Bisubstrate and prodrug approaches for the rational design of new DXR nhibitors: Novel antimicrobial and antitubercular drugs – ANTITUB

We propose to undertake research activities aimed at getting new insights into the development of unexplored types of DXR bisubstrate inhibitors. The design of new bisubstrate analog ligands is an effective strategy to enhance the potency and specificity in enzyme inhibition for certain enzymes. To perform this project, we will employ computational approaches, including molecular modeling and dynamics simulations to characterize the protein/ biligand inhibitor interactions in order to optimize the design and the synthesis of appropriate bisubstrates. In a first approach, we will design bisubstrate inhibitors that simultaneously target the substrate and the NADPH cofactor binding sites. In a second approach, based on in situ fragment-based drug design, we will explore the in situ molecular formation of irreversible bisubstrate inhibitors. Appropriate and complementary bioorthogonal biligands will be designed to exploit in situ click chemistry that employs the target enzyme itself to catalyze the synthesis of a well-suited inhibitor. With the aim of overcoming the microbial bioavailability and mycobacterial impermeability of DXP inhibitors, synthesis of prodrugs of DXR inhibitors will be conceptualized to act as a site-specific drug delivery and to allow the development of mutual prodrugs with synergetic action with or without additional benefit. Compounds will be tested on the isolated E. coli and M. smegmatis DXR and kinetic data will be determined. Growth inhibition will be tested on non-pathogenic bacteria e.g. E. coli and M. smegmatis and on pathogenic bacteria e.g. M. tuberculosis. The antibiotic activity of the synthetic inhibitors on M. tuberculosis-infected macrophages will be investigated by the use of a confocal fluorescence-based imaging method. Moreover, to observe the effects of drug treatment on the cell wall and direct structural change in the cell wall after drug exposure, we will use high-resolution magic angle spinning solid-state NMR spectroscopy.

MD simulations of DXR in complex with DXP, the natural substrate of DXR and FOM inhibitor We carried were carried out using crystal structures available in the Protein Data Bank. From this work, important interactions that were then used to define energetic filters for subsequent docking calculations were identified. From the in silico support, several appropriate and complementary bioorthogonal ligands such as azide and cycloalkynes were designed as well as bi-substrat. A serie of azide compounds linked to the nitrogen atome was synthesized and inhibited E. coli DXR. The synthesis of constrained alkynes being tedious, a range of alkynes tagged analogs of the NADPH cofactor based on docking, was carried out. No DXR inhibition was observed with theses ligands. Except to the triazole bi-substrats synthetized via a click reaction, all the design bi-subtrats were inefficient on DXR. We investigated the possibilities to target a hydrophobic pocket of the DXR active site. Based on the docking a series of commercial compounds has been selected and a series of new bi-substrates has been synthetized. These compounds inhibited the DXR as efficiently as fosmidomycin. Phosphate analog of the fosmidomycin, the fosfoxacin, and its derivatives have been synthesized and tested on the DXR of E.coli and M. smegmatis. The inhibition of the DXR depends on the microorganism DXRs. To shed light on these results, molecular dynamics have been performed. About 40 prodrugs of fosfoxacine derivatives have been synthesized. Very interesting results were observed with 14 prodrugs as they efficiently inhibited the growth of M. smegmatis and P.falciparum. Phosphoramidate prodrugs were also prepared but they are inactive on the growth of both strains probably due to the absence of the essential enzymes for prodrug hydrolysis. To develop and settle the parameter of HR-MAS NMR, experiments have been carried out with intact bacteria before and after treatment with an efficient antitubercular.

Other perspectives, besides the structural and biophysics studies include the continuation of the computational studies of ligand binding by DXR. Many question remain to be address, including more specific mechanistic detail on ligand binding and enzymatic reaction, the role and mechanism of large-scale active-site loop conformations changes.

To study the feasibility the click reactioninside the DXR active site, the synthesis of constrained alkynes has to be pursue. The interesting results obtained by targeting a hydrophobic pocket located in the active site of the DXR, open new perspectives for the design of novel inhibitors of the DXR. To conduct growh inhibition on mycobacteria, all developped compounds should be prepare as prodrugs. To increase the efficiency of therapeutic agents, mutual prodrug and bis-progrug strategiesof DXR inhibitors have to be developped.
Our results open new perspectives in the actual context of resistance. It is therefore urgent to find innovative targets for new antimicrobial drug. Therefore, it would be interesting to test our compounds on bacteria classified by the WHO as pathogenic priority (Shigella dysenteriae, Hélicobacter pylori..).

1- Synthesis and biological evaluation of phosphate isosters of fosmidomycin and analogs as inhibitors of Escherichia coli and Mycobacterium smegmatis 1-deoxyxylulose 5-phosphate reductoisomerases.
Munier M, Tritsch D, Krebs F, Esque J, Hemmerlin A, Rohmer M, Stote RH, Grosdemange-Billiard C.*
Bioorg Med Chem., 2017, 25, 684-689.

