Introduction

Alphaviruses, a genus of arthropod-borne viruses, continue to pose significant threats to human health, particularly in regions where their vectors are prevalent1,2. Chikungunya virus (CHIKV), which causes chronic arthritis, has infected millions of individuals in the Americas, Africa, and Asia3. Recent outbreaks in New England of Eastern Equine Encephalitis virus (EEEV), whose symptoms can often be fatal or lead to long-term neurological damage, have led to multiple public health warnings and heightened surveillance efforts4. Other alphaviruses of concern include Venezuelan Equine Encephalitis virus (VEEV) and Mayaro virus (MAYV). The endemic global CHIKV infections and localized EEEV outbreaks not only underscore the persistent risk of alphavirus infections but also highlight the urgent need for effective therapeutic interventions, as there are currently no approved antiviral drugs specifically targeting these viruses5. Among the most promising therapeutic targets is the non-structural protein 2 protease (nsP2pro), an alphaviral cysteine protease that plays a crucial role in the virus life cycle6.

Potent and selective high-quality chemical probes, as defined by Arrowsmith et al.7, are valuable tools in biological research as they allow scientists to investigate the functions of specific proteins and biological pathways, providing insights into disease mechanisms and aiding in drug discovery. Cysteine proteases are an established family of druggable enzymes that are often targeted using covalent inhibitors8. Although inhibitors that target the catalytic cysteine often show potent inhibition, a challenge remains to achieve selectivity against the remaining proteome to minimize off-target effects. In their recent review, Hartung et al.9 outlined several key criteria for high-quality covalent chemical probes, emphasizing the need for specificity in target engagement. A primary requirement, in addition to potency, selectivity, and the absence of moieties that could lead to promiscuity or assay interference, is that the chemical probe should have a well-defined site of covalent interaction, labeling the primary target without affecting other proteins.

We have previously described the identification of (E)−5-(2-ethoxyphenyl)-N-(3-(methylsulfonyl)allyl)−1H-pyrazole-3-carboxamide (RA-0002034, 1) as a covalent inhibitor of nsP2 cysteine proteases with potent antialphaviral activity (Fig. 1)10. Although 1 met many of the criteria for a covalent chemical probe, it lacked chemical stability due to cyclization by an intramolecular aza-Michael reaction to an inactive cyclic dihydropyrazolo[1,5-a]pyrazin-4(5H)-one 211, which occurred slowly at physiological pH and rapidly under basic conditions. Furthermore, commercial samples of 1 were often contaminated with varying levels of 2 that could only be quantified by NMR or extended HPLC runs11. In this report, we describe the full characterization of the isoxazole analog 3 that maintains potency for inhibition of nsP2pro, demonstrates remarkable proteome-wide selectivity, and has the chemical stability required of a high-quality chemical probe. We also describe a high-quality negative control analog 4 that retains the isoxazole core and covalent warhead but has drastically reduced nsP2pro inhibition.

Results and discussion

Chemical probe characterization

Chemical properties.

A systematic structure–activity study of the 5-membered heterocyclic core in 1 was previously performed to identify bioisosteric replacements of the pyrazole with improved chemical stability12. Switching nitrogen to oxygen led to the identification of (E)−3-(2-ethoxyphenyl)-N-(3-(methylsulfonyl)allyl)isoxazole-5-carboxamide (SGC-NSP2PRO-1, 3) in which the isoxazole was no longer able to undergo intramolecular cyclization (Fig. 1). Isoxazole 3 was isolated as a free flowing white solid with m.p. 178 °C that was stable when stored at room temperature for 6 months. Isoxazole 3 was also stable in solution across a pH range from 3–12 with no evidence of degradation or cyclization (Supplementary Figure S1). Additional characterization of 3 showed that it had aqueous kinetic solubility of 20 µM, which was 500-fold above its IC50 for inhibition of nsP2pro12, and showed no evidence of aggregation by dynamic light scattering (Supplementary Figure S2). For measurement of intrinsic reactivity of the vinyl sulfone, 3 demonstrated a t1/2of 70 min in the presence of 5 mM GSH (Supplementary Figure S3), well above the minimum half-life recommended for covalent inhibitors9.

Fig. 1
figure 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.

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Biological activity

SGC-NSP2PRO-1 (3) was a potent covalent inhibitor of the CHIKV nsP2 protease with IC50= 40 nM12. To further characterize the kinetic properties of 3 as a covalent inhibitor, time dependent inactivation of CHIKV nsP2pro was measured at multiple inhibitor concentrations (Fig. 2a). Analysis of the kinetic inactivation data produced a kinact/Ki ratio of 5950 M–1 s–1, demonstrating that isoxazole 3 was an efficient nonpeptide covalent cysteine protease inhibitor (Fig. 2a). Isoxazole 3 also demonstrated potent inhibition of alphaviral replication using the CHIKV-nLuc and VEEV-nLuc reporter viruses with EC50= 0.05 and 0.5 µM, respectively12. To further profile 3 for potential pan-alphaviral activity it was tested for the reduction viral titer against infectious isolates of CHIKV, MAYV, and VEEV. Isoxazole 3 demonstrated a dose dependent 5–9 log decrease in viral titer (Fig. 2b) against this range of New and Old World alphaviruses indicating its potential for pan-antialphaviral efficacy. Isoxazole 3 was also active when assayed against an EEEV-nLuc replicon (Supplementary Figure S4). Importantly, 3 did not display cellular toxicity at concentrations up to 10 µM in human cells (A549ACE2 and HEK293) after 48 h exposure (Supplementary Figure S5).

Fig. 2
figure 2

(a) Isoxazole 3 inhibits CHIKV nsP2 protease activity in a time-dependent manner. 1.5-fold serial dilutions were used to determine Ki, kinact, and kinact/Ki 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.

