Letters Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Bioactivity and Mode of Action of Bacterial Tetramic Acids Martin Klapper,† André Paschold,† Shuaibing Zhang,† Christiane Weigel,‡ Hans-Martin Dahse,§ Sebastian Götze,† Simona Pace,∥ Stefanie König,∥ Zhigang Rao,∥ Lisa Reimer,† Oliver Werz,∥ and Pierre Stallforth*,†
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†
Independent Junior Research Group Chemistry of Microbial Communication, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany ‡ Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany § Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany ∥ Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, 07743, Jena, Germany S Supporting Information *
ABSTRACT: Microbially produced 3-acyltetramic acids display a diverse range of biological activities. The pyreudiones are new members of this class that were isolated from bacteria of the genus Pseudomonas. Here, we performed a structure−activity relationship study and determined their mode of action. An efficient biomimetic synthesis was developed to synthesize pyreudione A. Pyreudiones and synthetic analogs thereof were tested for their amoebicidal, antibacterial, antiproliferative, and cytotoxic activities. The length of the alkyl side chain and the nature of the amino acid residues within the tetramic acid moiety strongly affected activity, in particular against mycobacteria. The mode of action was shown to correlate with the ability of pyreudiones to act as protonophores. Removal of the acidic proton by methylation of pyreudione A resulted in a loss of bioactivity.
M
(8; C12-TA) can be formed from N-acyl homoserine lactones (AHLs), e.g., 7 (Figure 1B), which are important quorum sensing autoinducers in Gram-negative bacteria.13 Most tetramic acids, however, are typically biosynthesized by the joint actions of a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS)14 or a hybrid PKSNRPS15 from an activated acyl precursor and an α-amino acid.6 Interestingly, in the case of the pyreudiones, a monomodular NRPS is sufficient to condense L-proline with oxo-acyl precursors (Figure 1B).10,11 Eventually, a Dieckmanntype cyclization furnishes the characteristic tetramic acid moiety. Reports of biomimetic synthetic approaches featuring a latestage Dieckmann-type cyclization are known, including solid phase syntheses.16 Most synthetic strategies, however, commence with the common pyrrolidine-2,4-dione moiety, which is subsequently modified (as depicted in Figure 2C).5,17 The first synthesis of pyreudione A (1), B (2), and alkyl side chain analogs (11−14) was conducted in a similar fashion: starting from Weinreb amide (9), the pyrrolizidine-1,3-dione
icrobial natural products and synthetic derivatives thereof are indispensable sources of lead structures in the development of anti-infectives.1 An understanding of their modes of action is key to modulating their activity and to effectively using them as drugs.2 Structure−activity relationship studies are the prerequisite to increasing the desired bioactivity and to decreasing unwanted toxic side effects. For instance, the inhibitor of bacterial type I signal peptidase in Gram-negative bacteria, G0775, was developed based on the Streptomycesderived natural product class of arylomycins.3,4 Tetramic acids are an important class of natural products exhibiting numerous biological activities.5 Their wide range of activities is mirrored in the diversity of organisms that are known to produce this class of compounds.6 These include marine sponges (e.g., aurantosides),7 fungi (e.g., tenuazonic acid),8 and bacteria (e.g., magnesidin).9 Recently, we discovered the amoebicidal pyreudiones 1−6 (Figure 1A), which are produced by the soil bacterium Pseudomonas f luorescens HKI0770.10,11 These acyltetramic acids allow the producing organisms to prevent amoebal predation and were studied in the context of microbial predator−prey relationships.12 The biogenesis of tetramic acids can proceed via different routes. For instance, 3-acyl tetramic acids such as 3-(1hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione © XXXX American Chemical Society
Received: May 16, 2019 Accepted: July 9, 2019
A
DOI: 10.1021/acschembio.9b00388 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
