Viewpoint pubs.acs.org/jmc
Bacterial Iron Uptake Pathways: Gates for the Import of Bactericide Compounds Isabelle J. Schalk*,†,‡ and Gaeẗ an L. A. Mislin†,‡ †
Université de Strasbourg, UMR7242, ESBS, Boulevard Sébastien Brant, F-67413 Illkirch, France CNRS, UMR7242, ESBS, Boulevard Sébastien Brant, F-67413 Illkirch, France
‡
ABSTRACT: Bacterial resistance to most antibiotics in clinical use has reached alarming proportions. A challenge for modern medicine will be to discover new antibiotics or strategies to combat multidrug resistant bacteria, especially Gramnegative bacteria for which the situation is particularly critical. Vectorization of bactericide compounds by siderophores (iron chelators produced by bacteria) is a promising strategy able to considerably increase the efficacy of drugs. Such a Trojan horse strategy can also extend activity of specific Gram-positive antibiotics to Gram-negative bacteria.
R
consequently a key nutriment for bacterial proliferation and in the infection process. To access iron during infections, bacteria produce siderophores, small organic compounds (MW between 200 and 2000 Da) with a very high affinity for iron(III).2 Siderophores are secreted into the bacteria’s environment where they scavenge iron from the host and the resulting complexes, and thus iron is transported back into the bacteria.3 Siderophores with very different chemical structures have been described (Figure 1A) and are classified into three main families depending on the iron-chelating group: hydroxamates, catecholates, and carboxylates. Currently more than 500 different siderophores are known.2,4 Iron-loaded siderophores are transported back into bacteria by specific cell wall transporters.5 In Gram-negative bacteria, iron is delivered by siderophores either to the bacterial periplasm or to the cytoplasm depending on the siderophore pathway and bacterium.3 Consequently, Trojan horse strategies using siderophores as vectors should deliver drugs either to the bacterial periplasm or to the cytoplasm according to the system exploited. There has been work since the 1980s on these iron-uptake pathways to develop Trojan horse strategies where antibiotics are covalently linked to siderophores. The idea is that each time a bacterium internalizes a ferric ion, a molecule of drug is transported as well. Some bacteria themselves have developed such Trojan horse strategies with antibiotics covalently linked to siderophores to eliminate competitors. These natural compounds are called sideromycins, and archetypes are albomycins (Figure 1B), natural antibiotics produced by Streptomyces. The chelating part of albomycins is structurally similar to ferrichrome, a siderophore used by E. coli. After iron
ecently, the World Health Organization (WHO) published its first ever list of antibiotic-resistant “priority pathogens” that it predicts will in the very near future pose substantial threats to human health. The situation is particularly critical for Gram-negative bacteria: the outer membrane of these bacteria acts as a permeability barrier against many antimicrobials. Indeed, the Gram-negatives Acinetobacter, Pseudomonas and various Enterobacteriaceae (including Klebsiella, E. coli, Serratia, and Proteus) are all high up in this WHO list. If we do not take urgent measures, we will soon enter a postantibiotic era in which common infections and small wounds will, as in the past, kill. This has major implications for other fields of medicine, notably transplantation and surgery more generally. It is clearly urgent to develop new antibiotics, vaccines and other strategies to fight bacteria. This is a real challenge for medicinal chemistry over the coming years, and almost no new classes of relevant antibiotics have reached the market these last few decades.1 In this context, vectorization of antibiotics is a promising strategy as illustrated by the work of the team of M. J. Miller reported in this issue.13 The strategy involves developing chemically derivatized antibiotics or antibiotic vectors that target defined bacterial cells: specific recognition and transport mechanisms are exploited to cross bacterial cell wall ensuring effective assimilation into the microorganisms. The team of M. J. Miller developed a vectorized analogue of daptomycin, an antibiotic only active against Gram-positive bacteria. The resulting vectorized daptomycin has potent activity against multidrug-resistant strains of A. baumannii (a Gram-negative pathogen) both in cultures and in infection models. The publication in this issue of the journal (see ref 13) used bacterial siderophore-dependent iron-uptake pathways as gateways for a vectorization strategy. Iron is a cofactor of enzymes involved in important biological processes and © XXXX American Chemical Society
Received: April 10, 2017
A
DOI: 10.1021/acs.jmedchem.7b00554 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Viewpoint
Figure 1. A. Examples of siderophores. Enterobactin (1) produced by E. coli; Ferrichrome (3) produced by Ustilago sphaerogena; Pyochelin (2) and Pyoverdine (4) produced by P. aeruginosa. B. Examples of natural microcins. Albomycins (5, 6, and 7) produced by Streptomyces species; Microcin E492 produced Klebsiella pneumonia (8). B. Trojan horse compounds currently in clinical trial. MC-1 (9), BAL30072 (10), and S-649266 or Cefiderocol (11).
