Periodic Reporting for period 1 - BLISS (Beta-Lactamase Inhibitors Synthesised through in Situ click chemistry)
Reporting period: 2022-10-16 to 2024-10-15
The BLISS (Beta-Lactamase Inhibitors Synthesised through in Situ click chemistry) project aims to directly address the critical global challenge of antimicrobial resistance (AMR), specifically targeting β-lactamases, enzymes responsible for deactivating β-lactam antibiotics. AMR is a growing threat to public health, as β-lactam antibiotics are among the most widely used and effective treatments for bacterial infections. The production of β-lactamases, particularly serine β-lactamases (SBLs), is the primary mechanism behind the resistance of Gram-negative bacteria to these antibiotics. The development of novel β-lactamase inhibitors (BLIs) is essential to overcoming this resistance and restoring the efficacy of β-lactam antibiotics.
BLISS focuses on the use of Kinetic Target-Guided Synthesis (KTGS), a breakthrough approach, to allow for the rapid and cost-effective discovery of potent and selective boronic acid transition-state inhibitors (BATSIs). In this project, β-lactamases are used as a template to drive the identification of inhibitors directly from a library of reagents, potentially reducing the time and resources typically required in traditional drug discovery methods. This approach enables the generation of BATSIs specifically targeting a range of β-lactamases involved in AMR, with promising efficiency.
This multidisciplinary project aims to achieve key milestones in the discovery of novel BATSIs. Using KTGS, several boronic acid-based inhibitors were identified and demonstrated strong activity against different β-lactamase variants, including KPC-2, a critical enzyme in resistant bacterial strains. These inhibitors exhibited the potential to restore the activity of β-lactam antibiotics, highlighting their promise for further development in AMR treatment strategies. Importantly, the KTGS platform also allowed for the identification of BATSIs with minimal resource use, enhancing the overall cost-effectiveness of the drug development process.
The primary objectives of BLISS are:
1. Investigate the use of KTGS to generate potent BATSIs targeting β-lactamases involved in AMR;
2. Discover novel and effective BLIs capable of restoring the efficacy of β-lactam antibiotics;
The outcomes of BLISS hold the potential to aid future AMR treatment strategies, contributing to an efficient alternative method for discovering novel BLIs and ensuring the continued effectiveness of β-lactam antibiotics. In addition to its scientific impact, BLISS contributes to the European Union strategic goals by addressing the pressing need for novel antimicrobial agents and offering a cost-effective approach to drug discovery, potentially reducing healthcare costs associated with AMR. This work also aligns with broader societal and economic objectives by combating a global health threat and fostering innovation in pharmaceutical development.
BLISS focuses on the use of Kinetic Target-Guided Synthesis (KTGS), a breakthrough approach, to allow for the rapid and cost-effective discovery of potent and selective boronic acid transition-state inhibitors (BATSIs). In this project, β-lactamases are used as a template to drive the identification of inhibitors directly from a library of reagents, potentially reducing the time and resources typically required in traditional drug discovery methods. This approach enables the generation of BATSIs specifically targeting a range of β-lactamases involved in AMR, with promising efficiency.
This multidisciplinary project aims to achieve key milestones in the discovery of novel BATSIs. Using KTGS, several boronic acid-based inhibitors were identified and demonstrated strong activity against different β-lactamase variants, including KPC-2, a critical enzyme in resistant bacterial strains. These inhibitors exhibited the potential to restore the activity of β-lactam antibiotics, highlighting their promise for further development in AMR treatment strategies. Importantly, the KTGS platform also allowed for the identification of BATSIs with minimal resource use, enhancing the overall cost-effectiveness of the drug development process.
The primary objectives of BLISS are:
1. Investigate the use of KTGS to generate potent BATSIs targeting β-lactamases involved in AMR;
2. Discover novel and effective BLIs capable of restoring the efficacy of β-lactam antibiotics;
The outcomes of BLISS hold the potential to aid future AMR treatment strategies, contributing to an efficient alternative method for discovering novel BLIs and ensuring the continued effectiveness of β-lactam antibiotics. In addition to its scientific impact, BLISS contributes to the European Union strategic goals by addressing the pressing need for novel antimicrobial agents and offering a cost-effective approach to drug discovery, potentially reducing healthcare costs associated with AMR. This work also aligns with broader societal and economic objectives by combating a global health threat and fostering innovation in pharmaceutical development.
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.
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.
The BLISS project achieved the first successful application of Kinetic Target-Guided Synthesis (KTGS) for developing β-lactamase inhibitors (BLIs). The study demonstrated that β-lactamases can act as effective scaffolds for in situ click chemistry, enabling the generation of BATSIs. Screening with AmpC and KPC-2 scaffolds led to the identification of eight BLI candidates, including some compounds with inhibitory activity in the nanomolar range. These candidates also displayed encouraging MIC values, indicating potential clinical relevance.
Future research will focus on improving the selectivity of KTGS with β-lactamases as templates and optimising warhead designs to enhance potency and efficacy.
As the first project to explore KTGS with boronic acids for BLIs, BLISS marks a significant step forward in addressing β-lactamase-mediated antibiotic resistance.
In summary, the project has established a strong foundation, and further advancements hold promise for impactful innovations in combating antimicrobial resistance.
Future research will focus on improving the selectivity of KTGS with β-lactamases as templates and optimising warhead designs to enhance potency and efficacy.
As the first project to explore KTGS with boronic acids for BLIs, BLISS marks a significant step forward in addressing β-lactamase-mediated antibiotic resistance.
In summary, the project has established a strong foundation, and further advancements hold promise for impactful innovations in combating antimicrobial resistance.