Structure-guided design of small-molecule therapeutics against RSV disease

Abstract

In the United States, respiratory syncytial virus (RSV) is responsible for the majority of infant hospitalizations resulting from viral infections, as well as a leading source of pneumonia and bronchiolitis in young children and the elderly. In the absence of vaccine prophylaxis or an effective antiviral for improved disease management, the development of novel anti-RSV therapeutics is critical. Several advanced drug development campaigns of the past decade have focused on blocking viral infection. These efforts have returned a chemically distinct panel of small-molecule RSV entry inhibitors, but binding sites and molecular mechanism of action appeared to share a common mechanism, resulting in comprehensive cross-resistance and calling for alternative druggable targets such as viral RNA-dependent RNA-polymerase complex. Areas Covered: In this review, the authors discuss the current status of the mechanism of action of RSV entry inhibitors. They also provide the recent structural insight into the organization of the polymerase complex that have revealed novel drug targets sites, and outline a path towards the discovery of next-generation RSV therapeutics. Expert opinion: Considering the tremendous progress experienced in our structural understanding of RSV biology in recent years and encouraging early results of a nucleoside analog inhibitor in clinical trials, there is high prospect that new generations of much needed effective anti-RSV therapeutics will become available for clinical use in the foreseeable future.

Keywords: RNA-dependent RNA-polymerase; Respiratory syncytial virus; small molecule antiviral; viral replication; virus entry.

Figure 1

Schematic of the RSV life cycle. (1) Infection commences with viral attachment, fusion of the viral envelope with target membranes, and entry. (2) The viral nucleocapsid genome and RdRp complex are released into the cytoplasm. (3) Together with host factors, the RdRp complex begins primary transcription to synthesize viral mRNAs. (4) Viral proteins are synthesized, glycoproteins are transported to the plasma membrane through the host cell secretory system. (5) Switch of RdRp to replicase mode results in the production of full-length antigenomes of positive polarity. (6) Antigenomes then serve as templates for the creation of progeny genomic RNA. (7) Newly synthesized genomes template the majority of viral mRNAs through secondary transcription. (8) For particle assembly, progeny genomes with associated RdRp components are shuttled to budding sites at the plasma membrane. (9) Virion assembly. The virus entry machinery and the RdRp complex were identified as molecular targets of a large proportion of developmental drug candidates.

Figure 2

Fusion pore formation through class I viral fusion proteins. (A) The F protein initially folds into a metastable prefusion conformation. (B) Once proteolytically matured and activated, the prefusion F head domain undergoes a series of major conformational changes, resulting in the assembly of an elongated trimeric coil (HR-A), which propels the fusion peptide towards the target membrane. (C) Heptad repeats proximal to the transmembrane domain (HR-B) start to fold onto the HR-A central helix during F hairpin formation, generating fusion dimples in the lipid bilayers. (D & E) Continued hairpin formation leads to the assembly of the 6HB through zippering of the HR-B domains into the grooves of the HR-A triple helix. Extreme negative curvature is introduced into the approaching bilayers, resulting in the disarray of lipids in the outer monolayers and ultimately merger of the outer layers in a hemifusion intermediate. (F) Transmembrane domains and fusion peptides are brought into close proximity, leading to the opening of a fusion pore. Creation of a productive fusion pore by paramyxovirus F proteins is not mandatorily dependent on full closure of the 6HB [100]. Images of protein structures were generated in Pymol. PDB codes: prefusion F:4MMS; postfusion F:3RRT

Figure 3

Structural model of the RSV fusion protein. (A) The RSV F protein in the prefusion conformation. Shown are a ribbon model of the native prefusion F trimer (left), a single monomer (center), and a monomer with known resistance mutations (magenta) in the 392–401 and 486–489 microdomains (right). (B) The F protein in the thermodynamically stable postfusion conformation. Fields of view as described for (A). (C) Schematic of the domain architecture of the RSV fusion protein. Proteolytic maturation of newly synthesized F results in the liberation of the F₁ and F₂ subunits. F₁ harbors the fusion peptide (FP; blue), heptad-repeat A (HR-A; red), connecting domains 1 and 2 (yellow), heptad-repeat B (HR-B; green), a transmembrane domain (TM; olive), and a short cytoplasmic or luminal tail (CP; tan). F₂ remains an integral structural component of the trimer. This subunit contains the signal peptide (SP; grey) and a heptad-repeat C (HR-C; cyan) domain. (D) Schematic of the prefusion RSV F trimer overlaid with entry inhibitor binding locations (orange) and resistance hot spots (magenta spheres). For clarity, only one monomer was color-coded by domain structure as outlined in (C). Images of protein structures were generated in Pymol. PDB codes: prefusion F:4MMS; postfusion F:3RRT; Inhibitor bound F: 5EA3, 5EA4, 5EA5, 5EA6, 5EA7.

Figure 4

Structural models of the RSV N protein and the nucleocapsid. (A) Structure of the helical RSV nucleocapsid showing the encapsidated RNA in red and individual N protein protomers in dark and light blue. (B) Surface representation of a RSV N monomer (left). Rotation of the N monomer reveals the RSV P protein binding site (red sphere) and resistance mutations against RSV604 (yellow). (C) The RSV N protein is composed of two core domains, NTD (blue) and CTD (tan), which enclose the viral RNA genome (red). Terminal extensions from the NTD and CTD domains, the N- and C-arm, respectively, interact with adjacent N subunits in the helical nucleocapsid structure. Images of protein structures were generated in Pymol. PDB codes:4BKK(RSV N), 4UCB(RSV NTD-P).

Figure 5

Structure of a mononegavirales L protein and homology models of the RSV L protein generated based on the coordinates reported for the related VSV L structure [65]. (A) Structure of the VSV L protein shown as surface (left) and ribbon (right) models. Red spheres in the ribbon model highlight the active site for phosphodiester bond formation. (B) The VSV L structure displayed five main structural units: polymerase (cyan), capping (green), connector domain (yellow), methyltransferase (orange), and C–terminal domain (red). (C) Sequence analysis revealed six conserved regions (CR I–VI) in the L proteins. (D) Insertion analysis has shown that paramyxovirus L proteins can be further divided into three large regions (LRI-III). The location of the polymerase active site is shown in (B–D) as red lines, mutations that confer resistance to the RSV nucleoside analog inhibitor ALS-8176 are shown as black crosses. (E) Homology models of the RSV L protein based off the VSV L structure. A surface representation (left) and a model of the internal organization (right) are shown. The active site for phosphodiester bond formation and the ALS-8176 resistance mutations are highlighted as magenta and yellow spheres, respectively. Arrows indicate channels for the template (black) and newly synthesized (magenta) RNA strands. Images of protein structures were generated in Pymol; PDB code:5A22 (VSV L). Homology models were generated using SWISS-MODEL.