Siderophore–antibiotic conjugates: exploiting iron uptake to deliver drugs into bacteria

  • I.J. Schalk
    Correspondence
    Corresponding author. I. J. Schalk, UMR 7242, ESBS, Blvd Sébastien Brant, BP 10412, F-67413, Illkirch, Strasbourg, France.
    Affiliations
    Université de Strasbourg, UMR7242, ESBS, Bld Sébastien Brant, F-67413 Illkirch, Strasbourg, France

    CNRS, UMR7242, ESBS, Bld Sébastien Brant, F-67413 Illkirch, Strasbourg, France
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Open ArchivePublished:April 09, 2018DOI:https://doi.org/10.1016/j.cmi.2018.03.037

      Graphical abstract

      The growing problem of bacterial antibiotic resistance makes the discovery and development of new antibiotics increasingly urgent. Otherwise, we will be forced back to a time when common infections were potentially life-threatening. Growing bacterial antibiotic resistance will also affect other fields of medicine: for example, rendering simple surgical procedures or cancer chemotherapy problematic. This problem is the most pronounced for Gram-negative bacteria such as Acinetobacter, Pseudomonas, and various members of the Enterobacteriaceae. The number of new antibiotics currently in development is insufficient to combat this growing threat.
      One of the main hurdles for antibiotics against Gram-negative bacteria is the need for the drugs to cross the outer membrane. A currently proposed strategy to bypass the impermeability of the Gram-negative cell wall is the use of nutrient-import transporters to transport the antibiotics. The most often proposed pathways are the siderophore-dependent pathways for the uptake of iron, a metal essential for bacterial growth. Siderophores are small organic chelators (molecular weight 200–2000 Da) synthesized by bacteria to scavenge iron from their environment and transport it to the bacterial cytoplasm via specific uptake pathways that involve specific outer-membrane transporters in Gram-negative bacteria [
      • Hider R.
      Siderophore mediated absorption of iron.
      ,
      • Schalk I.J.
      • Mislin G.L.
      • Brillet K.
      Structure, function and binding selectivity and stereoselectivity of siderophore-iron outer membrane transporters.
      ]. Antibiotics can be covalently linked to siderophores [
      • Klahn P.
      • Brönstrup M.
      Bifunctional antimicrobial conjugates and hybrid antimicrobials.
      ,
      • Mislin G.L.
      • Schalk I.J.
      Siderophore-dependent iron uptake systems as gates for antibiotic Trojan horse strategies against Pseudomonas aeruginosa.
      ] and, following iron scavenging, the siderophore–antibiotic hybrid is recognized by bacterial ferri-siderophore uptake pathways. The antibiotic uses the siderophore as a Trojan horse to exploit ferri-siderophore uptake transporters to gain entry into the bacteria. Thus, each time a siderophore transports an iron ion, it also transports an antibiotic molecule.
      When fighting for resources, microorganisms also produce siderophore–antimicrobial hybrids (called sideromycins or microcins) to compete with other species and dominate a given niche. Examples of natural sideromycins are albomycins, ferrimycins, danomycins, and salmycins, isolated mainly from streptomycetes or actinomycetes [
      • Mislin G.L.
      • Schalk I.J.
      Siderophore-dependent iron uptake systems as gates for antibiotic Trojan horse strategies against Pseudomonas aeruginosa.
      ]. Microcins (mostly isolated from enteric bacteria) are ribosomally synthesized antibiotic peptides that undergo a posttranslational maturation process, including the addition of a siderophore moiety.
      Given the potent antibiotic properties of natural sideromycins, several academic laboratories and pharmaceutical companies have developed siderophore–antibiotic hybrids. β-Lactam antibiotics, especially cephalosporins, have been the most widely used carriers for siderophore conjugates, but antibiotics from other classes—such as spiramycin, vancomycin, and norfloxacin—have also been used [
      • Klahn P.
      • Brönstrup M.
      Bifunctional antimicrobial conjugates and hybrid antimicrobials.
      ,
      • Mislin G.L.
      • Schalk I.J.
      Siderophore-dependent iron uptake systems as gates for antibiotic Trojan horse strategies against Pseudomonas aeruginosa.
      ]. In many cases, the activity of the siderophore–antibiotic conjugates in vitro on Gram-negative bacteria was higher than that of the same non-modified antibiotics under bacterial growth conditions of iron deprivation. Moreover, a cephalosporin S-649266, also called cefiderocol (developed by Shionogi and GSK) and using such a Trojan horse strategy to enter bacteria, is now in clinical development [
