and in vitro evaluation of a phosphonate prodrug: ..
"Synthesis and Invitro Evaluation of a Phosphonate Prodrug ..
In conclusion, we have improvedon the synthesis of a novel STAT6inhibitor and show that it potently inhibits the phosphorylation ofSTAT6 in human bronchial epithelial cells stimulated with either IL-4or IL-13. Key to the synthesis is having the phophonate oxygens protectedprior to coupling to the amine. Furthermore, preactivation of thecinnamate building block as pentachlorophenyl ester improved couplingyields compared to the published method. As mentioned, this classof inhibitor has not been evaluated and shown to inhibit STAT6 todate. Given the importance of this transcription factor in asthmaand allergic lung disease, our promising results suggest that targetingthe SH2 domain with phosphatase-stable, cell-permeable phosphopeptidemimetic prodrugs is a potential treatment modality for this disease.
Synthesis and in vitro evaluation of fluorescent and magnetic ..
Because the biological activity has notbeen reported, prodrug 1 was evaluated for its abilityto inhibit STAT6 Tyr641 phosphorylationin intact Beas-2B cells. Compound 1 was added to cellsfrom a DMSO stock solution. Cells were preincubated with 1 for 2 h and were then stimulated with IL-4 or IL-13 for 1 h. Levelsof phospho-STAT6(Y641) and total STAT6 proteins were estimated byWestern blotting of the cell lysates (Figure A). Prodrug 1 inhibited pSTAT6 phosphorylation at 1–10μM in both IL-4 and IL-13 stimulated Beas-2B cells. This resultsuggests that 1 enters cells, the POM groups are cleaved,and the resulting phosphonate binds to the SH2 domain of STAT6 therebypreventing recruitment to the cytokine receptors and subsequent phosphorylationby JAKs. The free phosphate version of 1 was reportedto inhibit the binding of a fluorescently tagged phosphopeptide at Prodrug 1 at concentrations up to 10 μMwas found not to be toxic to Beas-2B cells in a 72 h MTS assay (Figure C). The duration of pSTAT6 inhibition was assayedby treating Beas-2B cells with a single dose of 1 (10 μM). Cellswere incubated for predefined time intervals and were stimulated withIL-4 for 1 h prior to cell lysis. Western blot analysis showed thatnearly complete inhibition of phospho-STAT6(Y641) occurred at 2 hand was maintained up to 48 h (Figure B).The reduction in pSTAT6 was not due to toxicity as the MTS assay showednot effect on proliferation.