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An elevated intracellular NADH:NAD+ ratio, or 'reductive stress', has been associated with multiple diseases, including disorders of the mitochondrial electron transport chain. As the intracellular NADH:NAD+ ratio can be in near equilibrium with the circulating lactate:pyruvate ratio, we hypothesized that reductive stress could be alleviated by oxidizing extracellular lactate to pyruvate. We engineered LOXCAT, a fusion of bacterial lactate oxidase (LOX) and catalase (CAT), which irreversibly converts lactate and oxygen to pyruvate and water. Addition of purified LOXCAT to the medium of cultured human cells with a defective electron transport chain decreased the extracellular lactate:pyruvate ratio, normalized the intracellular NADH:NAD+ ratio, upregulated glycolytic ATP production and restored cellular proliferation. In mice, tail-vein-injected LOXCAT lowered the circulating lactate:pyruvate ratio, blunted a metformin-induced rise in blood lactate:pyruvate ratio and improved NADH:NAD+ balance in the heart and brain. Our study lays the groundwork for a class of injectable therapeutic enzymes that alleviates intracellular redox imbalances by directly targeting circulating redox-coupled metabolites.

Oligomers composed of β(3)-amino acid residues and a mixture of α- and β(3)-residues have emerged as proteolytically stable structural mimics of α-helices. An attractive feature of these oligomers is that they adopt defined conformations in short sequences. In this manuscript, we evaluate the impact of β(3)-residues as compared to their α-amino acid analogs in prenucleated helices. Our hydrogen-deuterium exchange results suggest that heterogeneous sequences composed of "αααβ" repeats are conformationally more rigid than the corresponding homogeneous α-peptide helices, with the macrocycle templating the helical conformation having a significant influence.

α-Helices constitute the largest class of protein secondary structures and play a major role in mediating protein−protein interactions. Development of stable mimics of short α-helices would be invaluable for inhibition of protein−protein interactions. This Account describes our efforts in developing a general approach for constraining short peptides in α-helical conformations by a main-chain hydrogen bond surrogate (HBS) strategy. The HBS α-helices feature a carbon−carbon bond derived from a ring-closing metathesis reaction in place of an N-terminal intramolecular hydrogen bond between the peptide i and i + 4 residues. Our approach is centered on the helix−coil transition theory in peptides, which suggests that the energetically demanding organization of three consecutive amino acids into the helical orientation inherently limits the stability of short α-helices. The HBS method affords preorganized α-turns to overcome this intrinsic nucleation barrier and initiate helix formation. The HBS approach is an a...

Alpha-helices constitute the largest class of protein secondary structures and play a major role in mediating protein-protein interactions. Development of stable mimics of short alpha-helices would be invaluable for inhibition of protein-protein interactions. This Account describes our efforts in developing a general approach for constraining short peptides in alpha-helical conformations by a main-chain hydrogen bond surrogate (HBS) strategy. The HBS alpha-helices feature a carbon-carbon bond derived from a ring-closing metathesis reaction in place of an N-terminal intramolecular hydrogen bond between the peptide i and i + 4 residues. Our approach is centered on the helix-coil transition theory in peptides, which suggests that the energetically demanding organization of three consecutive amino acids into the helical orientation inherently limits the stability of short alpha-helices. The HBS method affords preorganized alpha-turns to overcome this intrinsic nucleation barrier and initiate helix formation. The HBS approach is an attractive strategy for generation of ligands for protein receptors because placement of the cross-link on the inside of the helix does not block solvent-exposed molecular recognition surfaces of the molecule. Our metathesis-based synthetic strategy utilizes standard Fmoc solid phase peptide synthesis methodology, resins, and reagents and provides HBS helices in sufficient amounts for subsequent biophysical and biological analyses. Extensive conformational analysis of HBS alpha-helices with 2D NMR, circular dichroism spectroscopies and X-ray crystallography confirms the alpha-helical structure in these compounds. The crystal structure indicates that all i and i + 4 C=O and NH hydrogen-bonding partners fall within distances and angles expected for a fully hydrogen-bonded alpha-helix. The backbone conformation of HBS alpha-helix in the crystal structure superimposes with an rms difference of 0.75 A onto the backbone conformation of a model alpha-helix. Significantly, the backbone torsion angles for the HBS helix residues fall within the range expected for a canonical alpha-helix. Thermal and chemical denaturation studies suggest that the HBS approach provides exceptionally stable alpha-helices from a variety of short sequences, which retain their helical conformation in aqueous buffers at exceptionally high temperatures. The high degree of thermal stability observed for HBS helices is consistent with the theoretical predictions for a nucleated helix. The HBS approach was devised to afford internally constrained helices so that the molecular recognition surface of the helix and its protein binding properties are not compromised by the constraining moiety. Notably, our preliminary studies illustrate that HBS helices can target their expected protein receptors with high affinity.

We report the design, synthesis, and characterization of a short peptide trapped in a pi-helix configuration. This high-energy conformation was nucleated by a preorganized pi-turn, which was obtained by replacing an N-terminal intramolecular main chain i and i + 5 hydrogen bond with a carbon-carbon bond. Our studies highlight the nucleation parameter as a key factor contributing to the relative instability of the pi-helix and allow us to estimate fundamental helix-coil transition parameters for this conformation.