NRPS Substrate Promiscuity Leads to More Potent Antitubercular

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NRPS Substrate Promiscuity Leads to More Potent Antitubercular Sansanmycin Analogues Yunying Xie,*,‡ Qiang Cai,‡ Hao Ren, Lifei Wang, Hongzhang Xu, Bin Hong, Linzhuan Wu, and Ruxian Chen* Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China S Supporting Information *

ABSTRACT: Sansanmycins, members of the uridyl peptide antibiotics, are assembled by nonribosomal peptide synthetases (NRPSs), the substrate promiscuity of which results in the diversity of products. Further exploration of the NRPSs’ substrate promiscuity by reinvestigating sansanmycin producer strain led to the isolation and structural elucidation of eight new uridyl peptides, sansanmycins H−O (1−8). Among them, sansanmycin L, containing a 6-OH-bicyclic residue and Phe3 first found at the position AA3, exhibited activity against M. tuberculosis H37Rv with an MIC value of 2 μg/mL, 8-fold more potent than that of the major compound, sansanmycin A (MIC = 16 μg/mL).

S

ansanmycins1−5 are a group of uridyl peptide antibiotic (UPAs) produced by Streptomyces sp. SS, sharing a common structure with pacidamycins,6 mureidomycins,7 and napsamycins.8 They have a unique structure, a 3′-deoxyuridyl attached via an unusual exocyclic enamide to a tetrapseudopeptide backbone. The pseudopeptide chain displayed interesting double reversal due to the β-peptidation of the (2S,3S)-N-methyl-2,3-diaminobutyric acid (DABA) and a ureido linkage.9 The uridyl peptide antibiotics target a clinically unexploited MraY (translocase I) to block the synthesis of the cell wall10 and have good antibacterial activity against highly refractory pathogens Pseudomonas aeruginosa, Mycobacterium tuberculosis H37Rv, and multi-drug-resistant M. tuberculosis strains.1,11,12 Recently, the biosynthetic gene clusters for pacidamycins,9,13 napsamycins,14 and sansanmycins15,16 were identified and characterized, indicating that the assembly of the pseudopeptide chain is catalyzed by nonribosomal peptide synthetases (NRPSs) with highly dissociated modules. The biosynthetic enzymes of UPAs show striking substrate flexibility combined by marked stringency against certain residues.17−19 Remarkable flexibility is shown at the C-terminus of natural UPAs, where tryptophan is replaced with phenylalanine or m-tyrosine, while the N-terminus is more conserved, with almost only m-tyrosine © XXXX American Chemical Society and American Society of Pharmacognosy

or its derivative bicyclic tetrahydroisoquinoline attached to the β-amino group of DABA. In addition, the residue at the position AA3 of UPAs varies with the producer strain, e.g., alanine in pacidamycins, methionine in mureidomycins and napsamycins, and methionine or leucine in sansanmycins. NRPS responsible for the assembly of sansanmycin also showed substrate flexibility and specification, and seven related compounds, sansanmycins A−G, have been obtained from the same producer strain Streptomyces sp. SS. Reinvestigation of the NRPS substrate promiscuity led to isolation and structural elucidation of eight new uridyl peptides, sansanmycin H−O (1−8), with more potent antitubercular activity. Details of the isolation, structure elucidation, and antibacterial activity of these compounds are reported herein. The supernatant of the fermentation broth of sansanmycin producer strain Streptomyces sp. SS was subjected to column chromatography on D4006 macroporous resin, Toyopearl DEAE 650 M, and reversed-phase silica gel (ODS) guided by bioassay and HPLC-UV to yield eight new uridyl peptides, sansanmycins H−O (1−8). Sansanmycin H (1) is a white powder with a molecular formula of C38H49N8O12S, which is Received: February 16, 2014

