CHEMICAL PROBES AND INHIBITORS FOR POLYPEPTIDES OF SARS CORONAVIRUSES

Abstract

The present invention relates to a compound which can be used in the treatment of infections caused by SARS coronaviruses, e.g. by blocking the active site of the proteases 3CL.sup.pro and PL.sup.pro. The compound can be used as inhibitor or label as such or can be used in screening methods for profiling other inhibitors. Moreover, the present invention relates to a method of producing the compound.

Claims

1. A compound represented by the following general formula (1), ##STR00011## wherein one of R.sup.1 to R.sup.5 is selected from the group consisting of an azide group, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a biotin group, an aryl group, and a heteroaryl group, —NX.sup.1X.sup.2, —OX.sup.3, —SX.sup.4, —C(O)X.sup.5, —C(O)NX.sup.6X.sup.7, —COOX.sup.8, and —SO.sub.3X.sup.9, wherein X.sup.1 to X.sup.9 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, and a heteroaryl group, wherein the one of R.sup.1 to R.sup.5 contains an azide group, an alkynyl group, a biotin group, or a fluorophoric group; the remaining four of R.sup.1 to R.sup.5 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a halogen atom, —NE.sup.1E.sup.2, —NO.sub.2, —CN, —OE.sup.3, —SE.sup.4, —C(O)E.sup.5, —C(O)NE.sup.6E.sup.7, —COOE.sup.8, and —SO.sub.3E.sup.9, wherein E.sup.1 to E.sup.9 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, and a heteroaryl group, and wherein R.sup.1 to R.sup.5 may bind to each other to form one or more rings; R.sup.6, R.sup.7, and R.sup.10 to R.sup.12 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a halogen atom, —NA.sup.1A.sup.2, —NO.sub.2, —CN, —OA.sup.3, —SA.sup.4, —C(O)A.sup.5, —C(O)NA.sup.6A.sup.7, —COOA.sup.8, and —SO.sub.3A.sup.9, wherein A.sup.1 to A.sup.9 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, and a heteroaryl group, and wherein R.sup.6 and R.sup.7 and/or R.sup.10 to R.sup.12 may bind to each other to form one or more rings; and R.sup.8 and R.sup.9 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, and a heteroaryl group; or a pharmaceutically acceptable salt thereof; and wherein the alkyl groups, the cycloalkyl groups, the alkenyl groups, the cycloalkenyl groups, the alkynyl groups, the aryl groups, and the heteroaryl groups may independently be (further) substituted or unsubstituted and the alkyl groups, the alkenyl groups, and the alkynyl groups may independently be branched or linear.

2. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier, excipient or diluent.

3. A method for the treatment and/or prevention of an infection or condition in a subject, comprising administering an effective amount of the compound according to claim 1 to the subject.

4. The method of claim 3, wherein the infection or condition is caused by a SARS coronavirus.

5. A production method for producing a compound represented by the following general formula (1), ##STR00012## wherein the method comprises reacting a compound represented by the following general formula (2) ##STR00013## with a compound represented by the following general formula (3), ##STR00014## wherein one of R.sup.1 to R.sup.5 is selected from the group consisting of an azide group, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, a biotin group, an aryl group, and a heteroaryl group, —NX.sup.1X.sup.2, —OX.sup.3, —SX.sup.4, —C(O)X.sup.5, —C(O)NX.sup.6X.sup.7, —COOX.sup.8, and —SO.sub.3X.sup.9, wherein X.sup.1 to X.sup.9 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, and a heteroaryl group, wherein the one of R.sup.1 to R.sup.5 contains an azide group, an alkynyl group, a biotin group, or a fluorophoric group; the remaining four of R.sup.1 to R.sup.5 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a halogen atom, —NE.sup.1E.sup.2, —NO.sub.2, —CN, —OE.sup.3, —SE.sup.4, —C(O)E.sup.5, —C(O)NE.sup.6E.sup.7, —COOE.sup.8, and —SO.sub.3E.sup.9, wherein E.sup.1 to E.sup.9 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, and a heteroaryl group, and wherein R.sup.1 to R.sup.5 may bind to each other to form one or more rings; R.sup.6, R.sup.7, and R.sup.10 to R.sup.12 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a halogen atom, —NA.sup.1A.sup.2, —NO.sub.2, —CN, —OA.sup.3, —SA.sup.4, —C(O)A.sup.5, —C(O)NA.sup.6A.sup.7, —COOA.sup.8, and —SO.sub.3A.sup.9, wherein A.sup.1 to A.sup.9 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, and a heteroaryl group, and wherein R.sup.6 and R.sup.7 and/or R.sup.10 to R.sup.12 may bind to each other to form one or more rings; R.sup.8, R.sup.9, and R.sup.14 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, and a heteroaryl group; and R.sup.13 is selected from the group consisting of a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a trifluorophenyloxy group, a difluorophenyloxy group, a fluorophenyloxy group, a phenyloxy group, a pentafluorophenylthio group, a tetrafluorophenylthio group, a trifluorophenylthio group, a difluorophenylthio group, a fluorophenylthio group, a phenylthio group, a trinitrophenyloxy group, a dinitrophenyloxy group, a nitrophenyloxy group, a N-succinimidyloxy group, and a benzotriazolyloxy group; and wherein the alkyl groups, the cycloalkyl groups, the alkenyl groups, the cycloalkenyl groups, the alkynyl groups, the aryl groups, and the heteroaryl groups may independently be (further) substituted or unsubstituted and the alkyl groups, the alkenyl groups, and the alkynyl groups may independently be branched or linear.

