Myung-Ryul Lee

An apoptosis-inducing small molecule that binds to heat shock protein 70.

Apoptosis (or programmed cell death) is a fundamental biological process that regulates a variety of normal physiological processes, ranging from development to aging. Damaged or unwanted cells in organisms are removed by the intrinsic and/or extrinsic apoptotic pathways. The intrinsic apoptotic pathway occurs by the release of cytochrome c from mitochondria. The extrinsic apoptotic pathway is caused by the binding of death ligands, such as TNF (tumor necrosis factor), Fas, and TRAIL (TNF-related-apoptosis-inducing ligand), to their corresponding receptors. Although programmed cell death is involved in a number of key biological phenomena, aberrant apoptosis results in diverse human diseases. For example, the dysregulation of apoptosis disrupts tissue homeostasis by prolonging cell survival and contributes to the progression of diverse human tumors. In addition, retarded apoptosis causes the elimination of autoreactive lymphocytes to fail, leading to autoimmunity. Moreover, excessive apoptosis results in cell-loss disorders such as neurodegenerative (Alzheimer,s and Parkinson,s diseases) and cardiovascular diseases. Since apoptosis is involved in both normal physiology and various human diseases, research on apoptosis has become a central area in basic biological studies and in the development of therapeutic agents. Small molecules that either induce or prevent apoptotic cell death have significant potential as therapeutic agents to treat apoptosis-related diseases. In addition, these agents could also be employed to understand the roles that apoptotic regulatory proteins play in biological processes. Herein we describe a novel apoptosis-inducing small molecule which interacts with Hsc70 and Hsp70. Cell-based screening with a small molecule library is an attractive approach to identify bioactive compounds that regulate protein functions in cells or affect processes such as cell differentiation or morphology. We applied this approach to select molecules with apoptosis-inducing activity, using a recently prepared imidazole library on a solid support to identify bioactive compounds that induce interesting cellular events (Scheme 1a). The amine-conjugated diethylene glycol linker was introduced into the library for facile solid-phase synthesis and the identification of target protein(s) by affinity chromatography. To search for molecules that induce apoptosis in cells, 216 imidazole derivatives (1 mm) were incubated with the highly proliferative P19 embryonic carcinoma cell line for 3 h and subsequently treated with a mixture of annexin V-fluorescein (0.5 mgmL ) and propidium iodide (PI, 2 mgmL ) to rapidly screen for apoptosis inducers. The exposure of phosphatidylserine on the outer leaflet of the cell plasma membrane is a key feature of the early stages (2–4 h) of apoptosis. Phosphatidylserine can be detected fluorescently by using annexin Vfluorescein. Propidium iodide (PI) can be used to monitor membrane-perturbed cells which result from the plasma membrane becoming permeable (a feature of necrosis) or late-stage apoptosis. Therefore, the combined use of annexin V-fluorescein and PI allows for the rapid evaluation of apoptosis in cells treated with the compound library. In our screen, compounds that exhibited positive annexin V and negative PI staining in P19 cells after 3 h incubation were selected as inducers of apoptosis. However, compounds that showed positive annexin Vand positive PI staining in the cells were not selected as “hits” because it is possible that the treated cells underwent necrosis rather than apoptosis. One compound apoptozole-linker (Az-linker) showed a high level of positive annexin V and negative PI staining in P19 cells (Scheme 1b). For further studies, Az (without linker) was resynthesized and purified (see the Supporting Information). Scheme 1. Structures of a) an imidazole library (see the Supporting Information for substituents R–R) and b) apoptozole (Az).

Fabrication of carbohydrate chips and their use to probe protein|[ndash]|carbohydrate interactions

Carbohydrate microarrays have received considerable attention as an advanced technology for the rapid analysis of carbohydrate–protein interactions. This protocol provides detailed procedures for the preparation of carbohydrate microarrays by immobilizing hydrazide-conjugated carbohydrates on epoxide-derivatized glass slides. In addition, we describe the use we make of these microarrays in glycomics research. Unlike other techniques that require large amounts of samples and long assay times, carbohydrate microarrays are used to carry out the rapid assessment of a number of carbohydrate-recognition events with tiny amounts of carbohydrate samples. Furthermore, the microarray technology is also utilized for the rapid assay of enzyme activities. We are able to routinely prepare carbohydrate microarrays within 12 h by using hydrazide-conjugated carbohydrates and apply these microarrays for the studies of glycan–protein interactions within 8 h.

