Abstract
(R)-9-(2-Phosphonylmethoxypropyl)adenine (PMPA) is an acyclic nucleoside phosphonate that has been shown to be effective in the treatment of AIDS although it has a shorter separation between the adenine and phosphorus than dideoxy-AMP and dAMP. By using pre-steady state kinetic methods, we examined the incorporation of the diphosphate of PMPA, 2',3'-dideoxyadenosine 5'-triphosphate (ddATP), and dATP catalyzed by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, an exonuclease-deficient T7 DNA polymerase (T7 exo-), and wild-type rat DNA polymerase beta in order to evaluate the selectivity of PMPA as an antiviral inhibitor. With a DNA/DNA or DNA/RNA 22/43-mer duplex, the diphosphate of PMPA (PMPApp) is as effective as ddATP in reactions catalyzed by HIV-1 reverse transcriptase in that both analogs have similar substrate specificity constants (kp/Kd) which are only 5-fold lower than dATP. In contrast, PMPApp is a much weaker inhibitor of the reaction catalyzed by T7 exo- (with the DNA/DNA 22/43-mer duplex) in that PMPApp has a 5 x 10(-4)-fold lower kp/Kd than ddATP and dATP. The lower kp/Kd of PMPApp is due to a 1000-2000-fold lower incorporation rate (kp) and a 35-45-fold lower binding constant (Kd). Similarly, PMPApp is 800-fold less inhibitory toward polymerase beta with the DNA/DNA 22/43-mer duplex, whereas in studies with a single nucleotide gapped DNA (22-20/43-mer) PMPApp is 13-fold less inhibitory than ddATP. Although parallel studies will need to be performed using appropriate human polymerases, these results begin to define the mechanistic basis for the reported lower toxicity of PMPA in the treatment of AIDS.
Highlights
The human immunodeficiency virus (HIV)1 is a retrovirus and a causative agent of the Acquired Immunodeficiency Syn
Kinetic Pathway of Nucleotide Incorporation—We studied the kinetics for incorporation of dATP, dideoxyadenosine 5-triphosphate (ddATP), and PMPApp using pre-steady state kinetic methods
Single nucleotide incorporation catalyzed by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) [19, 23], T7 DNA polymerase [18], and rat DNA polymerase  [24, 25] follows the kinetic pathway shown in Equation 1, where kobs is the observed rate of nucleotide incorporation, kp is the maximum rate of nucleotide incorporation, and Kd represents the nucleotide equilibrium dissociation
Summary
Materials—The chemicals and where they were purchased are as follows: [␥-32P]ATP, ICN (Costa Mesa, CA); dATP and ddATP, Sigma; acetylated bovine serum albumin, Life Technologies, Inc.; calf intestine alkaline phosphatase and T4 polynucleotide kinase, New England Biolabs (Beverly, MA); polyacrylamide and Biospin columns, Bio-Rad. The concentrations of HIV-1 RT, T7 exoϪ, and rat DNA polymerase  reported in this paper were corrected based on active-site titration as described previously [18, 19]. T7 exoϪ was preincubated with DNA 22/43-mer, E. coli thioredoxin, and DTT in T7 buffer containing 40 mM Tris chloride (pH 7.5 at 20 °C), 50 mM sodium chloride, 1 mM EDTA, 1 mM DTT, and 0.1 mg/ml bovine serum albumin as described [18]. All experiments using rat DNA polymerase  were carried out at 37 °C in pol  buffer containing 50 mM Tris chloride (pH 8.0 at 37 °C), 50 mM potassium chloride, 0.2 mg/ml bovine serum albumin, 1 mM DTT, 10 mM magnesium chloride, 0.1 mM EDTA, and 5% glycerol (v/v). Data from measurement of Kd of dNTP (dATP, ddATP, and PMPApp) were fitted to a hyperbolic equation: kobs ϭ kp[dNTP]/{[dNTP] ϩ Kd}, where kp is the maximum rate of dNTP incorporation
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