6. A screening method comprising a contacting step (a) of bringing at least one compound according to claim 1 or a salt thereof in contact with (wild type) protease(s) PL.sup.pro and/or 3CL.sup.pro of a SARS coronavirus.

7. The method according to claim 6, wherein the method comprises a further contacting step (al) of bringing the at least one compound represented by the general formula (1) or a salt thereof in contact with modified protease(s) PL.sup.pro and/or 3CL.sup.pro of a SARS coronavirus.

8. The method according to claim 6, wherein the contacting step is carried out in vitro.

9. The method according to claim 6, wherein the method further comprises, after the contacting step (a), a step (b) of adding a compound selected from the group consisting of a fluorophore-azide, a fluorophore-alkyne, a biotin-azide, or a biotin-alkyne.

10. The method according to claim 6, wherein the method further comprises an analyzing step (c) of analyzing as to whether the compound according to general formula (1) reacted with the protease(s).

11. The method according to claim 6, wherein the method comprises a further contacting step (a0) of bringing the (wild type) protease PL.sup.pro and/or 3CL.sup.pro of a SARS coronavirus in contact with at least one compound to be screened before the contacting step (a) with the compound according to general formula (1).

12. The compound according to claim 1, wherein each of R.sup.2 to R.sup.11 is a hydrogen atom.

13. The compound according to claim 1, wherein R.sup.12 is selected from a n-propyl group, an iso-propyl group, an iso-butyl group, a 2,5-dichlorophenyl group, a 3,5-bis(trifluoromethyl)phenyl group, and a group of the following formulas (5) to (7) ##STR00015##

14. The compound according to claim 1, wherein the compound of the general formula (1) is selected from the compounds of the following formulas (10) to (18) ##STR00016## ##STR00017## ##STR00018##

15. The method according to claim 8, wherein the contacting step is carried out in one or more cells.

Description

[0067] The figures show:

[0068] FIG. 1: Genome architecture of SARS-CoV-2. Genes encoding structural proteins are highlighted in black. The cleavage sites of PL.sup.pro and 3CL.sup.pro in the polyprotein are marked with arrows.

[0069] FIG. 2: Synthesis of one compound of general formula (2) (LS-probe).

[0070] FIG. 3: Method of ligand selection and screening using a compound according to general formula (2) (LS-probe). Wt=wild type; m=mutant.

[0071] FIG. 4: Method of competitive inhibitor profiling against PL.sup.pro and 3CL.sup.pro.

[0072] FIG. 5: Cleavage assays for purified 3CL.sup.pro using fluorogenic (7-amino-4-methylcoumarin, AMC) peptide substrates. a, Using different enzyme concentrations from 50 nM to 500 nM. b, Using peptidic substrate concentrations between 5 μM and 250 μM. c, With a different peptidic substrate in the range from 5 μM to 250 μM. For b and c fluorescence intensities with and without enzyme overlap at 125 μM.

[0073] FIG. 6: Example for the ligand selection screening with live cell labelling of wild type (wt) and active site mutant (m) by the LS probe library at 20 μM. Representative data of three independent replicates.

[0074] FIG. 7: Quantification of wild type labelling intensities (wt) normalized to DMSO controls and specificity (wt/m) of LS probes at 20 μM. Specific probes exhibit a high wild type to mutant ratio (n=3). Criss-cross pattern: not determined.

[0075] FIG. 8: Concentration series of selected probes for labelling of 3CL.sup.pro and PL.sup.pro.

[0076] FIG. 9: Selectivity of compounds of formulas (13) and (15) at 20 μM in live cell labelling of proteases 3CL.sup.pro and PL.sup.pro, respectively, in the background of a native E. coli proteome. Flu: fluorescence gels; Coo: Coomassie stained gels. Representative gels of three independent replicates.

[0077] FIG. 10: Comparison of the in situ labelling of 3CL.sup.pro of SARS-CoV-1 and SARS-CoV-2 in live heterologously expressing E. coli cells. The probes yield equal labelling intensity and specificity for the two 3CL.sup.pro homologues.

[0078] FIG. 11: Labelling of 3CL.sup.pro with compound of formula (13) and PL.sup.pro with compound of formula (15) in the background of the native proteome of HepG2 cell lysates at 20 μM probe concentration. Controls (A heat) give the unspecific background labelling of the heat denatured proteome. Representative results of three independent experiments.

[0079] FIG. 12: Half-maximal inhibitory concentrations (IC.sub.50s) determined from curve fittings of quantitative competitive labelling experiments. a, With compounds M26 (=SalB) and X.sup.05 inhibiting labelling of 3CL.sup.pro by compound of formula (13) (IC.sub.50=12 uM for M26=SalB; IC.sub.50=43 μM for X05). b, With compounds M03 and X05 inhibiting labelling of PL.sup.pro by compound of formula (15) (IC.sub.50=26 μM for M03; IC.sub.50=81 μM for X05).

