Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain

• Published in: The FEBS journal 2009-03, Vol.276 (6), p.1546-1553
• Main Authors: Guranowski, Andrzej, Starzyńska, Elżbieta, Pietrowska-Borek, Małgorzata, Rejman, Dominik, Blackburn, George M
• Format: Article
• Language: English
• Subjects: adenine nucleotide analogs; Ap4A hydrolases; Ap₄A hydrolases; dinucleoside polyphosphates; modified pyrophosphate substrates; stable pyrophosphate analogs
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2009.06882.x

Dinucleoside polyphosphates (NpnN's; where N and N' are nucleosides and n = 3-6 phosphate residues) are naturally occurring compounds that may act as signaling molecules. One of the most successful approaches to understand their biological functions has been through the use of NpnN' analogs. Here, we present the results of studies using novel diadenosine polyphosphate analogs, with an oxymethylene group replacing one or two bridging oxygen(s) in the polyphosphate chain. These have been tested as potential substrates and/or inhibitors of the symmetrically acting Ap₄A hydrolase [bis(5'-nucleosyl)-tetraphosphatase (symmetrical); EC 3.6.1.41] from E. coli and of two asymmetrically acting Ap₄A hydrolases [bis(5'-nucleosyl)-tetraphosphatase (asymmetrical); EC 3.6.1.17] from humans and narrow-leaved lupin. The six chemically synthesized analogs were: ApCH₂OpOCH₂pA (1), ApOCH₂pCH₂OpA (2), ApOpCH₂OpOpA (3), ApCH₂OpOpOCH₂pA (4), ApOCH₂pOpCH₂OpA (5) and ApOpOCH₂pCH₂OpOpA (6). The eukaryotic asymmetrical Ap₄A hydrolases degrade two compounds, 3 and 5, as anticipated in their design. Analog 3 was cleaved to AMP (pA) and β,γ-methyleneoxy-ATP (pOCH₂pOpA), whereas hydrolysis of analog 5 gave two molecules of α,β-oxymethylene ADP (pCH₂OpA). The relative rates of hydrolysis of these analogs were estimated. Some of the novel nucleotides were moderately good inhibitors of the asymmetrical hydrolases, having Ki values within the range of the Km for Ap₄A. By contrast, none of the six analogs were good substrates or inhibitors of the bacterial symmetrical Ap₄A hydrolase.