Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase

Sara T Gebre,Scott A Cameron,Lei Li,Y.S Babu,Vern L Schramm

doi:10.1074/jbc.m117.801779

Abstract

Purine nucleoside phosphorylase (PNP) is part of the human purine salvage pathway. Its deficiency triggers apoptosis of activated T-cells, making it a target for T-cell proliferative disorders. Transition-state analogues of PNP bind with picomolar (pm) dissociation constants. Tight-binding PNP inhibitors show exceptionally long lifetimes on the target enzyme. We solve the mechanism of the target residence time by comparing functional off-rates in vitro and in vivo We report in vitro PNP-inhibitor dissociation rates (t½) from 3 to 31 min for seven Immucillins with dissociation constants of 115 to 6 pm Treatment of human erythrocytes with DADMe-Immucillin-H (DADMe-ImmH, 22 pm) causes complete inhibition of PNP. Loss of [14C]DADMe-ImmH from erythrocytes during multiple washes is slow and biphasic, resulting from inhibitor release and rebinding to PNP catalytic sites. The slow phase gave a t½ of 84 h. Loss of [14C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t½ of 1.6 h by preventing rebinding. Thus, in human erythrocytes, rebinding of DADMe-ImmH is 50-fold more likely than diffusional loss of the inhibitor from the erythrocyte. Humans treated with a single oral dose of DADMe-ImmH in phase 1 clinical trials exhibit regain of PNP activity with a t½ of 59 days, corresponding to the erythropoiesis rate in humans. Thus, the PNP catalytic site recapture of DADMe-ImmH is highly favored in vivo We conclude that transition-state analogues with picomolar dissociation constants exhibit long lifetimes on their targets in vivo because the probability of the target enzyme recapturing inhibitor molecules is greater than diffusional loss to the extracellular space.

Highlights

Analyses of drug efficacy have considered the significance of the dissociation rate constant or off-rate and its correla
As the lifespan of mouse erythrocytes is ϳ25 days, regain of Purine nucleoside phosphorylase (PNP) activity can be attributed to erythropoiesis rather than diffusional loss of DADMe-ImmH
Transition-state analogue inhibitors of human PNP bind with picomolar dissociation constants and cause slow-onset tight-binding inhibition. Residence times of these inhibitors on the PNP target are on the timescale of minutes with in vitro enzyme-inhibitor complexes

Summary

Introduction

Analyses of drug efficacy have considered the significance of the dissociation rate constant or off-rate (koff) and its correla-. The Albert Einstein College of Medicine and Victoria University of Wellington own patents for the Immucillins as purine nucleoside phosphorylase inhibitors. 1 Present address: Ferrier Research Institute, Victoria University of Wellington, Lower Hutt 5010, New Zealand. Residence time is an important factor in determining biological efficacy and can be determined from half-life measurements obtained from the inhibitor off-rates [4]. In mice, a single oral dose of the PNP inhibitor DADMe-ImmH (Fig. 1) gave inhibition in circulating erythrocytes for the life of the cells, whereas koff in solution predicted a much shorter efficacy [4]. Extended inhibition on the target suggests a reduced koff rate under physiological conditions, cellular accumulation of inhibitor, covalent inhibition, or frequent rebinding events. Rebinding contributions to long-term inhibition have been mathematically treated and are known to contribute to cell receptor-ligand interactions (6 – 8)

Results

Discussion

Conclusion