CORM-3 induces DNA damage through Ru(II) binding to DNA.

When the 'CO-releasing molecule-3', CORM-3 (Ru(CO)3Cl(glycinate)), is dissolved in water it forms a range of ruthenium complexes. These are taken up by cells and bind to intracellular ligands, notably thiols such as cysteine and glutathione, where the Ru(II) reaches high intracellular concentrations. Here, we show that the Ru(II) ion also binds to DNA, at exposed guanosine N7 positions. It therefore has a similar cellular target to the anticancer drug cisplatin, but not identical, because Ru(II) shows no evidence of forming intramolecular crossbridges in the DNA. The reaction is slow, and with excess Ru, intermolecular DNA crossbridges are formed. The addition of CORM-3 to human colorectal cancer cells leads to strand breaks in the DNA, as assessed by the alkaline comet assay. DNA damage is inhibited by growth media containing amino acids, which bind to extracellular Ru and prevent its entry into cells. We conclude that the cytotoxicity of Ru(II) is different from that of platinum, making it a promising development target for cancer therapeutics.

N-terminal domain of prion protein directs its oligomeric association.

The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar ß-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined ß-sheet-rich oligomer, containing ∼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23-90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91-231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein.

Interactions between the protein barnase and co-solutes studied by NMR.

Protein solubility and stability depend on the co-solutes present. There is little theoretical basis for selection of suitable co-solutes. Some guidance is provided by the Hofmeister series, an empirical ordering of anions according to their effect on solubility and stability; and by osmolytes, which are small organic molecules produced by cells to allow them to function in stressful environments. Here, NMR titrations of the protein barnase with Hofmeister anions and osmolytes are used to measure and locate binding, and thus to separate binding and bulk solvent effects. We describe a rationalisation of Hofmeister (and inverse Hofmeister) effects, which is similar to the traditional chaotrope/kosmotrope idea but based on solvent fluctuation rather than water withdrawal, and characterise how co-solutes affect protein stability and solubility, based on solvent fluctuations. This provides a coherent explanation for solute effects, and points towards a more rational basis for choice of excipients.

Pharmacological chaperone for the structured domain of human prion protein.

In prion diseases, the misfolded protein aggregates are derived from cellular prion protein (PrP(C)). Numerous ligands have been reported to bind to human PrP(C) (huPrP), but none to the structured region with the affinity required for a pharmacological chaperone. Using equilibrium dialysis, we screened molecules previously suggested to interact with PrP to discriminate between those which did not interact with PrP, behaved as nonspecific polyionic aggregates or formed a genuine interaction. Those that bind could potentially act as pharmacological chaperones. Here we report that a cationic tetrapyrrole [Fe(III)-TMPyP], which displays potent antiprion activity, binds to the structured region of huPrP. Using a battery of biophysical techniques, we demonstrate that Fe(III)-TMPyP forms a 11 complex via the structured C terminus of huPrP with a K(d) of 4.5 ± 2 µM, which is in the range of its IC(50) for curing prion-infected cells of 1.6 ± 0.4 µM and the concentration required to inhibit protein-misfolding cyclic amplification. Therefore, this molecule tests the hypothesis that stabilization of huPrP(C), as a principle, could be used in the treatment of human prion disease. The identification of a binding site with a defined 3D structure opens up the possibility of designing small molecules that stabilize huPrP and prevent its conversion into the disease-associated form.

Folding kinetics of the human prion protein probed by temperature jump.

Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (approximately 3,000 s(-1)), with the extrapolated folding rate constant at approximately 20 degrees C in physiological conditions reaching 20,000 s(-1). By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant phi-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix-helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrP(C) conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state that is not compact (m value analysis gives a beta(t) value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible effect on stability but still led to formation of aberrant conformations during folding implies an easily perturbed folding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal dominant condition, 20 of the 33 amino acid replacements occur in the helix-2/3 sequence.

High-affinity tamoxifen analogues retain extensive positional disorder when bound to calmodulin.

Using a combination of NMR and fluorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High-affinity binding (Kd∼300 nM) saturates with a 2:1 idoxifene:CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-affinity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2:1 idoxifene:CaM complex illustrates how high-affinity binding occurs without the loss of extensive positional dynamics.

