Introduction

Chemical investigations of bacterial symbionts associated with eukaryotic hosts have yielded structurally and functionally diverse small molecules that have pivotal roles in regulating host–bacteria interactions. The insect pathogenic (entomopathogenic) bacteria of the Photorhabdus genus have emerged as one such prolific source of novel bioactive metabolites.1, 2, 3 These bacteria engage in a mutualistic symbiosis with the entomopathogenic nematodes of the family Heterorhabditidiae.4, 5 Infective juvenile nematodes carry Photorhabdus in their intestinal tracts, penetrate susceptible insect larvae in the soil and regurgitate Photorhabdus in the insect circulatory fluid (hemolymph). The bacteria stochastically switch between two major phenotypic variants, the mutualistic M-form associated with colonization of the nematode and the pathogenic P-form associated with producing diverse cytotoxins, nematode development signals, innate immunomodulators and anti-microbials.6 During infection and insect consumption, the bacteria exponentially proliferate, the nematodes consume the resulting bacterial biomass and the insect dies from septicemia. The nematodes proceed through their developmental cycle, and ultimately, newly formed infective juveniles emerge from the insect carcass and hunt for new insect larval prey.

Of the growing number of bioactive small molecules identified from the Photorhabdus genus, several have been shown to regulate key biological functions associated with the complex tripartite bacteria–nematode–insect relationship.1, 2, 3 For example, a variety of multipotent stilbene metabolites have been identified from Photorhabdus species, which serve as nematode development signals, invertebrate innate immunosuppressants and anti-microbial defense compounds against microbial competitors.7, 8, 9 Additionally, a tyrosine-derived virulence factor, rhabduscin, containing an isonitrile functional group displayed potent inhibitory activity against phenoloxidase, an enzyme known to be a crucial component of the insect’s innate immune system, and was required for virulence at physiologically relevant inocula.10 Indeed, genomic analyses of individual Photorhabdus species indicate a much larger number of biosynthetic gene clusters, many of which have not been characterized to date.1, 2, 3

As part of our efforts to discover new bioactive small molecules from bacterial symbionts, here we focused on the strain Photorhabdus luminescens TT01 grown in a hemolymph-mimetic bacterial culture medium. The medium was based on the remarkably high concentrations of the 20 free proteinogenic amino acids (35.01 g l−1 of free amino acids) in the hemolymph of their larval host Galleria mellonella.11 Through HPLC/UV/MS-guided fractionation, NMR-based structural elucidation and HR-ESI-QTOF-MS analysis, we characterized the chemical structures of two previously unknown bacterial pyrazinone metabolites, which we named lumizinones A (1) and B (2), together with two linear N-acetyl dipeptides (3 and 4). Here, we describe their isolation from the P-form phenotypic variant of P. luminescens, their structure elucidation and their in vitro calpain protease inhibitory activity.

Results and discussion

P. luminescens subsp. laumondii strain TT0112 was cultivated on Luria-Bertani agar plate at 30 °C for 48 h. A single colony was inoculated into 5 ml of the hemolymph-mimetic bacterial growth medium and then incubated at 30 °C on a rotary shaker (250 r.p.m.). After 2 days, the culture broth was centrifuged (3000 r.p.m., 15 min), the supernatant was extracted with 10 ml of ethyl acetate and the organic layer was dried under reduced pressure. Crude ethyl acetate-soluble materials were analyzed by C18 HPLC connected to a low-resolution ESI-MS system. HPLC/UV/MS data analysis displayed the presence of two distinct peaks 1 and 2, eluting at tR 14.29 min ([M+H]+ m/z 259) and tR 15.48 min ([M+H]+ m/z 293), respectively (Figure 1). The peaks shared a similar UV absorption spectrum with λmax of 220, 280 and 330 nm, suggesting the presence of a pyrazinone-type chromophore (Supplementary Figure S1).13 To further characterize the two metabolites, a 12 L scale aerobic cultivation of P. luminescens TT01 was initiated in the same medium at 30 °C for 48 h, and the whole culture was extracted two times with equal volumes of ethyl acetate (total 24 L). The organic fraction was dried in vacuo to yield 2.0 g of crude material. The ethyl acetate extract (2.0 g) was subjected to silica flash column chromatography using a hexane-ethyl acetate-methanol solvent composition followed by reversed-phase HPLC purification, which yielded pure compounds 1 (2.2 mg, lumizinone A) and 2 (1.5 mg, lumizinone B) (Figure 2).

