Metabolite analysis and compound isolation

The preparation of the protoplasts of A. nidulans and transformation are described in the Supplementary Methods. For small-scale metabolite analysis in A. nidulans, transformants containing the desired plasmids were selected from CD sorbitol agar (2% glucose as carbon source) appropriately supplemented with riboflavin, uracil and/or pyridoxine. CD-ST agar was inoculated with spores and incubated at 28 °C for 4 days. The agar was then collected and extracted with acetone for 30 min with sonication. After centrifugation, the supernatant (200 μl) was concentrated and resuspended in methanol (100 μl). The sample was subjected to LC–QTOF (quadrupole time-of-flight) analysis with an Agilent 6545 QTOF equipped with a reverse-phase column (Agilent Poroshell, 120 EC-C18; 2.7 μm, 3.0 × 50 mm) using positive-mode electrospray ionization with 1% acetonitrile in H2O (containing 0.1% formic acid) for the first 2 min, then a linear gradient of 1–95% for 9 min and finally 95% acetonitrile for 3 min with a flow rate of 0.6 ml min−1. The data were collected and analyzed using MassHunter 10.0 (Agilent). Some LC–MS analyses were performed on an Agilent LC–MSD iQ (Agilent InfinityLab Poroshell 120 Aq-C18; 2.7 μm, 100 Å, 2.1 × 100 mm) using positive-mode and negative-mode electrospray ionization with a linear gradient of 1–99% acetonitrile in H2O supplemented with 0.1% (v/v) formic acid in 13.25 min followed by 99% acetonitrile for 3 min with a flow rate of 0.6 ml min−1. The data were collected and analyzed using OpenLab CDS 2.4 (Agilent). For large-scale analysis, A. nidulans transformants were inoculated on 40 plates each containing 50 ml of CD-ST agar and were placed in a 28 °C incubator for 3–4 days. After 4 days, the solid agar cultures were cut into small pieces and extracted extensively with acetone. The residual was loaded on a normal-phase CombiFlash system and subjected to flash chromatography with a gradient of CH2Cl2 and methanol for initial separation. Metabolites of interest, tracked by analytical high-performance LC (HPLC) and LC–MS, were purified from the corresponding fractions by reverse-phase semipreparative HPLC with a COSMOSIL column with a flow rate of 4 ml min−1 of solvents A (H2O with 0.1% trifluoroacetic acid) and B (acetonitrile). NMR spectra were obtained with a Bruker AV500 spectrometer with a 5-mm dual cryoprobe at the UCLA Molecular Instrumentation Center. (1H-NMR, 500 MHz; 13C-NMR, 125 MHz). High-resolution mass spectra were also recorded on a Agilent 6545 QTOF high-resolution MS instrument (UCLA Molecular Instrumentation Center). The mass and NMR spectra were analyzed using MassHunter 10.0 (Agilent) and MestReNova-9.0.1 (Mestrelab Research), respectively.

Protein expression and purification

The intron-free open reading frames encoding Cp1A, Cp1B, Cp1D, Cp2B, Cp2C and Cp2D were amplified by PCR using complementary DNA from the corresponding A. nidulans transformant as a template and ligated to linear expression vector pET28a by Gibson assembly (New England Biolabs) according to the manufacturer’s protocol. The mfaA gene sequence was obtained from the National Center for Biotechnology Information database, was codon-optimized on the basis of the codon preference of E. coli and synthesized by Integrated DNA Technologies (IDT). The gene encoding CHU (polyphosphate kinase) was also synthesized by IDT. The plasmids were then transformed into E. coli BL21(DE3) individually and grown overnight in 5 ml of Luria–Bertani (LB) medium with 50 μg ml−1 kanamycin at 37 °C. The overnight cultures were used as seed cultures for 1 L of fresh LB medium containing 50 μg ml−1 kanamycin and incubated at 37 °C until the OD600 reached 0.8. The cultures were cooled on ice before the addition of 0.1 mM isopropyl-β-d-thiogalactopyranoside (GoldBio) to induce protein expression. The expression was performed at 16 °C for 20 h at 220 rpm. E. coli cells were harvested by centrifugation at 5,200g for 15 min and resuspended in 30 ml of A10 buffer (50 mM sodium phosphate buffer, 150 mM NaCl and 10 mM imidazole, pH 8.0) containing one tablet of Pierce protease inhibitor (Thermo Fisher Scientific). The cell suspension was lysed on ice by sonication and the lysate was centrifuged at 17,000g for 30 min at 4 °C to remove the insoluble cellular debris. Recombinant 6×His-tagged proteins were purified at 4 °C from corresponding soluble fractions by affinity chromatography with Ni-NTA agarose resin (GE Healthcare). Briefly, recombinant proteins on the resin was initially washed with wash buffer A1 (50 mM sodium phosphate, 150 mM NaCl and 10 mM imidazole, pH 8.0) until no protein was detected in the eluent using the Bradford reagent. Then, the same procedure was repeated with wash buffer A2 (50 mM sodium phosphate, 150 mM NaCl and 20 mM imidazole, pH 8.0). The target protein was eluted by elution buffer A (50 mM sodium phosphate, 150 mM NaCl and 250 mM imidazole, pH 8.0). The purified proteins were concentrated and exchanged into storage buffer (50 mM sodium phosphate, 200 mM NaCl and 10% glycerol, pH 8.0) with an Amicon Ultra concentrator (30-kDa cutoff; Merck Millipore). SDS–PAGE was performed to check the protein purity and a Bradford protein assay (Bio-Rad) was used to calculate protein concentration with BSA (Sigma) as the standard. The proteins were aliquoted and stored at −80 °C until used in in vitro assays. The plasmids used for protein purification are listed in Supplementary Table 3. Results of SDS–PAGE analysis are presented in Supplementary Figs. 7 and 15.

