De novo peptide or protein design starts with a flexible three-dimensional protein backbone and involves the search for all amino acid sequences that will fold into such a template ([1]-[3]) and exhibit higher stability/specificity. It is of paramount importance to designing improved inhibitors, engineering proteins with higher stability, conferring novel properties to catalytic sites of enzymes, and speeding up the drug discovery process [5].

Our current de novo protein design framework consists of two stages: (i) in silico sequence selection, and (ii) fold validation. We proved that our O(n²) in silico sequence selection model was superior in computational performance than several other O(n) formulations in literature [4]. Recently, we have devised two new sequence selection models that map sequences onto a highly flexible template with multiple structures [6]. One applied the single structure algorithm on a weighted average of the multiple structures, and the other employed binary distance bin variables to account for each of the structures, but imposed constraints on C^α position pairs (i,k) and (k,p) to eliminate solutions which would otherwise suggest physically impossible structures [6]. Our de novo design methodology incorporates true backbone flexibility because it considers conformations of all possible combinations of continuous dihedral angle and C^α-C^α distance values between bounds, which can be determined from the crystal or solution structures.

Confirmed by experimental data, our framework already proved to be highly successful in redesigning Compstatin, a 13-residue therapeutic peptide that inhibits Complement 3 and is used for treating unregulated complement activation [1]-[2]. In this work, the framework will be extended to the redesign of larger systems of C3a and an HIV-1 gp41 inhibitor. C3a is a 77-residue cleavage component of Complement 3 that is generated during the complement activation cascade. De novo design of C3a was based upon both the X-ray crystal structure elucidated by Huber et al. [7] and our own structures of the protein generated using molecular dynamics simulation [8]. The design was aimed at obtaining an antagonist that binds to the C3aR receptor better than C3a. In view of the multiple structures corresponding to the design template for C3a, the novel formulations using both a weighted average structure and binary distance bin variables were employed in the design work. Sequences for the antagonist were predicted and are being synthesized for experimental validation by our research collaborators.

The design template for the anti-HIV inhibitor is constituted by the crystal structure of the inhibitor-gp41 complex ellucidated by Peter Kim's group (PDB code:1GZL), who found a potent crosslinked 14-residue peptide that binds to HIV-1 gp41 with an IC₅₀ value of 35μM [9]. Upon binding to gp41, the inhibitor prevents exposure of the fusion peptide and the subsequent viral entry. Our goal is to find a more potent peptide than the 14-residue inhibitor via our de novo protein design framework. Since only one crystal structure resolved by Kim et al. [9] for the inhibitor-glycoprotein complex is used, our basic sequence selection model for single template structure suffices in this work [6]. Fold specificities were confirmed on a full-atomistic scale using an approximate fold validation method aided by CYANA and TINKER [8]. Sequences for the novel inhibitors were predicted and are currently tested experimentally.

[1]- J.L. Klepeis and C.A. Floudas and D. Morikis and C.G. Tsokos and E. Argyropoulos and L. Spruce and J.D. Lambris. "Integrated Computational and Experimental Approach for Lead Optimization and Design of Compstatin Variants with Improved Activity." J. Am. Chem. Soc. 125 (2003): 8422-8423.

[2]- J.L. Klepeis and C.A. Floudas and D. Morikis and C.G. Tsokos and J.D. Lambris. "Design of Peptide Analogs with Improved Activity Using a Novel de Novo Protein Design Approach."Ind. Eng. Chem. Res. 43 (2004): 3817-3826.

[3]- C.A. Floudas and H.K. Fung and S.R. McAllister and M. Mönnigmann and R. Rajgaria. "Advances in Protein Structure Prediction and De Novo Protein Design: A Review." Chem. Eng. Sci. 61 (2006): 966-988 .

[4]- H.K. Fung and S. Rao and C.A. Floudas and O. Prokopyev and P.M. Pardalos and F. Rendl. "Computational Comparison Studies of Quadratic Assignment Like Formulations for the In Silico Sequence Selection Problem in De Novo Protein Design." J. Comb. Optim. 10 (2005): 41-60 .

[5]- C.A. Floudas. "Research Challenges, Opportunities and Synergism in Systems Engineering and Computational Biology." AIChE J.51 (2005): 1872-1884.

[6]- 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 (2006): in print.

[7]- R. Huber and H. Scholze and E.P. Paques and J. Deisenhofer. "Crystal Structure Analysis and Molecular Model of Human C3a Anaphylatoxin." Hoppe-Seylers Z Physiol. Chemie. 361 (1980): 1389-1399.

[8]- H.K. Fung and M.S. Taylor and C.A. Floudas and D. Morikis and J.D. Lambris. "Redesigning Complement 3a into Flexible Templates from Both X-ray Crystallography and Molecular Dynamics Simulation." (2006): in preparation.

[9]- S.K. Sia and P.A. Carr and A.G. Cochran and V.N. Malashkevich and P.S. Kim. "Short Constrained Peptides that Inhibit HIV-1 Entry." Proc. Nat. Acad. Sci. 99 (2002): 14664-14669.

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