Structure of a mutant human purine nucleoside phosphorylase with the prodrug, 2-fluoro-2'-deoxyadenosine and the cytotoxic drug, 2-fluoroadenine

Abstract

A double mutant of human purine nucleoside phosphorylase (hDM) with the amino acid mutations Glu201Gln:Asn243Asp cleaves adenosine-based prodrugs to their corresponding cytotoxic drugs. When fused to an anti-tumor targeting component, hDM is targeted to tumor cells, where it effectively catalyzes phosphorolysis of the prodrug, 2-fluoro-2'-deoxyadenosine (F-dAdo) to the cytotoxic drug, 2-fluoroadenine (F-Ade). This cytotoxicity should be restricted only to the tumor microenvironment, because the endogenously expressed wild type enzyme cannot use adenosine-based prodrugs as substrates. To gain insight into the interaction of hDM with F-dAdo, we have determined the crystal structures of hDM with F-dAdo and F-Ade. The structures reveal that despite the two mutations, the overall fold of hDM is nearly identical to the wild type enzyme. Importantly, the residues Gln201 and Asp243 introduced by the mutation form hydrogen bond contacts with F-dAdo that result in its binding and catalysis. Comparison of substrate and product complexes suggest that the side chains of Gln201 and Asp243 as well as the purine base rotate during catalysis possibly facilitating cleavage of the glycosidic bond. The two structures suggest why hDM, unlike the wild-type enzyme, can utilize F-dAdo as substrate. More importantly, they provide a critical foundation for further optimization of cleavage of adenosine-based prodrugs, such as F-dAdo by mutants of human purine nucleoside phosphorylase.

Figure 1

(A) Overall structure of trimeric hDM. In each monomer, β-sheets are in yellow and loops are in green. α-helices are shown in red, except for the two helices F and H of each monomer shown in navy. F-dAdo is shown as sticks. (B) One subunit of hDM complexed with F-dAdo is superimposed on one subunit of hPNP complexed with guanosine (1RFG) and unliganded hPNP (1ULA). hDM is shown in navy, hPNP complexed with guanosine is green, and uncomplexed hPNP is red. In F-dAdo and guanosine, shown as sticks, carbons are in yellow and green, respectively. In both molecules, nitrogens are navy, oxygens red, and fluorine is light blue. The flexible loop, 241–265, is indicated by an asterisk.

Figure 2

Interaction of hDM with F-dAdo, phosphate/sulfate, and F-Ade. In hDM, carbons are shown in light gray, oxygens in red, and nitrogens in navy. Water molecule is shown as red sphere. In F-dAdo and F-Ade, Fluorine is shown in light blue and carbons in yellow and green, respectively. Sulfate is shown in yellow. (A) A close overview of hDM binding to F-dAdo and sulfate which is bound to the phosphate binding site. (B) A close overview of hDM binding to F-Ade. (C) Base binding pocket of hDM complexed to F-dAdo or F-Ade superimposed. (D) Base binding pocket of hDM complexed with F-dAdo, shown in yellow is superimposed on the structure of hPNP complexed with guanosine, shown in pink (1RFG). (E and F) Simulated annealing F_o-F_c omits maps calculated with CNS. Blue and red contours drawn at +3.5 and −3.5 sigma levels.

Figure 3

Proposed mechanism for cleavage of F-dAdo to F-Ade by hDM.