A

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the same as mureidomycin A.7 The ESIMS/MS spectrum of 1 also showed the same fragments as mureidomycin A. The 1H NMR spectrum of 1, however, displayed a different aromatic pattern from mureidomycin A,7,20 with a tyrosine (δ 7.09 (2H, d), 6.80 (2H, d)) and an m-tyrosine residue (δ 7.23 (1H, t), 6.78 (1H, d), 6.75 (1H, d), 6.72/6.70 (1H, s)) instead of two m-tyrosine residues (δ 7.08 (1H, t), 7.00 (1H, t), 6.64 (1H, d), 6.62 (1H, d), 6.57 (1H, d), 6.56 (2H, m), 6.53 (1H, d)). Despite spectral complexities caused by rotational conformers, a characteristic spectrum of uridyl peptide antibiotics,6 the anomeric (δ 6.03/6.07), olefinic (δ 5.93/5.98), and uracil protons (δ 5.54/5.89, 7.00/7.35) of rotamers, was easily recognized, which indicated the presence of an unusual nucleoside. Three methyl proton signals of rotamers at δ 3.00/2.69 (s, −NCH3), 2.05/2.04 (s, −SCH3), and 1.19/0.59 (d, −CHCH3) suggested the amino acid residues DABA and methionine were present. All of the above and an extra upfield carbonyl group were confirmed by interpretation of 13C NMR, 1 H−1H COSY, HSQC, and HMBC data of 1 (Table 1). The fragment sequence of 1 was proposed using the HMBC correlations (Figure 1) from relevant N-CH3, α, or olefinic protons to neighboring carboxylic carbons. First, the N-CH3 signal (δH 3.00/2.69) of DABA showed a cross-peak to the carboxylic carbon signal of the m-Tyr (δC 178.6/179.5) residue, establishing that the m-Tyr is attached to DABA via a βpeptidation. The olefinic proton (δH 5.93/5.98) of the unusual uridine and the α proton (δH 4.60/4.48) of DABA signals were individually correlated to the amide carbon signals of DABA (δC 170.5/170.1) and Met (δC 177.4) residues. These correlations enabled assignment of an unusual partial sequence of uridine−DABA−Met for 1. The carboxylic carbon signal (δC 181.8) of the Tyr residue appeared downfield compared to the others, indicating a free carboxylic carbon group of the Tyr residue. Additionally, the overlapped α protons (δH 4.24) of Met and Tyr residues showed a cross-peak to the independent upfield carbonyl carbon (δC 161.3/161.1), deducing the Cterminal sequence of Met−CO−Tyr for 1. The connectivity between structural fragments was further confirmed by ESIMS/ MS data for the protonated molecular ion, m/z 841 (Figure 2). The absolute configurations of all the amino acid residues were preliminarily assigned as the natural S-configuration to 1 on biogenetic grounds, given that it is the analogue of pacidamycins, the absolute configuration of which has been established by total synthesis.21 Sansanmycin I (2) has the same molecular formula of C44H50N9O11 and ESIMS/MS fragments with sansanmycin C,2 but a longer retention time on a C18 column than sansanmycin C, which hinted at the replacement of methionine sulfoxide in sansanmycin C with the more hydrophobic phenylalanine in 2. Compared with the 1H NMR spectrum of sansanmycin C, that of 2 showed five extra aromatic protons at δ 7.10 (m, 2H), 7.26 (m, 2H), and 7.25 (m, 1H) and a lost methyl proton signal at δ 2.74 (s, −SO−CH3), which further confirmed the above hypothesis. Interpretation of the 2D NMR and ESIMS/MS data (see Table S1 and Figures S2 and S14 in the Supporting Information) also confirmed this proposed structure. Sansanmycins J (3) and K (4) have the same molecular formula of C39H49N8O12S, 12 mass units greater than that of 1, attributed to the formation of tetrahydroisoquinoline from formaldehyde and m-tyrosine in compound 1. In support of this hypothesis, the 1H NMR (D2O, pD = 8.0) data for 3 and 4 (see Tables S2 and S3 in the Supporting Information) proved to be very similar to those of 1, with significant differences limited to

Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data for Sansanmycin Ha extra signals due to conformers multiplicity