[0080] FIG. 13: Inhibition of PL.sup.pro determined by an enzyme activity assay using the fluorogenic substrate Arg-Leu-Arg-Gly-Gly-AMC. a, Inhibition of PL.sup.pro activity by X05 (IC.sub.50=44 μM). b, Inhibition of PL.sup.pro activity by M03 (IC.sub.50=10 μM). c, Inhibition of PL.sup.pro activity by compound of formula (15) (IC.sub.50=58 μM).

[0081] FIG. 14: Inhibition curves of 3CL.sup.pro inhibitors determined by quantification of fluorescent labeling intensity by compound of formula (13) (n=3). Lith: lithospermic acid; Ros: rosmarinic acid; SalA: salvianolic acid A; SalC: salvianolic acid C; SalB: salvianolic acid B.

[0082] FIG. 15: Examples of fluorescent gels with competitive labelling of 3CL.sup.pro.

[0083] FIG. 16: IC.sub.50 values of the most active inhibitors quantified by competitive fluorescence labeling.

[0084] FIG. 17: Competitive labelling with concentration series of extracts of the roots of Salvia miltiorrhiza.

[0085] The present invention will be further illustrated in the following examples without being limited thereto.

EXPERIMENTAL PROCEDURES

General

Click Reactions

[0086] Click chemistry was performed using 40 μL cell lysate, 2 μL of a 0.65 mM tetramethylrhodamine (TAMRA) azide stock in DMSO, 4 μL of a 1.66 μM TBTA (tris((1-benzyl-4-triazolyl)methyl)amine) stock in tert-butanol/DMSO (8:2 v/v). To start the cycloaddition 2 μL of a freshly prepared 52 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) stock in water and 2 μL of a 50 mM CuSO.sub.4 stock solution in water were added. The samples were incubated for 1 h at room temperature before quenching with 50 μL 2×SDS loading buffer (63 mM Tris-HCl, 10% (v/v) glycerol, 2% (w/v) SDS, 0.0025% (w/v) bromophenol blue, 10% (v/v) p-mercaptoethanol; dissolved in water).

SIDS-PAGE and In-Gel Fluorescence Scanning

[0087] Before performing SDS-PAGE the samples were incubated for 10 mi at 95° C. and subsequently centrifuged down. SDS gels containing 10% acrylamide and an aqueous solution of 37.5:1 acrylamide and N,N′-methylenebisacrylamide were used with a PeqLab system and run at 75 mA per gel. Visualization was done by in-gel fluorescence scanning of the tetramethylrhodamine (TAMRA) dye (Fusion-FX7). Equal protein content and separation in SDS-gels was confirmed by Coomassie staining (InstantBlue™, expedeon).

Example 1: Synthesis of an LS-Probe

[0088] One LS-probe of the formula (19) was synthesized according to the following procedure (cf. FIG. 2).

2-tert-Butoxycarbonylamino-3-[4-(prop-2-ynyloxy)phenyl]-propionic acid propargyl ester (19-2)

[0089] The reaction was adapted from a protocol described in the literature (Polic, V. & Auclair, K. Allosteric Activation of Cytochrome P450 3A4 via Progesterone Bioconjugation. Bioconjug Chem 28, 885-889 (2017)). 6 g of (21.3 mmol, 1 eq) N-tert-Butoxycarbonyl-tyrosine (19-1) and 9 g of (56.1 mmol, 3 eq) K.sub.2O.sub.3 were suspended in 30 mL dry DMF under N2 flow. After stirring for 10 min at room temperature 7.9 mL (73.1 mmol, 3.5 eq) of an 80% solution propargyl bromide in toluene was slowly added. The solution was left to react for 18 h at room temperature. 150 mL H.sub.2O were used to quench the reaction. The mixture was extracted with diethyl ether, washed with distilled water and brine. The combined organic layers were dried over MgSO.sub.4 before solvent evaporation in vacuo. The yellow oil was used in the next step without any further purification (7.3 g, 100%).

[0090] .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm): 1.42 (s, 9H, (CH.sub.3).sub.3C—), 2.51 (m, 2H, —CO.sub.2CH.sub.2CCH, -PhOCH.sub.2CCH), 3.07 (m, 2H, -PhCH.sub.2CH—), 4.59-4.93 (m, 1H, -PhCH.sub.2CH—), 4.64-4.80 (m, 2H, —CO.sub.2CH.sub.2CCH, -PhOCH.sub.2CCH), 6.90 (m, 2H, aromat.), 7.09 (m, 2H, aromat.)

2-Amino-3-[4-(prop-2-ynyloxy)phenyl]-propionic acid propargyl ester (19-2b)

[0091] The reaction was adapted from a protocol described in the literature (Polic, V. & Auclair, K. Allosteric Activation of Cytochrome P450 3A4 via Progesterone Bioconjugation. Bioconjug Chem 28, 885-889 (2017)). To 180 mL of MeOH on an ice bath, 21 mL (294 mmol, 15 eq) acetyl chloride were slowly added. The solution was left to stir for 10 mi at 0° C. 7 g of (19-2) were added and the solution was allowed to warm to room temperature. After 2 h the solvent was evaporated in vacuo to give the pure product as slightly brownish-white powder (4.1 g, 100%).

