Synthesis of an Antimicrobial Enterobactin-Muraymycin Conjugate for Improved Activity Against Gram-Negative Bacteria

Overcoming increasing antibiotic resistance requires the development of novel antibacterial agents that address new targets in bacterial cells. Naturally occurring nucleoside antibiotics (such as muraymycins) inhibit the bacterial membrane protein MraY, a clinically unexploited essential enzyme in peptidoglycan (cell wall) biosynthesis. Even though a range of synthetic muraymycin analogues has already been reported, they generally suffer from limited cellular uptake and a lack of activity against Gram-negative bacteria. We herein report an approach to overcome these hurdles: a synthetic muraymycin analogue has been conjugated to a siderophore, i. e. the enterobactin derivative EntKL, to increase the cellular uptake into Gram-negative bacteria. The resultant conjugate showed significantly improved antibacterial activity against an efflux-deficient E. coli strain, thus providing a proof-ofconcept of this novel approach and a starting point for the future optimisation of such conjugates towards potent agents against Gram-negative pathogens

Entwicklung von artifiziellen Siderophoren, antibakteriellen Wirkstoffen und Siderophor-Wirkstoff-Konjugaten gegen pathogene Bakterien

The growing number of pathogenic bacteria, resistant to commonly used antibiotics, lead to a serious threat to global health systems. Especially, Gram-negative pathogens provide big challenges in drug development due to the effective permeation barrier provided by their complex cell envelope. As a result, many potential drugs displaying an antibacterial activity against Gram-positive bacteria remain inactive against Gram-negative bacteria, although their biological targets are generally present. A smart approach to tackle this issue is based on the conjugation of antibiotics to siderophores to form siderophore-drug conjugates. Siderophores are small molecule iron chelators, possessing an active uptake mechanism over the cell envelope of Gram-negative bacteria and thereby acting as carrier for the translocation of antimicrobial drugs across the bacterial cell envelope. In the context of this work, (AcO)EntKL 147, which is an artificial analogue of the naturally occurring tris-catechol siderophore enterobactin 10, was synthesized via total synthesis and utilized for the formation of siderophore-cargo and siderophore-drug conjugates. In growth recovery assays under iron-limiting conditions, it was demonstrated that (AcO)EntKL 147 and a series of corresponding cargo-conjugates is capable to mediate iron-uptake into the cells of the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. These findings were further substantiated by fluorescent labeling experiments followed by confocal fluorescence microscopy with bacterial cultures of E. coli and P. aeruginosa utilizing siderophore-dye conjugates based on (AcO)EntKL 147. In a next step, it was shown that (AcO)EntKL 147 can also function as carrier in a siderophore-drug conjugate to either activate the antibacterial activity of the attached antibiotic against the Gram-negative bacteria E. coli or P. aeruginosa, respectively, or to enhance the activity compared to the parent drug. In the course of this work a total of 14 siderophore-drug conjugates based on (AcO)EntKL 147 were synthesized while six of them provided a significant antimicrobial activity against E. coli and/or P. aeruginosa and four of them exhibited an increased activity against at least one of the tested strains compared to parent antibiotic or enabled a turn-on in antimicrobial activity. Even if these results are preliminary as the total biological evaluation about the efficacy of the drug conjugates has not been finished yet, the gained information further encourages for closer investigation on enterobactin-drug conjugates based on (AcO)EntKL 147. A second subproject dealt with the semi-synthetic modification of the antituberculous agent griselimycin (GM) 329 in order to improve its activity against different mycobacteria and its pharmacokinetic properties. Therefore, the side chain of GM 329 was cleaved under acidic conditions to generate the cyclopeptide A 330 which was utilized as building block for the formation of GM-derivatives with modified side chains. By biological evaluation of the synthesized compounds, several structural motives improving the antimicrobial activity, the pharmacokinetic properties and significantly reducing a known off-target toxicity were identified. However, griselimycin 329 shows a reduced metabolic stability as well as a reduced antibacterial activity against different mycobacteria compared to its derivatives methylgriselimycin 470 and cyclohexylgriselimycin 471. Therefore, beneficial side chains found in the course of this work should be incorporated in the corresponding methylgriselimycin and cyclohexylgriselimycin derivatives via total synthesis. The total synthesis of these compounds was not part of this work but was conducted by my colleague Dr. Elmira Ghabraie. Consequently, the data obtained through this study provided a basis for the development of novel methyl- and cyclohexylgriselimycin derivatives which are potentially integrated in improved drug regimen against tuberculosis. A third subproject covered the modification of the antifungal polyene macrolide amphotericin B 540 to enable the formation of antifungal antibody