Thioamide Activation for the Tuberculosis World – Tea-4-Two

The first main task will develop efficient and drug-like boosters either fully specific of the new pathway, or with an inhibitory activity equilibrated on both the new pathway and EthA-R. We will provide medicinal chemistry to transform the initial hit into a lead for in vivo testing and subsequently into a preclinical candidate.
Task 1.1. Step-wise optimization by rational design
Task 1.2. Fragment-based optimization of second generation of boosters
Task 1.3. Inhibitory and binding mode of improved analogues in both series
Task 1.4. DMPK profile
The second main task will address the question of the biological efficacy of the compounds issued from Main Task 1. In the present context, efficacy implies not only the antibacterial activity against M. tuberculosis in vivo and against MDR-XDR strains, but also the capacity of bacteria to develop escape mechanisms.
Task 2.1. Bacterial escape mechanisms
Task 2.2. Activity of the boosters on MDR and XDR strains
Task 2.3. In vivo efficacy of the ETH-boosters combinations on a TB-mice model
The third main task will aim to unravel the bioactivation routes taken by ETH in this new pathway Task 3.1. Genetic manipulations to study EthAR and EthA2R2 pathways independently.
Task 3.2. Bioactivation of ETH by EthA2 detected by HRMAS-NMR and Mass spectroscopy
Task 3.3. Bioactivation of ETH by EthA2 characterize by Mass spectroscopy
Task 3.4. Global impact on cell envelope biosynthesis

1. We have obtained at this point of the project a good understanding of the mode of action of our second-generation compounds. These compounds circumvent the normal bioactivation pathway EthA-R, and thus revert ETH resistance in clinical strain mutated in EthA. We believe that this strategy, allowing a reversion of resistance, is a strong novelty in the history of antibiotherapy. We have submitted in May this collaborative work, which involve all partners of Tea-4-Two (except the NMR team which will be involved in the second step of this work), to a high impact magazine.
2. We have discovered that some compounds may use a third pathway of bioactivation which will be explored in the next period of Tea-4-Two
3. Successful animal experiments show that more than one lead compounds could be pushed in preclinical development. A couple of lead compound identified in this first period is now under pharmacological development. ADME has been done as well as some toxicology studies. This work will certainly opens perspectives to interesting publications.
We have discovered that regulation by EthR induces alternative initiation of transcription, a phenomenon recently observed, but far from being fully understood in bacteria. We think our data will help to progress in this understanding.

The objective of the program proposed hereafter is to validate a new concept of reprogramming anti-Tb drug bioactivation that offer the solution to the problem of EthA mutated strains insensitive to our first generation of boosters. There is no doubt that in case of success of this project, GSK and Bioversy will be open to negociate licencing agreements that will greatly extent the boosting concept of Ethionamide to all TB strains, including MDR, XDR and EthR bacteria. This work is, to our knowledge, the first demonstration of the feasibility of the reversion of acquired resistance in bacteria.

The results of this first part of the project are under review in a high impact journal.
Other related publications: Palladium-free Sonogashira-type cross-coupling reaction of bromoisoxazolines or N-alkoxyimidoyl bromides and alkynes. N.P. Probst, B. Deprez, N. Willand, Tetrahedron Letters, Volume 57, Issue 10, 9 March 2016, Pages 1066–1070
Communications:
A. Baulard. Invited speaker at the Swiss tropical Institute for the annual TbX meeting. Le 27 Mai 2015: Boosting ETH as a new anti-TB therapeutic approach
A. Baulard. Invited Speaker at the «Institut des Sciences de la Vie« of the University of Louvain, 25th of March 2016, Belgium: Resetting innate and acquired
drug resistance in Mycobacterium tuberculosis.
N. Willand. Invited speaker at In silico drug design meeting, 30 Novembre 2015, Villeneuve d'Ascq, France.. New tricks for old drugs: using Fragment-based Drug Discovery and in silico screening for the discovery.
N. Willand. Invited speaker at SCT-DMCCB Joint Meeting, 16 October 2015, Dijon, France. Using Aged Drugs in New Ways, the Concept of Boosting Antibiotics.
N. Willand. Invited speaker at Université de Namur, Belgique, 25 September 2015, New tricks for old antibiotics.
N. Willand. Invited speaker at Duhram University, 25 Février 2015, Boosting antibiotics using transcriptional regulators: how an old drug can become a new drug.
Patents:
COMPOSES DE TYPE SPIROISOXAZOLINE AYANT UNE ACTIVITE POTENTIALISATRICE DE L'ACTIVITE D'UN ANTIBIOTIQUE, 26.06.2014, PCT/EP2013/077706
HÉTÉROCYCLES AZOTÉS SATURÉS ET N-ACYLÉS POTENTIALISANT L'ACTIVITÉ D'UN ANTIBIOTIQUE ACTIF CONTRE LES MYCOBACTÉRIES, 26.06.2014, PCT/EP2013/077732
Spiroisoxazoline Compounds Having an Activity Potentiating the Activity of an Antibiotic, 03.12.2015 US, 28.10.2015 EP
COMPOSES DE TYPE SPIROISOXAZOLINE AYANT UNE ACTIVITE POTENTIALISATRICE DE L'ACTIVITE D'UN ANTIBIOTIQUE, 28.10.2015 EP, 14.09.2015 CR, 30.07.2015 SG

