A semiautomated multiparameter approach for anti-HIV drug screening.
We are implementing a series of complementary assays for initial follow-up confirmation and prioritization of new active anti-HIV compounds identified by the U.S. National Cancer Institute's large-scale in vitro primary anti-HIV screen. Two different kinds of cellular viability assays, in addition to specific assays for total cellular DNA content, supernatant reverse transcriptase activity, p24 core antigen production and the synthesis of infectious HIV virions are all performed from a single well of a 96-well microtiter plate containing human host cells infected with HIV. Antiviral activities of several known prototype HIV inhibitors including 3'-azido,3'-deoxythymidine, 2',3'-dideoxycytidine, dextran sulfate and phorbol myristate acetate were compared in these multiparameter assays as a means of validation. Procedures to automate the method optimally, as well as to maximize the safety of the technicians working with HIV and HIV-infected cells have been emphasized. The resulting semiautomated, highly reproducible battery of assays yields a maximum amount of antiviral and cytotoxicity information from a minimum amount of sample. This is especially crucial when analyzing new synthetic compounds and natural product extracts or fractions where the available amounts of sample may be very limited.
Gulakowski
R J
RJ
Laboratory of Drug Discovery Research and Development, NCI-FCRDC, Frederick Cancer Research and Development Center, Maryland.
McMahon
J B
JB
Staley
P G
PG
Moran
R A
RA
Boyd
M R
MR
D000998:Antiviral Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005069:Evaluation Studies as Topic; D005452:Fluoresceins; D006678:HIV / Q000187:drug effects* / Q000302:isolation & purification; D016655:HIV Core Protein p24 / Q000032:analysis; D015658:HIV Infections / Q000188:drug therapy; D006801:Humans; D007211:Indoles; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D013601:T-Lymphocytes / Q000187:drug effects / Q000382:microbiology; D013778:Tetrazolium Salts; D014773:Virology / Q000379:methods
D000998:Antiviral Agents; D005452:Fluoresceins; D016655:HIV Core Protein p24; D007211:Indoles; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; C007293:DAPI; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012194:RNA-Directed DNA Polymerase