Indolopyridones Inhibit Human Immunodeﬁciency Virus Reverse Transcriptase with a Novel Mechanism of Action (cid:1)

We have discovered a novel class of human immunodeﬁciency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone com- pounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed “hot spots” for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-speciﬁc footprinting experiments revealed and high-perfor- mance liquid chromatography-puriﬁed primers (Genset Oligos). Plasmids were sequenced to conﬁrm that they contained the desired mutations. Mutant viruses were created by recombination of the mutant protease-RT sequence with a protease-RT-deleted HIV-1 HXB2 proviral clone (10). Antiviral assays. The antiviral activity on different HIV-1 strains, HIV-2 ROD and SIV Mac251, was determined in a cell-based virus replication assay. Here MT4-LTR-EGFP cells (150,000 cells/ml) are infected (multiplicity of infection [MOI] of 0.01) in the presence or absence of different inhibitor concentrations. After 3 days of incubation, the amount of virus replication is quantiﬁed by measuring the EGFP ﬂuorescence and expressed as the 50% effective concen- tration (EC 50 ). The toxicity of inhibitors is determined in parallel on mock-infected MT4 cells (150,000 cells/ml) stably transformed with a CMV-EGFP reporter gene and cultured in the presence or absence of test compound concentrations. After 3 days of incubation, cell proliferation is quantiﬁed by mea- suring the EGFP ﬂuorescence and expressed as CC 50 values (cytotoxic concentration of drug which reduced the viable cell number by 50%). Determination of anti-HIV-1 activity and toxicity in CEM cells was done by an (MTT)-based col- orimetric cytopathic effect protection assay toxicity in a PBMC-based antiviral assay with a p24- based readout Genotyping and subtype determination. Genotypic analysis was performed by automated population-based full-sequence analysis (ABI PRISM BigDye Ter-minator cycle sequencing). Sequencing results are reported as amino acid changes compared to the HIV-1 wild-type (HXB2) reference sequence (19). Subtypes were determined by heteroduplex mobility assay (6) or sequencing. In vitro selection experiments. MT4-LTR-EGFP cells infected with HIV-1 wild-type strain IIIB at an MOI of 0.01 to 0.001 times the 50% cell culture infectious dose per cell in the presence of INDOPY-1 at three times the EC 50 . This corresponded to an initial infection rate of (cid:5) 1%. Cultures were subculti- vated in the presence of the same concentration of inhibitor every 3 to 4 days and microscopically examined for signs of virus replication. At 100% virus break- through, the supernatant was collected and stored as a new virus strain. These new virus strains were used to infect fresh cultures in the presence of a higher concentration of INDOPY-1. The procedure was repeated up to a concentration of 2.4 (cid:4) M. Viral breakthrough was assessed by microscopic scoring of the cytopathogenicity and virus-induced ﬂuorescence. A viral breakthrough of 100% was deﬁned as microscopic evidence of extensive viral replication in all cell clusters (full cytopathic effect). Samples for genotyping were obtained from the harvested supernatant.