Spin labelled nitrosoureas and triazenes and their non-labelled clinically used analogues--a comparative study on their physicochemical properties and antimelanomic effects.

Physicochemical properties, such as half life time (tau0.5), alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of spin labeled (containing nitroxyl free radical moiety) amino acid nitrosoureas, synthesized in our laboratory, have been studied and compared to those of the antitumor drug N'-cyclohexyl-N-(2-chloroethyl)-N-nitrosourea (lomustine, CCNU). We have shown that the introduction of amino acid moieties and the replacement of cyclohexylamine with nitroxyl moiety leads to a faster decomposition, higher alkylating, lower carbamoylating activity, better antimelanomic activity and lower general toxicity, when compared to those of CCNU. It was also established that spin labeled triazenes, previously synthesized by us, were more stable in phosphate saline than their nonlabeled analogue, 5-(3,3-dimethyltriazene-1-yl)-imidazole-4-carboxamide (dacarbazine, DTIC). A higher cytotoxicity to B16 melanoma cells than to YAC-1 and lymphocytes was demonstrated for all spin labeled triazenes, in comparison with DTIC. An assumption has been made to explain the lower general toxicity of the spin labeled nitrosoureas compared to that of CCNU. Based on the results presented, we accept that a new trend for synthesis of more selective and less toxic nitrosourea and triazene derivatives as potential antimelanomic drugs might be developed.

Essential binding and functional domains of human bleomycin hydrolase.

Bleomycin hydrolase (BH) is unusual among cysteine proteinases because it appears to form multihomomeric structures, inactivates the antitumor glycopeptide bleomycin, and contains a unique C-terminal amino acid sequence. We now demonstrate intrinsic endopeptidase activity associated with human BH (hBH) using artificial substrates and intracellular dimerization of hBH using a yeast two-hybrid assay. To determine domains important for homomeric interactions and catalysis, we constructed N- and C-terminal deletion mutants and identified an N-terminal region (hBH1-82) that interacted with two nonoverlaping hBH domains: one near the N-terminus (hBH14-103) and another neighboring the C-terminus (hBH358-455). In vitro hBH aggregated with a molecular mass of 235 kD corresponding to a homotetramer and the C-terminus was critical for this oligomerization since no tetramers were found when the last 40 amino acids were deleted. The penultimate 8 amino acids, which constitute a unique and highly conserved bleomycin hydrolase-like domain (BHYD), were essential for BH and aminopeptidase activity but not for endopeptidase activity or oligomer formation. Thus, the C-terminus of hBH has two independent roles controlling both the catalytic activity and oligomerization of hBH.