'Carbon-Monoxide-Releasing Molecule-2 (CORM-2)' Is a Misnomer: Ruthenium Toxicity, Not CO Release, Accounts for Its Antimicrobial Effects.

Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological 'gasotransmitter', in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (<0.1 mol CO per mol CORM-2). A strong negative correlation between viability and cellular ruthenium accumulation implies that ruthenium toxicity underlies biocidal activity. Exogenous amino acids and thiols (especially cysteine, glutathione and N-acetyl cysteine) protected bacteria against inhibition of growth by CORM-2. Bacteria treated with 30 µM CORM-2, with added cysteine and histidine, exhibited no significant loss of viability, but were killed in the absence of these amino acids. Their prevention of toxicity correlates with their CORM-2-binding affinities (Cys, Kd 3 µM; His, Kd 130 µM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 µM; GSSG, Kd 25,000 µM). The toxicity of low, but potent, levels (15 µM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data.

The H187R mutation of the human prion protein induces conversion of recombinant prion protein to the PrP(Sc)-like form.

Prion diseases are associated with a conformational switch in the prion protein (PrP) from its normal cellular form (denoted PrP(C)) to a disease-associated "scrapie" form (PrP(Sc)). A number of PrP(Sc)-like conformations can be generated by incubating recombinant PrP(C) at low pH, indicating that protonation of key residues is likely to destabilize PrP(C), facilitating its conversion to PrP(Sc). Here, we examine the stability of human PrP(C) with pH and find that PrP(C) fold stability is significantly reduced by the protonation of two histidine residues, His187 and His155. Mutation of His187 to an arginine, which imposes a permanently positively charged residue in this region of the protein, has a dramatic effect on the folding of PrP(C), resulting in a molecule that displays a markedly increased propensity to oligomerize. The oligomeric form is characterized by an increased ß-sheet content, loss of fixed side chain interactions, and partial proteinase resistance. Hence, the protonation state of H187 appears to be crucial in determining the conformation of PrP; the unprotonated form favors native PrP(C), while the protonated form favors PrP(Sc)-like conformations. These results are relevant to the pathogenic H187R mutation found in humans, which is associated with an inherited prion disease [also termed Gerstmann-Sträussler-Scheinker (GSS) syndrome] with unusual features such as childhood neuropsychiatric illness. Our data imply that the intrinsic instability of the PrP(C) conformation in this variant is caused by a positive charge at this site in the protein. This mutation is distinct from all those associated with GSS, which have much more subtle physical consequences. The degree of instability might be the cause of the unusually early onset of mental disturbance in affected individuals.

Allomorphy as a mechanism of post-translational control of enzyme activity.

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. ß-phosphoglucomutase (ßPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of ßPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In ßPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate ß-glucose 1,6-bisphosphate, whose concentration depends on the ß-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

A thiol-reactive Ru(II) ion, not CO release, underlies the potent antimicrobial and cytotoxic properties of CO-releasing molecule-3.

Carbon monoxide (CO)-releasing molecules (CORMs), mostly metal carbonyl compounds, are extensively used as experimental tools to deliver CO, a biological 'gasotransmitter', in mammalian systems. CORMs are also explored as potential novel antimicrobial drugs, effectively and rapidly killing bacteria in vitro and in animal models, but are reportedly benign towards mammalian cells. Ru-carbonyl CORMs, exemplified by CORM-3 (Ru(CO)3Cl(glycinate)), exhibit the most potent antimicrobial effects against Escherichia coli. We demonstrate that CORM-3 releases little CO in buffers and cell culture media and that the active antimicrobial agent is Ru(II), which binds tightly to thiols. Thus, thiols and amino acids in complex growth media - such as histidine, methionine and oxidised glutathione, but most pertinently cysteine and reduced glutathione (GSH) - protect both bacterial and mammalian cells against CORM-3 by binding and sequestering Ru(II). No other amino acids exert significant protective effects. NMR reveals that CORM-3 binds cysteine and GSH in a 1:1 stoichiometry with dissociation constants, Kd, of about 5 µM, while histidine, GSSG and methionine are bound less tightly, with Kd values ranging between 800 and 9000 µM. There is a direct positive correlation between protection and amino acid affinity for CORM-3. Intracellular targets of CORM-3 in both bacterial and mammalian cells are therefore expected to include GSH, free Cys, His and Met residues and any molecules that contain these surface-exposed amino acids. These results necessitate a major reappraisal of the biological effects of CORM-3 and related CORMs.