[0092] .sup.1H-NMR (D.sub.2O, 400 MHz) δ (ppm): 2.98 (t, J=2.4 Hz, 1H, —OCH.sub.2CCH), 3.30-3.09 (m, 2H, -PhCH.sub.2CH—), 3.99 (dd, 1H, J=7.8, J=5.2 Hz, -PhCH.sub.2CH—), 4.85 (d, 2H, J=2.4 Hz, —OCH.sub.2H), 7.10 (m, 2H, aromat.), 7.33 (m, 2H, aromat.).

O-Propargyl Tyrosine (19-3)

[0093] The reaction was adapted from a protocol described in the literature (Polic, V. & Auclair, K. Allosteric Activation of Cytochrome P450 3A.sup.4 via Progesterone Bioconjugation. Bioconjug Chem 28, 885-889 (2017)). To a mixture of 30 mL MeOH and 42 mL 2 M NaOH 5.6 g (20 mol, 1 eq) (19-2b) was added. The reaction was stirred for 17 h at room temperature. With concentrated HCl the pH of the mixture was adjusted to 7 and it was kept at 4° C. for 4 h. The precipitate was filtered off and dried in vacuo to give the pure product as light yellow powder (3.51, 17.1 mmol, 83%).

[0094] .sup.1H-NMR (D.sub.2O, 400 MHz) δ (ppm) 2.98 (m, 1H, -PhOCH.sub.2CCH), 3.12 (dd, 1H, J=14.9, J=8.1 Hz, -PhCH.sub.2CH—), 3.27 (dd, 1H, J=14.3, J=4.9 Hz, -PhCH.sub.2CH—), 4.00 (td, 1H, J=13.1, J=2.2 Hz, -PhCH.sub.2CH—), 4.85 (t, 2H, J=1.7 Hz, -PhOCH.sub.2CCH), 7.11 (m, 2H, aromat.), 7.32 (m, 2H, aromat.).

2-(2-Chloroacetamido)-3-(4-(prop-2-yn-1-yloxy)phenyl)propanoic acid (19-4)

[0095] The reaction was adapted from a protocol described in the literature (Beagle, L. K. et al. Efficient Synthesis of 2,5-Diketopiperazines by Staudinger-Mediated Cyclization. Synlett, 2337-2340 (2012)). 1.03 g (5 mmol) of (19-3) were suspended in 35 mL of dry THF under an N2 flow. 0.6 mL (1.5 eq) of chloroacetyl chloride were added. The suspension was stirred at reflux for 3 hours. The reaction was extracted with ethyl acetate and washed with distilled water and brine. The combined organic layers were dried over MgSO.sub.4 before solvent evaporation in vacuo. The yellow crystals were purified via column chromatography (DCM:MeOH=9:1) to give the pure product as light yellow crystals (1.19 g, 85%).

[0096] .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm): 2.52 (t, 1H, J=2.2 Hz, —OCH.sub.2CCH), 3.09-3.23 (m, 2H, -PhCH.sub.2CH—), 4.05 (d, J=2.0 Hz, 2H, —OCH.sub.2CCH), 4.68 (d, J=2.5 Hz, 2H, —CH—Cl.sub.2), 4.86 (m, 1H, -PhCH.sub.2CH—), 6.94 (d, 2H, J=8.6 Hz, aromat.), 7.11 (d, J=8.4 Hz, 2H, aromat.).

[0097] ESI-HRMS: m/z=296.0685 [M+H].sup.+, calc. for C.sub.14H.sub.14ClNO.sub.4+H.sup.+=296.0684.

Perfluorophenyl 2-(2-chloroacetamido)-3-(4-(prop-2-yn-1-yloxy)phenyl)propanoate (19, LS-probe)

[0098] The reaction was adapted from a protocol described in the literature (Liu, Y. et al. Building Nanowires from Micelles: Hierarchical Self-Assembly of Alternating Amphiphilic Glycopolypeptide Brushes with Pendants of High-Mannose Glycodendron and Oligophenylalanine. J Am Chen Soc 138, 12387-94 (2016)). 0.92 g (3.2 mmol) of (19-4) and 0.72 g (3.6 mmol) of pentafluorophenol were dissolved in 80 mL dry DCM under nitrogen. The solution was stirred on ice for 20 mi before adding 40 mg of DMAP (0.32 mmol) and 0.74 g DCC (3.6 mol). The suspension was stirred over night at room temperature. The reaction was quenched with 4 mL 3N HC. The resulting solution was kept at 4° C. over night. The precipitate was filtered off over celite and washed with cold DCM. Afterwards the filtrate was washed with saturated NaHCO.sub.3 solution and distilled water, dried over MgSO.sub.4 before the solvent was evaporated in vacuo. The solid was purified by flash chromatography (ethyl acetate, hexane) to give 0.93 g (63%) of the pure product as pale yellow crystals.

[0099] .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ (ppm): 2.51 (t, 1H, J=1.8 Hz, —OCH.sub.2CCH), 3.11-3.23 (m, 2H, -PhCH.sub.2CH—), 4.05 (d, J=4.5 Hz, 2H, —OCH.sub.2CCH), 4.68 (d, J=2.1 Hz, 2H, —CH—Cl.sub.2), 4.87 (dt, J=6.32, J=7. Hz, 1H, -PhCH.sub.2CH—), 6.94 (d, 2H, J=8.5 Hz, aromat.), 6.96 (s, 1H, —NH—), 7.11 (d, J=8.7 Hz, 2H, aromat.).