conjugates. Accordingly, the amphotericin-B-derivatives AMP-PEG4-N3 544 and AMP Lys PEG4 N3 545 were synthesized. Following biological evaluation revealed that AMP-PEG4-N3 544 has lost its antifungal properties while the evaluation of AMP Lys PEG4 N3 545 is still pending. If AMP Lys PEG4 N3 545 maintains the biological activity of amphotericin B 540, it will be used as precursor for an antifungal antibody conjugate.Die wachsende Zahl pathogener Bakterien, die gegen gängige Antibiotika resistent sind, stellt eine ernste Bedrohung für die globalen Gesundheitssysteme dar. Vor allem Gram-negative Pathogene stellen aufgrund der wirksamen Permeationsbarriere, die durch ihre komplexe Zellhülle gebildet wird, eine große Herausforderung für die Wirkstoffentwicklung dar. Diese hat zur Folge, dass viele potenzielle Wirkstoffe, die eine antibakterielle Wirkung gegen Gram-positive Bakterien aufweisen, gegen Gram-negative Bakterien inaktiv bleiben, obwohl ihre biologischen Zielstrukturen im Allgemeinen vorhanden sind. Ein intelligenter Ansatz zur Lösung dieses Problems basiert auf der Konjugation von Antibiotika mit Siderophoren, um Siderophor-Wirkstoff-Konjugate zu bilden. Siderophore sind niedermolekulare Eisen-Chelatoren, die einen aktiven Aufnahmemechanismus über die Zellhülle von Gram-negativen Bakterien besitzen und dadurch als Transportmolekül (Carrier) für die Translokation von antimikrobiellen Wirkstoffen in Bakterienzellen fungieren können. Im Rahmen dieser Arbeit wurde (AcO)EntKL 147, ein artifizielles Analogon des natürlich vorkommenden Triscatechol-Siderophors Enterobactin 10, mittels Totalsynthese hergestellt und für die Bildung von Siderophor-Cargo- und Siderophor-Wirkstoff-Konjugaten verwendet. Durch biologische Evaluation mittels Wachstum-Wiederherstellungs-Experimenten in Eisen-limitiertem Medium konnte gezeigt werden, dass (AcO)EntKL 147 und eine Reihe von abgeleiteten Cargo-Konjugaten die Eisenaufnahme in die Zellen der Gram-negativen Bakterien Escherichia coli und Pseudomonas aeruginosa vermitteln können. Diese Ergebnisse wurden durch Fluoreszenzmarkierungsexperimente mit anschließender konfokaler Fluoreszenzmikroskopie an Bakterienkulturen von E. coli und P. aeruginosa unter Verwendung von Siderophor-Farbstoff-Konjugaten auf Basis von (AcO)EntKL 147 weiter untermauert. In einem nächsten Schritt wurde gezeigt, dass (AcO)EntKL 147 auch als Carrier in einem Siderophor-Wirkstoff-Konjugat fungieren kann, um entweder die antibakterielle Aktivität des angehängten Antibiotikums gegen die Gram-negativen Bakterien E. coli bzw. P. aeruginosa zu aktivieren oder die Aktivität im Vergleich zum Ausgangsstoff zu erhöhen. Dafür wurden insgesamt 14 Siderophor-Wirkstoff-Konjugate auf Basis von (AcO)EntKL 147 synthetisiert, von denen sechs eine signifikante antimikrobielle Aktivität gegen E. coli und/oder P. aeruginosa aufwiesen und vier eine erhöhte Aktivität gegen mindestens einen der getesteten Stämme im Vergleich zum Ausgangsstoff zeigten oder einen Turn-on der antimikrobiellen Aktivität ermöglichten. Auch wenn es sich hierbei um vorläufige Ergebnisse handelt, da die vollständige biologische Bewertung der Wirksamkeit der Wirkstoffkonjugate noch nicht abgeschlossen ist, ermutigen die gewonnenen Informationen zu einer genaueren Untersuchung von Enterobactin-Wirkstoff-Konjugaten auf Basis von (AcO)EntKL 147. Ein zweites Teilprojekt befasste sich mit der semi-synthetischen Derivatisierung des Antituberkulosemittels Griselimycin (GM) 329, um dessen Aktivität gegen verschiedene Mykobakterien sowie seine pharmakokinetischen Eigenschaften zu verbessern. Dafür wurde die Seitenkette von GM 329 unter sauren Bedingungen abgespalten, um das Cyclopeptid A 330 zu erzeugen, welches als Baustein für die Bildung von GM-Derivaten mit modifizierten Seitenketten verwendet wurde. Durch biologische Evaluation der hergestellten Verbindungen konnten mehrere Strukturmotive identifiziert werden, welche die antimikrobielle Aktivität und die pharmakokinetischen Eigenschaften verbessern und eine bekannte Off-Target-Toxizität deutlich verringern. Allerdings weist GM 329 im Vergleich zu seinen Derivaten Methylgriselimycin (MGM) 470 und Cyclohexylgriselimycin (CGM) 471 eine geringere metabolische Stabilität sowie eine geringere antibakterielle Aktivität gegen verschiedene Mykobakterien auf. Daher sollten die im Rahmen dieser Arbeit gefundenen vorteilhaften Seitenketten über eine Totalsynthese in die entsprechenden MGM- und CGM-Derivate eingebaut werden. Die Totalsynthese dieser Verbindungen war nicht Teil dieser Arbeit, sondern wurde von meiner Kollegin Dr. Elmira Ghabraie durchgeführt. Die im Rahmen dieser Studie gewonnenen Daten bilden somit die Grundlage für die Entwicklung neuer MGM und CGM-Derivate, die in eine verbesserte Therapie gegen Tuberkulose integriert werden könnten. Ein drittes Teilprojekt befasste sich mit der Modifizierung des antimykotischen Polyenmakrolids Amphotericin B 540, um die Bildung von antimykotischen Antikörperkonjugaten zu ermöglichen. Dementsprechend wurden die Amphotericin-B-Derivate AMP-PEG4-N3 544 und AMP-Lys-PEG4-N3 545 synthetisiert. Die anschließende biologische Bewertung ergab, dass AMP-PEG4-N3 544 seine antimykotischen Eigenschaften verloren hat, während die Evaluation von AMP-Lys-PEG4-N3 545 noch aussteht. Wenn AMP-Lys-PEG4-N3 545 über die biologische Aktivität von Amphotericin B 540 verfügt, wird es als Vorläufer für ein antimykotisches Antikörperkonjugat verwendet werden