To purify Cp1B for crystallization, the cell pellet of E. coli BL21(DE3) was resuspended in Tris buffer (50 mM Tris and 500 mM NaCl, pH 8.0) and lysed by sonication on ice. Cell debris was removed by centrifugation at 17,000g, 4 °C for 30 min. Recombinant proteins in the supernatant were purified using nickel–Sepharose resin (GE Healthcare) and initially washed with wash buffer B1 (50 mM Tris, 500 mM NaCl and 30 mM imidazole, pH 8.0) until no protein was detected in the eluent using the Bradford reagent. Then, the target protein was eluted by elution buffer B (50 mM Tris, 500 mM NaCl and 300 mM imidazole, pH 8.0). The elution containing the target protein was concentrated to 2 ml for size-exclusion chromatography. The protein was then loaded onto a size-exclusion chromatograph (GE Healthcare, Superdex 200) in buffer of 50 mM Tris pH 8.0 with 100 mM NaCl through the Bio-Rad chromatography system. The purified Cp1B was concentrated to 15 mg ml−1 for crystallization.

Enzymatic assays

To assay the activities of Cp1A and MfaA, 100-μl reactions were performed at 30 °C for 3 h in 50 mM sodium phosphate buffer (pH 8.0) containing 0.2 mM FeSO4, 2 mM αKG, 2 mM ascorbate, 1 mM substrate and 10 μM Cp1A or MfaA. The reaction mixture in the absence of protein was prepared as the negative control. Enzyme reactions were quenched by adding 100 μl of acetonitrile and centrifuged at 17,000g for 5 min, before being subjected to 3-NPH derivatization71. To perform 3-NPH derivatization for the detection of 1,2-dicarboxylic acid, all reagents for derivatization were freshly prepared before use. Then, 50 μl of reaction mixture was sequentially treated with 50 μl of 50 mM 3-NPH (Sigma) in methanol and H2O (70:30, v/v), 50 μl of 50 mM EDC (Oakwood Chemical) in methanol and H2O (70:30, v/v) and 50 μl of 7% v/v pyridine (Acros Organics) in methanol and H2O (70:30, v/v) and mixed thoroughly. Derivatization mixtures were incubated at 37 °C for 30 min and centrifuged at 17,000g for 10 min. The supernatant was subjected to LC–QTOF analysis with an Agilent 6545 QTOF equipped with a reverse-phase column (Agilent Poroshell, 120 EC-C18; 2.7 μm, 3.0 × 50 mm) using positive-mode electrospray ionization with 1% acetonitrile in H2O (containing 0.1% formic acid) for the first 2 min, then a linear gradient of 1–95% for 9 min and finally 95% acetonitrile for 3 min with a flow rate of 0.6 ml min−1. The data were collected and analyzed by MassHunter 10.0 (Agilent).

To assay the activities of Cp1B and Cp2B, assays were carried out in 50 mM sodium phosphate buffer (pH 8.0) or 50 mM HEPES buffer (pH 8.0). A typical reaction contained 25 μM enzyme, 5 mM (±)-t-ES or other dicarboxylic acid, 2.5 mM amino acid, 10 mM ATP and 10 mM MgCl2 in 100 μl of 50 mM sodium phosphate buffer (pH 8.0). After incubation at 30 °C for 16 h, the reaction was then quenched with 120 μl of acetonitrile and centrifuged at 17,000g for 5 min. The supernatant was subjected to LC–QTOF analysis with the same conditions as mentioned above or analyzed by an Agilent LC–MSD iQ with a reverse-phase column (Agilent InfinityLab Poroshell 120 Aq-C18; 2.7 μm, 100 Å, 2.1 × 100 mm) using positive-mode and negative-mode electrospray ionization with a linear gradient of 1–99% acetonitrile in H2O supplemented with 0.1% (v/v) formic acid with a flow rate of 0.6 ml min−1. The data were collected and analyzed by OpenLab CDS 2.4 (Agilent). The detailed gradient conditions are listed in the figure legends of the Supplementary Figures. The analytical percentage yields shown in Fig. 3c (heat map) were estimated from the standard curves of enzymatically prepared standards generated from peak areas at 204 nm by HPLC. To determine the diastereomeric ratio values, the sample was analyzed by chiral analytical HPLC with a CHIRALPAK IA-3 column (150 × 4.6 mm, 3 μm) at room temperature (flow rate 1 ml min−1, 40% acetonitrile in H2O with 0.1% trifluoroacetic acid).

