JCJC SVSE 5 - JCJC - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques

From fragments to inhibitors of MabA (FabG1) a new challenge to treat tuberculosis – 2FightTb

Fragment based approach for the discovery of inhibitors of MabA, an enzyme involved in the biosynthesis of Mycobacterium tuberculosis mycolic acids

Innovative medicinal chemistry approach involving the design and synthesis of low molecular weight molecules, the use of high-throughput assays for measuring enzymatic and bactericidal activities of compounds and ADME profiling

Discovery of the first MabA drug-like inhibitors to propose a new strategy to fight tuberculosis

Tuberculosis, a disease caused by Mycobacterium tuberculosis, causes more than 1.8 million deaths and 10.4 million new cases every year worldwide. The treatment of the disease involves long chemotherapy which causes many side effects, so it is necessary to find new therapeutic solutions. Mycolic acids are fatty acids that play an essential role in the architecture and permeability of the M. tuberculosis cell wall. Mycolic acid biosynthesis machinery, which is the target of several anti-tuberculosis drugs, is an important reservoir of attractive targets. The MabA protein (FabG1), involved in the mycolic acid biosynthesis, has been genetically shown to be essential for M. tuberculosis survival. The objective of the project is used a fragment-based approach to identify the first low-molecular-weight MabA inhibitors and to optimize their physicochemical, pharmacokinetic and pharmacodynamic properties for a proof of concept in mice.

The fragment-based approach represents a new way for the development of small molecules as drug candidates. This approach involves the use of compounds with a limited number of heavy atoms (non hydrogen) and a molecular weight lower than 300 g/mol. These characteristics give to these compounds better physicochemical properties, especially in terms of solubility, compared to lead-like or drug-like compounds from regular compound libraries. Moreover in fragment-based approaches the screening requires a lower number of compounds compared to conventional HTS approaches, and gives a higher success rate for identifying hits and generating lead compounds.
In order to identify molecules capable of inhibiting the MabA enzyme, a new high throughput enzymatic assay based on mass spectrometry has been developed and optimized for the use in 384-well plates. This assay consists in quantifying by LCMSMS the product of the enzymatic reaction in the presence of a potential inhibitor. A fragment-library was tested on the enzyme and the best MabA inhibitors were selected and validated by dose-response experiments. Analogs of these first inhibitors were synthesized and then tested on the enzyme. This allowed us to establish structure-activity relationships. A library of 240 compounds was synthesized in solid phase in order to rapidly increase the structural diversity of the compounds and to improve their solubility and their stability in a biological media. The best inhibitors were tested on M. tuberculosis in order to measure their ability to inhibit bacterial growth.

To identify molecules able to inhibit MabA enzyme, a new high-throughput enzymatic assay based on mass spectrometry was developed in 384-well plates.
Two screenings of 1280 and 2985 compounds were performed using this novel enzymatic assay.
The best inhibitors were selected and validated by dose-response experiments. This led to the identification of the first small-molecular-weight MabA inhibitors.
The syntheses of several original chemical series led to the first structure-activity relationships and the best inhibitors showed IC50s on the enzyme in the micromolar range.
These inhibitors have been tested on Mycobacterium tuberculosis and several compounds are capable of inhibiting bacterial growth.

This medchem project led to the identification of the first inhibitors of MabA (FabG1). These compounds showed good solubility (> 100 µM) and good plasma stability (t1 / 2> 6h) and microsomal stability (t1 / 2> 40 min) but a strong plasma protein binding (> 99.5%) which limits their use for an in vivo proof of concept. Novel pharmacomodulations are underway to increase the enzymatic and bacterial activity of the compounds and to optimize their physicochemical and pharmacokinetic properties. These compounds will be useful for designing new antibacterial therapies and overcoming resistance to existing drugs.

This work led to three publications:

Kussau, T., Flipo, M., Van Wyk, N., Viljoen, A., Olieric, V., Kremer, L., Blaise, M. Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis beta-ketoacyl-acyl carrier protein reductase MabA. Acta crystallographica. Section D, Structural biology,2018, 74(Pt 5): 383-393.

