(405f) De Novo Protein Design of Agonists and Antagonists of the C3a Receptor: Theoretical Predictions and Experimental Validation

Protein design, also known as the
inverse folding problem, seeks the amino acid sequence that will fold into a
given 3-dimensional template. The protein design problem exhibits degeneracy
due to the fact that many amino acid sequences fold into a given template. It
is therefore important to examine all the possible sequences for a given
template and rank them based upon specific properties that are being designed
(activity, specificity, etc.).

A de novo design framework with a ranking metric
based on fold specificities was applied to the design of C3a receptor (C3aR)
agonists and antagonists. The
design is based upon the structure of C3a, which activates C3aR. C3a is a 77-residue peptide that mediates the pro-inflammatory
activities in the human complement system and possibly has opposing
immunological roles in some cellular systems (34). Improper activation of the
complement system can cause tissue injury in various pathological conditions
and contributes to several immune diseases, including stroke, heart attack,
Alzheimer's disease, asthma, rheumatoid arthritis, and rejection of
xenotransplantation. The
crystal structure resolved by Huber et al. [1], as well as flexible template
structures generated by MD simulations with explicit solvation via water
molecules were employed as the design template.

The framework consists of two
stages: a sequence selection stage and a validation stage. The sequence
selection stage produces a rank-ordered list of amino acid sequences with the
lowest energies by solving an integer programming sequence selection model [2].
The sequence selection model incorporates backbone flexibility into the design
process by utilizing the set of structures obtained from the MD simulations
with implicit solvation and with explicit water molecules. In doing so, the
pairwise energy between two residues can take on a range of values depending
upon the range of distances obtained from the structures.

The second stage re-ranks the
sequences from stage one using either a fold specificity or an approximate
binding affinity [3-4]. Since structural information of the C3a:C3aR complex
was unknown, only fold specificity calculations could be employed. Fold specificity
measures how likely a given sequence will fold into the design template
structure [5]. Thus the design was driven by the hypothesis that structure
implies function, and novel sequences of C3a that adopt the C3a fold are
potential candidates for C3aR agonists or antagonists.

Hundreds of sequences were
generated and ranked using the de novo protein design framework. The top seven
sequences were selected for synthesis and experimental validation. Of the seven designed peptides tested,
two were prominent agonists while two others were prominent antagonists. The
agonists showed a up to a five-fold improvement in EC50 compared with the previously
discovered "superagonist" by Ember et al. [6], with EC50s of 33.4 and 66.16 nM.
The antagonists were also very potent, with IC50s of 26.13 and 63.04 nM.

This
work highlights the success of the protein design framework. In this case,
structural information on the binding site was unknown, so the design was
driven by fold specificities. Since structure often implies function, this is a
good metric to use when structural binding data is unavailable.

[1] Huber,
R., H. Scholze, E. Paques, and J. Deisenhofer, 1980. Crystal Structure Analysis
and Molecular Model of Human C3a Anaphylatoxin. Hoppe Seylers Zeitschrifft fuer
Physiologische Chemie 361:1389–1399.

[2] H. K. Fung, M. S. Taylor, and C. A. Floudas. Novel Formulations for
the sequence selection problem in de novo protein design with flexible
templates. Optim. Methods & Software,
22:51-71, 2007.

[3] Bellows,
M. L., H. K. Fung, C. A. Floudas, A. Lopez de Victoria, and D. Morikis, 2010.
New Compstatin Variants Through Two De Novo Protein Design Frameworks. Biophys
J 98:2337–2346.

[4] Bellows,
M. L., M. S. Taylor, P. A. Cole, L. Shen, R. F. Siliciano, H. K. Fung, and C.
A. Floudas, 2010. Discovery of entry inhibitors for HIV-1 via a new de novo
protein design framework. Biophys J 99:3445–3453.

[5] Fung,
H. K., C. A. Floudas, M. S. Taylor, L. Zhang, and D. Morikis, 2008. Toward
Full-Sequence De Novo Protein Design with Flexible Templates for Human
Beta-Defensin-2. Biophys. J. 94:584–599.

[6] Ember,
J., N. Johansen, and T. Hugli, 1991. Designing Synthetic Superagonists of C3a
Anaphylatoxin. Biochem. 30:3603–3612.