Because of the insolubility of Cp1C when expressed from E. coli BL21(DE3), the decarboxylase Cp2C from cp2 cluster was expressed for characterization instead. In vitro assays of Cp2C were performed in 50 μl of 50 mM sodium phosphate buffer (pH 8.0), containing 50 μM Cp2C, 100 μM PLP, 2 mM l-amino acid such as l-ornithine, l-lysine and l-arginine at 30 °C. After 1 h at 30 °C, all reactions were quenched with 50 μl of acetonitrile, centrifuged at 17,000g for 5 min, subjected to dansyl derivatization with dansyl chloride (Tokyo Chemical Industry) and analyzed by LC–MS. Dansyl derivatization was performed by adding 50 μl of 1 M borate buffer (pH 8.0) and dansyl chloride solution (10 mM final concentration). After incubation at 30 °C for 1 h, the resultant reaction mixture was centrifuged at 17,000g for 5 min. The resultant supernatant was subjected to LC–QTOF analysis using the same gradient methods described above (Supplementary Fig. 34).

To assay the activities of Cp1D, assays were performed in 50 mM sodium phosphate buffer (pH 8.0) or 50 mM HEPES buffer (pH 8.0). A typical reaction contains 10 μM or 25 μM Cp1D, 2 mM or 2.5 mM monoamide such as 14 or 15, 2.5 mM or 5 mM amine, 10 mM ATP and 10 mM MgCl2 in 100 μl of 50 mM sodium phosphate buffer (pH 8.0). After incubation at 30 °C for 16 h, the subsequent sample preparation and LC–QTOF or LC–MS (Agilent LC–MSD iQ) analysis followed the same methods as described for the Cp1B reaction. The detailed gradient conditions for LC–MS analysis are shown in each figure legend in the Supplementary Figures. The analytical percentage yields shown in Fig. 4 were estimated from the standard curves generated at λ = 204 nm of purified 15-b15 (for 15-b1 to 15-b17, 15-b28, 15-b39 and 15-b40), 15-b24 (for 15-b21 to 15-b23), 15-b27 (for 15-b25 to 15-b26) or 15-b37 (for 15-b34 to 15-b36). For kinetics analysis of Cp1D and Cp2D for the amidation, a similar procedure to that mentioned above was used except for various concentrations of a single diastereomer (2S,3S)-t-ES-Ile or (2R,3R)-t-ES-Ile. Briefly, 100 µl of reaction mixture in 50 mM sodium phosphate buffer (pH 8.0) contained 10 mM MgCl2, 10 mM ATP, 5 mM agmatine, 4 μM Cp1D or Cp2D, various concentrations of (2S,3S)-t-ES-Ile or (2 R,3 R)-t-ES-Ile. The reaction was incubated at 30 °C for 5 min and quenched with 100 µl of acetonitrile, which was subjected to LC–MS analysis. The apparent kinetic constants are derived from the formation of the corresponding product (velocity) versus substrate concentration data using a nonlinear regression fitting method with GraphPad Prism 9.

To generate Cp1B mutants, the plasmid pML8010 containing the wild-type cp1B gene was used as the template for PCR-based site-directed mutagenesis. DNA sequencing was used to confirm the identities including the mutated positions of the expression plasmids. Following expression and purification, Cp1B mutants were subjected to activity assays as described above. Reactions were performed at 30 °C for 20 min in 100 μl of 50 mM sodium phosphate buffer (pH 8.0). Reaction components were 25 μM Cp1B or mutants, 5 mM (±)-t-ES, 2.5 mM l-Ile, 10 mM ATP and 10 mM MgCl2. The relative activities of Cp1B mutants were calculated by setting the activity of the wild type at 100%, quantified by the formation of 14.

Stepwise enzymatic assay with Cp1A/MfaA and Cp1B

Purified Cp1A or MfaA was added to 50 ml of 50 mM sodium phosphate buffer (pH 8.0) containing 0.2 mM FeSO4, 2 mM αKG, 2 mM ascorbate, 1 mM fumaric acid and 10 μM Cp1A or MfaA at 30 °C for 16 h. The enzyme was then removed by Amicon concentrators (Millipore). Subsequently 10 μM Cp1B, 2.5 mM l-isoleucine, ATP cofactor (10 mM) and MgCl2 (10 mM) were added, followed by incubation at 30 °C for 16 h. The enzyme in reaction solution was again removed by ultrafiltration. The filtrate was adjusted to pH ~3 with 4 M H2SO4 solution and extracted with ethyl acetate. Organic solvent was removed under reduced pressure and further purified using semipreparative HPLC. Purified product was subjected to NMR analysis and analysis using chiral analytical HPLC with a CHIRALPAK IA-3 column (150 × 4.6 mm, 3 μm) at room temperature (flow rate 1 ml min−1, 40% acetonitrile in H2O with 0.1% trifluoroacetic acid).

Coupled activity assay with Cp1B and Cp1D

The coupled activity assay for Cp1B with Cp1D was typically performed in 100 μl of 50 mM sodium phosphate buffer (pH 8.0) containing 25 μM Cp1B, 25 μM Cp1D, 5 mM (±)-t-ES, 2.5 mM amino acid, 5 mM amine, 10 mM ATP cofactor and 10 mM MgCl2. The reaction mixture was incubated at 30 °C for 16 h before quenching the reaction. The subsequent sample preparation and LC–MS analysis followed the same methods as described for the Cp1B reaction.

