11 research outputs found
Oligomerization of a Glucagon-like Peptide 1 Analog: Bridging Experiment and Simulations
AbstractThe glucagon-like peptide 1 (GLP-1) analog, liraglutide, is a GLP-1 agonist and is used in the treatment of type-2 diabetes mellitus and obesity. From a pharmaceutical perspective, it is important to know the oligomerization state of liraglutide with respect to stability. Compared to GLP-1, liraglutide has an added fatty acid (FA) moiety that causes oligomerization of liraglutide as suggested by small-angle x-ray scattering (SAXS) and multiangle static light scattering (MALS) results. SAXS data suggested a global shape of a hollow elliptical cylinder of size hexa-, hepta-, or octamer, whereas MALS data indicate a hexamer. To elaborate further on the stability of these oligomers and the role of the FA chains, a series of molecular-dynamics simulations were carried out on 11 different hexa-, hepta-, and octameric systems. Our results indicate that interactions of the fatty acid chains contribute noticeably to the stabilization. The simulation results indicate that the heptamer with paired FA chains is the most stable oligomer when compared to the 10 other investigated structures. Theoretical SAXS curves extracted from the simulations qualitatively agree with the experimentally determined SAXS curves supporting the view that liraglutide forms heptamers in solution. In agreement with the SAXS data, the heptamer forms a water-filled oligomer of elliptical cylindrical shape
Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life
Glucagon-like peptide 1 (GLP-1) is
a small incretin hormone stimulated
by food intake, resulting in an amplification of the insulin response.
Though GLP-1 is interesting as a drug candidate for the treatment
of type 2 diabetes mellitus, its short plasma half-life of <3 min
limits its clinical use. A strategy for extending the half-life of
GLP-1 utilizes the long half-life of human serum albumin (HSA) by
combining the two via chemical conjugation or genetic fusion. HSA
has a plasma half-life of around 21 days because of its interaction
with the neonatal Fc receptor (FcRn) expressed in endothelial cells
of blood vessels, which rescues circulating HSA from lysosomal degradation.
We have conjugated GLP-1 to C34 of native sequence recombinant HSA
(rHSA) and two rHSA variants, one with increased and one with decreased
binding affinity for human FcRn. We have investigated the impact of
conjugation on FcRn binding affinities, GLP-1 potency, and pharmacokinetics,
combined with the solution structure of the rHSA variants and GLP-1âalbumin
conjugates. The solution structures, determined by small-angle X-ray
scattering, show the GLP-1 pointing away from the surface of rHSA.
Combining the solution structures with the available structural information
about the FcRn and GLP-1 receptor obtained from X-ray crystallography,
we can explain the observed <i>in vitro</i> and <i>in vivo</i> behavior. We conclude that the conjugation of GLP-1
to rHSA does not affect the interaction between rHSA and FcRn, while
the observed decrease in the potency of GLP-1 can be explained by
a steric hindrance of binding of GLP-1 to its receptor
Investigations of albumin-insulin detemir complexes using molecular dynamics simulations and free energy calculations
Insulin detemir is a lipidated insulin analogue that obtains a half-life extension by oligomerization and reversible binding to human serum albumin. In the present study, the complex between a detemir hexamer and albumin is investigated by an integrative approach combining molecular dynamics (MD) simulations, molecular mechanics PoissonâBoltzmann surface area (MM-PBSA) free energy calculations, and dynamic light scattering (DLS) experiments. Recent reported small-angle X-ray scattering data could not unambiguously resolve the exact binding site of detemir on albumin. We therefore applied MD simulations to deduce the binding site and key proteinâprotein interactions. MD simulations were started from initial complex structures based on the SAXS models, and free energies of binding were estimated from the simulations by using the MM-PBSA approach for the different binding positions. The results suggest that the overlapping FA3âFA4 binding site (named FA4) is the most favorable site with a calculated free energy of binding of â28 Âą 6 kcal/mol and a good fit to the reported SAXS data throughout the simulations. Multiple salt bridges, hydrogen bonds, and favorable van der Waals interactions are observed in the binding interface that promote complexation. The binding to FA4 is further supported by DLS competition experiments with the prototypical FA4 ligand, ibuprofen, showing displacement of detemir by ibuprofen. This study provides information on albuminâdetemir binding on a molecular level, which could be utilized in a rational design of future lipidated albumin-binding peptides
Self-Interaction of Human Serum Albumin:A Formulation Perspective
In the present
study, small-angle X-ray scattering (SAXS) and static light scattering
(SLS) have been used to study the solution properties and self-interaction
of recombinant human serum albumin (rHSA) molecules in three pharmaceutically
relevant buffer systems. Measurements are carried out up to high protein
concentrations and as a function of ionic strength by adding sodium
chloride to probe the role of electrostatic interactions. The effective
structure factors (Seff) as a function
of the scattering vector magnitude q have been extracted
from the scattering profiles and fit to the solution of the OrnsteinâZernike
equation using a screened Yukawa potential to describe the double-layer
force. Although only a limited q range is used, accurate
fits required including an electrostatic repulsion element in the
model at low ionic strength, while only a hard sphere model with a
tunable diameter is necessary for fitting to high-ionic-strength data.
The fit values of net charge agree with available data from potentiometric
titrations. Osmotic compressibility data obtained by extrapolating
the SAXS profiles or directly from SLS measurements has been fit to
a 10-term virial expansion for hard spheres and an equation of state
for hard biaxial ellipsoids. We show that modeling rHSA as an ellipsoid,
rather than a sphere, provides a much more accurate fit for the thermodynamic
data over the entire concentration range. Osmotic virial coefficient
data, derived at low protein concentration, can be used to parameterize
the model for predicting the behavior up to concentrations as high
as 450 g/L. The findings are especially important for the biopharmaceutical
sector, which require approaches for predicting concentrated protein
solution behavior using minimal sample consumption
Small-Angle X-ray Scattering Data in Combination with RosettaDock Improves the Docking Energy Landscape
We
have performed a benchmark to evaluate the relative success
of using small-angle X-ray scattering (SAXS) data as constraints (hereafter
termed SAXS<sub>constrain</sub>) in the RosettaDock protocol (hereafter
termed RosettaDock<sub>SAXS</sub>). For this purpose, we have chosen
38 protein complex structures, calculated the theoretical SAXS data
for the protein complexes using the program CRYSOL, and then used
the SAXS data as constraints. We further considered a few examples
where crystal structures and experimental SAXS data are available.
SAXS<sub>constrain</sub> were added to the protocol in the initial,
low-resolution docking step, allowing fast rejection of complexes
that violate the shape restraints imposed by the SAXS data. Our results
indicate that the implementation of SAXS<sub>constrain</sub> in general
reduces the sampling space of possible proteinâprotein complexes
significantly and can indeed increase the probability of finding near-native
protein complexes. The methodology used is based on rigid-body docking
and works for cases where no or minor conformational changes occur
upon binding of the docking partner. In a wider perspective, the strength
of RosettaDock<sub>SAXS</sub> lies in the combination of low-resolution
structural information on protein complexes in solution from SAXS
experiments with proteinâprotein interaction energies obtained
from RosettaDock, which will allow the prediction of unknown three-dimensional
atomic structures of proteinâprotein complexes