17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

STRUCTURAL BASIS FOR K65R FUNCTION: TENOFOVIR RESISTANCE, REDUCED NUCLEOTIDE INCORPORATION AND EXCISION ANTAGONISM

Antivir Ther. 2008; 13(Suppl. 3):A44 (abstract no. 39)

K Das^1,2, R Bandwar^1,2, KL White³, JY Feng³, SG Sarafianos^1,2, S Tuske^1,2, X Tu^1,2, AD Clark, Jr^1,2, PL Boyer⁴, BL Gaffney², RA Jones², MD Miller³, SH Hughes⁴ and E Arnold^1,2
1Center for Advanced Biotechnology and Medicine (CABM), Rutgers University, Piscataway, NJ, USA; ²Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA; ³Gilead Sciences, Inc., Foster City, CA, USA; ⁴HIV Drug Resistance Program, NCI-Frederick, MD, USA

BACKGROUND: K65R is a primary reverse transcriptase (RT) mutation selected in HIV-infected patients taking antiretroviral regimens containing tenofovir (TFV) or other nucleoside reverse transcriptase inhibitors. The mutant RT has a reduced rate of dNTP incorporation and significantly decreased excision of zidovudine (AZT).

METHODS: Using X-ray crystallography, we determined the crystal structures of K65R mutant RT with double-stranded DNA (27:21-mer) in complexes with tenofovir diphosphate (TFV-DP) at 3.0 Å and dATP at 3.5 Å resolution, respectively.

RESULTS: The crystal structures reveal the molecular mechanism of resistance by the K65R mutation, which is distinct from the steric hindrance mechanism caused by M184V and the ATP-mediated excision mechanism used by thymidine analogue mutations (TAMs). The K65R mutation forms a molecular platform through the interactions of R65 with the conserved amino acid residue R72. The platform can restrict the conformational adaptability of both amino acid residues that is required for DNA polymerization by RT, which can explain the lower rate of nucleotide incorporation by the K65R mutant as well as its decreased excision rate. The amino acid residues R72 and R65 have alternative rotameric conformations in the TFV-DP-bound structure of K65R RT when compared with the dATP-bound structure, which is apparently responsible for discriminating TFV-DP from dATP and allowing for resistance.

DISCUSSION: The R65+R72 platform interfaces with M184 and TAMs at two distinct sites. If the K65R and M184V mutations co-exist, the side chains of R72 and V184 would stack like walls on either side of the ribose ring of a bound dNTP, which further stabilizes the pocket and may explain the lower rate of dNTP incorporation and excision by the double-mutant that results in partial TFV resensitization. The K70R TAM, if it were to coemerge with K65R, would add additional restrictions to the R65+R72 platform that might be detrimental for dNTP binding, ATP binding (for excision) and/or nucleotide incorporation, which may explain the rare appearance of K65R+K70R mutations. Other TAMs, such as T215Y, which enhance binding of ATP for excision, are antagonistic with K65R because the R65+R72 platform would hinder the proper positioning of the phosphates of ATP, thus decreasing excision and leading to no enhancement of resistance.

2008-06-10
39

Copyright © 2008 - International Medical Press Ltd.. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the International Medical Press Ltd. 2-4 Idol Lane, London EC3R 5DD UK.