(MIC) of 0.4 μg mL−1 for pyreudione analog 14 (C12 side chain),11 as well as a strong amoebicidal activity. In this study, we provide a strategic structure−activity relationship study of pyreudiones as well as insights into their mode of action. Thus, first the biological activity of previously isolated pyreudiones 1−6 as well as derivatives of the predominant bacterial product, pyreudione A (1), was studied in detail. All isolated pyreudiones and previously synthesized alkyl side chain analogs were tested for their antimicrobial activity with respect to methicillin-resistant Staphylococcus aureus (MRSA), M. aurum, and M. vaccae (Table 1). Consistent with our previous hypothesis that the lipophilicity of this compound family correlates with activity, pyreudione derivative 14 with the longest alkyl side chain (C12) was most potent against mycobacteria, and also against MRSA (MIC = 3.12 μg mL−1). Notably, a Z-configured double bond in the C12-alkyl side chain in pyreudione D (4) reduced the antimicrobial activity. Compared to pyreudione B (2; C10) and the corresponding C12 alkyl side chain analog 14, pyreudione A (1; C8) was only moderately active in these antimicrobial tests. These trends are mirrored by the reported amoebicidal activities, indicating that the length of the side chain is crucial for both amoebicidal and antibacterial activities.10 Furthermore, the amoebicidal effect of the pyreudiones may indicate a general toxicity for eukaryotic cells including human cells. Thus, their antiproliferative effect (growth inhibitory concentration, GI50) was tested in this study using the human cell lines HUVEC and K-562, while cytotoxicity (cytotoxic concentration, CC50) was determined with respect to human HeLa cells (Table 1). Pyreudione analogs 11−13 with short side chains (C2−C6) and the more polar hydroxylated pyreudione I (6) did not show any
Figure 1. (A) Isolated pyreudiones 1−6.10,11 (B) Biosyntheses of tetramic acids derived from N-acylhomoserine lactones (AHLs), e.g., 7 (left),13 and of pyreudione A, 1 (right).11
(10) was accessed by a base-mediated Dieckmann-type cyclization, followed by an acylation using activated fatty acids (Figure 2A).10 Since analogs with shorter chain lengths, 11−13 (two to six carbon atoms in the side chain), displayed no amoebicidal activity with respect to Dictyostelium discoideum AX2 (Table 1), a correlation between the lipophilicity and the antimicrobial activity was suggested.10 First antimicrobial tests revealed a high level of activity against mycobacteria (M. aurum and M. vaccae) with a minimal inhibitory concentration
Figure 2. Synthetic approaches toward pyreudiones and analogs. Note that pyreudiones and analogs display the characteristic keto−enoltautomerism (depicted exo-Z-enol is most prevalent).22 (A) Synthesis toward pyreudione A (1), (B) 2, and alkyl side chain analogs 11−14.10 (B) Synthesis of glycine analog 18.20 (C) Synthesis of pyreudione A amino acid analogs 24−27.23,24 (D) Biomimetic synthesis toward pyreudione A (1). B
DOI: 10.1021/acschembio.9b00388 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology Table 1. Bioactivity Data of the Strategic SAR Study (All Values Given in μg mL−1) compound pyreudione pyreudione pyreudione pyreudione pyreudione pyreudione
C2 (11) C4 (12) C6 (13) A (1) B (2) C12 (14)
pyreudione C (3) pyreudione D (4) D-pyreudione
A (28) pyreudione E (5) pyreudione I (6) 18 24 25 26 27 31
(Gly) (L-Ala) (L-Phe) (D-Phe) (L-Trp) (Me-Pyr A)
chain
IC50
length
D.d.a
2 4 6 8 10 12
>100e >100e >100e 4e 1e 2e
10 12
1 5
8 8 8
4 6h >100h
8 8 8 8 8 8
35 >100 5 5 >100 >100
MIC MRSAb
M.v.c
saturated side chain analogs -f -f f -f -f -f -g 25 25 3.12 3.12 0.4h unsaturated side chain analogs 50 1.56 12.5 0.78 ring analogs -i -i 50 12.5h >100 >100 amino acid analogs >100 25 50 12.5 25 6.25 25 6.25 25 6.25 >100 25
GI50
GI50
CC50
M.a.d
HUVEC
K-562
HeLa
-f -f -f 50 6.25 0.4h
>50 >50 >50 43 24 19
>50 >50 >50 28 28 20
>50 >50 >50 35 42 39
3.12 3.12
16 >50
13 >50
16 50
-i 12.5h >100
34 -i >50
28 -i >50
31 -i >50
25 12.5 3.12 6.25 6.25 25
>50 >50 30 30 >50 >50
>50 40 35 23 25 >50
46 33 32 28 34 ≥50
a
With respect to D. discoideum AX2. bWith respect to methicillin-resistant S. aureus MRSA 134/94 R9. cWith respect to M. vaccae 10670 M4. With respect to M. aurum SB66. eAs previously reported.10 fNo activity in initial antimicrobial tests against these strains. gOnly tested against nonresistant Staphylococcus aureus 511 B3 (MIC = 100 μg mL−1). hAs previously reported.11 iNot determined.
