Fragment-Based Discovery of Irreversible Covalent Inhibitors of Cysteine Proteases Using Chlorofluoroacetamide Library

Abstract

Fragment-based approach combined with electrophilic reactive compounds is a powerful strategy to discover novel covalent ligands for protein target. However, the promiscuous reactivity often interferes with identification of the fragments possessing specific binding affinity to the targeted protein. In our study, we report the fragment-based covalent drug discovery using the chemically tuned weak reactivity of chlorofluoroacetamide (CFA). We constructed a small fragment library composed of 30 CFA-appended compounds and applied it to the covalent ligand screening for cysteine protease papain as a model protein target. Using the fluorescence enzymatic assay, we identified CFA-benzothiazole 30 as a papain inhibitor, which was found to irreversibly inactivate papain upon enzyme kinetic analysis. The formation of the covalent papain-30 adduct was confirmed using electrospray ionization mass spectrometry analysis. The activity-based protein profiling (ABPP) experiment using an alkynylated analog of 30 (i.e., 30-yne) revealed that 30-yne covalently labeled papain with high selectivity. These data demonstrate potential utility of the CFA-fragment library for de novo discovery of target selective covalent inhibitors.

Introduction

Irreversible inhibition of protein function by covalent modification with electrophilic compounds has potential advantages over reversible inhibition, including potent and sustained pharmacological activity^1–6) and overcoming drug resistance.^7–9) The development of covalent inhibitors has been discouraged in the pharmaceutical industry owing to their latent toxicity associated with indiscriminate reaction with off-target biomolecules.¹⁰⁾ However, the recent advances in targeted covalent inhibitors (TCIs) illuminates the benefits of selective covalent targeting of disease-associated proteins.^11–13) Several TCIs targeting cancer-associated tyrosine kinases such as epidermal growth factor receptor, and Bruton’s tyrosine kinase have been clinically approved for cancer treatment.^7–9,14,15) These TCIs possess an acrylamide-type Michael acceptor as the electrophilic reactive group (warhead) and irreversibly inhibit tyrosine kinase activity through covalent capture of the cysteine residue within the ATP-binding pocket. Currently, most of the TCIs have been developed using a structure-guided approach wherein an electrophilic warhead is incorporated into an affinity ligand for the targeted protein at an appropriate position rationally determined from the structural information of protein–ligand complex.

Fragment-based drug discovery (FBDD) is another powerful strategy to find novel ligands for the protein of interest.¹⁶⁾ FBDD focuses on low molecular weight fragments (typically 120–250 Da), enabling fast discovery of drug leads by exploring broader chemical space and structural diversity with a smaller library size as opposed to conventional high-throughput screening techniques. However, it is challenging to detect the weak reversible interactions between small fragments and their protein targets. A combination of drug-like fragments and covalent warheads would offer an attractive solution to this major obstacle in FBDD, as protein–ligand interactions could be easily detected owing to the irreversible nature of the covalent modification. In this regard, disulfide tethering¹⁷⁾ is the most representative example that has been successfully applied to various protein targets including oncogenic K-Ras^G12C protein.¹⁸⁾ Other electrophiles such as vinyl sulfones and acrylates are also employed in the tethering approach.^19,20)

Although proven to be a valid strategy in various cases, the specificity of covalent ligand screening remains a concern, since potency is reflected by covalent bond formation with the protein target, and fragments with high promiscuous reactivity, rather than with specific affinity to the targeted binding site, might also be detected as potent hits. This risk can be minimized using mildly reactive electrophiles. Chloroacetamide is a privileged covalent warhead, successfully applied in the LC/MS-based screening against purified proteins²¹⁾ and MS-based chemical proteomics in native cellular conditions.²²⁾ However, chloroacetamides may not be suitable as a drug development candidate due to its high intrinsic reactivity. Recently, we have introduced chlorofluoroacetamide (CFA) as a new class of thiol-reactive warheads which show much weaker reactivity compared to chloroacetamides.^23,24) In the present study, we report the assembly of small fragment library composed of 30 CFA derivatives and its application to covalent FBDD screening against papain as a model protein target. The results of fluorescence screening assay showed that CFA-benzothiazole 30 acts as an irreversible inhibitor for papain. The formation of papain-30 covalent adduct was confirmed using electrospray ionization (ESI)-MS analysis. Activity-based protein profiling (ABPP) experiment using the alkynylated analogue of 30 (i.e., 30-yne) revealed that CFA probe irreversibly binds to papain in a highly selective manner even under crude lysate conditions. These results demonstrate the potential utility of CFA-fragment library for de novo development of target selective covalent inhibitors.

Results and Discussion

Assembly of the CFA-Fragment Library

Since the utility of CFA as a reactive warhead for covalent ligands has not been studied previously, very few CFA-appended fragments were available for purchase from commercial venders. We therefore initiated our study to assemble the CFA-fragment library by incorporating a CFA unit into amine derivatives. Commercially available low molecular weight amines were conjugated to sodium chlorofluoroacetate using propylphosphonic anhydride (T3P^®) as the coupling agent (Table 1). All the CFA-fragments obtained had molecular weight below 300 Da and the majority of the fragments fulfilled the ‘rule of three,’²⁵⁾ a principle that suggests favorable properties of compounds in FBDD, where the number of hydrogen bond (HB) donors is ≤3, the number of HB acceptors is ≤3, and C Log P is ≤3. In addition to the structural diversity of the amine fragments, we also included several sets of the regioisomers possessing the same structural scaffold since disposition of warhead is crucial for efficient reaction with targeted cysteine thiol, especially in the case of weakly reactive CFA.