A systematic review of prion therapeutics in experimental models.

Prion diseases are transmissible, invariably fatal, neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy and scrapie in animals. A large number of putative treatments have been studied in experimental models over the past 30 years, with at best modest disease-modifying effects. The arrival of variant CJD in the UK in the 1990s has intensified the search for effective therapeutic agents, using an increasing number of animal, cellular and in vitro models with some recent promising proof of principle studies. Here, for the first time, we present a comprehensive systematic, rather than selective, review of published data on experimental approaches to prion therapeutics to provide a scientific resource for informing future therapeutics research, both in laboratory models and in clinical studies.

Enhanced ligand affinity for receptors in which components of the binding site are independently mobile.

Using calmodulin antagonism as a model, it is demonstrated that, under circumstances in which binding sites are motionally independent, it is possible to create bifunctional ligands that bind with significant affinity enhancement over their monofunctional counterparts. Suitable head groups were identified by using a semiquantitative screen of monofunctional tryptophan analogs. Two bifunctional ligands, which contained two copies of the highest-affinity head group tethered by rigid linkers, were synthesized. The bifunctional ligands bound to calmodulin with a stoichiometry of 1:1 and with an affinity enhancement over their monofunctional counterparts; the latter bound with a stoichiometry of 2:1 ligand:protein. A lower limit to the effective concentrations of the domains of calmodulin relative to each other (0.2-2 mM) was determined. A comparable effective concentration was achieved for bifunctional ligands based on higher-affinity naphthalene sulphonamide derivatives.

Variant Creutzfeldt-Jakob disease: pathology, epidemiology, and public health implications.

Prion diseases, or transmissible spongiform encephalopathies, include Creutzfeldt-Jakob disease (CJD) in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. These neurodegenerative diseases are invariably fatal and can be transmitted by inoculation or dietary exposure. They are associated with the accumulation of an altered, disease-associated form of the normal prion protein. Pathologically, prion diseases result in neuronal cell death and a characteristic spongiform appearance of the brain tissue. The emergence of a variant form of CJD (vCJD) in the United Kingdom in 1996 has been causally and experimentally linked to the UK BSE epidemic in the 1980s and early 1990s. The finding that BSE is transmissible to different animal species, unlike previously characterized prion diseases such as sheep scrapie, has raised enormous public health concerns worldwide. Although it is not yet possible to gauge the size of a potential vCJD epidemic, preliminary data indicate a significant dietary exposure to BSE-infected material in Britain and wider implications of the transmissibility of prion diseases. The threat to public health has intensified research efforts to understand the molecular basis of prion diseases, understand their transmission between species, improve methods of diagnosis, and develop therapeutic strategies for treatment and prevention of disease. In this review, we summarize current data on the pathology of BSE and vCJD and the epidemiology of vCJD, and we outline public health implications based on these data, emphasizing preventative measures and areas of research for screening and diagnosis.

Conformational properties of beta-PrP.

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a beta-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126-227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

The residue 129 polymorphism in human prion protein does not confer susceptibility to Creutzfeldt-Jakob disease by altering the structure or global stability of PrPC.

There are two common forms of prion protein (PrP) in humans, with either methionine or valine at position 129. This polymorphism is a powerful determinant of the genetic susceptibility of humans toward both sporadic and acquired forms of prion disease and restricts propagation of particular prion strains. Despite its key role, we have no information on the effect of this mutation on the structure, stability, folding, and dynamics of the cellular form of PrP (PrP(C)). Here, we show that the mutation has no measurable effect on the folding, dynamics, and stability of PrP(C). Our data indicate that the 129M/V polymorphism does not affect prion propagation through its effect on PrP(C); rather, its influence is likely to be downstream in the disease mechanism. We infer that the M/V effect is mediated through the conformation or stability of disease-related PrP (PrP(Sc)) or intermediates or on the kinetics of their formation.