Cellular metabolism in lymphocytes of a novel thioether-phospholipid-AZT conjugate with anti-HIV-1 activity.
We previously synthesized a thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-hexadecylthio-2-methoxypropyl)-phosphothymidine, CP-102) with potent anti-HIV-1 activity and significant reduction in cell cytotoxicity compared to AZT alone. To study the cellular metabolism of the conjugate compound we synthesized a double-tritium-labeled thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-[9,10-3H]-S-octadecylthio-2-O-methoxypropyl)-phosphothymidine-[methyl-3H], [3H]CP-102). The intracellular radioactive metabolic products of [3H]CP-102 treated human lymphoblastoid CEM-SS cells were analyzed by HPLC and thin-layer chromatography. Results of this investigation provide evidence that a putative intracellular lipid cleavage enzyme metabolizes [3H]CP-102 to form a thioetherdiglyceride compound that migrates with an authentic 1-S-octadecyl-2-O-methyl-thioglycerol standard on TLC. The thioetherdiglyceride metabolite did not react with the ninhydrin reagent indicating it did not contain a primary amine such as that found on serine or ethanolamine containing phospholipids. Also, the product did not contain a phosphatidic acid group based on migration characteristics in the TLC plate. The other major hydrophilic metabolite was 3'-azido-3'-deoxythymidine-[methyl-3H]-monophosphate (AZT-MP) with lesser amounts of AZT, AZT-DP and AZT-TP. In summary, the best interpretation of these data is that the thioetherphospholipid-AZT conjugate, [3H]CP-102, is cleaved by a putative intracellular lipid cleavage enzyme to release a thioetherdiglyceride compound and AZT-MP. The resulting AZT-MP was either dephosphorylated to AZT or sequentially phosphorylated to AZT-DP and, ultimately, to AZT-TP, the known inhibitory metabolite against HIV-1 reverse transcriptase. Phospholipid-nucleoside conjugates may provide a unique approach for developing anti-HIV-1 prodrugs that do not have a strict requirement for a nucleoside kinase for initial activation of the prodrug to an antiviral form.
Kucera
G L
GL
Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA. gkucera@wfubmc.edu
Goff
C L
CL
Iyer
N
N
Morris-Natschke
S
S
Ishaq
K S
KS
Wyrick
S D
SD
Fleming
R A
RA
Kucera
L S
LS
D019380:Anti-HIV Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D054306:Dideoxynucleotides; D015497:HIV-1 / Q000187:drug effects; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D010743:Phospholipids / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D014316:Tritium; D015215:Zidovudine / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000031:analogs & derivatives
C437603:3'-azido-3'-deoxy-5'-(1-hexadecylthio-2-methoxypropyl)phosphothymidine; D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; D010743:Phospholipids; D014316:Tritium; D015215:Zidovudine