Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases

Abstract

Dinucleoside 5 ,5 -P 1 ,P n -polyphosphates, and particularly the diadenosine compounds, have been implicated in extracellular purinergic signalling and in various intracellular processes, including DNA metabolism, tumour suppression and stress responses. If permitted to accumulate, they may also be toxic. One approach to understanding their function is through the various specific degradative enzymes that regulate their levels. Eight adenosine-5 -O-phosphorylated polyols (derivatives of glycerol, erythritol and pentaerythritol) and 11 adenosine-5 -O-phosphorothioylated polyols (derivatives of glycerol, erythritol, pentaerythritol, butanediol and pentanediol) have been tested as inhibitors of specific diadenosine tetraphosphate (Ap 4 A) hydrolases. Of these two groups of novel nucleotides, the adenosine-5 -O-phosphorothioylated polyols were generally stronger inhibitors than their adenosine-5 -O-phosphorylated counterparts. 1,4-Di(adenosine-5 -O-phosphorothio) erythritol appeared to be the strongest inhibitor of (asymmetrical) Ap 4 A hydrolases (EC 3.6.1.17) from both lupin and human, with K i values of 0.15 µM and 1.5 µM respectively. Of eight adenosine-5 -Ophosphorylated polyols, 1,4-di(adenosine-5 -O-phospho) erythritol was the only compound that inhibited the lupin enzyme. Two derivatives of pentaerythritol, di(adenosine-5 -O-phosphorothio)-di(phosphorothio) pentaerythritol and tri(adenosine-5 -Ophosphorothio)-phosphorothio-pentaerythritol, proved to be the strongest inhibitors of the prokaryotic (symmetrical) Ap 4 A hydrolase (EC 3.6.1.41) so far reported. The estimated K i values were 0.04 µM and 0.08 µM respectively. All of these inhibitors were competitive with respect to Ap 4 A. These new selectively acting Ap 4 A analogues should prove to be valuable tools for further studies of Ap 4 A function and of the enzymes involved in its metabolism.

Structure and Substrate-binding Mechanism of Human Ap4A Hydrolase

“…Human Ap 4 Aase can bind a wide range of ligands including various adenosine-5Ј-phosphorylated and adenosine-5Ј-phosphorothioylated polyols (37) and in addition can hydrolyze AZT-p 4 A (3Ј-azido-3Ј-deoxythymidine(5Ј)tetraphospho(5Ј)-adenosine) (38) and 5-phosphoribosyl 1-pyrophosphate (39). By comparing the structure of human Ap 4 Aase to the structure of the animal and plant counterparts from C. elegans and L. angustifolius, respectively, together with the results from the fragmentation assay, mechanistic and evolutionary insights into unraveling the binding characteristics of these enzymes can be established.…”

Di-Adenosine Tetraphosphate (Ap4A) Metabolism Impacts Biofilm Formation byPseudomonas fluorescensvia Modulation of c-di-GMP-Dependent Pathways

“…The reaction mixtures were incubated at 30°C for 1 h before heat treatment at 65°C for 15 min. The reaction mixtures were then separated and visualized using a thin-layer chromatography (TLC) method essentially as described previously (14). Briefly, 5-l portions of reaction mixtures were spotted onto TLC plates (with an aluminum backing and precoated with silica gel containing a fluorescent indicator; Merck catalog no.…”

Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain

“…Only analogs 3 and 4 acted as competitive inhibitors, with K i values of 2.2 μ m ( 3 ) and 1.5 μ m ( 4 ) for the lupin enzyme and of 2.1 μ m ( 3 ) and 2.5 μ m ( 4 ) for the human counterpart. These K i values lie in the range of the K m values for Ap 4 A: 2.5 μ m for the narrow‐leaved lupin [25] and 2 μ m for the human enzyme [16].…”

“…P α ‐Chiral phosphorothioate analogs of Ap 4 A have been used to show that the yeast Ap 4 A phosphorylase forms an enzyme–AMP intermediate [14], whereas a complex of a methylene analogue of Ap 4 A, AppCH 2 ppA, with the (asymmetrical) Ap 4 A hydrolase from Caenorhabditis elegans , was used to determine the 3D structure of the enzyme–substrate complex [15]. Some nondegradable analogs appeared to be extremely strong inhibitors of the Ap 4 A hydrolases; two adenosine‐5′‐ O ‐phosphorothioylated pentaerythritols are strong inhibitors of the (symmetrical) Ap 4 A hydrolase from Escherichia coli (with K i values of 0.04 and 0.08 μ m ) [16], and methylene analogues of adenosine 5′‐tetraphosphate (p 4 A) strongly inhibited the asymmetrically acting Ap 4 A hydrolases with K i values in the nanomolar range [17]. Finally, potential medical application has been demonstrated for AppCHClppA, a competitive inhibitor of ADP‐induced platelet aggregation, which plays a central role in arterial thrombosis and plaque formation [18], and for [ 18 F]AppCHFppA, which appeared to be useful in imaging of positron‐emission tomography to detect atherosclerotic lesions and, hence, promising for the noninvasive characterization of vascular inflammation [19].…”