Maolin Guo

Interactions of organometallic anticancer agents with nucleotides and DNA

Three classes of organometallic complexes, metallocene diacidos Cp2MX2(M=Ti, V, Nb, Mo, Re), ferricenium salts and organotin compounds have been reported to exhibit impressive in vivo or in vitro antitumour activities against a range of tumour cell lines. Titanocene dichloride is currently on the phase II clinical trials. Biological studies suggest that DNA is one of the primary intracellular targets of the metallocene dihalides but their mechanism of action is poorly understood. By using various modern techniques, the binding modes of Cp2TiCl2, Cp2VCl2, Cp2NbCl2, Cp2MoCl2, Cp2ZrCl2, Cp2Fe+X−, (CH3)2SnCl2, (C2H5)2SnCl2, (C2H5)2SnCl2(phen) with DNA and nucleotides in aqueous solution have been investigated. The results show that all the above organometallic agents exhibit high affinity to phosphate group of nucleotides. In aqueous solution, 5′GMP(or 5′dGMP), 5′AMP, 5′CMP, 5′TMP with Cp2TiCl2 or Cp2MoCl2 form chelate complexes in which both base nitrogen atom and phosphate oxygen atom of nucleotides bind to the metal center; whereas the other organometallics may bind to dGMP via only the phosphate group. The interactions between Cp2TiCl2, Cp2ZrCl2, (CH3)2SnCl2 or (C2H5)2SnCl2 with calf thymus DNA suggest that Cp2TiCl2(aq) may bind to both the base ring sites and the phosphate backbone of DNA; while other organometallics(aq) bind to DNA via only the phosphate group. In addition, the relationship between DNA binding property of metal anticancer complex and their anticancer activity is discussed and a hypothesis named ‘Two-Pole Complementary Principle’ is also put forward and some criteria are suggested as well.

Bacterial heme-transport proteins and their heme-coordination modes

Efficient iron acquisition is critical for an invading microbes survival and virulence. Most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. Utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. Once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in order to release the iron. Some Gram-negative bacteria also secrete extracellular heme-binding proteins called hemophores, which function to sequester heme from the environment. The heme-transport genes are often genetically linked as gene clusters under Fur (ferric uptake regulator) regulation. This review discusses the gene clusters and proteins involved in bacterial heme acquisition, transport and processing processes, with special focus on the heme-coordination, protein structures and mechanisms underlying heme-transport.

Iron-binding and anti-Fenton properties of baicalein and baicalin

Baicalein and baicalin, the major bioactive compounds found in the Chinese herb Scutellaria baicalensis, have been shown to be effective against cancer, bacterial infections and oxidative stress diseases. However, little is known about their mechanisms of action. To probe whether iron homeostasis modulation may play a role in their bioactivity, we have investigated their iron binding characteristics under physiologically relevant conditions. A 2:1 baicalein-ferrous complex was readily formed in 20mM phosphate buffer, pH 7.2, with a binding constant approximately 2-9 x 10(11)M(-2), whereas a 1:1 baicalein-ferric complex was formed, under the same conditions, with an apparent binding constant approximately 1-3 x 10(6)M(-1). Baicalein appears to bind the ferrous ion more strongly than ferrozine, a well known iron(II) chelator. Using (1) H NMR and Zn(2+) and Ga(3+) as probes, the iron-binding site on baicalein was elucidated to be at the O6/O7 oxygen atoms of the A-ring. No binding was observed for baicalin under the same NMR conditions. Furthermore, baicalein strongly inhibits the Fe-promoted Fenton chemistry via a combination of chelation and radical scavenging mechanism while baicalin can provide only partial protection against radical damage. These results indicate that baicalein is a strong iron chelator under physiological conditions and hence may play a vital role in modulating the bodys iron homeostasis. Modulation of metal homeostasis and the inhibition of Fenton chemistry may be one of the possible mechanisms for herbal medicine.

Holo- and Apo-bound Structures of Bacterial Periplasmic Heme-binding Proteins

An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an “in” position where it can coordinate the heme iron to an “out” orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg228 in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg228, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B12-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B12, compared with ligands for FhuD, a peptide siderophore.

Titanium(IV) targets phosphoesters on nucleotides: implications for the mechanism of action of the anticancer drug titanocene dichloride

Abstract. Reactions between the anticancer drug titanocene dichloride (Cp2TiCl2) and various nucleotides and their constituents in aqueous solution or N,N-dimethylformamide (DMF) have been investigated by 1H and 31P NMR spectroscopy and in the solid state by IR spectroscopy. In aqueous solution over the pH* (pH meter reading in D2O) range 2.3–6.5, CMP forms one new species with Ti(IV) bound only to the phosphate group. In acidic media at pH*<4.6, three species containing titanocene bound to the phosphate group of dGMP, AMP, dTMP and UMP are formed rapidly. The bases also appear to influence titanocene binding. Only one of these Ti(IV)-bound species can be detected in the pH* range of 4.6–6.5 in each case. The order of reactivity towards Cp2TiCl2(aq) at pH* ca. 3 is GMP>TMP≈AMP>CMP. At pH*>7.0, hydrolysis of Cp2TiCl2 predominated and little reaction with the nucleotides was observed. Binding of deoxyribose 5′-phosphate and 4-nitrophenyl phosphate to Cp2TiCl2(aq) via their phosphate groups was detected by 31P NMR spectroscopy, but no reaction between Cp2TiCl2(aq) and deoxyguanosine, 9-ethylguanine or deoxy-D-ribose was observed in aqueous solution. The nucleoside phosphodiesters 3′,5′-cyclic GMP and 2′,3′-cyclic CMP did not react with Cp2TiCl2(aq) in aqueous solution; however, in the less polar solvent DMF, 3′,5′-cyclic GMP coordination to {Cp2Ti}2+ via its phosphodiester group was readily observed. Binding of titanocene to the phosphodiester group of the dinucleotide GpC was also observed in DMF by 31P NMR. The nucleoside triphosphates ATP and GTP reacted more extensively with Cp2TiCl2(aq) than their monophosphates; complexes with bound phosphate groups were formed in acidic media and to a lesser extent at neutral pH. Cleavage of phosphate bonds in ATP (and GTP) by Cp2TiCl2(aq) to form inorganic phosphate, AMP (or GMP) and ADP (or GDP) was observed in aqueous solutions. In addition, titanocene binding to ATP was not inhibited by Mg(II), but the ternary complex titanocene-ATP-Mg appeared to form. These reactions contrast markedly with those of the drug cisplatin, which binds predominantly to the base nitrogen atoms of nucleotides and only weakly to the phosphate groups. The high affinity of Ti(IV) for phosphate groups may be important for its biological activity.