d
amoebicidal activity (IC50 (D. discoideum) = 35 μg mL−1) and a lower cytotoxicity (CC50 (HeLa) = 45.6 μg mL−1) in comparison to bicyclic pyreudione A (1), while the antimycobacterial activity remained (MIC = 25 μg mL−1). On the basis of these results, we further varied the nature of the amino acid moiety. For the L-alanine (24), L-phenylalanine (25), D-phenylalanine (26), and L-tryptophan (27) analogs, the respective pyrrolidine-2,4-dione core structures 19−22 were obtained in a first step, which could subsequently be acylated with different fatty acids as previously described.10 Briefly, in a single reaction sequence, the respective Bocprotected amino acid was condensed with Meldrum’s acid (MA), decarboxylated, cyclized, and deprotected using trifluoroacetic acid (TFA).17,23 To obtain pyreudione A (1; C8) analogs, the core structures 19−22 were acylated with octanoic acid (23) using equimolar amounts of 4-dimethylaminopyridine (DMAP) to achieve a C3 acylation (Figure 2C).22,24 Amino acid analogs 24−27 were tested for their antimicrobial, antiproliferative, and cytotoxic activities. Interestingly, the L-alanine- and L-tryptophan-derived pyreudione analogs 24 and 27, respectively, were not amoebicidal (IC50 (D. discoideum) > 100 μg mL−1) but retained their antimycobacterial activity (MIC = 6.25 μg mL−1 for 27), while their cytotoxicity was similar in comparison to pyreudione A (1; Table 1). Only the L- and D-phenylalanine analogs 25 and 26 displayed amoebicidal activity comparable to pyreudione A (1; IC50 (D. discoideum) = 5 μg mL−1). Notably, the amoebicidal activities for the L- and Denantiomers were identical, which was also the case for pyreudione A (1) and its enantiomer 28 derived from Dproline (Figure 2A).10 While being more potent than pyreudione A (1), the antimycobacterial activity, antiproliferative effect, and cytotoxicity were similar for both L- and Dphenylalanine analogs 25 and 26 (Table 1). These
antiproliferative/cytotoxic activities (GI50, CC50 > 50 μg mL−1). Pyreudione D (4) displayed only modest cytotoxicity (CC50 (HeLa) = 49.8 μg mL−1). Pyreudione C (3), with an additional double bond conjugated to the exoenol, showed the largest cytotoxic effect (CC50 (HeLa) = 16.0 μg mL−1). However, in comparison to doxorubicin, a very potent chemotherapeutic agent (GI50 (K-562) = 0.2 μg mL−1, CC50 (HeLa) = 0.08 μg mL−1),18 all pyreudiones are only moderately active at most. While being about 2 orders of magnitude more active against mycobacteria, the cytotoxicity of the C12 pyreudione alkyl side chain analog 14 was comparable to that of pyreudione A (1 ; CC50 (HeLa) = 38.9 μg mL−1 versus 34.9 μg mL−1, respectively). Next, we investigated the influence of the amino acid moiety on the bioactivity, while keeping the alkyl side chain length constant (C8). Thus, different synthetic approaches toward tetramic acids were pursued and the resulting pyreudione analogs were tested for their bioactivities (Table 1). In general, pyrrolizidine alkaloids are well-known cytotoxic agents.19 Hence, the effect of the bicyclic system regarding activity was investigated. These pyrrolizidine alkaloids are a [3.3.0] bicyclic system, i.e. they consist of two fused five-membered rings. Altering the type of ring system to a [4.3.0] system containing a six-membered ring derived from L-pipecolic acid fused with a five-membered ring (previously isolated and synthesized pyreudione E (5), Figure 1A)11 led to increased antibacterial activity while the amoebicidal activity did not change. Disruption of the bicyclic system by removing one ring was investigated next. In the synthesis of pyreudione derivatives, we thus exchanged L-proline with other amino acids. Glycine derivative 18 was first synthesized (Figure 2B).20 Briefly, N-acetyl−glycine (15) was activated and first acylated with 3-oxo ester (16)21 and subsequently cyclized with sodium ethoxide. Interestingly, glycine analog 18 showed a lower C
