Stefanie+Schwemlein's+Proposal

Mercury is known to be a toxic substance, as exposure to this heavy metal can detrimentally affect the brain, kidney, and lungs. The major sources of mercury in the environment are from atmospheric emissions of mercury from a number of industrial processes including the combustion of coal and of fossil fuels, municipal and medical waste incinerators, along with natural processes such as volcanic eruptions and emissions from the ocean. Controversy has arisen concerning the use of dental amalgams, which contain mercury, and much debate has recently been surfacing regarding the use of thimerosal, a preservative that contains mercury, in vaccines, as well as regarding the risk of over-exposure to organic mercury through the consumption of fish. Due to the harmful effects of mercury toxicity, which can range from the development of neurodegenerative disorders to mercury buildup in the body and fatality, it is crucial that better forms of treatment for mercury toxicity be studied. Mercury toxicity can be a result of exposure to any of the substance’s different forms. The elemental form of mercury is a liquid metal that can volatilize, and subsequently a person may inhale the vapor, which would then be absorbed by the lungs and enter the bloodstream. This lipid-soluble form of mercury is able to cross the blood-brain barrier and become oxidized into inorganic mercury (Hg2+). Elemental mercury is found in dental amalgams and some thermometers, among other things. Inorganic mercury (Hg2+) is formed upon the metabolizing of elemental mercury or organic mercury. It is often found in the brain in this metabolized form, as it cannot cross the blood-brain barrier in its normal form. Exposure to the final form of mercury, organic mercury, most often occurs through consumption of methylmercury in fish and through injection of ethylmercury in thimerosal in vaccines. Exposure to any of these forms of mercury for extended periods of time or in high amounts results in high levels of toxicity.

The main current pharmacological treatment for heavy metal toxicity is chelation therapy. Chelating agents are ligands that form bonds with metal ions, therefore making those ions inert. The chelate complex that is formed between chelating agents and metal ions can subsequently be excreted without causing damage to the body. Current clinical chelation therapy of mercury poisoning generally uses thiol compounds such as dimercaptosuccinic acid (DMSA), dimercaptopropane-sulfonic acid (DMPS) (cite). Cystein (Cys) and N-acetylcysteine (NAC) are commonly administered for hemodialysis (removal of waste products from blood when the kidneys are not functioning properly). However, there are a number of problems associated with using these chelate agents as treatments. For example, while cysteine binds with a high affinity to Hg ((Ka = (5.7±2.6) x 1011 M-1)), the complex that forms between cysteine and Hg2+ can be transported by the amino acid transport system, ascribed to the phenomenon of molecular mimicry, and deposit the Hg2+ into the brain or other organs (cite). The application of cysteine therefore would exacerbate the toxicity. Additionally, cysteine is catabolized in the gastrointestinal tract and in the blood plasma, so its use in chelation therapy is not favorable. DMPS and DMSA are dithiols used as chelators in the treatment of various heavy-metal toxicities. Their binding affinities to Hg2+ (DMSA: Ka = (2.7±0.1) x 109 M-1; DMPS: Ka = (2.0±0.4) x 109 M-1) are significantly weaker than that of cysteine (Ka = (5.7±2.6) x 1011 M-1), so it is critical that better chelating agents be used in the treatment of mercury and other heavy-metal toxicities.

We hypothesize that peptide ligands containing cysteine will bind mercury (II) and form complexes that are larger than the cysteine-Hg(II) complexes, and will hence evade transportation across the blood-brain barrier via amino acid carriers. Therefore, the purpose of this study is to evaluate the binding affinities of some cysteine peptide ligands with Hg(II), and to compare the structural size of these complexes with that of the Cys-Hg(II) complexes. The thermodynamic parameters and binding affinities associated with those bindings will be determined by isothermal titration calorimetry (ITC). The structural size of the various complexes will be assessed by using…

We will evaluate the binding affinities of some cysteine dipeptides and a naturally occurring cysteine pentapeptide, phytochelatin-2, for mercury (II). We will determine the binding affinities and associated themodynamic parameters of the interactions of mercury (II) with the dipeptides of Cys containing histidine (His), for its imidazole-N-donor atom, and tryptophan (Trp), for its potentialeletrostatic cation-pi interactions. We anticipate improved chemical stability towards peptidases by using alternating D- and L-stereochemistry. His-// D //-Cys and Trp-// D //-Cys will be used as model cyteine dipeptides in this study. Phytochelatin-2 (γ-Glu-Cys-γ-Glu-Cys-Gly) is a natural heavy-metal chelator found in some plants, fungi, and algae. They contain atypical peptide bonds, which will evade degredation by peptidases. Because these chelators are found in nature, their potential use for the treatment of heavy-metal poisoning is highly desirable.

Computational Proposal Following the experimental portion of the project, it is important to model the Hg2+ Cys S-conjugates computationally in order to compare the complexes that are formed with the dipeptides with the complexes formed with the main clinical treatments of mercury toxicity, DMSA, DMPS, and cysteine. The binding affinity of our dipeptides to Hg2+ is significantly stronger than that of DMSA and DMPA, as shown in the experimental ITC data, but it is significantly weaker than that of cysteine. Using cysteine as a as a chelating treatment for mercury toxicity is counterproductive though, as the cystein-mercury complex that forms can cross the amino acid transport system and the blood brain barrier, as a result of molecular mimicry. Therefore, in order to show that the dipeptides would be a more suitable chelation treatment for mercury toxicity than cysteine, we will show through computer modeling that the peptide complexes display a size and shape too dissimilar to the cysteine complexes to cross the amino acid transport system. Our ITC data show that dipeptides display strong binding with Hg2+ at molar ratios of 1 and 1.5. In addition, we will be modeling phytochelatin-2 (γ-Glu-Cys-γ-Glu-Cys-Gly) and the chelation complex between phytochelatin-2 and Hg2+. 