(666c) Predicting the Mitochondrial Toxicity of Nucleoside-Analogue Antiretroviral Drugs Via Stochastic Modeling

Nucleoside-analogue Reverse Transcriptase Inhibitors (NRTIs) are an important class of drugs used for the treatment of HIV infection. Although NRTIs have been effective in controlling viral populations below the detection limit, their prolonged use leads to several medical complications. Clinical and molecular biological evidences suggest that NRTIs cause toxic side-effects by interfering with normal mitochondrial function. Since NRTIs are structurally similar to the natural deoxynucleotides (dNTPs), they competitively inhibit the mitochondrial DNA (mtDNA) replication and transcription processes. The objective of this work is to investigate the individual and synergistic effects of NRTIs on these two processes using a stochastic computational model.

Human DNA polymerase-γ is the enzyme responsible for the replication of mtDNA. Early studies on NRTI-induced toxicity led to the ‘DNA pol-γ hypothesis’, which postulates that the inhibition of pol-γ by NRTIs causes mtDNA depletion and thereby mitochondrial dysfunction. Since the NRTIs lack the 3'-OH group required for the elongation of mtDNA, their incorporation into the strand by pol-γ leads to termination of mtDNA replication. However, recent findings indicate that the inhibition of the human mitochondrial RNA polymerase (POLRMT) is as important as that of pol-γ, if not more (Mallon et al, 2005). POLRMT is the enzyme responsible for the transcription of mtDNA into three unique preliminary RNA strands. The effects of NRTIs and AVRNs (antiviral ribonucleosides) on these two polymerases in different types of cells are crucial for the understanding of clinical toxicity.

The known mechanism of polymerase chain reaction is modeled and simulated using Gillespie's algorithm, a kinetic Monte Carlo method. At each location on the mtDNA strand, the polymerases can perform one of these actions: (1) Addition of the correct nucleotide (2) Addition of an incorrect nucleotide (non-Watson-Crick pair) (3) Addition of an incorrect sugar (4) Addition of a NRTI (5) Addition of an AVRN (6) Excision of the previously added nucleotide (7) Disassociation of the polymerase from the strand (8) Association of the polymerase to the strand. The simulation stops either when a strand is completely replicated or transcribed, or when the polymerase disassociates and fails to re-associate. The occurrence of the latter is more probable when a NRTI or AVRN was added in the previous step and was failed to be removed.

Apart from obtaining a quantitative measure of clinical toxicity, the model is used to determine the sensitivity of drug response to various parameters. The model serves to help experimentalists in identifying key factors and parameters for measurement. In future, the model will be extended to include the mitochondrial translation machinery to predict the under-expression of OXPHOS proteins.