Effects of trace metal compounds on HIV-1 reverse transcriptase - An in vitro study

The effect of 44 different metal ions (Ag+, Al3+, AsO2-, Au3+, Ba2+ Be2+, B i3+, Cd2+, Ce3+, Co2+, CrO42-, Cr3+, CS+, CU2+, Fe3+, Fe2+, Ga3+ Ge4+, Hg2, Ir4+, La3+, Li+, Mn2+, Mo6+, Ni2+, Os4+, Pb2+, Pt4+, Rb+, Rh3+, Sb5+, SeO 42-, SeO32-, Sn2+, Sr2+, Th4+, TI+, UO22+, VO3-, VO2+, WO42-, Y3+, Zn2+ and Zr4+) On the activity of the reverse transcriptase (RT) of the human immun odeficiency 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 r eaction with increasing amounts of enzyme; (3) an incubation temperature of 37 degrees C and an incubation time of 1 h were chosen after preliminary s tudies in a wide range of temperature and time. At an incubation temperatur e greater than or equal to 40 degrees C, there was a dramatic loss of enzym atic activity. In addition, when RT alone was preincubated for 1 h at 5 deg rees C, 25 degrees C, and 37 degrees C, there was a large (83%) loss of act ivity at 37 degrees C as compared to that at 5 degrees C. These results are indicative of enzyme thermolability, which is higher in the absence of sub strates. The effect of metal ions on RT activity was tested using two diffe rent metal salt concentrations (10(-4) M and 10(-5) M). Under such experime ntal conditions, the presence of five metal ions (Pt4+, Ag+, Rh3+, Zn2+, an d Hg2+) decreased the RT activity in a dose-response fashion. The observed order of effectiveness with respect to inhibition was Pt4+ > Ag+ > Rh3+ > Z n2+ = Hg2+. Estimated mean inhibitory concentrations (IC50) were 7.8 mu M f or (NH4)(2)PtCl6, 14.1 mu M for AgNO3, 46.8 mu M for RhCl3, 53.7 mu M for Z n(SO)(4), and 56.2 mu M for Hg(NO3)(2). Because these data are of the same order of magnitude as the corresponding values related to other RT inhibito rs used in anti-AIDS therapy, metal compounds or their derivatives could gi ve an interesting contribution in the development of new RT inhibitors for clinical use.