Periodic Reporting for period 2 - MepAnti (Exploiting the methylerythritol phosphate pathway as a source of drug targets for novel anti-infectives)
Berichtszeitraum: 2022-04-01 bis 2024-09-30
“Exploiting the methylerythritol phosphate pathway as a source of drug targets for novel anti-infectives” focuses on the anti-microbial resistance (AMR) problem. It is a unique network across Europe that combines leading scientists from academia and industry with the aim to train the next generation of scientists in the anti-infective drug-discovery field. Bacteria are expected to be the next global killer, but despite the awareness of the potential danger from AMR bacteria, few drug-makers have addressed this growing concern and very few new developments in antibiotic portfolios from pharmaceutical companies have been achieved. Therefore, common bacterial pathogens will continue to develop resistance to antibiotics. The MepAnti project aims to tackle this global problem by using underexplored targets that will afford compounds endowed with a novel mode of action (MoA). In fact, the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is entirely absent in humans, while it is essential for medically relevant pathogens (e.g. Plasmodium falciparum, Mycobacterium tuberculosis, Escherichia coli, Pseudomonas aeruginosa and other Gram-negative bacteria). This pathway has been already validated as a potential source of novel anti-infective agents, but to date, only a few inhibitors have been reported. The current ITN program is organized into three different work packages (WP 1–3) that despite being thematically different, are complementary and overlap with each other. Specifically, WP 1 aims to elucidate the structural features of the constituent enzymes of the MEP pathway and to confirm the binding mode of inhibitors through complex crystal structures of inhibitors and proteins. The main objective of WP 2 is the design and synthesis of potent and selective inhibitors with concomitant multiparameter optimization. Subsequently, the compounds synthesized in WP 2 will be tested and further validated in relevant pathogens in WP 3. This collaborative network with an intersectoral composition will enable the identification of new promising compounds to combat the global problem of emerging resistance.
To conclude, the project has achieved several significant outcomes:
- High-resolution structures were solved for key MEP pathway enzymes from pathogenic organisms. This enabled the identification of unique binding sites for selective inhibition.
- Design and synthesis of multiple novel inhibitors with promising in vitro activity against pathogens. The best candidates showed potential for further development.
- Mechanistic studies have revealed innovative modes of action, such as irreversible inhibition, enhancing the compounds' potential for therapeutic application.
Besides the scientific advancements, MepAnti has equipped the next generation of researchers with multidisciplinary skills in structural biology, medicinal chemistry, and microbiology.
As a next step, the identified inhibitors will be further optimized relative to potency, selectivity, and pharmacokinetics, to ultimately develop clinical candidates.
To conclude, the project has achieved several significant outcomes:
- High-resolution structures were solved for key MEP pathway enzymes from pathogenic organisms. This enabled the identification of unique binding sites for selective inhibition.
- Design and synthesis of multiple novel inhibitors with promising in vitro activity against pathogens. The best candidates showed potential for further development.
- Mechanistic studies have revealed innovative modes of action, such as irreversible inhibition, enhancing the compounds' potential for therapeutic application.
Besides the scientific advancements, MepAnti has equipped the next generation of researchers with multidisciplinary skills in structural biology, medicinal chemistry, and microbiology.
As a next step, the identified inhibitors will be further optimized relative to potency, selectivity, and pharmacokinetics, to ultimately develop clinical candidates.
The work performed in WP 1 led to solving the crystal structures of most of the MEP pathway enzymes from several homologues. This includes the Mycobacterium tuberculosis (Mtb) DXPS, Plasmodium falciparum (Pf) DXPS, Pseudomonas aeruginosa (Pa) IspD, Klebsiella pneumoniae (Kp) IspD, PaIspE, Aquifex aeolicus (Aa) IspE, PaIspF, PaIspH, MtbIspH, and Escherichia coli (Ec) IspH. Additionally, several co-crystallization trials with inhibitors and substrates have been done resulting in complex structures of inhibitors with MtbDXR and PaDXPS while AaIspE was solved in complex with ATP. Optimum conditions for protein expression, purification and crystallization were established for several targets.
