Synthesis and Biological Activity of PTEN-Resistant Analogues of Phosphatidylinositol 3,4,5-Trisphosphate

Abstract

The activation of phosphatidylinositol 3-kinase (PI 3-K) and subsequent production of PtdIns(3,4,5) P 3 launches a signal transduction cascade that impinges on a plethora of downstream effects on cell physiology. Control of PI 3-K and PtdIns(3,4,5)P 3 levels are important therapeutic targets in treatments for allergy, inflammation, cardiovascular, and malignant human diseases. We designed metabolically-stabilized, i.e., phosphatase resistant, analogues of PtdIns(3,4,5)P 3 as probes for longlived potential agonists or potentially antagonists for cellular events mediated by of PtdIns(3,4,5) P 3 . In particular, two types of analogues were prepared containing phosphomimetics that would be selectively resistant to the lipid 3-phosphatase PTEN. The total asymmetric synthesis of the 3-phosphorothioate-PtdIns(3,4,5)P 3 and 3-methylenephosphonate-PtdIns(3,4,5)P 3 analogues is described. These two analogues showed differential binding to PtdIns(3,4,5)P 3 binding modules, and both were potential long-lived activators that mimicked insulin action in sodium transport in A6 cells.The phosphoinositide 3-kinase (PI 3-K) signaling pathway contains important therapeutic targets in human pathophysiology. 1,2 Phosphatidylinositol-3,4,5 -triphosphate (PtdIns(3,4,5) P 3 ) is a ubiquitous signaling lipid found in higher eukaryotic cells 3 and activates a plethora of downstream cellular processes. 4 These signaling events include cell proliferation and transformation, 5 cell shape and motility, 6 and insulin action and alteration of glucose transport. 7 PtdIns(3,4,5)P 3 -regulated signaling is modulated by the lipid 3-phosphatase PTEN 8 and SH2 domain-containing inositol 5-phosphatase SHIP. 9A metabolically-stabilized (ms) analogue of PtdIns(3,4,5)P 3 that resists lipid 3-and 5-phosphatases would have numerous applications in understanding the role of PtdIns(3,4,5)P 3 in cell physiology. The ms-PtdIns(3,4,5)P 3 analogues could separate the activation of signal transduction from the degradation of the signal by phosphatase action in cells. This chemical biology approach to dissection of the PI 3-K pathway is complementary to the use of siRNA knockdowns or genetic knockouts for PTEN and SHIP. We focused first on a 3-stabilized PtdIns(3,4,5)P 3 analog, i.e., one resistant to hydrolysis by PTEN, and we selected two stabilized phosphomimetic isosteres to replace the 3-phosphate of PtdIns(3,4,5)P 3 .gprestwich@pharm.utah.edu. Phosphorothioates are phosphomimetics that show reduced rates of enzyme-mediated hydrolysis. 10 However, the replacement of P = O by P = S also affects the pKa of the phosphate and removes a H-bond acceptor. 11,12 For example, the phosphorothioate analogue of PtdIns (3)P had reduced binding activity for cognate binding proteins, due in part to reduced Hbonding. 13 We hypothesized that a 3-phosphorothioate of PtdIns(3,4,5)P 3 could be either an antagonist or a long-lived agonist in the PI 3-K signaling pathway, because of reduced dephosphorylation by PTEN. Moreover, the methylenephosphonate analogue of PtdIns(3)...

Synthesis and Biological Activity of Metabolically Stabilized Cyclopentyl Trisphosphate Analogues of D‐myo‐Ins(1,4,5)P3

“…Crystallization of the crude product from EtOH (130 mL) gave 8 as a fine white solid (3.740 g, 95.9 %). (10). A mixture of 9 (1.28 g, 2.59 mmol), benzaldehyde dimethyl acetal (0.43 mL, 2.86 mmol), TsOH (49.0 mg, 0.26 mmol) in DMF (10 mL) was stirred for 1 h at 50 8C.…”

“…We have previously employed phosphorothioate and methylenephosphonates to prepare phosphatase-resistant analogues of phosphoinositides [8][9][10] and lysophosphatidic acid. [11] In each case, the analogues were less susceptible to acidic or enzymatic cleavage and some bound to cognate binding proteins with reduced affinity.…”

PTEN inhibitors cause a negative inotropic and chronotropic effect in mice

“…Akt has been shown to promote cell survival in various cell types including cardiomyocytes (Fujio et al, 2000; Matsui and Rosenzweig, 2005). PIP3 is very sensitive to PTEN at the plasma membrane (Das et al, 2003); however, its analog 3-phosphorothioate-PtdIns (3,4,5)P3 (3-PT-PIP3) is resistant to PTEN enzymatic activity and generates insulin-like effects (Zhang et al, 2006). …”

5‐Stabilized Phosphatidylinositol 3,4,5‐Trisphosphate Analogues Bind Grp1 PH, Inhibit Phosphoinositide Phosphatases, and Block Neutrophil Migration

“…We recently described the preparation and activity of the 3-phosphatase-resistant, metabolically-stabilized (ms) analogues 1 and 2 (Figure 1) of PtdIns(3,4,5)P 3 that were both stable to degradation by PTEN and acted as inhibitors of PTEN (13). One of these analogues incorporated a single phosphorothioate (PT) substituent, and an additional study examined the chemistry and biological activity of the 3,4,5-trisphosphorothioate analogue 3 of PtdIns(3,4,5)P 3 (14).…”

“…Third, we explored the effects of the exogenously-delivered the 3-, 5-, and 3,4,5-stabilized PtdIns(3,4,5)P 3 analogues on complement factor C5a-mediated polarization and migration of wild-type murine neutrophils. Finally, the long-lived agonist activities of the 5-PT ( 4 ) and 5-MP ( 5 ) analogues in mimicking insulin action in sodium transport in A6 cells were compared to previous data for the 3-PT ( 1 ), 3-MP ( 2 ), and 3,4,5-PT 3 ( 3 ) analogues (13,14). …”