Leptin is a four-helix bundle: secondary structure by NMR

AbstractLeptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N -labeled) have been assigned and its secondary structure reveals it to be a four-helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short-helix cytokine family. A three-dimensional model was built verifying the mechanical consistency of the identified elements with a short-helix cytokine core

Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase

The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of ≈22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data

Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319.

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7-14 days resulted in a 25-50% lowering of plasma glucose coupled with a 10-30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans

Treatment of DIO mice with either FGF21 or LY2405319 improves metabolic dysfunction.

<p>Male DIO mice were treated with FGF21 or LY2405319 at various concentrations, as indicated, by constant infusion for 14 days. Changes in body weight (A), fat mass (B) cumulative food intake (C), and blood glucose (D) were determined for the duration of treatment. Values are reported as mean ± SEM. (*), p<0.01 as compared to vehicle.</p

Summary of <i>in vitro</i> activities of FGF21 and LY2405319.

<p>CI: 95% confidence interval;</p>*<p><i>P</i>≤0.05.</p

LY2405319 bioactivity is comparable to wild type FGF21.

<p>LY2405319 and FGF21 bioactivities were compared in mouse 3T3-L1/Klb fibroblasts (A) and differentiated mouse 3T3-L1 adipocytes (B) by monitoring 2-Deoxy-D-[U-¹⁴C]-glucose accumulation and in human HepG2 hepatoma cells by examining relative expression of GLUT1 mRNA (C) after incubation for 3 h or 1 h, respectively, with various concentrations of FGF21 (black squares) or LY2405319 (red triangles). Mean values ± SEM are presented as fold relative to treatment with vehicle alone.</p