Effects of small molecular inhibitors on the binding between HIV-1 reverse transcriptase and DNA as revealed by SPR biosensor

Abstract

Surface plasmon resonance (SPR) biosensor technique was applied to analyze the human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT) binding to DNA template–primer (T–P) duplexes. The kinetics including the initial binding and the subsequent conformation change of the protein–DNA complex in the absence and presence of various chemicals were determined. The effects of three nucleoside-based reverse transcriptase inhibitors (NRTIs) and three non-nucleoside-based reverse transcriptase inhibitors (NNRTIs) on the RT–DNA interaction were analyzed based on the conformation change model. Efavirenz (EFV) slightly affected the binding kinetics of the RT–DNA interaction and the binding affinity ( K A = 2.99 × 10 8 M −1) is lower than that in the absence of the inhibitor ( K A = 5.61 × 10 8 M −1). Nevirapine (NVP) induced RT to bind tightly to DNA duplexes followed by a conformation change step. The action of an unsaturated fatty acid, nervonic acid (NA) was similar to NVP. The dideoxynucleosides, whether complementary (‘correct’) or non-complementary (‘incorrect’) to the bases of 3′-end of template–primer DNA, showed no effect on the binding of RT to DNA. However, the initial association and dissociation rates for the RT–DNA interaction are greater in the presence of ‘correct’ dideoxynucleosides than that of in the presence ‘incorrect’ ones, indicating slight preference of RT to ‘correct’ bases. The azido group of AZT (3′-azido-3′-deoxythymidine) enhanced the dissociation of RT from DNA in the initial binding step, where the absence of which resulted in a 34-fold increase in the overall binding affinity ( K A = 1.94 × 10 10 M −1).