Cellular metabolism in lymphocytes of a novel thioether–phospholipid–AZT conjugate with anti-HIV-1 activity

• Published in: Antiviral research Vol. 50; no. 2; pp. 129 - 137
• Main Authors: Kucera, Gregory L, Goff, Christy L, Iyer, Nathan, Morris-Natschke, Susan, Ishaq, Khalid S, Wyrick, Steven D, Fleming, Ronald A, Kucera, Louis S
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.05.2001; Elsevier
• Subjects: Anti-HIV Agents - chemical synthesis; Anti-HIV Agents - chemistry; Anti-HIV Agents - metabolism; Anti-HIV-1 activity; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiviral agents; Biological and medical sciences; Cells, Cultured; Cellular metabolism; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dideoxynucleotides; HIV-1 - drug effects; Human immunodeficiency virus 1; Humans; Lymphocytes - metabolism; Lymphocytes of a novel thioether–phospholipid–AZT conjugate; Medical sciences; nucleosides; Pharmacology. Drug treatments; Phospholipids - chemical synthesis; Phospholipids - chemistry; Phospholipids - metabolism; Tritium; Zidovudine - chemical synthesis; Zidovudine - chemistry; Zidovudine - metabolism; TBAP, tetrabutylammonium dihydrogen phosphate; Cellular metabolism; Lymphocytes of a novel thioether–phospholipid–AZT conjugate; HIV, human immunodeficiency virus; Anti-HIV-1 activity; PBS, phosphate buffered saline; AZT, 3′-azido-3′-deoxythymidine; AZT-MP, 3′-azido-3′-deoxythymidine monophosphate; AIDS, acquired immunodeficiency syndrome; AZT-TP, 3′-azido-3′-deoxythymidine triphosphate; HPLC, high pressure liquid chromatography; AZT-DP, 3′-azido-3′-deoxythymidine diphosphate; Thiol; Targeting; Ether; HIV-1 virus; Retroviridae; Phospholipid; Metabolism; Lentivirus; Virus; Dideoxynucleoside; Antiviral; Human immunodeficiency virus; Conjugated compound; Chemical synthesis; Lymphocyte; Pyrimidine nucleoside; Zidovudine
• ISSN: 0166-3542; 1872-9096
• DOI: 10.1016/S0166-3542(01)00137-1

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.