COLORIMETRIC ASSAYS FOR EVALUATION OF THE MODE OF ACTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NONNUCLEOSIDE REVERSE-TRANSCRIPTASE INHIBITORS

Four non-nucleoside reverse transcriptase (RT) inhibitors, 9-CI-TIBO r ahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)im 5,1-jk)(1,4)-benzodi azepin-2(1H)-thione)], nevi rapine 11-cyclopropyl-4-methyl-dipyrido[2, 3-b:2',3'-e]-[1 ,4]diazepin-6-one), MSA-300 nyl)cyclopropyl]-N'-(5-chl oropyrid-2-yl)-thiourea) and delavirdine ethanesulphonamido-1H-indol-2 -yl-carbonyl)-4-[3-(1 -methylethylamino)pyridinyl] piperazine) were an alysed for the mode of action of their inhibition of human immuno-defi ciency virus type 1 (HIV-1) RT in three different assays utilizing a 9 6-well microtitre plate format, with solid-phase conjugated poly(ra) a s template. These were: (i) direct RT assay for determination of IC50 values of RT inhibitors; (ii) RT template/primer binding inhibition (B IG) assay, for measuring the effect of various substances on the RT ac tivity binding to template/primer; (iii) RT protein ELISA, for measuri ng RT protein binding to template/primer with a monoclonal antibody re active against a peptide in the RNase H region. MSA-300 and delavirdin e gave the lowest IC50 values, ranging from 0.17 mu M to 0.24 mu M for MSA-300 and from 0.12 mu M to 0.38 mu M for delavirdine, whereas high er IC50 values of approximately 20 mu M 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/pri mer to RT protein. Their inhibition of RT activity was not due to prev ention of RT binding to template/primer. TIBO, nevirapine and delavird ine bound to RT reversibly and they bound more tightly to RT template/ primer ternary than to RT template binary complex. MSA-300 showed a co mparatively high affinity for the enzyme. The utility of the three ass ays in relation to screening and analysis of RT inhibitory substances is discussed.