Figure 1
figure 1

Detection of compounds 1–4 from organic extracts of P. luminescens TT01. UV traces of crude extracts (a) and compounds 1–4 (b–e) were monitored at 210 nm.

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Figure 2
figure 2

Chemical structures of compounds 1–4.

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Lumizinone A (1) was isolated as a colorless solid. The molecular formula was determined to be C15H18N2O2 (obsd [M+H]+ m/z 259.1447, calcd 259.1447) based on HR-ESI-QTOF-MS data, indicating that the chemical structure of 1 contains 8° of unsaturation (Supplementary Figure S2). The NMR-based structural characterization was achieved by the interpretation of 1H and 2D spectral data (gCOSY, gHSQC and gHMBC) (Supplementary Figures S3–S6). Briefly, the 1H NMR spectrum of 1 recorded in methanol-d4 displayed characteristics of two aromatic resonances (δH 7.06 (2H, m), 6.74 (2H, m)), an olefinic methine proton (δH 7.04 (1H, brs)), two methylene signals (δH 3.73 (2H, s), 2.56 (2H, d, J=7.2 Hz)), a methine proton (δH 2.13 (1H, m)) and a doublet methyl signal (δH 0.91 (6H, d, J=6.7 Hz)) (Table 1). The HSQC spectrum of 1 indicated that all of the protons were directly bonded to carbons. The COSY cross-peaks between H-9/H-13 (δH 7.06) and H-10/12 (δH 6.74) along with proton coupling constant (J=8.5 Hz) suggested the presence of a para-substituted benzene ring and a long-range COSY correlation from H-9/H-13 (δH 7.06) to a singlet methylene H-7 (δH 3.73) allowed us to establish a benzyl moiety. Subsequently, observed COSY correlations from doublet methyl protons H-3′/H-4′ (δH 0.91) to an aliphatic methylene proton H-1′ (δH 2.56) also supported construction of an isobutyl partial structure. The presence of two partial structures were further supported by the analysis of HMBC NMR spectral data. The HMBC correlations from H-9/H-13 (δH 7.06) to a hydroxylated aromatic carbon signal C-11 (δC 156.3) and a methylene carbon C-7 (δC 34.7), and from H-10/H-12 (δH 6.74) to a quaternary carbon C-8 (δC 126.8) established a para-hydroxybenzyl group. The other isobutyl partial structure was determined by the additional HMBC correlations from H-3′/H-4′ (δH 0.91) to C-1′ (δC 41.0). Construction of the pyrazinone ring was achieved by the HMBC correlations from H-1′ (δH 2.56) to an amide carbonyl C-2 (δC 156.8) and from a singlet methylene proton H-7 (δH 3.73) to a quaternary carbon C-6 (δC 139.3) and an olefinic methine carbon C-5 (δC 121.4). Finally, the HMBC correlations from an olefinic methine H-5 (δH 7.04) to C-7 (δC 34.7) and C-3 (δC 156.7) unambiguously constructed the 2(1H)-pyrazinone substituted with a para-hydroxybenzyl moiety and an isobutyl group at C-6 and C-3, respectively (Figure 3). Additionally, the structures were further supported by comparison with previously reported 1H and 13C chemical shifts of other 3,6-disubstituted pyrazinones.14 Therefore, the structure of 1 was assigned as 6-(4-hydroxybenzyl)-3-isobutylpyrazin-2(1H)-one.

