- 12:30 PM

De Novo Protein Design of Complement 5a

Ho Ki Fung and Christodoulos A. Floudas. Department of Chemical Engineering, Princeton University, Engineering Quadrangle, Princeton, NJ 08544

Complement 5a is a 74-residue peptide cleaved from the N-terminal region of the α-chain of complement protein C5. Upon binding of C5a to the C5a receptor, a G protein-coupled receptor, most of the proinflammatory signaling is induced. The level of C5a is regulated by carboxypeptidase, which cleaves the Arg at position 74 and renders the peptide inactive. If the regulation is compromised, excessive prolonged production of C5a will occur which can result in a number of diseases including adult respiratory distress syndrome (ARDS), Alzheimer's disease, cystic fibrosis, rheumatoid arthritis, ischemic heart disease, etc. In view of the crucial role of C5a in regulating the complement cascade, efforts to treat inflammatory diseases have been focused on developing antagonists to the C5a receptor. They are reviewed in refs. [1] and [2] and are based on the C-terminal region of C5a as the design template for designing peptides with variable lengths. So far two potent C5a receptor antagonists have been reported: Me-FKPdChaWr by Merck and a hexapeptidic macrocycle Ac-F[OPdChaWR] by Finch et al.[3]. They have IC50's of 100 nM and 20 nM respectively against a maximum concentration of 100 nM of human polymorphonuclear leukocyte C5a receptors [3].

In this work, we use our de novo protein design framework to design antagonists to C5a receptor. Our framework consists of two stages: (i) in silico sequence selection, and (ii) fold specificity calculation. It has been successfully applied to the full-sequence design of human beta-defensin-2 [4] and the design of the oligomerization interface of G protein-coupled receptors [5]. We split our study of C5a into three different cases. The first case involves the redesign of the cyclic antagonist Ac-F[OPdChaWR] based on its ten NMR structures as the flexible design templates [6]. In the second case we design C-terminal hexapeptides and decapeptides based on the templates of the twenty NMR structures of C5a elucidated by Zhang et al. [7]. The third case deals with the redesign the three binding sites of C5a to C5a receptor proposed by Huber-Lang et al. [8]. Since all design backbone templates have multiple structures, we will employ both the weighed average and distance bin sequence selection models in our design [9], which are developed explicitly to handle backbone flexibility. As far as fold validation is concerned, we are applying a highly computationally efficient method, CYANA, on a full-atomistic scale driven by the AMBER forcefield, together with a local energy minimization package TINKER, for calculating fold specificities of the new sequences to the native fold.

[1] D. Morikis and J.D. Lambris. " Structural Aspects and Design of Low-Molecular-Mass Complement Inhibitors." Biochem. Soc. Trans. 30 (2002): 1026-1036.

[2] D. Ricklin and J.D. Lambris. "Complement-Targeted Therapeutics." Nature Biotechnology 25 (2007): 1265-1275.

[3] A.M. Finch and A.K. Wong and N.J. Paczkowski and S.K. Wadi and D.J. Craik and D.P. Fairlie and S.M. Taylor. "Low-Molecular-Weight Peptidic and Cyclic Antagonists of the Receptor for the Complement Factor C5a." J. Med. Chem. 42 (1999): 1965-1974.

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

[5] M.S. Taylor and H.K. Fung and R. Rajgaria and M. Filizola and H. Weinstein and C.A. Floudas. "Mutations Affecting the Oligomerization Interface of G-Protein-Coupled Receptors Revealed by a Novel De Novo Protein Design Framework." Biophys. J. 94 (2008): 2470-2481.

[6] L. Zhang and B. Mallik and D. Morikis. "Structural Study of Ac-Phe-[Orn-Pro-dCha-Trp-Arg], a Potent C5a Receptor Antagonist, by NMR." unpublished work (2007).

[7] X. Zhang and W. Boyar and M.J. Toth and L. Wennogle and N.C. Gonnella. "Structural Definition of the C5a C Terminus by Two-Dimensional Nuclear Magnetic Resonance Spectroscopy." Proteins 28 (1997): 261-267.

[8] M.S. Huber-Lang and J.V. Sarma and S.R. McGuire and K.T. Lu and V.A. Padgaonkar and E.M. Younkin and R.F. Guo and C.H. Weber and E.R. Zuiderweg and F.S. Zetoune and P.A. Ward. "Structure-Function Relationships of Human C5a and C5aR." J. Immunol. 170 (2003): 6115-6124.

[9] H.K. Fung and 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 (2007): 51-71.