The drug-discovery process was initiated using different approaches: structure-, ligand- and fragment-based drug design; classical structure–activity relationship (SAR) studies and also more innovative named protein-templated techniques (i.e. dynamic combinatorial chemistry and kinetic target-guided synthesis). This use of a unique combination of various hit-identification strategies increased the chances of finding successful candidates in a short time. Therefore, each ESR in WP 2, built a structurally diverse collection of hits using a platform of various hit-identification strategies. Currently, we have promising inhibitors for each of the MEP pathway enzymes in hand that are undergoing multiparameter optimization en route to the clinic. Patent applications will be filed once the clear frontrunner for each target has emerged.
WP 3 is continuously monitoring the in vitro and whole-cell activities of the compounds synthesised by the ESR of WP2. In the case of ESR 10 and ESR 11 that work with Mycobacterium tuberculosis and Plasmodium falciparum, both have been trained to work with bacterial pathogens under BSL3 conditions. ESR 10 which is working with M. tuberculosis got the approval to work with human primary cells and, infection experiments with Mtb-infected primary human macrophages were successfully started to determine the activity against intracellular mycobacteria. In parallel to the in vitro testing, ESR 11 established the isopentenyl diphosphate (IDP) rescue assay in P. falciparum to prove the target engagement of the most promising MEP inhibitors.
Progress was also achieved in the proteomic approaches that will help target the identification of the most potent compounds.
The key results, including high-resolution structures, validated inhibitors, and novel chemical scaffolds, will provide a solid foundation for the development of clinical candidates targeting AMR pathogens. The intersectoral collaborations will ensure that the outcomes are transferable to both academic and industrial settings.
Several dissemination activities have been used to reach the scientific community including publications in high-impact journals and presentations at international conferences. In addition, MepAnti engaged in several outreach activities such as Famelab competition, newspaper articles and open days at universities, which helped to raise public awareness about AMR and the importance of anti-infective research.
The drug-discovery process was initiated using different approaches: structure-, ligand- and fragment-based drug design; classical structure–activity relationship (SAR) studies and also more innovative named protein-templated techniques (i.e. dynamic combinatorial chemistry and kinetic target-guided synthesis). This use of a unique combination of various hit-identification strategies increased the chances of finding successful candidates in a short time. Therefore, each ESR in WP 2, built a structurally diverse collection of hits using a platform of various hit-identification strategies. Currently, we have promising inhibitors for each of the MEP pathway enzymes in hand that are undergoing multiparameter optimization en route to the clinic. Patent applications will be filed once the clear frontrunner for each target has emerged.
WP 3 is continuously monitoring the in vitro and whole-cell activities of the compounds synthesised by the ESR of WP2. In the case of ESR 10 and ESR 11 that work with Mycobacterium tuberculosis and Plasmodium falciparum, both have been trained to work with bacterial pathogens under BSL3 conditions. ESR 10 which is working with M. tuberculosis got the approval to work with human primary cells and, infection experiments with Mtb-infected primary human macrophages were successfully started to determine the activity against intracellular mycobacteria. In parallel to the in vitro testing, ESR 11 established the isopentenyl diphosphate (IDP) rescue assay in P. falciparum to prove the target engagement of the most promising MEP inhibitors.
Progress was also achieved in the proteomic approaches that will help target the identification of the most potent compounds.
The key results, including high-resolution structures, validated inhibitors, and novel chemical scaffolds, will provide a solid foundation for the development of clinical candidates targeting AMR pathogens. The intersectoral collaborations will ensure that the outcomes are transferable to both academic and industrial settings.
Several dissemination activities have been used to reach the scientific community including publications in high-impact journals and presentations at international conferences. In addition, MepAnti engaged in several outreach activities such as Famelab competition, newspaper articles and open days at universities, which helped to raise public awareness about AMR and the importance of anti-infective research.
This research program goes beyond the state-of-the-art since it tackles attractive and underexplored targets from the MEP pathway in a unique way. The interdisciplinary scientific network is focusing on i) structural features of the constituted enzymes ii) design and synthesis of potent and selective inhibitors iii) cell-based testing against important pathogens with in vitro ADME-T profiling. Ultimately, the knowledge around the MEP pathway will be enhanced and compounds to bridge the problem known as “the translational gap” will be obtained. The final clinical candidate(s) will constitute a breakthrough in the anti-infective field.