[0100] .sup.13C-NMR (CDCl.sub.3, 400 MHz) δ (ppm): 36.93 (PhCH.sub.2CH—), 42.81 (—CH.sub.2Br), 53.73 (-PhCH.sub.2CH—), 56.33 (—OCH.sub.2CCH), 76.09 (—OCH.sub.2CCH), 77.96 (—OCH.sub.2CCH), 115.74 (aromat.), 130.83 (aromat.), 157.48 (aromat.), 166.60 (—COO—), 174.96 (—NCO—).

[0101] .sup.19F-NMR (CDCl.sub.3, 400 MHz) δ (ppm): −152.67 (d, 2F, ortho), −157.57 (t, 1F, para), −162.42 (t, 2F, meta).

Example 2 Recombinant Proteins

[0102] Both 3CL.sup.pro and PL.sup.pro likely liberate themselves from the polyprotein by cleaving their respective N- and C-terminal sequences as described for the homologous sequences of SARS-CoV-1. Hereby dimerization and maturation of 3CL.sup.pro by N-terminal self-cleavage is required for full activation of the protease and the ability of trans-processing of other non-structural proteins. Dimeric crystal structures of mature 3CL.sup.pro show that the N-terminus of one 3CL.sup.pro monomer is in close proximity to the active site pocket of the other monomer and the N-terminus is cleaved between the short consensus sequence Leu-Gln and Ser already during expression. Molecular modelling of the flexible peptide sequence prior to cleavage indicated that the first amino acids before the cleavage site occupy a part of the active site pocket which is in agreement with mechanistic models of protease maturation. It was reasoned that inhibitors targeting the protease prior to full activation could be of great value for drug development against SARS-CoV-2. Thus, there was constructed a 3CL.sup.pro version fused with a non-cleavable N-terminal Strep-tag 11. Modelling predicted an identical behavior of the fusion peptide at the active site compared to the native sequence of wild type prior to cleavage. Codon optimized sequences of the 3CL.sup.pro and PL.sup.pro domains of nsp3 and nsp5 of SARS-CoV-2 were ultimately cloned into an IPTG inducible vector and heterologously expressed in Escherichia coli. Proteins were either purified via affinity chromatography or used in situ in the native cell of the expression system. Confirming the predictions regarding the N-terminal modification, the purified 3CL.sup.pro version indeed was inactive in a protease assay using oligopeptide substrates linked to a fluorogenic 7-amino-4-methylcoumarin group (FIG. 5). This represented the unique opportunity to validate the utility of the LS-ABPP (LS-activity-based protein profiling) strategy against PL.sup.pro and the pre-activation stage of 3CL.sup.pro of SARS-CoV-2 and demonstrate its versatility for customizing probes to different targets. In order to discover probes with specificity for the active site of the proteases, there were also constructed the corresponding active site mutants Cys145Ala (3CL.sup.pro) and Cys114Ala (PL.sup.pro). The experimental details are given in the following.

Plasmid Preparation

[0103] The gene coding for the herein used proteins (3CL.sup.pro, PL.sup.pro and their mutants) were custom synthesized and constructed in a pET-51b(+) plasmid (GenScript, New Jersey) for protein expression in E. col BL21 cells. The expression vector was IPTG inducible with ampicillin marker and N-terminal Strep-tag II with cloning site KpnI-BamHI.

Transformation of Plasmids

[0104] For preparation of competent cells, 1 mL of an overnight culture of E. coli BL21 cells was inoculated in 25 mL of LB medium and kept at 37° C. and 180 rpm. At an OD.sub.600 of 0.5 the cells were transferred into a 50 mL tarson tube and kept on ice for 20 min. Centrifugation at 6000 rpm and 4° C. for 10 mi was performed. The supernatant was discarded and the cell pellet resuspended into 20 mL ice cold calcium chloride (50 mM). After an incubation time of 20 min on ice the cells were centrifuged at 6000 rpm for 10 min at 4° C. and the supernatant discarded. The cell pellet was again resuspended in 4.25 mL of calcium chloride solution (50 mM) as well as 0.75 mL of glycerol. Aliquots of 200 μL were made and directly put into liquid nitrogen before storing them at −80° C.

[0105] An aliquot of competent E. coli BL21 cells was thawed on ice. 1 μL of the corresponding plasmid (50 ng/mL) was added to the cells and mixed by tabbing the tube three times. After incubation on ice for 30 min the cells were heat shocked at 42° C. for 45 sec. Afterwards the tube was placed on ice for 3 min. 900 μL LB media was added to the cells and they were kept at 37° C. for 1 h whilst shaking at 550 rpm. Centrifugation at 8000 rpm and 4° C. for 2 mi was performed. The supernatant was discarded and the cell pellet resuspended. The cell suspension was streaked on ampicillin plates (100 μg/L), which were kept at 37° C. overnight. The next day colonies could be picked for overnight cultures which could be used to prepare glycerol stocks.

