Structural insights into the inhibition of cytosolic 5'-nucleotidase II (cN-II) by ribonucleoside 5'-monophosphate analogues

Abstract

Cytosolic 5'-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5'-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues.

Figure 1. Chemical structures of the ribonucleoside phosphonate analogues evaluated for their inhibition potential against cN-II activity (compound number from 1 to 25) and designed derivatives that could not be evaluated in vitro (virtual compounds 26 to 32).

Figure 2. Schematic representation of the chemical retrosynthetic pathways used for the studied derivatives.

Figure 3. Average docking score of the studied derivatives and natural nucleoside monophosphates classified either by ranking score from the highest to the lowest (A) or by compound number (B).

Figure 4. Docking results obtained with the ribonucleoside phosphonate analogues.

(A) Docking pose of derivative 23 (dark blue) compared to that of IMP (cyan thick sticks); IMP binding site is shown by surface representation and secondary structure elements of cN-II are in grey. (B) Docking poses of compounds 19 (red) and 21 (green) compared to that of IMP. Atomic details of the interaction between IMP (C) or derivative 19 (D) or 21 (E) or 23 (F) and protein residues; magnesium ion is represented in green ball model, aspartic residues are shown in yellow sticks and other residues are shown in pink sticks model. Distances between IMP and protein residues are indicated in Å. The black arrow in panels D, E and F indicates the nucleobase/ribose displacements inducing a weakening of the binding to aromatic residues.

Figure 5. (A) Docking poses for middle score analogues, 9 (purple) and 11 (yellow) showing an increased distance between their base and Tyr 210.

(B) Binding modes obtained for the lowest score compounds, 7 (grey) and 14 (dark blue); distances between the nucleobase of 14 and aromatic residues (His 209, Phe 157) are indicated in Å while compound 7 adopted a fully inverted orientation. (C) Importance of the β-hydroxyl group for the binding of the hypoxanthine analogues to cN-II: docking poses obtained with compound 27 (orange) lacking the β-hydroxyl group (ball model) and compound 23 (dark blue). Water molecules are shown in ball and stick model and distance in Å.

Figure 6. Inhibition of the nucleotidase activity by the studied phosphonate analogues.

IMP was used as substrate and the cN-II activity was measured according to the inorganic phosphate release.

Figure 7. Inhibition of the nucleotidase activity by different types of cN-II inhibitors: β-hydroxyphosphonate analogues 14, 19, 23 and fludarabine monophosphate (F-ara-AMP).

Compound 14 was used as a negative control.