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Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
A Trojan-Horse Strategy Including a Bacterial Suicide Action for the Efficient Use of a Specific Gram-Positive Antibiotic on Gram-Negative Bacteria Isabelle J. Schalk* Université de Strasbourg, CNRS, UMR7242, ESBS, Boulevard Sébastien Brant, F-67413 Illkirch, Strasbourg, France ABSTRACT: In the alarming context of rising bacterial antibiotic resistance, there is an urgent need to discover new antibiotics or increase and/or enlarge the activity of those currently in use. The need for new antibiotics is even more urgent in the case of Gram-negative bacteria, such as Acinetobacter, Pseudomonas, and Enterobacteria, which have become resistant to many antibiotics and have an outer membrane with very low permeability to drugs. Vectorization of antibiotics using siderophores may be a solution to bypass such a bacterial wall: the drugs use the iron transporters of the outer membrane as gates to enter bacteria in a Trojan-horse strategy. Designing siderophore−antibiotics that can cross outer membranes has become almost routine, but their transport across the inner membrane is still a limiting step, as well as a strategy that allows dissociation of the antibiotic from the siderophore once inside the bacteria. Liu et al. (J. Med. Chem. 2018, DOI: 10.1021/acs.jmedchem.8b00218) report the synthesis of a siderophore−cephalosporin compound and demonstrate that β-lactams, such as cephalosporins, can serve as β-lactamasetriggered releasable linkers to allow intracellular delivery of Gram-positive antibiotics to Gram-negative bacteria.
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increase their activity or render them active against a larger range of pathogens.2,3 The group of M. Miller had the highly original idea of synthesizing a drug conjugate consisting of a siderophore linked to a cephalosporin with an attached oxazolidinone, a specific Gram-positive antibiotic: siderophore−cephalosporin−oxazolidinone conjugate. After uptake of this compound across the outer membrane of Gram-negative bacteria via the ferri-siderophore uptake transporters, the cephalosporin component is rapidly hydrolyzed by periplasmic β-lactamase to release the oxazolidinone, which can then translocate across the inner membrane and reach its target (ribosomes) in the bacterial cytoplasm (Figure 1). The siderophore−cephalosporin−oxazolidinone conjugate is active against clinical isolates of Acinetobacter baumannii (Gramnegative bacterium), as well as strains that produce large amounts of ADC-1 β-lactamase, a bacterial enzyme that provides multiresistance to β-lactam antibiotics, such as penicillins, cephalosporins, cephamycins, and carbapenems. Liu et al. were able to render a specific Gram-positive antibiotic active against Gram-negative bacteria because of a vectorization strategy coupled to an original antibiotic release strategy involving cephalosporin hydrolysis by a periplasmic enzyme of the target bacteria.1 Gram-negative bacteria are often less sensitive to antibiotics because of their outer membrane, which is an efficient permeability barrier against toxic molecules, including many antimicrobials.4 Liu et al. were able to bypass this barrier by using bacterial iron uptake pathways to import the antibiotics across the Gram-negative outer membrane. Iron acquisition by bacteria involves the production of small organic iron chelators called siderophores, which have a very strong affinity for iron.5 These compounds (MW between 200 and 2000 Da) scavenge
acterial antibiotic resistance is currently a major threat to world health and food safety. As this public health problem progresses, it will quickly exhaust our therapeutic options. It may affect every person of any age in any region of the world in the near future. Such antibiotic resistance will also compromise the progress of modern medicine in areas such as organ transplantation, surgery, and chemotherapy. According to the World Health Organization (WHO), if nothing is done, the world will enter a postantibiotic era, in which current infections and minor wounds will be able to kill once again. The WHO also published its first list of “priority pathogenic agents” that are resistant to antibiotics, listing 12 families of the most threatening bacteria for human health. The most critical group contains multiresistant Gram-negative bacteria: Acinetobacter, Pseudomonas, and Enterobacteria. These bacteria have become resistant to many antibiotics, including carbapenems and the third generation of cephalosporins, the best