Mutations in Adenosine Deaminase-like (ADAL) Protein Confer Resistance to the Antiproliferative Agents N6-Cyclopropyl-PMEDAP and GS-9219

Background/Aim: GS 9219 is a double prodrug of antiproliferative nucleotide analog 9-(2-Phosphonylmetho- xyethyl)guanine (PMEG), with potent in vivo efficacy against various hematological malignancies. This study investigates the role of adenosine deaminase-like (ADAL) protein in the intracellular activation of GS-9219. Materials and Methods: A cell line resistant to 9-(2-Phosphonylmethoxyethyl)-N 6 - cyclopropyl-2,6-diaminopurine (cPrPMEDAP), an intermediate metabolite of GS-9219, was generated and characterized. Results: The resistant cell line was cross-resistant to cPrPMEDAP and GS-9219, due to a defect in the deamination of cPrPMEDAP to PMEG. Mutations in the ADAL gene (H286R and S180N) were identified in the resistant cells that adversely-affected its enzymatic activity. Introduction of the wild-type ADAL gene re-sensitized resistant cells to both cPrPMEDAP and GS-9219. Conclusion: The ADAL protein plays an essential role in the intracellular activation of GS- 9219 by catalyzing the deamination of cPrPMEDAP metabolite to PMEG. Mutations affecting the activity of ADAL confer resistance to both GS-9219 and its metabolite cPrPMEDAP. In addition to their well-established broad-spectrum antiviral activity, some of the acyclic nucleoside phosphonate analogs are potent antitumor agents (1). 9-(2-Phosphonylmetho-xyethyl) guanine (PMEG) is one such example as it exhibits potent antiproliferative effects in multiple in vitro and in vivo models. In cells, PMEG is transformed to an active phosphorylated metabolite PMEG diphosphate (PMEGpp) that acts as a potent inhibitor of nuclear DNA polymerases α, δ and e, blocking DNA synthesis and repair via effective incorporation and chain termination (2, 3). GS-9219 was designed as a novel double prodrug of PMEG consisting of the N 6 -substituted prodrug of PMEG, 9-(2-phospho-nylmethoxyethyl)-N 6 -cyclopropyl-2,6- diaminopurine (cPrPMEDAP), and two symmetrical ethyl- alanine phosphonoamidate groups that mask the charged hydrophilic phosphonate moiety. The N 6 -cyclopropyl prodrug moiety allows for a specific intracellular activation, limiting the plasma exposure to the parent compound PMEG that can