δC

uracil-2 uracil-4 uracil-5

N−CO−N CO−N CH

155.3 179.6 105.3

uracil-6

CH

143.2

sugar-1 sugar-2 sugar-3

O−CH−N O−CH CH2

sugar-4 sugar-5 DABA-1 DABA-2

>C −CH CO-N CH

DABA-3 DABA-4

CH CH3

DABA-N-CH3 Met-1 Met-2 Met-3

N−CH3 CO−N CH CH2

32.9 177.4 55.9 33.8

Met-4 Met-S-CH3 m-Tyr-1 m-Tyr-2 m-Tyr-3

CH2 CH3 CO−N CH CH2

32.2 17.1 178.6 54.8 42.3

m-Tyr-1′ m-Tyr-2′ m-Tyr-3′ m-Tyr-4′

ArC ArCH Ar−C−O ArCH

141.4 119.0 158.9 117.0

m-Tyr-5′

ArCH

133.0

m-Tyr-6′

ArCH

124.0

ureido Tyr-1 Tyr-2 Tyr-3

N−CO−N CO-N CH CH2

161.3 181.8 59.7 40.4

Tyr-1′ Tyr-2′

ArC ArCH

132.5 133.5

Tyr-3′ Tyr-4′ Tyr-5′ Tyr-6′

ArCH Ar−C−O ArCH ArCH

118.1 157.0 118.1 133.5

positionb

96.1 75.7 35.7 147.0 99.7 170.5 58.4 53.8 16.1

δH (J, Hz)

δC

δH (J, Hz)

170.8 5.54, d (9.6) 7.00, d (9.6) 6.03, s 4.53, m 2.67, m 2.99, m 5.93, s 4.60, d (10.8) 4.89, m 1.19, d (7.8) 3.00, s 4.24, 1.87, 1.95, 2.49, 2.05,

m m m m s

4.04, m 2.51, m 2.89, m

142.6 96.4 75.4 35.9

99.3 170.1 58.8 56.2 16.6 30.7

33.6

5.89, d (9.6) 7.35, d (9.0) 6.07, s 4.40, m 2.63, m 2.87, m 5.98, s 4.48, d (11.4) 4.14, m 0.59, d (7.8) 2.69, s

1.87, m 1.95, m 2.04, s

179.5 54.4 43.6

6.72, s

4.24, m 2.73, m 2.96, m 6.70, s

158.8 6.78, d (9.0) 7.23, t (9.0) 6.75, d (9.0) 161.1 4.24, m 2.83, m 3.02, m

43.6

2.73, m 2.96, m

7.09, d (9.6) 6.80, d 6.80, d 7.09, d (9.6)

a The spectra were recorded in D2O. The chemical shifts (δ) are given in ppm. bAbbreviations for the structural units are Tyr = tyrosine, Trp = tryptophan, DABA = 2-amino-3-methylaminobutyric acid, Met = methionine.

replacement of the m-tyrosine in 1 [δH 7.23 (1H, t), 6.78 (1H, d), 6.75 (1H, d), 6.72/6.70 (1H, s), 4.04/4.24 (1H, m), 2.51/ 2.73 (1H, m), 2.89/2.96 (1H, m)] with a 6-hydroxyB

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same as 3 and 4, attributed to the formation of tetrahydroisoquinoline from formaldehyde and m-tyrosine. These proposed structures were further confirmed by NMR and ESIMS/MS data (see Tables S4−S7 and Figures S5−S8, S23, S26, S29, and S32 in the Supporting Information). Antibacterial Activities. Compounds 1−8 were tested for antibacterial activity against M. tuberculosis H37Rv and Pseudomonas aeruginosa 11 (Chart 1), showing that 5, bearing a 6-OH-bicyclic residue and Phe3 first found at the position AA3, had the highest anti-TB activity, with an MIC value of 2 μg/mL, 8-fold more potent than the major compound sansanmycin A. Compound 1 differs from other uridyl peptides containing m-Tyr4 or Trp4 in that it contains Tyr4 and showed slightly more anti-TB potency than sansanmycin A. The 6hydroxytetrahydroisoquinoline-containing compounds 3, 5, and 7 (MIC = 16, 2, and 8 μg/mL respectively) showed higher antiTB activity than 8-hydroxytetrahydroisoquinoline-containing compounds 4, 6, and 8 (MIC ≥ 16 μg/mL). It was deduced that the position of the hydroxyl group at the tetrahydroisoquinoline was also an important factor, which might interrupt the ammonium ion of the N-terminus to bind at the MraY active site in place of the Mg2+ cofactor.22 Although more than 30 uridyl peptide compounds have been created via the natural flexibility of their NRPS-mediated biosynthesis pathway, sansanmycins H-M (1−6) differ markedly from the known uridyl peptide compounds by virtue of having Tyr4 but not m-Tyr4, Phe4, or Trp4 as the C-terminal amino acid and Phe3 instead of Met3, Ala3, or Leu3 in known uridyl peptide antibiotics.19 The unique substrate promiscuity profile of NRPS responsible for the assembly of sansanmycins might lead to novel variants by a precursor-directed method.