Table 1. Preparation of CFA-Fragments 1–30 by T3P-Mediated Coupling^a)

a) Reaction conditions: Amine (1.0 eq), sodium chlorofluoroacetate (1.5 eq), and DIPEA (2.0 eq) in CH₂Cl₂ were treated with T3P (50% solution in AcOEt) (1.5 eq) for indicated time at ambient temperature. b) Isolated yields.

Fragment-Based Drug Discovery Screening of Papain Inhibitors

To test the utility of CFA-fragment library in fragment-based covalent ligand discovery, we selected papain as a model protein target. Papain possesses a highly nucleophilic cysteine residue (Cys25) in its active site, which catalyzes hydrolysis of substrate peptide bond in combination with His159 and Asp175.²⁶⁾ Among proteases, papain is a representative cysteine protease belonging to papain family, which also includes various mammalian proteases such as cathepsins. We hypothesized that the efficiency of CFA fragment library could be demonstrated by selective targeting of the catalytic cysteine residue, which is conserved across large family of proteases. It is also known that papain shares a high structural homology with several disease-associated cysteine proteases including cathepsin K (46% sequence identity with papain), a promising target for the treatment of osteoporosis.²⁷⁾ Therefore, our study may act as a potential clue for discovery of selective covalent inhibitor for clinically relevant cysteine proteases.

Initially, we screened CFA-fragments 1–30 against papain (0.5 µM) by an enzymatic assay using the fluorophore-containing peptide substrate (Fig. 1a). While most CFA-fragments showed no inhibitory activity against papain after 2 h incubation even at 100 µM, we found that CFA-fragments 23, 29, and 30 moderately inhibited cysteine protease activity of papain under the same conditions. Among them, fragment 30 possessing 2-amino-6-ethoxybenzothiazole scaffold showed the most potent inhibitory activity (approx. 70% inhibition at 100 µM). The enzyme activity of papain was almost recovered upon incubation with 10 µM of 30, indicating that IC₅₀ value of 30 is in the order of ten micro-molars. Interestingly, fragment 22, which possesses the same benzothiazole scaffold as 30, showed no inhibitory activity. This result suggests that the 6-alkoxy group of 30 plays an important role for its inhibitory activity against papain.

Fig. 1. (a) Screening of Inhibitory Activity of CFA-Fragments 1–30 against Papain

Papain (0.5 µM) was incubated with each CFA-fragment (10 or 100 µM) in 40 mM HEPES (pH 7.0) containing 200 µM TCEP (37 °C, 2 h) before Bz-Arg-AMC (0.8 µM) was added. Bz-Arg-AMC: Benzoyl-L-arginine 4-methyl-coumaryl-7-amide. Data is represented as mean ± standard deviation (n = 3). (b) Time-dependent inactivation of papain by CFA-fragment 30 at varying concentrations. (c) Kitz–Wilson double-reciprocal plot of the inhibition of papain by CFA-fragment 30.

To gain insight into the inhibition mode the CFA-fragments 23, 29, and 30, we next performed the enzyme kinetic analysis according to the method reported previously.²⁰⁾ Relative activity of papain (3.84 µM) was evaluated in the presence of different concentrations of CFA-fragments (0–500 µM) for 0–60 min period. Fragments 23 and 29 did not exhibit time- and concentration-dependent increase in papain inhibition, implying that these compounds reversibly inhibited the papain activity. In contrast, CFA-benzothiazole 30 displayed time- and concentration-dependent inhibition (Fig. 1b), indicating that 30 irreversibly inactivated the cysteine protease activity of papain. The apparent second-order inhibition rate constant (k_inact/K_i) value, a common evaluation metric for describing the potency of irreversible inhibitors, was determined to be 0.354 M⁻¹ s⁻¹ for 30 by Kitz–Wilson double-reciprocal plot (Fig. 1c). This value is comparable to those of reported non-peptidic fragments possessing an acrylate moiety as the warhead (k_inact/K_i = 0.46–1.23 M⁻¹ s⁻¹).²⁰⁾

Investigation of the Covalent Adduct Formation

To directly observe the covalent modification of papain by CFA-fragment 30, we assessed the formation of covalent adduct by LC/MS analysis (Fig. 2). In the analysis of untreated papain, the peak observed at 8.56 min showed molecular weight of 23421 Da (m/z = 1802.6, z = 13) by ESI-MS analysis, which corresponds to the theoretical molecular weight of the active form of papain (23422 Da, 212 amino acids containing three disulfide bonds). When papain (10 µM) was incubated with 30 (100 µM, 8 h), a new peak appeared at 8.79 min, the molecular weight of which was 23674 Da (m/z = 1822.1, z = 13). The 253 Da mass shift suggests that 30 covalently modified papain with 1 : 1 stoichiometry. Since papain possesses Cys25 as a single free cysteine residue, we speculate that 30 forms a covalent bond with this reactive cysteine, in turn effectively inhibiting the protease activity of papain in the fluorescence assay.