The FeMoco-deficient MoFe Protein Produced by a nifH Deletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

The His-tag MoFe protein expressed by thenifH deletion strain Azotobacter vinelandiiDJ1165 (ΔnifH MoFe protein) was purified in large quantity. The α2β2 tetrameric ΔnifH MoFe protein is FeMoco-deficient based on metal analysis and the absence of the S = 3/2 EPR signal, which arises from the FeMo cofactor center in wild-type MoFe protein. The ΔnifH MoFe protein contains 18.6 mol Fe/mol and, upon reduction with dithionite, exhibits an unusually strong S = 1/2 EPR signal in the g ≈ 2 region. The indigo disulfonate-oxidized ΔnifH MoFe protein does not show features of the P2+ state of the P-cluster of the ΔnifB MoFe protein. The oxidized ΔnifH MoFe protein is able to form a specific complex with the Fe protein containing the [4Fe-4S]1+ cluster and facilitates the hydrolysis of MgATP within this complex. However, it is not able to accept electrons from the [4Fe-4S]1+ cluster of the Fe protein. Furthermore, the dithionite-reduced ΔnifH MoFe can be further reduced by Ti(III) citrate, which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.

Iron Chelators as Potential Therapeutic Agents for Parkinson’s Disease

Parkinsons disease (PD) is a neurological disorder characterized by the progressive impairment of motor skills in patients. Growing evidence suggests that abnormal redox-active metal accumulation, caused by dysregulation, plays a central role in the neuropathology of PD. Redox-active metals (e.g. Fe and Cu) catalyze essential reactions for brain function. However, these metals can also participate in the generation of highly toxic free radicals that can cause oxidative damage to cells and ultimately lead to the death of dopamine-containing neurons. The emergence of redox-active metals as key players in the pathogenesis of PD strongly suggests that metal-chelators could be beneficial in the treatment of this condition. This mini-review summarizes major recent developments on natural, synthetic iron chelating compounds and hydrogen peroxide-triggered prochelators as potential candidates for PD treatment.

A novel protein-mineral interface

Transferrins transport Fe3+ and other metal ions in mononuclear-binding sites. We present the first evidence that a member of the transferrin superfamily is able to recognize multi-nuclear oxo-metal clusters, small mineral fragments that are the most abundant forms of many metals in the environment. We show that the ferric ion–binding protein from Neisseria gonorrhoeae (nFbp) readily binds clusters of Fe3+, Ti4+, Zr4+ or Hf4+ in solution. The 1.7 Å resolution crystal structure of Hf–nFbp reveals three distinct types of clusters in an open, positively charged cleft between two hinged protein domains. A di-tyrosyl cluster nucleation motif (Tyr195-Tyr196) is situated at the bottom of this cleft and binds either a trinuclear oxo-Hf cluster, which is capped by phosphate, or a pentanuclear cluster, which in turn can be capped with phosphate. This first high-resolution structure of a protein–mineral interface suggests a novel metal-uptake mechanism and provides a model for protein-mediated mineralization/dissimilation, which plays a critical role in geochemical processes.NOTE: In the version of this article initially published online, the institution affiliations were assigned incorrectly because of a mistake that occurred during production. The correct affiliations for all authors are as follows: Dmitriy Alexeev1, Haizhong Zhu2, Maolin Guo2,3, Weiqing Zhong2,4, Dominic J.B. Hunter2, Weiping Yang2,3, Dominic J. Campopiano2 and Peter J. Sadler2. All of the footnotes (corrected) are as follows: 1Institute of Cell and Molecular Biology, Michael Swann Building, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK; 2School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK; 3Current address: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA; and 4Current address: School of Pharmacy, Second Military Medicine University, Shanghai 200433, China. We apologize for any inconvenience this may have caused. This mistake has been corrected in the HTML and print version of the article.

A Turn‐on Fluorescent Sensor for Imaging Labile Fe3+ in Live Neuronal Cells at Subcellular Resolution

An eye for an iron: A highly sensitive, selective and reversible turn-on Fe(3+) sensor for imaging labile Fe(3+) in live cells at subcellular resolution is reported. The sensor can respond to changes in intracellular Fe(3+) levels and was used to image endogenous chelatable Fe(3+) in live human neuroblastoma SH-SY5Y cells, with two Fe(3+) pools being identified in mitochondria and endosomes/ lysosomes for the first time.

A novel profluorescent probe for detecting oxidative stress induced by metal and H2O2 in living cells

A profluorescent probe that has no fluorescent response to H(2)O(2), iron or copper ions but can be readily activated in the presence of both H(2)O(2) and Fe (or Cu) ion has been developed; the probe is capable of detecting oxidative stress promoted by Fe (or Cu) and H(2)O(2) (i.e. the Fenton reaction conditions) in living cells.