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Target engagement

The CHIKV nsP2pro domain contains six cysteines in its protein sequence. Mass spectrometry was performed to demonstrate target engagement of isoxazole 3 with the CHIKV nsP2pro active site catalytic cysteine, corresponding to C478 using the numbering from full length nsP2. Purified nsP2pro was incubated with 1 µM of 3 in DMSO for 30 min, followed by tryptic digestion and LC–MS/MS analysis (n = 2). A single modification corresponding to the mass shift of vinyl sulfone 3 (+ 350.094) was identified at C478 on the ANVCWAK tryptic peptide (corresponding to aa 475–481 in nsP2), resulting from addition of the cysteine side chain to the β-carbon of the electrophilic warhead (Supplementary File S1 and Supplementary Figure S6). This modification was only identified in the samples treated with 3 and was absent in the DMSO controls. None of the other five nsP2pro cysteine residues were modified demonstrating the specificity of 3 for the active site catalytic C478 of CHIKV nsP2pro.

Cysteine protease selectivity

Isoxazole 3 was screened against a panel of 20 human cysteine proteases and three viral cysteine proteases at 10 µM in duplicate (Fig. 3 and Supplementary Table S1). Compared to control inhibitors, 3 demonstrated no significant inhibition against this panel of cathepsins, ubiquitin specific peptidases (USPs), other human cysteine proteases, and viral papain-like cysteine proteases. The data indicated that β-aminomethyl vinyl sulfone warhead in 3 was > 30-fold selective for CHIKV nsP2pro over the other cysteine proteases.

Fig. 3
figure 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.

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Proteome-wide selectivity. Isoxazole 3 was assessed in HEK293 cells by two independent chemoproteomics experiments: TAMRA labeling and biotin pulldown.

Fluorescence-based chemoproteomics. The selectivity of isoxazole 3 in human cells was evaluated by fluorescence-based chemoproteomics (Fig. 4). Clickable isoxazole VS was synthesized, as well as the corresponding fluorescent probe (TVS) containing a TAMRA dye (Fig. 4a). VS was a potent inhibitor of the CHIKV nsP2pro with IC50 = 65 nM and also demonstrated inhibition of CHIKV-nLuc replication with EC50 = 33 nM (Supplementary Figure S7). As a positive control, the fluorescent TVS demonstrated efficient labelling of purified full length nsP2 as indicated by fluorescence imaging of the expected band at 42.9 kDa (Fig. 4b, lane P). Human HEK293 cell lysates were incubated with VS (10 µM, 30 min), followed by the sequence of click reaction with the TAMRA fluorophore, separation of proteins on a denaturing polyacrylamide gel, and fluorescence imaging. In the HEK293 cells lysate, no human proteins were observed to be labeled by VS following TAMRA click reaction (Fig. 4B, lane A2). Incubation of cell lysates with VS, with addition of nsP2 protein, led to efficient TAMRA labeling of nsP2 (Fig. 4b, lane B2) with no labelling of any other proteins from the HEK293 cell lysate. In a control reaction, no TAMRA labelling of nsP2 was observed when the active site cysteine was blocked by preincubation with VSC lacking an alkyne (Fig. 4b, lane B1). As an additional positive control, clickable chloroacetamide CA was evaluated in the same TAMRA labelling experiments. Extensive labeling of the cell lysates was seen with CA (Fig. 4b, lane C2), and pre-incubation with VSC had no effect in reducing this labeling (Fig. 4b, lane C1). Purified nsP2 was also labeled in spiked samples of the HEK293 cell lysate (Fig. 4B, lane D2). Notably, pre-incubation with VSC did not block TAMRA labelling of the spiked nsP2 by CA (Fig. 4b, lane D1), suggesting that labelling might also occur on one or more of the 5 non-active site cysteines and providing evidence of promiscuous labelling by CA that was not seen with VS. Notably, a simple vinyl sulfone (VSS, Supplementary Figure S8) lacking the isoxazole core did not show strong labelling of either nsP2 or other proteins in HEK293 cell lysates, which was further evidence of the lack of promiscuity of the vinyl sulfone warhead. These TAMRA labeling experiments demonstrated the selectivity of 3, combing a vinyl sulfone warhead with an isoxazole core, for nsP2pro with an absence of labelling of other proteins in HEK293 cell lysate.

Fig. 4
figure 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.

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HEK Lysate Pull-Down. To further investigate the human proteome-wide selectivity of isoxazole 3, biotin-streptavidin pulldown experiments were conducted using HEK293 lysates to enrich for potential off-target proteins. Alkyne-modified isoxazole VS was incubated with HEK293 cell lysates for 30 min followed by click reaction with biotin azide. The mixture was incubated with magnetic streptavidin beads for 15 min to capture biotin-labelled proteins. The beads were washed with 1% SDS, 8 M urea, and 20% acetonitrile to remove non-specific binding proteins. The enriched proteins were released from the streptavidin beads by heating in SDS buffer. The proteins were run on a denaturing polyacrylamide gel and silver staining did not indicate any highly enriched proteins (Fig. 5a, lane 2 vs. 1), further supporting the apparent high selectivity of isoxazole 3 against the proteins in the HEK293 cell lysate. To further analyze for potential off-target proteins, lanes 1 and 2 were subjected to trypsin digestion and LC–MS/MS analysis. Proteins were identified and quantified using MaxQuant software and the resulting list was filtered to remove common contaminates from sample handling. Using an enrichment cut-off of log2fold-change > 2 (lane 2 over 1) and number of unique peptides > 213, a total of 14 proteins were identified representing candidate off-targets and non-specific proteins that were captured by the streptavidin beads (Supplementary File S2). To account for non-specific proteins captured by the beads, a parallel biotin-streptavidin pull down experiment was performed using cell lysate that was pre-incubated with isoxazole 3 (10 µM) for 30 min prior to addition of VS (Fig. 5a, lane 4). This control experiment indicated that proteins with up to fivefold enrichment in comparison to the cell lysate alone (lane 4 over 1) could be designated non-specific binding proteins, including all 14 of the candidate off-targets (Fig. 5b). A third biotin-streptavidin pull down was performed using HEK293 cell lysate that was spiked with purified nsP2 protein (4 µg) as a positive control (Fig. 5a, lane 3). In this experiment, LC–MS/MS analysis identified the control nsP2 protein, which was enriched sevenfold, as well as three candidate off-target proteins with > fivefold enrichment: a dynein heavy chain (DYNC1H1), a chromatid cohesion protein (PDS5A), and an apolipoprotein (APOD). Notably, none of these three candidate off-targets were identified in the biotin-streptavidin pull down with VS alone (Fig. 5a, lane 2), indicating that they are also unlikely to be specific off-targets of the vinyl sulfone. The combined results (Fig. 5b) demonstrate the remarkable proteome-wide selectivity of the vinyl sulfone in isoxazole 3, with no human cysteine proteases or other enzymes identified as off-targets.

