Achieving the ultimate physiological goal in transition state analogue inhibitors for purine nucleoside phosphorylase

Genetic deficiency of human purine nucleoside phosphorylase (PNP) causes T-cell immunodeficiency. The enzyme is therefore a target for autoimmunity disorders, tissue transplant rejection and T-cell malignancies. Transition state analysis of bovine PNP led to the development of immucillin-H (ImmH), a powerful inhibitor of bovine PNP but less effective for human PNP. The transition state of human PNP differs from that of the bovine enzyme and transition state analogues specific for the human enzyme were synthesized. Three first generation transition state analogues, ImmG (Kd = 42 pM), ImmH (K_d = 56 pM), and 8-aza-ImmH (K_d = pM), are compared with three second generation DADMe compounds (4′-deaza-1′-aza-2′ -deoxy-1′-(9-methylene)-immucillins) tailored to the transition state of human PNP. The second generation compounds, DADMe-ImmG (K_d = 7 pM), DADMe-ImmH (K_d = 16 pM), and 8-aza-DADMe-ImmH (K_d = 2.0 nM), are superior for inhibition of human PNP by binding up to 6-fold tighter. The DADMe-immucillins are the most powerful PNP inhibitors yet described, with Km/K_d ratios up to 5,400,000. ImmH and DADMe-ImmH are orally available in mice; DADMe-ImmH is more efficient than ImmH. DADMe-ImmH achieves the ultimate goal in transition state inhibitor design in mice. A single oral dose causes inhibition of the target enzyme for the approximate lifetime of circulating erythrocytes.