Background:  Inhibition of HIV reverse transcriptase (RT) is the backbone of current antiretroviral therapies. We have identified a new class of HIV RT inhibitors that block the polymerization reaction via a mechanism of action that is clearly different from that of current nucleoside- (NRTI), nucleotide- (NtRTI) or non-nucleoside RT inhibitors (NNRTI).

Methods:  Antiviral assays were performed in MT4 cells with an LTR-reporter gene read-out. RT enzymatic activity was determined using a standard DNA polymerization assay with a DNA/RNA primer-template and radio-labeled nucleotides. Enzyme kinetics was studied using the Michaelis-Menten approach under conditions of saturating primer-template concentrations. DNA footprinting-techniques were used to determine the translocation of RT on its primer-template.

Results:  Screening of a random chemical library using a cell-based antiviral assay led to the discovery of a new class of HIV-1 inhibitors with a non-nucleoside/nucleotide structure. Compound-1, a prototype of this class, displayed good potency against HIV-1 (EC₅₀ = 30 nM) and time-of-addition experiments and enzymatic assays proved that it inhibits the reverse transcription step. A reduced susceptibility of < 10-fold was observed when Compound-1 was tested on HIV-2. Enzyme kinetics studies showed that Compound-1 is a competitive inhibitor of nucleotide incorporation by HIV-1 RT and HIV-2 RT. Three other biochemical features differentiate this compound from current NNRTI:  the presence of physiological concentrations of ATP in an enzymatic assay increased the potency of Compound-1 more than 10-fold (IC₅₀ = 30 nM); inhibition of RT-mediated polymerization was observed only when using p(rA)p(dT) or p(rG)p(dC) as the primer-template, and not in the presence of p(rC)p(dG) or p(rU)p(dI); and an RT translocation assay showed that Compound-1 forces the enzyme to move on its primer-template in the same way as when a dNTP binds the enzyme.

Conclusions:  Antiviral assays and biochemical experiments showed that Compound-1 inhibits HIV RT by a mechanism of action that is clearly different from that of current NNRTI and N(t)RTI. The competitive mode of RT inhibition and the effect on enzyme translocation suggest that Compound-1 blocks the DNA polymerization step by binding to the active site of HIV RT and inhibiting nucleotide binding. We therefore propose nucleotide-competing reverse transcriptase inhibitors (NcRTI) as a name for this new class of HIV inhibitors.

Keywords: New Antiretroviral Agents; Reverse Transcriptase Inhibitors; Enzymology