Fig. 2. (a) The LC Analysis of Papain (Top) and ESI-MS Data of the Peak Observed at 8.56 min (Bottom)

The mass peak (m/z = 1802.6, z = 13, MW 23421) corresponds to papain (theoretical MW 23,422 Da). The mass peak (m/z = 1805.2, z = 13) is assigned to be S-sulfinylated papain (MW 23455 Da). (b) The LC analysis (top) of papain treated with CFA-fragment 30 and ESI-MS data of the peak observed at 8.79 min (bottom). The mass peak (m/z = 1822.1, z = 13) is assigned to be the covalent adduct of papain with CFA-fragment 30 (MW 23674 Da).

Evaluation of Covalent Labeling Using an Activity-Based Probe

To further assess the covalent adduct formation between papain and CFA-fragment, we designed 30-yne as an activity-based probe for papain,²⁸⁾ with the expectation that the replacement of the ethoxy group of 30 by the propargyloxy group would not largely affect the inhibitory activity (Fig. 3). In the initial experiment, commercially available papain (0.1 mg/mL) was treated with 30-yne for 1 h at 37°C under neutral aqueous conditions. Proteins covalently modified by 30-yne were conjugated to rhodamine-azide as the reporter fluorophore under copper-catalyzed azide-alkyne cycloaddition (CuAAC) conditions and detected by in-gel fluorescence analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We observed a fluorescent band at approx.25 kDa that corresponds to the major protein band in Coomassie Brilliant Blue (CBB) staining (Fig. 3a). The intensity of this fluorescence band was found to be increased depending on the concentration of 30-yne (12.5–50 µM). Conversely, the fluorescent band completely disappeared by pretreatment of papain with E-64 (100 µM, 1 h), a known irreversible inhibitor of papain (Fig. 3b). Since E-64 reacts with the catalytic Cys25 residue of papain at its epoxide moiety,²⁹⁾ it can be strongly speculated that 30-yne reacted with papain at this reactive cysteine. Pretreatment with 30 (100 µM, 1 h) resulted in partial blockade of papain labeling by 30-yne, suggesting that 30-yne is a good surrogate probe of 30. We also performed fluorescent labeling experiment of papain in the presence of crude Escherichia (E.) coli lysate (Fig. 3c). The E. coli lysate, which was prepared by sonication in phosphate buffer (pH 6.2), was mixed with the activated papain (0.1 mg/mL) and the mixture was treated with 30-yne (37°C, 1 h). Upon CBB staining, we observed a number of proteins derived from E. coli in the mixture. By contrast, in-gel fluorescence analysis revealed that 30-yne selectively labeled papain in concentration-dependent manner. No obvious off-target band was observed even at 50 µM of 30-yne, suggesting that CFA-benzothiazole is highly selective toward papain.

Fig. 3. (a) Concentration-Dependent Labeling of Papain Using 30-yne; Papain (0.1 mg/mL) Was Treated with 30-yne (12.5–50 µM) in 50 mM Sodium Phosphate (pH 6.2) Containing 120 mM NaCl and 1 mM DTT for 1 h at 37 °C; (b) Competitive Labeling of Papain; Papain (0.1 mg/mL) Was Pretreated with Dimethyl Sulfoxide (DMSO) or Inhibitor (E-64 or 30) (100 µM) for 1 h Followed by 30-yne (25 µM, 1 h, 37 °C); (c) Fluorescent Labeling of Papain (0.1 mg/mL) in the Presence of E. coli Lysate

Conclusion

To summarize, we assembled a small set of electrophile-fragment library composed of 30 CFA derivatives and demonstrated its utility in fragment-based discovery of novel covalent inhibitors for cysteine protease. Based on the enzyme assay, we identified CFA-benzothiazole 30 as the covalent inhibitor for papain. Covalent modification of papain by 30 was further confirmed by ESI-MS. We also demonstrated that alkynylated probe 30-yne serves as a selective covalent probe that can evaluate state of activity of papain even under the crude lysate conditions. It is apparent that the high target selectivity of the 30-yne is benefited from the chemically tuned weak reactivity of CFA. Although the direct structural evidence is yet to be provided, our data strongly suggest that the CFA group of 30 covalently modified the catalytic Cys25 of papain. We anticipate that further structural modification of 30 would lead to a more potent CFA inhibitor for cysteine proteases.

Current covalent drug discovery strategies mainly target less-conserved, noncatalytic cysteines in order to achieve high target selectivity. Our result suggests that CFA-based FBDD approach is useful for targeting proteins possessing highly reactive catalytic cysteine, which may include the main protease of severe acute respiratory syndrome (SARS)-CoV-2 (COVID-19).³⁰⁾ We envision that the FBDD approach employing a large set of CFA compounds will contribute to de novo development of highly selective covalent ligand toward a wide variety of proteins, which might be difficult by the existing approaches using more reactive electrophiles.

