Meng Wu

A Europium‐Ion‐Based Luminescent Sensing Probe for Hydrogen Peroxide

A bright idea: A 15-fold increase in fluorescence intensity occurs when the complex formed between Eu3+ and the antibiotic tetracycline binds to hydrogen peroxide at neutral pH. The complex can be used in the determination of the concentration of H2O2, the activity of oxidases, the concentration of glucose, and also in an optical sensor for H2O2.

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.

Transient receptor potential canonical (TRPC) channels are Ca2+-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca2+ rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca2+ rise with an IC50 value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5′-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10–20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.

C-terminal binding: An expanded repertoire and function of 14-3-3 proteins

Amino and carboxyl termini are unique positions in a polypeptide. They tend to be exposed in folded three dimensional structures. Diversity and functional significance of C‐terminal sequences have been appreciated from studies of PDZ and PEX domains. Signaling 14‐3‐3 protein signaling by recognizing phosphorylated peptides plays a critical role in a variety of biological processes, including oncogenesis. The preferential binding of 14‐3‐3 to phosphorylated C‐terminal sequences, mode III, provides a means of regulated binding and considerably expands the substrate repertoire of 14‐3‐3 interaction partners.

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Determination of the activity of catalase using a europium(III)-tetracycline-derived fluorescent substrate

A one-step method is described for the fluorometric determination of the activity of the enzyme catalase (EC 1.11.1.6.), based on the finding that H(2)O(2) in the europium (III)-tetracycline-hydrogen peroxide system is consumed by catalase. This is accompanied by a large decrease in both fluorescence intensity and decay time. The limit of detection (LOD; at S/N=3) for catalase at 30 degrees C for a 10-min kinetic assay is 1.0 unit/mL, with a linear range from 1.0 to 10 unit/mL. At an incubation time of 30 min at 37 degrees C for a one-point assay, the LOD is 0.046 unit/mL, with a linear range from 46 to 400 munit/mL. The assay was performed on microtiterplates and is fully compatible with existing plate readers. It is a one-step, simple, and sensitive method suitable for both continuous kinetic and one-point detections, does not require the addition of other substrates, and works best at neutral pH (with an optimum at pH 6.9). The reagent has the typical spectral features of a europium-ligand complex including a large Stokes shift (210 nm), a red line-like emission (centered at 616 nm), and a decay time in the microsecond domain. It is also the first europium-based probe that is compatible with the 405-nm diode laser. In summary, the new assay provides distinct advantages over direct ultraviolet detection and over the two-reagent (peroxidase) method.

Selective Inhibition of the Kir2 Family of Inward Rectifier Potassium Channels by a Small Molecule Probe: The Discovery, SAR, and Pharmacological Characterization of ML133

The K(ir) inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (K(ir)1-K(ir)7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective K(ir)2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of K(ir)2.1 function. Here we report one potent K(ir)2.1 inhibitor, ML133, which inhibits K(ir)2.1 with an IC(50) of 1.8 μM at pH 7.4 and 290 nM at pH 8.5 but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on K(ir)1.1 (IC(50) > 300 μM) and displays weak activity for K(ir)4.1 (76 μM) and K(ir)7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the K(ir) family reported to date. Because of the high homology within the K(ir)2 family-the channels share a common design of a pore region flanked by two transmembrane domains-identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent K(ir) inhibitors. Using chimeric channels between K(ir)2.1 and K(ir)1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of K(ir)2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of K(ir)1.1 to those of K(ir)2.1 (N171D and C175I) transplants ML133 inhibition to K(ir)1.1. Together, the combination of a potent, K(ir)2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of K(ir)2 inward rectifier potassium channels.

14-3-3 protein interacts with Huntingtin-associated protein 1 and regulates its trafficking.

HAP1 (Huntingtin-associated protein 1) consists of two alternately spliced isoforms (HAP1A and HAP1B, which have unique C-terminal sequences) and participates in intracellular trafficking. The C terminus of HAP1A is phosphorylated, and this phosphorylation was found to decrease the association of HAP1A with kinesin light chain, a protein involved in anterograde transport in cells. It remains unclear how this phosphorylation functions to regulate the association of HAP1 with trafficking proteins. Using the yeast two-hybrid system, we found that HAP1 also interacts with 14-3-3 proteins, which are involved in the assembly of protein complexes and the regulation of protein trafficking. The interaction of HAP1 with 14-3-3 is confirmed by their immunoprecipitation and colocalization in mouse brain. Moreover, this interaction is specific to HAP1A and is increased by the phosphorylation of the C terminus of HAP1A. We also found that expression of 14-3-3 decreases the association of HAP1A with kinesin light chain. As a result, there is less HAP1A distributed in neurite tips of PC12 cells that overexpress 14-3-3. Also, overexpression of 14-3-3 reduces the effect of HAP1A in promoting neurite outgrowth of PC12 cells. We propose that the phosphorylation-dependent interaction of HAP1A with 14-3-3 regulates HAP1 function by influencing its association with kinesin light chain and trafficking in neuronal processes.

Time-Resolved Luminescence Imaging of Hydrogen Peroxide Using Sensor Membranes in a Microwell Format:

We demonstrate an optical imaging scheme for hydrogen peroxide in a microwell-based format using the europium(III) tetracycline complex as the fluorescent probe, which is incorporated into a polyacrylonitrile-co-polyacrylamide polymer matrix. The resulting sensor membranes are integrated into a 96-microwell plate. Hydrogen peroxide can be visualized by means of time-resolved luminescence lifetime imaging. The imaging system consists of a fast, gated charge-coupled device (CCD) camera and a pulsed array of 96 light emitting diodes (LEDs). Fluorescence lifetime images are acquired in different modes (rapid lifetime determination, RLD, and phase delay rationing, PDR) and compared with conventional intensity-based methods with respect to sensitivity and the dynamic range of the sensor. The lowest limits of detection can be achieved by the RLD method. The response time of the sensor is comparatively high, typically in the range of 10 to 20 minutes, but the response is reversible. The largest signal changes are observed at pH values between 6.5 and 7.5.

Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels

Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1–KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1–KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1–KCNE1 channels.

Time-Resolved Fluorescent Imaging of Glucose

A method for the fluorescent imaging of glucose is described that is based on the detection of enzymatically produced hydrogen peroxide, using the europium(III) tetracycline complex as the fluorescent probe incorporated into a hydrophilic polymer layer. Coadsorption of glucose oxidase (GOx) makes these sensor layers respond to the hydrogen peroxide produced by the GOx-assisted oxidation of glucose. The hydrogel layers are integrated into a 96-microwell plate for a parallel and simultaneous detection of various samples. Glucose is visualized by means of time resolved luminescence lifetime imaging. Unlike in previous methods, the determination of H2O2 does not require the addition of peroxidase or a catalyst to form a fluorescent product. The lifetime-based images obtained are compared with conventional fluorescence intensity-based methods with respect to sensitivity and the dynamic range of the sensor layer. The main advantages provided by this sensing scheme for H2O2 include reversibility, applicability at neutral pH, and the straightforwardness of the transducer system and the imaging device.