Figure 1. Selected 2D NMR correlations for 1.

Figure 2. ESIMS/MS data of the parent ion peak (m/z 841) of 1.

tetrahydroisoquinoline in 3 [δH 6.99 (1H, d, 8.4 Hz) 6.72/6.77 (1H, d, 8.9/8.4 Hz), 6.54/6.63 (1H, s), 3.88 (1H, d, 15.5 Hz), 3.97 (1H, d, 16.4 Hz), 3.88 (1H, m), 2.66 (2H, m)] and 8hydroxytetrahydroisoquinoline in 4 [(δH 7.06 (1H, m), 6.67 (1H, d, 7.7 Hz), 6.51 (1H, d, 7.7 Hz), 4.09 (1H, d, 17.1 Hz), 3.76 (1H, d, 17.0 Hz), 3.87 (1H, dd, 4.3 Hz, 9.9 Hz), 2.69 (2H, m)]. The 2D NMR and ESIMS/MS data for 3 and 4 (see Figures S3, S4, S17, and S20 in the Supporting Information) also revealed diagnostic correlations supportive of the proposed structures. Sansanmycins L (5) and M (6) have the same molecular formula of C45H50N9O11, and sansanmycins N (7) and O (8) have the same molecular formula of C41H50N9O11S, 12 mass units greater than that of 2 and sansanmycin A, respectively, the

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EXPERIMENTAL SECTION

General Experimental Procedures. UV data were recorded on a Shimadzu UV-2550 spectrophotometer. NMR data were acquired with Varian Mercury 600 spectrometers using D2O as solvent (pD = 8.0). ESIMS and ESIMS/MS data were recorded on a Finnigan LTQ XT ion trap mass spectrometer, and HRESIMS data were obtained using a Finnigan LTQ Orbitrap XT mass spectrometer. HPLC analyses were performed on an Agilent 1200 instrument using an XBridge C18 column (4.6 × 150 mm, 3.5 μm) on a binary LC system (solvent A: 0.1% (w/v) (NH4)2CO3), solvent B: methanol; flow rate, 1 mL min−1; 0−40 min, 20−45% B (linear gradient), 40−50 min, 45% B; UV detection at 254 nm and oven temperature at 40 °C). HPLC purifications were carried out using a YMC-Pack ODS-A column (250