Experimental

Chemistry

Reagents and solvents were obtained from commercial suppliers and used without further purification, unless otherwise stated. Reactions were carried out under a positive atmosphere of nitrogen, unless otherwise stated. Reactions were monitored by TLC carried out on Merck TLC Silica gel 60 F₂₅₄, using shortwave UV light as the visualizing agent and phosphomolybdic acid in EtOH and heat as developing agent. Flash column chromatography was performed using Kanto Chemical Silica gel 60 N (spherical, 40–50 µm). ¹H-NMR spectra were recorded on Varian Unity Plus 400 MHz spectrometer or Bruker Avance III HD 500 MHz spectrometer and were calibrated using residual undeuterated solvent as the internal references (CDCl₃: 7.26 ppm; dimethyl sulfoxide (DMSO)-d₆: 2.50 ppm). The following abbreviations were used to explain NMR peak multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Low-resolution mass spectra were recorded on Bruker micrOTOF focus II mass spectrometer using electrospray ionization time-of-flight (ESI-TOF) reflectron experiments.

Typical Procedure for the Preparation of CFA-Fragments: 2-Chloro-2-fluoro-N-(5-hydroxy-naphthalen-1-yl)acetamide (1)

To a stirred solution of 5-amino-1-naphthol (126 mg, 0.792 mmol) and sodium chlorofluoroacetate (159 mg, 1.18 mmol) in dichloromethane (8 mL) was added T3P (50 wt% solution in AcOEt, 701 µL, 1.18 mmol) and N,N-diisopropylethylamine (DIPEA) (273 µL, 1.57 mmol) at 0°C. After stirred at ambient temperature for 1 h, the reaction mixture was diluted with water and extracted thrice with CHCl₃. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexane/AcOEt = 3 : 1) to afford the title compound (37.2 mg, 18% yield) as a pale purple solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.31 (1H, br s), 8.14 (1H, d, J = 8.5 Hz), 7.98 (1H, d, J = 7.5 Hz), 7.51 (1H, t, J = 8.0 Hz), 7.41–7.36 (2H, m), 6.88–6.82 (1H, m), 6.55 (1H, d, J = 51.0 Hz); ESI-MS m/z: 276.02 (M + Na)⁺ (Calcd for C₁₂H₉ClFNO₂Na: 276.02).

2-Chloro-2-fluoro-N-(4-phenoxyphenyl)acetamide (2)

White solid. ¹H-NMR (400 MHz, CDCl₃) δ: 7.83 (1H, br s), 7.52 (2H, d, J = 8.8 Hz), 7.32 (2H, t, J = 8.0 Hz), 7.10 (1H, t, J = 8.0 Hz), 7.04–6.96 (4H, m), 6.41 (1H, d, J = 51.2 Hz); ESI-MS m/z: 302.04 (M + Na)⁺ (Calcd for C₁₄H₁₁ClFNO₂Na: 302.04).

2-Chloro-2-fluoro-N-(2-phenoxyphenyl)acetamide (3)

Colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ: 8.61 (1H, br s), 8.41 (1H, dd, J = 8.0, 1.5 Hz), 7.41–7.36 (2H, m), 7.20–7.02 (5H, m), 6.88 (1H, dd, J = 8.0, 1.5 Hz), 6.40 (1H, d, J = 50.5 Hz); ESI-MS m/z: 302.01 (M + Na)⁺ (Calcd for C₁₄H₁₁ClFNO₂Na: 302.04).

2-Chloro-2-fluoro-N-(3-phenoxyphenyl)acetamide (4)

Yellow oil. ¹H-NMR (500 MHz, CDCl₃) δ: 7.85 (1H, br s), 7.40–7.29 (4H, m), 7.28–7.24 (1H, m), 7.14 (1H, t, J = 7.5 Hz), 7.04 (2H, d, J = 7.5 Hz), 6.87–6.82 (1H, m), 6.39 (1H, d, J = 51.0 Hz); ESI-MS m/z: 302.03 (M + Na)⁺ (Calcd for C₁₄H₁₁ClFNO₂Na: 302.04).

2-Chloro-N-(3-chloro-4-methylphenyl)-2-fluoroacetamide (5)

White solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.85 (1H, br s), 7.66 (1H, d, J = 2.5 Hz), 7.34 (1H, dd, J = 8.5, 2.5 Hz), 7.22 (1H, d, J = 8.5 Hz), 6.41 (1H, d, J = 51.0 Hz), 2.36 (3H, s); ESI-MS m/z: 257.98 (M + Na)⁺ (Calcd for C₉H₈Cl₂FNONa: 257.99).

2-Chloro-2-fluoro-N-[4-(pyridin-4-yl)phenyl]acetamide (6)

Yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.67 (2H, d, J = 6.0 Hz), 8.13 (1H, br s), 7.73 (2H, d, J = 9.0 Hz), 7.68 (2H, d, J = 9.0 Hz), 7.50 (2H, d, J = 6.0 Hz), 6.45 (1H, d, J = 51.0 Hz); ESI-MS m/z: 265.06 (M + H)⁺ (Calcd for C₁₃H₁₁ClFN₂O: 265.05).

