Discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as inhibitors of the human poly(A)-selective ribonuclease Caf1

Abstract

Eukaryotic mRNA contains a 3' poly(A) tail, which plays important roles in the regulation of mRNA stability and translation. Well-characterized enzymes involved in the shortening of the poly(A) tail include the multi-subunit Ccr4-Not deadenylase, which contains the Caf1 (Pop2) and Ccr4 catalytic components, and poly(A)-specific ribonuclease (PARN). Two Mg(2+) ions present in the active sites of these ribonucleases are required for RNA cleavage. Here, we report the discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as (sub)micromolar inhibitors of Caf1.

Keywords: Caf1/CNOT7; Deadenylase; Mg(2+) dependent nuclease; PARN; Ribonuclease.

Graphical abstract

Figure 1

Discovery of 3-hydroxy-pyrimidine-2,4-dione compounds as inhibitors of the Caf1 ribonuclease. (A) Possible interaction modes of 3-hydroxy-pyrimidine-2,4-dione compounds with two divalent metal ions in the active site of Caf1. (B) Structure and activity of compounds selected from the Open Chemical Repository Collection (NCI, Bethesda). IC₅₀ values refer to inhibition of Caf1. Also indicated is the standard error of the mean (n = 3).

Figure 2

Molecular modelling of inhibitors in the active site of Caf1. Complex crystal structures of human Caf1 (a; PDB 4GMJ.B) and PARN (b; PDB 2A1R) show that the cognate poly(A) ligand (stick models) likely binds in a similar manner to the active site (protein surface with Mg²⁺ ions as green spheres), despite the fact that the active sites in Caf1 and PARN have somewhat different shapes. In (c) it can be seen that the top-ranking poses of the most potent compounds 5j (magenta), 8e (cyan), and 8j (green) adopt very similar conformations upon docking to the 4GMJ.B model, whereas several poses with similar scores were recorded for lead compound 5a (yellow), whereof only some (one shown) were similar to the bound conformations of the more potent compounds. Probable polar binding contacts of the most potent compound 8j with the Caf1 active site include a cation–π interaction between one of the Mg²⁺ ions and the phenyl ring of the inhibitor, as well as H-bonds and polar interactions involving the 3° amine and one of the carbonyl O and the OH of the N-hydroxyimide group as indicated (d; dashed lines). Furthermore, 8j makes numerous van-der-Waals contacts with residues (labelled in d) of the active site cavity.

Figure 3

Inhibition of the ribonuclease activities of (A) Caf1, (B) PARN and (C) Ccr4 by 3,7-N-substituted-1-hydroxy-3,7-dihydro-1H-purine-2,6-diones. Reactions contained no enzyme (control), complete reactions (enzyme) or complete reactions in the presence of the indicated compound. Compounds were used at 30 μM (5a–e, 8a–d, 8f), 10 μM (5h–k), or 3 μM (8e, 8j). Error bars represent the standard deviation.

Scheme 1

Synthesis of 7-substituted 1-hydroxy-3,7-dihydro-1H-purine-2,6-diones. Reagents and conditions: (a) ethyl 2-bromoacetate, CHCl₃, rt, 2 h (37–79%); (b) (ethoxymethylene)cyanamide, THF, Δ, 16 h, then KOBu^t, EtOH, Δ, 2 h (46–80% over 2 steps); (c) CDI, PhMe, Δ, 2 h, then O-allylhydroxylamine, aq NaOH, EtOH, Δ, 2 h (21–61% over 2 steps); (d) Pd(OAc)₂, PPh₃, HCOOH, EtOH–H₂O (8:2), 80 °C, 2 h (23–55%); (e) CDI, PhMe, Δ, 2 h, then O-benzylhydroxylamine, aq NaOH, EtOH, Δ, 2 h (74% over 2 steps); (f) R²X (X = Br or I), K₂CO₃, DMF, 80 °C, 2–12 h, (67–98%); (g) H₂, 10% (w/w) Pd(C), CH₂Cl₂–MeOH (1:9) (33–53%); (h) urea, 2-methoxyethanol, 190 °C, 24 h, then aq NaOH, Δ, 3 h (33%). For definitions of R¹ and R² refer to Tables 1–3.

Scheme 2

Synthesis of 6-hydroxy-1-phenethyl-1,4-dihydro-5H-imidazo[4,5-b]pyridin-5-one 13. Reagents and conditions: (a) CHCl₃, KOBu^t, THF–DMF (2:1), −100 °C, 15 min, then CaCO₃, H₂O, 75 °C, 48 h, then H₂, 10% (w/w) Pd(C), EtOAc–MeOH–AcOH (18:1:1), 55 psi, 18 h (60% over 3 steps); (b) ethyl allyloxyacetate, (Me₃Si)₂NLi, THF, −78 °C to rt over 16 h (36%); (c) Pd(OAc)₂, PPh₃, HCOOH, EtOH–H₂O (8:2), 80 °C, 2 h (33%).