Georatusin, a Specific Antiparasitic Polyketide–Peptide Hybrid from

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

pubs.acs.org/OrgLett

Georatusin, a Specific Antiparasitic Polyketide−Peptide Hybrid from the Fungus Geomyces auratus Yi-Ming Shi,† Christian Richter,‡ Victoria L. Challinor,† Peter Grün,† Antonio Girela del Rio,§ Marcel Kaiser,⊥ Anja Schüffler,# Meike Piepenbring,§ Harald Schwalbe,‡ and Helge B. Bode*,†,∥ †

Merck-Stiftungsprofessur für Molekulare Biotechnologie, Fachbereich Biowissenschaften, ‡Institut für Organische und Chemische Biologie, Zentrum für Biomolekulare Magnetische Resonanz, §Department of Mycology, Fachbereich Biowissenschaften, Biologicum, and ∥Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt, 60438 Frankfurt am Main, Germany ⊥ Swiss Tropical and Public Health Institute Parasite Chemotherapy and University of Basel, 4051 Basel, Switzerland # Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF), 67663 Kaiserslautern, Germany S Supporting Information *

ABSTRACT: Georatusin (1), featuring a highly reduced, methylated polyketide moiety fused to a tryptophan by an amide and ester bond forming a 13-membered ring, was produced by the soil fungus Geomyces auratus. An HMQC−COSY spectrum was measured to build up the connectivities despite the overlapping proton signals. DQF-COSY, HETLOC, J-HMBC, and ROESY were implemented to determine the relative configuration of the flexible moiety. Georatusin (1) shows specific antiparasitic activities against Leishmania donovani and Plasmodium falciparum without obvious cytotoxicity. The biosynthesis of 1 was also proposed.

F

ungi make up a taxon of eukaryotic organisms with probably more than 1.5 million estimated species,1 diverging from the other two eukaryotic kingdoms (animals and plants). Living in multispecies communities, fungi produce small molecules as antibiotics and signaling compounds for intercellular communication,2 interacting with the host and other microorganisms, and defeating other microbial competitors to thrive in complex ecosystems.3 The vast repertoire of fungal natural products with their unusual modes of action4 has inspired generations of researchers to explore new chemical entities and search for small-molecule probes and therapeutic agents. This includes the penicillin antibiotics, the immunosuppressant cyclosporin, and the cholesterol-reducing drug lovastatin.4,5 However, despite the high value of the identified natural products and the wide occurrence of fungi in all different environments, 100 9.1 1.6 >100

0.005 2.7 1.2 0.008 0.008

a The controls differ for each tested organism. Melarsoprol, benznidazole, miltefosine, chloroquine, and podophyllotoxin were used as controls for T. brucei rhodesiense, T. cruzi, L. donovani, P. falciparum, and mammalian L6 cells, respectively.

Because of the pathogenicity of G. destructans and the widespread occurrence of the Geomyces genus, not only in bat hibernacula but also commonly in soil and cool environments,10 it is tempting to speculate that the specific antiparasitic function of georatusin (1) might be essential to help the fungi establish their ecological niche. The discovery of the unique structure and specific bioactivity of 1 provides new insight into the metabolic capabilities and ecological significance of Geomyces and clearly warrants the exploration of this genus in the future. With respect to the biosynthesis of highly reduced polyketide−peptide hybrids from fungi, compounds such as aspyridone A,15 cytochalasan,16 and thermolide A (Scheme 1A)17 have been described in the past decade. Because no Geomyces genomes are available in the NCBI database, the biosynthetic pathway of georatusin [1 (Scheme 1B)] was proposed on the basis of the established paradigms17−20 and the genome sequences of Metarhizium species, among which Metarhizium acridium is a known producer of metacridamides.14 Antismash analysis21 of genome sequences from 11 Metarhizium species led to the identification of at least 16 biosynthesis gene clusters encoding two different polyketide− peptide synthases (Figure S21).22 The clusters encode either a single polyketide−peptide megasynthetase hybrid with a terminal R domain and a trans-acting ER (Figure S21, type I) or two separate enzymes encoding a PKS and an NRPS with a C-terminal domain (Figure S21, type II). Consequently, there are two conceivable pathways for forming the macrolide of georatusin (1). (1) The R domain reductively releases an amino aldehyde that is attacked by OH-22 followed by oxidation (pathway a, Scheme 1B), as presented in the lactonization of thermolide.17 (2) The C-terminal domain offloads the thioester-tethered amide via cyclization to form the lactone (pathway b, Scheme 1B).19 Another interesting feature of the proposed biosynthesis of 1 is the recruitment of a Damino acid rather than incorporation of an L-amino acid, in contrast to the case for metacridamides. To the best of our knowledge, thermolide analogues from thermophilic fungi17,20 are the only other examples for the occurrence of a D-amino acid in such natural products. However, the mechanism for Damino acid selection or epimerization has not yet been elucidated. It might be possible that the A domain is responsible for the exclusive incorporation of a D-tryptophan provided by an epimerase encoded elsewhere in the genome. C