Table 1 1H and 13C NMR spectral data of lumizinone A (1) and B (2) in CD3ODa
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Figure 3
figure 3

Key COSY (bold) and HMBC (arrow) correlations of compounds 1 and 2.

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Lumizinone B (2) was also isolated as a colorless solid. The molecular formula was determined to be C18H16N2O2 (obsd [M+H]+ m/z 293.1286, calcd 293.1290) based on HR-ESI-QTOF-MS data, indicating that the chemical structure of 2 contains 12° of unsaturation (Supplementary Figure S2). Similar to 1, 1H NMR suggested the presence of a para-hydroxybenzyl ring; however, the constitution of an additional benzyl ring was supported instead of the isobutyl group in 1 (Supplementary Figure S7). The gross structure of 2 was established by analysis of the COSY and HMBC spectral data (Supplementary Figures S8–S10). COSY correlations from H-3′/ H-7′ (δH 7.27) to H-5′ (δH 7.15) and a singlet methylene proton H-1′ (δH 3.99) supported the presence of the benzyl ring system, which was confirmed by HMBC correlations. Finally, the HMBC correlation from H-1′ (δH 3.99) to C-2 (δC 156.5) allowed the substitution of the benzyl group at C-3 (Figure 3). Therefore, the structure of 2 was assigned as 3-benzyl-6-(4-hydroxybenzyl)pyrazin-2(1H)-one.

Because wild-type P. luminescens uses an invertible promoter switch to stochastically regulate formation of the M- and P-form phenotypic variants, we assessed lumizinone production in two genetically engineered strains of P. luminescens where the promoter was locked in an ON (M-form) or OFF (P-form) orientation.6 Lumizinone production was only detected in the pathogenic P-form phenotypic variant, unambiguously establishing the cellular state for the production of these metabolites (Figure 4).

Figure 4
figure 4

Extracted ion counts chromatograms of lumizinones A (1) and B (2) from genetically engineered Photorhabdus luminescens strains locked in the phenotypic M-form and P-form.

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In the course of isolating lumizinones A (1) and B (2), two compounds 3 and 4 were also isolated and identified by the analysis of NMR and HR-ESI-QTOF-MS spectral data (Supplementary Figures S11–S17). Examination of the 1H and 2D NMR (gCOSY and gHMBC) of 3 and 4 combined with Marfey’s analysis unambiguously allowed for their structures to be assigned as N-acetyl-l-leucyl-l-tyrosine (3) and N-acetyl-l-leucyl-l-methionine (4), respectively. The connectivity of an N-acetyl group was established by two- and three-bond long-range HMBC correlations toward a carbonyl carbon from each α-proton in the leucine amino-acid and methyl protons in the acetyl group, and the absolute configurations of amino-acid constituents in compounds 3 and 4 were determined by Marfey’s analysis (Supplementary Figure S18).15 These compounds have previously been described as hydrolytic peptide products of the carboxypeptidase enzyme.16

Many natural products derived from microorganisms share a 3,6-disubstituted 2(1H)-pyrazinone core, most of which are classified by constitution of different amino-acid residues, such as valine-tyrosine (tyrvalin), valine-phenylalanine (phevalin) and valine-leucine (leuvalin) metabolites.17 The lumizinones isolated from P. luminescens are structurally distinct from other 3,6-disubstituted 2(1H)-pyrazinone metabolites, in which they are derived from a combination of tyrosine-leucine (1) or tyrosine-phenylalanine (2). The representative cyclic dipeptide phevaline was originally isolated from a terrestrial Streptomyces sp. in the course of activity-based screening for protease inhibitors.14 Interestingly, aureusimine metabolites including phevaline (aureusimine B) were also isolated from the human pathogen Staphylococcus aureus.18 More recently, it has been reported that phevaline may have a potential role in S. aureus biofilm formation.19 Consequently, we also screened the metabolite profile of the dual insect–human pathogen Photorhabdus asymbiotica. However, lumizinone metabolites (1 and 2) were not detected under the conditions of our experiment (Supplementary Figure S19). Nor could a homolog of the central aureusimine nonribosomal peptide synthetase be identified in the reported genomes of P. luminescens TT01 or P. asymbiotica.20, 21 It is currently unclear how the lumizinones are biosynthesized in P. luminescens.