Fig. 5
figure 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.

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Negative control analog

The methoxymethyl (MOM) derivative of pyrazole 1 (MOM-1, Supplementary Figure S9) had been nominated previously as a negative control of pyrazole 110. However, MOM-1 demonstrated residual inhibition of CHIKV nsP2pro at 10 µM and robust inhibition of CHIKV-nLuc replication at 1 µM that severely limited its utility as a negative control in cells. MOM-1 contained an acid labile protecting group that may degrade to generate the potent nsP2pro inhibitor pyrazole 1, which may be one reason for its antiviral activity in cells. Thus, we sought to identify a high-quality negative control analog of 3 with improved chemical stability and without residual nsP2pro inhibition. Analysis of the structure–activity relationships for inhibition of CHIKV nsP2pro12 identified (E)−3-(2-ethoxyphenyl)-N-isopropyl-N-(3-(methylsulfonyl)allyl)isoxazole-5-carboxamide (SGC-NSP2PRO-1N, 4) as a candidate negative control compound (Fig. 1). Isoxazole 4 contained an isopropyl substituent on the amide nitrogen but retained the critical vinyl sulfone covalent warhead as mandated in the covalent probe criteria9. The structure–activity relationship of the β-amido methyl vinyl sulfone chemotype12 had previously demonstrated that addition of an alkyl substituent on the amide nitrogen led to a reduction in activity as an nsP2pro inhibitor. In enzyme assays, 4 demonstrated very weak CHIKV nsP2pro inhibition with > 100-fold reduction in potency compared to isoxazole 3 (Fig. 6a). In addition, the isopropyl-substituted analog 4 was fully inactive in the CHIKV-nLuc replication assay at concentrations up to 10 µM (Fig. 6b). Isoxazole 4 was stable upon prolonged storage and in solution across a wide range of pH (Supplementary Figure S1) and thus meets the criteria9 of a high-quality negative control analog for the chemical probe 3.

Fig. 6
figure 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.

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Chemistry

Chemical probe and negative control

Synthesis of isoxazole 3 was accomplished as previously reported12. Negative control analog 4 was synthesized in two steps starting from (E)−3-bromo-1-(methylsulfonyl)prop-1-ene 5. Substitution of the bromine by 2-propanamine afforded (E)-N-isopropyl-3-(methylsulfonyl)prop-2-en-1-amine 6, which was followed by amide coupling with 7 to yield (E)−3-(2-ethoxyphenyl)-N-isopropyl-N-(3-(methylsulfonyl)allyl)isoxazole-5-carboxamide 4 (Fig. 7).

Fig. 7
figure 7

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

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Analogs for chemoproteomics

The clickable derivative VS containing an alkyne handle was synthesized by modification of the previously described methods (Fig. 8)12. Claisen condensation of acetophenone 8 with diethyl oxalate in presence of sodium hydride followed by cyclization with hydroxylamine hydrochloride yielded the isoxazole 9. Incorporation of the alkyne handle was achieved by propargylation of the hydroxy group to give 10. Saponification and amide coupling yielded the clickable derivative VS. A control analog VSC lacking the clickable alkyne group was synthesized by amide coupling reaction of the commercially available unsubstituted phenyl analog with the vinyl sulfone warhead. The pre-clicked control TVS was synthesized by reaction of VS with the TAMRA dye using a Cu-catalyzed azide-alkyne [3 + 2] cycloaddition reaction (Fig. 8). Clickable chloroacetamide (CA) was synthesized by the previously described method10.

Fig. 8
figure 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.

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Discussion

In this study, we describe the full characterization of isoxazole 3 as a high-quality covalent chemical probe for CHIKV nsP2pro. Isoxazole 3 meets all of the state-of-the-art requirements of a covalent chemical probe including fast kinetics of enzyme inactivation, proteome-wide selectivity, and potent cellular activity without toxicity9. Notably, vinyl sulfone 3 demonstrated pan-antialphaviral activity in infected cells and will be a powerful tool to study the role of nsP2pro in the replication, propagation, and virulence of New and Old World alphaviruses that remain a global public health threat3,14. A negative control analog 4 was identified that retained the structural features of the inhibitor 3, including the vinyl sulfone covalent warhead, but demonstrated > 100-fold reduction in potency against CHIKV nsP2pro and was inactive in antiviral assays. The β-aminomethyl vinyl sulfone 3 demonstrated remarkable selectivity for the nsP2 cysteine protease and showed no activity against a large panel of cathepsins, ubiquitin specific peptidases, other human cysteine proteases, and viral Papain-like proteases. Chemoproteomic profiling from HEK293 cells using two orthogonal techniques further demonstrated the remarkable proteome-wide selectivity profile of 3.

Vinyl sulfones are often listed as highly reactive covalent warheads that are promiscuous cysteine electrophiles15,16. However, there is a distinct difference in electrophilicity between internal vinyl sulfones as found in 1 and 3 and external vinyl sulfones which are found in many of the promiscuous electrophiles. Three internal vinyl sulfones are currently under clinical development: K777, a parasite cysteine protease inhibitor17; VVD-133214, a WRN helicase inhibitor18; and rigosertib, a multi-kinase inhibitor targeting PI3K and PLK19. In contrast, no external vinyl sulfone is currently listed in clinical development. K777, a peptidyl-styryl sulfone, demonstrated selective targeting of cathepsin B and L in Vero E6 cells20. VVD-133214, a methyl vinyl sulfone, selectively engaged C727 located in the helicase domain of WRN and demonstrated proteome-wide selectivity over other cellular proteins18. Rigosertib, a benzyl styryl sulfone, was a selective non-ATP-competitive PLK1 and PI3K inhibitor21but also interacted with RAS signaling proteins22 and its full proteome selectivity data has not been reported. We now add the nsP2 cysteine protease inhibitors 1 and 3 as examples of internal vinyl sulfones that act as covalent modifiers of their target enzyme with high proteome-wide selectivity. These examples demonstrate that internal vinyl sulfones are warheads that warrant additional study as chemotypes for development of covalent cysteine protease inhibitors and candidate drug molecules15.

