Characterization of resistance to a potent D-peptide HIV entry inhibitor.
BACKGROUND
PIE12-trimer is a highly potent D-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (> 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket.


RESULTS
Using deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data.


CONCLUSIONS
These data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified.
['Smith|Amanda R|AR|', 'Weinstock|Matthew T|MT|', 'Siglin|Amanda E|AE|', 'Whitby|Frank G|FG|', 'Francis|J Nicholas|JN|', 'Hill|Christopher P|CP|', 'Eckert|Debra M|DM|', 'Root|Michael J|MJ|', 'Kay|Michael S|MS|0000-0003-3186-9684']
D019380:Anti-HIV Agents D002460:Cell Line D024882:Drug Resistance, Viral D015658:HIV Infections D015497:HIV-1 D006801:Humans D009154:Mutation D010455:Peptides D053586:Virus Internalization
D-peptide PIE12-trimer Hydrophobic pocket gp41 Viral passaging Compensatory mutation gp120 Viral fitness HIV entry inhibition Viral resistance mutation