Previously, phevaline was shown to harbor calpain protease inhibitory activity (half-maximal inhibitory concentration (IC50=1.3 μm).14 Calpains are a Ca2+-dependent family of intracellular cysteine proteases that are widely distributed in eukaryotic cells and tissues.22, 23 Calpain is a cytoplasmic heterodimer composed of a catalytic subunit (80 kDa) and a regulatory subunit (30 kDa). Calpain 1 (μ-calpain) and calpain 2 (m-calpain), two representative calpain isoforms, are activated by micromolar and millimolar Ca2+ concentrations within the cells, respectively. Calpains have crucial roles in numerous physiological and pathological process in the cells. For example, calpains catalyze the hydrolysis of a variety of substrate proteins that are associated with physiological processes, such as signal transduction, cell proliferation and differentiation, apoptosis, membrane fusion and platelet activation. However, hyperactivation of calpains by elevation of Ca2+ concentration could lead to various pathological processes including ischemia, brain injury, cancer and neurological disorders such as Alzheimer’s disease.24 Owing to the known calpain inhibitory activity of phevaline, the inhibitory activities of compounds 1–4 against calpain protease were evaluated in an established luminescence assay. Lumizinone A (1) displayed an inhibitory effect against calpain with an IC50 value of 3.9 μm (Figure 5), whereas compounds 2–4 were not active in this assay (IC50 >100.0 μm). Cysteine proteases have been implicated in the activation of the nuclear factor-κB inflammatory signaling pathway in the model invertebrate Drosophila melanogaster.25 While we speculate that such cysteine protease inhibition could serve an immunomodulatory role during insect pathogenesis, which could be supported by the observation that lumizinone metabolites are only detected in the pathogenic P-form (Figure 4), further experiments are required to determine whether the lumizinones provide an ecological benefit to P. luminescens during its multipartite lifecycle.

Figure 5
figure 5

Calpain protease inhibitory activity of lumizinone A (1).

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In summary, we cultivated P. luminescens TT01 in a bacterial medium mimicking substrate features in natural hemolymph of the host insect G. mellonella and isolated two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two linear N-acetylated dipeptides (3 and 4). The chemical structures of 1–4 were established by the analysis of NMR and HR-ESI-QTOF-MS spectral data. NMR-based structural characterization demonstrated that 1 and 2 share a pyrazinone ring system constituted with para-hydroxyl benzyl and isobutyl or benzyl, respectively. Compound 1 showed single-digit, μm-level inhibitory activity against a model cysteine protease (calpain, IC50=3.9 μm). Our results expand the chemical repertoire of the bacterial symbiont P. luminescens and raise intriguing new questions regarding the potential roles of pyrazinones in host–bacteria interactions.

