Lalla A. Ba

Selenium‐ and Tellurium‐Containing Multifunctional Redox Agents as Biochemical Redox Modulators with Selective Cytotoxicity

Various human diseases, including different types of cancer, are associated with a disturbed intracellular redox balance and oxidative stress (OS). The past decade has witnessed the emergence of redox-modulating compounds able to utilize such pre-existing disturbances in the redox state of sick cells for therapeutic advantage. Selenium- and tellurium-based agents turn the oxidizing redox environment present in certain cancer cells into a lethal cocktail of reactive species that push these cells over a critical redox threshold and ultimately kill them through apoptosis. This kind of toxicity is highly selective: normal, healthy cells remain largely unaffected, since changes to their naturally low levels of oxidizing species produce little effect. To further improve selectivity, multifunctional sensor/effector agents are now required that recognize the biochemical signature of OS in target cells. The synthesis of such compounds provides interesting challenges for chemistry in the future.

Synthesis and Selective Anticancer Activity of Organochalcogen Based Redox Catalysts

Many tumor cells exhibit a disturbed intracellular redox state resulting in higher levels of reactive oxygen species (ROS). As these contribute to tumor initiation and sustenance, catalytic redox agents combining significant activity with substrate specificity promise high activity and selectivity against oxidatively stressed malignant cells. We describe here the design and synthesis of novel organochalcogen based redox sensor/effector catalysts. Their selective anticancer activity at submicromolar and low micromolar concentrations was established here in a range of tumor entities in various biological systems including cell lines, primary tumor cell cultures, and animal models. In the B-cell derived chronic lymphocytic leukemia (CLL), for instance, such compounds preferentially induce apoptosis in the cancer cells while peripheral blood mononuclear cells (PBMC) from healthy donors and the subset of normal B-cells remain largely unaffected. In support of the concept of sensor/effector based ROS amplification, we are able to demonstrate that underlying this selective activity against CLL cells are pre-existing elevated ROS levels in the leukemic cells compared to their nonmalignant counterparts. Furthermore, the catalysts act in concert with certain chemotherapeutic drugs in several carcinoma cell lines to decrease cell proliferation while showing no such interactions in normal cells. Overall, the high efficacy and selectivity of (redox) catalytic sensor/effector compounds warrant further, extensive testing toward transfer into the clinical arena.

Redox active secondary metabolites.

Various secondary metabolites from plants, bacteria and fungi are redox active and able to modulate the intracellular redox equilibrium in living cells. Many of these compounds behave as antioxidants, yet some of them also cause oxidative modifications, which may ultimately result in cell death. Natural isothiocyanates and xanthohumol, for instance, appear to act specifically in and against cells with a disturbed redox balance, such as certain cancer cells. Similarly, polysulfane and pyocyanin derivatives employ the glutathione antioxidant defense system of cells to generate a lethal cocktail of reactive oxygen species. Together, these redox-modulating metabolites provide promising new leads to target selectively certain cancer cells. They may also be useful in the treatment of autoinflammatory diseases.

The Organotelluride Catalyst (PHTE)2NQ Prevents HOCl-Induced Systemic Sclerosis in Mouse

Systemic sclerosis (SSc) is a connective tissue disorder characterized by skin and visceral fibrosis, microvascular damage, and autoimmunity. HOCl-induced mouse SSc is a murine model that mimics the main features of the human disease, especially the activation and hyperproliferation rate of skin fibroblasts. We demonstrate here the efficiency of a tellurium-based catalyst 2,3-bis(phenyltellanyl)naphthoquinone ((PHTE)(2)NQ) in the treatment of murine SSc, through its selective cytotoxic effects on activated SSc skin fibroblasts. SSc mice treated with (PHTE)(2)NQ displayed a significant decrease in lung and skin fibrosis and in alpha-smooth muscle actin (α-SMA) expression in the skin compared with untreated mouse SSc animals. Serum concentrations of advanced oxidation protein products, nitrate, and anti-DNA topoisomerase I autoantibodies were increased in SSc mice, but were significantly reduced in SSc mice treated with (PHTE)(2)NQ. To assess the mechanism of action of (PHTE)(2)NQ, the cytotoxic effect of (PHTE)(2)NQ was compared in normal fibroblasts and in mouse SSc skin fibroblasts. ROS production is higher in mouse SSc fibroblasts than in normal fibroblasts, and was still increased by (PHTE)(2)NQ to reach a lethal threshold and kill mouse SSc fibroblasts. Therefore, the effectiveness of (PHTE)(2)NQ in the treatment of mouse SSc seems to be linked to the selective pro-oxidative and cytotoxic effects of (PHTE)(2)NQ on hyperproliferative fibroblasts.

Flavonoid-DNA binding studies and thermodynamic parameters.

Interactional studies of new flavonoid derivatives (Fl) with chicken blood ds.DNA were investigated spectrophotometrically in DMSO-H2O (9:1 v/v) at various temperatures. Spectral parameters suggest considerable binding between the flavonoid derivatives studied and ds.DNA. The binding constant values lie in the enhanced-binding range. Thermodynamic parameters obtained from UV studies also point to strong spontaneous binding of Fl with ds.DNA. Viscometric studies complimented the UV results where a small linear increase in relative viscosity of the DNA solution was observed with added optimal flavonoid concentration. An overall mixed mode of interaction (intercalative plus groove binding) is proposed between DNA and flavonoids. Conclusively, investigated flavonoid derivatives are found to be strong DNA binders and seem to be promising drug candidates like their natural analogues.

