Several alkylating agent prodrugs with antitumor activity target the O-6 position of guanine residues inDNA. These include the chloroethylating agents Cloretazine, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[(1-|(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) and KS119W (the water-soluble form of KS119),carmustine (BCNU) and lomustine (CCNU) and the methylating agents temozolomide (TMZ), procarbazine,dacarbazine (DTIC) and streptozocin. The chloroethylating agents are the most potent because thealkylation of the O-6 position of guanine in DNA leads to the formation of a l-^-deoxycytidinyO^A/1-deoxyguanosyl)ethane (G-C) DNA cross-link, Of the chloroethylating drugs, Cloretazine and KS119 are by alarge margin the most specific for the O-6 position of guanine. All of the chloroethylating and methylatingagents are susceptible to the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) whichstoichiometrically transfers alkyl and methyl groups from the O-6 position of guanine to cysteine 145 of theAGT molecule by flipping the guanine O-6 adduct out of the DNA helix into a binding pocket in the AGTmolecule. The alkylated form of AGT is rapidly degraded by the proteasomal system and the DNA isrestored to'its native state; this action represents the primary mechanism of tumor and host tissue resistanceto Cloretazine, KS119, BCNU and CCNU. O6-Benzylguanine (O6-BG) is among the most potent knowninhibitors of AGT; this agent reacts with AGT to form S-benzylcysteine in the active site of the protein,depleting AGT and increasing the sensitivity of both tumor and host cells to agents that chloroethylate andmethylate the O-6 position of guanine in DNA. Relatively non-toxic doses of O6-BG have been shown inboth cell systems and patients to deplete the AGT content of tumors. This action sensitizes cell systems andtumors in vivo to BCNU; however, since AGT levels are also depleted by O6-BG in normal tissues, an 80%reduction in the dosage of BCNU is required, leading to an ineffective therapeutic dosage level of BCNU.These findings imply that methodology that selectively depletes AGT in tumor tissue relative to normaltissues is required to circumvent AGT induced tumor resistance to guanine O-6 alkylating agents. Toaccomplish this we propose to use the hypoxic tumor cell fraction present in solid tumors, which is a majorsite of tumor vulnerability, to selectively activate prodrugs to generate potent inhibitors of AGT. The primaryoverall objective is the selection of a prodrug for eventual clinical development to use in combination with O-6 guanine chloroethylating and methylating agents. The analog selected must deplete AGT selectively orpreferentially in solid tumors, thereby permitting usage in sequential combination of close to full therapeuticdosage of the alkylating agent employed without enhanced myelosuppression or toxicity to other normaltissue.
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