Our research interest lies in Biological Inorganic Chemistry, in particular, metal in biology and medicine including design, synthesis of metalloagents as antimicrobials (or resistance breakers), and antivirals. Our particular interest is on bismuth drugs from mechanistic study to medicinal application. To understand the mechanism of action of metalloagents, we have established metalloproteomic approaches e.g. GE-ICP-MS/LC-GE-ICP-MS, fluorescence based approach, together with proteomics, metabolomics and transcriptomics, providing a platform for understanding the role of metals in biology and medicine at a system level. We are also interested in the structure and function of metalloproteins, in particular those that may serve as potential targets of metallodrugs.
Metal in medicine
Bismuth compounds have long been used in clinic for the treatment of various diseases, in particular, for Helicobacter pylori infection. Knowledge on their mechanisms of action enables new medicinal application of bismuth drugs to be unveiled.
Bismuth drugs as anti-SARS-CoV-2 agents
Bismuth drugs have been shown to suppress SARS-CoV-2 replication and relieve virus-associated pneumonia in Syrian hamsters. Ranitidine bismuth citrate exhibits low cytotoxicity and protected SARS-CoV-2 infected cell with a high selectivity index of 975. The drug likely targets viral helicase to inhibit viral replication and has clinical potential for the treatment of COVID-19. The work has been published in Nature Microbiology, 2020, 5, 1439–1448.
Bismuth drugs as resistant breakers of metallo-β-lactamases
In addition, bismuth drugs also have potentials to be repositioned for combating antimicrobial resistance (AMR). As bismuth compounds could inhibit metallo-β-lactamases, e.g. NDM-1, through displacement of zinc cofactor from the active sites of the enzyme, thus restoring activity of β-lactam antibiotics. The bismuth bound structure of NDM-1 has also been resolved. This study demonstrates a high potential of Bi(III) compounds as the first broad-spectrum B1 MBL inhibitors to treat MBL-positive bacterial infection in conjunction with existing carbapenems. This work has been published in Nature Communications, 2018, 9, 439.
Bismuth drugs exhibit low toxicity to host cells
Selective toxicity of bismuth drugs to H. pylori but bot host is unveiled for the first time. It has been demonstrated to be attributable to glutathione and multidrug resistance protein transporter mediate a self-propelled disposal of bismuth antiulcer drug. A model was derived to elucidate the uptake of the metallodrug, and which may readily be extended to other drug candidates. (Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 3211-3216). This work was highly recommended and commented by Prof. Peter Sadler. (Proc. Natl. Acad. Sci. U.S.A., 2015, 112, 4187-4188)
Auranofin as a dual inhibitor of metallo-β-lactamases and mobilized colistin resistance
A clinically used anti-rheumatic drug auranofin was identified as a dual inhibitor of metallo-β-lactamases (MBLs) and mobilized colistin resistance (MCRs). It could irreversibly abrogate both enzyme activity via the displacement of Zn(II) cofactors from their active sites, and synergize with antibiotics on killing a broad spectrum of carbapenem and/or COL resistant bacterial strains. Combination of auranofin with colistin rescues all mice infected by Escherichia coli co-expressing MCR-1 and New Delhi metallo-β-lactamase 5 (NDM 5). The work has been piblished in Nature Communications, 2020, 11, 5263.
Combating AMR by combination of gallium with acetate
Mechanism studies of Ga(III) allowed its potential application for combating AMR. Combination of Ga(III) with acetate inhibited growth of antibiotic resistant Pseudomonas aeruginosa cells and the effectiveness is validated in murine skin infection models. This is owing to enhanced uptake of Ga(III), reduced TCA cycle flow and bacterial respiration. This work has been published in Chemical Science 2019, 10, 6099-6106. The article is selected as the "pick of the week".
Metallomics and metalloproteomics: from methodology to application
Metallomics and metalloproteomics are emerging fields complementary to genomics and proteomics, the frontier of biological inorganic chemistry. We are studying the role, uptake, transport and storage of selected metals essential for protein functions. Targets and binding proteins of metallodrugs can be identified using a wide variety of state-of-the-art techniques such as proteomics, metallomics and structural biology. We have established different metalloproteomic approaches, allowing comprehensive identification of metal-binding proteins at proteome-wide scale.
In this laboratory, we have established an approach namely GE-ICP-MS to simultaneously detect metals and their associated proteins. This method is complementary to LA-ICP-MS and synchrotron X-ray fluorescence spectrometry (SXFS) but is more convenient for faily usage. Using this approach, we have mined potential targets of bismuth drugs from H. pylori which provide guidance for rational drug design. (Angew. Chem. Int.Ed., 2013, 52, 4916-4920)
The method was further extended to two dimensional, namely LC-GE-ICP-MS to enhance the resolution of protein separation by combining with liquid chromatography. The 2D LC-GE-ICP-MS enables 34 silver binding proteins to be identified from E. coli. By integration with metabolomics, bioinformatics and systemic biology, the first dynamic snapshot on antimicrobial action of silver in E. coli was unveiled. Silver primarily damages multiple enzymes in glycolysis and tricarboxylic acid (TCA) cycle, leading to the stalling of the oxidative branch of the TCA cycle and an adaptive metabolic divergence to the reductive glyoxylate pathway, then further damages the adaptive glyoxylate pathway and suppresses the cellular oxidative stress responses, causing systemic damages and death of the bacterium. The related works have been published in PLoS Biology 2019 17(6): e3000292; Chemical Science 2019;10,7193-7199; Chemical Science, 2020, 11, 11714-11719.
