Periodic Reporting for period 1 - NovInDXS (Development of novel inhibitors of the anti-infective target DXS using dynamic combinatorial chemistry (DCC))
Reporting period: 2018-03-01 to 2020-02-29
Antimicrobial resistance (AMR) is emerging as a serious threat to global public health as bacteria develop new resistance mechanisms and extremely drug-resistant strains are emerging. The current slow speed of drug development and disinvestment of the pharmaceutical industry in the field of anti-infectives is aggravating this problem. In this alarming situation, innovative strategies for drug development and novel targets for which inhibitors with an unprecedented mode of action can be developed are urgently required.
NovInDXS aims at the development of selective and potent inhibitors of the important and underexplored anti-infective target 1 deoxy d xylulose-5-phosphate synthase (DXS), an enzyme from the 2C-methyl-d-erythritol 4-phosphate pathway (MEP pathway) that is absent in humans but is essential for medically relevant pathogens. Despite substantial efforts dedicated to the discovery of inhibitors for DXS, to date, very few active compounds have been reported and none of them fulfils the requirements as an ideal candidate for further development.
To address these issues NovInDXS used target-directed dynamic combinatorial chemistry (tdDCC) for the identification as well as for the optimisation of new inhibitors of DXS. This new application of tdDCC exhibits a potential of identifying potent hits in less time compared to the traditional medicinal chemistry approach.
NovInDXS aims at the development of selective and potent inhibitors of the important and underexplored anti-infective target 1 deoxy d xylulose-5-phosphate synthase (DXS), an enzyme from the 2C-methyl-d-erythritol 4-phosphate pathway (MEP pathway) that is absent in humans but is essential for medically relevant pathogens. Despite substantial efforts dedicated to the discovery of inhibitors for DXS, to date, very few active compounds have been reported and none of them fulfils the requirements as an ideal candidate for further development.
To address these issues NovInDXS used target-directed dynamic combinatorial chemistry (tdDCC) for the identification as well as for the optimisation of new inhibitors of DXS. This new application of tdDCC exhibits a potential of identifying potent hits in less time compared to the traditional medicinal chemistry approach.
The NovInDXS project started with establishing the first DCC experiment for identification of novel ligands for the anti-infective target DXS. For this study, we used acylhydrazone formation as a reversible reaction of choice and the DXS enzyme from Deinococcus radiodurans (drDXS) as a target protein.
To maximise the chances of finding potent ligands for DXS, we decided to use heterocyclic building blocks for acylhydrazone formation in DCC, as they are fundamental structural motifs in a plethora of bioactive compounds. The initial choice of heterocyclic building blocks was based on their structural similarities with the existing hits and/or with the cofactor thiamine diphosphate (ThDP) of the enzyme DXS. The molecular docking study of all possible acylhydrazones in the active site of the enzyme drDXS suggested that these products are accommodated in the active site of the enzyme drDXS.
We performed an adaptive tdDCC experiment (DCC-1) by reacting several aldehydes with hydrazides in the presence (protein-templated) and absence (blank) of the target drDXS. The comparison of these tdDCC experiments revealed the amplification of five acylhydrazone products in the protein-templated DCC. These five hits showed moderate binding affinity (KD) ranging from 55−270 μM for the enzyme drDXS and 50−152 μM for the truncated homologue from Mycobacterium tuberculosis dmtDXS. Instead of a traditional medicinal-chemistry approach, we decided to employ tdDCC to optimise these hits. We assumed that using the common structural motifs from the first hits as building blocks in the second round of tdDCC (DCC-2), along with new aldehyde and/or hydrazide counterparts would provide better chances of identifying improved hits. Using this innovative approach we were able to identify five new hits with improved binding affinities (KD, = 7–150 μM for drDXS and 10−110 μM for dmtDXS). To further support our hypothesis that ‘tdDCC can be used for the optimisation of inhibitors/hits’, we performed the third round of tdDCC (DCC-3 and DCC-4) using the same approach. We aimed at studying to some extent the traditional medicinal chemistry approach driven by structure−activity relationships, by including a range of heterocyclic building blocks to cover a wide chemical space with good structural diversity. On the contrary to the traditional medicinal chemistry approach, where all possible acylhydrazone products from these three DCLs should be synthesised and tested for their biological activity, we let the protein select its best binders and synthesised only the amplified derivatives. When tested the eleven hits from these tailored libraries for their binding affinities for drDXS and dmtDXS, most of the hits show substantial improvements in binding affinity compared to the hits from the first and second round of tdDCC experiments. Five hits showed single-digit micromolar affinities (2–8 μM) for drDXS. Similarly, two hits showed 6 and 7 μM binding affinity for dmtDXS. Furthermore, we tested these hits for their enzymatic activity against drDXS and selected hits against Mycobacterium tuberculosis DXS, as well as evaluated their antibacterial activity against Escherichia coli TolC. We observed significant improvement in enzymatic activity of hits from IC50 value of 51 ± 3 μM in the first round of DCC to IC50 = 34 ± 4 μM in the third round of DCC, and antibacterial-activity from no inhibition in the first round of DCC to MIC value of 14 ± 4 μM in the third round of DCC.
We also confirmed the binding mode of the identified hits by molecular docking, competition assays with cofactor and substrates, and competition DCC experiments with cofactor and known substrate inhibitor. These findings suggest that most of the identified hits from DCL-1 are cofactor ThDP and substrate(s) competitors.
