Conference Reports for NATAP

ICAAC 47th Interscience Conference on Antimicrobial Agents and Chemotherapy Sept 17-20, 2007 Chicago, ILL. Back

Novel 2'-Fluoro Substituted Nucleotide HIV Reverse Transcriptase Inhibitor GS-9148 Exhibits Low Potential for Mitochondrial Toxicity In Vitro       Reported by Jules Levin ICAAC Sept 17-20, 2007, Chicago   G Laflamme, D Grant, C Boojamra, L Zhang, H Hui, J Chen, R Mackman, A Ray, and T Cihlar Gilead Sciences, Inc., Foster City, CA   INTRODUCTION Mitochondrial toxicity is an important etiological factor associated with various adverse symptoms observed in patients treated with some nucleoside HIV reverse transcriptase inhibitors (NRTIs)1,2.   GS-9148 (Fig. 1) is a novel nucleotide HIV RT inhibitor with in vitro antiretroviral activity against both wild-type and NRTI-resistant strains (M184V, K65R, L74V, multiple TAMs)3. It has been rationally designed to minimize the mitochondrial toxicity of its structural precursor d4AP4 by incorporating a 2'-fl uoro (2'-F) substitution into its 2',3'-dideoxy-2', 3'-didehydro-ribose ring.   Here we report on the in vitro mitochondrial toxicity profile of GS-9148 and its orally bioavailable prodrug GS-9131 (Fig. 1) that is currently undergoing clinical evaluation.   AUTHOR CONCLUSIONS GS-9148 was rationally designed to minimize the mitochondrial toxicity of its precursor d4AP   Similar to previously characterized 2'-F-2',3'-dideoxynucleosides9, the substitution of d4AP with 2'-F decreased the inhibiting potential towards DNA pol _, reducing the potential for mitochondrial toxicity   Unlike d4AP and some marketed NRTIs (ddI, d4T, ddC), neither GS-9148 nor its prodrugs deplete mtDNA in HepG2 liver cells   A minimal effect of GS-9148 on the production of lactate by HepG2 cells was observed. In contrast, treatment with ddI increased lactate production   The present in vitro study suggests that similar to tenofovir, GS-9148 and its prodrug GS-9131 have low potential for mitochondrial toxicity   METHODS Composer homology modeling module in Sybyl v.7.3 was used to optimize the sequence alignment and 3D model of human DNA polymerase _ based on the X-ray structure of T7 DNA polymerase5.   The steady-state inhibition of DNA polymerase _ by active di-/triphosphate metabolites was characterized using the incorporation assay with [3H]dATP and activated calf-thymus DNA6.   Relative levels of mitochondrial DNA (mtDNA) in HepG2 cells treated with tested compounds were determined by sequential DNA hybridization7 using 32P labeled cytochrome c and _-actin DNA probes and/or by quantitative PCR using delta-delta CT method8 for cytochrome b and _ -actin gene fragment amplification.   HepG2 cells were incubated with tested compounds for 14 and 21 days. The relative levels of mtDNA and production of lactate were determined by DNA hybridization and a spectrophotometric quantification, respectively. Results are means ± SD from a representative experiment performed in duplicate.