DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

Huntington's disease (HD) is caused by an expansion of a CAG trinucleotide sequence that encodes a polyglutamine tract in the huntingtin (Htt) protein. Expansion of the polyglutamine tract above 35 repeats causes disease, with the age of onset inversely related to the degree of expansion above this number. Growing evidence suggests that mitochondrial function is compromised during HD pathogenesis, but how this occurs is not understood. We examined mitochondrial properties of HeLa cells that expressed green fluorescent protein (GFP)- or FLAG-tagged N-terminal portions of the Htt protein containing either, 17, 28, 74 or 138 polyglutamine repeats. Immunofluorescence staining of cells using antibodies against Tom20, a mitochondrion localized protein, revealed that cells expressing Htt proteins with 74 or 138 polyglutamine repeats were more sensitized to oxidative stress-induced mitochondria fragmentation and had reduced ATP levels compared with cells expressing Htt proteins with 17 or 28 polyglutamine repeats. By measuring changes in fluorescence of a photoactivated GFP protein targeted to mitochondria, we found that cells expressing red fluorescent protein (RFP)-tagged Htt protein containing 74 polyglutamine repeats had mitochondria that displayed reduced movement and fusion than cells expressing RFP-Htt protein with 28 polyglutamine repeats. Overexpression of Drp-1(K38A), a dominant-negative mitochondria-fission mutant, or Mfn2, a protein that promotes mitochondria fusion, suppressed polyglutamine-induced mitochondria fragmentation, the reduction of ATP levels and cell death. In a Caenorhabditis elegans model of HD, we found that reduction of Drp-1 expression by RNA interference rescued the motility defect associated with the expression of Htt proteins with polyglutamine repeats. These results suggest that the increase in cytotoxicity induced by Htt proteins containing expanded polyglutamine tracts is likely mediated, at least in part, by an alteration in normal mitochondrial dynamics, which results in increased mitochondrial fragmentation. Furthermore, our results suggest that it might be possible to reverse polyglutamine-induced cytotoxicity by preventing mitochondrial fragmentation.

Expanded polyglutamine (polyQ) tracts are associated with the induction of protein aggregation and cause cytotoxicity in nine different neurodegenerative disorders. Here, we report that ubiquilin suppresses polyQ-induced protein aggregation and toxicity in cells and in an animal model of Huntington's disease. Overexpression of ubiquilin in HeLa cells and primary neurons reduced aggregation of polyQ-containing proteins and cell death induced by overexpression of a green fluorescent protein (GFP)-huntingtin fusion protein containing 74 polyQ repeats [GFP-Htt(Q74)], in a dose-dependent manner. Moreover, overexpression of ubiquilin suppressed oxidative stress-induced cell death in HeLa cell lines stably expressing GFP-Htt(Q74). In contrast, knockdown of ubiquilin expression in these cell lines was associated with increases in DNA fragmentation, caspase activation, GFP-fusion protein aggregation, and cell death. Caenorhabditis elegans lines expressing GFP-Htt fusion proteins in body wall muscle displayed a polyQ repeat length-dependent decrease in body movement compared with wild-type animals. RNA interference of the C. elegans ubiquilin gene exacerbated the motility defect, whereas overexpression of ubiquilin prevented, and could rescue, loss of worm movement induced by overexpression of GFP-Htt(Q55). These results suggest that ubiquilin might be a novel therapeutic target for treating polyQ diseases.

Loss of ubiquilin or erasin activates ER stress, increases accumulation of polyubiquitinated proteins, and shortens lifespan in worms.