SYNTHESIS OF A PHOTOAFFINITY ANALOG OF 3'-AZIDOTHYMIDINE, 5-AZIDO-3'-AZIDO-2',3'-DIDEOXYURIDINE - INTERACTIONS WITH HERPESVIRUS THYMIDLNE KINASE AND CELLULAR ENZYMES

Long term administration of 3'-azidothymidine (AZT) for the treatment of AIDS has led to detrimental clinical side effects in some patients, the biochemical causes of which are still being delineated. Base-subs tituted, azido-nucleotide photoaffinity analogs have routinely proven to be effective tools for identifying and characterizing nucleotide-ut ilizing enzymes. Therefore, we have synthesized 5-azido-3'-azido-2',3' -dideoxyuridine, which is a potential photoaffinity analog of two huma n immunodeficiency virus drugs, AZT and 3'azido-2',3'-dideoxyuridine. A partially purified herpes simplex virus type 1 thymidine kinase and [gamma-P-32]ATP were used to make an AZT monophosphate analog, [P-32]5 -azido-3'-azido-2',3'-dideoxyuridine monophosphate. The photoaffinity properties of this analog were initially tested with herpes simplex vi rus type 1 thymidine kinase. Photoaffinity labeling of this enzyme was saturable (half-maximal, 30 mu M) and could be specifically inhibited by AZT, AZT monophosphate, thymidine, and thymidine monophosphate. Ph otolabeling of rat Liver microsomal membranes was also done, and sever al membrane proteins that interact with AZT monophosphate were identif ied. The antiviral and cytotoxic activities of 5-azido-3'-azido-2',3'- dideoxyuridine were determined using human immunodeficiency virus, typ e 1 strain IIIB and an AZT drug-resistant strain in human T lymphocyte H9 cells.