Effects of trace metal compounds on HIV-1 reverse transcriptase

Abstract

The effect of 44 different metal ions (Ag⁺, Al³⁺, AsO ^-₂ , Au³⁺, Ba²⁺, Be²⁺, Bi³⁺, Cd²⁺, Ce³⁺, Co²⁺, CrO ^2-₄ , Cr³⁺, Cs⁺, Cu²⁺, Fe³⁺, Fe²⁺, Ga³⁺, Ge⁴⁺, Hg²⁺, Ir⁴⁺, La³⁺, Li⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Os⁴⁺, Pb²⁺, Pt⁴⁺, Rb⁺, Rh³⁺, Sb⁵⁺, SeO ^2-₄ , SeO ^2-₃ , Sn²⁺, Sr²⁺, Th⁴⁺, Tl⁺, UO^2/2+, VO^3/-, VO²⁺, WO ^2-₄ , Y³⁺, Zn²⁺, and Zr⁴⁺) on the activity of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV-1) was investigated in vitro. For this study, the RT activity assay was carried out by means of an enzyme-linked immunosorbent assay (ELISA) kit, using the template/primer hybrid poly(A) · oligo(dT)₁₅, which required some modifications: (1) possible interfering metal chelators (such as EDTA) in the original lysis buffer were avoided, and a new buffer (50 mM Tris-NO₃, pH 7.8) was used throughout; (2) an amount of 2 ng of RT per well was considered to be optimal after checking the linearity of the reaction with increasing amounts of enzyme; (3) an incubation temperature of 37‡C and an incubation time of 1 h were chosen after preliminary studies in a wide range of temperature and time. At an incubation temperature ≥40‡C, there was a dramatic loss of enzymatic activity. In addition, when RT alone was preincubated for 1 h at 5‡C, 25‡C, and 37‡C, there was a large (83%) loss of activity at 37‡C as compared to that at 5‡C. These results are indicative of enzyme thermolability, which is higher in the absence of substrates. The effect of metal ions on RT activity was tested using two different metal salt concentrations (10^-4 M and 10^-5 M). Under such experimental conditions, the presence of five metal ions (Pt⁴⁺, Ag⁺, Rh³⁺, Zn²⁺, and Hg²⁺) decreased the RT activity in a dose-response fashion. The observed order of effectiveness with respect to inhibition was Pt⁴⁺ > Ag⁺ > Rh³⁺ > Zn²⁺ = Hg²⁺. Estimated mean inhibitory concentrations (IC₅₀) were 7.8 ΜM for (NH₄)₂PtCl₆, 14.1ΜM for AgNO₃, 46.8ΜM for RhCl₃, 53.7ΜM for Zn(SO)₄, and 56.2 ΜM for Hg(NO₃)₂. Because these data are of the same order of magnitude as the corresponding values related to other RT inhibitors used in anti-AIDS therapy, metal compounds or their derivatives could give an interesting contribution in the development of new RT inhibitors for clinical use.