Generierung bizyklischer Peptide als aktivitätsbasierte Sonden für Proteasen aus Mycobacterium tuberculosis mittels Phagen-Display und ihre Anwendung in der in vivo Bildgebung
Biochemie
Parasitologie und Biologie der Erreger tropischer Infektionskrankheiten
Zusammenfassung der Projektergebnisse
A major clinical need for the management of bacterial infections, such as tuberculosis, is the development of imaging agents, e.g. for non-invasive monitoring of a treatment response. The aim of this project was to design chemical probes for hydrolytic enzymes from bacterial pathogens such as Mycobacterium tuberculosis (Mtb) and Staphylococcus. aureus that could be translated into non-invasive imaging agents for bacterial infections. Initially, we aimed to develop activity-based probes against virulence-associated M. tuberculosis proteases (Hip1, MarP) that are based on a bicyclic peptide (BCP) scaffold selected by phage display. Activitybased probes are small molecules that irreversibly bind to the active site of target enzymes thus labeling targets with an integrated tag, e.g. a radioisotope for PET-imaging, or a fluorophore for optical imaging. In order to be suitable for imaging applications, probes are required to be highly selective for their bacterial targets. The phage-display dependent selection of BCPs had previously been shown to generate highly potent and selective binders to individual serine proteases. In this work, we modified traditional selection protocols to include a reactive electrophile into the small molecule linker used for BCP cyclization. This modification would allow for the enrichment of BCPs that irreversibly bind to the active site of a target protease. However, pilot experiments indicated that for the chosen targets this strategy would not be able to identify BCPs with the inhibitory potency required for the design of chemical probes. We therefore pursued an alternative target-based strategy employing a combination of substrate profiling and library screening for small molecule inhibitors to guide the rational design of chemical probes for the Mtb protease ‘Hydrolase important for pathogenesis 1’ (Hip1, also known as Rv2224c). Using a variety of substrate profiling methods we identified natural and non-natural amino acid sequences preferred by Hip1 enabling the design of a highly potent and selective Hip1 reporter substrate that produces a Hip1-dependent fluorescence signal in live M. tuberculosis and M. tuberculosis-infected macrophages. Work is ongoing to translate this probe into a quenched fluorescent substrate suitable for in vivo optical imaging of tuberculosis in mice. Furthermore, we screened a chemical library of ~500 serine-reactive small molecules in an enzymatic Hip1 assay identifying a non-selective chloroisocoumarin-based inhibitor. Optimization of this scaffold using the Hip1 selectivity data allowed us to develop a Hip1-selective irreversible inhibitor able to block endogenous Hip1 activity in live M. tuberculosis. Given the role of Hip1 in M. tuberculosis virulence, analogs of this inhibitor are being tested for their anti-tuberculosis properties. Furthermore, we are preparing fluorescent and radioactively labelled analogs as Hip1-specific activity-based probes for non-invasive in vivo imaging studies. We complemented this target-based strategy by an unbiased probe discovery approach for bacterial pathogens that would identify suitable probe targets based on their biological accessibility and expression levels in a cell-based chemical screen without the need for target selection a priori. To achieve this goal we established a competitive activity-based protein profiling platform in live S. aureus as a model organism. This approach identified two selective inhibitors against two previously uncharacterized, serine hydrolases from S. aureus. We identified the corresponding targets by mass spectrometry as Sa02844 and putative tributyrin esterase and we were able to translate the irreversible chloroisocoumarin-based Sa02844 inhibitor into Staphylococcus spp.-selective optical probes as assessed by comparative bacterial labeling experiments and fluorescence microscopy in macrophage coculture assays (manuscript in preparation). A near-infrared Sa02844 probe is currently being tested as a non-invasive imaging tool in several mouse models of S. aureus infection. This or derived chemical probes against S. aureus might be of great clinical use for non-invasive imaging of S. aureus biofilms on medical implants, endocarditis, and other infections.
Projektbezogene Publikationen (Auswahl)
- ACS Infect Dis, 2016, 2 (11):807-815, Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis
Lentz CS, Ordonez AA, Kasperkiewicz P, La Greca F, O’Donoghue AJ, Schulze CJ, Powers JC, Craik CS, Drag M, Jain SK, Bogyo M
(Siehe online unter https://doi.org/10.1021/acsinfecdis.6b00092)