Overproduction of Proteins

[0106] Overnight cultures for cellular assays were prepared by taking a small amount of a bacterial cryo-stock (15% glycerol, stored at −80° C.) and inoculating them in 5 mL LB in sterile 13 mL polypropylene tubes (Sarstedt, ref 62.515.028), supplemented with antibiotics as indicated and grown for 14-16 h at 37° C. (180 rpm). 1 mL of a corresponding overnight culture was inoculated in 50 mL LB medium containing 100 μL/mL ampicillin and kept at 37° C. and 180 rpm. At an OD.sub.600 of 0.3 the protein expression was induced by adding 0.2 μg/mL of an IPTG solution (1 M). The cells were incubated at 37° C. and 180 rpm for 2 h before centrifugation at 4000 rpm for 20 min at 4° C.

Affinity-Purification of Proteins

[0107] For purifying the proteins overexpression was performed in a scale of 3 L as described. The cell culture was centrifuged down at 4000 rpm for 20 mi at 4° C. The supernatant was discarded and the cell pellet washed with 15 mL PBS before centrifuging at 4000 rpm for 20 min at 4° C. The cells were lysed by ultrasound treatment (25% amplitude, 0.5 s ON, 2.1 s OFF, 20 pulses, Branson Digital Sonifier). Afterwards samples were centrifuged for 1 h at 4000 rpm and 4° C. The supernatant was transferred into a new 50 mL flask and put on ice before performing affinity purification via Strep-tag II on an ÄKTA start (GE Healthcare) using StrepTrap HP columns (GE Healthcare). Standard Bradford protocols were used to calculate the concentration of the protein fractions. 200 μL aliquots of 0.4 g/mL protein were frozen in liquid nitrogen before storing them at −80° C.

[0108] Enterokinase digest of 3CL.sup.pro

[0109] For cleavage of the N-terminal Strep-tag II, 2 μL enterokinase (0.3 mg/mL, Boehringer Mannheim) was added to 25 μL purified recombinant 3L.sup.pro (0.8 mg/mL) in phosphate-free buffer (50 mM Tris-HCl, 1 mM EDTA, pH=0.3) and the mixture was incubated at 37° C. for 3.5 h.

Example 3: LS-Probe Modification with Ligands (FIG. 3B) and In Vitro Robe Labelling

[0110] The LS probe was reacted individually with a selection of different amine containing ligands in microliter scale. Time-resolved .sup.19F NMR spectra helped to optimize the reaction conditions and revealed complete conversion after 60 min even for the least reactive aromatic amines. Although a quantitative reaction was expected, potential residues of unreacted probe were removed using amino-functionalized polystyrene beads to prevent unspecific protein reactivity. Subsequently, the reaction mixture was lyophilized and dissolved in DMSO before direct application to protein labeling experiments. The LS-probe modification was conducted by the following general protocol and the compounds of the formulas (10) to (18) were successfully prepared by said protocol.

[0111] In a 1.5 mL micro reaction tube were added 2.5 μL of 5 M pyridine (in DMSO), 2.5 μL of a probe stock (1 mM in butyl acetate) as well as 2.5 μL of the corresponding amine containing ligand (1 mM in DMSO) to result in a final concentration of 50 μM in the cell suspension. The mixture was incubated for 1 h at room temperature. Quenching was performed by adding 50 μL butyl acetate to the reaction mixture and pipetting the entire solution into a fresh 1.5 mL micro reaction tube containing 5 mg of (aminomethyl)polystyrene beads (70-90 mesh, Sigma-Aldrich). After incubation for 15 min the supernatant was transferred into a fresh 1.5 mL micro reaction tube. The beads were washed with 50 μL of butyl acetate and the supernatants were combined. The pooled solution was dried at high vacuum. 2 μL of DMSO were used to dissolve the reacted probe. For dose down experiments the same procedure as for the reaction of a LS-probe and ligand was used. To get a dose down of a final concentration of 50 μM, 20 μM, 10 μM, 5 μM, 1 μM and 0.1 μM in 50 μL the respective amount of ligand and probe was used.

In Vitro Probe Labelling of Proteins in Lysates and Purified Protein (FIG. 3C)

[0112] The respective protein aliquot (0.4 mg/mL) was thawed on ice. Reaction of LS probe and ligands was performed as described. The reacted probe was dissolved in 2 μL DMSO and added with 8 μL PBS to 10 μL protein solution before incubation for 30 min at 400 rpm and 37° C. After incubation Click Chemistry was performed.

Example 4: In Situ Probe Labelling of Proteins in Live E. coli Cells (FIG. 3D) and In Situ Competitive Profiling