Chart 1. Structures and Activity of Sansanmycin Analogues

C

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× 20 mm, 5 μm; eluted with 0.1% (w/v) (NH4)2CO3−MeOH; flow rate, 5 mL min−1; UV detection at 254 nm and oven temperature at 40 °C) on a Shimadzu 20A HPLC system. Anion exchange chromatography was performed using Toyopearl DEAE 650 M (GE Healthcare). Fermentation, Extraction, and Isolation. Streptomyces sp. SS, the sansanmycin A producer strain, was maintained at −80 °C as a spore suspension in glycerol and cultivated on an ISP2 slant at 28 °C for 7 days. With pieces of well-grown agar cultures of Streptomyces sp. SS, 10 × 500 mL Erlenmeyer flasks were precultured, each flask containing 100 mL of a production medium consisting of glucose 3%, starch 0.5%, peptone 0.6%, (NH4)2SO4 0.7%, and CaCO3 0.2%. The inoculated flasks were incubated on a rotary shaker (220 rpm) at 28 °C for 48 h. The obtained preculture was used to inoculate 20 × 5 L Erlenmeyer flasks (each with 50 mL of preculture), each containing 1 L of production medium, and incubated at 28 °C and 220 rpm. After 5 days the culture broth was harvested. A 40 L amount of fermentation broth was obtained by repeated fermentation. The fermentation broth was filtered to remove the mycelia, 35 L of filtrate thus obtained was applied on a column of macroporous absorbant resin 4006 (5 L, 7.8 × 110 cm), and after washing with 25 L of water, the active absorbed materials were eluted with 20 L of 20% (FI) and 20 L of 40% (FII) aqueous acetone. Antibiotic activity was determined by a paper-disk agar diffusion assay using Pseudomonas aeruginosa on Mueller-Hinton medium. Then FI was chromatographed on Toyopearl DEAE 650 M (column 3.8 × 100 cm) eluted with Tris-HCl (20 mM, pH 8.5) plus NaCl and monitored by UV to yield sansanmycin H (1; white powder, 208 mg) and crude sansanmycin N along with another fraction, FI-A (see Supporting Information Figure S1). Purification of crude sansanmycin N using HPLC (YMC-Pack ODS-A 5 μm, 250 × 20 mm column, 0.1% (w/v) (NH4)2CO3−MeOH; flow rate, 5 mL min−1) afforded sansanmycin N (7; white powder, 11 mg). Further fractionation and purification of fraction FI-A using HPLC yielded sansanmycin J (3; white powder, 7.8 mg) and sansanmycin K (4; white powder, 8.0 mg), and further fractionation and purification of fraction FII following Figure S1 gave sansanmycin A (9; white powder, 413 mg), sansanmycin O (8; white powder, 15.2 mg), sansanmycin I (2; white powder, 32.7 mg), and sansanmycin L (5; white powder, 6.3 mg) along with sansanmycin M (6; white powder, 11.1 mg). Sansanmycin H (1): white powder; UV (MeOH) λmax (log ε) 256 (4.35); 1H NMR (600 MHz) and 13C NMR (150 MHz) in D2O (pD 8.0), see Table 1; EPIMS m/z 841 [M + H]+; HR-ESIMS m/z 841.31850 [M + H]+ (calcd for C38H49O12N8S, 841.31852); EPIMS/ MS data see Figure 2. Sansanmycin I (2): white powder; UV (MeOH) λmax (log ε) 258 (4.34) 220 (4.70); 1H NMR (600 MHz) and 13C NMR (150 MHz) in D2O (pD 8.0), see Table S1 (Supporting Information); ESIMS m/z 880 [M + H]+; HR-ESIMS m/z 880.36243 [M + H]+ (calcd for C44H50N9O11, 880.36243); ESIMS/MS data see Figure S14 (Supporting Information). Sansanmycin J (3): white powder; UV (MeOH) λmax (log ε) 256 (4.17); 1H NMR (600 MHz) and 13C NMR (150 MHz) in D2O (pD 8.0), see Table S2 (Supporting Information); ESIMS m/z 853 [M + H]+; HR-ESIMS m/z 853.31869 [M + H]+ (calcd for C39H49O12N8S, 853.31852); ESIMS/MS data see Figure S17 (Supporting Information). Sansanmycin K (4): white powder; UV (MeOH) λmax (log ε) 256 (4.23); 1H NMR (600 MHz) and 13C NMR (150 MHz) in D2O (pD 8.0), see Table S3 (Supporting Information); ESIMS m/z 853 [M + H]+; HR-ESIMS m/z 853.31859 [M + H]+ (calcd for C39H49O12N8S, 853.31852); ESIMS/MS data see Figure S20 (Supporting Information). Sansanmycin L (5): white powder; UV (MeOH) λmax (log ε) 256 (4.18); 1H NMR (600 MHz) and 13C NMR (150 MHz) in D2O (pD 8.0), see Table S4 (Supporting Information); ESIMS m/z 892 [M + H]+; HR-ESIMS m/z 892.36253 [M + H]+ (calcd for C45H50O11N9, 892.36243); ESIMS/MS data see Figure S23 (Supporting Information). Sansanmycin M (6): white powder; UV (MeOH) λmax (log ε) 256 (4.20) 220 (4.59); 1H NMR (600 MHz) and 13C NMR (150 MHz) in D2O (pD 8.0), see Table S5 (Supporting Information); ESIMS m/z

