Dual Inhibition of Human Parainfluenza Type 3 and Respiratory Syncytial Virus Infectivity with a Single Agent

Abstract

Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) cause lower respiratory infection in infants and young children. There are no vaccines for these pathogens, and existing treatments have limited or questionable efficacy. Infection by HPIV3 or RSV requires fusion of the viral and cell membranes, a process mediated by a trimeric fusion glycoprotein (F) displayed on the viral envelope. Once triggered, the pre-fusion form of F undergoes a series of conformational changes that first extend the molecule to allow for insertion of the hydrophobic fusion peptide into the target cell membrane and then refold the trimeric assembly into an energetically stable post-fusion state, a process that drives the merger of the viral and host cell membranes. Peptides derived from defined regions of HPIV3 F inhibit infection by HPIV3 by interfering with the structural transitions of the trimeric F assembly. Here we describe lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F that potently inhibit infection by both HPIV3 and RSV. The lead peptide inhibits RSV infection as effectively as does a peptide corresponding to the RSV HRC domain itself. We show that the inhibitors bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F with high affinity. Co-crystal structures of inhibitors bound to the HRN domains of HPIV3 or RSV F reveal remarkably different modes of binding in the N-terminal segment of the inhibitor.

Effects of Single α-to-β Residue Replacements on Recognition of an Extended Segment in a Viral Fusion Protein

“…[33][34][35] A crystal structure of VIQKI co-assembled with HPIV3-HRN revealed a six-helix bundle assembly similar to the HRC/HRN assembly in the post-fusion conformation of the HPIV3 F protein ( Figure 1B). 30 As expected, two distinct conformations were observed within the VIQKI peptide, an α-helix spanning residues corresponding to 457-484 of the F protein, and an extended conformation for residues corresponding to 449-456. Our studies focused on backbone modifications in the extended portion of VIQKI (Figure 1…”

“…This strategy requires molecules that can engage large surfaces on target proteins, a goal that is generally difficult to achieve with small molecules (MW<1000). [1][2][3] Clinically useful inhibitors of pathogenic protein-protein interactions are typically engineered proteins or large peptides. Antibodies and other large proteins are expensive to produce.…”

“…4 Smaller polypeptides offer the advantage of chemical synthesis but suffer from rapid degradation by endogenous proteases. [1][2][3] One strategy for retaining the benefits of medium-sized polypeptides while minimizing proteolytic susceptibility is to incorporate non-natural subunits into the peptidic backbone. [5][6][7][8][9][10][11][12] The challenge inherent in this approach is to maintain target protein engagement despite backbone alteration.…”

“…[27][28][29] This study began with a previously reported peptide derived from the HRC domain of HPIV3, designated VIQKI ( Figure 1C), which blocks infection by both HPIV3 and respiratory syncytial virus (RSV). [30][31][32] These two viruses are leading causes of fatal lower respiratory infections in young children. [33][34][35] A crystal structure of VIQKI co-assembled with HPIV3-HRN revealed a six-helix bundle assembly similar to the HRC/HRN assembly in the post-fusion conformation of the HPIV3 F protein ( Figure 1B).…”

Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

“…Counting drugs in clinical use, drug candidates in clinical trials or under formal development, and early-stage experimental compounds, a large number of direct-acting entry inhibitors blocking pneumoor paramyxovirus infection have been identified. Chemically, these include biopharmaceuticals such as neutralizing antibodies [14,37,[51][52][53][54] synthetic peptides [55,56], and chemically synthesized small molecules [57][58][59][60][61]. However, the anti-RSV nAb palivizumab is the only F protein-targeting drug approved for human use, and a number of small-molecule candidates that have advanced to clinical testing are likewise directed against RSV.…”