DOI: 10.1021/acschembio.9b00388 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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the carbon NMR of all possible products (see Supporting Information).28 According to DP4 probabilities, 3-methoxy-2octanoyl-5,6,7,7a-tetrahydro-3H-pyrrolizin-1-one (31) is the obtained isomer with 99.95% confidence. Methylated pyreudione A (31) neither was amoebicidal (IC 50 (D. discoideum) > 100 μg mL−1 ) nor showed antiproliferative activity (GI50 > 50 μg mL−1, Table 1). However, weak cytotoxic activity (CC50 (HeLa) ≥ 50 μg mL−1) and low antimycobacterial activity were still detectable (MIC = 25 μg mL−1), which can potentially be attributed to a still possible interference with the membrane integrity.26 To examine the proposed mode of actions as a protonophore, pyreudione A (1) was subjected to a protonophore assay (using pH-sensitive Lysotracker red DND 99) where it exhibited similar activity to that of the well-known protonophore carbonylcyanid-m-chlorphenylhydrazon (CCCP, Figure 3B).29 The result for methylated pyreudione A (31), however, was similar to the negative control (Supporting Information Figure S5). These data imply that pyreudione A (1) actively transports protons into lysosomes along with intralysosomal acidification, suggesting protonophoric properties. In summary, pyreudiones and synthetic analogs were tested for their biological activities, and their mode of action was investigated. The length of the alkyl side chain of the pyreudiones as well as the nature of the amino acid moiety strongly influence the activity. As a general trend, it was observed that the length of the alkyl chain positively correlates with various bioactivities. Certain aromatic amino acid analogs show particularly strong activity, especially against mycobacteria. Furthermore, an efficient biomimetic synthesis was developed to synthesize pyreudione A (1) that allows facile access to even more analogs. Our results regarding the mode of action of the pyreudiones are in good agreement with a protonophore activity. In a protonophore assay pyreudione A (1) acidified lysosomes with comparable activity as the wellknown protonophore CCCP. Accordingly, methylation of pyreudione A (1) removed its activity. Overall, the natural product class of pyreudione alkaloids was characterized regarding the bioactivity and mode of action.
observations point toward an unspecific mode of action of pyreudiones and analogs rather than a specific interaction with distinct target proteins. Reports on the mode of action of the structurally similar tetramic acids reutericyclin25 and C12-TA (8),26 which dissipate the pH gradient and the membrane potential, suggest that pyreudiones and analogs could also act as protonophores. Capping of the acidic proton of the tetramic acid moiety by methylation should thus result in a loss of activity. To this end, a more reliable synthesis toward the natural product pyreudione A (1) was established for subsequent modification via methylation. Hence, a two-step biomimetic synthesis with an improved total yield was developed that allowed access to greater amounts of pyreudione A (1). Briefly, N-acylation of Lprolinemethylester (29) with 3-oxoester (16) in the presence of DMAP at high temperatures furnished 30. A subsequent base-mediated Lacey−Dieckmann cyclization under anhydrous conditions yielded pyreudione A (1) in 52% yield over two steps (Figure 2D). The enantiomeric purity was retained, as the optical rotation of the synthetic product [α]25 D = −38.8 (c = 0.3 in MeOH) corresponds to that of the natural product.10 While methylation with methyl iodide or trimethylsilyl diazomethane was not successful, use of dimethyl sulfate27 yielded methylated pyreudione A (31; Figure 3A). Hetero-
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.9b00388. Figure 3. Studies on the mode of action of pyreudiones. (A) Methylation of pyreudione A 1. (B) Mode of action of pyreudione A 1 as protonophore. Pyreudione A 1 exhibited similar activity as the well-known protonophore carbonylcyanid-m-chlorphenylhydrazon (CCCP).
