Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens.

Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that undergoes phenotypic variation and lives in mutualistic association with nematodes of the family Heterorhabditidae. The pair infects and kills insects, and during their coordinated lifecycle, the bacteria produce an assortment of specialized metabolites to regulate its mutualistic and pathogenic roles. As part of our search for new specialized metabolites from the Photorhabdus genus, we examined organic extracts from P. luminescens grown in an amino-acid-rich medium based on the free amino-acid levels found in the circulatory fluid of its common insect prey, the Galleria mellonella larva. Reversed-phase HPLC/UV/MS-guided fractionation of the culture extracts led to the identification of two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two N-acetyl dipeptides (3 and 4). The lumizinones were produced only in the phenotypic variant associated with nematode development and insect pathogenesis. Their chemical structures were elucidated by analysis of 1D and 2D NMR and high-resolution ESI-QTOF-MS spectral data. The absolute configurations of the amino acids in 3 and 4 were determined by Marfey's analysis. Compounds 1-4 were evaluated for their calpain protease inhibitory activity, and lumizinone A (1) showed inhibition with an IC50 (half-maximal inhibitory concentration) value of 3.9 μm.

INTRODUCTION

Chemical investigations of bacterial symbionts associated with eukaryotic hosts have yielded structurally and functionally diverse small molecules that play 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.[1-3] 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 antimicrobials.[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.[1-3] For example, a variety of multipotent stilbene metabolites have been identified from Photorhabdus species, which serve as nematode development signals, invertebrate innate immunosuppressants, and antimicrobial defense compounds against microbial competitors.[7-9] 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.[1-3]

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 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 TT01[12] was cultivated on an LB 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 (3,000 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 high-performance liquid chromatography (HPLC) connected to a low-resolution electrospray ionization mass spectrometry (ESIMS) 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). Two 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 twice 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

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

Figure 2

Chemical structures of compounds 1–4

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 degrees 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-hydroxy benzyl 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-hydroxy benzyl moiety and an isobutyl group at C-6 and C-3, respectively (Figure 3). Additionally, the structures were further supported by comparison to 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 CD3ODd

	Lumizinone A (1)				Lumizinone B (2)
No.	δCa	type	δHb	Mult (J in Hz)	δCa	type	δHb	Mult (J in Hz)
1		NH				NH
2	156.8	C			156.5	C
3	156.7	C			155.8	C
4		N				N
5	121.4	CH	7.04	br s	121.4	CH	7.04	br s
6	139.4	C			139.7	C
7	34.7	CH2	3.73	s	34.6	CH2	3.72	s
8	126.8	C			126.7	C
9	129.5	CH	7.06	m	129.5	CH	7.05	m
10	115.1	CH	6.74	m	115.1	CH	6.72	m
11	156.3	C			156.2	C
12	115.1	CH	6.74	m	115.1	CH	6.72	m
13	129.5	CH	7.06	m	129.5	CH	7.05	m
1′	41.0	CH2	2.56	d (7.2 Hz)	38.2	CH2	3.99	s
2′	26.5	CH	2.13	m	137.5	C
3′	21.4	CH3	0.91	d (6.7 Hz)	128.6	CH	7.27	d (7.5 Hz)
4′	21.4	CH3	0.91	d (6.7 Hz)	127.8	CH	7.23	t (7.5 Hz)
5′					125.9	CH	7.15	t (7.2 Hz)
6′					127.8	CH	7.23	t (7.5 Hz)
7′					128.6	CH	7.27	d (7.5 Hz)

chemical shifts of 13C were determined by HSQC and HMBC.

600 MHz for 1H NMR data.

chemical shifts were not assigned by overlapping signal.

methanol-d4 (δH 3.29, δC 47.7).

Figure 3

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

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 degrees of unsaturation (Supplementary Figure S2). Similar to 1, 1H NMR suggested the presence of a para-hydroxy benzyl ring, however, the constitution of an additional benzyl ring was supported instead of the isobutyl group in 1 (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 wildtype P. luminescens employs 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

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

In the course of isolating lumizinones A (1) and B (2), two compounds 3 and 4 were also isolated and identified by 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 play 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 (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 play 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, hyper-activation 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), while compounds 2–4 were not active in this assay (IC50 >100.0 μM). Cysteine proteases have been implicated in the activation of the NF-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

Calpain protease inhibitory activity of lumizinone A (1)

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

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 (LB) 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, 250 × 4.6 mm, 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 35cc (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 35cc (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 mm) 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 mm, flow rate 4.0 ml min−1) with a gradient elution from 10% to 100% aqueous acetonitrile, and the metabolites were finally purified over a Phenyl-Hexyl 5μm column (9.4 × 250 mm) 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 min and tR 29.31 min, respectively.

Silica fraction 5 containing compounds 3 and 4 were subjected to a Waters Sep-Pak® Vac 35cc (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 mm, flow rate 10.0 ml min−1) and Phenomenex Luna C18 (2) 10 μm column (10.0 × 250 mm, 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 ε) 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 ε) 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. Compounds 3 (0.5 mg) and 4 (0.3 mg) were hydrolyzed in 500 μl of 6N 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 1N NaHCO3. The reaction mixture was heated at 80 °C for 3 min and quenched with 50 μl of 2N HCl. The derivatized materials were then diluted to 300 μl with 50% aqueous acetonitrile for LC/MS analysis. 10 μl of the materials was analyzed by the single quadrupole LC/MS system equipped with a Phenomenex Kinetex C18 (100Å) 5 μm (4.6 × 250 mm) column using a flow rate (0.7 ml min−1) and a solvent system of water and acetonitrile containing 0.1% formic acid. The retention times of derivatized amino acids were as follows; gradient: 0–40 min, 20–60% acetonitrile, L-Leu 26.45 min, D-Leu 30.17 min; gradient: 0–30 min, 20–35% acetonitrile, L-Tyr 11.92 min, D-Tyr 12.06 min; gradient: 0–40min, 40–100% acetonitrile; L-Met 7.25 min, 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 LB 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 (USA) HT-4X evaporation system for 2 h. The samples were re-suspended 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 mm, 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, 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 ppm 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) according to the manufacturer’s instructions. Luminescence was monitored using an Envision® multimode plate reader (PerkinElmer). Stock solutions of compounds were prepared in 10% DMSO and stored at −20 °C prior to use, and the compounds and enzyme were resuspended in a buffer solution composed of 10 mM HEPES (pH 7.2), 10 mM DTT, 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.