[0113] The probes obtained in Example 3 were screened in situ against 3CL.sup.pro and PL.sup.pro expressed in intact E. coli cells and their corresponding active site mutants Cys145Ala (3CL.sup.pro) and Cys114Ala (PL.sup.pro). (FIG. 3D). To this aim, the cells were incubated with 20 μM of the ligand modified probes obtained in Example 3 for one hour, followed by cell lysis and click chemistry with tetramethylrhodamine (TAMRA) azide to append a fluorescent reporter tag. After gel electrophoresis by SDS-PAGE, fluorescence imaging revealed probe labeling of the enzymes (FIG. 3D). The probes resulted in strongly labelled bands of 3CL.sup.pro and PL.sup.pro. The probes were further examined for their specificity by comparing labelling of wild type (wt) versus active site mutant (m) proteins (FIG. 6). Fluorescence intensities were quantified relative to the DMSO control and probes with a ratio wt/m>2 were considered specific. The probes of Example 3 exhibited great specificity for labelling only the wild type but not mutant 3CL.sup.pro and PL.sup.pro (FIG. 7). Compounds (13) and (14) showed particular specificity for labelling of 3CL.sup.pro, Compounds (10) to (12) and (15) were particular specific for PL.sup.pro It was thus possible to identify complementary probes for the two proteases. To estimate their sensitivity, there were performed labelling experiments with the most specific probes in concentration dependence. Strikingly, 3CL.sup.pro was labelled by compound (13) and PL.sup.pro by compounds (12) and (15) as the most sensitive probes at concentrations as low as 1 μM (FIG. 8). Interestingly, overproduced 3CL.sup.pro and PL.sup.pro were the only bands that compounds (13) and (15) labelled in live E. coli cells emphasizing the selectivity of the probes in the background of a native proteome (FIG. 9). The experiments were conducted according to the following protocol.

[0114] After overexpressing a protein in E. coli, for each sample 1 mL of the induced bacterial culture was transferred into a 1.5 mL Eppendorf tube. The cells were pelleted by centrifugation (4000 rpm, 7 min, 4° C.), washed with 50 μL PBS before re-suspending them in 48 μL PBS. The reacted and quenched probes, solved in 2 μL DMSO were added to the cell suspension before incubation at 400 rpm at 37° C. for 1 h. After incubation the cells were pelleted by centrifugation (4000 rpm, 5 min, 4° C.) washed with 50 μL PBS and resuspended in 50 μL PBS. The cell suspension was stored at −80° C. before further processing. After thawing the samples, they were lysed by ultrasound treatment (10% amplitude, 0.5 s ON, 1 s OFF, 10 pulses, Branson Digital Sonifier). The resulting lysates were used for click chemistry and SDS-PAGE. After fluorescence scanning, Coomassie staining was applied to compare protein concentrations in the gel and validate the experiments.

In Situ Competitive Profiling

[0115] The respective protein was overproduced in E. coli BL21 cells as described before. Reaction of LS probe and ligand was performed as described with 0.25 μL of LS-probe (1 mM), ligand (1 mM) and 5 M pyridine per sample. In the meantime, 1 μL of competitive molecule (10 mM) was incubated with 10 μL of the protein aliquot and 49 μL cell suspension for 1 h at 25° C. and 400 rpm. Each sample of the reacted probe was dissolved in 0.25 μL DMSO and added to the cell suspension before incubation for 1 h at 400 rpm and 37° C. The cells were further treated like described for in situ labeling with LS-probes.

Example 5: Comparing Homologues of 3CL.SUP.pro

[0116] In order to verify that the LS-probe strategy also allows to label 3CL.sup.pro of the closely related SARS-CoV-1, another member of the Sarbecovirus subgenus, there was also expressed its wild type and mutant sequences and performed LS probe labelling experiments in situ by the protocol given in Example 4. Labelling intensity and specificity for the active site of both 3CL.sup.pro homologs were virtually identical for all tested compounds (cf. FIG. 10), demonstrating robustness even across different viral strains.

Example 6: LS-Probe Labelling of Virus Proteases in Background of HepG2 Proteomes

[0117] The potential application of the probes to label the proteases in the background of a native human proteome was also investigated. Lysates of human hepatocellular carcinoma (HepG2) cells were used and supplemented with different concentrations of purified 3CL.sup.pro and PL.sup.pro. Applying compound of formula (13) for 3CL.sup.pro and compound of formula (15) for PL.sup.pro at 20 μM followed by click chemistry resulted in the detection of both proteases as strong bands at protease concentrations down to 77 μg/mL (FIG. 11). Only relatively few additional off-target bands were labelled in HepG2 cell lysates suggesting that these probes could be employed to label and detect the activity of SARS-CoV-2 proteases in the background of a complex proteome. The following procedures were applied for the experiments.

[0118] Human hepatocellular carcinoma HepG2 (ATCC, HB-8065) were maintained in DMEM (4.5 g/L glucose, pyruvate) (Gibco), supplemented with 10% fetal bovine serum (PAA Laboratories) and penicillin/streptomycin (25 μg/mL each) at 37° C., 5% CO.sub.2 (Gibco). Cells were seeded in T75 flasks at a density of 60.000 cells/cm.sup.2 and allowed to proliferate for 3 days. For the experiments, 2×10.sup.8 cells were harvested and lysed by sonication in PBS (3×25% amplitude, 0.5 s ON, 2.1 s Off, 20 pulses).

[0119] The respective protein aliquot (0.4 mg/mL) as well as the HepG2 cell lysate aliquot were thawed on ice. Reaction of the LS-probe was performed as described before with a final concentration of the reacted probe of 20 μM. Samples for the protein dose down containing 10 μg, 5 μg, 1 μg, 0.5 μg and 0.1 μg of protein were prepared with 40 μL of HepG2 cell lysate (1.5 mg/mL), the dissolved and reacted LS-probe and PBS to give a volume of 65 μL. Incubation of 30 min at 400 rpm and 37° C. was performed before click reaction.