Fluorescence based approach for imaging proteins
We have developed membrane permeable Ni2+-NTA-based fluorescent probes by conjugation of nitrilotriacetate (NTA) moiety with a fluorophore and arylazide, followed by coordination with Ni2+ ions. This is the first probe of such type to be able to enter live cells for imaging proteins. By varying fluorophore, a series of probes with blue, red and green colors have been obtained. Importantly, through coordination of different metal ions, a metal tunable probes could be achieved, allowing tracking metal associated proteins from live cells. The related works have been published in Proc. Natl. Acad. Sci.U.S.A., 2015, 112, 2948-2953; ACS Sensor 2019, 4, 5, 1190–1196; Journal of Material Chemistry B, 2017,5, 1166-1173. The work was highlighted by Nature Biotechnology (Nat. Biotech., 2015, 33, 359-359.), recommended in F1000 Prime and reported by Asian Scientist Magazine and Asia Research News.
Integrative omic approach for MoA studies of metallodrugs
By integration of in-house metalloproteomics with quantitative proteomics, bismuth binding proteins and bismuth-regulated proteins have been mined at a proteome-wide scale. A total of 63 Bi-binding proteins and 119 Bi-regulated proteins were identified from H. pylori, which provides rich sources for in depth analysis of biological pathways disrupted by bismuth drugs. Subsequent bioinformatics analysis and bioassays enable a holistic view on the mode of action of bismuth drugs against H. pylori. Based on this study, it is concluded that sustainable antimicrobial activity of bismuth drugs against H. pylori and low likelihood to develop resistance to bismuth drugs by H. pylori was attributed to the multi-targeted mode of action of a bismuth drug (Chemical Science. 2017, 8, 4626-4633). Moreover, transcriptomics and metabolomics were also integrated with metalloproteomics, allowing detailed analysis of cellular response of H. pylori to bismuth drugs (Chemical Science, 2018, 9, 7488-7497).
Structure and function of metalloproteins
It has been estimated that a quarter to one-third of proteins are metalloproteins. Metal ions in metalloproteins are critical to the protein's function, structure or stability. In fact, numerous essential biological functions require metal ions. Our particular interest lies in a cysteine-rich metalloprotein, neuronal growth inhibitory factor (GIF or metallothionein III) and histidine-rich proteins (e.g. Hpn, Hpnl, HspA) as well as nickel homeostasis related metalloproteins. We have solved the three dimensional structures of the MT-3, HpHypA, HpSlyD. Potential protein targets of bismuth drugs in H. pylori have been extensively studied.
Full scenario of nickel translocation via accessory proteins to urease
We discovered that UreG is a nickel-dependent GTPase, the activity of which is stimulated by potassium and ammonium bicarbonate ions. The two metallochaperones form a (UreE)2-(UreG)2 complex in the presence of GTP and magnesium ions. We also found that UreE acts as a scaffold for the recruitment of GTP to UreG and that UreE takes the nickel ion from an accessory protein HypA to pass it along to UreG. The study expands our horizons on the molecular details of nickel translocation among metallochaperones UreE, UreG, and HypA, which further extends our knowledge on the urease maturation process. (J Biol Chem., 2015, 290, 12474-12485.) This paper was chosen as the paper of the work by JBC. J. Biol. Chem., 2015, 290, 12486.
UreG as new target for the development of urease inhibitor
Urease is a virulence factor of H. pylori and the design of urease inhibitors is extremely challenging owing to its deeply buried active site and specific substrate. We showed that bismuth inhibits urease activity through disrupting urease maturation process, i.e., nickel insertion to apo-urease, via binding and functional interference of UreG. This suggests that UreG serves as a novel target for the design of urease inhibitors, which was further validated by virtual screening and bioassays. The work was published in PLoS Biol, 2018, 16, e2003887.
A zinc-binding site by negative selection induces metallodrug susceptibility in an essential chaperonin
Metal complexes could play a useful role in restricting the mutation of Helicobacter pylori, a bacterium that causes stomach ulcers, and in inhibiting bacterial growth. Using a metallomic approach, a novel zinc-binding site originated from negative selection, was discovered. Since biological features by negative selection are usually inert to change during evolution, this study sheds light on a promising field whereby medicines can be designed or improved to specifically target the residues that uniquely evolved in pathogenic proteins so as to retard the emergence of drug resistance. (Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 4943-4948.)
Nickel chaperone protein HypA structure and its interaction with metallo-GTPase HypB
The solution structure of zinc-bound HypA (Zn-HypA) was determined by NMR spectroscopy together with molecular dynamics simulated annealing. Zn-HypA folds into two domains, including a zinc domain and a nickel domain with a mixed alpha/beta structure. HypB forms a complex with HypA with a low affinity. Such interactions were also observed in vivo using a GFP-fragment reassembly technique. Nickel is transferred from HypA to the conserved metal binding site of HypB through a specific protein-protein interaction. HypB undergoes nickel-dependent dimerization with enhanced e GTPase activity. The conformational change of HypB coupled with GTP hydrolysis triggers the nickel donation from HypB to its downstream receptor, e.g. the large subunit of [NiFe]-hydrogenase. (J. Am. Chem. Soc., 2009, 131, 10031-10040 ; J. Biol. Chem., 2012, 287, 6753-6763.)