Tuberculosis(TB) is still a scourge of mankind. TB treatment regimen uses old first line drugs, for 6–9 months. Poor patient adherence leads to the repeated exposure of the bacterium to sub-inhibitory drug concentrations that select for resistance. As a result, multiple drug-resistant TB (MDR-TB) strains have now emerged. This has increased the use of 2nd line drugs together with their lower therapeutic index and side effects that undoubtedly favour the emergence of extremely drug resistant TB (XDR-TB). Recent efforts have led to the identification of promising compounds, including the recently conditionally approved bedaquiline and delamanid, but the anti-TB drug pipeline remains insufficiently filled.
An alternative therapeutic strategy. The originality of our innovative approach arises from the peculiar observation that a significant number of anti-TB antibiotics are prodrugs, bioactivated by mycobacterial enzymes. In particular, bioactivation of ethionamide (ETH) is performed by the monooxygenase EthA.
Moreover we discovered that this bioactivation process is not fully exploited as EthA is under the control of the repressor EthR. In 2009, we proposed to inhibit EthR by drug-like molecules to stimulate production of EthA, which will boost ETH bioactivation. Results demonstrated that our lead compound was able to triple the activity of ETH in a TB-infected mice model (Willand et al. 2009 Nature Med.).
Stumbling block. Ethionamide is an essential pillar for the successful treatment of MDR-TB as recently reassessed by a large meta-analysis (Ahuja et al. 2012 PLoS Med.). Unfortunately, the extensively use of ETH associated to its serious side effect at the recommended dose, has led to ETH resistant strains mutated in EthA. Whereas very efficient to boost the efficacy of ETH against drug sensitive and MDR bacteria, our innovative approach was logically ineffective to kill ethA mutated clinical strains.
Reprogramming ETH bioactivation. During our efforts of chemical diversification, we serendipitously identified 2nd generation of boosters awakening a normally silent enzymatic pathway that we proved also able to bioactivate ethionamide, however, independently of EthA. The genetic organization of this pathway is a replica of the EthR-A regulon, as it is composed of a TetR Type regulator (now called EthR2) and of an oxydoreductase (now called EthA2). We have now demonstrated that our 2nd generation of boosters inhibits EthR2, releasing the production of EthA2 to bioactivate ETH. In combination with ETH, our new boosters were shown highly active against all TB strains tested so far, including MDR strains mutated in EthA.
The main objectives of the Tea-4-Two program will be to optimize our second-generation of boosters to develop strong, specific and safe EthR2 inhibitors, which associated to ETH will offer a completely novel therapeutic alternative, either as a first line drug combination, or to treat MDR- and XDR-TB. Efforts will also focussed on the mechanisms of bioactivation of ETH through this new pathway, and on the role of the pathway in the physiology of the bacteria. Escape mechanisms will be explored, not only to decipher the mode of action but also to predict the frequency and the mechanism of resistance in the prospect of clinical applications.
Drug boosting through inactivation of resistance mechanisms has been used for antibiotics in the ß-lactam family. ß-lactams are commonly prescribed for the treatment of many infections in combination with clavulanic acid, an inhibitor of ß-lactamases that are key determinants of ß-lactam resistance. This approach has extended the life of ß-lactams for more than 30 years. The Tea-4-Two approach push forward this concept, as our new boosters, on one hand, circumvent the resistance mechanism developed so far by ETH resistant clinical strains, and on the other hand, drastically increase the sensitivity of all tested TB strains, opening perspectives of dose and side effect reduction.