Dik-Lung Ma

A Highly Selective Luminescent Switch‐On Probe for Histidine/Histidine‐Rich Proteins and Its Application in Protein Staining

The luminescence sensing of histidine and histidine-rich proteins plays a pivotal role in biochemistry and molecular biology, in particular when both temporal and spatial resolution are required. An abnormal level of histidine-rich proteins is an indicator for many diseases, such as advanced liver cirrhosis, AIDS, renal disease, asthma, pulmonary disorders, thrombotic disorders, f] and malaria. Some analyses of histidine and histidine-rich proteins have been developed in conjunction with immunoassay and colorimetric detection methods. The most commonly used method for the detection of histidine and histidine-rich proteins in biological samples is chromatography, which is usually performed through the combination of an effective separation technique, such as thin-layer chromatography, gas chromatography, or HPLC, followed by UV/Vis or fluorescence spectroscopy. The use of high-performance capillary electrophoresis with a derivation reagent has also been reported. However, the aforementioned methods are generally tedious, laborious, and, most importantly, expensive for routine detection in a biochemistry laboratory. Although numerous studies have dealt with the detection of histidine or histidine-rich proteins, studies on the use of luminescent probes for this purpose remain sparse. Notable examples include research by Fabbrizzi and co-workers, who developed competitive noncovalent fluorescence turn-on probes for histidine in the form of dizinc(II) or dicopper(II) macrocyclic complexes, which recognize histidine through the formation of an imidazolate bridge between the two dizinc(II) or dicopper(II) centers; however, the resulting noncovalent ensemble may be less stable than a covalently linking sensory system, and the complexity of the synthetic process makes it difficult to implement in a convenient manner. Photoluminescent iridium(III) complexes have emerged as a topical area of interest in inorganic photochemistry and phosphorescent materials for optoelectronic and luminescence signaling applications. Significant changes in the photophysical behavior and emission properties of iridium(III) complexes may be induced by the presence of biomolecules. Luminescent transition-metal complexes for protein staining, such as the luminescent ruthenium complex known as SYPRO Ruby dye, have been reported previously. However, despite its high sensitivity and its broad dynamic range, the use of SYPRORuby dye is limited, as it is sold only as a formulated solution; therefore, it is not possible to optimize the dye for a particular electrophoresis protocol and protein. In this context, the luminescent cyclometalated iridium(III) solvent complex [Ir(ppy)2(solv)2] + (1; ppy= 2phenylpyridine, solv=H2O or CH3CN) has received particular attention for the following reasons: 1) [Ir(ppy)2(OH2)2] , which contains weakly bound solvent ligands, may bind covalently to amino acids/proteins through a ligand substitution reaction with the OH2 ligand; 2) an intriguing solvent/ media dependence of the emission properties of [Ir(ppy)2(OH2)2] + has been observed; 3) [Ir(ppy)2(OH2)2] + can be synthesized conveniently and rapidly; 4) the use of organic solvents is not required for the optimal sensing of amino acids/proteins with [Ir(ppy)2(OH2)2] , and the iridium complex is readily soluble and stable in aqueous staining solutions. In this study, [Ir(ppy)3] (2) was also prepared for comparative studies, as its binding with proteins was expected to be largely hydrophobic in nature. Herein, we describe the luminescent switch-on probe [Ir(ppy)2(solv)2] + (1) for histidine/histidinerich proteins and demonstrate its utility in protein staining. The Ir complexes 1-CF3SO3 and 2 (Figure 1a) were prepared according to a previously reported method. The structure of 1-ClO4 was established by X-ray crystallography and is depicted in Figure 1b, and the crystal-packing diagrams are given in the Supporting Information. The metal–ligand bonding parameters for 1-ClO4 are comparable to those reported previously for cyclometalated iridium(III) complexes. The complex 1-CF3SO3 (50 mm) is weakly emissive in phosphate buffered saline (PBS). In the presence of histidine (His), 1-CF3SO3 exhibits an intense emission at lmax= 505 nm, the intensity of which reaches saturation level at [His]/[Ir] 4 (Figure 2a). A plot of I/I0 versus [His]/[1-CF3SO3] (I and I0 are emission intensities with and without His) shows an up-to180-fold intensity enhancement at ratios [His]/[1-CF3SO3] 4:1. The luminescence response of 1-CF3SO3 to various other [*] Dr. D.-L. Ma, Dr. W.-L. Wong, W.-H. Chung, F.-Y. Chan, P.-K. So, Dr. T.-S. Lai, Z.-Y. Zhou, Prof. Y.-C. Leung, Prof. K.-Y. Wong Department of Applied Biology and Chemical Technology, Central Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University Hung Hom, Kowloon, Hong Kong (China) Fax: (+1)852-2364-9932 E-mail: bcedmond@polyu.edu.hk bckywong@inet.polyu.edu.hk

