The BLISS project undertook extensive research with the primary goal of discovering BATSIs for combating antimicrobial resistance (AMR). Below is a summary of the scientific activities and key achievements:
Objective 1: Investigate β-Lactamases (KPC-2 and AmpC) as Scaffolds for KTGS
1) Assessment of β-lactamases suitability for KTGS: A systematic analysis of β-lactamases was conducted to evaluate their compatibility with KTGS. Structural and physicochemical characteristics, including active site size and accessibility, were assessed to identify optimal scaffolds for in situ click chemistry.
2) Synthesis of azido-functionalized boronic acids: Six derivatives, including benzyl and acyclic boronic acids, were designed, synthesised, characterised and tested for inhibitory activity against a pool of β-lactamases. A m-substituted benzyl boronic acid demonstrated potent inhibition, validating its role as an effective warhead for KTGS.
3) Development of an alkyne library: A 90-component library of structurally diverse alkynes was created from commercially available compounds and implemented with several molecules prepared by organic synthesis. This library aims to facilitate multicomponent KTGS, enhancing the exploration of chemical space.
4) Optimisation of KTGS reaction conditions: A robust protocol was established, yielding a reproducible amplification coefficient (AC) of ≥3 compared to negative controls, ensuring efficient triazole product formation under optimised conditions.
In this part, it was validated the use of KPC-2 and AmpC as scaffolds for KTGS. Moreover, it was identified a lead azido-functionalized benzyl boronic acid (warhead) with strong inhibitory activity against both enzymes. Eventually, a reliable workflow for KTGS-based inhibitor design was established.
Objective 2: Discover Novel Inhibitors for KPC-2 and AmpC
1) KTGS screening: Triazole-based inhibitors were generated by reacting azido-functionalised boronic acids with alkyne library components using KPC-2 and AmpC as scaffold in multicomponent KTGS experiments.
2) Inhibitors testing: The inhibitor obtained from the KTGS screenings were tested against KPC-2 and AmpC to evaluate their inhibitory potential.
In this section, three novel triazole inhibitors for KPC-2 (values: Ki 0.7–1.5 μM) and five for AmpC, including a lead compound with a of Ki 600 nM were identified through KTGS. These results provided insights into active site constraints of KPC-2 and AmpC, paving the way for improved inhibitor design.
Objective 3: Identify Highly Active BATSI for In Vivo Testing
1) Antibacterial Efficacy Testing: KTGS-derived inhibitors were evaluated using microbroth dilution assays against clinically relevant bacterial strains producing β-lactamases. Compounds were assessed for their ability to reduce the minimum inhibitory concentration (MIC) of β-lactam antibiotics in combination treatments.
It was achieved up to 8-fold MIC reductions for certain KTGS-derived compounds when combined with antibiotics, demonstrating enhanced bacterial inhibition. Also, the functional inhibition of β-lactamases by KTGS-derived inhibitors was confirmed, validating the approach as a viable tool for BATSI discovery.
This research demonstrated the potential of KTGS to generate novel BATSIs targeting KPC-2 and AmpC. While in vitro no inhibitors surpassed the baseline activity of the warheads under current conditions, substantial progress was made in establishing KTGS as a resource-efficient platform.