DOI: 10.1021/acs.orglett.8b00293 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

Scheme 1. (A) Known Highly Reduced Polyketide−Peptide Hybrids from Fungi Exemplified by Thermolide A, Metacridamide A, and Aspyridone A and (B) Proposed Biosynthetic Pathway for Georatusin (1)a

a

ER may be defective and complemented by a trans-acting ER as shown in Figure S21 (type I).

■

(4) (a) Keller, N. P.; Turner, G.; Bennett, J. W. Nat. Rev. Microbiol. 2005, 3, 937−947. (b) Schueffler, A.; Anke, T. Nat. Prod. Rep. 2014, 31, 1425−1448. (5) Brakhage, A. A.; Schroeckh, V. Fungal Genet. Biol. 2011, 48, 15− 22. (6) Grundmann, F.; Kaiser, M.; Schiell, M.; Batzer, A.; Kurz, M.; Thanwisai, A.; Chantratita, N.; Bode, H. B. J. Nat. Prod. 2014, 77, 779−783. (7) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866−876. (8) Hu, K.; Westler, W. M.; Markley, J. L. J. Biomol. NMR 2011, 49, 291−296. (9) (a) Bifulco, G.; Dambruoso, P.; Gomez-Paloma, L.; Riccio, R. Chem. Rev. 2007, 107, 3744−3779. (b) Kawahara, T.; Izumikawa, M.; Takagi, M.; Shin-Ya, K. Org. Lett. 2012, 14, 4434−4437. (c) Sikorska, J.; Hau, A. M.; Anklin, C.; Parker-Nance, S.; Davies-Coleman, M. T.; Ishmael, J. E.; McPhail, K. L. J. Org. Chem. 2012, 77, 6066−6075. (d) Rodriguez, J.; Nieto, R. M.; Blanco, M.; Valeriote, F. A.; Jimenez, C.; Crews, P. Org. Lett. 2014, 16, 464−467. (10) Lorch, J. M.; Meteyer, C. U.; Behr, M. J.; Boyles, J. G.; Cryan, P. M.; Hicks, A. C.; Ballmann, A. E.; Coleman, J. T.; Redell, D. N.; Reeder, D. M.; Blehert, D. S. Nature 2011, 480, 376−378. (11) Lorch, J. M.; Lindner, D. L.; Gargas, A.; Muller, L. K.; Minnis, A. M.; Blehert, D. S. Mycologia 2013, 105, 237−252. (12) Li, Y.; Sun, B.; Liu, S.; Jiang, L.; Liu, X.; Zhang, H.; Che, Y. J. Nat. Prod. 2008, 71, 1643−1646. (13) Parish, C. A.; de la Cruz, M.; Smith, S. K.; Zink, D.; Baxter, J.; Tucker-Samaras, S.; Collado, J.; Platas, G.; Bills, G.; Diez, M. T.; Vicente, F.; Peláez, F.; Wilson, K. J. Nat. Prod. 2009, 72, 59−62. (14) Krasnoff, S. B.; Englich, U.; Miller, P. G.; Shuler, M. L.; Glahn, R. P.; Donzelli, B. G.; Gibson, D. M. J. Nat. Prod. 2012, 75, 175−180. (15) Bergmann, S.; Schumann, J.; Scherlach, K.; Lange, C.; Brakhage, A. A.; Hertweck, C. Nat. Chem. Biol. 2007, 3, 213−217. (16) Schümann, J.; Hertweck, C. J. Am. Chem. Soc. 2007, 129, 9564− 9565. (17) Niu, X.; Chen, L.; Yue, Q.; Wang, B.; Zhang, J.; Zhu, C.; Zhang, K.; Bills, G. F.; An, Z. Org. Lett. 2014, 16, 3744−3747. (18) Boettger, D.; Hertweck, C. ChemBioChem 2013, 14, 28−42. (19) (a) Gao, X.; Haynes, S. W.; Ames, B. D.; Wang, P.; Vien, L. P.; Walsh, C. T.; Tang, Y. Nat. Chem. Biol. 2012, 8, 823−830. (b) Zhang, J.; Liu, N.; Cacho, R. A.; Gong, Z.; Liu, Z.; Qin, W.; Tang, C.; Tang, Y.; Zhou, J. Nat. Chem. Biol. 2016, 12, 1001−1003. (20) Guo, J. P.; Zhu, C. Y.; Zhang, C. P.; Chu, Y. S.; Wang, Y. L.; Zhang, J. X.; Wu, D. K.; Zhang, K. Q.; Niu, X. M. J. Am. Chem. Soc. 2012, 134, 20306−20309.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00293. Detailed experimental procedures, NMR and ECD spectra, and PKS-NRPS biosynthetic gene clusters from Metarhizium (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yi-Ming Shi: 0000-0001-6933-4971 Harald Schwalbe: 0000-0001-5693-7909 Helge B. Bode: 0000-0001-6048-5909 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Y.-M.S. is supported by a Postdoctoral Research Fellowship from the Alexander von Humboldt Foundation. This work was funded in part by the state of Hesse as part of the LOEWE research cluster Integrated Fungal Research (IPF). The authors thank the following colleagues from the Goethe Universität Frankfurt: Miguel Rosas for isolating the fungal strain, Jascha Weisenborn for providing a preliminary identification, Florian Hennicke for managing the strain collection, and Dr. Krishna Saxena for ECD measurement.