2-Chloro-2-fluoro-N-[4-(pyridin-3-yl)phenyl]acetamide (7)

Yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.85 (1H, d, J = 2.0 Hz), 8.61 (1H, dd, J = 5.0, 1.5 Hz), 8.06 (1H, br s), 7.90 (1H, dt, J = 8.0, 2.0 Hz), 7.72 (2H, d, J = 9.0 Hz), 7.61 (2H, d, J = 9.0 Hz), 7.40 (1H, dd, J = 8.0, 5.0 Hz), 6.46 (1H, d, J = 51.0 Hz); ESI-MS m/z: 265.06 (M + H)⁺ (Calcd for C₁₃H₁₁ClFN₂O: 265.05).

2-Chloro-2-fluoro-N-[3-(pyridin-4-yl)phenyl]acetamide (8)

White solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.67 (2H, d, J = 6.0 Hz), 8.32 (1H, br s), 7.97 (1H, d, J = 1.5 Hz), 7.61 (1H, dt, J = 7.5, 2.0 Hz), 7.52–7.46 (4H, m), 6.46 (1H, d, J = 51.0 Hz); ESI-MS m/z: 265.06 (M + H)⁺ (Calcd for C₁₃H₁₁ClFN₂O: 265.05).

2-Chloro-2-fluoro-N-[3-(pyridin-3-yl)phenyl]acetamide (9)

Pale-brown solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.86 (1H, d, J = 2.0 Hz), 8.62 (1H, d, J = 5.0, 2.0 Hz), 8.15 (1H, br s), 7.92 (1H, dt, J = 8.0, 2.0 Hz), 7.88 (1H, t, J = 2.0 Hz), 7.62–7.59 (1H, m), 7.50 (1H, t, J = 8.0 Hz), 7.45–7.39 (2H, m), 6.46 (1H, d, J = 51.0 Hz); ESI-MS m/z: 265.05 (M + H)⁺ (Calcd for C₁₃H₁₁ClFN₂O: 265.05).

2-Chloro-2-fluoro-N-(quinolin-6-yl)acetamide (10)

White solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.89 (1H, dd, J = 4.0, 1.5 Hz), 8.39 (1H, d, J = 2.5 Hz), 8.20 (1H, br s), 8.18 (1H, d, J = 8.5 Hz), 8.13 (1H, d, J = 9.0 Hz), 7.67 (1H, dd, J = 9.0, 2.5 Hz), 7.44 (1H, dd, J = 8.5, 4.0 Hz), 6.49 (1H, d, J = 51.0 Hz); ESI-MS m/z: 239.03 (M + H)⁺ (Calcd for C₁₁H₉ClFN₂O: 239.04).

2-Chloro-2-fluoro-N-(quinolin-8-yl)acetamide (11)

White solid. ¹H-NMR (400 MHz, CDCl₃) δ: 10.71 (1H, br s), 8.85 (1H, d, J = 4.0 Hz), 8.73 (1H, d, J = 7.2 Hz), 8.18 (1H, d, J = 8.4 Hz), 7.62–7.53 (3H, m), 6.53 (1H, d, J = 50.8 Hz); ESI-MS m/z: 239.04 (M + H)⁺ (Calcd for C₁₁H₉ClFN₂O: 239.04).

2-Chloro-2-fluoro-N-(quinolin-5-yl)acetamide (12)

White solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.98 (1H, dd, J = 4.0, 1.5 Hz), 8.34 (1H, br s), 8.20 (1H, d, J = 8.5 Hz), 8.09 (1H, d, J = 8.5 Hz), 7.90 (1H, d, J = 7.5 Hz), 7.75 (1H, t, J = 7.5 Hz), 7.50 (1H, d, J = 8.5, 4.0 Hz), 6.56 (1H, d, J = 51.0 Hz); ESI-MS m/z: 239.04 (M + H)⁺ (Calcd for C₁₁H₉ClFN₂O: 239.04).

2-Chloro-2-fluoro-N-(quinolin-3-yl)acetamide (13)

Orange solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.88 (1H, d, J = 2.5 Hz), 8.81 (1H, d, J = 2.5 Hz), 8.40 (1H, br s), 8.10 (1H, d, J = 8.5 Hz), 7.85 (1H, d, J = 8.5 Hz), 7.71 (1H, t, J = 7.5 Hz), 7.60 (1H, t, J = 7.5 Hz), 6.52 (1H, d, J = 50.5 Hz); ESI-MS m/z: 239.05 (M + H)⁺ (Calcd for C₁₁H₉ClFN₂O: 239.04).

2-Chloro-2-fluoro-N-(4-hydroxy-6-methylpyrimidin-2-yl)acetamide (14)

Pale-brown solid. ¹H-NMR (400 MHz, CDCl₃) δ: 6.39 (1H, d, J = 51.0 Hz), 5.95 (1H, s), 2.26 (3H, s); ESI-MS m/z: 242.00 (M + Na)⁺ (Calcd for C₇H₇ClFN₃O₂Na: 242.01).

2-Chloro-2-fluoro-N-(5-methoxy-4-methylpyrimidin-2-yl)acetamide (15)

White solid. ¹H-NMR (400 MHz, DMSO-d₆) δ: 11.08 (1H, s), 7.08 (1H, d, J = 52.0 Hz), 6.55 (1H, s), 3.90 (3H, s), 2.33 (3H, s); ESI-MS m/z: 256.06 (M + Na)⁺ (Calcd for C₈H₉ClFN₃O₂Na: 256.02).