Materials and methods

General experimental procedures

UV/Vis spectra were obtained on an Agilent Cary 300 UV-visible spectrophotometer (Agilent, Santa Clara, CA, USA) with a path length of 10 mm. 1H and 2D- (gCOSY, gHSQC and gHMBC) NMR spectral data were measured on an Agilent 600 MHz NMR spectrometer (Agilent) equipped with a cold probe, and the chemical shifts were recorded as δ values (p.p.m.). Low-resolution HPLC/MS data were measured using an Agilent 6120 single quadrupole LC/MS system (Agilent). High-resolution ESI-MS data were obtained using an Agilent iFunnel 6550 QTOF instrument (Agilent) fitted with an ESI source coupled to an Agilent 1290 Infinity HPLC system (Agilent). Flash column chromatography was carried out on Waters Sep-Pak Vac 35 cm3 (10 g) C18 or silica cartridges (Waters, Milford, MA, USA). The isolation of metabolites was performed using an Agilent Prepstar HPLC system (Agilent) with an Agilent Polaris C18-A 5 μm (21.2 × 250 mm2) column (Agilent), a Phenomenex Luna C18(2) (100 Å) 10 μm (10.0 × 250 mm2) column (Phenomenex, Torrance, CA, USA) and an Agilent Phenyl-Hexyl 5 μm (9.4 × 250 mm2) column (Agilent).

Cultivation and extraction

The hemolymph-mimetic bacterial growth medium was made up of yeast extract (5 g l−1) and the amino-acid concentrations found in the hemolymph of the insect host G. mellonella.11 Single colonies of P. luminescens TT01 first grown on Luria-Bertani agar (10 g l−1 tryptone, 5 g l−1 yeast extract, 10 g l−1 sodium chloride, 18 g l−1 agar) were inoculated into 5 ml of the hemolymph-mimetic medium and incubated at 30 °C at 250 r.p.m. After 2 days, supernatants of cell culture broth were extracted with ethyl acetate (2 × 5 ml) in 14 ml polypropylene round-bottom tubes, and the ethyl acetate-soluble layers were evaporated under reduced pressure. Crude materials were dissolved in 200 μl of 100% methanol and then monitored on an Agilent 6120 single quadrupole LC/MS system (Column; Phenomenex Kinetex C18 column (Phenomenex), 250 × 4.6 mm2, 5 μm, flow rate; 0.7 ml min−1, mobile phase composition; water and acetonitrile (ACN) containing 0.1% formic acid; analysis method; 0–30 min, 10–100% ACN; hold for 5 min, 100% ACN; 1 min, 100–10% ACN). For larger-scale cultivation, a P. luminescens TT01 seed culture (12 × 5 ml) was transferred into 12 × 1 L of hemolymph-mimetic medium in 4 L Elenmeyer flasks. After 3 days, the combined whole culture broth was extracted with 24 L ethyl acetate, and the organic-soluble layer was dried by rotary evaporation to yield a combined crude extract (2.0 g).

Isolation of metabolites

Crude materials (2.0 g) were subjected to a Waters Sep-Pak Vac 35 cm3 (10 g) silica cartridge and separated using a step gradient with the following solvent composition: Fraction 1, hexane:EtOAc=10:1 (v v−1); Fraction 2, hexane:EtOAc=1:1 (v v−1); Fraction 3, 100% EtOAc (v v−1); Fraction 4: EtOAC:MeOH=20:1 (v v−1); Fraction 5: EtOAc:MeOH=1:1 (v v−1); Fraction 6: 100% MeOH (v v−1). Fraction 3 containing compounds 1 and 2 was further separated over a Waters Sep-Pak Vac 35 cm3 (10 g) C18 cartridge with the following step gradient: 20, 40, 60, 80 and 100% MeOH in water (v v−1). Separation of the resulting 60% MeOH fraction was performed using an Agilent Prepstar HPLC system with an Agilent Polaris C18-A 5 μm (21.2 × 250 mm2) column (flow rate 10.0 ml min−1) using a 1 min fraction collection time window. The combined fraction 42+43 was fractionated using a Phenomenex Luna C18 (2) 10 μm column (10.0 × 250 mm2, flow rate 4.0 ml min−1) with a gradient elution from 10 to 100% aqueous ACN, and the metabolites were finally purified over a Phenyl-Hexyl 5μm column (9.4 × 250 mm2) with a general gradient system (10–100% aqueous methanol for 30 min, 4 ml min−1). Compounds 1 and 2 were eluted at tR 26.74 and 29.31 min, respectively.

