       Document 0701
 DOCN  M9590701
 TI    The use of immobilized substrates to identify and characterize
       inhibitors of hiv-1 integrase
 DT    9509
 AU    Hazuda D; Felock P; Hastings J; Uncapher C; Wolfe A; Department of
       Antiviral Research, Merck & Co., West Point, PA
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Session III, speakers'
       abstracts - unpaged). Unique Identifier : AIDSLINE AIDS/95920018
 AB    In the life cycle of HIV-1, a crucial step in replication is the
       integration of a DNA copy of the viral genome into the genome of the
       host cell. The process of integration requires a series of
       endonucleolytic and DNA strand transfer steps which are catalyzed by a
       virally encoded protein, integrase. An assay for the DNA strand transfer
       activity of integrase was developed using immobilized oligonucleotides
       to represent HIV LTR donor substrates. Integrase processes the 3' end of
       the immobilized donor; cleavage of a biotinylated target DNA substrate
       and strand transfer results in covalent linkage between the biotinylated
       target and the immobilized LTR. The reaction product is readily detected
       using an avidin-linked calorimetric assay. This system has been used to
       screen for potential inhibitors of the enzyme, as well as to study the
       dynamics of the interaction between HIV-1 integrase and the LTR. In the
       presence of divalent cation, integrase binds the immobilized LTR,
       forming a strand transfer competent complex which is stable in the
       absence of added target DNA. Assembly as well as stability of the
       complex is unaffected by donor substrate processing. The addition of an
       appropriate target DNA substrate to preassembled complexes leads to
       rapid catalysis. Small molecular weight inhibitors of the enzyme have
       been identified which (1) inhibit the reaction only if added at the time
       of complex assembly, or (2) are also efficacious in inhibiting the
       strand transfer of the preassembled complexes. Strand transfer activity
       cannot be restored by removing the compounds prior to the addition of
       target DNA substrate, suggesting that the first class of inhibitors
       works by inhibiting assembly, while the latter may disrupt preassembled
       complexes. Given the apparent stability of preintegration complexes in
       HIV-infected cells, the ability to inhibit preassembled complexes may be
       advantageous in developing integrase inhibitors which are effective
       antiviral chemotherapeutics.
 DE    Antiviral Agents/PHARMACOLOGY  Calorimetry  Cations, Divalent  DNA
       Nucleotidyltransferases/*ANTAGONISTS & INHIB/METABOLISM  DNA,
       Viral/METABOLISM  Drug Design  HIV Long Terminal Repeat
       HIV-1/*ENZYMOLOGY  Molecular Weight  Substrate Specificity  MEETING
       ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

