Identification of the molecular target(s) of anticancer metal complexes is a formidable challenge since most of them are unstable toward ligand exchange reaction(s) or biological reduction under physiological conditions. Gold(III) meso-tetraphenylporphyrin (gold-1 a) is notable for its high stability in biological milieux and potent in vitro and in vivo anticancer activities. Herein, extensive chemical biology approaches employing photo-affinity labeling, click chemistry, chemical proteomics, cellular thermal shift, saturation-transfer difference NMR, protein fluorescence quenching, and protein chaperone assays were used to provide compelling evidence that heat-shock protein 60 (Hsp60), a mitochondrial chaperone and potential anticancer target, is a direct target of gold-1 a in vitro and in cells. Structure-activity studies with a panel of non-porphyrin gold(III) complexes and other metalloporphyrins revealed that Hsp60 inhibition is specifically dependent on both the gold(III) ion and the porphyrin ligand.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Incorporation of nickel ions to the active sites of urease and hydrogenase is prerequisite for the appropriate functions of the metalloenzymes. Such a process requires the participation of several accessory proteins. Interestingly, some of them are shared by the two enzymes in their maturation processes. In this work, we characterized the molecular details of the interaction of metallochaperones UreE and HypA in Helicobacter pylori. We show by chemical cross-linking and static light scattering that the UreE dimer binds to HypA to form a hetero-complex i.e. HypA-(UreE)2. The dissociation constant (Kd) of the protein complex was determined by ITC to be 1 μM in the absence of nickel ions; whereas binding of Ni(2+) but not Zn(2+) to UreE resulted in ca. one fold decrease in the affinity. The putative interfaces on HypA unveiled by NMR chemical shift perturbation were found mainly at the nickel binding domain and in the cleft between α1 and β1/β6. We also identified that the C-domain of UreE, in particular the C-terminal residues of 158-170 are indispensable for the interaction of UreE and HypA. Such an interaction was also observed intracellularly by GFP-fragment reassembly assay. Moreover, we demonstrated using a fluorescent probe that nickel is transferred from HypA to UreE via the specific protein-protein interaction. Deletion of the C-terminus (residues 158-170) of UreE abolished nickel transfer and led to a significant decrease in urease activity. This study provides direct in vitro and in vivo evidence as well as molecular details of nickel translocation mediated by protein-protein interaction.

Glutathione and multidrug resistance protein (MRP) play an important role on the metabolism of a variety of drugs. Bismuth drugs have been used to treat gastrointestinal disorder and Helicobacter pylori infection for decades without exerting acute toxicity. They were found to interact with a wide variety of biomolecules, but the major metabolic pathway remains unknown. For the first time (to our knowledge), we systematically and quantitatively studied the metabolism of bismuth in human cells. Our data demonstrated that over 90% of bismuth was passively absorbed, conjugated to glutathione, and transported into vesicles by MRP transporter. Mathematical modeling of the system reveals an interesting phenomenon. Passively absorbed bismuth consumes intracellular glutathione, which therefore activates de novo biosynthesis of glutathione. Reciprocally, sequestration by glutathione facilitates the passive uptake of bismuth and thus completes a self-sustaining positive feedback circle. This mechanism robustly removes bismuth from both intra-and extracellular space, protecting critical systems of human body from acute toxicity. It elucidates the selectivity of bismuth drugs between human and pathogens that lack of glutathione, such as Helicobacter pylori, opening new horizons for further drug development.bismuth | drug selectivity | glutathione | MRP | positive feedback

Metals and metalloids are crucial for life and indispensable for a series of biological processes. [1] It is estimated that a quarter to one third of all proteins require metals to carry out their functions, and roughly half of the known enzymes uses a particular metal as a cofactor. [2] In spite of the prevalence and importance of metalloproteins, they are generally poorly characterized in many organisms. A recent study demonstrated that the microbial metalloproteome is much more extensive and diverse than we presently know. [3] Currently, it is impossible to predict, genome-wide, the numbers and types of metals used by organisms and to define any metalloproteome until the proteins are fully characterized owing to diverse and poorly recognized metal coordination sites. Moreover, metals/metalloids have long been used for therapeutic purposes, for example, arsenic trioxide for the treatment of acute promyelocytic leukemia. [4] The detailed molecular mechanisms, however, are still not fully understood owing to the complex functions of metals in biological systems.[5] A robust and convenient approach, by which metals/metalloids can be mapped to their associated proteins proteome-wide is urgently needed.[6] Such a methodology will improve our understanding of the molecular mechanisms of metal-dependent biological processes and profoundly promote metallomics research, [7] an integrated biometal science complementary to genomics and proteomics.[8] Gel electrophoresis has been one of the commonly used methods for separation and analysis of proteins based on their molecular mass and charge; however, it fails to provide information on metal identity and content for metalloproteins. The lack of convenient subsequent methods for specific metal detection confines its application on providing metalrelated information of corresponding proteins. Although laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) [9] and synchrotron X-ray fluorescence spectrometry (SXFS) [10] have been used for the identification of metalbinding proteins on gels and in tissues/organs, either compromised sensitivity originating from the sample introduction system or limited accessibility to the synchrotron facility prevents their routine applications. Other strategies such as metal isotope radioautography, which employs unique metal isotopes to label metalloproteins, are also very inconvenient for daily usage. [11] Herein, a new strategy based on column-type gel electrophoresis coupled with a metal-specific detection system, that is ICP-MS, was developed (Figure 1 a), allowing both metals and their associated proteins to be examined comprehensively. Since the strategy can be used to analyze and at the same time to separate and isolate proteins, it can readily be applied to not only detect metalloproteins and/or metalbound proteins with a sensitivity at the femtomole level, but also conveniently integrate current proteomics with metallomics. We further showed the bismuth profile in cell lysates of Helicobacter pylori upon treatme...

Repurposing bismuth drugs against the key oral pathogenPorphyromonas gingivalisin planktonic, biofilm, and intracellular states for reconciling the immuno-inflammatory responses.

A macrocyclic ruthenium(III) complex [Ru (N O )Cl ]Cl (Ru-1) is reported as an inhibitor of angiogenesis and an anti-tumor compound. The complex is relatively non-cytotoxic towards endothelial and cancer cell lines in vitro, but specifically inhibited the processes of angiogenic endothelial cell tube formation and cancer cell invasion. Moreover, compared with known anti-cancer ruthenium complexes, Ru-1 is distinct in that it suppressed the expression of vascular endothelial growth factor receptor-2 (VEGFR2), and the associated downstream signaling that is crucial to tumor angiogenesis. In addition, in vivo studies showed that Ru-1 inhibited angiogenesis in a zebrafish model and suppressed tumor growth in nude mice bearing cancer xenografts.

Identification of the molecular target(s) of anticancer metal complexes is aformidable challenge since most of them are unstable towardl igand exchange reaction(s) or biological reduction under physiological conditions.Gold(III) meso-tetraphenylporphyrin (gold-1 a)i sn otable for its high stability in biological milieux and potent in vitro and in vivo anticancer activities.H erein, extensive chemical biology approaches employing photo-affinity labeling,click chemistry, chemical proteomics,cellular thermal shift, saturation-transfer difference NMR, protein fluorescence quenching, and protein chaperone assays were used to provide compelling evidence that heat-shockprotein 60 (Hsp60), amitochondrial chaperone and potential anticancer target, is ad irect target of gold-1 a in vitro and in cells.S tructure-activity studies with ap anel of non-porphyrin gold(III) complexes and other metalloporphyrins revealed that Hsp60 inhibition is specifically dependent on both the gold(III) ion and the porphyrin ligand.