Dual-locked chemical reporters based on cephalosporin to detect drug-resistant bacteria for research and diagnosis
Keywords: Humanities and Sciences.
Chemistry.

Optical imaging offers real-time, non-invasive detection of biomolecules in cells, tissues, and whole organisms. Numerous 'always-on' probes may target or bind biomarkers to correlate signals with molecular states or biological activities. However, since they continuously emit signals regardless of their interactions with the targets, the signal specificity is unsatisfied, and the signal-to-background ratio is low. Probes that fluoresce or bioluminescence only when they interact with a biomarker of interest have a higher signal-to-background ratio and lower limit of detection, allowing to improve optical imaging capabilities in vitro and in vivo. Nevertheless, this one-to-one molecular design may still give 'false positive' signals owing to off-target effect in a complex biological environment. Alternatively, dual-locked probes are particularly intriguing due to their enhanced specificity and multiplex detection capabilities. Dual-locked probes are a unimolecular molecule that respond to two distinct inputs with one or more outputs. In contrast to simple single-locked probes, these probes are selectively activated by the correct combination and sequence of triggers. In this way, they can improve the contrast and generate better sensitivity and specificity. Throughout my doctoral studies, I have endeavored to develop chemical reporters leveraging the dual-lock strategy to detect antibiotic-resistant bacteria from clinical samples. One of the major mechanisms by which these bacteria confer resistance to β-lactam antibiotics is the production of β-lactamase enzymes. Here, I describe the multidisciplinary efforts in the development of dual-locked and activatable probes to detect drug-resistant bacteria based on the molecular scaffold of cephalosporins, a class of β-lactam antibiotics. Chapter 1 details the design, synthesis, and assessment of a dual targets-locked fluorogenic probe (CDG-Tre) that permits fluorescence labeling of single living Bacillus Calmette-Guérin (BCG) cells inside macrophages. CDG-Tre (1.8) labeling necessitates both signal activation by BlaC, a β-lactamase expressed by Mycobacterium Tubercolusis (Mtb), and signal retention by the Ag85s-mediated trehalose metabolic pathway. We performed enzymatic, bacterial, and cellular studies to demonstrate that our dual-targets (BlaC and Ag85s) locked strategy offers greater selectivity for mycobacteria over other clinically relevant Corynebacterineae suborder species and enables single live bacteria labeling inside macrophages. Chapter 2 investigates the dual-signal lock strategy to design highly sensitive bioluminogenic probes. The thesis first discusses the construction of the dual-signal locked probe (D-Bluco) by attaching a dabcyl quencher to a single-locked biolumilogeic probe based on a β-Lactamase responsive cephalosporin structure and a luciferin moiety. The quencher is hypothesized to further suppress the background emission of the single-locked system and improve the sensitivity of the probe. Its performance was compared with a single-locked probe without a quencher. The quenching process of D-Bluco and its sensitivity in detecting bacteria that express β-lactamases were then evaluated. Chapter 3 presents the development of Raman probes to detect the activity of extended-spectrum β-lactamases (ESBLs). This dualplex Raman system composed of two Raman probes for target recognition and gold nanostar for amplification of high-quality Raman signals. These two Raman porbes, R1G and R3G, response only in the presence of 1st (broad-spectrum β-lactamases) and 3rd generation cephalosporins (extended-spectrum β-lactamases), respectively. The design and synthesis the Raman probes and their ability to differentiate between broad-spectrum and ESBL β-lactamases were discussed