9 research outputs found
Charge-Based Inhibitors of Amylin Fibrillization and Toxicity
To test the hypothesis that electrostatic repulsion is an important force opposing amyloid fibril assembly, we designed peptides that substitute strings of positively or negatively charged residues into the sequence of the amyloidogenic hormone amylin, which contributes to type 2 diabetes pathology. Arg-1 and Arg-2 substitute four positively charged arginines for segments that in structural models of amylin fibrils form the end of strand β1 and the beginning of strand β2, respectively. Mem-T substitutes negatively charged aspartates for the peptide segment with the largest avidity for membranes. All three charge-loaded peptides fibrillize poorly on their own and inhibit fibril elongation of WT-amylin at physiological ionic strength. The inhibition of WT-amylin fibril elongation rates is salt-dependent indicating that the analogs act through electrostatic interactions. Arg-1 protects against WT-amylin cytotoxicity towards a MIN6 mouse model of pancreatic β-cells, and Arg-2 protects at higher concentrations, whereas Mem-T has no effect. The most effective variant, Arg-1, inhibits WT-amylin fibril elongation rates with an IC50 of ~1 µM and cytotoxicity with an IC50 of ~50 µM, comparable to other types of fibrillization inhibitors reported in the literature. Taken together, these results suggest that electrostatic interactions can be exploited to develop new types of inhibitors of amyloid fibrillization and toxicity
NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products
Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (DM) of 3.3. The 2D 1H-13C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1H, 15 ppb for 13C and 98% peaks with equivalent peak height. Finally, 2D 1H-15N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics
pH Dependence of Amylin Fibrillization
In type 2 diabetics, the hormone
amylin misfolds into amyloid plaques
implicated in the destruction of the pancreatic β-cells that
make insulin and amylin. The aggregative misfolding of amylin is pH-dependent,
and exposure of the hormone to acidic and basic environments could
be physiologically important. Amylin has two ionizable residues between
pH 3 and 9: the α-amino group and His18. Our approach to measuring
the p<i>K</i><sub>a</sub> values for these sites has been
to look at the pH dependence of fibrillization in amylin variants
that have only one of the two groups. The α-amino group at the
unstructured N-terminus of amylin has a p<i>K</i><sub>a</sub> near 8.0, similar to the value in random coil models. By contrast,
His18, which is involved in the intermolecular β-sheet structure
of the fibrils, has a p<i>K</i><sub>a</sub> that is lowered
to 5.0 in the fibrils compared to the random coil value of 6.5. The
lowered p<i>K</i><sub>a</sub> of His18 is due to the hydrophobic
environment of the residue, and electrostatic repulsion between positively
charged His18 residues on neighboring amylin molecules in the fibril.
His18 acts as an electrostatic switch inhibiting fibrillization in
its charged state. The presence of a charged side chain at position
18 also affects fibril morphology and lowers amylin cytotoxicity toward
a MIN6 mouse model of pancreatic β-cells. In addition to the
two expected p<i>K</i><sub>a</sub> values, we detected an
apparent p<i>K</i><sub>a</sub> of ∼4.0 for the amylin-derived
peptide NAc-SNNÂFÂGÂAÂILSS-NH<sub>2</sub>, which
has no titratable groups. This p<i>K</i><sub>a</sub> is
due to the pH-induced ionization of the dye thioflavin T. By using
alternative methods to follow fibrillization such as the dye Nile
Red or turbidimetry, we were able to distinguish between the titration
of the dye and groups on the peptide. Large differences in reaction
kinetics were observed between the different methods at acidic pH,
because of charges on the ThT dye, which hinder fibril formation much
like the charges on the protein
Application of Ultra-Centrifugation and Bench-Top 19F NMR for Measuring Drug Phase Partitioning for the Ophthalmic Oil-in-Water Emulsion Products
Mechanism of Amylin Fibrillization Enhancement by Heparin*
We characterized the interaction of amylin with heparin fragments of defined length, which model the glycosaminoglycan chains associated with amyloid deposits found in type 2 diabetes. Binding of heparin fragments to the positively charged N-terminal half of monomeric amylin depends on the concentration of negatively charged saccharides but is independent of oligosaccharide length. By contrast, amylin fibrillogenesis has a sigmoidal dependence on heparin fragment length, with an enhancement observed for oligosaccharides longer than four monomers and a leveling off of effects beyond 12 monomers. The length dependence suggests that the negatively charged helical structure of heparin electrostatically complements the positively charged surface of the fibrillar amylin cross-β structure. Fluorescence resonance energy transfer and total internal reflection fluorescence microscopy experiments indicate that heparin associates with amylin fibrils, rather than enhancing fibrillogenesis catalytically. Short heparin fragments containing two- or eight-saccharide monomers protect against amylin cytotoxicity toward a MIN6 mouse cell model of pancreatic β-cells
Dynamic α-Helix Structure of Micelle-bound Human Amylin*
Amylin is an endocrine hormone that regulates metabolism. In patients
afflicted with type 2 diabetes, amylin is found in fibrillar deposits in the
pancreas. Membranes are thought to facilitate the aggregation of amylin, and
membrane-bound oligomers may be responsible for the islet β-cell toxicity
that develops during type 2 diabetes. To better understand the structural
basis for the interactions between amylin and membranes, we determined the NMR
structure of human amylin bound to SDS micelles. The first four residues in
the structure are constrained to form a hairpin loop by the single disulfide
bond in amylin. The last nine residues near the C terminus are unfolded. The
core of the structure is an α-helix that runs from about residues
5–28. A distortion or kink near residues 18–22 introduces pliancy
in the angle between the N- and C-terminal segments of the α-helix.
Mobility, as determined by 15N relaxation experiments, increases
from the N to the C terminus and is strongly correlated with the accessibility
of the polypeptide to spin probes in the solution phase. The spin probe data
suggest that the segment between residues 5 and 17 is positioned within the
hydrophobic lipid environment, whereas the amyloidogenic segment between
residues 20 and 29 is at the interface between the lipid and solvent. This
orientation may direct the aggregation of amylin on membranes, whereas
coupling between the two segments may mediate the transition to a toxic
structure