Flavivirus nonstructural proteins and replication complexes as antiviral drug targets

Abstract

Many flaviviruses are well-known pathogens, such as dengue, Zika, Japanese encephalitis, and yellow fever viruses. Among them, dengue viruses cause global epidemics and threaten billions of people. Effective vaccines and antivirals are in desperate need. In this review, we focus on the recent advances in understanding viral nonstructural (NS) proteins as antiviral drug targets. We briefly summarize the experimental structures and predicted models of flaviviral NS proteins and their functions. We highlight a few well-characterized inhibitors targeting these NS proteins and provide an update about the latest development. NS4B emerges as one of the most promising drug targets as novel inhibitors targeting NS4B and its interaction network are entering clinical studies. Studies aiming to elucidate the architecture and molecular basis of viral replication will offer new opportunities for novel antiviral discovery. Direct-acting agents against dengue and other pathogenic flaviviruses may be available very soon.

Figure 1.. Molecular and structural flavivirology.

(A) Genome organization of the flavivirus genome. Flaviviral RNA is composed of a 5′UTR, one single open reading frame, and a 3′UTR. Translation of the flavivirus genomes produces a single polyprotein, later cleaved to structural and non-structural proteins by both host (in yellow) and viral NS2B-3 (in black) proteases. Capsid protein (C protein), pre-membrane protein (prM protein), and envelope protein (E protein) are structural proteins, whereas the remaining (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are non-structural proteins (B) Diagrammatic representation of the membrane topology of the flaviviral polyprotein, as well as the polyprotein cleavage sites. NS2A, NS2B, NS4A, and NS4B contain transmembrane domains, while NS1 contains conserved residues that interact with the ER membrane [19]. NS5 is indirectly associated with the ER membrane through binding to NS3, which is in turn bound to transmembrane proteins NS2A, NS4A, and NS4B [124]. (C) Respective solved protein structures predicted models, and their structural organization in the ER membrane are illustrated. Each structure is annotated with a representative PDB ID, where appropriate, and visualized in cartoon. AlphaFold 2 (AF2) was used to predict the structures for NS2A, NS4A, and NS4B transmembrane proteins as they remain unresolved. NS1 (PDB: 4O6B) consists of a homodimer, annotated as NS1/A and NS1/B. The diagram is rotated 90 degrees to depict NS1 interaction with the membrane. NS2B-NS3 protease (NS3pro) interacts with E60 (2-sulfanylidene-1,3-thiazolidin-4-one) (PDB: 6L50). NS2B-3, NS4A, 2k peptide and NS4B were predicted with high confidence by AF2 (pLDDT score > 70). NS2B anchors the NS2B-3 complex to the ER membrane. While NS4A has been predicted with high confidence, the structural organization within the membrane appears to differ from the literature, with the C-terminus of AF2-predicted NS4A residing in the ER lumen. NS5 (PDB: 5DTO) consists of an N-terminal MTase and a C-terminal RNA-dependent RNA polymerase (RdRp). Figures were created with Biorender.

Figure 2.. The landscape of NS1 structures.

(A) Dimeric NS1 cartoon model in a simulated transparent map at 2.5 Å at a low contour of 0.2. One of the monomers is colored by its three-domain architecture, a hydrophobic β-roll (residues 1–29, orange), an a/b wing (residues 38–151, blue), and a rigid β-ladder (residues 181–352, cyan). The dimer has a distinct crossed shape with the wings extending from the central β-ladder which has an extended β-sheet that faces the β-roll as the membrane-associated surface (side view) and a “spaghetti loop” on the opposite polar outer face that lacks structured elements. (B) Tetrameric and (C) hexameric recombinant secreted NS1 (sNS1) forms as confirmed by Bo et al. (2021) cryoEM structures [1], modelled using the dimeric NS1 structure coupled with a simulated transparent map at 2.5 Å at the high contour of 1.0 and at 6.0 Å at the low contour of 0.3 respectively. The second NS1 dimer is coloured in yellow and blue for each monomer chain. The third NS1 dimer for the hexamer model is coloured in light and dark purple. (D) Immunoaffinity-purified sNS1 form from infected cell cultures as reported by Chew et al (2022) [2]. Model of sNS1wt: Fab56.2 predicted structures rigid body fitted in the CryoEM map (transparent grey, contoured at 0.14) with a correlation value of 0.75 to the fitted regions (map simulated from atoms at 5 Å). The antibody Fab56.2 domain is coloured in dark and light green for the Heavy (H) and Light (L) chains respectively. The predicted apoA-I, major protein component of HDL, model is coloured in intervals of grey and light purple representing its 11 and 22 residue alpha helical repeats. ApoA-I model is fitted in the spherical map region with its orientation informed by cross-linking mass spectrometry. (E) Comparison of the NS1 binding by anti-Denv Fab56.2 to earlier published NS1:Fab structures namely 2B7 (PDB ID: 6WER) [3] and 1G5.3 (PDB ID: 7BSC) overlayed on the lipid membrane cartoon for the side view and the top view shown at the bottom depicting the differing angles that they bind to the β-ladder. Figure prepared using ChimeraX.

Figure 3.. Selected flavivirus Protease Inhibitors.

(A) Structure of a small molecule compound 9, formula 4-(NH2CH2)-Ph, in complex with the NS2B-NS3 protease from Dengue 2 virus. (B) Structure of a cyclic peptide compound 12 in complex with the NS2B-NS3 protease from Zika virus. Colour code: compounds light blue, partially unfolded NS2B cofactor magenta, and NS3 protease domain yellow.

Figure 4.. Predicted protein structure of NS4B and its inhibitors.

(A) Comparison between NS4B topology derived from biochemical data, adapted from Bhardwaj, et al., and topology of the AF2 model. (B) Schematic of AF2-predicted structural organisation of NS4B in the ER membrane, with proposed transmembrane domains based on biochemical studies labelled and colored in dark green and cytoplasmic facing regions coloured in cyan. Sites of interaction between NS4B and the compounds JNJ-A07, NITD-688, and BDAA are annotated. JNJ-A07 alters the conformation of loop 3 of NS4B, occurring between TM3 and TM4. BDAA, a small molecule inhibitor of YFV, binds P219 in TM5 of NS4B. NITD-688 has also been identified to bind to NS4B. (C) Effect of JNJ-A07 binding on the interaction of NS2B3-NS4B. JNJ-A07 blocks NS3-NS4B interaction and complex formation but does not disrupt existing NS3-NS4B complexes. Created with Biorender.

Figure 5.. Structure of NS5 MTase-RdRp and its inhibitors.

(A) Overall structure of DENV3 full length NS5. MTase domain is colored in yellow. Fingers Palm and Thumb domains of the RdRp are colored in green, cyan, and pink. Linker region is colored in orange. (B) View of the RdRp active site, marked with a triangle. The allosteric inhibitors binds a novel pocket on the Thumb domain next to the active site. Chemical structure of the (C) Compound 29 [114], (D) NITD008. (E) AT-752.