Silica fraction 5 containing compounds 3 and 4 were subjected to a Waters Sep-Pak Vac 35 cm3 (10 g) C18 cartridge using the following step gradient: 20, 40, 60, 80 and 100% MeOH in water (v v−1). The 40% methanol fraction was subsequently separated using an Agilent Prepstar HPLC system with Agilent Polaris C18-A 5 μm (21.2 × 250 mm2, flow rate 10.0 ml min−1) and Phenomenex Luna C18 (2) 10 μm column (10.0 × 250 mm2, flow rate 4.0 ml min−1), yielding compounds 3 (tR 11.64 min) and 4 (tR 12.18 min), respectively.

Lumizinone A (1): Colorless solid; UV (CH3OH) λmax (log ɛ) 330 (3.81), 280 (sh, 3.31), 210 (3.88) nm; 1H and 13C NMR spectra (see Table 1); HR-ESI-QTOF-MS [M+H]+ m/z 259.1447 (calcd for C15H19N2O2, 259.1447).

Lumizinone B (2): Colorless solid; UV (CH3OH) λmax (log ɛ) 326 (3.64), 280 (sh, 3.31), 223, (3.83), 202 (4.07) nm; 1H and 13C NMR spectra, (see Table 1); HR-ESI-QTOF-MS [M+H]+ m/z 293.1286 (calcd for C18H17N2O2, 293.1290).

N-acetyl-l-leucyl-l-tyrosine (3): Colorless solid; 1H NMR (CD3OD, 600 MHz) δ 7.01 (2H, d, J=8.5 Hz, H-5 and H-9), 6.67 (2H, d, J=8.5 Hz, H-6 and H-8), 4.56 (1H, dd, J=8.2, 5.2 Hz, H-2), 4.38 (1H, dd, J=9.6, 5.6 Hz, H-11), 3.08 (1H, dd, J=14.0, 5.2 Hz, H-3), 2.89 (1H, dd, J=14.0, 8.2 Hz, H-3), 1.93 (3H, s, H-17), 1.60 (1H, m, H-13), 1.54-1.44 (2H, m, H-12), 0.93 (3H, d, J=6.6 Hz, H-14), 0.88 (3H, d, J=6.6 Hz, H-15), 13C NMR (CD3OD, 125 MHz) δ 173.1 (C-10), 173.0 (C-1), 171.9 (C-16), 155.9 (C-7), 129.9 (C-5, C-9), 127.3 (C-4), 114.6 (C-6, C-8), 53.5 (C-2), 51.4 (C-11), 40.1 (C-12), 35.9 (C-3), 24.3 (C-13), 21.7 (C-14), 20.8 (C-17), 20.5 (C-15); HR-ESI-QTOF-MS [M+H]+ m/z 337.1763 (calcd for C17H25N2O5, 337.1763).

N-acetyl-l-leucyl-l-methionine (4): Colorless solid; 1H NMR (CD3OD, 600 MHz) δ 4.49 (1H, dd, J=9.0, 4.4 Hz, H-2), 4.38 (1H, dd, J=9.8, 5.5 Hz, H-7), 2.56 (1H, ddd, J=13.8, 9.0, 5.0 Hz, H-4), 2.50 (1H, m, H-4), 2.13 (1H, dddd, J=13.7, 9.0, 7.2, 4.5 Hz, H-3), 2.06 (3H, s, H-5), 1.96 (3H, s, H-13), 1.95 (1H, m, H-3), 1.69 (1H, m, H-9), 1.57 (2H, m, H-8), 0.96 (3H, d, J=6.6 Hz, H-10), 0.92 (3H, d, J=6.6 Hz, H-11), 13C NMR (CD3OD, 125 MHz) δ 173.7 (C-1), 173.4 (C-6), 171.7 (C-12), 51.5 (C-2), 51.7 (C-7), 40.3 (C-8), 31.0 (C-3), 29.6 (C-4), 24.3 (C-9), 21.8 (C-10), 20.8 (C-13), 20.4 (C-11), 13.7 (C-5); HR-ESI-QTOF-MS [M+H]+ m/z 305.1531 (calcd for C13H25N2O4S, 305.1535).