Cyclometalated Platinum(II) Complexes as Highly Sensitive Luminescent Switch‐On Probes for Practical Application in Protein Staining and Cell Imaging

Protein-staining platinum: The luminescent switch-on characteristic of the platinum(II) complex can be utilized for staining a series of proteins in sodium dodecyl sulfate-polyarcylamide gels, to give emissive gel images directly under UV light (see figure). The detection sensitivity for BSA protein is down to 6.0 ng, revealing potential practical applications of luminescent platinum(II) complexes in the luminescent signaling of biomolecules.

Cyclometalated gold(III) complexes with N-heterocyclic carbene ligands as topoisomerase I poisons

A panel of stable [Au(R-C--N--C)(N-heterocyclic carbene)](+) complexes displays prominent in vitro anticancer properties; [Au(C--N--C)(IMe)]CF(3)SO(3) (1, IMe = 1,3-dimethylimidazol-2-ylidene) significantly poisons topoisomerase I in vitro and suppresses tumor growth in nude mice model.

Stable Anticancer Gold(III)–Porphyrin Complexes: Effects of Porphyrin Structure

In the design of physiologically stable anticancer gold(III) complexes, we have employed strongly chelating porphyrinato ligands to stabilize a gold(III) ion [Chem. Commun. 2003, 1718; Coord. Chem. Rev. 2009, 253, 1682]. In this work, a family of gold(III) tetraarylporphyrins with porphyrinato ligands containing different peripheral substituents on the meso-aryl rings were prepared, and these complexes were used to study the structure-bioactivity relationship. The cytotoxic IC(50) values of [Au(Por)](+) (Por=porphyrinato ligand), which range from 0.033 to >100 microM, correlate with their lipophilicity and cellular uptake. Some of them induce apoptosis and display preferential cytotoxicity toward cancer cells than to normal noncancerous cells. A new gold(III)-porphyrin with saccharide conjugation [Au(4-glucosyl-TPP)]Cl (2a; H(2)(4-glucosyl-TPP)=meso-tetrakis(4-beta-D-glucosylphenyl)porphyrin) exhibits significant cytostatic activity to cancer cells (IC(50)=1.2-9.0 microM) without causing cell death and is much less toxic to lung fibroblast cells (IC(50)>100 microM). The gold(III)-porphyrin complexes induce S-phase cell-cycle arrest of cancer cells as indicated by flow cytometric analysis, suggesting that the anticancer activity may be, in part, due to termination of DNA replication. The gold(III)-porphyrin complexes can bind to DNA in vitro with binding constants in the range of 4.9 x 10(5) to 4.1 x 10(6) dm(3) mol(-1) as determined by absorption titration. Complexes 2a and [Au(TMPyP)]Cl(5) (4a; [H(2)TMPyP](4+)=meso-tetrakis(N-methylpyridinium-4-yl)porphyrin) interact with DNA in a manner similar to the DNA intercalator ethidium bromide as revealed by gel mobility shift assays and viscosity measurements. Both of them also inhibited the topoisomerase I induced relaxation of supercoiled DNA. Complex 4a, a gold(III) derivative of the known G-quadruplex-interactive porphyrin [H(2)TMPyP](4+), can similarly inhibit the amplification of a DNA substrate containing G-quadruplex structures in a polymerase chain reaction stop assay. In contrast to these reported complexes, complex 2a and the parental gold(III)-porphyrin 1a do not display a significant inhibitory effect (<10%) on telomerase. Based on the results of protein expression analysis and computational docking experiments, the anti-apoptotic bcl-2 protein is a potential target for those gold(III)-porphyrin complexes with apoptosis-inducing properties. Complex 2a also displays prominent anti-angiogenic properties in vitro. Taken together, the enhanced stabilization of the gold(III) ion and the ease of structural modification render porphyrins an attractive ligand system in the development of physiologically stable gold(III) complexes with anticancer and anti-angiogenic activities.