Detection of ADP from Cp1B assay

The reaction mixtures (200 μl) in 100 mM Tris-HCl buffer (pH 8.0) contained 0.25 μM Cp1B, 10 mM ATP, 12 mM MgCl2, 300 μM reduced nicotinamide adenine dinucleotide (NADH), 500 μM phosphoenolpyruvic acid (PEP), 41 U per ml pyruvate kinase (PK; Sigma), 59 U per ml lactate dehydrogenase (LDH; Sigma), 10 mM KCl with 1 mM ES or other acid donors and 5 mM l-Phe. PK and LDH were stored in 10 mM HEPES (pH 7.0) with 100 mM KCl and 0.1 mM EDTA with 50% glycerol. PK requires potassium ion as an essential cofactor for its activity. The reaction mixture was incubated at 30 °C and the consumption of NADH was monitored continuously for 60 min with a TECAN M200 plate reader by measuring the absorbance at 340 nm. The consumption of NADH reflects the formation of ADP upon Cp1B-catalyzed formation of acyl-phosphate. Therefore, the phosphorylation activity of Cp1B toward each substrate was derived from the consumption of NADH (the formation of ADP). The phosphorylation activity of Cp1B for (2S,3S)-t-ES was set as 100% activity to calculate the relative activity for the other substrates. The results are shown in Extended Data Fig. 6a.

For kinetics analysis of Cp1B for the phosphorylation, the similar procedure mentioned above was used except for various concentrations of (2S,3S)-t-ES being used. Briefly, the reaction mixtures (100 μl) in 100 mM Tris-HCl buffer (pH 8.0) contained 1.0 μM Cp1B, 10 mM ATP, 12 mM MgCl2, 300 μM NADH, 500 μM PEP, 41 U per ml PK (Sigma), 59 U per ml LDH (Sigma) and 10 mM KCl with various concentrations (0.04 mM to 1 mM) of (2S,3S)-t-ES and 5 mM l-Phe. The reaction mixture was incubated at 30 °C and the consumption of NADH at 10 min was used to derive the reaction rate (velocity) for phosphorylation. Kinetic constants (apparent) were derived from velocity versus substrate concentration data using a nonlinear regression fitting method with GraphPad Prism 9. The result is shown in Extended Data Fig. 6b.

Hydroxamate-based colorimetric assay

A hydroxamate-based colorimetric assay53 was used to test substrate specificity toward N-succinyl amino acids for ABS Cp1D or Cp2D (ref. 72). The reaction was performed in 150 μl of 50 mM Tris buffer (pH 8.0) containing 20 μM Cp1D or Cp2D, 15 mM ATP, 5 mM N-succinyl amino acid substrate, 200 mM hydroxylamine and 10 mM MgCl2. The reaction was quenched after incubation for 8 h at 30 °C by addition of equivalent volume (150 μl) of stopping solution (10% (w/v) FeCl3 and 3.3% (w/v) trichloroacetic acid dissolved in 0.7 M HCl). The precipitated enzyme was removed by centrifugation, 200 μl of the supernatant was transferred to a 96-well plate and the absorbance of the ferric-hydroxamate complex at 540 nm was measured by a Tecan M200 plate reader. The absorbance at 540 nm was used to calculate the relative activity shown in Extended Data Fig. 8b and the absorbances of N-succinyl-l-Leu and N-succinyl-l-Tyr after subtracting that of the negative control (without Cp1D or Cp2D) were set as 100% activity for Cp1D and Cp2D, respectively.

Enzymatic synthesis of t-ES amino acids

To obtain (2S,3S)-t-ES amino acids, a 20 ml reaction in 50 mM sodium phosphate buffer (pH 8.0) containing purified 2.5 μM Cp1B or Cp2B with 5 mM (±)-t-ES, 2.5 mM amino acid, 10 mM ATP and 10 mM MgCl2 was performed at 30 °C for 16 h. The protein was removed by Amicon concentrators (Millipore) and the concentrate was washed twice with three volumes of water. The filtrate was combined and was carefully adjusted to pH 2–3 with 4 M H2SO4 solution. The acidified filtrate was further extracted with an equal volume of ethyl acetate twice. The combined organic layer was washed with brine, dried over MgSO4 and filtered. The solvent was evaporated in vacuo to give a crude mixture that was further purified by HPLC (water and acetonitrile, both supplemented with 0.1% trifluoroacetic acid) on a Cosmosil C18 AR-II column (5.0 µm, 10 × 250 mm; Nacalai Tesque) to afford the products with varying isolated yields. The isolated yield for each compound is shown in Fig. 3c. All isolated compounds were characterized by NMR (Supplementary Tables 17–54 and Supplementary Figs. 92–281).