Synthetic procedures, 13NMR calculations, experimental procedure for bioactivity tests, NMR spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
nuclear multiple bond correlation (HMBC) NMR excluded methylation at the former exoenol, since 2J and 3J couplings of methylene protons 2′ and 3′ in the side chain to a ketone carbon (δ 195.9 ppm) were observed (see HMBC zoom-in, Supporting Information). A selective NOESY recorded at the chemical shift of the O-methyl group (δ 3.17 ppm, see Supporting Information) revealed a coupling of the O-methyl group with the protons at the 5- and 7a-positions, indicating that the methylation occurred at the carbamide carbonyl. This result could be confirmed by computational methods modeling
ORCID
Pierre Stallforth: 0000-0001-7260-9921 Author Contributions
M.K., O.W., and P.S. designed the study. M.K., A.P., S.Z., M.K., C.W., H.-M.D., S.G., S.P., S.K., Z.R., and L.R. performed experiments. M.K., A.P., S.Z., O.W., and P.S. analyzed data. M.K., O.W., and P.S. wrote the manuscript. All authors have given approval to the final version of the manuscript. D
DOI: 10.1021/acschembio.9b00388 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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tetramate macrolactams from phylogenetically diverse bacteria. Proc. Natl. Acad. Sci. U. S. A. 107, 11692−11697. (16) Romoff, T. T., Ma, L., Wang, Y. W., and Campbell, D. A. (1998) Solid phase synthesis of 3-acyl-2,4-pyrrolidinediones (3-acyl tetramic acids) via mild cyclative cleavage. Synlett 1998, 1341−1342. (17) Jouin, P., Castro, B., and Nisato, D. (1987) Stereospecific Synthesis of N-Protected Statine and Its Analogs Via Chiral Tetramic Acid. J. Chem. Soc., Perkin Trans. 1, 1177−1182. (18) Yang, W. J., Soares, J., Greninger, P., Edelman, E. J., Lightfoot, H., Forbes, S., Bindal, N., Beare, D., Smith, J. A., Thompson, I. R., Ramaswamy, S., Futreal, P. A., Haber, D. A., Stratton, M. R., Benes, C., McDermott, U., and Garnett, M. J. (2012) Genomics of Drug Sensitivity in Cancer (GDSC): a resource for therapeutic biomarker discovery in cancer cells. Nucleic Acids Res. 41, D955−D961. (19) Robertson, J., and Stevens, K. (2017) Pyrrolizidine alkaloids: occurrence, biology, and chemical synthesis. Nat. Prod. Rep. 34, 62− 89. (20) Petroliagi, M., and Igglessi-Markopoulou, O. (1997) An efficient synthesis of novel N-acetyl-3-alkanoyl and 3-dienoyl tetramic acids. J. Chem. Soc., Perkin Trans. 1, 3543−3548. (21) Arp, J., Götze, S., Mukherji, R., Mattern, D. J., Garcia-Altares, M., Klapper, M., Brock, D. A., Brakhage, A. A., Strassmann, J. E., Queller, D. C., Bardl, B., Willing, K., Peschel, G., and Stallforth, P. (2018) Synergistic activity of cosecreted natural products from amoebae-associated bacteria. Proc. Natl. Acad. Sci. U. S. A. 115, 3758− 3763. (22) Jeong, Y. C., and Moloney, M. G. (2011) Synthesis of and tautomerism in 3-acyltetramic acids. J. Org. Chem. 76, 1342−1354. (23) Hosseini, M., Kringelum, H., Murray, A., and Tønder, J. E. (2006) Dipeptide analogues containing 4-ethoxy-3-pyrrolin-2-ones. Org. Lett. 8, 2103−2106. (24) Sengoku, T., Nagae, Y., Ujihara, Y., Takahashi, M., and Yoda, H. (2012) A Synthetic Approach to Diverse 3-Acyltetramic Acids via O- to C-Acyl Rearrangement and Application to the Total Synthesis of Penicillenol Series. J. Org. Chem. 77, 4391−4401. (25) Cherian, P. T., Wu, X., Maddox, M. M., Singh, A. P., Lee, R. E., and Hurdle, J. G. (2015) Chemical modulation of the biological activity of reutericyclin: a membrane-active antibiotic from Lactobacillus reuteri. Sci. Rep. 4, 4721−4730. (26) Lowery, C. A., Park, J., Gloeckner, C., Meijler, M. M., Mueller, R. S., Boshoff, H. I., Ulrich, R. L., Barry, C. E., Bartlett, D. H., Kravchenko, V. V., Kaufmann, G. F., and Janda, K. D. (2009) Defining the Mode of Action of Tetramic Acid Antibacterials Derived from Pseudomonas aeruginosa Quorum Sensing Signals. J. Am. Chem. Soc. 131, 14473−14479. (27) Shestak, O. P., and Novikov, V. L. (2010) Synthesis of coruscanones A and B, metabolites of Piper coruscans, and related compounds. Russ. Chem. Bull. 59, 81−90. (28) Smith, S. G., and Goodman, J. M. (2010) Assigning Stereochemistry to Single Diastereoisomers by GIAO NMR Calculation: The DP4 Probability. J. Am. Chem. Soc. 132, 12946− 12959. (29) Goldsby, R. A., and Heytler, P. G. (1963) Uncoupling of Oxidative Phosphorylation by Carbonyl Cyanide Phenylhydrazones 0.2. Effects of Carbonyl Cyanide M-Chlorophenylhydrazone on Mitochondrial Respiration. Biochemistry 2, 1142−1147.
We are grateful for financial support from the Leibniz Association, Aventis Foundation (Ph.D. fellowship to M.K.), and the Deutsche Forschungsgemeinschaft (DFG) STA1431/ 2-1 and CRC1127 ChemBioSys. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We thank A. Perner and H. Heinecke (HKI Jena) for MS and NMR measurements.
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