Biomimetic Enterobactin Analogue Mediates Iron-Uptake and Cargo Transport into E. coli and P. aeruginosa

The design, synthesis and biological evaluation of the artificial enterobactin analogue EntKL and several fluorophore-conjugates thereof are described. EntKL provides an attachment point for cargos such as fluorophores or antimicrobial payloads. Corresponding conjugates are recognized by outer membrane siderophore receptors of Gram-negative pathogens and retain the natural hydrolyzability of the tris-lactone backbone, known to be key for uptake into the cytosol. Initial density-functional theory (DFT) calculations of the free energies of solvation (ΔG(sol)) and relaxed Fe-O force constants of the corresponding [Fe-EntKL]3- complexes indicated a similar iron binding constant compared to natural enterobactin (Ent). The synthesis of EntKL was achieved via an iterative assembly based on a 3-hydroxylysine building block over 14 steps with an overall yield of 3%. A series of growth recovery assays under iron-limiting conditions with Escherichia coli and Pseudomonas aeruginosa mutant strains that are defective in natural siderophore synthesis revealed a potent concentration-dependent growth promoting effect of EntKL similar to natural Ent. Additionally, four cargo-conjugates differing in molecular size were able to restore growth of E. coli indicating an uptake into the cytosol. P. aeruginosa displayed a stronger uptake promiscuity as six different cargo-conjugates were found to restore growth under iron-limiting conditions. Imaging studies utilizing BODIPYFL-conjugates, demonstrated the ability of EntKL to overcome the Gram-negative outer membrane permeability barrier and thus deliver molecular cargos via the bacterial iron transport machinery of E. coli and P. aeruginosa