In summary, we describe the full characterization of SGC-NSP2PRO-1 (3) as a high-quality chemical probe and SGC-NSP2PRO-1N (4) as a negative control analog that can be used to study the biological role of the viral nsP2pro. Isoxazole vinyl sulfone 3 meets the full range of biological and chemical criteria recently proposed for a high-quality chemical probe (Table 1)9. Isoxazole 3 has poor pharmacokinetic properties in mice, which limits its utility to in vitro and cellular studies. However, its remarkable potency and proteome-wide selectivity make it an ideal chemical probe for study of alphaviral replication and virulence in cell culture.

Table 1 Summary of the nsP2pro chemical probe (3) and negative control analog (4).
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Methods

General chemistry methods

All reactions were conducted in oven-dried glassware under a dry nitrogen atmosphere unless otherwise specified. All reagents and solvents were obtained from commercial sources and used without further purification. No unexpected safety hazards were encountered during the synthesis. Analytical thin layer chromatography (TLC) was performed on pre-coated silica gel plates (200 μm, F254 indicator), visualized under UV light or by staining with iodine and KMnO₄. Column chromatography utilized pre-loaded silica gel cartridges on a Biotage automated purification system. 1H and 13C NMR spectra were recorded in DMSO-d₆ and CD3CN at 400/500/700 and 101/126/176 MHz, respectively, on a Bruker spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane for 1H NMR, with major peaks designated as s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). High-resolution mass spectrometry (HRMS) analyses were performed at the UNC Department of Chemistry Mass Spectrometry Core Laboratory using a Q Exactive HF-X mass spectrometer. Liquid chromatography-mass spectrometry (LC–MS) was conducted on an Agilent 1290 Infinity II LC System with an Agilent Infinity Lab PoroShell 120 EC-C18 column (30 °C, 2.7 μm, 2.1 × 50 mm), employing a 5 − 95% CH₃CN in water eluent, with 0.2% (v/v) formic acid as the modifier and a flow rate of 1 mL/min. Preparative high-performance liquid chromatography (HPLC) was executed using an Agilent 1260 Infinity II LC System equipped with a Phenomenex C18 column (PhenylHexyl, 30 °C, 5 μm, 75 × 30 mm), with a 5 − 95% CH₃CN in water eluent and 0.05% (v/v) trifluoroacetic acid as the modifier, at a flow rate of 30 mL/min. Analytical HPLC data were recorded on a Waters Alliance HPLC with a PDA detector or an Agilent 1260 Infinity II series with a PDA detector (EC-C18, 100 mm × 4.6 mm, 3.5 μm), using a 10 − 90% CH₃CN in water eluent at a flow rate of 1 mL/min. The final compounds were confirmed to be > 95% pure by HPLC analysis. Isoxazole 3, 3-(2-ethoxyphenyl)isoxazole-5-carboxylic acid (7), and clickable chloroacetamide CAwere synthesized following the reported procedures10,12. 6-TAMRA azide, (N-(9-(5-((6-azidohexyl)carbamoyl)−2-carboxyphenyl)−6-(dimethylamino)−3H-xanthen-3-ylidene)-N-methylmethanaminium), was purchased from Vector Laboratories (catalog number CCT-1246).

(E)−3-(2-ethoxyphenyl)-N-isopropyl-N-(3-(methylsulfonyl)allyl)isoxazole-5-carboxamide (4)

To a stirred solution of (E)−3-bromo-1-(methylsulfonyl)prop-1-ene (5, 0.5 g, 2.5 mmol, 1.0 eq.) in DCM (10 mL) were added propan-2-amine (0.15 g, 2.5 mmol, 1.0 eq.) and Cs2CO3 (1.23 g, 3.77 mmol, 1.5 eq.) at 0 °C and stirred at 25 °C for 2 h. On completion of the reaction based on TLC and LCMS, the mixture was filtered through celite bed and concentrated under vacuum to afford crude (E)-N-isopropyl-3-(methylsulfonyl)prop-2-en-1-amine (6) (0.41 g, 92% yield) as a colorless liquid. MS (ESI) m/z: 178.2 [M + H]+.

To a stirred solution of carboxylic acid 7 (0.25 g, 1.07 mmol, 1.0 eq.) in DMF (5.0 mL) were added amine 6 (0.23 g, 1.3 mmol, 1.2 eq.), TBTU (0.41 g, 1.2 mmol, 1.2 eq.), DIPEA (0.4 mL, 2.1 mmol, 2.0 eq.) and the reaction was stirred at 25 °C for 2 h. On completion of the reaction based on TLC and LCMS, the mixture was diluted with water (30 mL), organic layer extracted with EtOAc (3 × 50 mL), washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude was purified by preparative HPLC to afford (E)−3-(2-ethoxyphenyl)-N-isopropyl-N-(3-(methylsulfonyl)allyl)isoxazole-5-carboxamide (4) (0.05 g, 12%) as a white sticky solid. 1H NMR (400 MHz, DMSO-d6): δ 7.89 (d, J = 7.7 Hz, 1H), 7.55 – 7.45 (m, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.02 (s, 1H), 6.80 (s, 2H), 4.25 (q, J = 7.0 Hz, 5H), 2.99 (s, 3H), 1.44 (t, J = 7.0 Hz, 3H), 1.22 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, DMSO-d6): δ 165.9, 160.8, 159.3, 155.4, 142.4, 132.4, 131.1, 127.2, 120.8, 114.7, 112.9, 103.2, 64.1, 50.1, 42.2, 40.6, 20.8, 19.7, 14.5. HRMS (ESI) m/z: [M + H]+ calculated for C19H25N2O5S: 393.1484, found 393.1481. HPLC purity (254 nm) > 99%.