Methyl 4-(2-Chloro-2-fluoroacetamido)-3-methylbenzoate (16)

Off-white solid. ¹H-NMR (400 MHz, CDCl₃) δ: 8.08 (1H, d, J = 8.8 Hz), 7.96–7.84 (3H, m), 6.44 (1H, d, J = 51.0 Hz), 3.89 (3H, s), 2.34 (3H, s); ESI-MS m/z: 282.03 (M + Na)⁺ (Calcd for C₁₁H₁₁ClFNO₃Na: 282.03).

2-Chloro-2-fluoro-N-(1H-indazol-4-yl)acetamide (17)

White solid. ¹H-NMR (400 MHz, CDCl₃) δ: 8.19 (1H, br s), 8.11 (1H, s), 7.67 (1H, d, J = 7.2 Hz), 7.43–7.36 (2H, m), 6.50 (1H, d, J = 51.2 Hz); ESI-MS m/z: 250.02 (M + Na)⁺ (Calcd for C₉H₇ClFN₃ONa: 250.02).

2-Chloro-2-fluoro-N-(1H-indazol-5-yl)acetamide (18)

Pale-brown solid. ¹H-NMR (400 MHz, CDCl₃) δ: 8.12 (1H, s), 8.07 (1H, s), 7.97 (1H, br s), 7.49 (1H, d, J = 8.8 Hz), 7.43 (1H, d, J = 8.8 Hz), 6.45 (1H, d, J = 51.0 Hz); ESI-MS m/z: 250.02 (M + Na)⁺ (Calcd for C₉H₇ClFN₃ONa: 250.02).

2-Chloro-2-fluoro-N-(1H-indazol-6-yl)acetamide (19)

Pale-yellow solid. ¹H-NMR (400 MHz, DMSO-d₆) δ: 12.98 (1H, br s), 10.73 (1H, br s), 8.10 (1H, s), 8.00 (1H, s), 7.72 (1H, d, J = 8.8 Hz), 7.21 (1H, dd, J = 8.8, 1.6 Hz), 6.88 (1H, d, J = 49.0 Hz); ESI-MS m/z: 250.00 (M + Na)⁺ (Calcd for C₉H₇ClFN₃ONa: 250.02).

N-[(1H-Indazol-5-yl)methyl]-2-chloro-2-fluoroacetamide (20)

White solid. ¹H-NMR (400 MHz, DMSO-d₆) δ: 13.0 (1H, br s), 9.19 (1H, br s), 8.03 (1H, s), 7.63 (1H, s), 7.50 (1H, d, J = 8.0 Hz), 7.27 (1H, d, J = 8.4 Hz), 6.77 (1H, d, J = 49.0 Hz), 4.43 (2H, d, J = 5.6 Hz); ESI-MS m/z: 264.04 (M + Na)⁺ (Calcd for C₁₀H₉ClFN₃ONa: 264.03).

2-Chloro-2-fluoro-N-(thiazol-2-yl)acetamide (21)

White solid. ¹H-NMR (400 MHz, CDCl₃) δ: 11.42 (1H, s), 7.57 (1H, d, J = 3.6 Hz), 7.09 (1H, d, J = 3.6 Hz), 6.64 (1H, d, J = 50.0 Hz); ESI-MS m/z: 194.97 (M + H)⁺ (Calcd for C₅H₅ClFN₂O: 194.98).

2-Chloro-2-fluoro-N-(6-fluorobenzo[d]thiazol-2-yl)acetamide (22)

White solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.79 (1H, dd, J = 9.0, 4.5 Hz), 7.54 (1H, dd, J = 7.5, 2.5 Hz), 7.22 (1H, td, J = 9.0, 2.5 Hz), 6.56 (1H, d, J = 50.5 Hz); ESI-MS m/z: 284.96 (M + Na)⁺ (Calcd for C₉H₅ClF₂N₂OSNa: 284.97).

2-Chloro-2-fluoro-N-(2-phenylthiazol-5-yl)acetamide (23)

Pale-yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.84 (1H, br s), 7.95–7.90 (2H, m), 7.77 (1H, s), 7.49–7.41 (3H, m), 6.54 (1H, d, J = 50.5 Hz); ESI-MS m/z: 292.99 (M + Na)⁺ (Calcd for C₁₁H₈ClFN₂OSNa: 292.99).

2-Chloro-2-fluoro-N-(4-methyl-2-phenylthiazol-5-yl)acetamide (24)

Yellow solid. ¹H-NMR (400 MHz, CDCl₃) δ: 8.06 (1H, br s), 7.89–7.86 (2H, m), 7.43–7.39 (3H, m), 6.52 (1H, d, J = 50.8 Hz), 2.45 (3H, s); ESI-MS m/z: 307.00 (M + Na)⁺ (Calcd for C₁₂H₁₀ClFN₂OSNa: 307.01).

2-Chloro-2-fluoro-N-[(2-phenylthiazol-5-yl)methyl]acetamide (25)

White solid. ¹H-NMR (400 MHz, CDCl₃) δ: 7.91–7.89 (2H, m), 7.73 (1H, s), 7.43–7.41 (3H, m), 6.70 (1H, br s), 6.33 (1H, d, J = 51.2 Hz), 4.74 (2H, d, J = 5.6 Hz); ESI-MS m/z: 307.00 (M + Na)⁺ (Calcd for C₁₂H₁₀ClFN₂OSNa: 307.01).

