Background: Nitric oxide (•NO) is a free radical involved in numerous physiological and pathological processes in mammals. NO is produced endogenously by a family of enzymes known as NO synthases (NOS). NO is chemically unreactive towards most bioorganic compounds, but it can rapidly and spontaneously auto-oxidize to yield the highly reactive species, N2O3, which covalently modifies (nitrosates) free thiol and amino groups. High levels of NO synthesis and nitrosation are generally associated with immune response against bacteria and viruses. Recently we showed that nonpolar molecules of •NO and O2 are absorbed from aqueous solution and concentrated in a small volume of hydrophobic cores of various proteins. Within such “micelles” catalysis of •NO oxidation and formation of nitrosating N2O3 occurs. We also demonstrated that hydrophobic derivatives of 5-aminonaphtalenesulfonamides (ANSA) can be efficiently solubilized by various proteins and suppress their enzymatic activity upon nitrosation. Protein inactivation occurs due to irreversible cross-linking between the functional amino acid side chains and the aryldiazonium cation, which forms upon nitrosation of the NH2 group of ANSA in the protein hydrophobic interior.

Description of Project: Many bacteria (if not all) produce significant amounts of NO endogenously. They are also persistently attacked by exogenous NO produced by macrophages during immune response. Additionally, tumor cells are characterized by the increased level of NOS expression and •NO production. Based on these observation, we propose that our ANSA derivatives will preferentially target bacterial or tumor cells in the diseased organism. Our preliminary data show that hydrophobic ANSAs posses a strong cytotoxic effect towards both gram negative and positive bacteria and also prostate tumor cell lines. This effect depends on the level of NO that is produced endogenously or provided via external sources such as activated macrophages, NO-donors, or aqueous NO solution. To further increase the specificity of ANSA towards infectious bacteria or cancer cells, various peptides have been covalently attached to the NH2 group of ANSA. Blocking of the NH2 group with the peptidyl ligand will prevent it from being converted into the aryldiazonium cation upon reaction with N2O3. Thus the peptide will keep ANSA inactive and nontoxic even in the presence of •NO. Cleavage of such a peptide by proteases specifically expressed by bacteria or in tumors activates ANSA cytotoxicity.

Applications: 1) Treatment of bacterial, viral or fungal infections; 2) Treatment of resistant strains of pathogens; 3) Treatment of tumors; 4) Destruction of specific cytotoxins or enzymes.

Patent Status: U.S. patent application has been filed.

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