(E)-N-(3-(methylsulfonyl)allyl)−3-(2-(prop-2-yn-1-yloxy)phenyl)isoxazole-5-carboxamide (VS)

To a stirred solution of 1-(2-hydroxyphenyl)ethan-1-one (8) (5 g, 36.8 mmol, 1.0 eq.) in toluene (200 mL) was added NaH (7.4 g, 184.1 mmol, 5.0 eq.) portion-wise at 0 °C, and the reaction was stirred at 25 °C for 0.5 h. Then diethyl oxalate (7.5 mL, 55.2 mmol, 1.5 eq.) was added and the reaction was stirred at 60 °C for 2 h. On completion of the reaction based on TLC and LCMS analysis, the reaction was poured into water (200 mL), quenched by 1 N HCl solution till pH ~ 3, extracted with EtOAc (2 × 200 mL), combined organic layers dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude was purified by combiflash (eluted with 20% EtOAc in hexane) to afford ethyl 4-(2-hydroxyphenyl)−2,4-dioxobutanoate (4.6 g, 57% yield) as colorless oil. MS (ESI) m/z: 237.4 [M + H]+. The product was dissolved in EtOH (50 mL), hydroxylamine hydrochloride (2.7 g, 38.9 mmol, 2.0 eq.) was added and the reaction was stirred at 90 °C for 6 h. On completion of the reaction based on TLC and LCMS, the mixture was diluted with water (200 mL), organic layer extracted with EtOAc (2 × 200 mL), washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude was purified by combiflash (eluted with 10% EtOAc in hexane) to afford ethyl 3-(2-hydroxyphenyl)isoxazole-5-carboxylate (9) (1.4 g, 30% yield) as a white solid. MS (ESI) m/z: 234.4 [M–H]–.

To a stirred solution of 9 (1.4 g, 6.0 mmol, 1.0 eq.) in DMF (14 mL) were added K2CO3 (1.7 g, 12.0 mmol, 2.0 eq.) and 3-bromoprop-1-yne (0.9 g, 7.2 mmol, 1.2 eq.). The reaction mixture was stirred at 25 °C for 16 h. On completion of the reaction based on TLC and LCMS, the mixture was diluted with water (10 mL), organic layer extracted with EtOAc (2 × 10 mL), washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude was purified by combiflash (eluted with 30% EtOAc in hexane) to afford ethyl 3-(2-(prop-2-yn-1-yloxy)phenyl)isoxazole-5-carboxylate (10) (0.7 g, 43% yield) as white solid. MS (ESI) m/z: 272.1 [M + H]+.

To a stirred solution of 10 (0.7 g, 2.6 mmol, 1.0 eq.) in MeOH (7 mL) were added NaOH (0.3 g, 5.2 mmol, 2.0 eq.) and water (4 mL). The reaction mixture was stirred at 25 °C for 1 h. On completion of the reaction based on TLC and LCMS, the reaction mixture was diluted with water (20 mL), acidified by 1N HCl till pH ~ 3, extracted with DCM (2 × 20 mL), combined organic layers dried over anhydrous Na2SO4, filtered, and concentrated to afford 3-(2-(prop-2-yn-1-yloxy)phenyl)isoxazole-5-carboxylic acid (0.6 g, 88% yield). MS (ESI) m/z: 244.2 [M–H]-. To a stirred solution of this carboxylic acid (0.2 g, 0.8 mmol, 1.0 eq.) in pyridine (2.0 mL) were added (E)−3-(methylsulfonyl)prop-2-en-1-amine12 (0.1 g, 0.8 mmol, 1.0 eq.), TBTU (0.4 g, 1.2 mmol, 1.5 eq.) and the reaction was stirred at 25 °C for 1 h. On completion of the reaction based on TLC and LCMS, the mixture was diluted with water (10 mL), organic layer extracted with EtOAc (2 × 10 mL), washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The resulting crude was purified by combiflash (eluted with 50% EtOAc in hexane), followed by preparative HPLC to afford (E)-N-(3-(methylsulfonyl)allyl)−3-(2-(prop-2-yn-1-yloxy)phenyl)isoxazole-5-carboxamide (VS) (0.08 g, 27% yield) as a white solid: m.p. 173 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.24 (t, J = 5.8 Hz, 1H), 7.90 (dd, J = 8.1, 1.7 Hz, 1H), 7.62 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.44 (dd, J = 8.5, 1.2 Hz, 1H), 7.36 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.18 (s, 1H), 6.80 (d, J = 1.6 Hz, 2H), 4.82 (d, J = 2.4 Hz, 2H), 4.14 (dd, J = 5.9, 2.4 Hz, 2H), 3.51 (t, J = 2.4 Hz, 1H), 3.00 (s, 3H). 13C NMR (100 MHz, DMSO-d6): 159.5, 150.7, 148.2, 143.1, 142.1, 131.8, 130.5, 125.7, 122.6, 118.1, 117.0, 98.2, 80.3, 77.5, 61.6, 42.1, 39.2. HRMS (ESI) m/z: [M + H]+ calculated for C17H17N2O5S: 361.0858, found 361.0845. HPLC purity (254 nm) > 99%.

(E)-N-(9-(2-carboxy-4-((6-(4-((2-(5-((3-(methylsulfonyl)allyl)carbamoyl)isoxazol-3-yl)phenoxy)meth-yl)−1H-1,2,3-triazol-1-yl)hexyl)carbamoyl)phenyl)−6-(dimethylamino)−3H-xanthen-3-ylidene)-N-methyl methanaminium (TVS)

