) Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolytic cleavage of purine nucleosides to form the free base and ribose 1-phosphate. The nucleoside cleavage reaction can be utilized to activate prodrugs such as 6-methyl-2'-deoxypurine riboside (MePdR). MePdR is non-toxic while the cleavage product, 6-methypurine (MeP), is highly cytotoxic. The differences in substrate specificity between human and E. coli PNPs can be utilized to selectively activate MePdR to MeP in tumor cells. Human PNP is trimeric, with 32 kD subunits, and is specific for 6-oxopurines, such as guanosine and inosine. E. coli PNP is hexameric, with 25 kD subunits, and cleaves 6-aminopurines, such as adenosine, as well as 6-oxopurines. Only E. coli PNP efficiently cleaves MePdR. By transfecting tumor cells with the E. coli PNP gene, the prodrug can be selectively activated, thus concentrating the release of cytotoxic MeP to the desired site of action. Sorscher and coworkers have successfully demonstrated this concept in vivo, which forms the basis for this NCDDG application. To further optimize prodrug activation, we propose to design new prodrug/enzyme combinations in which the prodrug is resistant to low level cleavage by human PNP and cleavage by intestinal flora. We will use the 3-D structures of E. coli PNP and human PNP, with and without ligands, to design new prodrugs and to engineer enzymes that specifically cleave the prodrug. New prodrugs will be synthesized by Drs. Secrist and Montgomery in Laboratory Program 3, new enzymes will be prepared by Dr. Sorscher in Laboratory Program 1 and biochemical activities will be determined by Dr. Parker in Laboratory Program 2. Crystallographic and modeling studies will be performed in Laboratory Program 4. We envision that the process of designing new enzyme/prodrug combinations will be iterative -- as is the case for structure-based drug design -- in which cycles of modeling, synthesis, biochemical analysis and crystallographic analysis are repeated to achieve the desired level of optimization.
Showing the most recent 10 out of 29 publications
