Discovery of entry inhibitors for HIV-1 via a new de novo protein design framework

Abstract

A new (to our knowledge) de novo design framework with a ranking metric based on approximate binding affinity calculations is introduced and applied to the discovery of what we believe are novel HIV-1 entry inhibitors. 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 by solving an integer programming sequence selection model. The validation stage consists of fold specificity and approximate binding affinity calculations. The designed peptidic inhibitors are 12-amino-acids-long and target the hydrophobic core of gp41. A number of the best-predicted sequences were synthesized and their inhibition of HIV-1 was tested in cell culture. All 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, which is a 3-15-fold improvement over the native sequence, depending on the donor. In addition, the best sequence equally inhibited wild-type and Enfuvirtide-resistant virus strains.

Figure 1

Overview of the de novo design framework.

Figure 2

Crystal structure of C14linkmid in complex with the hydrophobic core of gp41, PDB code: 1GZL. The diaminoalkane crosslinker and the hydrophobic core of gp41, consisting of residues Leu²⁹, Leu³⁰, Leu³², Thr³³, Val³⁴, Trp³⁵, Gly³⁶, Ile³⁷, Lys³⁸, Leu⁴⁰, and Gln⁴¹are colored blue (the residues are numbered according to their position in 1GZL). Figure created using PyMOL v0.99 (73).

Figure 3

Inhibition of entry of wild-type HIV into primary CD⁴⁺ T cells by the top five mutant sequences and the native sequence. Experiments were done in duplicate with cells from two donors and at two peptide concentrations, 50 μM and 500 μM. Replication (% of control) was determined by single-round infectivity assay with GFP-encoding recombinant HIV. (Error bars) Span of values obtained.

Figure 4

Inhibition of entry of wild-type and gp41 N43D mutant HIV into primary CD⁴⁺ T cells by SQ435 (A and B) and Enfuvirtide (C). Replication (% of control) was determined by single-round infectivity assay with GFP-encoding recombinant HIV. Individual donors illustrated with unique symbols and separate curves to illustrate the donor-to-donor variability in potency. (Solid symbol) Wild-type virus. (Open symbol) N43D mutant. (Dashed lines) Aggregate IC₅₀ curve across all donors, and two different dash styles distinguish the two viruses used.

Figure 5

Inhibition of entry of HIV bearing five different envelopes into primary CD⁴⁺ T cells by SQ435 (A) and Enfuvirtide (B). Envelopes tested include CXCR4-tropic HXB2, two CCR5-tropic reference envelopes YU-2 and SF162, and two clinically isolated CCR5-tropic envelopes C98v1e4 and C109v1e4. Replication (% of control) was determined by a single-round infectivity assay with GFP-encoding recombinant HIV. Each envelope isolate is illustrated with a unique symbol and separate curve. (Dashed lines) Aggregate IC50 curve across all clinical isolates.