Example 7: Competitive Screening for Identifying Enzyme Inhibitors

[0120] Since the probes exhibited sensitive and active site-specific labelling of PL.sup.pro as well as of the pre-activation stage of 3CL.sup.pro, their potential as chemical tools for the competitive screening of enzyme inhibitors was investigated. There were searched over 1000 structures of commercially available food grade additives, natural products, and protease inhibitors and manually selected 44 compounds with electrophilic motifs such as aldehydes, Michael acceptors, epoxides, and esters that could potentially react covalently with the nucleophilic active site cysteine of 3CL.sup.pro and PL.sup.pro. The electrophilic compound library was prepared in form of DMSO stocks and individually pre-incubated for an initial screening with the purified proteases followed by labelling with the compound of formula (13) for 3CL.sup.pro and the compound of formula (15) for PL.sup.pro. Successful inhibitors would block the active site and thereby prevent subsequent probe labelling which could be read out by lowered in-gel fluorescence. Indeed, at an initial concentration of 200 μM some compounds even abolished probe labelling. Then there was quantified the fluorescence signal relative to the control and compounds that resulted in more than 50% inhibition of competitive labelling were selected to investigate their dose response relationship. Half maximal inhibitory concentrations (IC.sub.50s) were calculated from curve fittings of concentration-dependent quantitative competitive labelling. Phenethyl isothiocyanate, which is produced from its precursor gluconasturtiin by vegetables of the Brassicaceae family, scored against both proteases likely due to unspecific thiol reactivity of isothiocyanates, but only led to incomplete inhibition (X05, FIG. 12). In contrast, curcumin (M03) almost completely inhibited labelling of PL.sup.pro with an IC.sub.50 of 26 μM and was inactive against 3CL.sup.pro (FIG. 12). However, curcumin is a well-known pan-assay interference (PAIN) compound and as such of no particular interest. Both X05 and M03 inhibited enzyme activity of PL.sup.pro with a fluorogenic peptide substrate, confirming the validity of the competitive screening approach (FIG. 13).

[0121] More interesting was the activity of salvianolic acid B (M26, SalB), which inhibited labelling of 3CL.sup.pro with an IC.sub.50 of 12 μM (FIGS. 14 and 15). Thus, there was focused on compounds with a closely related caffeic acid ester motif. All compounds showed a clear dose response behavior whereby rosmarinic acid (Ros) and salvianolic acid A (SalA) were the most active with IC.sub.50 values of 10 μM and 4.8 μM, respectively. Interestingly, salvianolic acid C (SalC), which differs from SalA only by a hydroxyl group locked into a benzofuran ring, was considerably less active with an so of 91 μM (FIGS. 15 and 16). Also, the closely related lithospermic acid (Lith) only exhibited an IC.sub.50 of 32 μM. These results demonstrate a fine-tuned structure activity relationship of salvianolic acid derivatives for the inhibition of 3CL.sup.pro. Since SalA and the other potent salvianolic acid derivatives are produced by the plant Salvia miltiorrhiza (red sage), there were prepared and tested extracts of its dried roots in a competitive labelling assay with compound of formula (13) against 3CL.sup.pro and there was observed potent inhibition down to 1 mg/mL (FIG. 17). The following procedures were used for the above experiments.

In Vitro Competitive Profiling

[0122] The respective protein aliquot (0.4 mg/mL) was thawed on ice. 10 μL of protein were used per reaction and pipetted into a 1.5 mL micro reaction tube together with 9.6 μL of PBS as well as 0.4 μL of the competitive compound (10 mM). The proteins were incubated for 30 min at 25° C. and 400 rpm. Reaction of probe and ligand was done as described with 0.1 μL LS-probe (1 mM), ligand (1 mM) and pyridine (5 M) per sample. The reacted probe was solved in 0.1 μL DMSO and pipetted to the proteins and incubated for 30 min at 37° C. and 400 rpm. Afterwards Click Chemistry was performed directly.

[0123] For dose down experiments the same procedure as for in vitro competitive profiling with LS-probes was performed. Final concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 10 μM, 5 μM and 1 μM of the competitive compound were used with 5 μM of the reacted probe.

Preparation of Root Extracts from Red Sage (Salvia miltiorrhiza)

[0124] For preparation of crude extracts, a protocol for purification of salvianolic acids was followed (Dong, J., Liu, Y., Liang, Z. & Wang, W. Investigation on ultrasound-assisted extraction of salvianolic acid B from Salvia miltiorrhiza root. Ultrason Sonochem 17, 61-5 (2010)). 10 g commercially available Salvia miltiorrhiza root powder was suspended in 200 mL 60% ethanol (20 mL per 1 g powdered root) and subjected to sonication for 1 h. The mixture was filtered and the flow-through was concentrated in vacuo below 40° C. Subsequent lyophilization yielded 4.916 g of crude extract as a brown solid. To further concentrate the extract, 4.5 g crude extract were resuspended in 50 mL H.sub.2O and acidified with HCl to pH=2. The mixture was extracted five times with ethyl acetate, the combined organic phases were dried over MgSO.sub.4 and the solvent was evaporated, yielding 0.535 g of a dark red solid.