Identification of Small Molecules Aborting Resistance to TB prodrugs – SMARt-TB

WP1: “Medicinal chemistry and hit to lead optimization”. In this work-package we focus on the chemical optimization of SMARt molecules. Hits are optimized, in order to improve their potency, their physicochemical properties and their PK profile. Improvement of potency is indeed essential to confirm the target engagement of the compounds in vitro.
In parallel with increasing the potency of the compounds, improvement of their physicochemical and PK properties will be necessary to carry the project up to the in vivo proof of concept.
WP2: “Target discovery and validation”. This part of the program is addressing the essential question of the identification of the targets of the SMARt-PRE molecules. Transcriptomics has already open interesting avenues and hypothesis regarding the mode of action of SMARt-PRE molecules. In a parallel process, SMARt-PRE combined with pretomanid, will be used to select for resistant mutants.
WP3: “In vitro profiling and in vivo proof of concept”. Test will be performed in oxygen and nutrient starvation models of TB. The potential of the boosters/prodrugs combinations will be evaluated in vitro on clinical isolates, including MDR and XDR strains. The mechanism and the frequency of resistance will also be evaluated in vitro. Finally, we will challenge this concept with optimized SMARt-PRE molecules, used in combination with pretomanid, in an acute and in a chronic mouse model of TB.

M. tb strains were used to select for resistant mutants to the Pretomanid-SMARt-PRE combination. A total of eight clones were selected for DNA extraction and whole genome sequencing. RNA sequencing was performed on M. tb treated with SMART-PRE alone or in combination with pretomanid. Pretomanid metabolism in combination with SMARt-PRE was studied by LC-MS/MS in wild type, and resistant M. tb strains. The impact on the bacteria cell-wall is also under investigation. The extended chemical modulation and diversification of the hit SMARt-PRE has been done with the synthesis of more than 150 analogues. The potency of the compounds has been evaluated in combination with Pretomanid on resistant and sensitive strains. The study of the structure-activity relationships has led to compounds with potency in the nanomolar range. Further chemical explorations are still ongoing. Nine compounds have been selected for further profiling.

The biological achievements and the successful medchem optimization in the SMARt-PRE series will be published soon. An in vivo proof of concept is also planned.

Oral communications:
1. Léo Faïon, Aurore Dreneau, Kamel Djaout, Marion Prieri, Rosangela Frita, Catherine Piveteau, Benoit Deprez, Marion Flipo, Alain Baulard, Nicolas Willand; Structure-Activity Relationship Studies Within The First Chemical Family Of Boosters Of The Nitroimidazole Anti-Tb Drug Pretomanid; 4th Drug Discovery Day - From Concept to Scale up, Lille, France,17th Dec 2019.

2. Aurore Dreneau, Léo Faïon, Kamel Djaout, Marion Prieri, Rosangela Frita, Catherine Piveteau, Benoit Deprez, Marion Flipo, Alain Baulard, Nicolas Willand; Discovery, Optimization and Biological Evaluation Of The First Boosters Of The Nitroimidazole Anti-TB Drug Pretomanid, 27th SCT Young Research Fellow Meeting, Caen, France, January 29-31 2020.

Posters:
1. Aurore Dreneau, Léo Faïon, Kamel Djaout, Marion Prieri, Rosangela Frita, Catherine Piveteau, Benoit Deprez, Marion Flipo, Alain Baulard, Nicolas Willand; Discovery, Optimization and Biological Evaluation Of The First Boosters Of The Nitroimidazole Anti-TB Drug Pretomanid, 4th Drug Discovery Day - From Concept to Scale up, Lille, France,17th Dec 2019.

2. Léo Faïon, Aurore Dreneau, Kamel Djaout, Marion Prieri, Rosangela Frita, Catherine Piveteau, Benoit Deprez, Marion Flipo, Alain Baulard, Nicolas Willand; Structure-Activity Relationship Studies Within The First Chemical Family Of Boosters Of The Nitroimidazole Anti-Tb Drug Pretomanid; 27th SCT Young Research Fellow Meeting, Caen, France, January 29-31 2020.

In the past decade, we have seen a dramatic increase worldwide in the number of antibiotic-resistant bacteria, against which the effective therapeutic arsenal is gradually disappearing. Tuberculosis is particularly concerned by the emergence of multidrug-resistant strains. These resistances very often force doctors to prolong the treatment and to adapt the antibiotic cocktail used during treatment, which further delays or even precludes healing. In addition, while strains resistant to all antibiotics are exceptional, they raise concerns of a future TB epidemic with restricted therapeutic options. There is therefore a real medical need for new therapeutic approaches to fight more effectively against resistance. The treatment of tuberculosis is based on the use of a large number of pro-antibiotics including pyrazinamide (PZA), delamanid, and maybe soon pretomanid, which have the particularity of being active against dormant bacilli. Nevertheless, the future of PZA is seriously compromised by the high rate of circulating resistant strains, and resistance to the nitro-imidazoles pretomanid and delamanid is already documented, even though these molecules are still under clinical evaluation.
The SMARt-TB project propose a new way of fighting resistance to PZA and to nitro-imidazoles in tuberculosis, a concept we recently proved possible with SMARt compounds reverting resistance to ethionamide. We have now used an innovative phenotypic screening involving bacteria resistant to PZA and to nitro-imidazoles, and we have identified small molecules called SMARtPZA and SMARtPRE (Small Molecules Aborting Resistance) that allow to revert resistance to these antibiotics. Transcriptomic experiments already allow us to infer the mode of action of SMARtPRE hits, which obviously stimulate an alternative bioactivation pathway of nitro-imidazoles.
The main objectives of the SMARt-TB project are (1) to synthetize highly efficient analogs of the SMARt hits identified by screening, (2) to characterize the molecular basis of the mode of action of our SMARt molecules, (3) to identify the bioactivation process of the pro-antibiotics through the new awakened pathways, (4) to develop "drug-like" lead-molecules to evaluate the therapeutic potential in mouse models infected with PZA- or nitroimidazole- resistant TB strains. Our objectives after 42 months are therefore to identify and validate new bioactivation pathways of PZA and pretomanid, and to allow the development of combinations of drug-like molecules to revert resistance to these two major anti-tuberculosis drugs.
If SMARt-TB confirms further the potential of reprogramming mycobacteria to bio-activate essential prodrugs, this strategy may progressively lead to a new era where resistance can be reverted, and maybe prevented.