These results are published recently in the preprint [R. P. Jumde, et al, https://doi.org/10.26434/chemrxiv.12681761.v1](opens in new window) and submitted for publication in an internationally renowned peer-reviewed scientific journal.
To maximise the chances of finding potent ligands for DXS, we decided to use heterocyclic building blocks for acylhydrazone formation in DCC, as they are fundamental structural motifs in a plethora of bioactive compounds. The initial choice of heterocyclic building blocks was based on their structural similarities with the existing hits and/or with the cofactor thiamine diphosphate (ThDP) of the enzyme DXS. The molecular docking study of all possible acylhydrazones in the active site of the enzyme drDXS suggested that these products are accommodated in the active site of the enzyme drDXS.
We performed an adaptive tdDCC experiment (DCC-1) by reacting several aldehydes with hydrazides in the presence (protein-templated) and absence (blank) of the target drDXS. The comparison of these tdDCC experiments revealed the amplification of five acylhydrazone products in the protein-templated DCC. These five hits showed moderate binding affinity (KD) ranging from 55−270 μM for the enzyme drDXS and 50−152 μM for the truncated homologue from Mycobacterium tuberculosis dmtDXS. Instead of a traditional medicinal-chemistry approach, we decided to employ tdDCC to optimise these hits. We assumed that using the common structural motifs from the first hits as building blocks in the second round of tdDCC (DCC-2), along with new aldehyde and/or hydrazide counterparts would provide better chances of identifying improved hits. Using this innovative approach we were able to identify five new hits with improved binding affinities (KD, = 7–150 μM for drDXS and 10−110 μM for dmtDXS). To further support our hypothesis that ‘tdDCC can be used for the optimisation of inhibitors/hits’, we performed the third round of tdDCC (DCC-3 and DCC-4) using the same approach. We aimed at studying to some extent the traditional medicinal chemistry approach driven by structure−activity relationships, by including a range of heterocyclic building blocks to cover a wide chemical space with good structural diversity. On the contrary to the traditional medicinal chemistry approach, where all possible acylhydrazone products from these three DCLs should be synthesised and tested for their biological activity, we let the protein select its best binders and synthesised only the amplified derivatives. When tested the eleven hits from these tailored libraries for their binding affinities for drDXS and dmtDXS, most of the hits show substantial improvements in binding affinity compared to the hits from the first and second round of tdDCC experiments. Five hits showed single-digit micromolar affinities (2–8 μM) for drDXS. Similarly, two hits showed 6 and 7 μM binding affinity for dmtDXS. Furthermore, we tested these hits for their enzymatic activity against drDXS and selected hits against Mycobacterium tuberculosis DXS, as well as evaluated their antibacterial activity against Escherichia coli TolC. We observed significant improvement in enzymatic activity of hits from IC50 value of 51 ± 3 μM in the first round of DCC to IC50 = 34 ± 4 μM in the third round of DCC, and antibacterial-activity from no inhibition in the first round of DCC to MIC value of 14 ± 4 μM in the third round of DCC.
We also confirmed the binding mode of the identified hits by molecular docking, competition assays with cofactor and substrates, and competition DCC experiments with cofactor and known substrate inhibitor. These findings suggest that most of the identified hits from DCL-1 are cofactor ThDP and substrate(s) competitors.
These results are published recently in the preprint [R. P. Jumde, et al, https://doi.org/10.26434/chemrxiv.12681761.v1](opens in new window) and submitted for publication in an internationally renowned peer-reviewed scientific journal.
Although tdDCC has been successfully employed as a hit-identification strategy for few known targets, its potential has never been tested for challenging targets like the TPP-dependent enzyme DXS, for which limited potent inhibitors have been identified. Importantly, developing inhibitors for this underexplored target can help in finding urgently needed new anti-infectives with a novel mode of action. As highlighted above, the use of (chiral)heterocycles in combination with DCC maximised the chances of identifying hits of enzyme DXS in this project. The use of tdDCC in drug discovery has been limited to the initial hit-identification process. NovInDXS demonstrated an unprecedented use of tdDCC technique for identification as well as subsequent optimisation of novel inhibitors of anti-infective target DXS. Along with the molecular docking and competition assays, we further exploited tdDCC technique to identify the mode of inhibition of identified inhibitors in NovInDXS.
The NovInDXS, along with the development of the researcher, also targets socio-economic benefits of the European Union. The results obtained in the NovInDXS project is highly important for the healthcare industries and the researchers working on developing new anti-infectives. The European research community and industry working on this topic will significantly benefit from the outcome of the NovInDXS research, which is highlighted in ChemRxiv. The new strategies developed during this project can find application in other medicinal chemistry projects. Overall, some of the identified new heterocyclic inhibitors of DXS in NovInDXS may constitute a suitable starting point for developing pre-clinical candidates to be further developed as potential new and relatively non-toxic anti-infective drugs.
The NovInDXS, along with the development of the researcher, also targets socio-economic benefits of the European Union. The results obtained in the NovInDXS project is highly important for the healthcare industries and the researchers working on developing new anti-infectives. The European research community and industry working on this topic will significantly benefit from the outcome of the NovInDXS research, which is highlighted in ChemRxiv. The new strategies developed during this project can find application in other medicinal chemistry projects. Overall, some of the identified new heterocyclic inhibitors of DXS in NovInDXS may constitute a suitable starting point for developing pre-clinical candidates to be further developed as potential new and relatively non-toxic anti-infective drugs.