■

REFERENCES

(1) Hawksworth, D. L. Mycol. Res. 2001, 105, 1422−1432. (2) (a) Hornby, J. M.; Jensen, E. C.; Lisec, A. D.; Tasto, J. J.; Jahnke, B.; Shoemaker, R.; Dussault, P.; Nickerson, K. W. Appl. Environ. Microbiol. 2001, 67, 2982−2992. (b) Hogan, D. A. Eukaryotic Cell 2006, 5, 613−619. (3) Kusari, S.; Hertweck, C.; Spiteller, M. Chem. Biol. 2012, 19, 792− 798. D

DOI: 10.1021/acs.orglett.8b00293 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters (21) Blin, K.; Wolf, T.; Chevrette, M. G.; Lu, X.; Schwalen, C. J.; Kautsar, S. A.; Suarez Duran, H. G.; de los Santos, E. L. C.; Kim, H. U.; Nave, M.; Dickschat, J. S.; Mitchell, D. A.; Shelest, E.; Breitling, R.; Takano, E.; Lee, S. Y.; Weber, T.; Medema, M. H. Nucleic Acids Res. 2017, 45, W36−W41. (22) (a) Sbaraini, N.; Guedes, R. L.; Andreis, F. C.; Junges, A.; de Morais, G. L.; Vainstein, M. H.; de Vasconcelos, A. T.; Schrank, A. BMC Genomics 2016, 17, 736. (b) Gao, Q.; Jin, K.; Ying, S.-H.; Zhang, Y.; Xiao, G.; Shang, Y.; Duan, Z.; Hu, X.; Xie, X.-Q.; Zhou, G.; Peng, G.; Luo, Z.; Huang, W.; Wang, B.; Fang, W.; Wang, S.; Zhong, Y.; Ma, L.-J.; St. Leger, R. J.; Zhao, G.-P.; Pei, Y.; Feng, M.-G.; Xia, Y.; Wang, C. PLoS Genet. 2011, 7, e1001264.

E

DOI: 10.1021/acs.orglett.8b00293 Org. Lett. XXXX, XXX, XXX−XXX