VS (5.5 mg, 0.015 mmol, 1.0 eq.) was mixed with 6-TAMRA azide, 10.0 mg, 0.016 mmol, 1.1 eq.), CuSO4.5H2O (0.4 mg, 0.0015 mmol, 0.1 eq.), and Na-ascorbate (0.6 mg, 0.003 mmol, 0.2 eq.) in DMSO (2.0 mL) for 1 h at 25 °C. On completion of the click reaction based on TLC and LCMS, solvent was removed in vacuo and the crude was purified by preparative HPLC to afford the desired pre-clicked vinyl sulfone (TVS) as a pink oil (5.0 mg, 36% yield). 1H NMR (700 MHz, CD3CN): δ 8.30 (d, J = 8.3 Hz, 1H), 8.08 (dd, J = 8.3, 1.8 Hz, 1H), 7.94 – 7.89 (m, 2H), 7.82 – 7.74 (m, 2H), 7.52 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H), 7.38 (t, J = 5.9 Hz, 1H), 7.35 – 7.26 (m, 2H), 7.13 – 7.05 (m, 3H), 6.87 (dd, J = 9.5, 2.5 Hz, 2H), 6.79 (dt, J = 15.2, 4.5 Hz, 1H), 6.75 (d, J = 2.4 Hz, 2H), 6.57 (dt, J = 15.2, 1.9 Hz, 1H), 5.17 (s, 2H), 4.30 (t, J = 6.8 Hz, 2H), 4.09 (ddd, J = 6.2, 4.5, 1.9 Hz, 2H), 3.29 (q, J = 6.5 Hz, 2H), 3.21 (s, 10H), 2.86 (s, 3H), 2.51 (s, 1H), 1.83 (p, J = 7.1 Hz, 2H), 1.48 (p, J = 7.0 Hz, 2H), 1.34 – 1.28 (m, 3H), 1.27 – 1.21 (m, 3H). 13C NMR (176 MHz, CD3CN): δ 166.6, 166.2, 160.9, 160.0, 158.4, 158.3, 151.9, 148.7, 144.7, 143.9, 143.6, 139.5, 135.1, 133.9, 132.5, 131.9, 131.4, 129.7, 129.6, 126.6, 124.9, 123.8, 119.8, 118.8, 116.6, 115.2, 114.4, 99.7, 97.2, 68.5, 50.6, 42.9, 41.3, 41.2, 40.3, 40.1, 30.6, 29.8, 26.6, 26.5. HRMS (ESI) m/z: [M + H]+ calculated for C48H52N8O9S: 916.3572, found 916.3529. HPLC purity (254 nm) > 99%.

(E)-N-(3-(methylsulfonyl)allyl)−3-phenylisoxazole-5-carboxamide (VSC)

To a stirred solution of 3-phenylisoxazole-5-carboxylic acid (0.2 g, 1.1 mmol, 1.0 eq.) and TBTU (0.5 g, 1.6 mmol, 1.5 eq.) in pyridine (5 mL) was added (E)−3-(methylsulfonyl)prop-2-en-1-amine12 (0.18 g, 1.1 mmol, 1.0 eq.) and the reaction was stirred at 25 °C for 2 h. On completion of the reaction (based on TLC and LCMS analysis), the reaction was poured into water, extracted with EtOAc, combined organic layers dried over anhydrous Na2SO4, filtered, and concentrated. The crude was purified by preparative HPLC to afford (E)-N-(3-(methylsulfonyl)allyl)−3-phenylisoxazole-5-carboxamide (VSC) as a white solid (0.15 g, 34% yield,): m.p 164 °C. 1H NMR (500 MHz, DMSO-d6): δ 9.39 (t, J = 5.8 Hz, 1H), 7.96 – 7.91 (m, 2H), 7.69 (s, 1H), 7.55 (dt, J = 4.6, 2.8 Hz, 3H), 6.86 – 6.77 (m, 2H), 4.15 (dd, J = 5.7, 3.1 Hz, 2H), 3.01 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 163.9, 162.6, 155.7, 142.1, 130.7, 130.5, 129.3, 127.8, 126.7, 104.9, 42.1, 38.9. HRMS (ESI) m/z: [M + H]+ calculated for C14H15N2O4S: 307.0753, found 307.0741. HPLC purity (254 nm) > 99%.

General biological methods

The CHIKV nsP2pro (UniProt ID: F2YI08), CHIKV-nLuc viral replication, VEEV-nLuc viral replication, and alphavirus (CHIKV, MAYV, and VEEV) titer reduction assays were performed using the reported protocols10.

nsP2pro binding kinetics

Time-dependent enzyme inhibition experiment was performed as described by Mons et al.23 An internally quenched peptide substrate derived from the CHIKV 1/2 cleavage site (QLEDRAGA/GIIETPRG) was designed with an N-terminal QSY®−21 quencher and a C-terminal Cy5 fluorophore (Lifetein). For time-dependent enzyme inhibition experiment, 5 µL of peptide substrate (20 µM) was added to 1.5-fold serially diluted isoxazole 3, and the reaction was initiated by the addition of 5 µL CHIKV nsP2 protease (300 nM). The reaction progress was monitored and read continuously over 2 h using a PerkinElmer Envision 2105 plate reader, using excitation and emission wavelengths of 620 nm and 685 nm respectively, and using a dichroic mirror with a cutoff at 658 nm. Raw data were normalized to background fluorescence and the time course plots were fit with Equation 1 to determine kobs (kobs = (kinact x [I]) / (Ki + [I]) and replotted against inhibitor concentration to determine Ki and kinact values for isoxazole 3.

Equation 1: \(F_t=\frac{v_i}{k_{obs}}[1-e^-k_{obs}{t}]\) where Ft = time-dependent signal resulting from product formation (in AU), vi = initial product formation velocity (in AU/s), t = incubation time after enzyme addition (in s), kobs = observed rate of time-dependent inhibition from initial to final (in s−1).

EEEV-nLuc replicon assay

To generate an EEEV-nLuc replicon plasmid construct (pEEEV-V105-nLuc-Rep), the EEEV strain V105 cDNA sequence (GenBank KP282670), encoding the nanoluciferase (nLuc) gene sequence (GenBank AFI79295) in place of the viral structural genes, was cloned downstream of the 26S subgenomic promoter and upstream of the 3’ UTR in a manner identical to the viral structural gene open reading frame. The EEEV V105 cDNA sequence was modified to match type-specific consensus sequences in the 5’ and 3’ UTRs that were missing from the strain sequence as previously described24. EEEV-V105-nLuc replicon RNA encoding the viral nonstructural proteins and the nLuc gene was synthesized by in vitro transcription by mMessage mMachine SP6 transcription (Invitrogen; AM1340) using linearized, sequence-confirmed pEEEV-V105-nLuc-Rep plasmid as a template. Proper synthesis of viral replicon RNA was confirmed by gel electrophoresis and the RNA concentration was measured by UV spectrometry. Low-passage normal human dermal fibroblasts (ATCC PCS-201–012) that had been maintained at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle medium (Gibco; 11,965–084) supplemented with 10% fetal bovine serum and 100 U/mL penicillin–streptomycin were seeded into black 96-well plates (Greiner Bio-One; 655,086) at a density of 5.7 × 103 cells per well in a volume of 100 μl and cultured overnight. The next day, 1 μg of EEEV-V105-nLuc replicon RNA was transfected into the cells using Lipofectamine MessengerMAX Reagent (Invitrogen; LMRNA015) according to the manufacturer’s instructions. Following RNA transfection, the transfection medium was removed and replaced with culture medium supplemented with twofold serial dilutions of 3 (0.16–20 μM). Control cells were maintained in an equal volume of culture medium supplemented with DMSO. At 24 h post-treatment, nLuc activity was quantified using the Nano-Glo Luciferase Assay System (Promega; N1120) according to the manufacturer’s instructions. Luminescence was measured on a Tecan infinite M plex plate reader with automatic attenuation luminescence setting and 1000 ms integration time.