2-Chloro-2-fluoro-N-(3-phenylisoxazol-5-yl)acetamide (26)

Yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.87 (1H, br s), 7.85–7.80 (2H, m), 7.50–7.46 (3H, m), 6.83 (1H, s), 6.52 (1H, d, J = 50.5 Hz); ESI-MS m/z: 277.02 (M + Na)⁺ (Calcd for C₁₁H₈ClFN₂O₂Na: 277.02).

N-[2-(1H-Indol-3-yl)ethyl]-2-chloro-2-fluoroacetamide (27)

Orange solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.05 (1H, br s), 7.62 (1H, d, J = 8.0 Hz), 7.40 (1H, d, J = 8.0 Hz), 7.23 (1H, td, J = 8.0, 1.0 Hz), 7.15 (1H, td, J = 8.0, 0.5 Hz), 7.07 (1H, d, J = 2.0 Hz), 6.37 (1H, br s), 6.26 (1H, d, J = 51.0 Hz), 3.75–3.60 (2H, m), 3.05 (2H, t, J = 6.5 Hz); ESI-MS m/z: 277.06 (M + Na)⁺ (Calcd for C₁₂H₁₂ClFN₂ONa: 277.05).

N-Adamantan-1-yl-2-chloro-2-fluoroacetamide (28)

White solid. ¹H-NMR (500 MHz, CDCl₃) δ: 6.15 (1H, d, J = 51.5 Hz), 5.91 (1H, br s), 2.12 (3H, br s), 2.04 (6H, d, J = 3.5 Hz), 1.70 (6H, t, J = 3.5 Hz); ESI-MS m/z: 268.09 (M + Na)⁺ (Calcd for C₁₂H₁₇ClFNONa: 268.09).

(E)-2-Chloro-2-fluoro-N-[4-(phenyldiazenyl)phenyl]acetamide (29)

Orange solid. ¹H-NMR (500 MHz, CDCl₃) δ: 8.05 (1H, br s), 7.98 (2H, d, J = 9.0 Hz), 7.91 (2H, d, J = 7.0 Hz), 7.76 (2H, d, J = 9.0 Hz), 7.54–7.46 (3H, m), 6.45 (1H, d, J = 51.0 Hz); ESI-MS m/z: 314.03 (M + Na)⁺ (Calcd for C₁₄H₁₁ClFN₃ONa: 314.05).

2-Chloro-N-(6-ethoxybenzo[d]thiazol-2-yl)-2-fluoroacetamide (30)

Off-white solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.71 (1H, d, J = 9.0 Hz), 7.30 (1H, d, J = 2.5 Hz), 7.08 (1H, dd, J = 9.0, 2.5 Hz), 6.55 (1H, d, J = 50.0 Hz), 4.10 (2H, q, J = 7.0 Hz), 1.46 (3H, t, J = 7.0 Hz); ESI-MS m/z: 310.99 (M + Na)⁺ (Calcd for C₁₁H₁₀ClFN₂O₂SNa: 311.00).

Preparation of 2-Chloro-2-fluoro-N-[6-(prop-2-yn-1-yloxy)benzo[d]thiazol-2-yl]acetamide (30-yne)

To a stirred solution of 2-amino-6-hydroxybenzothiazole (501 mg, 3.01 mmol) and K₂CO₃ (1.02 g, 7.41 mmol) in dry DMF (10 mL) was added propargyl bromide (240 µL, 3.17 mmol) dropwise at ambient temperature. After stirred overnight, the reaction mixture was diluted with AcOEt and sat. NaHCO₃. The organic phase was separated and the aqueous phase was extracted twice with AcOEt. The combined organic layers were washed twice with sat. NaHCO₃ and brine, dried over MgSO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (CHCl₃/MeOH = 40 : 1) to give 6-(prop-2-yn-1-yloxy)benzo[d]thiazol-2-amine (274 mg, 45% yield) as a grey solid. To a stirred solution of 6-(prop-2-yn-1-yloxy)benzo[d]thiazol-2-amine (52.1 mg, 0.255 mmol) and sodium chlorofluoroacetate (58.5 mg, 0.435 mmol) in dry dichloromethane (1 mL) was added T3P (50 wt% solution in AcOEt, 219 µL, 0.367 mmol) and DIPEA (128 µL, 0.735 mmol) at ambient temperature. After stirred for 1 h, the reaction mixture was diluted with sat. NaHCO₃ and extracted thrice with AcOEt. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexane/AcOEt = 3 : 1) to afford 30-yne (66.8 mg, 88% yield) as an off-white solid. ¹H-NMR (500 MHz, CDCl₃) δ: 7.75 (1H, d, J = 9.0 Hz), 7.43 (1H, d, J = 2.5 Hz), 7.16 (1H, dd, J = 9.0, 2.5 Hz), 6.55 (1H, d, J = 50.5 Hz), 4.77 (2H, d, J = 2.5 Hz), 2.56 (1H, t, J = 2.5 Hz); ESI-MS m/z: 320.99 (M + Na)⁺ (Calcd for C₁₂H₈ClFN₂O₂SNa: 320.99).

