(442f) A New De Novo Approach for Optimizing Peptides That Inhibit HIV-1 Entry

Until recently, treatment of HIV has been based on nucleoside analog reverse transcriptase and protease inhibitors, the cost of which is overwhelming. Drawbacks of these types of anti-HIV drugs also include patient intolerance with antiviral cocktails and drug resistance of the viral variants [1]. Inhibiting HIV's entry into the host cell offers a promising alternative for the drug mechanism. So far there is only one anti-HIV drug that targets viral entry being available in the market, although several others are progressing through clinical trials [1]. It is a 36-residue linear peptide called enfuvirtide, or Fuzeon, marketed by Roche/Trimeris. Novel approaches for de novo protein design [2] can further optimize the therapeutic peptide and in this work, we present the design of a potent 14-residue crosslinked peptidic inhibitor that functions by the same mechanism as Fuzeon's.

In our work we applied our new de novo protein design framework, which is split into two stages: (i) in silico sequence selection, and (ii) fold validation. Sequence selection was performed by an energy minimization model which mapped sequences onto a 3-dimensional backbone template and picked the sequences with the lowest energies ([3]-[5]). 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 from the stage one model. Our framework treats true protein backbone flexibility [6] explicitly by the use of discretized energies over distance bins in stage one and the consideration of all possible conformations of continuous dihedral angle and C^α-C^α distance values between preset bounds in stage two. Confirmed by experimental data, the 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 ([3]-[4]).

The design template for the anti-HIV entry peptidic inhibitor was from a crystal structure of the inhibitor bound to the hydrophobic pocket of gp41, an evelope glycoprotein of the virus which mediates fusion of the viral and cellular membranes. The crystal structure was ellucidated by Peter Kim's group (PDB code:1GZL). They found a potent crosslinked 14-residue peptide that bound to HIV-1 gp41 with an IC₅₀ value of 35μM [7]. Upon binding of the inhibitor to gp41, the virus will not be able to fuse with the host cell for infection. Our goal was to find a more potent peptide through our de novo protein design framework that bound to gp41 with higher specificity. Since only one crystal structure resolved by Kim et al. [7] for the inhibitor-glycoprotein complex was used, our sequence selection model for single template structure sufficed in this work, although models to handle the more usual case of a design template with multiple structures have also been developed [8]. Fold specificities were validated on a full-atomistic scale using CYANA and TINKER. Sequences for the novel inhibitors were predicted and are currently tested experimentally.

[1]- P.A. Galanakis and G.A. Spyroulias and A. Rizos and P. Samolis and E. Krambovitis. "Conformational Properties of HIV-1 gp120/V3 Immunogenic Domains."Curr. Med. Chem. 12 (2005): 1551-1568.

[2]- 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 .

[3]- 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.

[4]- 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.

[5]- 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.

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

[7]- 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.

[8]- 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." J. Comb. Optim., (2006): accepted for publication.