Modulation of Oxidative Phosphorylation with IM156 Attenuates Mitochondrial Metabolic Reprogramming and Inhibits Pulmonary Fibrosis.

Metabolic reprogramming of the myofibroblast plays a fundamental role in the pathogenesis of fibrosing interstitial lung diseases. Here, we characterized the in vitro and in vivo metabolic and antifibrotic effects of IM156, an oxidative phosphorylation (OXPHOS) modulator that acts by inhibiting protein complex 1. In vitro, IM156 inhibited transforming growth factor ß (TGFß)-dependent increases in mitochondrial oxygen consumption rate and expression of myofibroblast markers in human pulmonary fibroblasts without altering cell viability or adding to TGFß-induced increases in the extracellular acidification rate. IM156 significantly increased cellular AMP-activated protein kinase (AMPK) phosphorylation and was 60-fold more potent than metformin. In vivo, chronic oral administration of IM156 was highly distributed to major peripheral organs (i.e., lung, liver, kidney, heart) and had significant dose-related effects on the plasma metabolome consistent with OXPHOS modulation and AMPK activation. IM156 increased glycolysis, lipolysis, ß-oxidation, and amino acids and decreased free fatty acids, tricarboxylic acid cycle activity, and protein synthesis. In the murine bleomycin model of pulmonary fibrosis, daily oral administration of IM156, administered 7 days after lung injury, attenuated body/lung weight changes and reduced lung fibrosis and inflammatory cell infiltration. The plasma exposures of IM156 were comparable to well tolerated doses in human studies. In conclusion, the metabolic and antifibrotic effects of IM156 suggest that OXPHOS modulation can attenuate myofibroblast metabolic reprogramming and support testing IM156 as a therapy for idiopathic pulmonary fibrosis and other fibrotic diseases. SIGNIFICANCE STATEMENT: Fibrosing interstitial lung diseases have a poor prognosis, and current antifibrotic treatments have significant limitations. This study demonstrates that attenuation of fibrogenic metabolic remodeling, by modulation of oxidative phosphorylation with IM156, prevents myofibroblast phenotype/collagen deposition and is a potentially effective and translational antifibrotic strategy.

ROSETTA-informed design of structurally stabilized cyclic anti-amyloid peptides.

ß-amyloid oligomers are thought to be the most toxic species formed en route to fibril deposition in Alzheimer's disease. Transthyretin is a natural sequestering agent of ß-amyloid oligomers: the binding site to ß-amyloid has been traced to strands G/H of the inner ß-sheet of transthyretin. A linear peptide, with the same primary sequence as the ß-amyloid binding domain on transthyretin, was moderately effective at inhibiting ß-amyloid fibril growth. Insertion of a ß-turn template and cyclization greatly increased stability against proteolysis and improved efficacy as an amyloid inhibitor. However, the cyclic peptide still contained a significant amount of disorder. Using the Simple Cyclic Peptide Application within ROSETTA as an in silico predictor of cyclic peptide conformation and stability, we investigated putative structural enhancements, including stabilization by disulfide linkages and insertion of a second ß-turn template. Several candidates were synthesized and tested for secondary structure and ability to inhibit ß-amyloid aggregation. The results demonstrate that cyclization, ß-sheet structure and conformational homogeneity are all preferable design features, whereas disulfide bond formation across the two ß-strands is not preferable.

Retinol-Binding Protein Interferes with Transthyretin-Mediated ß-Amyloid Aggregation Inhibition.

ß-Amyloid (Aß) aggregation is causally linked to Alzheimer's disease. On the basis of in vitro and transgenic animal studies, transthyretin (TTR) is hypothesized to provide neuroprotection against Aß toxicity by binding to Aß and inhibiting its aggregation. TTR is a homotetrameric protein that circulates in blood and cerebrospinal fluid; its normal physiological role is as a carrier for thyroxine and retinol-binding protein (RBP). RBP forms a complex with retinol, and the holoprotein (hRBP) binds with high affinity to TTR. In this study, the role of TTR ligands in TTR-mediated inhibition of Aß aggregation was investigated. hRBP strongly reduced the ability of TTR to inhibit Aß aggregation. The effect was not due to competition between Aß and hRBP for binding to TTR, as Aß bound equally well to TTR-hRBP complexes and TTR. hRBP is known to stabilize the TTR tetrameric structure. We show that Aß partially destabilizes TTR and that hRBP counteracts this destabilization. Taken together, our results support a mechanism wherein TTR-mediated inhibition of Aß aggregation requires not only TTR-Aß binding but also destabilization of TTR quaternary structure.

Transthyretin variants with improved inhibition of ß-amyloid aggregation.

Aggregation of ß-amyloid (Aß) is widely believed to cause neuronal dysfunction in Alzheimer's disease. Transthyretin (TTR) binds to Aß and inhibits its aggregation and neurotoxicity. TTR is a homotetrameric protein, with each monomer containing a short α-helix and two anti-parallel ß-sheets. Dimers pack into tetramers to form a hydrophobic cavity. Here we report the discovery of a TTR mutant, N98A, that was more effective at inhibiting Aß aggregation than wild-type (WT) TTR, although N98A and WT bound Aß equally. The N98A mutation is located on a flexible loop distant from the putative Aß-binding sites and does not alter secondary and tertiary structures nor prevent correct assembly into tetramers. Under non-physiological conditions, N98A tetramers were kinetically and thermodynamically less stable than WT, suggesting a difference in the tetramer folded structure. In vivo, the lone cysteine in TTR is frequently modified by S-cysteinylation or S-sulfonation. Like the N98A mutation, S-cysteinylation of TTR modestly decreased tetramer stability and increased TTR's effectiveness at inhibiting Aß aggregation. Collectively, these data indicate that a subtle change in TTR tetramer structure measurably increases TTR's ability to inhibit Aß aggregation.