Description du sujet de thèse

Mycobacteria comprise numerous species, including strict, slow-growing pathogens such as Mycobacterium tuberculosis (Mtb), M. leprae or M. marinum, and opportunistic pathogens, either slow-growing, such as M. avium or M. kansaii, or fast-growing, such as M. abscessus (Mab).
Mtb is the causative agent of tuberculosis (TB) and claimed an estimated 1.6 million deaths in 2021, a number that has been increasing for the first time in a decade, as the result of the COVID-19 pandemic. In the case of non-resistant Mtb, current therapy involves a two-month treatment with four drugs (isoniazid, rifampicin, pyrazinamide and ethambutol) followed by a four-month regimen with isoniazid and rifampicin. Because of treatment duration and secondary effects, strict observance is not always met which favours the emergence of resistance. The proportion of multidrug resistant TB (MDR-TB), defined as being resistant to both isoniazid and rifampicin, in patients previously treated for TB is 20 %, but may exceed 50 % in certain countries. XDR-TB is defined as MDR-TB which is also resistant to at least one drug among fluoroquinilones, bedaquiline and linezolid. In such cases, treatment may require up to 20 months and up to seven drugs administered simultaneously. The rate of success in treatment of MDR-TB, although improving, remains below 60 %. In the case of XDR-TB, success rate can be as low as 22 %. The drugs used as first line treatment of TB are old: the most recent, rifampicine, is in use since 1963. Only three new molecules have been introduced in the clinic to combat Mtb in the last 60 years: bedaquiline in 2012, delamanid in 2014 and pretomanid in 2019. However, resistance has already emerged. There is a real need for new treatments in order to reduce the incidence of TB.
Mycobacterium abscessus (Mab) is a deleterious opportunistic pathogen commonly found in adult cystic fibrosis patients and also involved in nosocomial soft tissues and skin infections. Currently, treatment of Mab infections involves macrolides (azithromycin or clarithromycin), often combined to various antibiotics (amikacin, carbapenems…) depending on the sensitivity profile of the strain. These treatments are often ineffective and Mab's intrinsic and acquired resistance to many antibiotics has earned the bacterium with the title of “antibiotic nightmare”. As for all mycobacteria, this intrinsic resistance is in part attributed to the mycolic acid-containing cell envelope, which is present in all mycobacteria. In the case of Mab, this intrinsic resistance is exacerbated by the presence of multiple antibiotic- and target-modifying enzymes. As a result, active drugs on Mtb are mostly ineffective in the case of Mab. Moreover, the drug discovery pipeline is scarcely populated and most of the compounds currently investigated are actually repurposed compounds or reformulation of existing drugs. Even more so than in the case of Mtb, new drugs are urgently needed to handle the increased incidence of Mab infections for fragile patients (immunocompromised, cystic fibrosis) as well as for the general population.
InhA, an enzyme of the FAS-II system
Mab and Mtb have in common essential enzymes belonging to the FAS-II system required for the biosynthesis of mycolic acids, which represent privileged targets for the discovery of new anti-TB drugs. Among these essential enzymes, InhA catalyzes the reduction of trans-2-enoyl-ACP substrates and is the primary target of the first-line anti-TB drug INH. INH acts as a prodrug requiring activation by the catalase-peroxidase KatG. Mutations in the katG gene and in the promotor region of inhA are the major causes for INH resistance. Different classes of direct inhibitors of Mtb-InhA, requiring no prior activation by KatG, have been discovered, which include (in alphabetic order): benzothiophenes, diazaborines, pyridomycins, pyridones (notably the 4-hydroxy-2-pyridone NITD-916 lead compound, see below), pyrrolidine carboxamides, thiadiazoles, and triclosan (TCL) derivatives. However, none of these compounds has been approved for clinical use so far. Fragment-based drug discovery methods have also been used and led to the discovery of a nanomolar inhibitor of Mtb-InhA, but disappointingly without any activity against Mtb. Therefore, there is currently a need to identify new inhibitors that are active on the pathogens.
Phosphopantetheinyl transferases are relevant drug targets
Phosphopantetheinyl transferases (PPTases) catalyse the transfer of the 4'-phosphopantetheinyl (Ppant) group of coenzyme A (CoA) to a conserved serine residue of the carrier protein (CP) domain of fatty acid synthases (FAS), polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS). These megasynthases, which can exist as large multidomain polypeptides (type I) or as an assembly of distinct proteins (type II), are responsible for the biosynthesis of a broad range of lipids and complex organic compounds. The transfer of Ppant to the CP domain allows the substrate to be attached by a thioester bond and shuttled to the different catalytic sites of the synthases. This is a mandatory activation step for all FAS, PKS and NRPS. PPTases are classified in three families: PPTases from family I, generally named AcpS, primarily activate FAS-I systems, while those from family II activate secondary metabolite biosynthetic pathways and FAS-II. PPTases from family III are actually domains in yeast and fungi FAS-I megasynthases.