2- Toward the design of novel antimicrobials: Synthesis and biological evaluation of phosphate isoster of fosmidomycin and analogs. PharmaMed-2016, International Conference on Medicinal and Pharmaceutical Chemistry,
Dubai (UAE),5-7 décembre 2016,

3- Original approach for the synthesis of deoxy-xylulose phosphate reductoisomerase (DXR) inhibitors: access to bi-ligands. A. Dreneau, D.Tritsch, M. Rohmer & C. Grosdemange-Billiard
53ième Semaine d’Etude en Chimie Organique,
Sulniac, (France), 24 Mai-9Juin 2016

4- Prodrug approach for the rational design of new deoxyxylulose phosphate reducto-isomerase (DXR) inhibitors: potential antitubercular drugs.
M.Munier, D.Tritsch, M. Rohmer and C. Grosdemange-Billiard, 16th Tetrahedron Symposium - Challenges in Bioorganic & Organic Chemistry, Berlin, (Allemegne), 16-19 Juin 2015

5- Molecular dynamic study of ligand recognition by DXR: implication for the design of new antibiotic.
Fanny Krebs, Catherine Grosdemange-Billiard, Roland Stote.
Groupe thématique Enzymes-Groupe Graphisme et Modélisation Moléculaire (GT enzymes-GCMM),
Sète (France) 25-28 Mai 2015

6- From the discovery of a novel isoprenoid biosynthesis pathway to the design of new antimicrobials.
C. Grosdemange-Billiard
BIT’s 5th Annual International Congress of Medchem,
Suzhou (Chine), 18-20 novembre 2014.

Today, almost all important microbial infections throughout the world, such as tuberculosis, malaria, nosocomial diseases, are becoming resistant to antibiotics. Antimicrobial multi-drug resistance has been called one of the world's most pressing public health problems. It is therefore urgent to find innovative targets for new antimicrobial drugs. Proteins involved in isoprenoid biosynthesis represent such targets. Isoprenoids are found in all living organisms and are essential for all bacteria. The alternative mevalonate-independent methylerythritol phosphate (MEP) pathway for the biosynthesis of isoprenoids, which is present in many pathogenic bacteria e.g. Mycobacterium tuberculosis, M. leprae, as well as in opportunistic pathogens e.g. enterobacteria, Acinetobacter spp., Pseudomonas spp., and present in the parasitic Plasmodium species responsible for malaria, but absent in human represents an attractive target for the design and development of new antimicrobials. Accordingly, all enzymes of the MEP pathway represent potential targets for the design of a novel and unexplored types of antibacterial and antiparasitic drugs, with minimal side effects expected for the patient.
The aim of this proposal is to combine the knowledge and expertise of different research groups in biocomputing, enzymology, biochemistry, biophysics and organic synthesis to design and to develop novel types of antimicrobial molecules that inhibit DXR, the second enzyme of the MEP pathway. DXR contains a divalent metal cation such as Mg2+ in its active site and utilizes NADPH as cofactor. We propose to undertake research activities aimed at getting new insight into the development of unexplored types of DXR bisubstrate inhibitors. The design of new bisubstrate analog ligands is an effective strategy to enhance the potency and specificity in enzyme inhibition for certain enzymes. To perform this project, we will employ computational approaches, including molecular modeling and dynamics simulations to characterize the protein/ biligand inhibitor interactions in order to optimize the design and the synthesis of appropriate bisubstrates. Two parallel approaches will be used. First, we will design bisubstrate inhibitors that simultaneously target the substrate and the NADPH cofactor binding sites. Based on in situ fragment-based drug design, we will explore the in situ molecular formation of irreversible bisubstrate inhibitors. Appropriate and complementary bioorthogonal billigands will be designed to exploit in situ click chemistry that employs the target enzyme itself to catalyze the synthesis of a well-suited inhibitor. The second approach is related to microbial bioavailability and mycobacterial impermeability overcoming of DXP inhibitors. Synthesis of prodrugs of DXR inhibitors will be conceptualized with the aim of overcoming those problems towards DXR inhibitors, to act as a site-specific drug delivery and to allow the development of mutual prodrugs with synergetic action with or without additional benefit. Compounds will be tested on the isolated E. coli and M. smegmatis DXRs, and kinetic data will be determined. Growth inhibition will be tested on non-pathogenic bacteria e.g. E. coli and M. smegmatis and on pathogenic bacteria e.g. M. tuberculosis. The antibiotic activity of the synthetic inhibitors on M. tuberculosis-infected macrophages will be investigated through the use of a confocal fluorescence-based imaging method. Moreover, to observe the effects of drug treatment on the cell wall and direct structural change in the cell wall after drug exposure, we will use high-resolution magic angle spinning solid-state NMR spectroscopy.
The generated knowledge will lay on the foundation for the development of novel types of antimicrobial and antitubercular drugs, which are urgently needed to combat the global threat posed by the multi-drug resistance of bacteria and parasitic protozoa.