Structural Insights into the Mechanisms of Action of Functionally Distinct Classes of Chikungunya Virus Nonstructural Protein 1 Inhibitors

Abstract

Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (6'-fluoro-homoneplanocin A [FHNA], its 3'-keto form, and 6'-β-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-of-their-kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S-adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the ring-aperture membrane-binding and oligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target.

Keywords: Chikungunya virus; GTP; SAM; antivirals; binding pocket; capping; inhibitors; molecular docking; nsP1; resistance.

FIG 1

Chemical structures of the CHIKV nsP1-targeting compounds used in this study.

FIG 2

Cross-resistance analysis of compound-resistant CHIKV strains with mutations in nsP1. The sensitivities of wt CHIKV and the nsP1 G230R K299E, S454G W456R, and P34S mutants to various nsP1-targeting inhibitors were assessed in CPE reduction assays in Vero E6 cells. The mechanistically unrelated compound favipiravir, which targets nsP4, was included as a control. Data are expressed as cell viability relative to the viability of uninfected cells and represent means ± standard deviations from at least two independent experiments performed in quadruplicate (n = 8).

FIG 3

Inhibitory effects of selected compounds on the enzymatic activity of purified wt SFV or CHIKV nsP1 in a biochemical assay measuring the formation of the covalent [³²P]m⁷GMP-nsP1 reaction intermediate. (A) wt SFV nsP1 was incubated with [α-³²P]GTP and 100 μM SAM and was treated with increasing doses (50 μM to 1 mM) of inhibitors. SFV nsP1 D64A was used as a negative control. VC, solvent control. (B) wt SFV nsP1 was incubated with [α-³²P]GTP with or without 50 μM sinefungin (SIN) in the presence or absence of 100 μM SAM. SFV nsP1 D64A was used as a negative control. (C) wt CHIKV nsP1 was incubated with [α-³²P]GTP and 10 μM SAM and was treated with increasing doses (0.5 to 32 μM) of inhibitors. (D) wt CHIKV nsP1 was incubated with [α-³²P]GTP and 10 μM SAM and was treated with increasing doses (125 μM to 1 mM) of sinefungin. (E) wt CHIKV nsP1 was incubated with [α-³²P]GTP and 10 μM SAM in the presence (left) or absence (right) of DTT and increasing concentrations (12.5 to 500 μM) of FHNA. In all cases, the covalent [α-³²P]m⁷GMP-nsP1 intermediate was visualized after overnight exposure of the PhosphorImager screen. Coomassie blue staining with GelCode blue reagent was used to demonstrate the loading of equal protein quantities.

FIG 4

Predicted binding pockets in the CHIKV nsP1 oligomeric structure. (A) Top view of the nsP1 dodecameric ring. Shown are the main binding pocket (pocket 1, in orange) and secondary binding pocket (pocket 2, in blue) predicted by the ICM Pocket Finder method. (B) Front view of the nsP1 dodecameric ring. (C) The predicted main binding pocket is elongated and contains two binding sites virtually divided by the catalytic residues H37 and D63, which correspond to the SAM- and GTP-binding sites. Three consecutive nsP1 protomers, with subscripts n, n+1, and n–1, were used to define these pockets within the nsP1 complex (PDB code 6Z0V).

FIG 5

Suggested binding mode of the endogenous ligands GTP (orange) and SAM (green) in the catalytic pocket of CHIKV nsP1 (gray). The known catalytic residues H37 and D63 are not directly involved in binding, most likely due to the available conformation of the CHIKV nsP1 cryo-EM structure (PDB code 6Z0V), where D63 is deeply buried in the protein structure.

FIG 6

Key amino acid residues linked to resistance to CHIKV nsP1 inhibitors, mapped to the CHIKV nsP1 cryo-EM structure (PDB code 6Z0V), represented as three consecutive nsP1 protomers: n, n+1, and n–1. Residues are color-coded as follows: green, catalytic residues H37 and D63; blue, residues P34 and T246, involved in resistance to the MADTP series; orange, residues G230 and K299, conferring resistance to the FHNA series. Residues F450 to S458 (purple) flank the area where residues S454 and W456, involved in resistance to the CHVB series, would be located. Orange and green dotted volumes represent the proposed binding pockets of GTP and SAM, respectively, for reference.

FIG 7

Predicted binding mode of the CHVB and MADTP series in the CHIKV nsP1 SAM-binding site of the main binding pocket (PDB code 6Z0V). (A) CHVB-066 (purple) and CHVB-032 (pink) in complex with CHIKV nsP1, occupying the SAM-binding site (green dots). Both CHVB compounds form hydrogen bonds with Y154 and A155 (in yellow) and share a binding mode. The strength of hydrogen bonds is represented by the diameter of the sphere. (B) MADTP-372 (blue) in complex with CHIKV nsP1, occupying the SAM-binding site (green dots). This compound forms hydrogen bonds with Y154 and A155 (in yellow). P34 (in blue) is the main residue responsible for MADTP compound resistance. T246 (in blue) is an additional residue that, upon mutation, causes some level of resistance to MADTP.

FIG 8

Binding mode of the FHNA series in the CHIKV nsP1 secondary binding pocket. (A) The FHNA series is suggested to bind to a secondary binding pocket (pocket 2) in the RAMBO domain of CHIKV nsP1 (PDB code 6Z0V) outside of the main binding pocket. (B) The defined binding pocket is flanked by residues G230_n and K299_n–1. FHNA and FHA have similar binding modes that are not shared by the inactive analogue FMA.