Interactions of polysulfanes with components of red blood cells

Traditionally, the activity of most polysulfanes has been associated with the redox behaviour of the sulfur-sulfur bond. Here we show that polysulfanes, such as diallyltri- and tetrasulfide, also interact with cellular membranes and certain metalloproteins. Together, multiple interactions with various biological targets may explain best the biological activity of such compounds.

Targeting the disturbed redox equilibrium in chronic lymphocytic leukemia by novel reactive oxygen species-catalytic 'sensor/effector' compounds

Precursor transformation, clonal sustenance, and therapeutic resistance in cancer are significantly mediated by deregulated reactive oxygen species (ROS), which primarily act as DNA-stressors. Here, we demonstrate that elevated ROS in chronic lymphocytic leukemia (CLL) may represent a promising therapeutic target. We designed organochalcogens, which, based on a ‘sensor/effector’ principle, would confer selective cytotoxicity through the generation of intolerably high ROS levels preferentially in CLL cells, as these carry a high-level redox burden. Our novel compounds show an encouraging profile of efficient induction of apoptosis, low normal cell toxicity, and promising chemotherapy synergism. These findings warrant further mechanistic and preclinical studies of this therapeutic principle in CLL.

Open Season for Hunting and Trapping Post-translational Cysteine Modifications in Proteins and Enzymes

During the last decade, numerous mammalian redox proteins and enzymes have been discovered that act together to control various cellular processes ranging from cell proliferation to differentiation and apoptosis. 2] Many of these proteins contain redox-active cysteine residues, which besides their catalytic functions fulfil important roles in redox sensing and signalling. In these proteins, function and activity are often controlled by specific post-translational cysteine modifications. Together, these proteins and enzymes form the “cellular thiolstat”, an extensive and highly significant network able to sense changes in the intracellular redox environment and to trigger a measured, appropriate and rapidly reversible response (e.g. , in the form of gene expression, antioxidant defence or by inducing apoptosis). The thiol(ate) group of cysteine is ideal for this kind of reversible redox signalling. 9] It is readily, rapidly and reversibly Sthiolated, S-nitrosated or oxidized to various sulfur chemotypes by a range of different redox mechanisms (including thiol/ disulfide exchange, electron transfer, radical reactions and electrophilic attacks). Such reactions often result in dramatic changes in the structure, function and activity of the proteins/ enzymes affected, and can also contribute quite considerably to controlled, reversible protein–protein interactions. Table 1 provides a brief and necessarily incomplete overview of the most common cysteine modifications encountered in human cells. Since these modifications do not occur at random but rather critically depend on the accessibility and redox sensitivity of individual cysteine residues, a gradual, differentiated and measured cellular response is possible. As Dalle-Donne and colleagues have rightly pointed out, such modifications could well form the basis for “life and death decisions of the cell”. Despite the importance of post-translational cysteine modifications in catalysis and redox signalling, we still know little about the kind, extent and dynamics of these modifications in living cells. This lack of knowledge has recently been highlighted in a study by Winther and colleagues, who demonstrated that cysteine residues in proteins and enzymes, rather than glutathione (GSH), are the prime targets of oxidizing species, such as diamide. Traditionally, GSH has been considered to be the major, if not the only, sacrificial intracellular thiol being targeted by oxidants. It now appears that GSH shares this role with protein-based thiols, the oxidation of which in turn plays a significant role in cellular signalling and in controlling the cellular stress response. This apparent lack of knowledge, however, is not coincidental. Several factors complicate the in vitro as well as in vivo study of cysteine modifications in proteins. First of all, sulfur is a true redox chameleon that occurs in numerous oxidation states and modifications (so-called “chemotypes”) in most living cells. In the sulfiredoxin (Srx)–peroxiredoxin (Prdx) system alone, six sulfur chemotypes are present as integral parts of catalysis and redox sensing; among them are thiols, a sulfenic acid, various disulfides, a sulfinic acid, a thiosulfinate and a rather exotic sulfinic phosphoryl ester. 3, 12] Just considering—and measuring—thiols and disulfides in proteins is therefore not enough: In order to gain a complete picture, several sulfur chemotypes need to be identified and quantified, some of which are highly reactive, notoriously unstable or otherwise difficult to track down. To complicate matters further, the sulfur atom in these chemotypes is disappointingly “silent”, that is, most traditional analytical techniques, such as UV–visible spectrophotometry, NMR or EPR spectroscopy are often insufficient to describe these sulfur species. This lack of characteristic signals seriously complicates an easy detection of individual cysteine modifications in proteins and enzymes, especially inside intact cells. The alternative, that is, isolating cysteine proteins from cells and analysing them in vitro, is equally dissatisfying, since sulfur chemotypes are often unstable and decompose or react readily with their environment during isolation and subsequent analysis. While techniques such as X-ray crystallography are, at least in principle, able to differentiate between the various cysteine modifications, they are also prone to the formation of artefacts. This has been highlighted by the circumstances surrounding the discovery of a sulfinic acid modification in Prdx by Littlechild and colleagues in 2000. At the time, the sulfinic acid detected was initially considered to be an artefact caused by oxidation during the lengthy crystallization procedure (J. A. Littlechild, personal communication). It is therefore reasonable to approach the (bio)chemistry surrounding the cellular thiolstat with small, but decisive steps. From a signalling perspective, three—mostly reversible—posttranslational thiol modifications are of particular interest : Sthiolation of cysteine residues resulting in (mixed) disulfides, Snitrosation resulting in S-nitrosothiols, and S-hydroxylation [a] Prof. C. Jacob, Dr. L. A. Ba Division of Bioorganic Chemistry, School of Pharmacy University of Saarland Campus, 66123 Saarbr cken (Germany) Fax: (+ 49) 681-302-3464 E-mail : c.jacob@mx.uni-saarland.de