Hugues Prevet, Marion Flipo, Pascal Roussel, Benoit Deprez, Nicolas Willand. Microwave-assisted synthesis of functionalized spirohydantoins as 3-D privileged fragments for scouting the chemical space Tetrahedron Letters 2016, 57, 2888

Ngoc Chau Tran, Heleen Dhondt, Marion Flipo, Benoit Deprez, Nicolas Willand. Synthesis of functionalized 2-isoxazolines as three-dimensional fragments for fragment-based drug discovery. Tetrahedron Letters 2015, 56, 4119.

Nine posters were presented at national and international conferences. Two posters were rewarded with the Best Poster Award at the Young Researchers Days (2016 et 2018).

According to most recent data, tuberculosis (TB), a disease caused by Mycobacterium tuberculosis, accounts for more than 1.7 million deaths and 8.8 million new cases each year worldwide. The treatment of the disease involves chemotherapy, which has to be administered for six months or up to four years for treating multi-drug-resistant strains (MDR-TB). In that context, therapy is often associated with serious side-effects, which reduce patient compliance and thus lead to high rates of recurrence and mortality. Molecules that are active against previously unexploited targets are obviously needed, and recent efforts have led to the approval of Bedaquiline, a selective inhibitor of mycobacterial ATP synthase. However, new drugs with different modes of action are still needed in order to shorten the therapies and stop the emergence of resistance.
Mycolic acids are very long-chain fatty acids playing an essential role in the architecture and permeability of the envelope of M. tuberculosis. The mycolic acid biosynthetic machinery, which is the target of first and second line antitubercular drugs, still represents an important reservoir of attractive targets. The protein called MabA, also named FabG1, is involved in mycolic acids biosynthesis and catalyses the NADPH-specific reduction of long chain beta-ketoacyl derivatives. This enzyme has genetically been shown to be essential for M. tuberculosis survival and it represents a target of choice to start a drug discovery program. The main objectives of the 2FightTb project are to use highly innovative approaches to find and develop new low-molecular-weight inhibitors of MabA. These compounds will be useful to design novel therapies against tuberculosis, to shorten treatment and to overcome drug resistance.
The input of the project is the recent discovery by the principal investigator of a series of small molecular fragments binding to MabA. The goal set out to pursue is the design of drug-like inhibitors of MabA through fragment evolution guided by a variety of complementary assays. To reach the goal, a focussed multidisciplinary consortium that gathers the required expertise and technical resources will be coordinated by the principal investigator. The work plan, inspired from a previous project (Willand et al. Nature Med. 2009, Flipo et al. J. Med. Chem. 2011 and 2012), includes structure-based drug design, guided by X-ray crystallography and in silico modeling, combined with target-based and cell-based activity assays performed at high throughput on the Equipex Imaginex platform and ADME profiling. This project is challenging because it requires a significant work across the boundaries between chemistry, physics and microbiology. However, we strongly believe that is feasible. Indeed, the principal investigator has demonstrated the chemical tractability of MabA through the screening of a fragment library and the consortium has already worked together successfully to produce drug candidates.
At the end of the project we plan to deliver a lead compound that fits the following minimum acceptable profile: a small molecule, soluble in aqueous media, that selectively inhibits MabA, with an IC50 in the nanomolar range, active on sensitive and resistant M. tuberculosis strains with adequate plasma and microsomal stability for in vivo proof of concept.
MabA has not yet been used successfully as a drug target but the goal of the project is worth the personal and financial investment. Indeed, small MabA inhibitors, hitting an unprecedented target should kill sensitive and resistant Mycobacterium tuberculosis strains.

Project coordinator

Madame Marion FLIPO (Institut Pasteur de Lille U761) – marion.flipo@univ-lille.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


IPL Institut Pasteur de Lille U761

Help of the ANR 255,000 euros
Beginning and duration of the scientific project: January 2014 - 42 Months

Useful links

Explorez notre base de projets financés



ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter