reversibly photoswitched between an active spiropyran (SP) isomer and a less active merocyanine (MC) isomer upon irradiation with UV and visible light, respectively, both in solution and on a surface of a microstructured optical fiber (MOF). The most potent inhibitor in the series (SP-3 b) has a C-terminal phenylalanyl-based α-ketoester group and inhibits α-chymotrypsin with a Ki of 115 nM. An analogue containing a C-terminal Weinreb amide (SP-2 d) demonstrated excellent stability and photoswitching in solution and was attached to the surface of a MOF. The SP isomer of Weinreb amide 2 d is a competitive reversible inhibitor in solution and also on fiber, while the corresponding MC isomer was significantly less active in both media. The ability of this new class of spiropyran-based protease inhibitor to modulate enzyme activity on a MOF paves the way for sensing applications.

Dual sensor for Cd(II) and Ca(II): selective nanoliter-scale sensing of metal ions.

The first selective, dual sensor for Ca(2+) and Cd(2+) capable of detection at 100 pM concentrations was designed and synthesized. The experimental observations made for the MC-cation complexes and the selectivity of compounds 1 and 2 with Ca(2+) and Cd(2+) ions were further explored using density functional theory. A first step toward a nanoliter-scale dip sensor for the dual sensing of Ca(2+) and Cd(2+) was demonstrated using microstructured optical fiber as the sensing platform which is important for ion sensing in confined spaces such as the medium surrounding cell clusters. In addition, this system displays picomolar sensitivity for these ions, with an added ability to reproducibly turn ion-binding on/off.

Microstructured optical fibers and live cells: a water-soluble, photochromic zinc sensor.

A new biologically compatible Zn(II) sensor was fabricated by embedding a Zn(II) sensing spiropyran within the surface of a liposome derived from Escherichia coli lipids (LSP2). Solution-based experiments with increasing Zn(II) concentrations show improved aqueous solubility and sensitivity compared to the isolated spiropyran molecule (SP2). LSP2 is capable of sensing Zn(II) efflux from dying cells with preliminary data indicating that sensing is localized near the surface membrane of HEK 293 cells. Finally, LSP2 is suitable for development into a nanoliter-scale dip-sensor for Zn(II) using microstructured optical fiber as the sensing platform to detect Zn(II) in the range of 100 ρM with minimal photobleaching. Existing spiropyran based sensing molecules can thus be made biologically compatible, with an ability to operate with improved sensitivity using nanoscale liquid sample volumes. This work represents the first instance where photochromic spiropyran molecules and liposomes are combined to create a new and multifunctional sensing entity for Zn(II).

New cholesterol esterase inhibitors based on rhodanine and thiazolidinedione scaffolds.

We present a new class of inhibitors of pancreatic cholesterol esterase (CEase) based on 'priviledged' 5-benzylidenerhodanine and 5-benzylidene-2,4-thiazolidinedione structural scaffolds. The lead structures (5-benzylidenerhodanine 4a and 5-benzylidene-2,4-thiazolidinedione 4b) were identified in an in-house screening and these inhibited CEase with some selectivity over another serine hydrolase, acetylcholinesterase (AChE) (4a, CEase IC(50)=1.76 µM vs AChE IC(50)=5.14 µM and 4b, CEase IC(50)=5.89 µM vs AChE IC(50) >100 µM). A small library of analogs (5a-10a) containing a core amino acid in place of the glycerol group of the lead structures, was prepared to explore other potential binding interaction with CEase. These analogs inhibited CEase with IC(50) values ranging from 1.44 to 85 µM, with the majority exhibiting some selectivity for CEase versus AChE. The most potent compound of the library (10a) had 17-fold selectivity over AChE. We also report molecular docking (with CEase) and detailed kinetic analysis on the amino acid analogs to further understand the associated structure-activity relationships.

Fluorescence-based aluminum ion sensing using a surface-functionalized microstructured optical fiber.

The first microstructured optical fiber-based sensor platform for aluminum ions using a surface-attached derivative of lumogallion (3), a known fluorescence-based indicator, has been fabricated. These fibers allow for strong evanescent field interactions with the surrounding media because of the small core size while also providing the potential for real-time and distributed measurements. The fluorescence response to aluminum ions was first demonstrated by applying the procedure to glass slides. This was achieved through the covalent attachment of the fluorophore to a polyelectrolyte-coated glass surface and then to the internal holes of a suspended-core microstructured optical fiber to give an effective aluminum sensor. Whereas the sensor platform reported is fabricated for aluminum, the approach is versatile, with applicability to the detection of other ions.

Designing inhibitors against fructose 1,6-bisphosphatase: exploring natural products for novel inhibitor scaffolds.

Natural products often contain unusual scaffold structures that may be elaborated by combinatorial methods to develop new drug-like molecules. Visual inspection of more than 128 natural products with some type of anti-diabetic activity suggested that a subset might provide novel scaffolds for designing potent inhibitors against fructose 1,6-bisphosphatase (FBPase), an enzyme critical in the control of gluconeogenesis. Using in silico docking methodology, these were evaluated to determine those that exhibited affinity for the AMP binding site. Achyrofuran from the South American plant Achyrocline satureoides, was selected for further investigation. Using the achyrofuran scaffold, inhibitors against FBPase were developed. Compounds 15 and 16 inhibited human liver and pig kidney FBPases at IC50 values comparable to that of AMP, the natural allosteric inhibitor.

A library of novel allosteric inhibitors against fructose 1,6-bisphosphatase.

The identification of a proper lead compound for fructose 1,6-bisphosphatase (FBPase) is a critical step in the process of developing novel therapeutics against type-2 diabetes. Herein, we have successfully generated a library of allosteric inhibitors against FBPase as potential anti-diabetic drugs, of which, the lead compound 1b was identified through utilizing a virtual high-throughput screening (vHTS) system, which we have developed. The thiazole-based core structure was synthesized via the condensation of alpha-bromo-ketones with thioureas and substituents on the two aryl rings were varied. 4c was found to inhibit pig kidney FBPase approximately fivefold better than 1b. In addition, we have also identified 10b, a tight binding fragment, which can be use for fragment-based drug design purposes.

Design, synthesis, and bioactivity of novel inhibitors of E. coli aspartate transcarbamoylase.

A series of inhibitors of the aspartate transcarbamoylase, an enzyme involved in pyrimidine nucleotide biosynthesis, has been synthesized. These inhibitors are analogues of a highly potent inhibitor of this enzyme, N-phosphonacetyl-L-aspartate (PALA). Analogues have been synthesized with modifications at the alpha- and beta-carboxylates as well as at the aspartate moiety. The ability of these compounds to inhibit the enzyme was evaluated. These studies, with functional group modified PALA derivatives, showed that amide groups can be a useful substitute of the carboxylate in order to reduce the charge on the molecule, and indicate that the relative position of the functional group in the beta-position is more critical than the nature of the functional group. Some of the molecules synthesized here are potent inhibitors of the enzyme.

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.