Proteomic and transcriptomic study on the action of a cytotoxic saponin (Polyphyllin D): Induction of endoplasmic reticulum stress and mitochondria‐mediated apoptotic pathways

Polyphyllin D (PD) is a potent cytotoxic saponin found in Paris polyphylla. In the present study, bioinformatic, proteomic and transcriptomic analyses were performed to study the mechanisms of action of PD on human nonsmall cell lung cancer (NSCLC) cell line (NCI‐H460). Using a gene expression‐based bioinformatic tool (connectivity map), PD was identified as a potential ER stress inducer. Our proteomic and transcriptomic analyses revealed that PD treatment led to upregulation of typical ER stress‐related proteins/genes including glucose‐regulated protein 78 (BiP/GRP78) and protein disulfide isomerase (PDI). In particular, elevated expression of C/EBP homologous transcription factor (chop) and activation of caspase‐4 occurred at early time point (8 h) of PD treatment, signifying an initial ER stress‐mediated apoptosis. Induction of tumor suppressor p53, disruption of mitochondrial membrane, activation of caspase‐9 and caspase‐3 were detected upon prolonged PD treatment. Collectively, these data revealed that PD induced the cytotoxic effect through a mechanism initiated by ER stress followed by mitochondrial apoptotic pathway. The ability of activating two major pathways of apoptosis makes PD an attractive drug lead for anticancer therapeutics.

A selective oligonucleotide-based luminescent switch-on probe for the detection of nanomolar mercury(II) ion in aqueous solution

An oligonucleotide-based luminescent platinum(II) switch-on probe has been developed for selective detection of nanomolar Hg(2+) ions.

Identification of natural product Fonsecin B as a stabilizing ligand of c-myc G-quadruplex DNA by high-throughput virtual screening

Fonsecin B has been identified as stabilizing ligand of c-myc G-quadruplex DNA using high-throughput virtual screening of a natural product database, and inhibited Taq polymerase-mediated DNA extension in vitro through stabilization of the G-quadruplex secondary structure.

Discovery of a drug-like G-quadruplex binding ligand by high-throughput docking.

There has been considerable interest in the study of G-quadruplex DNA owing to its involvement in the regulation of telomerase activities. 2] Human telomeric DNA is composed of a repeating double-stranded [TTAGGG/CCCTAA]n sequence except in the 3’-terminal region, which consists of a singlestranded tandem [TTAGGG] repeat sequence over several hundred bases. In normal somatic cells, approximately 100 bases are lost in each cell division, and after the telomeres have been shortened to a critical threshold, the cell undergoes apoptosis. In cancer cells, telomeric length is maintained by telomerase, and telomerase activity is expressed in >90% of tumor cell lines, but in relatively few normal cell types. Therefore, the inhibition of telomerase activity by ligand-induced stabilization of G-quadruplexes has become an attractive strategy for developing new anticancer drugs. Planar aromatic molecules with scaffolds that have extended delocalized pelectron systems such as cationic porphyrins, 2,7] BRACO19, 9-anilinoproflavin, 7] triazines, pentacyclic acridines, and telomestatin 9b,10a] are known to bind to and stabilize Gquadruplex DNA, resulting in anti-telomerase activity. This gives rise to telomere shortening and suppression of cell growth, ultimately leading to cell death. Recently, we also demonstrated by molecular modeling studies that quindoline derivatives have the ability to stabilize the G-quadruplex structure in c-myc. However, most reported small-molecule Gquadruplex stabilizers have extended planar structures that result in poor bioavailability. Virtual screening of chemical databases by molecular docking is one of the most powerful approaches to discover smallmolecule inhibitors. The major advantage of virtual screening of drug-like compounds is that chemical diversity is generated without the need for chemical synthesis ; confirmed hits identified in a screen could be used to guide further synthesis and quantitative structure–activity relationship analysis. Abagyan and co-workers recently demonstrated the applicability of high-throughput virtual screening of a marketed drug database in the identification of anti-androgen scaffolds. Inspired by this promising result, we extended the scope of identifying G-quadruplex DNA binding ligands through the virtual screening of a drug-like compound database. To develop a highthroughput screening platform for G-quadruplex DNA stabilizing ligands, a computer model was constructed by using the X-ray crystal structure of the intramolecular human telomeric G-quadruplex DNA (PDB code: 1KF1). It is common to use X-ray crystal structures for virtual screening of novel compounds from large databases because X-ray crystallography generally provides a larger amount of high-quality experimental data than NMR spectroscopy, and thus crystal structures are thought to provide a more accurate depiction. NMR structures are solved in a more biologically relevant environment; however, they provide a dynamic representation of the biomolecule when used as a collection. In the current study, the NMR structure of the intramolecular human telomeric G-quadruplex DNA in K solution (PDB code: 2GKU) is different from the X-ray crystal structure; the DNA strands are oriented in a (3+1) direction in the NMR structure, whereas the X-ray structure shows an all parallel direction, and as such, studies on the structure of the intramolecular human telomere quadruplex in physiological K solution have raised extreme controversy. Tan and co-workers recently reported the intramolecular human telomere quadruplex to adopt a parallel-stranded conformation in the noncrystalline state in K solution under molecular crowding conditions, as the K crystal structure quadruplex does. We report herein a new drug-like compound identified through in silico screening that is an effective stabilizer of Gquadruplex DNA. This compound also possesses high selectivity for G-quadruplex versus duplex DNA. Over 100000 compounds in a drug-like database that passed the Lipinski filters were screened in silico. The continuously flexible ligands were docked to a grid representation of the receptor and assigned a score reflecting the quality of the complex according to the ICM method (Molsoft). The bestscoring molecule in this new class of drug-like hits, 1H-pyrazole-3-carboxy-4-methyl-5-phenyl-(1H-indol-3-ylmethylene)hydrazide, was evaluated for its ability to stabilize G-quadruplex DNA (Figure 1). To the best of our knowledge, compound 1 has not yet been reported to stabilize G-quadruplex DNA.