Enzymatic synthesis of (2S,3S)-t-ES-Phe-amine

The 15-ml reactions in 50 mM sodium phosphate buffer (pH 8.0) containing 2.0 mM 15, 5.0 mM amine donor and 2.5 μM Cp1D were performed at 30 °C for 16 h. Protein was removed by Amicon concentrators (Millipore) and the concentrate was washed twice with three volumes of water. For reactions containing b20, b29, b31, b32 and b41 as amine donors, the filtrate was evaporated in vacuo. The residue was dissolved in DMF and further purified with HPLC with a Cosmosil column (Nacalai Tesque, 5C18-AR-II; 10 × 250 mm) with a flow rate of 4 ml min−1 of solvents A (H2O with 0.1% trifluoroacetic acid) and B (acetonitrile with 0.1% trifluoroacetic acid). For other amine donors containing hydrophobic functional groups, the filtrate was combined and the pH was adjusted to around 2–3 with 4 M H2SO4 solution. The acidified filtrate was further extracted with equal volume ethyl acetate twice. The combined organic layer was washed with brine, dried over MgSO4 and filtered. The solvent was evaporated in vacuo to give a crude mixture that was further purified by HPLC (water and acetonitrile, both supplemented with 0.1% trifluoroacetic acid) on a Cosmosil, C18 AR-II column (5.0 µm, 10 × 250 mm; Nacalai Tesque) to afford the corresponding product with varying isolated yields as shown in Fig. 4. All compounds were characterized by NMR (Supplementary Tables 55–68 and Supplementary Figs. 282–351).

Enzymatic synthesis of cysteine protease inhibitors

A large-scale 20-ml reaction in 50 mM sodium phosphate buffer (pH 8.0) containing 5 mM (±)-t-ES, 2.5 mM l-amino acid, 2.5 mM amine donor, 2.5 μM Cp1B and 2.5 μM Cp1D was carried out at 30 °C for 16 h. The protein was removed by Amicon concentrators (Millipore) and the concentrate was washed two times with three volumes of water. For diamine or alkanoamine as amine donor, the filtrate was evaporated in vacuo and then directly subject to reverse-phase CombiFlash system (Teledyne) with a gradient of acetonitrile in H2O (0–5 min, 0–5% acetonitrile; 5–10 min, 5–20% acetonitrile; 10–20 min, 20–60% acetonitrile; 20–25 min, 60% B; 25–35 min, 100% B). Fractions containing target compound were combined and further purified with HPLC with a Cosmosil column (Nacalai Tesque, 5C18-AR-II; 10 × 250 mm) with a flow rate of 4 ml min−1 of solvents A (H2O with 0.1% trifluoroacetic acid) and B (acetonitrile with 0.1% trifluoroacetic acid). For other amine donors containing hydrophobic functional groups, the filtrate was combined and pH was adjusted to around 2–3 with 4 M H2SO4 solution. The acidified filtrate was further extracted with an equal volume of ethyl acetate for two times. The combined organic layer was washed with brine, dried over MgSO4 and filtered. The solvent was evaporated in vacuo to give a crude mixture that was further purified by HPLC. The isolated yield for each compound is shown in Fig. 5e. For example, 8.5 mg of E-64c was obtained with 54% isolated yield from a one-pot reaction. The spectroscopic and physical properties of E-64c were identical to those reported in the literature73. Isolated yields for other compounds were as follows: (2S,3S)-t-ES-a9-b7, 6.5 mg and 35% yield; (2S,3S)-t-ES-a9-b13, 6.5 mg and 40% yield; (2S,3S)-t-ES-a10-b9, 4.8 mg and 28% yield; (2S,3S)-t-ES-a10-b14, 6.7 mg and 37% yield; (2S,3S)-t-ES-a10-b26, 5.7 mg and 31% yield; (2S,3S)-t-ES-Leu-b44, 8.7 mg and 45% yield. All compounds were characterized by NMR (Supplementary Tables 69–74 and Supplementary Figs. 40, 41 and 352–381).

Chemoenzymatic synthesis of cysteine protease inhibitor

To chemoenzymatically synthesize selective cathepsin L inhibitor CLIK-148, a large-scale 20-ml reaction in 50 mM sodium phosphate buffer (pH 8.0) containing 5 mM (±)-t-ES, 2.5 mM l-Phe, 2.5 mM dimethylamine (b28), 2.5 μM Cp1B and 2.5 μM Cp1D was carried out at 30 °C for 16 h. Protein was removed by following the same procedure as mentioned above. The subsequent filtrate acidification and extraction followed the same methods as described for amine donors with a hydrophobic terminal. Solvent was evaporated in vacuo to give a crude mixture. The crude mixture was then dissolved in DMF, before adding 2-(2-aminoethyl) pyridine (Combi-Blocks; 1.2 equivalents), HATU (Combi-Blocks; 1.2 equivalents) and triethylamine (Sigma; 3 equivalents) at 0 °C. The resulting mixture was stirred at room temperature until all substrates were consumed. The reaction mixture was applied to reverse-phase HPLC chromatography using a Cosmosil column (Nacalai Tesque, 5C18 MS-II; 10 × 250 mm; flow rate of 4 ml min−1, acetonitrile in H2O with 0.1% trifluoroacetic acid) to yield 10.7 mg of CLIK-148 (52% total isolated yield).