(i) Each bacterial species produces and uses specific siderophores: aerobactin, yersiniabactin, enterobactin, and salmochelin for Klebsiella pneumonia; enterobactin, salmochelin, aerobactin, and yersiniabactin for Escherichia coli; and acinetobactin, fimsbactin, and baumannoferrins for Acinetobacter baumanii. Consequently, it will be possible to selectively target bacterial species by using appropriately chosen siderophores for vectorizing the antibiotic. This narrow selectivity, in addition to other advantages, reduces the risk of antibiotic resistance. (ii) The large diversity in the chemical structures of siderophore constitutes a valuable chemical collection for the design of specific siderophore−antibiotic conjugates. A large number of siderophore−antibiotic conjugates have already been described. The the most effective are currently based on catechol or hydroxamate siderophore analogues (DOI: 10.1021/acs.jmedchem.7b00102 in this issue13). (iii) Vectorization is a potential solution for any chemical compound with a strong inhibitory effect on biological systems but unable to enter bacteria by diffusion. Pharmaceutical companies and academic laboratories have plenty of such compounds. This strategy can also be used to adapt Grampositive only antibiotics for an uptake into the periplasm of Gram-negative bacteria and consequently extends their activity
chelation, the siderophore moiety allows the whole albomycin molecule to enter target bacteria via ferrichrome transporters. The presence of the chelating part thereby substantially increases the antibiotic activity of the molecule. Many other natural compounds of this type have been described, and they are all powerful antibiotics. The efficient and selective antibiotic activities of sideromycins against competing bacteria encouraged several research teams to develop Trojan horse approaches using bacterial iron uptake pathways as gateways. Various groups, and especially that of M. J. Miller, have made substantial contributions to the development of such compounds by covalently coupling antibiotics to natural siderophores or synthetic siderophore analogues (Figure 2). Several such compounds are now in clinical phases of development: MC16 a monobactam (Pfizer in 2012), BAL300727 a monosulfactam (Basilea Pharmaceutica in 2010), and a cephalosporin (S-649266 also called cefiderocol)8 (Shionogi in 2015 and GSK, respectively) (Figure 1C). Current work on antibiotic vectorization and bacterial iron acquisition suggests that Trojan horse strategies using siderophore-uptake pathways are promising, and the following points have emerged. B
DOI: 10.1021/acs.jmedchem.7b00554 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Viewpoint
Figure 2. The Gram-negative bacterial cell wall is composed of an outer and an inner membrane separated by the periplasm. To import iron, essential for growth, bacteria secrete small iron(III) chelators called siderophores. The reabsorption of ferri-siderophore complexes involves specific outer and inner membrane transporters. The iron is delivered either into the bacterial periplasm or cytoplasm depending on the nature of the siderophore and the bacterial species. These systems can be exploited by Trojan horse strategies, using siderophores to deliver drugs either into the bacterial periplasm or cytoplasm. In some ferri-siderophore uptake pathways, the iron is released from the siderophore by a mechanism involving degradation of the chelator; this could be an asset for certain types of Trojan horse strategies using siderophores as vectors.
not affected, uptake of the conjugates across the outer membrane into bacterial periplasm is likely to be efficient. (v) The mechanisms of iron release from the siderophores in the bacterial periplasm and cytoplasm often involve hydrolysis of the siderophore (the case for enterobactin and salmochelin).11,12 This is potentially useful for vectorization strategies involving the degradation of the vector to generate or release a siderophore-free drug inside the bacterium. The bottleneck step in these Trojan horse strategies for Gram-negative bacteria is the uptake of siderophore−antibiotic conjugates across the bacterial inner membrane. Large molecules crossing the inner membrane use either ABC transporters or proton-motive-dependent permeases.3 Most siderophore−antibiotic conjugates developed so far are retained in the periplasm: they are not transported further in across the inner membrane. It is still unclear how to design siderophore− antibiotic conjugates able to cross this barrier. Consequently, most successful Trojan horse strategies have been based on antibiotics with periplasmic targets, such as β-lactams. Siderophore−antibiotic conjugates with cytoplasmic targets will only become valuable once we understand and can exploit the various molecular mechanisms and structures involved in the translocation of ferri-siderophores from the bacterial cell surface to the cytoplasm. There is an urgent need for progress in this field. In conclusion, bacterial siderophore-dependent iron-uptake pathways are strongly expressed during infections and play a key role during the infection processes. These systems are promising for the import of drugs into bacteria, thereby overcoming the impermeability of the bacterial wall, especially that of Gram-negative bacteria. The development of such Trojan horse strategies requires governments to make appropriate political decisions and invest in the development
to this category of pathogens. This is nicely illustrated by the work published in ref 13 (in this issue): Ghosh et al. made daptomycin (an antibiotic exclusively active against Grampositives) active against a multiresistant Acinetobacter baumanii (a Gram-negative bacterium). Gram-positive bacteria have a single lipid membrane surrounded by a cell wall, which is a thick layer of peptidoglycan and lipoteichoic acid. Gramnegatives have, in addition, a protective outer membrane. Gram-positives are therefore more sensitive than Gramnegative bacteria to antibiotics. (iv) Work with sideromycins and synthetic siderophore− antibiotic conjugates has shown that the antibiotic moiety can be more bulky than the siderophore moiety without seriously impeding uptake of the whole molecule across the outer membrane of Gram-negative bacteria. This was confirmed by the work of Ghosh et al., in which daptomycin is larger and bulkier than the vector fimsbactin. Another example is microcin MccE492 (Figure 1B) produced by Klebsiella pneumonia, where the siderophore transports an 80-amino-acid bactericide peptide into the target bacterial periplasm. The channel formed by the ferri-siderophore outer membrane transporters during the process of uptake across the bacterial outer membrane apparently has a large diameter, and the compound transported does not seem to interact in a specific manner with the transport machinery.9,10 The specific, and critical, step in the transport of sideromycins or siderophore−antibiotic conjugates across bacterial outer membranes is clearly the recognition of the siderophore moiety by the highly specific binding site of the outer membrane transporters. Therefore, for effective siderophore−antibiotic conjugates, it is important to identify where on the siderophore an antibiotic can be linked without affecting its recognition by the membrane transporters. If recognition is C
DOI: 10.1021/acs.jmedchem.7b00554 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Viewpoint
and in Vivo. J. Med. Chem. 2017, DOI: 10.1021/acs.jmedchem.7b00102.
of such new antibiotics. This in turn requires elucidating the molecular and cellular mechanisms involved in these ironuptake mechanisms and thereby facilitating the work of medicinal chemists.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 33 3 68 85 47 19. Fax: 33 3 68 85 48 29. E-mail:
[email protected]. ORCID
Isabelle J. Schalk: 0000-0002-8351-1679 Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Tommasi, R.; Brown, D. G.; Walkup, G. K.; Manchester, J. I.; Miller, A. A. ESKAPEing the Labyrinth of Antibacterial Discovery. Nat. Rev. Drug Discovery 2015, 14 (8), 529−542. (2) Hider, R. C.; Kong, X. Chemistry and Biology of Siderophores. Nat. Prod. Rep. 2010, 27 (5), 637−657. (3) Schalk, I. J.; Guillon, L. Fate of Ferrisiderophores after Import across Bacterial Outer Membranes: Different Iron Release Strategies Are Observed in the Cytoplasm or Periplasm Depending on the Siderophore Pathways. Amino Acids 2013, 44 (5), 1267−1277. (4) Boukhalfa, H.; Crumbliss, A. L. Chemical Aspects Od Siderophore Mediated Iron Transport. BioMetals 2002, 15 (4), 325−339. (5) Schalk, I. J.; Mislin, G. L. A.; Brillet, K. Structure, Function and Binding Selectivity and Stereoselectivity of Siderophore-Iron Outer Membrane Transporters. Curr. Top. Membr. 2012, 69, 37−66. (6) McPherson, C. J.; Aschenbrenner, L. M.; Lacey, B. M.; Fahnoe, K. C.; Lemmon, M. M.; Finegan, S. M.; Tadakamalla, B.; O’Donnell, J. P.; Mueller, J. P.; Tomaras, A. P. Clinically Relevant Gram-Negative Resistance Mechanisms Have No Effect on the Efficacy of MC-1, a Novel Siderophore-Conjugated Monocarbam. Antimicrob. Agents Chemother. 2012, 56 (12), 6334−6342. (7) Page, M. G. P.; Dantier, C.; Desarbre, E. In Vitro Properties of BAL30072, a Novel Siderophore Sulfactam with Activity against Multiresistant Gram-Negative Bacilli. Antimicrob. Agents Chemother. 2010, 54 (6), 2291−2302. (8) Kohira, N.; West, J.; Ito, A.; Ito-Horiyama, T.; Nakamura, R.; Sato, T.; Rittenhouse, S.; Tsuji, M.; Yamano, Y. In Vitro Antimicrobial Activity of a Siderophore Cephalosporin, S-649266, against Enterobacteriaceae Clinical Isolates, Including Carbapenem-Resistant Strains. Antimicrob. Agents Chemother. 2016, 60 (2), 729−734. (9) Schalk, I. J.; Mislin, G. L.; Brillet, K. Structure, Function and Binding Selectivity and Stereoselectivity of Siderophore-Iron Outer Membrane Transporters. Curr. Top. Membr. 2012, 69, 37−66. (10) Schons, V.; Atkinson, R. A.; Dugave, C.; Graff, R.; Mislin, G. L.; Rochet, L.; Hennard, C.; Kieffer, B.; Abdallah, M. A.; Schalk, I. J. The Structure-Activity Relationship of Ferric Pyoverdine Bound to Its Outer Membrane Transporter: Implications for the Mechanism of Iron Uptake. Biochemistry 2005, 44 (43), 14069−14079. (11) Lin, H.; Fischbach, M. A.; Liu, D. R.; Walsh, C. T. In Vitro Characterization of Salmochelin and Enterobactin Trilactone Hydrolases IroD, IroE, and Fes. J. Am. Chem. Soc. 2005, 127 (31), 11075− 11084. (12) Schalk, I. J.; Guillon, L. Fate of Ferrisiderophores after Import across Bacterial Outer Membranes: Different Iron Release Strategies Are Observed in the Cytoplasm or Periplasm Depending on the Siderophore Pathways. Amino Acids 2013, 44 (5), 1267−1277. (13) Ghosh, M.; Miller, P. A.; Mollmann, U.; Claypool, W. D.; Schroeder, V. A.; Wolter, W. R.; Suckow, M.; Yu, H.; Li, S.; Huang, W.; Zajicek, J.; Miller, M. J. Targeted Antibiotic Delivery: Selective Siderophore Conjugation with Daptomycin Confers Potent Activity against Multidrug Resistant Acinetobacter baumannii Both in Vitro D
DOI: 10.1021/acs.jmedchem.7b00554 J. Med. Chem. XXXX, XXX, XXX−XXX
