(152b) Discovery of Entry Inhibitors for HIV-1: Predictions Via a Novel De Novo Protein Design Framework 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 new de novo design framework with a ranking metric based on approximate binding affinity calculations is introduced. The framework consists of two stages: a sequence selection stage and a binding affinity calculation 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 [1]. The sequence selection model incorporates backbone flexibility into the design process by utilizing the set of structures obtained from NMR experiments. In doing so, the pair-wise energy between two residues can take on a range of values depending upon the range of distances obtained from the structures.

The second stage employs Monte Carlo simulations to first predict the structure (using Rosetta Abinitio [2]) of the sequences from stage one and then to perform docking simulations (using Rosetta Dock [3]) between the new sequence and the target protein. A rotamerically-based ensemble of structures for the new peptide, the target protein, and the peptide-protein complex is generated using Rosetta Design [4], and is used to calculate approximate molecular partition functions of the new peptide, the target protein, and the peptide-protein complex. Using these approximate partition functions, an approximate binding affinity is calculated [5]. The more accurate the partition functions are, the more precise the binding affinity will be.

Recent work outlined the progress and setbacks that have occurred in the pursuit of an HIV vaccine and the advances and options for controlling HIV. Excitement has been generated for the development of entry and fusion inhibitors: these drugs block the virus from merging with T cells. The first and only FDA-approved HIV fusion inhibitor, Fuzeon, was developed by Roche and Trimeris and is in use since 2003. Despite its success as an inhibitor, it is a 36 amino acid helical peptide and its relatively long length makes the drug difficult to manufacture and very costly. As a result, it has three critical weaknesses: (a) it can be easily degraded by proteolytic enzymes in the blood leading to short life times of 2hrs in vivo; (b) it has a high cost of production due to its size (a year's supply costs $20,000 per patient); and (c) it lacks oral bioavailability resulting in inconvenient dosage form and schedule.

We focused on fusion inhibitors and discovery of short peptide-based inhibitors of HIV-1 entry into host cells using the de novo design framework. These peptidic inhibitors consist of 12 amino acids and target the core hydrophobic pocket of gp41. The choice of the epitope is based on: (i) the success of enfuvirtide (Fuzeon), (ii) the work on short constrained C-peptides that inhibit HIV-1 gp41, and (iii) the crystal structure elucidated on the best inhibitor complexed with the gp41 hydrophobic core [6]. This crystal structure provides us with an excellent flexible protein-peptide backbone structural template to de novo design novel gp41 inhibitors.

A number of the best-predicted sequences were synthesized and their inhibition of HIV-1 was tested in vitro. All of the peptides examined showed inhibitory activity when compared with no drug present, and the novel peptide sequences outperformed the native template sequence used for the design. The best sequence showed micromolar inhibition. In addition, all peptides were shown to be non-toxic, maintaining their full viability at 500 μM.

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