Targeting a binding pocket within the trimer-of-hairpins: small-molecule inhibition of viral fusion

Abstract

Trimeric class I virus fusion proteins undergo a series of conformational rearrangements that leads to the association of C- and N-terminal heptad repeat domains in a "trimer-of-hairpins" structure, facilitating the apposition of viral and cellular membranes during fusion. This final fusion hairpin structure is sustained by protein-protein interactions, associations thought initially to be refractory to small-molecule inhibition because of the large surface area involved. By using a photoaffinity analog of a potent respiratory syncytial virus fusion inhibitor, we directly probed the interaction of the inhibitor with its fusion protein target. Studies have shown that these inhibitors bind within a hydrophobic cavity formed on the surface of the N-terminal heptad-repeat trimer. In the fusogenic state, this pocket is occupied by key amino acid residues from the C-terminal heptad repeat that stabilize the trimer-of-hairpins structure. The results indicate that a low-molecular-weight fusion inhibitor can interfere with the formation or consolidation of key structures within the hairpin moiety that are essential for membrane fusion. Because analogous cavities are present in many class I viruses, including HIV, these results demonstrate the feasibility of this approach as a strategy for drug discovery.

Fig. 1.

Specific labeling of F₁ by [¹²⁵I]BMS-356188. (A) The structures of BMS-433771 and [¹²⁵I]BMS-356188. (B) Immunoprecipitation of affinity-labeled F₁ polypeptide from intact virus by using either α-RSV polyclonal IgG to all RSV antigens (lane 3) or a mAb to F₁ (lane 2). The F₁ M_r (48,051) is indicated. Lane 1 is an α-RSV immunoprecipitation of [³⁵S]Met-labeled RSV. (C) BMS-433771 inhibits [¹²⁵I]BMS-356188 affinity labeling of the F₁. The α-F₁ protein immunoprecipitation analysis was performed as described for B. Increasing concentrations of BMS-433771 were used, as indicated below each lane. The [¹²⁵I]BMS-356188 concentration is 4 nM. (D) CNBr cleavage of the F₁ polypeptide after affinity labeling and immunoprecipitation. Lane 1, uncleaved F₁; lane 2, CNBr-cleaved F₁. M_r markers are indicated. Sequence of the CNBr peptide is illustrated with the HR-N region in red. (E) Endoprotease Glu-C digest of affinity-labeled F₁ compared with CNBr fragment of the same preparation. The proposed sequence of the Glu-C peptide is shown with the HR-N portion in red. (F) Affinity labeling of the HR-N57 peptide. HR-N57 (8 μM) was labeled with 10 nM [¹²⁵I]BMS-356188 (lane 1), in the presence of 1 μM of BMS-433771 (lane 2), with 50 μM RFI-461 (lane 3), with 50 μM RFI-461 in presence of 1 μM BMS-433771 competitor (lane 4), and in 20% trifluoroethanol (lane 5). The HR-N57 sequence is shown in red.

Fig. 3.

Covalent attachment site of [¹²⁵I]BMS-356188 within the HR-N hydrophobic pocket. (A) Sequence analysis of affinity-labeled HR-N42. Percent of total [¹²⁵I] counts recovered for each amino acid sequencing cycle is indicated. Tyr-198 is the most prevalently labeled amino acid (blue). (B) Molecular dynamic simulation scores for the docking of BMS-433771 into the hydrophobic pocket with dock 4.0.(C) Model (pose 6) of [¹²⁵I]BMS-356188 within the HR-N hydrophobic cavity (green). The photoreactive diazerine (white box) is oriented toward Tyr-198 (red surface). HR-N α-helices A and E and selected amino acids are indicated.

Fig. 2.

HR-N27, HR-N42, and HR-N57 peptide affinity labeling, CD spectra, and hydrophobic heptad repeat pattern. (A) HR-N peptides (8 μM) were labeled with 40 nM [¹²⁵I]BMS-356188 in the presence of 50 μM RFI-461 and analyzed by 15% SDS/PAGE. HR-N27, HR-N42, and HR-N57 labeling results and sequences are indicated. Efficient affinity labeling was seen for HR-N42 and HR-N57 only. (B) CD spectra for HR-N peptides in PBS alone. RFI-461 is omitted because compound autofluorescence interferes with CD measurements. The α-helical signatures are exhibited by HR-N42 and HR-N57 only. (C) Heptad repeat amino acids indicated for the three HR-N peptides. The a and d positions of hydrophobic amino acids shown to participate in timer formation are predicted from the helical wheel projection.

Fig. 4.

Proposed model for the mechanism of action of this inhibitor series. (A) The HR-C (white stick display; Phe-483, Phe-488, and Ile-492 aa side chains highlighted in yellow) association with the HR-N hydrophobic pocket (green surface display; Tyr-198 is shown in red). The HR-C amino acids Phe-483 and Phe-488 bind in the HR-N hydrophobic pocket. (B) The fusion inhibitor, BMS-433771, residing in the HR-N pocket, blocks the HR-C Phe-483, Phe-488, and Ile-492 associations.