Cysteine protease selectivity assays

Cysteine protease inhibition assays were performed in duplicate at BPS Bioscience Inc., San Diego, CA. A solution of 3 ten-fold higher than the final concentration was prepared with 10% DMSO in assay buffer and 5 µL of the dilution was added to a 50 µL reaction so that the final concentration of DMSO was 1% in all of the reactions. All control samples, including background and no compound controls, also contain 1% DMSO. The compounds were pre-incubated in duplicate at room temperature for 30 min in a mixture containing assay buffer, enzyme, and isoxazole 3. For cathepsin assays, the enzymatic reactions were conducted in duplicate at room temperature for 30 min in a 100 µL mixture containing 50 mM MES buffer, pH 5.0, 100 mM NaCl, 5 mM DTT, a cathepsin substrate, a cathepsin enzyme, and isoxazole 3. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 microplate reader. For cathepsin D and E, fluorescence intensities were measured at an excitation of 330 nm and an emission of 390 nm using a Tecan Infinite M1000 microplate reader. For deubiquitinase assays, the enzymatic reactions were conducted in duplicate at room temperature for 30 min in a 50 µL mixture containing 50 mM Tris–HCl, pH 7.4, 0.5 mM EDTA, 0.05% Tween 20, 1 mM DTT, 100 nM Ubiquitin-AMC substrate, a deubiquitinase enzyme, and isoxazole 3. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 microplate reader. For MALT1 assay, the MALT1 and inhibitor 3 were pre-incubated for 30 min at room temperature. Then, the enzymatic reactions were begun in duplicate with the addition of the substrate at room temperature for 30 min. This reaction contained a 50 µL mixture containing 50 mM HEPES, pH 8.0, 150 mM NaCl, 1 M Na Citrate, 0.05% CHAPS, 20 µM Ac-LRSR-AMC, MALT1 enzyme, and inhibitor 3. For the Papain-like protease (SARS-CoV-2) the assay kit was pre-incubated in duplicate at 37 °C, while for the 3CL Protease (MERS-CoV) and 3CL Protease (B.1.1.529, Omicron Variant) (SARS-CoV-2) the assay kits were pre-incubated in duplicate at room temperature for 30 min in a mixture containing assay buffer, enzyme, and 3. After 30 min, the enzymatic reactions were initiated by the addition of the substrate. The enzymatic reaction proceeded for 30 min to 4 h at either 37 °C or room temperature, respectively. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 microplate reader. The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of 3, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of the enzyme, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity = (F-Fb)/(Ft-Fb), where F = the fluorescent intensity in the presence of 3.

nsP2 target engagement

5 µL of purified nsP2 protease (2 mg/mL, 2.33 µM) was diluted in 93 µL of reaction buffer (25 mM HEPES pH 7.3, 1 mM DTT, 1% DMSO). 2 µL of a 50 µM stock solution in buffer of isoxazole 3 (final concentration 1 µM) or 2 µL of DMSO (prepared in the same dilutions as for the compound) was added and the mixture incubated at 24 °C for 30 min. 100 μL of each sample were reduced with 5 mM DTT at 37 ºC for 45 min and alkylated with 15 mM iodoacetamide in the dark for 45 min. Samples were digested with trypsin (Promega, 1:20 w/w) overnight at 37 ºC. The resulting peptide samples were acidified to 0.1% trifluoracetic acid, desalted using C18 spin columns (Pierce), and the eluates were dried via lyophilization. The samples were analyzed by LC–MS/MS using an Easy nLC 1200 coupled to a QExactive HF mass spectrometer (Thermo Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id × 25 cm, 2 μm particle size) (Thermo Scientific) and separated over a 120 min method. The gradient for separation consisted of 5–38% mobile phase B at a 250 nL/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN. The QExactive HF was operated in data-dependent mode where the 15 most intense precursors were selected for subsequent fragmentation. Resolution for the precursor scan (m/z 350–1600) was set to 60,000 with a target value of 3 × 106 ions. MS/MS scans resolution was set to 15,000 with a target value of 1 × 105 ions, 100 ms injection time. The normalized collision energy was set to 27% for HCD. Dynamic exclusion was set to 30 s, peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and ≥ 8 were excluded. Raw data was first processed using PMi-Byonic (Protein Metrics, version 5.1.1) with an open search to detect any unexpected deviation from the hypothesized mass shift corresponding to the isoxazole 3 (+ 351.10147 Da). Files were searched against the CHIKV-nsP2 sequence, and a wildcard open search was selected with a mass shift range of 250–450 Da mass shift on cysteine residues. Results were filtered for > 250 PSM Score, < 0.001 protein-aware posterior error probability, and > 10 modification score. A high-confidence mass shift of + 350.094, likely corresponding to the isoxazole 3, was detected in the treated samples but not the controls. Data was then processed using Proteome Discoverer (Thermo Scientific, version 3.1) and searched against a reviewed Uniprot E.coli BL21 database (containing 4,156 sequences; downloaded April 2023), appended with the CHIKV-nsP2 sequence, and a common contaminants database (MaxQuant, 245 sequences), using the Sequest HT search algorithm within Proteome Discoverer. Enzyme specificity was set to trypsin, up to two missed cleavage sites were allowed; Isoxazole 3 (+ 351.10147, + 350.094 Da) and carbamidomethylation of Cys, as well as oxidation of Met were set as variable modifications. A precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.02 Da were used. Label-free quantification (LFQ) was enabled. Peptides were filtered based on a 1% false discovery rate (FDR). PSM fragment ion data was exported from Proteome Discoverer and inputted into the Interactive Peptides Spectral Annotator tool25.