To chemoenzymatically synthesize selective cathepsin C inhibitor E-64c hydrazide, a large-scale 40-ml reaction in 50 mM sodium phosphate buffer (pH 8.0) containing 5 mM (±)-t-ES, 2.5 mM l-norleucine (a3), 2.5 mM tryptamine (b44), 2.5 μM Cp1B and 2.5 μM Cp1D was carried out at 30 °C for 16 h. The removal of protein and the compound extraction were performed following the same procedure as mentioned above. Solvent was evaporated in vacuo to give a crude mixture. The crude mixture was then dissolved in DMF, before adding tert-butyl 1-butylhydrazine-1-carboxylate (Aaron Chemicals; 2 equivalents), HATU (Combi-Blocks; 2.4 equivalents) and triethylamine (Sigma; 10 equivalents) at 0 °C. The resulting mixture was stirred at room temperature overnight. The reaction mixture was applied to reverse-phase HPLC chromatography using a Cosmosil column (Nacalai Tesque, Cosmosil 3PBr; 10 × 250 mm; flow rate of 4 ml min−1, acetonitrile in H2O with 0.1% formic acid) to yield 20.0 mg of the Boc-protected E-64c hydrazide (36% isolated yield). Then, 20 μl of trifluoroacetic acid was added to the solution of 8.0 mg of the Boc-protected E-64c hydrazide in 200 μl of dichloromethane. The reaction mixture was stirred at room temperature. After 4 h, the solvent was evaporated in vacuo to give a crude mixture. The crude mixture was applied to reverse-phase HPLC chromatography using a Cosmosil column (Nacalai Tesque, 5C18-AR-II; 10 × 250 mm; flow rate of 4 ml min−1, acetonitrile in H2O with 0.1% trifluoroacetic acid) to yield 4.5 mg of the cathepsin C inhibitor, E-64c hydrazide (26% total isolated yield).

Inhibition assay of cysteine cathepsin proteases

The cathepsin B inhibitor screening assay57 uses the ability of cathepsin B to cleave the synthetic AMC (7-amino-4-methylcoumarin)-based peptide substrate to release AMC, which can be quantified using a fluorometer or fluorescence microplate reader. In the presence of a cathepsin B inhibitor, the cleavage of the substrate is reduced or abolished, resulting in a decrease or total loss of the AMC fluorescence. Recombinant human procathepsin B (R&D systems) was activated to mature cathepsin B by incubation at 37 °C for 20 min in activation buffer (20 mM sodium acetate pH 5.5, 1 mM EDTA, 5 mM DTT and 100 mM NaCl). Cathepsin B activity was then assayed in final buffer conditions of 0.04 ng μl−1 cathepsin B, 40 μM Z-Phe-Arg-AMC (Sigma), 40 mM citrate phosphate (pH 5.5), 1 mM EDTA, 100 mM NaCl, 5 mM DTT and 0.01% Brij at 37 °C. Cleavage of Z-Phe-Arg-AMC to generate fluorescent AMC was monitored and the relative fluorescence units (RFU; excitation, 360 nm; emission, 460 nm) were recorded over a period of 30 min using an Infinite M200 PRO multimode microplate reader (Tecan). Two time points (T1 and T2) were chosen in the linear range of the plot to obtain the corresponding values for the fluorescence (RFU1 and RFU2). Slopes for inhibitor samples and enzyme control were calculated by dividing the net ΔRFU (RFU2 − RFU1) values by ΔT (T2 − T1). The percentage relative inhibition was calculated using the following equation (Eq. 1):

$$\% \,}\,}=\frac}\,}\,}\,}-}\,}\,}}}}\,}\,}\,}}\times 100$$

(1)

To test the effect of amino acid donor toward the inhibitory activity, t-ES-based compounds were obtained from coupled in vitro activity assays with 39 different amino acid donors while the amine donor was kept as agmatine. The typical assay contained 25 μM Cp1B, 25 μM Cp1D, 2 mM (±)-t-ES, 1 mM amino acid donor, 1 mM agmatine as the amine donor, 10 mM ATP and 10 mM MgCl2 in 100 μl of 50 mM sodium phosphate buffer (pH 8.0) at 30 °C for 16 h. The reaction was stopped by heat inactivation and centrifuged at 17,000g for 5 min. The supernatant was serially diluted and used (in theory, the final concentration of the corresponding E-64 analogs in the mixture was estimated to be less than 100 nM after serial dilution) for the cathepsin B inhibition assay.

To test the effect of amine donor toward the inhibitory activity, the t-ES-based compounds were obtained from coupled activity assays with 41 different amine donors while the amino acid used was a10. The assay contained 25 μM Cp1B, 25 μM Cp1D, 2 mM (±)-t-ES, 1 mM a10, 1 mM amine donor, 10 mM ATP and 10 mM MgCl2 in 100 μl of 50 mM sodium phosphate buffer (pH 8.0) at 30 °C for 16 h. The reaction was quenched and treated as mentioned above and the diluted sample was used for the cathepsin B inhibition assay.