892 [M + H]+; HR-ESIMS m/z 892.36254 [M + H]+ (calcd for C45H50O11N9, 892.36243); ESIMS/MS data see Figure S26 (Supporting Information). Sansanmycin N (7): white powder; UV (MeOH) λmax (log ε) 263 (4.08) 220 (4.44); 1H NMR (600 MHz) and 13C NMR (150 MHz) in D2O (pD 8.0), see Table S6 (Supporting Information); ESIMS m/z 876 [M + H]+; HR-ESIMS m/z 876.33463 [M + H]+ (calcd for C41H50O11N9S, 876.33450); ESIMS/MS data see Figure S29 (Supporting Information). Sansanmycin O (8): white powder; UV (MeOH) λmax (log ε) 259 (4.22) 220 (4.59); 1H NMR (600 MHz) and 13C NMR (150 MHz) in D2O (pD 8.0), see Table S7 (Supporting Information); ESIMS m/z 876.3 [M + H]+; HR-ESIMS m/z 876.33453 [M + H]+ (calcd for C41H50O11N9S, 876.33450); ESIMS/MS data see Figure S32 (Supporting Information). Antibacterial Assay. The MICs for antimycobacterial activity were determined by visual the microplate Alamar Blue assay (MABA).23 The bacterial strain Mycobacterium tuberculosis H37Rv was grown on 7H9 medium, and the final suspension of bacteria (in 7H9 medium) was 2 × 106 cfu/mL. All of the tested samples were dissolved in DMSO to make stock solutions of 20 mg/mL, and subsequent 2-fold dilutions were performed in 7H9 media (no Tween 80). Then serial dilutions were transferred to a 96-well microplate (Falcon 3072; Becton Dickinson, Lincoln Park, NJ, USA) in triplicate, and 100 μL of the bacterial suspension was added to each well, achieving a final volume of 200 μL (isoniazid and rifampicin were used as the positive controls). Additional control wells consisted of bacteria only (B) and medium only (M). Plates were incubated at 37 °C. Starting at day 5 of incubation, 20 μL of 10× Alamar Blue solution (Setotec Company) and 50 μL of 5% Tween 80 were added to one B well and one M well, and plates were reincubated at 37 °C. If the B wells became pink by 24 h, reagent was added to the entire plate. Plates were then incubated at 37 °C, and results were recorded at 24 h post-reagent addition. Visual MICs were defined as the lowest concentration of drug that prevented a color change. The MICs for P. aeruginosa were determined by a microdilution test following recommendations from the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS).24 The bacterial strain P. aeruginosa 11 was grown on Mueller-Hinton broth (MHB), and the final suspension of bacteria (in MHB medium) was 106 cells/mL. Tested samples (20 mg/mL as stock solution in DMSO and serial dilutions) were transferred to a 96-well plate in triplicate, and 100 μL of the bacterial suspension was added to each well, achieving a final volume of 200 μL (sansanmycin A was used as the positive control). After incubation at 37 °C for 24 h, the growth of the tested organism in the wells was detected by eye, and the MIC was defined as the lowest concentration that completely inhibits growth of the tested organism.

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ASSOCIATED CONTENT

S Supporting Information *

Workup procedure scheme; NMR data of 2−8, 1H and 13C NMR along with ESIMS/MS spectra of 1−8. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Y. Xie) Tel: +86 10 63010986. E-mail: [email protected]. cn. *(R. Chen) Tel/Fax: +86 10 63165276. E-mail: chrx888@ sohu.com. Author Contributions ‡

Y. Xie and Q. Cai contributed equally.

Notes

The authors declare no competing financial interest. D

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Note

ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (81273415), the Ministry of Science and Technology of China (2012ZX09301002-001018), and the Fundamental Research Funds for the Central Universities of China (2012N09).

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REFERENCES

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