A transient kinetic approach to investigate nucleoside inhibitors of mitochondrial DNA polymerase gamma

Abstract

Nucleoside analogs play an essential role in treating human immunodeficiency virus (HIV) infection since the beginning of the AIDS epidemic and work by inhibition of HIV-1 reverse transcriptase (RT), a viral polymerase essential for DNA replication. Today, over 90% of all regimens for HIV treatment contain at least one nucleoside. Long-term use of nucleoside analogs has been associated with adverse effects including mitochondrial toxicity due to inhibition of the mitochondrial polymerase, DNA polymerase gamma (mtDNA pol gamma). In this review, we describe our efforts to delineate the molecular mechanism of nucleoside inhibition of HIV-1 RT and mtDNA pol gamma based upon a transient kinetic approach using rapid chemical quench methodology. Using transient kinetic methods, the maximum rate of polymerization (k(pol)), the dissociation constant for the ground state binding (K(d)), and the incorporation efficiency (k(pol)/K(d)) can be determined for the nucleoside analogs and their natural substrates. This analysis allowed us to develop an understanding of the structure activity relationships that allow correlation between the structural and stereochemical features of the nucleoside analog drugs with their mechanistic behavior toward the viral polymerase, RT, and the host cell polymerase, mtDNA pol gamma. An in-depth understanding of the mechanisms of inhibition of these enzymes is imperative in overcoming problems associated with toxicity.

Figure 1

Comparison of Steady-state and Transient Kinetics

Figure 2

Schematic of a pulsed-quench-flow apparatus [21, 23].

Figure 3

Schematic of stopped-flow fluorescence apparatus.

Scheme 1

Scheme 2

Kinetic pathway for Pol γ polymerization. Schematic demonstrating rate constants for dNTP incorporation and removal by mtDNA Pol γ as determined by pre-steady state kinetic analysis. Figure adapted from [38].

Figure 4

Structures of clinically relevant antiviral and anticancer nucleoside analogs