available products to combat multiresistance bacteria. It is urgent and necessary to change the way we use antibiotics, on the one hand, to limit or even reverse the increase of antibiotic resistance and, on the other, to develop new strategies to fight bacteria with new antibiotic compounds. It takes approximately 15 years to develop an antibiotic with an approximate cost of 1.3 billion dollars. In addition, it is not profitable for pharmaceutical companies to develop such drugs because these molecules are used for short-term treatments. Consequently, only one new class of antibiotics has been developed in the past 30 years: the diarylquinolines, drugs treating tuberculosis by targeting mycobacterial ATP synthase. While awaiting the discovery of new classes of antibiotics, it may be important to optimize the activity spectrum of already used compounds. In this context, vectorization of antibiotics is a promising strategy, as illustrated by the work of the team of M. Miller,1 reported in this issue. The aim of such a strategy is to facilitate the entry of currently used or new antibiotics into bacterial cells and thus © XXXX American Chemical Society
Received: April 3, 2018
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DOI: 10.1021/acs.jmedchem.8b00522 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
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literature.2 The literature also clearly reveals that most Trojanhorse conjugates bearing an antibiotic with a cytoplasmic target are only poorly active against Gram-negative bacteria.2 Indeed, the two bottlenecks of antibiotic vectorization by siderophores are, first, the ability to cross the inner membrane and bring the antibiotic into the bacterial cytoplasm and, second, to design an efficient mechanism of intracellular antibiotic dissociation from the siderophore. The inner membrane uptake of ferrisiderophore complexes involves ABC or proton motive forcedependent transporters, for which only a few crystallographic structures are currently available and for which we have very little knowledge concerning their mechanism for the transport of ferri-siderophore complexes.10 The strategy of Liu et al. is elegant and powerful because it provides a solution to the two current bottlenecks of antibiotic vectorization by siderophores. They used cephalosporin as a hydrolyzable linker between the siderophore and the Grampositive antibiotic oxazolidinone. Once this siderophore− cephalosporin−oxazolidinone conjugate has been transported across the outer membrane, its cephalosporinase susceptibility allows release of the oxazolidinone moiety in the bacterial periplasm. The Gram-positive antibiotic trapped in the bacterial periplasm can cross the cytoplasmic membrane, as it does for Gram-positive bacteria, and reach its target in the bacterial cytoplasm. This is an original mechanism of antibiotic dissociation from the siderophore in the bacterial periplasm. The problem of the linker between the siderophore and the antibiotic component has been an obstacle to the development of siderophore−antibiotic conjugates for many years. The ideal linker should be resistant to extracellular conditions and stable during translocation promoted by the iron uptake systems but cleavable in the bacterial inner space. In addition, depending on the antibiotic vectorized, release must occur to avoid steric hindrance during the interaction of the antibiotic with its target to obtain an optimal interaction. Using cephalosporin as a linker and the likelihood that this compound will be hydrolyzed by a β-lactamase from the target bacteria is highly ingenious and singlehandedly solves the two problems of antibiotic vectorization by siderophores: antibiotic release from the siderophore and the ability of the antibiotic to cross the inner membrane. Overall, Liu et al. demonstrate that (i) oxazolidinone antibiotics are active against Gram-negative bacteria when they are efficiently delivered into bacteria, (ii) a Trojan-horse strategy using cephalosporin as a hydrolyzable linker works, and (iii) this strategy can deliver efficient drugs to even highly resistant bacteria, pathogens of major concern worldwide. This strategy can now be used for any Gram-positive antibiotic or drug with its target in the bacterial cytoplasm. Many highly promising in vitro inhibitors of key biological processes have been abandoned during development because they were unable to cross bacterial envelopes. The vectorization strategy proposed by Liu et al. could help them to enter bacteria and provide access to a new set of potential antibiotics.
