Achieving the Ultimate Physiological Goal in Transition State Analogue Inhibitors for Purine Nucleoside Phosphorylase

Andrzej Lewandowicz,Peter C Tyler,Gary B Evans,Richard H Furneaux,Vern L Schramm

doi:10.1074/jbc.c300259200

Andrzej Lewandowicz, Peter C Tyler + Show 3 more

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https://doi.org/10.1074/jbc.c300259200

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Abstract

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 (Kd = 56 pM), and 8-aza-ImmH (Kd = 180 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 (Kd = 7pM), DADMe-ImmH (Kd = 16 pM), and 8-aza-DADMe-ImmH (Kd = 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/Kd 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.

Highlights

From the ‡Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and §Carbohydrate Chemistry Team, Industrial Research Limited, Lower Hutt, New Zealand
Structure-based inhibitor design using early x-ray crystal structures of human purine nucleoside phosphorylase (PNP) led to a family of inhibitors with dissociation constants to 15 nM, but these were insufficient to cause the sustained elevation of blood deoxyguanosine needed to suppress division of activated T-cell populations [8, 9]
Transition state analogue inhibitor design based on the transition state structure of bovine PNP led to the design and synthesis of ImmH, a 23 pM inhibitor for the bovine enzyme but a 2–3-fold weaker inhibitor for the human enzyme [10, 11]

Summary

Introduction

From the ‡Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and §Carbohydrate Chemistry Team, Industrial Research Limited, Lower Hutt, New Zealand. Structure-based inhibitor design using early x-ray crystal structures of human PNP led to a family of inhibitors with dissociation constants to 15 nM, but these were insufficient to cause the sustained elevation of blood deoxyguanosine needed to suppress division of activated T-cell populations [8, 9]. Transition state analogue inhibitor design based on the transition state structure of bovine PNP led to the design and synthesis of ImmH, a 23 pM inhibitor for the bovine enzyme but a 2–3-fold weaker inhibitor for the human enzyme [10, 11]. In a mouse model of host versus graft disease, immucillin-H is as effective as cyclosporin in prolonging host survival [13] These favorable features of PNP inhibition have recently gained immucillin-H approval to enter phase I/II clinical trials against T-cell leukemia [14]

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