Biomimetic enterobactin analogue mediates iron-uptake and cargo transport into E. coli and P. aeruginosa

The design, synthesis and biological evaluation of the artificial enterobactin analogue Ent(KL) and several fluorophore-conjugates thereof are described. Ent(KL) provides an attachment point for cargos such as fluorophores or antimicrobial payloads. Corresponding conjugates are recognized by outer membrane siderophore receptors of Gram-negative pathogens and retain the natural hydrolyzability of the tris-lactone backbone. Initial density-functional theory (DFT) calculations of the free energies of solvation (Delta G(sol)) and relaxed Fe-O force constants of the corresponding [Fe-Ent(KL)](3-) complexes indicated a similar iron binding constant compared to natural enterobactin (Ent). The synthesis of Ent(KL) was achieved via an iterative assembly based on a 3-hydroxylysine building block over 14 steps with an overall yield of 3%. A series of growth recovery assays under iron-limiting conditions with Escherichia coli and Pseudomonas aeruginosa mutant strains that are defective in natural siderophore synthesis revealed a potent concentration-dependent growth promoting effect of Ent(KL) similar to natural Ent. Additionally, four cargo-conjugates differing in molecular size were able to restore growth of E. coli indicating an uptake into the cytosol. P. aeruginosa displayed a stronger uptake promiscuity as six different cargo-conjugates were found to restore growth under iron-limiting conditions. Imaging studies utilizing BODIPYFL-conjugates, demonstrated the ability of Ent(KL) to overcome the Gram-negative outer membrane permeability barrier and thus deliver molecular cargos via the bacterial iron transport machinery of E. coli and P. aeruginosa

Discovery of Aminoratjadone Derivatives as Potent Noncovalent CRM1 Inhibitors

Cancer cells frequently utilize elevated nuclear export to escape tumor suppression and gain proliferative advantage. Chromosome Region Maintenance 1 (CRM1/XPO1) mediates macromolecule nuclear export and plays an important role in tumorigenesis and progression. The clinical approval of its covalent inhibitor KPT-330 (Selinexor) validates the feasibility of targeting CRM1 to treat cancers. Here, we synthesized four aminoratjadone derivatives and found that two of them, KL1 and KL2, are noncovalent CRM1 inhibitors. The two compounds underwent spontaneous hydrolysis in aqueous buffers, and the resulting products were more active against CRM1. High-resolution crystal structures revealed the CRM1-binding mode of these compounds and explained the observed structure–activity relationships. In cells, KL1 and KL2 localized CRM1 in the nuclear periphery and led to depletion of nuclear CRM1, thereby inhibiting the nuclear export and growth of colorectal cancer cells at submicromolar concentrations. This work lays the foundation for further development of aminoratjadone-based noncovalent CRM1 inhibitors

Discovery of Aminoratjadone Derivatives as Potent Noncovalent CRM1 Inhibitors

Cancer cells frequently utilize elevated nuclear export to escape tumor suppression and gain proliferative advantage. Chromosome Region Maintenance 1 (CRM1/XPO1) mediates macromolecule nuclear export and plays an important role in tumorigenesis and progression. The clinical approval of its covalent inhibitor KPT-330 (Selinexor) validates the feasibility of targeting CRM1 to treat cancers. Here, we synthesized four aminoratjadone derivatives and found that two of them, KL1 and KL2, are noncovalent CRM1 inhibitors. The two compounds underwent spontaneous hydrolysis in aqueous buffers, and the resulting products were more active against CRM1. High-resolution crystal structures revealed the CRM1-binding mode of these compounds and explained the observed structure–activity relationships. In cells, KL1 and KL2 localized CRM1 in the nuclear periphery and led to depletion of nuclear CRM1, thereby inhibiting the nuclear export and growth of colorectal cancer cells at submicromolar concentrations. This work lays the foundation for further development of aminoratjadone-based noncovalent CRM1 inhibitors

Perfusion-induced redox differences in cytochrome c oxidase: ATR/FT-IR spectroscopy

AbstractAttenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow-exchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions