Vectorization of iridium(III) complexes using siderophore surrogates : a Trojan horse strategy against Gram-negative pathogenic bacteria – VECTRIUM

The expected Trojan horse conjugates are constituted of a siderophore analogue and an organometallic PS connected through a linker. Organic chemists from Partner 1 will design and synthesize of siderophore vectors-linker blocks. In the present project three siderophore-dependent iron uptake systems will be hijacked by our Trojan horse approach: those depending from pyochelin (Pch), pyoverdine (Pvd), enterobactin (Ent) and desferrioxamin (Dfo). Dfo is a commercialy available siderophore, already functionalized and therefore ready to conjugate with the PS. Pvd will be extracted form culture broth of Pseudomonas, purified and used to produce hemisynthetic conjugates. Finally, partner 1 described previously the synthesis of Pch and Ent analogues able to promote iron accumulation in the bacteria and vectorise xenobiotic into P. aeruginosa. Thus, vectors necessary for the project are easily accessible by adapting protocols reported by partner 1. CuAAC (click chemistry) will be primarily used to conjugate vectors and the ligand of organometallic PS.
Organometallic PS will be synthesized by Partner 2. In a first approach PS activable with blue light will be connected to the siderophore vector. However, blue light could be also deleterious for eucaryotic host cells and the development of PS able to be activated in the red spectrum will be an asset for the project. For this purpose, ligands will be screened to generate metal complexes exhibiting a maximum absorption around the 550 nm region with tail going to 600 nm. Structural modification of the best ligand is envisaged to generate an extra red-shift of the complex absorption wavelength.

Conjugates will be further evaluated for their photophysical and electrochemical properties and will be submitted to UV-visible spectroscopy and cyclic voltammetry to determine the absorption wavelength and consequently, the light source for excitation and the redox potential at the ground state. Then, the full photophysical characterization of the conjugates will also include the measurement of lifetime of the excited state as this parameter is also important value for the optimization of the activity of the PS. Finally, as some PS can be photo-unstable, the robustness of our compounds under irradiation, will also be evaluated in solvent/water mixtures and culture media.
The antibacterial activity (MICs) of Trojan horse conjugates will be assessed by Partner 3 on a panel of pathogenic bacteria in the presence/absence of light by the microdilution method as recommended by international guidelines (CLSI). A comparison of MICs obtained in rich (e.g., Mueller-Hinton, LB) and iron-depleted media will provide useful information on involvement of iron uptake systems. In all these experiments, conjugates will be compared with approved antibiotics used as reference. The ability of conjugates to promote iron acquisition will be confirmed using 55Fe uptake assays by Partner 1.

The target conjugates of our project consist of three elements: the siderophore vector, the metal complex connected by a spacer arm. In a first approach, to validate our hypotheses related to the synthesis, we used deferoxamine (Dfo), a commercial siderophore, inexpensive and already functionalized. We thus obtained the corresponding conjugates. These results validate the synthetic approach used, in particular the use of click chemistry (CuAAC) and made it possible to optimize the final purification of the compounds. In parallel, Partner 1, in collaboration with a team from the University of Hamburg, obtained the three-dimensional structure of the FoxA transporter of P. aeruginosa co-crystallized with a Dfo analogue (Nocardamin). These results should make it possible to conduct SAR studies to optimize the structure of our conjugates.
The first conjugates derived from Dfo have recently been tested for their intrinsic antibacterial activity, i.e. without light, on a panel of pathogenic bacteria. The majority of these molecules have shown activity on Gram-positive bacteria (up to 0.5 µg/ml), which seems independent of the iron concentration in the medium, suggesting siderophore-independent penetration into these pathogens. These molecules have no efficacy on Gram-negative bacteria in the absence of light and at the concentrations tested (= 64 µg/ml). The coming weeks should see the first tests under irradiation (blue LED light), not only on Dfo conjugates but also on the more promising ones based on catecholated siderophores, currently being synthesized. Indeed, analogues of the enterobactin siderophore (Ent) must now be conjugated with the metal complex before they can be tested, alone or in the presence of light. Preliminary work has been initiated in the field of absorption in the red spectrum of organometallic complexes by modification of the metal ligands. Several ligands of the literature have been chosen in order to achieve this goal. Some complexes have shown low solubilities in the usual organic solvents and others have led to the formation of by-products inseparable from the desired complex. Finally, ligands derived from coumarin have made it possible to obtain acceptable solubility properties, studies of photophysical properties are underway and structural modifications are envisaged in order to generate a greater bathochrome effect on the maximum absorption wavelength.

