Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases

Abstract

Nucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established both in vitro and in vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X-and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypass in vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases ␤, , , , , and Rev1 incorporating the active, 5-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40-to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X-and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.More than 30 million people worldwide are infected with the human immunodeficiency virus (HIV), which is the causative agent of AIDS. To manage the life-threatening effects of HIV replication, nucleoside reverse transcriptase inhibitors (NRTIs) have been a mainstay in effective combination antiretroviral therapy. NRTIs undergo phosphorylation by host cell kinases to be converted into their active di-or triphosphate (DP or TP) forms, which can serve as nucleotide substrates for HIV reverse transcriptase (RT). Incorporation of the NRTIs into the viral genome by HIV RT terminates the replication process due to the lack of a 3Ј-hydroxyl in these drugs. Unfortunately, host DNA polymerases (Pols), organized into the A, B, X, and Y families, are susceptible to drug inhibition, because these enzymes catalyze a nucleotidyl transfer reaction similar to that of HIV RT. Incorporation of the drug analog into human DNA will inhibit DNA replication and possibly lead to cell death and drug toxicity. A correlation has been established between the kinetics of nucleotide analog incorporation catalyzed by human DNA Pol ␥, an A-family member, and the observed clinical toxicity that presents as mitochondrial dysfunction (12,13,23,26). However, some drug toxicity occurs via Pol ␥-independent mechanisms (36, 52), such as the bone marrow toxicity as...

Presteady State Kinetic Investigation of the Incorporation of Anti-Hepatitis B Nucleotide Analogues Catalyzed by Noncanonical Human DNA Polymerases

“…As noted previously, PMPA-DP is a poor substrate for Pols κ, ι, and Rev1. 31 The kinetic parameters could only be measured for Pol κ because PMPA-DP incorporation was too inefficient to be observed with Pol ι and Rev1.…”

Insights into the Molecular Mechanism of Polymerization and Nucleoside Reverse Transcriptase Inhibitor Incorporation by Human PrimPol

“…The most severe NRTI-associated toxicities predominantly manifest in mitochondrial dysfunction (1)(2)(3)(4)(5)(6) and are attributed primarily to incorporation by the human mitochondrial DNA polymerase ␥ (mtDNA Pol ␥) (7)(8)(9). However, there are observed discrepancies between toxicity and the potential to inhibit mtDNA Pol ␥, suggesting alternative mechanisms and evaluation of additional host cell polymerases as potential perpetrators of antiviral toxicity (10,11).…”

“…All FDA-approved NRTIs are nucleoside analogs that lack a 3=-hydroxyl moiety to terminate DNA chain extension upon incorporation by RT into the growing proviral DNA. While significant health advances have been achieved with the use of NRTIs, the necessity for lifelong treatment to control HIV infection is limited by NRTI-associated toxicities that arise from virus-versus-host polymerase selectivity wherein NRTIs also serve as substrates for host polymerases (1,2).…”

HIV-1 Polymerase Inhibition by Nucleoside Analogs: Cellular- and Kinetic Parameters of Efficacy, Susceptibility and Resistance Selection

“…The therapeutic window of largely depends on molecular kinetic properties of the respective enzymes with regard to a particular inhibitor [10], [11]. therefore require high specificity for the targeted viral enzyme to allow for a clinical benefit.…”