Figure 1. Siderophore−cephalosporin−oxazolidinone conjugates capable of delivering the specific Gram-positive antibiotic oxazolidinone into Gram-negative bacteria.
iron in the bacterial environment and bring it back to the bacterial periplasm via specific ferri-siderophore TonB-dependent transporters (TBDT) embedded in the outer membranes.6 Siderophores are considered to be virulence factors and are produced along with their corresponding TBDT during infections.7 Liu et al. use a siderophore-dependent vectorization strategy also called a Trojan-horse strategy, in which antibiotics are covalently linked to a siderophore. Thus, the antibiotic is taken up by the bacteria each time an iron ion is transported across the outer membrane. The use of siderophores as vectors for the transport of antibiotics is well-documented in the literature and has even been developed by bacteria themselves to kill other microorganisms, allowing them to dominate a given niche.2,8 Many reviews have highlighted the advantages of such a Trojan-horse strategy.2 The efficiency of such an approach is highly dependent on the use of a strong iron chelator, such as catechol, for the siderophore part, to successfully compete with the natural siderophores of the pathogen for iron chelation.9 It has become relatively easy to design siderophore− antibiotic conjugates that can cross the bacterial outer membrane and reach the bacterial periplasm because of good knowledge about the mechanism of ferri-siderophore interaction with this family of transporters, gained from the large number of available X-ray structures of ferri-siderophore TBDT.6 TBDT are able to carry ferri-siderophore complexes, to which a compound (antibiotics) even larger than the siderophore has been linked, from the bacterial environment to the periplasm.2 Consequently, diverse successful siderophoredependent vectorization strategies for antibiotics, with their target in the bacterial periplasm, have been described in the
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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 B
DOI: 10.1021/acs.jmedchem.8b00522 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
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Notes
The author declares no competing financial interest.
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REFERENCES
(1) Liu, L. R.; Miller, P. A.; Vakulenko, S. B.; Stewart, N. K.; Boggess, W. C.; Miller, M. J. A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria To Commit Suicide with a Gram-Positive Antibiotic. J. Med. Chem. 2018, DOI: 10.1021/acs.jmedchem.8b00218. (2) Mislin, G. L.; Schalk, I. J. Siderophore-Dependent Iron Uptake Systems as Gates for Antibiotic Trojan Horse Strategies against Pseudomonas Aeruginosa. Metallomics: integrated biometal science 2014, 6 (3), 408−420. (3) Górska, A.; Sloderbach, A.; Marszałł, M. P. Siderophore-Drug Complexes: Potential Medicinal Applications of the “Trojan Horse” Strategy. Trends Pharmacol. Sci. 2014, 35 (9), 442−449. (4) Pagès, J.-M.; James, C. E.; Winterhalter, M. The Porin and the Permeating Antibiotic: A Selective Diffusion Barrier in Gram-Negative Bacteria. Nat. Rev. Microbiol. 2008, 6 (12), 893−903. (5) Hider, R. Siderophore Mediated Absorption of Iron. Struct. Bonding (Berlin) 1984, 58, 25. (6) 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. (7) Cornelis, P.; Dingemans, J. Pseudomonas aeruginosa Adapts Its Iron Uptake Strategies in Function of the Type of Infections. Front. Cell. Infect. Microbiol. 2013, 3, 75. (8) Rebuffat, S. Microcins in Action: Amazing Defence Strategies of Enterobacteria. Biochem. Soc. Trans. 2012, 40 (6), 1456−1462. (9) Gasser, V.; Baco, E.; Cunrath, O.; August, P. S.; Perraud, Q.; Zill, N.; Schleberger, C.; Schmidt, A.; Paulen, A.; Bumann, D.; Mislin, G. L.; Schalk, I. J. Catechol Siderophores Repress the Pyochelin Pathway and Activate the Enterobactin Pathway in Pseudomonas Aeruginosa: An Opportunity for Siderophore-Antibiotic Conjugates Development. Environ. Microbiol. 2016, 18 (3), 819−832. (10) 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.
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DOI: 10.1021/acs.jmedchem.8b00522 J. Med. Chem. XXXX, XXX, XXX−XXX
