Fluorination Influences the Bioisostery of Myo-Inositol Pyrophosphate Analogs

Abstract

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP₅ is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP₅ . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.

Keywords: fluorine; inositol pyrophosphates; phosphoramidite; phosphorylation; protein structures.

Figure 1

Inositol polyphosphates and their non‐hydrolysable analogs. (A) Abbreviated biosynthesis of inositol polyphosphates. (B–E) Structures of metabolically stable analogs of 5PP−IP₅.

Scheme 1

Synthesis of the fluorinated analog 5PCF₂P−IP₅. (A) Synthesis of the fluorinated combined phosphoramidite 1 was adapted from a recently reported strategy.[ ⁶ , ⁷ ] (B) In the first attempts to obtain 5PCF₂P−IP₅, inspired by our previous work on methylene bisphosphonate moieties, the difluoro−bisphosphonate moiety was appended on an early synthesis intermediate. (C) In the synthetic route that led to 5PCF₂P−IP₅, on the contrary, the difluoro−bisphosphonate moiety should be appended at the very end of the synthesis.

Figure 2

Representative NMR signals of the fluorinated phosphoramidite 1. Selected regions of (A) ³¹P NMR and (B) ¹⁹F NMR spectra of 1.

Figure 3

Interactions of 5PCF₂P−IP and 5PCF₂Am−IP₅ with Dipp1. (A) Stick models are used to depict 5‐PCF₂P−IP₅ and residues within polar‐bond range (<3.2 Å) in a crystal complex with DIPP1 (PDB code: 8G9 C); orange denotes phosphorus, red indicates oxygen, blue indicates fluoride, blue denotes nitrogen. The OMIT map is contoured at 2.5 σ and is shown in Figure S4. Four magnesium atoms are depicted as magenta spheres. (B) The orientation of 5PCF₂P−IP₅ in panel A (stick model with olive carbons) is superimposed upon 5PP−IP₅ (dark yellow carbons) in complex with DIPP1, as described in an earlier study ([37]; PDB code, 6WO7). (C) 5PCF₂Am−IP₅ (wheat‐colored carbons; C‐5 is numbered) in a crystal complex with DIPP1 (PDB code: 8G9D). The omit map is contoured at 2.5 σ. (D) Superimposition of 5PCF₂P−IP₅ in panel A upon 5PCF₂Am−IP₅ from panel C. (E), Superimposition of 5PCF₂Am−IP₅ from panel C upon 5PCF₂Am−IP₅ in a crystal complex with ScDdp1 (light green carbons). (F) Superimposition of 5PCF₂P−IP₅ from panel A with 5PCP−IP₅ in complex with DIPP1 (dark green carbons; [37], PDB code, 6WOG).

Figure 4

Interactions of 5PCF₂P−IP₅, 5PP−IP₅ and 5PCP−IP₅ with the PPIP5K^KD kinase domain. (A) Structural depiction of the catalytic pocket of the PPIP5K2^41–366/5PCF₂P−IP₅/AMPPNP crystal complex (PDB code: 8G9E). Colour‐coded tubes depict protein loops hosting residues that form polar contacts (broken black lines) with PPIP5K2^KD: yellow for the αβα domain, while blue and green denote different lobes from the ATP‐grasp domain (see [39]). 5PCF₂P−IP₅ and its interacting residues are depicted as colour‐coded stick models: carbons are olive, phosphorus is orange, oxygen is red, nitrogen is blue, and fluorine is cyan, the magenta sphere depicts magnesium, and the red spheres represent water molecules. The OMIT difference map for 5PCF₂P−IP₅ is contoured at 5.0 σ and shown in Figure S4. The broken red line depicts the 5.3 Å distance between the γ‐phosphate of AMPPNP and the closest oxygen from the 1‐phosphate. (B) Superimposition of PPIP5K2^41–366 protein complexes containing either 5PP−IP₅ (PDB=3T9D; depicted in light gray, 1‐ and 5‐carbons are labeled) or 5PCF₂P−IP₅ (color‐coded as in panel A). (C, D) IC₅₀ plots for inhibition of the reverse kinase activity of PPIP5K2^1–366, from a total of 6 (panel C) or 3 (panel D) independent experiments.

Figure 5

Analysis of ligand binding to the PPIP5K2^KD by ITC. Test solutions contained 7.5 μM of PPIP5K2^1–366 in the sample cell and 75 μM ligand in the syringe. Other details are provided in the Methods Section. Representative thermograms (1 of 3 replicates) are shown for titration of either (A) 5PP−IP₅; (B) 5PCPF₂P−IP₅; (C) 5PCP−IP₅; (D) 5PCF₂Am−IP₅; (E) 5PA−IP₅. (F) Corresponding thermodynamic data.

Figure 6

HPLC analysis of the products of PPIP5K‐mediated phosphorylation of 5PP−IP₅ and the various analogs. (A) Principle of the assay. PPIP5K2^1–366 was incubated with [³³P‐γ]ATP and either 5PP−IP₅ or one of the analogs (each of which is color‐coded as described in the figure), as described in Experimental Section. The [³³P]‐labeled reaction products were resolved in individual HPLC runs, and the elution data are incorporated into a single plot. (B) A representative data set is shown, from a total of 3 independent experiments.