15th International HIV Drug Resistance Workshop 13-17 June 2006, Sitges, Spain

THE IMPACT OF THE N43D RESISTANCE MUTATION ON ENFUVIRTIDE SENSITIVITY AND SIX-HELIX BUNDLE STRUCTURE IN COMBINATION WITH THE E137K POLYMORPHISM

Antivir Ther. 2006, 11:S55 (abstract no. 47)

X Bai, KL Wilson, JE Seedorff, D Ahrens, J Green, DK Davison, L Jin, SA Stanfield-Oakley, S Mosier, T Melby, ML Greenberg and JJ Dwyer
Trimeris, Inc., Morrisville, NC, USA

BACKGROUND: The fusion inhibitor (FI) Enfuvirtide (ENF) acts by binding to the gp41 heptad repeat 1 (HR1) region and preventing its interaction with the viral HR2 region. Treatment emergent resistance to ENF has been mapped to residues within HR1 and these mutations decrease susceptibility to ENF and substantially impair viral fitness. Patients with documented ENF mutations that remain on drug often maintain CD4⁺ cell benefits, perhaps related to reductions in viral fitness. N43D is a common ENF resistance mutation and we have observed that an existing E137K polymorphism increases the likelihood of developing N43D. We introduced N43D and E137K into peptide models and determined the structural effect of these mutations, separately and in combination, on the six-helix bundle by circular dichroism and X-ray crystallography.

METHODS: Site-directed mutagenesis (SDM) was used to construct viral envelopes and viruses bearing the N43D, E137K and N43D/E137K mutations. Effects on ENF susceptibility were determined in a cMAGI and pseudotype reporter virus assay. Stability of the HR1/HR2 bundle was assessed using circular dichroism and X-ray structures of mutant HR1/HR2 complexes were solved using molecular replacement.

RESULTS: At virological failure (VF) in the TORO trials, 22 patients (7.7%) had the N43D substitution; of these, 72.7% and 86.4% also had E137K at baseline and VF, respectively compared to 10.6% and 11.7%, respectively of all other patients). The N43D mutation conferred decreases in ENF susceptibility in vitro of 1.6-51 fold in different genetic backgrounds and assay systems, a decrease in bundle stability, and significant changes in the structure of the HR2. The presence of E137K partially restored the bundle stability lost with N43D, however structural defects in HR2 remained as did decreases in ENF susceptibility. We show that enhanced bundle stability coincides with formation of an ion pair between N43D and E137K.

CONCLUSIONS: The impact of N43D and E137K on the stability and structure of the six-helix bundle provide a mechanistic basis for the effect on ENF susceptibility and structural insights into potential compensatory mutations. These data also suggest a possible structural basis by which ENF resistance mutations may cause defects in viral fitness.

2006-06-13
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