Absolute configuration determination of amino acids

Standard d- and l-amino acids (leucine, methionine and tyrosine) were purchased from Sigma-Aldrich (St Louis, MO, USA). Compounds 3 (0.5 mg) and 4 (0.3 mg) were hydrolyzed in 500 μl of 6 n HCl at 110 °C for 1 h, and the reaction mixture was dried in vacuo or under purging of nitrogen gas. The hydrolysate was dissolved in distilled water and completely dried for 24 h in a Genevac HT-4X Evaporation System to remove excess acid. The hydrolyzed materials and standard amino acids were treated with 50 μl of a solution of Nα-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (FDAA) (10 mg ml−1 in acetone) followed by the addition of 100 μl of 1 n NaHCO3. The reaction mixture was heated at 80 °C for 3 min and quenched with 50 μl of 2 n HCl. The derivatized materials were then diluted to 300 μl with 50% aqueous ACN for LC/MS analysis. Ten microliters of the materials was analyzed by the single quadrupole LC/MS system equipped with a Phenomenex Kinetex C18 (100 Å) 5 μm (4.6 × 250 mm2) column using a flow rate (0.7 ml min−1) and a solvent system of water and ACN containing 0.1% formic acid. The retention times of derivatized amino acids were as follows: gradient: 0–40 min, 20–60% ACN, l-Leu 26.45 min, d-Leu 30.17 min; gradient: 0–30 min, 20–35% ACN, l-Tyr 11.92 min, d-Tyr 12.06 min; gradient: 0–40 min, 40–100% ACN; l-Met 7.25 min and d-Met 8.46 min.

Comparative chemical analysis of M- and P-form phenotypic variants

Genetically locked M- and P-form P. luminescens were individually cultivated on Luria-Bertani agar plates at 30 °C for 48 h. Single colonies were inoculated into 5 ml hemolymph-mimetic medium and cultivated under aerobic conditions in a shaking incubator (30 °C, 250 r.p.m.). After 72 h, the culture broths were centrifuged (20 min, 3000 r.p.m.), and the supernatants were then extracted with ethyl acetate (2 × 5 ml). The organic materials were dried under reduced pressure on a Genevac HT-4X Evaporation System (Genevac Inc, Gardiner, NY, USA) for 2 h. The samples were resuspended in 200 μl methanol, and 2 μl of sample was injected for HR-ESI-QTOF-MS analysis (Column; Phenomenex Kinetex C18 column, 250 × 4.6 mm2, 5 μm, flow rate; 0.7 ml min−1, mobile phase composition; water and ACN containing 0.1% formic acid; analysis method; 0–30 min, 5–100% ACN; hold for 5 min, 100% ACN; 1 min, 100–5% ACN). Extracted ion count chromatograms were extracted with m/z 259.1447 corresponding to lumizinone A and m/z 293.1286 corresponding to lumizinone B with a 10 p.p.m. mass window.

Calpain protease inhibitory assay

Calpain 1 (human plasma) was purchased from Sigma-Aldrich. The luminescence assay was performed using the Calpain-Glo Protease Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Luminescence was monitored using an Envision Multimode Plate Reader (Perkin-Elmer, Waltham, MA, USA). Stock solutions of compounds were prepared in 10% dimethyl sulfoxide and stored at −20 °C before use, and the compounds and enzymes were resuspended in a buffer solution composed of 10 mm HEPES (pH 7.2), 10 mm dithiothreitol, 1 mm EDTA and 1 mm EGTA. The compounds were serially diluted with an initial concentration of 200 μm across the row of a 96-well plate in triplicate.