Synthesis and Biological Activity of a Platinum(II) 6‐Phenyl‐2,2′‐bipyridine Complex and Its Dimeric Analogue

We have synthesized (pyridyl)‐(6‐phenyl‐2,2′‐bipyridine)platinum(II) hexafluorophosphate (1) and its corresponding dimer, μ‐N,N′‐bis(isonicotinyl)‐1,6‐hexanediamino bis‐[6‐phenyl‐2,2′‐bipyridine‐platinum(II)] dichloride (2). The DNA binding constants of 1 and 2 at 20 °C were determined by absorption titration to be 2.25×104 M−1 and 3.07×106 M−1, respectively. Compound 1 showed an AT preference, while 2 had no base preference. The binding site sizes of 2 for [poly(dA‐dT)]2, calf thymus DNA (ctDNA), and [poly(dG‐dC)]2, as determined by fluorescence titration, were 6.6, 4.0, and 2.8 bp, respectively. Compound 2 probably bound to [poly(dA‐dT)]2 through bisintercalation, and to [poly(dG‐dC)]2 by monointercalation. Binding of DNA by both complexes is favorable, since the binding free energies of 1 and 2 were estimated to be −5.8 and −8.7 kcal mol−1, respectively. The results of viscosity measurements and gel mobility shift assay demonstrated that binding of 1 and 2 caused DNA lengthening. The cytotoxicities of the complexes in various human cancer cell lines were determined by MTT assay. Complex 2 exhibited cytotoxicity comparable to that of cisplatin, and was more toxic than 1 by an order of magnitude.

Butyrate mediates nucleotide‐binding and oligomerisation domain (NOD) 2‐dependent mucosal immune responses against peptidoglycan

The interaction between digestive tract microbiological flora and food has an important influence on human health. Butyrate is produced during the fermentation of dietary fibres by intestinal bacteria and plays an important role in the regulation of mucosal immunity. In this report, we studied the impact of butyrate on the defence mechanism against the bacterial membrane component peptidoglycan (PGN). Butyrate was found to enhance PGN‐mediated IL‐8 and GRO‐α production. The expression of these chemokines required the activation of NF‐κB and was dependent on the concentrations of butyrate and PGN. Butyrate was found to up‐regulate nucleotide‐binding and oligomerisation domain (NOD) 2, but not NOD1 or TLR2. NOD2 up‐regulation was mediated by an increase in histone acetylation in the Nod2 promoter region, leading to enhanced PGN‐induced IL‐8 and GRO‐α secretion. Knockdown of NOD2 and TLR2 by siRNA significantly reduced PGN‐mediated chemokine production, suggesting that both NOD2 and TLR2 are required for maximal response. Our findings provide a better understanding of the mechanism by which butyrate regulates mucosal immunity for normal intestinal function. Based on the results of this study, we infer that dietary fibres can impact inflammatory bowel diseases.