In Mtb, two FAS and close to twenty PKS encoding genes were identified. They are involved in the synthesis of mycolic acids and of many other lipidic compounds found in the mycobacterium cell envelope, such as sulpholipids, di- and polyacil- trehaloses, phenolglycolipids. Besides their important role in the structuration of the cell envelope, many of these compounds, such as mycolic acids, trehalose dimycolate, phthiocerol dimycocerosate, were shown to be essential to the pathogenicity of Mtb. On the other hand, Mab contains 2 FAS systems and at least 15 genes encoding PKS or NRPS. Some of these genes are involved in the biosynthesis of mycolic acids or cell envelope lipids found in a wide variety of non-tuberculous mycobacterial species, such as glycopeptidolipids, trehalose pholyphleates or lipooligosaccharides. Mtb genome encodes two PPTases, one being dedicated to the FAS-I system (AcpS), while the other is responsible for the activation of FAS-II and of all PKS and NRPS (PptT). Thus, PptT has an essential role in the biosynthesis of major cell wall components and lipid virulence factors in Mtb. Our collaborators at IPBS have indeed demonstrated the essentiality of PptT for in vitro bacterial growth and for growth and persistence in the mouse model. This hypothesis was confirmed by the discovery of the amido urea compound 8918, a growth inhibitor of Mtb, which was shown to exert its bactericidal effect by binding to PptT.
The genome of Mab also encodes two PPTases, with PptAb being orthologous to PptT. PptT and PptAb share 69% sequence identity. These enzymes are therefore likely to have highly similar role in the biosynthesis of essential cell envelope components and multiple virulence factors. While PptT has been shown to be essential for Mtb growth in vitro and is required for its multiplication and persistence in the mouse infection model, PptAb was found to be necessary for the in vitro growth of Mab (C. Chalut, personal data). Mutations in the gene MAB_3570c, that encodes PptAb, indeed resulted in growth defects. PptT and PptAb are therefore potential therapeutic targets.

THESIS PROJECT
In tight collaboration with the groups of Yves Genisson and Christian Lherbet at the SPCMIB laboratory, we set up two ANR-financed research projects aiming at the design and the characterisation of inhibitors of InhA and PPTases of Mtb and Mab.
In the case of PPTases, a wealth of structural data hace been generated by a crystallographic screening of a fragment library: more than 70 structures of fragment-bound PptAb have been obtained. Based on this knowledge, a PhD student co-supervised by Y. Genisson and L. Maveyraud will design and synthetised evolution of these fragments, with the objective to obtain potent inhibitors of PPTases.
In the case of InhA, in collaboration with our group, the group of C. Lherbet have set up original synthetic approaches in order to explore possible un-exploited binding sites in the active site, the so-called minor portal. A PhD student co-supervised by C. Lherbet and L. Mourey with used the kinetic target-guided synthesis (KTGS) approach to generate new compounds with possible inhibitory activity. In this approach, mixture of reactive fragments is incubated in the presence of the protein, with the goal that the protein binding site will select specific fragment binding, resulting in specific reactions to occur.

In both projects, fragment evolution and fragment selection rely on information provided by structural biology and biophysics. This PhD project aims at providing such information in order to guide chemical synthesis and optimisation.
More specifically, the selected PhD student will have to optimise protein expression, purification and crystallisation in order provide pure proteins for crystallographic and biophysics experiments.
The PhD student will also be responsible for the structural biology part of the project: the obtention of crystals of protein-ligand complexes, data collection at synchrotron, structure determination and refinement. He/she will also be involved in the interpretation of structural information to guide the chemical synthesis.
The last part of the project will involve the characterisation of the protein-ligand interaction using biophysical technologies available at IPBS (spectral shift, TRIC, ITC…) and the determination of the inhibitory properties of the synthetised compounds with respect of the proteins of interest.

Contexte de travail

The successful candidate will join the Structural Biophysics Group, led by Lionel Mourey, at the Institute of Pharmacology and Structural Biology (IPBS, UMR 5089) in Toulouse. The 17 teams at the IPBS are at the forefront of discovering, characterising and exploiting new pharmacological pathways and targets in the fields of cancer, infectious diseases and inflammatory disorders, using molecular and cellular biology approaches, as well as in vivo experiments, including BSL-3-based studies on infectious diseases.
The Structural Biophysics group's activities range from fundamental to applied research in the fields of structural biology, biophysics and screening. Our expertise covers all stages from engineering to biochemical, biophysical and structural characterisation of complex macromolecular assemblies and macromolecule-ligand complexes of great interest to pharmaceutical and biotechnological research.
The project is funded by the ANR and is being carried out as part of a long-standing collaboration with the teams of Y. Génisson and C. Lherbet at the SPCMIB. The thesis will be jointly supervised by Laurent Maveyraud and Virginie Nahoum.

DESIRED PROFILE
Master's degree in structural biology, biophysics or protein biochemistry with knowledge in at least two of the following three areas:
- Protein biochemistry and purification methods
- Biophysical methods for characterising protein-ligand interactions
- X-ray crystallography
Desired practical experience:
- Expression and purification of recombinant proteins in E. coli
- Characterisation of proteins (DLS, DSF) and/or protein-ligand interactions (DSF, nanoDSF, thermophoresis, spectral shift, etc.)
- Protein crystallisation
- Determination of macromolecule structure by X-ray crystallography
Language:
Experienced level (minimum C1) in English and/or French.