Chemoselective ligation of maleimidosugars to peptides/protein for the preparation of neoglycopeptides/neoglycoprotein

Two types of maleimidosugars as thiol-selective carbohydrates, 1-maleimidosugars and acetyl-linked maleimidosugars, were efficiently synthesized. They were coupled to glutathione, Fas peptide and bovine serium albumin (BSA) to prepare the corresponding glycosylated peptides and a protein via stable thioether linkages in a chemoselective manner.

Fluorescent high-throughput screening of chemical inducers of neuronal differentiation in skeletal muscle cells

This protocol describes detailed procedures for the fluorescent high-throughput screening of small molecules that induce neurogenesis in cultures of skeletal muscle cells. The detection of neurogenesis relies on a fluorescent dye, FM 1-43, which is used to study the neuronal property of depolarization-induced synaptic vesicle recycling. Thus, small molecules with neurogenesis-inducing activity in skeletal muscle cells can be rapidly identified by measuring the fluorescence intensity of the treated cells using a fluorescent microplate reader. This protocol uses murine myoblast C2C12 cells for screening, which are readily available and relatively easy to culture. Neurogenesis of PC12 cells induced by nerve growth factor is employed as a positive control for this screening. The screening time for this protocol is 8 d, which also includes the procedure to detect depolarization-induced synaptic vesicle recycling using FM 1-43.

Design and biological evaluation of novel antioxidants containing N-t-butyl-N-hydroxylaminophenyl moieties

In order to develop therapeutic agents against neurodegenerative diseases, we designed novel antioxidants containing N-t-butyl-N-hydroxylaminophenyl moieties and evaluated in vitro and in vivo neuroprotective properties as well as anti-ischemic effects.

Di-oxanipecotic acids as more stable turn motifs than di-nipecotic acids

The folded structures of peptidomimetics containing dimers of oxanipecotic acid (Oxa) in loop segments were characterized and compared with those of the corresponding nipecotic acid (Nip)-based ones. According to structural studies using FT-IR and NMR spectroscopies, di-oxanipecotic acid adopted more stable turn conformations than di-nipecotic acid, and for tetramers, L,(S)-Oxa,(S)-Oxa,L and L,(S)-Oxa,(R)-Oxa,L formed hairpin-like structures but only L,(R)-Nip,(S)-Nip,L promoted the stable folded conformations.

Carbohydrate microarrays for enzymatic reactions and quantification of binding affinities for glycan-protein interactions.

Glycans are involved in a variety of physiological and pathological processes through interactions with proteins. Thus, the molecular basis of glycan-protein interactions provides valuable information on understanding biological phenomena and exploiting more effective carbohydrate-based therapeutic agents and diagnostic tools. Carbohydrate microarray technology has become a powerful tool for evaluating glycan-mediated biological events in a high-throughput manner. This technology is mostly applied for rapid analysis of glycans-protein interactions in the field of functional glycomics. In order to expand application areas of glycan microarrays, we have used carbohydrate microarrays for measurement of binding affinities between glycans and proteins and profiling of glycosyltransferase activities. The glycan microarrays used for these studies are constructed by immobilizing maleimide or hydrazide-conjugated glycans on the thiol or hydrazide-derivatized glass slides, respectively. This protocol describes the fabrication of carbohydrate microarrays and their applications to enzymatic reactions and determination of quantitative binding affinities.

Chemical microarrays constructed by selective attachment of hydrazide-conjugated substances to epoxide surfaces and their applications.

Microarray technology has received considerable attention for rapid analysis of biomolecular interactions and high-throughput screening to identify binding partners. An efficient and selective immobilization technique of substances on the surface is essential for successful construction of microarrays. Although a variety of immobilization methods have been exploited to prepare microarrays over the past decade, a superior technique needs to be developed for diverse applications. Recently, an efficient and simple method that relies on selective reactions between the hydrazide conjugated to substances and the epoxide derivatized on the solid surface was developed to fabricate chemical microarrays. Reactions between hydrazides with epoxides are highly selective in that they take place even in the presence of other potent nucleophiles such as amines and thiols. This technique is utilized to immobilize various substances such as small molecules, carbohydrates, and peptides to glass surfaces. The microarrays constructed by this immobilization method are used to evaluate protein binding to carbohydrates, peptides, and small molecules. In addition, the microarrays are also employed to determine binding affinities between proteins and binding partners as well as profiling of enzyme activities.

De novo design of non-hydrogen-bonded helical pseudopeptides composed of oxanipecotic acid oligomers

Ab initio calculation and circular dichroism experiments reveal that Oxa-oligomers adopted pronounced non-hydrogen-bonded helical structures.