Fung-Yi Chan

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

Benzothiazole-substituted benzofuroquinolinium dye: a selective switch-on fluorescent probe for G-quadruplex

A new switch-on fluorescent probe containing the natural product cryptolepine analogue benzofuroquinolinium moiety (binding scaffold) and a benzothiazole moiety (signalling unit) shows a remarkable fluorescence enhancement selective for the G-quadruplex nucleic acid structure. Binding studies revealed that the highly selective response of the fluorescent probe arises from end-stack binding to G-quadruplex.

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.

Identification of a New Class of FtsZ Inhibitors by Structure-Based Design and in Vitro Screening

The Filamenting temperature-sensitive mutant Z (FtsZ), an essential GTPase in bacterial cell division, is highly conserved among Gram-positive and Gram-negative bacteria and thus considered an attractive target to treat antibiotic-resistant bacterial infections. In this study, a new class of FtsZ inhibitors bearing the pyrimidine-quinuclidine scaffold was identified from structure-based virtual screening of natural product libraries. Iterative rounds of in silico studies and biological evaluation established the preliminary structure-activity relationships of the new compounds. Potent FtsZ inhibitors with low micromolar IC₅₀ and antibacterial activity against S. aureus and E. coli were found. These findings support the use of virtual screening and structure-based design for the rational development of new antibacterial agents with innovative mechanisms of action.

Rational design of berberine-based FtsZ inhibitors with broad-spectrum antibacterial activity.

Inhibition of the functional activity of Filamenting temperature-sensitive mutant Z (FtsZ) protein, an essential and highly conserved bacterial cytokinesis protein, is a promising approach for the development of a new class of antibacterial agents. Berberine, a benzylisoquinoline alkaloid widely used in traditional Chinese and native American medicines for its antimicrobial properties, has been recently reported to inhibit FtsZ. Using a combination of in silico structure-based design and in vitro biological assays, 9-phenoxyalkyl berberine derivatives were identified as potent FtsZ inhibitors. Compared to the parent compound berberine, the derivatives showed a significant enhancement of antibacterial activity against clinically relevant bacteria, and an improved potency against the GTPase activity and polymerization of FtsZ. The most potent compound 2 strongly inhibited the proliferation of Gram-positive bacteria, including methicillin-resistant S. aureus and vancomycin-resistant E. faecium, with MIC values between 2 and 4 µg/mL, and was active against the Gram-negative E. coli and K. pneumoniae, with MIC values of 32 and 64 µg/mL respectively. The compound perturbed the formation of cytokinetic Z-ring in E. coli. Also, the compound interfered with in vitro polymerization of S. aureus FtsZ. Taken together, the chemical modification of berberine with 9-phenoxyalkyl substituent groups greatly improved the antibacterial activity via targeting FtsZ.

Antimicrobial activity of a quinuclidine-based FtsZ inhibitor and its synergistic potential with β-lactam antibiotics

Filamenting temperature-sensitive mutant Z (FtsZ) is an essential cell division protein that cooperates in the formation of the cytokinetic Z-ring in most bacteria and has thus been recognized as a promising antimicrobial drug target. We have recently used a structure-based virtual screening approach to identify pyrimidine-linked quinuclidines as a novel class of FtsZ inhibitors. In this study, we further investigated the antibacterial properties of one of the most potent compounds (quinuclidine 1) and its synergistic activity with β-lactam antibiotics. Susceptibility results showed that quinuclidine 1 was active against multiple antibiotic-resistant bacterial strains including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium with minimal inhibitory concentrations of 24 μg ml−1. When quinuclidine 1 was combined with β-lactam antibiotics, synergistic antimicrobial activities against antibiotic-resistant strains of S. aureus were found. Further in vitro studies suggest that prevention of FtsZ protofilament formation by quinuclidine 1 impairs the formation of Z-ring, and thus inhibits bacterial division. These findings open a new approach for development of quinuclidine-based FtsZ inhibitors into potent antimicrobial agents.

