title: Colorimetric assays for evaluation of the mode of action of human immunodeficiency virus type 1 non-nucleoside reverse transcriptase inhibitors.
abstract: Four non-nucleoside reverse transcriptase (RT) inhibitors, 9-CI-TIBO [(+)-S-4,5,6,7-tetrahydro-9- chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo(4,5,1-jk)(1,4)- benzodiazepin-2(1H)-thione)], nevirapine (6,11-dihydro-11-cyclopropyl-4-methyl-dipyrido[2,3-b:2',3'-e]-[1,4]di azepin- 6-one), MSA-300 (N-[cis-2-(2-hydroxy-3-acetyl-6-methoxy-phenyl)-cyclopropyl]-N'- (5-chloropyrid-2-yl)-thiourea) and delavirdine ¿1-(5-methanesulphonamido-1H-indol-2-yl-carbonyl)-4-[3- (1-methylethylamino)pyridinyl]piperazine¿ were analysed for the mode of action of their inhibition of human immunodeficiency virus type 1 (HIV-1) RT in three different assays utilizing a 96-well microtitre plate format, with solid-phase conjugated poly(rA) as template. These were: (i) direct RT assay, for determination of IC50 values of RT inhibitors; (ii) RT template/primer binding inhibition (BIC) assay, for measuring the effect of various substances on the RT activity binding to template/primer; (iii) RT protein ELISA, for measuring RT protein binding to template/primer with a monoclonal antibody reactive against a peptide in the RNase H region. MSA-300 and delavirdine gave the lowest IC50 values, ranging from 0.17 microM to 0.24 microM for MSA-300 and from 0.12 microM to 0.38 microM for delavirdine, whereas higher IC50 values of approximately 20 microM were obtained for 9-CI-TIBO at all primer concentrations. None of the non-nucleoside substances had inhibiting effects on the binding of template, primer, or template/primer to RT protein. Their inhibition of RT activity was not due to prevention of RT binding to template/primer. TIBO, nevirapine and delavirdine bound to RT reversibly, and they bound more tightly to RT template/primer ternary than to RT template binary complex. MSA-300 showed a comparatively high affinity for the enzyme. The utility of the three assays in relation to screening and analysis of RT inhibitory substances is discussed.
authors.affiliation: Department of Medical Genetics, Uppsala University, Sweden. Xingwu.Shao@bio.embnet.se
authors.forename: X
authors.initials: X
authors.lastname: Shao
authors.forename: A S
authors.initials: AS
authors.lastname: Rytting
authors.forename: D H
authors.initials: DH
authors.lastname: Ekstrand
authors.forename: L
authors.initials: L
authors.lastname: Vrang
authors.forename: C F
authors.initials: CF
authors.lastname: Källander
authors.forename: J S
authors.initials: JS
authors.lastname: Gronowitz
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology*; D001569:Benzodiazepines / Q000494:pharmacology; D003124:Colorimetry; D020008:Delavirdine / Q000494:pharmacology; D004341:Drug Evaluation; D004797:Enzyme-Linked Immunosorbent Assay; D054303:HIV Reverse Transcriptase / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects*; D007093:Imidazoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D019829:Nevirapine / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013698:Templates, Genetic; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D019380:Anti-HIV Agents; D007093:Imidazoles; C116811:MSA 300; D011725:Pyridines; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D001569:Benzodiazepines; C067413:R-82913; D019829:Nevirapine; D020008:Delavirdine; D054303:HIV Reverse Transcriptase; D013890:Thiourea