Fluorescence-based chemoproteomics

HEK293 cells were lysed by sonication and the resulting lysates (~ 3.5 mg/mL) diluted in reaction buffer (25 mM HEPES pH 7.3, 1 mM DTT, 1% DMSO). 2 µL of a 250 µM stock solution in DMSO of clickable vinyl sulfone (VS) or clickable chloroacetamide (CA) were added to 40 µL of cell lysate (~ 140 µg total protein) and the mixtures were incubated at 24 °C for 30 min. Equal volumes of THPTA (5 mM stock in H2O) and CuSO4.H2O (10 mM stock in H2O) were premixed to generate the ligand-copper solution. For the click reaction, freshly prepared solutions of 10 µL 6-TAMRA azide (50 µM stock in buffer), 20 µL ligand-copper premix, and 10 µL sodium ascorbate (10 mM stock in H2O) were added successively to cell lysates, and the mixture was incubated in the dark at 24 °C for 1 h. On completion, 45 µL of the reaction was mixed with 45 µL of SDS-PAGE sample buffer [1:5 volumes of 10 × reducing agent (NuPAGE; cat. NP0009) to 2X SDS sample buffer (Novex, cat. LC2676)] and heated at 85 °C for 5 min. Samples were separated in a 4–12% Tris–Glycine SDS-PAGE (Novex, catalog number XP04125BOX). The TAMRA labeling was visualized on an Invitrogen iBrightFL1000 (515–545 nm excitation; 568–617 nm emission). Total protein was visualized following Coomassie staining. For reactions containing VSC, the competitor compound was added to the cell lysate (2 µL of a 240 µM stock solution in DMSO, final concentration 10 µM) and incubated at 24 °C for 30 min prior to the addition of the VS or CA. For reactions containing nsP2 protease, 4 µg of the purified protein was added to the cell lysate prior to the addition of any compound. Volumes of each reaction component is shown in Table 2.

Table 2 Volumes of chemoproteomics reaction components.
Full size table

HEK293 Cell lysate pull-down

For biotin-streptavidin pull-down experiments, 2 µL of a 300 µM DMSO stock solution of clickable vinyl sulfone VS was added to 50 µL of HEK293 cell lysate (~ 170 µg total protein) and the mixtures were incubated at 24 °C for 30 min. Equal volumes of THPTA (6 mM stock in H2O) and CuSO4.H2O (12 mM stock in H2O) were premixed to generate the ligand-copper solution. For the click reaction, freshly prepared solutions of 10 µL biotin-PEG3-azide (60 µM stock in buffer), 20 µL ligand-copper premix, and 10 µL sodium ascorbate (12 mM stock in H2O) were added successively to cell lysates, and the mixture was incubated in the dark at 24 °C for 1 h. On completion, the reaction mixture was added to 30 µL of pre-equilibrated (3 washes with reaction buffer) streptavidin magnetic beads (NEB, cat. S1420S) and incubated at 24 °C for 15 min. The supernatant was removed, and the beads washed with 100 µL of the following, successively: 3 × 1% SDS in H2O, 1 × reaction buffer, 3 × 8 M urea in H2O, 1 × reaction buffer, 3 × 20% acetonitrile in H2O, 1 × reaction buffer. 40 µL of SDS-PAGE sample buffer [1:5 volumes of 10X reducing agent (NuPAGE; catalog number NP0009) to 2X SDS sample buffer (Novex, cat. LC2676)] was added to the beads and the mixture heated at 85 °C for 5 min. Samples were separated in a 4–12% Tris–Glycine SDS-PAGE (Novex, catalog number XP04125BOX). For control experiments cell lysate diluted in reaction buffer was pre-incubated with 2 µL of 3 (final concentration 1 µM) at 24 °C for 30 min. 2 µL of VS (final concentration 1 µM) was then added and the mixture incubated at 24 °C for another 30 min. Click reaction was carried out using the procedure described above. A control sample consisting of 50 µL cell lysate, 10 µL buffer and 40 µL H2O was incubated with 30 µL of pre-equilibrated magnetic streptavidin beads at 24 °C for 15 min. The beads were washed and prepared following the procedure described above.

For mass spectrometry (MS) analysis, SDS-PAGE gel lanes were excised and cut into 1 mm cubes and destained overnight. The samples were reduced with 5 mM DTT and alkylated with 15 mM iodoacetamide, then digested with 0.5 µg/µL of trypsin overnight at 37 °C. Peptides were extracted from the gel and cleaned using ZipTip pipette tips. The samples were analyzed as duplicate injections by LC–MS/MS using an Easy nLC 1200 coupled to a QExactive HF mass spectrometer (Thermo Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id × 25 cm, 2 μm particle size) (Thermo Scientific) and separated over a 45 min gradient. The gradient for separation consisted of 5–38% mobile phase B at a 250 nL/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN. The QExactive HF was operated in data-dependent mode where the 15 most intense precursors were selected for subsequent fragmentation. Resolution for the precursor scan (m/z 350–1600) was set to 60,000 with a target value of 3 × 106 ions. MS/MS scans resolution was set to 15,000 with a target value of 1 × 105 ions, 100 ms injection time. The normalized collision energy was set to 27% for HCD. Dynamic exclusion was set to 30 s, peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and ≥ 8 were excluded. Raw data were processed using Proteome Discoverer (Thermo Scientific, version 3.1). Raw data were searched against a reviewed Uniprot human database (containing 20,355 sequences; downloaded January 2024), appended with a common contaminants database (MaxQuant, 245 sequences), and the CHIKV-nsP2 sequence. Enzyme specificity was set to trypsin, up to two missed cleavage sites were allowed; carbamidomethylation of Cys was set a static modification, oxidation of Met was set as a variable modification. A precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.02 Da were used. Label-free quantification (LFQ) was enabled. Data were filtered based on a 1%/5% peptide/protein FDR, a minimum of 2 peptides, and quantitation in min. 2 samples was required for further analysis. LFQ abundances were Log2 transformed and missing values were imputed from a normal distribution with width 0.3 and downshift of 1.8. Statistical analysis was performed in Perseus (version 1.6.14.0) using the imputed values. Student’s t-tests (unpaired) were performed for the inhibitor-treated to control comparisons, and a p-value < 0.05 was considered statistically significant. LFQ log2 fold change ratios were calculated, and an absolute Log2 ratio ± −1 was considered significant.