To date the conjugates derived from Dfo have been obtained and those derived from an analogue of the siderophore Ent are being synthesized. The purification of the Pvd and the synthesis of an analogue of the Pch, are in progress and should therefore make it possible to obtain the corresponding conjugates. Pch and Pvd are the two endogenous siderophores of our model organism, P. aeruginosa, and should therefore be the ultimate validation of our strategy at the end of the tests of antibacterial activity in the presence/absence of light. If these results support our strategy, we then want to develop a chemical library of siderophores with different combinations of three bidentate chelating groups in order to satisfy the octahedral hexadentate coordination of Fe(III). Conjugates with corresponding organometallic PS will thus be synthesized and evaluated. The most promising conjugates will then be studied to determine an eventual resistance. The origin and mechanism of these resistances will be also studied. This data is essential to develop conjugates with a longer shelf-life. Finally, for the most interesting conjugates, their toxicities will be evaluated on healthy hepatocytes to evaluate their therapeutic potential. Depending on the results obtained at the end of this process, the promoters of this project will submit the data to the TTO Conectus (Alsace) and apply for a maturation fund to evaluate in vivo the potential of our approach.

Vincent Normant, Inokentijs Josts, Lauriane Kuhn, Quentin Perraud, Sarah Fritsch, Philippe Hammann, Gaëtan L. A. Mislin, Henning Tidow, and Isabelle J. Schalk (2020). Nocardamine-Dependent Iron Uptake in Pseudomonas aeruginosa: Exclusive Involvement of the FoxA Outer Membrane Transporter. ACS Chem. Biol. 15, 10, 2741-2751. doi.org/10.1021/acschembio.0c00535

Pathogenic bacteria are a permanent threat for Humanity even the discovery of antibiotics led to the fade-out of mass epidemies. Nevertheless humanity faces more and more antibiotic-resistant bacterial strains. Thus, the constant development of innovative antibacterial strategies is crucial, especially against Gram-negative bacteria found in many lethal infections. The current antibiotic arsenal is mainly composed of organic compounds when organometallic derivatives appear to be reserved principally to cancer. However, the toxicity profiles of current last resort antibiotics (colistin, polymyxin, oxazolidinones) make now organometallic derivatives competitive in the benefit/risk balance. Moreover some of these organometallic species proved to have the photophysical properties making them potential photosensitizers (PS) useable in antibacterial photodynamic therapy (PDT). In this context iridium(III) complexes are promising candidates. However, these chemical entities are not able to cross the bacterial envelope of Gram-negative bacteria to reach their intracellular biological targets. Bacterial iron uptake systems are gates through the envelope. Siderophores are small iron(III) chelating molecules secreted by bacteria to promote iron acquisition. Siderophores are able to use bacterial iron uptake systems to cross bacterial membrane reaching therefore bacterial inner space. Siderophores could be thus used as vectors to promote accumulation of PS based on iridium(III) into Gram-negative bacteria using a so called Trojan horse strategy. The recognition of the ferric-siderophore by specific outer membrane transporters will lead to the uptake of the conjugate inside the bacterium. This approach should, at one and at the same time, increase the penetration of organometallic species and reduce the peripheral toxicity of vectorized metals for host cells. Vectors to be used in our approach will be analogs of enterobactin, ferrichrome and pyochelin, three siderophores commonly used by Gram-negative pathogens considered to be critical (WHO list). These vectors will be synthesized by Partner 1 (Dr. Gaëtan MISLIN, UMR7242, Illkirch) and conjugated to Ir(III) complexes prepared by partner 2 (Dr. Sylvain GAILLARD, ENSICaen, Caen). Partner 1 will assess the antibacterial activities (MIC) of the siderophore-Ir(III) conjugates in the presence, and in the absence, of light, on Pseudomonas aeruginosa (planktonic and biofilms). This pathogenic bacterium, responsible for serious infections, is known to be poorly permeable to many xenobiotics. A comparison of results obtained in rich and iron-depleted media will give an overview of metal conjugates activities under various physiological conditions and will provide useful informations on involvement of iron uptake systems. Iron acquisition is completely different in humans and in bacteria: iron homeostasis does not require the presence of siderophores. Actually, eukaryotic membranes are poorly permeable to the ferric siderophores selected in our approach. Thus, metal conjugates are not expected to promote intracellular penetration, and toxicity, of iridium(III) in human cells. To evaluate this point more promising compounds will be tested on a human lung fibroblast infection model in order to evaluate both the cytotoxicity of the conjugates and the robustness of our antibacterial strategy in the presence of eukaryotic cells. Finally, the conception of a perennial therapeutic approach must include a study of the mechanisms potentially involved in the emergence of resistance. To this end, partner 3 (Pr Patrick PLESIAT, Besançon) will investigate the strategies developed by bacteria resistant to our approach. Furthermore this partner also depends on the National Reference Center on Resistances and could enlarge the screening to a wide collection of bacteria including antibiotic-resistant strains and clinical isolates.