A covalent chemical probe for Chikungunya nsP2 cysteine protease with antialphaviral activity and proteome-wide selectivity

Abstract

Chikungunya is a mosquito-borne viral disease that causes fever and severe joint pain for which there is no direct acting drug treatments. Vinyl sulfone SGC-NSP2PRO-1 (3) was identified as a potent inhibitor of the nsP2 cysteine protease (nsP2pro) that reduced viral titer against infectious isolates of Chikungunya and other alphaviruses. The covalent warhead in 3 captured the active site C478 and inactivated nsP2pro with a k_inact/K_i ratio of 5950 M^-1 s^-1. The vinyl sulfone 3 was inactive across a panel of 23 other cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. A negative control analog SGC-NSP2PRO-1N (4) retained the isoxazole core and covalent warhead but demonstrated > 100-fold decrease in enzyme inhibition. Both 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. Vinyl sulfone 3 and its negative control 4 will find utility as high-quality chemical probes to study the role of the nsP2pro in cellular studies of alphaviral replication and virulence.

Keywords: Alphavirus; Antiviral; Chemical probe; Covalent inhibitor; Cysteine protease.

Fig. 1

nsP2pro inhibitor RA-0002034 (1) undergoes slow intramolecular cyclization to the inactive (2) Isoxazole SGC-NSP2PRO-1 (3) is chemically stable and a potent nsP2pro inhibitor. SGC-NSP2PRO-1N (4) is an inactive analog that retains the vinyl sulfone covalent warhead.

Fig. 2

(a) Isoxazole 3 inhibits CHIKV nsP2 protease activity in a time-dependent manner. 1.5-fold serial dilutions were used to determine K_i, k_inact, and k_inact/K_i ratio. (b) Isoxazole 3 demonstrated a 5–9 log decrease in viral titer against infectious isolates of CHIKV, MAYV, and VEEV alphaviruses. Data shown are averages ± SEM of two independent experiments.

Fig. 3

Activity of isoxazole 3 at 10 µM against 20 human and 3 viral cysteine proteases. The following were used as controls: E-64 (50 nM) Cathepsin B, K, L, S, and V; Cystatin C (300 nM) for Cathepsin F; Z-VRPR-FMK (10 nM) for MALT1; Ubiquitin-Aldehyde (0.1 nM) for UCHL3, (1 nM) for A20, UCHL1, USP1, USP14, (10 nM) for OTUD6B, USP2, USP5, USP7, USP8, USP10, USP20, and (1 µM) for Ataxin-3; GC376 (1 µM) for SARS2 Mpro; Protease Cocktail (10 µM) for MERS Mpro; and GRL0617 (10 µM) for SARS2 Plpro. Values are the average of duplicate determinations.

Fig. 4

TAMRA fluorescence chemoproteomics. (a). Chemical structures of chemoproteomics probes: vinyl sulfone (VS), vinyl sulfone control (VSC), chloracetamide (CA), TAMRA vinyl sulfone (TVS), and biotin vinyl sulfone (BVS). (b). Fluorescent imaging of SDS-PAGE following covalent labeling of purified nsP2 (lane P) by TVS or human cell lysates (lanes A–D) by VS or CA. In lanes 2, VS or CA were incubated with cell lysates prior to click reaction to append the TAMRA fluorophore. In lanes 1, prior to the click reaction, lysates were blocked with VSC. Lanes A and C contain HEK293 cell lysates (140 µg total protein each), whereas lanes B and D equivalent amounts of cell lysates were supplemented with purified CHIKV nsP2 (4 µg). M.Wt markers are indicated. The nsP2 band with calculated M.Wt. 42.9 kDa is marked while accounting for the slight curvature of the gel. An uncropped copy of the gel with membrane edges visible is shown in Supplementary Figure S22. All ligands were used at 10 µM.

Fig. 5

Streptavidin pull-down in HEK293 lysates by VS and biotin-azide click reaction. (a) Silver stain of SDS-PAGE gel loaded with HEK293 lysate (lane L) or proteins from streptavidin pull-down of cell lysate alone (lane 1). The VS pull-down was performed 3 times from the lysate and loaded onto the gel: VS alone (lane 2), VS alone from lysate spiked with nsP2 protein (lane 3), VS from lysate pre-blocked with 3 (lane 4). An uncropped copy of the gel with membrane edges visible is shown in Supplementary Figure S22. (b) Analysis of proteins identified from lanes 2–4 by LC–MS/MS. All identified proteins (▲); VS alone (◼); VS after pre-block with 3 (◌); VS alone from lysate spiked with nsP2 protein (⬤). Proteins with log₂ fold < 5 enrichment are shown in grey. Proteins with log₂ fold > 5 enrichment are shown in black, with nsP2 in red.

Fig. 6

Dose–response curves of isoxazole 3 and negative control analog 4. a. Inhibition of CHIKV nsP2pro following a 30 min incubation. Values are the mean of triplicate determinations. b. Inhibition of CHIKV-nLuc replication in human fibroblast MRC5 cells at 6 h post-inoculation with the virus. Values are the average of duplicate determinations.

Fig. 7

Synthesis of Negative Control 4. Reagents and conditions: (i) propan-2-amine, Cs₂CO₃; (ii) TBTU, DIPEA.

Fig. 8

Synthesis of clickable vinyl sulfone (VS) and pre-clicked vinyl sulfone control (TVS). Reagents and conditions: (i) diethyl oxalate, NaH; (ii) NH₂OH.HCl; (iii) 3-bromoprop-1-yne, K₂CO₃; (iv) NaOH; (v) (E)−3-(methylsulfonyl)prop-2-en-1-amine, TBTU, pyridine; (vi) 6-TAMRA azide, CuSO₄, Na ascorbate.