Kinetic analyses of synthesized inhibitors of cathepsin B were conducted to determine IC50 values. The concentrations of selected compounds and E-64 ranged from 2,174 nM to 0.27 nM. IC50 values were calculated as the inhibitor concentration that reduced cathepsin B activity by 50%. Kinetic assays were performed in a 96-well plate format with three independent replicates. Data analysis was conducted using Prism GraphPad software.

The inhibitory potencies of synthesized inhibitors toward cathepsins L (R&D systems) and K (R&D systems) were assessed. Cathepsin L (0.03 ng μl−1) and cathepsin K (0.10 ng μl−1) activities were assayed with 40 μM Z-Phe-Arg-AMC. The fluorogenic assays followed the same protocol as described for cathepsin B.

Crystallization of Cp1B, papain and papain–E-64 analogCrystallization of Cp1B

The protein concentration used for crystallization was 15 mg ml−1 (0.26 mM). The protein was incubated with ATP (final concentration: 0.26 mM), MgCl2 (final concentration: 0.26 mM) and (±)-t-ES (final concentration: 1.3 mM) for 30 min on ice, corresponding to the ratio of 1:1:1:5. The sitting-drop vapor diffusion method was used for the initial screening at 22 °C. The protein (1 μl) was then combined with the reservoir solution (1 μl) in a ratio of 1:1. The total volume of protein mixture was 2 μl, which was equilibrated against 50 μl of reservoir solution. Commercially available screen reagents including Index (Hampton Research), Crystal Screen (Hampton Research), Grid Screen (Hampton Research), Morpheus (Molecular Dimensions), JCSG (Molecular Dimensions) and NeXtal (Molecular Dimensions) were used. The crystals were observed after 1 week in the condition of 0.1 M MES–imidazole pH 6.5, 10% w/v PEG 20000, 20% v/v PEG MME 550, 0.02 M sodium l-glutamate, 0.02 M dl-alanine, 0.02 M glycine, 0.02 M dl-lysine HCl and 0.02 M dl-serine. The crystallization solution contained cryoprotectant. We did not perform any additional cryoprotection of the crystals before flash freezing. The crystals were flash-cooled and stored in liquid nitrogen.

Crystallization of papain

Twice-crystallized papain from papaya latex was purchased from Sigma (P4762) as a buffered aqueous suspension approximately 25 mg ml−1 in protein concentration. Aliquots of this suspension were mixed with methanol, at a 1:2 volume ratio of papain suspension to methanol, in the sample wells of sitting-drop crystallization trays allowing up to 30 μl of sample per well. For all crystallization experiments, a total volume of 15 μl was targeted, although crystals were successfully grown in up to 30-μl volumes. These drops were incubated against a reservoir solution containing 59% methanol and 889 mM NaCl. The crystal used for determination of the unliganded papain structure was grown as above; all others were grown from seeds. For seeding, crystals were propagated by crushing up previously grown papain crystals by repeated pipetting in their mother liquor and transferring small fragments to freshly prepared sitting drops using a strand of horsehair. Papain crystals would typically appear in this condition between 48 and 72 h without seeding but formed in 24 h if seeded. All crystals adopted a prismatic, diamond-shaped morphology.

Papain was also cocrystallized with 1 and analogs using the same protocol as above, with the addition of the chosen inhibitor compound dissolved in solution to the crystallization well. E-64 was purchased from Sigma (E3132) and dissolved to 1.25 mg ml−1 (3.5 mM) in 66% methanol. E-64c and E-64d were each purchased from Selleck Chemicals (S7392 and S7393) and dissolved to 1.25 mg ml−1 (3.7 mM) in 66% methanol. Approximately 1 mg of purified amine (2S,3S)-t-ES-a9-b7 was dissolved to 2.5 mg ml−1 (6.8 mM) in 66% methanol. For cocrystallization experiments with each compound, 2.4 μl of each compound solution was added to the crystallization well and mixed with protein solution. For these trials, the concentration of papain in each well was 0.35 mM, such that the estimated molar excess of inhibitor was 1.4× for 1, 1.5× for E-64d and 2.7× for (2S,3S)-t-ES-a9-b7. For all cocrystallization experiments, crystals were seeded with fragments of unliganded papain crystals as described above and appeared after approximately 24 h. Crystal development in the presence of 1 or its analogs was inconsistent without seeding.

Diffraction data collectionCp1B structure

The crystals were mounted on Mitegen loops and flash-frozen under a 100-K nitrogen stream. The data were collected at beamline 17-ID-2 at the National Synchrotron Light Source II. Diffraction data were collected at the wavelength of 0.97933 Å. The detector distance was 250 mm. Data were collected over oscillations of 0.25° per exposure. Whole datasets of 1,440 frames were collected in 30 s.

Papain structures

Crystals were mounted on Mitegen loops and flash-frozen under a 100-K nitrogen stream. Full X-ray diffraction datasets were acquired using a Rigaku FRE+ rotating-anode X-ray diffractometer using a Cu Kα source (emitting X-ray photons 1.54 Å in wavelength) and equipped with a Rigaku HTC detector. All rotation datasets were collected taking 2-min integrated exposures over oscillations of 0.5° per exposure totaling approximately 26 h of data collection per crystal, at a detector distance of either 78 or 74 mm. This configuration enabled visualization of reflections up to 1.4 Å in resolution at the detector edge.

