A-S Rytting

Baba M

Balzarini J

CFR Källander

De Clercq E

DHL Ekstrand

Ekstrand DHL

Goff SP

JS Gronowitz

L Vrang

X Shao

Four non-nucleoside reverse transcriptase (RT) inhibitors, 9-Cl-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(1 H)-thione)], nevirapine (6,11-dihydro-11-cyclopropyl-4-methyl-dipyrido[2,3- b:2′,3′- e]-[1,4]diazepin-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-1 H-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 IC50values 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 antibodyreactive against a peptide in the RNase H region. MSA-300 and delavirdine gave the lowest IC50values, ranging from 0.17 μM to 0.24 μM for MSA-300 and from 0.12 μM to 0.38 μM for delavirdine, whereas higher IC50values of approximately 20 μM were obtained for 9-Cl-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.</jats:p

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Antiviral Chemistry and Chemotherapy

Colorimetric Assays for Evaluation of the Mode of Action of Human Immunodeficiency Virus Type 1 Non-Nucleoside Reverse Transcriptase Inhibitors

Colorimetric Assays for Evaluation of the Mode of Action of Human Immunodeficiency Virus Type 1 Non-Nucleoside Reverse Transcriptase Inhibitors

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