Synthesis and characterization of non-hydrolysable diphosphoinositol polyphosphate second messengers

Abstract

The diphosphoinositol polyphosphates (PP-IPs) are a central group of eukaryotic second messengers. They regulate numerous processes, including cellular energy homeostasis and adaptation to environmental stresses. To date, most of the molecular details in PP-IP signalling have remained elusive, due to a lack of appropriate methods and reagents. Here we describe the expedient synthesis of methylene-bisphosphonate PP-IP analogues. Their characterization revealed that the analogues exhibit significant stability and mimic their natural counterparts very well. This was further confirmed in two independent biochemical assays, in which our analogues potently inhibited phosphorylation of the protein kinase Akt and hydrolytic activity of the Ddp1 phosphohydrolase. The non-hydrolysable PP-IPs thus emerge as important tools and hold great promise for a variety of applications.

Figure 1

The diphosphoinsitol polyphosphate biosynthetic pathway, in abbreviated form. Phosphorylation of inositol 1,4,5-triphosphate (IP₃) by inositol multikinase (IPMK) and inositol pentakisphosphate 2-kinase (IPPK) results in formation of inositol pentakisphosphate (IP₅) and inositol hexakisphosphate (IP₆). Both IP₅ and IP₆ are substrates for IP6K (inositol hexakisphosphate kinase) to yield the diphosphoinositol polyphosphates 5PP-IP₄ and 5PP-IP₅, respectively. The numbering of the ring positions is indicated in the IP₃ structure.

Figure 2

Comparison of the naturally occurring signalling molecule 5PP-IP₅ (1) with the analogues described in this study.

Figure 3

a) 5PP-IP₅ and 5PCP-IP₅ undergo a conformational change around pD 8.8. b) ¹H NMR titration curves for 5PP-IP₅ (hollow circles for H_4/6 and hollow diamonds for H_1/3) and 5PCP-IP₅ (filled circles for H_4/6 and filled diamonds for H_1/3). Between pD 8.4 and pD 9.2 peaks could not be assigned due to severe broadening of the resonances (see Figure S1).c) ³¹P NMR titrations curves for 5PP-IP₅ (hollow triangles for P₂) and 5PCP-IP₅ (filled triangles for P₂).

Figure 4

a) Binding of 5PP-IP₅ is proposed to stabilize Akt in an inactive conformation that cannot become phosphorylated by PDK1. b) Western-blots of Akt inhibition experiments. Akt phosphorylation at threonine 308 (p-Akt) was measured using a phosphospecific antibody. A Western-blot for total Akt was used as a loading control. IC₅₀ values were determined in three independent experiments, and the errors are indicated.

Figure 5

a) Ddp1 hydrolyzes diadenosine pentakisphosphate (Ap₅A) to adenosine monophosphate (AMP) and adenosine tetrakisphosphate (p₄A), which is subsequently further degraded. b) Ddp1 mediated Ap₅A hydrolysis is inhibited by 5PP-IP₅ and the analogues. IC₅₀ values for 5PP-IP₅, 5PCP-IP₅, 5PCP-IP₄ and 2Bz-5PCP-IP₄ were determined in three independent experiments and the inhibition curves are shown (see also Figure S4).

Scheme 1

Synthesis of non-hydrolysable PP-IP analogues. Reagents and conditions: (i) (bis-benzyloxyphosphoryl-methyl)phosphoryl chloride, KHMDS, THF, −78 °C to rt, overnight; (ii) NaOMe, MeOH, rt, overnight; (iii) H₂O, p-TsOH, acetone, overnight; (iv) N,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine, 1H-tetrazole, CH₃CN, 0 °C to rt, 36 h; (v) mCPBA, CH₃CN, 0 °C to rt, 3 h; (vi) H2, Pd black, NaHCO₃, t-BuOH/H₂O, rt, overnight; (vii) Conc. aq. NH₃, rt, 4 d; (viii) Dowex-H⁺.