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

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 PNPinhibitor dissociation rates (t1/2) 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 t1/2 of 84 h. Loss of [14C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t1/2 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 t1/2 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.

Original languageEnglish (US)
Pages (from-to)15907-15915
Number of pages9
JournalJournal of Biological Chemistry
Volume292
Issue number38
DOIs
StatePublished - Sep 22 2017

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Purine-Nucleoside Phosphorylase
Erythrocytes
T-cells
Catalytic Domain
T-Lymphocytes
Salvaging
Regain
Clinical Trials, Phase I
Erythropoiesis
Extracellular Space
Enzyme Inhibitors
Apoptosis
Molecules
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase. / Gebre, Sara T.; Cameron, Scott A.; Li, Lei; Babu, Y. S.; Schramm, Vern L.

In: Journal of Biological Chemistry, Vol. 292, No. 38, 22.09.2017, p. 15907-15915.

Research output: Contribution to journalArticle

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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 PNPinhibitor dissociation rates (t1/2) 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 t1/2 of 84 h. Loss of [14C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t1/2 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 t1/2 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.",
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T1 - Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase

AU - Gebre, Sara T.

AU - Cameron, Scott A.

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AU - Babu, Y. S.

AU - Schramm, Vern L.

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N2 - 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 PNPinhibitor dissociation rates (t1/2) 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 t1/2 of 84 h. Loss of [14C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t1/2 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 t1/2 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.

AB - 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 PNPinhibitor dissociation rates (t1/2) 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 t1/2 of 84 h. Loss of [14C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t1/2 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 t1/2 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.

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