Periodic Reporting for period 4 - SWEETBULLETS (Sweet Theranostics in Bitter Infections - Seek and Destroy) Reporting period: 2021-08-01 to 2022-09-30 Summary of the context and overall objectives of the project Bacterial infections are now a global threat demanding novel treatments due to the appearance of resistances against antibiotics at a high pace. The ESKAPE pathogens are those with highest importance in the EU and chronic infections due to biofilm formation are a particular task. Noninvasive pathogen-specific imaging of the infected tissue is not clinically available. Its successful implementation will enable the choice of appropriate therapy and boost efficacy. Furthermore, Gram- negative bacteria have a highly protective cellular envelope as an important resistance mechanism for drugs acting intracellularly, resulting in an alarmingly empty drug-pipeline.To overcome this gap, my team and established Lectin-directed Theranostics targeting pathogens via their extracellular carbohydrate-binding proteins at the site of infection for specific imaging and treatment. This was implemented for the highly resistant ESKAPE pathogen Pseudomonas aeruginosa.We established Sweet Imaging: The two lectins of P. aeruginosa, LecA and LecB, are involved in biofilm formation, a hallmark of chronic infections. First, we developed the first covalent LecA inhibitor that was conjugated to a fluorophore for the detection of biofilms. Then, we optimized the detection of P. aeruginosa by non-covalent LecA- and LecB-targeted fluorescent probes as pathogen-specific diagnostic tools. To this end, several probes were developed to target these extracellular lectins and demonstrated to stain P. aeruginosa biofilms in vitro. For LecA an activity boost to low-nanomolar affinity could be achieved by multivalency. In vitro, the divalent LecA-targeting imaging probe accumulated effectively in biofilms under flow conditions. Investigation of divalent LecA- and monovalent LecB-targeting imaging probes in an in vivo murine lung infection model revealed elevated probe accumulation in lungs of infected animals. These findings demonstrate the use of LecA- and LecB-targeting probes for the imaging of P. aeruginosa infections and suggest their potential as pathogen-specific diagnostics. Furthermore, we established Sweet Targeting, which is the delivery of antibiotics to the infection through covalent linking of lectin directing groups. The two extracellular P. aeruginosa-specific lectins LecA and LecB are key structural biofilm components and can be exploited for targeted drug delivery. By employing different antibiotics we assessed their bactericidal potency and targeting efficiency. Fluoroquinolones are effective antibiotics but linked to severe side effects. Biofilm-specific antibiotic delivery could locally increase drug concentration to break antimicrobial resistance and reduce the drug’s peripheral side effects. Two extracellular P. aeruginosa lectins, LecA and LecB, are essential structural components for biofilm formation and thus render a possible anchor for biofilm-targeted drug delivery. The standard-of-care drug ciprofloxacin suffers from severe systemic side effects and was therefore chosen for this approach. We synthesized several ciprofloxacin-carbohydrate conjugates and established a structure-activity relationship. Conjugation of ciprofloxacin to lectin probes enabled biofilm accumulation in vitro, reduced the antibiotic’s cytotoxicity, but also reduced its antibiotic activity against planktonic cells due to a reduced cell permeability and on target activity. This work defined the starting point for new biofilm/lectin-targeted drugs to modulate antibiotic properties and ultimately break antimicrobial resistance. Manufacturing of nano-carriers with surface exposed lectin-directed ligands was also performed that allow the noncovalent charging with antibiotics and avoids the possibly detrimental chemical modification of antibiotics. In vitro targeting was demonstrated using a newly developed flow cell system with immobilized lectins..In a last work package we aimed to develop Sweet SMART Targeting, i.e. conjugates as SMART drugs with a specific release of anti-biofilm lectin inhibitor and drug cargo upon contact with pathogen after the conjugation and development of linkers cleavable by pathogenic enzymes. In this work, several fluoroquinolones were conjugated to lectin probes by cleavable peptide linkers to yield lectin-targeted prodrugs. Mechanistically, these conjugates therefore remain non-toxic in the systemic distribution and will only be activated to kill once they have accumulated at the infection site. The synthesized prodrugs proved stable in presence of host blood plasma and liver metabolism, but rapidly released the antibiotic cargo in presence of P. aeruginosa in a self-destructive manner in vitro. Furthermore, the prodrugs showed good ADME properties and reduced toxicity in vitro, thus establishing the first lectin-targeted antibiotic prodrugs against P. aeruginosa. SWEETBULLETS established thus fundamentally novel lectin-directed theranostics to fight these deleterious infections. In future work, it will be rapidly extendable towards other ESKAPE pathogens, e.g. Klebsiella spp. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far My team and I at HZI have embarked on a systematic evaluation of lectin directing probes, linker types and imaging agents as well as antibiotic cargo. The successful proof of concept of the imaging of Pseudomonas biofilms has been provided and was published (Wagner, S. et al. Angew. Chem. Int. Ed. Engl. 2017). A large set of follow-up imaging agents has been prepared and tested in in vitro biofilm assays. To this end, we also improved the binding potency for for LecA and LecB (Mala et al., J Med Chem 2022, Zahorska et al., EP19306432.6 Zahorska et al, Angew. Chem. Int. Ed. Engl. 2022, Zahorska et al. ChemComm 2020, Sommer et al., J. Med. Chem. 2019, Metelkina et al., unpublished), and transformed these into conjugatable units for imaging and targeting. The imaging has been further improved by implementing a biofilm flow assay with nicely demonstrated the high specificity and robustness of the staining (Zahorska, Denig et al, unpublished). In the context of sweet targeting antibiotics, we prepared a large set of conjugates and analysed the relatively steep SAR of various linkers and lectin-directing groups on antimicrobial activity and demonstrated that the SMART targeting approach is suitable to deliver targeted and higjly active prodrugs (Meiers et al., J Med Chem 2020, Meiers et al., J Med. Chem. 2022, Meiers et al., EP21212989.4). Nanocarriers with lectin-directing groups have been prepared and were studied in a number of assays (Metelkina et al., J Mater Chem B, 2022). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) see above image.jpg