      • Saisho Y.
      • Katsube T.
      • White S.
      • Fukase H.
      • Shimada J.
      Pharmacokinetics, safety, and tolerability of cefiderocol, a novel siderophore cephalosporin for Gram-negative bacteria, in healthy subjects.
      ]. In the future, it may be interesting to apply this strategy to narrow-spectrum antibiotics specific to Gram-positive bacteria to investigate whether their vectorization to bypass the outer membrane could extend their actions to Gram-negative bacteria and widen their spectrum. Similarly, many highly promising in vitro inhibitors of key biological processes have been abandoned during development because they were unable to cross bacterial envelopes. A vectorization strategy of such compounds via siderophores could help them to enter bacteria and provide access to a new set of potential antibiotics.
      Current work on bacterial iron acquisition pathways has highlighted several aspects of this antibiotic vectorization by siderophores that make it promising. First, each bacterial species produces and uses specific siderophores, representing a chemical library of more than 500 siderophores with different chemical structures [
      • Hider R.
      Siderophore mediated absorption of iron.
      ]. It will thus be possible to selectively target bacterial species by using appropriately chosen siderophores for vectorizing the antibiotics. Such narrow selectivity may reduce the risk of antibiotic resistance. This large diversity of siderophore chemical structures also constitutes a valuable chemical collection for medicinal chemists to design different, highly specific siderophore–antibiotic conjugates. Furthermore, chemical structures of natural sideromycins or microcins show that the antibiotic portion can be bulkier than the siderophore portion without seriously affecting uptake of the entire molecule across the Gram-negative bacterial outer membrane [
      • Mislin G.L.
      • Schalk I.J.
      Siderophore-dependent iron uptake systems as gates for antibiotic Trojan horse strategies against Pseudomonas aeruginosa.
      ].
      An important element for effective uptake of siderophore–antibiotic conjugates across the outer membrane is to identify where an antibiotic can be linked to the siderophore without affecting its recognition by membrane transporters. More than 40 x-ray structures of ferri-siderophore outer membrane transporters have been resolved, and the design of effective siderophore–antibiotic conjugates for transport across Gram-negative outer membranes is no longer a limiting step; the uptake of the conjugates across the outer membrane into the bacterial periplasm is likely to be efficient as long as recognition is not affected [
      • Schalk I.J.
      • Mislin G.L.
      • Brillet K.
      Structure, function and binding selectivity and stereoselectivity of siderophore-iron outer membrane transporters.
      ]. Moreover, siderophores of Gram-negative bacteria can be classified into two categories: those delivering iron to the bacterial periplasm (which never reaches the cytoplasm) and those delivering iron to the bacterial cytoplasm (crossing both the outer and inner bacterial membranes) [
      • 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.
      ]. It is currently easy to design efficient siderophore–antibiotic compounds that can reach the bacterial periplasm and deliver drugs targeted to elements in this cell compartment. The major bottleneck in current vectorization strategies is the transport of siderophore–antibiotic conjugates across the inner bacterial membrane, which involves either ABC transporters or proton-motive-dependent permeases. The design of siderophore–antibiotic conjugates that can cross this barrier is still challenging, because of a lack of knowledge concerning the structures of inner membrane transporters, the mechanisms of channel formation, and the molecules involved. Thus, most successful siderophore-dependent vectorization strategies are based on antibiotics with periplasmic targets, such as β-lactams. Finally, the mechanisms of iron release from siderophores in the bacterial periplasm or cytoplasm often involve hydrolysis of the siderophore [
      • 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.
      ]. This is potentially an interesting point for vectorization strategies, which can be used to generate a siderophore-free drug inside the bacterium. Indeed, the presence of the siderophore portion may potentially inhibit the effective interaction of the drug with its target.
      Another important point to highlight in the development of antibiotic vectorization by siderophores is the competition that may exist between conjugates and native siderophores produced by the bacteria [
      • Gasser V.
      • Baco E.
      • Cunrath O.
      • August P.S.
      • Perraud Q.
      • Zill N.
      • et al.
      Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore–antibiotic conjugates development.
      ]. Such competition could contribute to a decreased efficacy of vectorized antibiotics in vivo as shown for a monobactam compound [
      • Tomaras A.P.
      • Crandon J.L.
      • McPherson C.J.
      • Banevicius M.A.
      • Finegan S.M.
      • Irvine R.L.
      • et al.
      Adaptation-based resistance to siderophore-conjugated antibacterial agents by Pseudomonas aeruginosa.
      ]. Different studies have underlined that the use of a strong iron chelator like a tricatechol increases significantly the chance of the conjugates to be competitive with the native siderophores for iron [
      • Gasser V.
      • Baco E.
      • Cunrath O.
      • August P.S.
      • Perraud Q.
      • Zill N.
      • et al.
      Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore–antibiotic conjugates development.
      ]. To more accurately evaluate the importance of native siderophores on conjugates' antibiotic activities, it is also important to use appropriate and different in vitro model systems to effectively recapitulate the host environment in order to properly assess the strength and potential antibiotic activities of siderophore conjugates [
      • Tomaras A.P.
      • Crandon J.L.
      • McPherson C.J.
      • Banevicius M.A.
      • Finegan S.M.
      • Irvine R.L.
      • et al.
      Adaptation-based resistance to siderophore-conjugated antibacterial agents by Pseudomonas aeruginosa.
      ].
      Siderophore–antibiotic compounds may also encounter bacterial resistance mechanisms, as shown for the monocarbam SMC-3176 for which a rapid adaptive resistance preventing entry via the siderophore-mediated iron uptake systems has been observed in Pseudomonas aeruginosa strains [
      • Kim A.
      • Kutschke A.
      • Ehmann D.E.
      • Patey S.A.
      • Crandon J.L.
      • Gorseth E.
      • et al.
      Pharmacodynamic profiling of a siderophore-conjugated monocarbam in Pseudomonas aeruginosa: assessing the risk for resistance and attenuated efficacy.
      ]. Indeed, bacteria often use several iron-uptake pathways and can switch between them to favour the most efficient, depending on the bacterial environment. Consequently, there is also an urgent need to investigate the molecular mechanisms involved in the regulation of expression of the various bacterial iron-uptake pathways to better understand and identify the factors that lead bacteria to switch between them, as they could also be interesting targets for new drugs. A better understanding of the mechanisms of interaction between siderophore and siderophore–antibiotic conjugates and their corresponding membrane transporters will also allow the design and synthesis of siderophore–antibiotic conjugates that target several siderophore membrane transporters from different iron-uptake pathways, reducing potential resistance due to the changing of iron-uptake pathways.
      In conclusion, successful siderophore-based vectorization strategies will require better knowledge of bacterial iron homeostasis in microbial communities, the mechanisms of interaction between siderophores and transporters, and the mechanisms and dynamics of transport across membranes. The development of such a strategy and its success will not be possible without substantial investment in fundamental research to better design siderophore–antibiotic hybrids.

      Transparency declaration

      Dr Schalk reports grants from Innovative Medicines Joint Undertaking under Grant Agreement N° 115525 through financial contributions from the European Union’s Seventh Framework Programme ( FP7/2007-2013 ) and contributions in kind from EFPIA companies (Basilea, GlaxoSmithKline, Sanofi, AstraZeneca), grants from Vaincre la Mucoviscidose ( RF20160501642 ) (French associations against cystic fibrosis), grants from Association Grégory Lemarchal ( RF20160501642 ) (French associations against cystic fibrosis), from Roche Pharmaceutical Research and Early Development Basel via the Roche Postdoctoral Fellowship (RPF) Program during the conduct of the study.

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