Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89

Abstract

The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity.

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS

“…IEC using high-salt concentrations was first described in the 1950s [12]. Long run times [11,13] and incompatibility with mass spectrometry, however, have limited its application for nucleotide quantification mostly to in vitro studies [14].…”

Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography–electrospray ionization mass spectrometry

“…The generated nucleosides were analyzed by using LC/UV, radioimmunoassay, and LC-MS/MS [3][4][5][6]. Strong anion-exchange HPLC has also been used to isolate and directly analyze nucleotides [7,8]. However, because of its long run time and requirement for radiometric detection, the application of the anion-exchange chromatographic method has been limited mainly to in vitro…”

“…Another challenge for rapid detection of the adenosine nucleotides is that low detection limits are often needed because the compounds are generally present at submicromolar levels in cells [13][14][15][16]. Mass spectrometry coupled with HPLC has become widely used for high-throughput analysis in pharmaceutical industry due to its high specificity and sensitivity [7,8,17,18]. The high selectivity of MS and MS/MS techniques provides great separation and detection power, making them attractive alternatives for the trace analysis of nucleotides.…”

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

“…ABC is first phosphorylated by adenosine phosphotransferase (AdoPT) into the cell to ABC monophosphate (Scheme 6), which is then converted to Carbovir monophosphate (CBV-MP), by the enzyme adenosine deaminase-like protein isoform 1 (ADAL1), recently identified to play a physiological role in the salvage routes of 6-substituted purine-or 2-aminopurine nucleoside monophosphate (Murakami et al, 2011). Finally, CBV-MP is anabolised to CBV-TP, a potent inhibitor of HIV-RT that acts both as a competitive inhibitor of dGTP for DNA incorporation and as a DNA chain terminator (Faletto et al, 1997;Stankova et al, 2012). Boyle et al (2012) reported the synthesis of ABC from the β-lactame (2) obtained by [2 + 2] cycloaddition of chlorosulphonyl isocyanate with cyclopentadiene (Scheme 5b) and subsequent Lipolase (a soluble preparation of Thermonyces lanuginosus lipase, produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism) catalysed kinetic resolution.…”