Structure-based Design, Synthesis, and Biological Evaluation of Isatin Derivatives as Potential Glycosyltransferase Inhibitors

Peptidoglycan glycosyltransferase (PGT) has been shown to be an important pharmacological target for the inhibition of bacterial cell wall biosynthesis. Structure‐based virtual screening of about 3 000 000 commercially available compounds against the crystal structure of the glycosyltransferase (GT) domain of the Staphylococcus aureus penicillin‐binding protein 2 (S. aureus PBP2) resulted in identification of an isatin derivative, 2‐(3‐(2‐carbamimidoylhydrazono)‐2‐oxoindolin‐1‐yl)‐N‐(m‐tolyl)acetamide (4) as a novel potential GT inhibitor. A series of 4 derivatives were synthesized. Several compounds showed more active antimicrobial activity than the initial hit compound 4, in particular 2‐(3‐(2‐carbamimidoylhydrazono)‐2‐oxoindolin‐1‐yl)‐N‐(3‐nitrophenyl)acetamide (4l), against Gram‐positive Bacillus subtilis and S. aureus with MIC values of 24 and 48 μg/mL, respectively. Saturation transfer difference (STD) NMR study revealed that there is a binding contact between 4l and the GT domain of S. aureus PBP2. Competitive STD‐NMR further proved that 4l and moenomycin A bind to GT domain in a competitive manner. Molecular docking study suggests a potential binding pocket of 4l in the GT domain of S. aureus PBP2. Taken together, compound 4l would provide a new scaffold for further development of potent GT inhibitors.

Validation of the AmpC β-Lactamase Binding Site and Identification of Inhibitors with Novel Scaffolds

AmpC β-lactamase confers resistance to β-lactam antibiotics in multiple Gram-negative bacteria. Therefore, identification of non-β-lactam compounds that inhibit the enzyme is considered crucial to the development of novel antibacterial therapies. Given the highly solvent-exposed active site, it is important to study the induced-fit movements and water-mediated interactions to improve docking accuracy and virtual screening enrichments in structure-based design of new AmpC inhibitors. Here, we tested multiple models of the AmpC binding site to investigate the importance of conserved water molecules and binding site plasticity on molecular docking. The results indicate that at least one conserved water molecule greatly improves the binding pose predictions and virtual screening enrichments of known noncovalent AmpC inhibitors. The best model was tested prospectively in the virtual screening of about 6 million commercially available compounds. Sixty-one chemically diverse top-scoring compounds were experimentally tested, which led to the identification of seven previously unknown inhibitors. These findings validate the essential features of the AmpC binding site for molecular recognition and are useful for further optimization of identified inhibitors.

Efficient Synthesis of Amine-Linked 2,4,6-Trisubstituted Pyrimidines as a New Class of Bacterial FtsZ Inhibitors

We have recently identified a new class of filamenting temperature-sensitive mutant Z (FtsZ)-interacting compounds that possess a 2,4,6-trisubstituted pyrimidine–quinuclidine scaffold with moderate antibacterial activity. Employing this scaffold as a molecular template, a compound library of amine-linked 2,4,6-trisubstituted pyrimidines with 99 candidates was successfully established by employing an efficient convergent synthesis designed to explore their structure–activity relationship. The results of minimum inhibitory concentration (MIC) assay against Staphylococcus aureus strains and cytotoxicity assay against the mouse L929 cell line identified those compounds with potent antistaphylococcal properties (MIC ranges from 3 to 8 μg/mL) and some extent of cytotoxicity against normal cells (IC50 ranges from 6 to 27 μM). Importantly, three compounds also exhibited potent antibacterial activities against nine clinically isolated methicillin-resistant S. aureus (MRSA) strains. One of the compounds, 14av_amine16, exhibited low spontaneous frequency of resistance, low toxicity against Galleria mellonella larvae, and the ability to rescue G. mellonella larvae (20% survival rate at a dosage of 100 mg/kg) infected with a lethal dose of MRSA ATCC 43300 strain. Biological characterization of compound 14av_amine16 by saturation transfer difference NMR, light scattering assay, and guanosine triphosphatase hydrolysis assay with purified S. aureus FtsZ protein verified that it interacted with the FtsZ protein. Such a property of FtsZ inhibitors was further confirmed by observing iconic filamentous cell phenotype and mislocalization of the Z-ring formation of Bacillus subtilis. Taken together, these 2,4,6-trisubstituted pyrimidine derivatives represent a novel scaffold of S. aureus FtsZ inhibitors.

A Thiazole Orange Derivative Targeting the Bacterial Protein FtsZ Shows Potent Antibacterial Activity

The prevalence of multidrug resistance among clinically significant bacteria calls for the urgent development of new antibiotics with novel mechanisms of action. In this study, a new small molecule exhibiting excellent inhibition of bacterial cell division with potent antibacterial activity was discovered through cell-based screening. The compound exhibits a broad spectrum of bactericidal activity, including the methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and NDM-1 Escherichia coli. The in vitro and in vivo results suggested that this compound disrupts the dynamic assembly of FtsZ protein and Z-ring formation through stimulating FtsZ polymerization. Moreover, this compound exhibits no activity on mammalian tubulin polymerization and shows low cytotoxicity on mammalian cells. Taken together, these findings could provide a new chemotype for development of antibacterials with FtsZ as the target.