Data processing, structure determination and refinementCp1B structure

Data frames in h5 file format were reduced in XDS and reflection intensities were scaled in XSCALE from the XDS package74. Data were converted to MTZ format using Pointless and resolution was determined by selecting the highest resolution shell where I/σI exceeded 2 using Aimless embedded in CCP4i2. The structure was solved by molecular replacement. The structure of Cp1B predicted using AlphaFold3 (ref. 75) (version 1; https://doi.org/10.5281/zenodo.14911266) was used as the search model by Phaser embedded in the PHENIX suite76,77,78. Refinement was performed using Refinement embedded in the PHENIX suite76,77. Briefly, Refinement was instructed to refine XYZ coordinates against both reciprocal space data and real-space maps, occupancies, individual B factors and translation, libration and screw parameters. No hydrogen atoms were modeled on any molecule. Three cycles of such refinement were performed before inspecting the agreement between the atomic coordinates and electron density map in Coot79 and building solvent molecules or adjusting side-chain positions to satisfy disagreements revealed in the difference Fourier map79. Water molecules were built manually and validated in Coot79. The statistics are summarized in Supplementary Table 4. The coordinates of the model were validated by Coot79. The images were drawn using PyMol.

Papain structures

Frames in OSC file format were reduced in XDS and reflection intensities were scaled in XSCALE and converted to MTZ format using XDSCONV74,80. Reflections extending to a resolution of 1.4–1.6 Å depending on the structure, determined by selecting for each dataset the highest resolution shell where completeness exceeded 90%, were included for integration, phasing and refinement. Phases were retrieved by molecular replacement using Phaser-MR through the PHENIX graphical user interface with a known X-ray diffraction structure of papain determined to 1.65-Å resolution (PDB 9PAP)76,77,78,81. For residue discrepancies between the protein sequence in this PDB entry and the sequence of papain reported in UniProt (P00784), the sequence from UniProt was adopted into the model for refinement.

Refinement of each structure was carried out in PHENIX. Briefly, PHENIX was instructed to refine XYZ coordinates against both reciprocal space data and real-space maps, occupancies and individual B factors. All protein and ligand atoms were treated as anisotropic in B-factor refinement if the structure was at least 1.4 Å in resolution, as for the papain–E-64d and papain–(2S,3S)-t-ES-a9-b7 structures, although solvent atoms remained isotropic. For all other structures, protein and ligand atoms were considered isotropic in B-factor refinement. No hydrogen atoms were modeled on any molecule. Three cycles of such refinement were run before inspecting the agreement between the atomic coordinates and electron density map in Coot and building solvent molecules or adjusting side-chain positions to satisfy disagreements revealed in the difference Fourier map79. Water molecules were built in positions marked by positive-difference Fourier peaks exceeding 3σ levels where hydrogen-bonding partners were present within 2.5–3.3 Å. Methanol molecules were only built at sites where a water molecule did not fully abolish the difference Fourier density upon subsequent refinement cycles and where the carbon atom of the methanol molecule would not exist within 3.3 Å of any other nonbonded atoms. Following model adjustments, the coordinates were saved and the same refinement protocol in PHENIX was repeated.

For structures of papain complexed with 1 or its analogs, prominent positive-difference Fourier density indicating the presence of a peptidic molecule bound to the active Cys25 typically developed after one or two of these iterations. The respective ligand was modeled into this density using pre-existing monomers in the CCP4 library for 1, E-64c and E-64d and a custom ligand built using Coot’s ligand builder for (2S,3S)-t-ES-a9-b7 (ref. 82). Restraints for (2S,3S)-t-ES-a9-b7 were generated using eLBOW in the PHENIX GUI, with the PDB file for the custom ligand modeled in Coot as input76,77,79,83. CIF files for each ligand were input as restraints for subsequent refinement of each respective complex structure. A bond length of 1.8 Å, with an allowed s.d. of 0.02 Å, was enforced for the covalent linkage between the sulfur atom of Cys25 and the ligand’s carbon C2 and the occupancy of all atoms in the ligand was refined in PHENIX as a single group76,77.

For the E-64d cocrystal structure only, we suspected that ester hydrolysis of this molecule might occur in the presence of polar solvents such as methanol or during binding of the epoxy warhead and we noted weak or absent density in all electron density maps corresponding to the possible leaving group. As such, the occupancies of C23 and C24 of E-64d were refined as a separate group from the rest of the ligand. As additional validation, ligand-omit maps were generated for each ligand-bound structure by deleting the ligand from the model and refining the original MTZ file naive to the presence of the ligand against it. Furthermore, composite omit 2Fo − Fc maps using simulated annealing were calculated in the PHENIX suite76,77 for each set of reflections against the corresponding final model to produce electron density maps less impacted by model bias84. For the E-64d cocrystal structure, where density in the 2Fo − Fc maps failed to cover all of the ligand’s aliphatic tail moieties, the pose of the ligand that was best accommodated by the density and gave the lowest Rfree in refinement was selected and a feature-enhanced 2Fo − Fc map was calculated in PHENIX from the original reflections and the optimized model to better justify ligand placement85.

Reporting summary

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