Stabilized phosphatidylinositol-5-phosphate analogues as ligands for the nuclear protein ING2: chemistry, biology, and molecular modeling

Abstract

The interaction of PtdIns(5)P with the tumor suppressor protein ING2 has been implicated in the regulation of chromatin modification. To enhance the stability of PtdIns(5)P for studies of the biological role in vivo, two phosphatase-resistant moieties were used to replace the labile 5-phosphate. The total asymmetric synthesis of the 5-methylenephosphonate (MP) and 5-phosphothionate (PT) analogues of PtdIns(5)P is described herein, and the resulting metabolically stabilized lipid analogues were evaluated in three ways. First, liposomes containing either the dioleoyl MP or PT analogues bound to recombinant ING2 similar to liposomes containing dipalmitoyl PtdIns(5)P, indicating that the replacement of the hydrolyzable 5-phosphate group does not compromise the binding. Second, the dioleoyl MP and PT PtdIns(5)P analogues were equivalent to dipalmitoyl PtdIns(5)P in augmenting cell death induced by a DNA double-strand break in HT1080 cells. Finally, molecular modeling and docking of the MP or PT analogues to the C-terminus PtdInsP-binding region of ING2 (consisting of a PHD finger and a polybasic region) revealed a number of complementary surface and electrostatic contacts between the lipids and ING2.

Figure 1

5-Methylenephosphonate and 5-Phosphothionate Analogues of PtdIns(5)P

Figure 2

PtdIns(5)P analogues are efficiently recognized by ING2. The SDS-PAGE gels showing the partitioning of the GST-fused C-terminal tail of ING2 between the supernatant fraction (S) and the liposome pellet (P). To better mimic native membranes, liposomes were prepared from phospholipids typically found in bilayers including PtdCho, PtdEtn, PtdSer in a ratio (65:26:9) or PtdCho, PtdEtn, PtdSer and the corresponding phosphoinositide in a ratio (54:22:9:15).

Figure 3

PtdIns(5)P and Analogues Augment Cell Death Induced by neocarzinostatin (NCS) Treatment. Cell death was measured in HT1080 cells treated with 10 μM of the indicated lipid or no lipid control, ± 45 nM NCS. The error bars represent the mean ± s.e.m. for four experiments.

Figure 4

(Panel A) Sequence Alignment of ING2 (202-281) with Related Family Members from PDB. The alignment was performed under the module of ALIGNMENT in MODELLER. (Panel B) Structural alignment of ING2 model (one of twenty models, red), 2g6q (white), 1x4i (blue), 1weu (purple), 1wes (cyan), and 1wen (yellow). PHD domains are the conserved fractions in the center of structures, end with a short helix and the following tails on left side are polybasic regions. (Panel C) Twenty ING2 models built by MODELLER. The multi-orientation coils on left side are polybasic C-termini.

Figure 5

Structural analysis of two representative models from 20 models generated by homology modeling. Panel A. Model 1. Panel B. Model 2.

Figure 6

Docking conformations of diC_18:1-PtdIns(5)P(red), diC_18:1-MP-PtdIns(5)P (2, green) and diC_18:1-PT-PtdIns(5)P (4, cyan) in surface of ING2 C-terminal model.

Scheme 1

Synthesis of 5-Methylenephosphonate Analogues of PtdIns(5) P. Reagents and conditions: (a) NaOMe, MeOH, r.t., 24 h; (b) dimethyl phosphonomethyltriflate, NaH, THF, 0 °C then r.t., 12 h; (c) TBAF_3H₂O, THF, r.t., 4 h; (d) 1H-tetrazole, CH₂Cl₂, Ar, r.t., 24 h; then t-BuOOH, 1 h; (e) TEA, BSTFA, MeCN, Ar, r.t., 24 h; (f) TMSBr, BSTFA, Ar, r.t., 10 h; (g) MeOH, r.t., 1 h.

Scheme 2

Synthesis of Intermediate 13. Reagents and conditions: (a) bis(2-cyanoethyl)diisopropylphosphorodiamidite, 1H-tetrazole, MeCN, Ar, r.t., 2 h; then sulfur, CS₂/Py (1:1), r.t., 3 h; (b) HF-Py, THF, r.t., 3 weeks.

Scheme 3

Synthesis of 5-Phosphothionate Analogues of PtdIns(5)P. Reagents and conditions: (a) TBAF_3H₂O, DMF, r.t., 6 h; (b) triethylsilyl chloride, imidazole, CH₂Cl₂, r.t., 15 h; (c) DIBAL-H, CH₂Cl₂, −78 °C, 2 h; (d) bis(2-cyanoethyl)diisopropylphosphorodiamidite, 1H-tetrazole, MeCN, Ar, r.t., 2 h; then sulfur, CS₂/Py (1:1), r.t., 6 h; (e) NH₄F, MeOH, r.t., 20 h; (f) 10a (or 10b), 1H-tetrazole, CH₂Cl₂, Ar, r.t., 24 h; then t-BuOOH, 1 h; (g) TEA, BSTFA, MeCN, Ar, r.t., 24 h; (h) TMSBr, BSTFA, Ar, r.t., 2 h; (i) MeOH, r.t., 1 h.