Papain Activity Screening

Papain (Sigma-Aldrich, P4762) (0.5 µM) in 40 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer (pH 7.0) was preactivated with 200 µM TCEP for 1 h in a 96-well plate. Activated papain was then incubated with the CFA-fragment (10 or 100 µM) at 37°C. After incubated for 2 h, Bz-Arg-AMC in HEPES buffer-DMSO (9 : 1) (0.8 µM) was added to the papain solution. After 1 h, the formation of 7-amino-4-methylcoumarin was measured by fluorescence (excitation: 355 nm, emission: 460 nm) with EnSpire (PerkinElmer, Inc.) multimode plate reader instrument.

Kinetic Analysis of Papain Inactivation

Papain (4.8 µM) in 50 mM sodium phosphate and 2 mM ethylenediaminetetraacetic acid (EDTA) at pH 6.2 was preactivated with 1 mM dithiothreitol (DTT) for 30 min. Activated papain (3.84 µM) in 4 : 1 mixture of 50 mM sodium phosphate and 2 mM EDTA at pH 6.2 and acetonitrile was then preincubated for 1 h with varying concentrations of the CFA-fragment. Every 15 min, 10 µL of the reaction mixture was added to a well of 96-well plate containing 100 µL of 4 : 1 mixture of 50 mM sodium phosphate and 2 mM EDTA at pH 6.2 and acetonitrile with 400 µM Cbz-Gly-ONp. p-Nitrophenol product formation was monitored by absorbance at 340 nm with PerkinElmer, Inc. EnSpire plate reader to determine the papain activity E. All reactions were performed in triplicate. The value of k_inact/K_i for the inhibitor was then determined according to the method described by Kitz and Wilson.³¹⁾ Briefly, the slopes of the plots of ln(100 × E/E₀) versus time were used to determine the pseudo-first-order inhibition constant k_app at a given concentration of the inhibitor (E₀: papain activity in the absence of the inhibitor). The second-order inhibition constant k_inact/K_i was then determined from the slope of the plot of the following equation: 1/k_app = K_i/k_inact·1/[inhibitor] + 1/k_inact.

LC/MS Analysis of Intact Proteins

The dialyzed proteins were analyzed by LC-MS (Dionex Ultimate 3000 HPLC system equipped with an autosampler, and EXACTIVE Plus mass spectrometer, Thermo) with column (Presto FF-C18 (3 × 75 mm), Imtakt) at 60°C, 0.1% TFA and acetonitrile (from 5 to 80% acetonitrile in 10 min) (200 µL/min). Samples were detected by ESI positive ion mode. Data acquisition and analysis were performed with Xcalibur software (version 2.2, Thermo).

Papain Labeling and in-gel Fluorescence Analysis

Papain (0.5 mg/mL) in 50 mM sodium phosphate (pH 6.2) was preactivated with 1 mM DTT for 30 min at 37°C. Activated papain solution (10 µL) was diluted with 50 mM sodium phosphate (pH 6.2) containing 150 mM NaCl and 1 mM DTT (40 µL) and treated with 30-yne in DMSO (2 µL). After incubated for 1 h at 37°C, to the mixture was added 100 mM N-ethylmaleimide solution (1 µL) and further incubated for 20 min. pH of the reaction mixture was adjusted to approx. 4.5 by adding 6.25% (v/v) AcOH solution (1.6 µL). The reaction mixture was subjected to CuAAC reaction with 12 µM rhodamine azide, 0.8 mM TCEP (Sigma-Aldrich), 80 µM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, Sigma-Aldrich) and 0.8 mM CuSO₄. The mixture was incubated for 30 min at 37 °C. After addition of 15 µL 5 × SDS-PAGE loading buffer, the mixture was further incubated for 30 min at 37 °C and the sample (12 µL) was analyzed with a 15% Acrylamide SDS-PAGE gel. The in-gel fluorescence imaging was performed using LAS-4000 lumino image analyzer (FUJI FILM). For the competition experiment, activated papain solution (0.1 mg/mL) was pretreated with an inhibitor (100 µM) for 1 h at 37°C before adding 30-yne.

Papain Labeling in the Presence of E. coli Lysate

E. coli pellet (approx. 2 g) was suspended in 10 mL lysis buffer (50 mM sodium phosphate, 150 mM NaCl, 1 mM DTT, pH 6.2) and lysed using a probe sonicator. The suspension was centrifuged (4°C, 13000 × g, 10 min) and the supernatant was collected. The lysate was diluted with four volumes of the lysis buffer. 40 µL of the diluted lysate was combined with activated papain solution (10 µL), treated with 30-yne, and processed for in-gel fluorescence analysis as described above.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems” (JSPS KAKENHI Grant No. JP17H06349) and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP18am0101091. N.S. acknowledges Grant-in-Aid for Young Scientists B (JSPS KAKENHI Grant No. JP17K15483), Grant-in-Aid for Scientific Research B (JSPS KAKENHI Grant No. 19H02854), and AMED under Grant Number JP20ak0101121 for their financial supports. ITbM is supported by the World Premier International Research Center Initiative, Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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