New selective Inhibitors for deciphering Lipid metabolism and virulence in M. tb – LipInTB

The activity-based protein profiling (ABPP) approach coupled with mass spectrometry using the alkyne (or azide)-probes will be used to label, enrich, isolate and further identify the enzymes targeted by our compounds.

Two robust experimental models based on ILI formation and degradation, thus focusing on the mycobacterial lipid metabolism during the dormancy and reactivation processes, will also be used to decipher the mode of acquisition and consumption of lipids by mycobacteria.

We have initially demonstrated that our two families of inhibitors block the extracellular and/or intracellular growth of M.tb by impairing the activity of various enzymes participating in important physiological processes related to the whole bacterial lipid metabolism.

The already selected inhibitors, displaying the best antimycobacterial activity, have been chemically modified by introduction of an alkyne (or azide) group. Such modification will allow the use of bio-orthogonal click chemistry reactions for the direct fishing of target proteins during the mycobacterial phases of active replication, latency and/or reactivation of the bacilli in infected foamy as well as non-foamy macrophages.

The penetration and localization of the inhibitor fluorescent probes; as well as activity-based protein profiling experiments with alkyne (or azide)-probes in host cells/mycobacteria alone, and in infected macrophages, are currently in progress.

Cavalier, J. F.; Spilling, C. D.; Durand, T.; Camoin, L.; Canaan, S. “Lipolytic enzymes inhibitors: A new way for antibacterial drugs discovery”. Eur. J. Med. Chem. 2021, 209, 112908. DOI: doi.org/10.1016/j.ejmech.2020.112908.

Madani, A.; Mallick, I.; Guy, A.; Crauste, C.; Durand, T.; Fourquet, P.; Audebert, S.; Camoin, L.; Canaan, S.; Cavalier, J. F. “Dissecting the antibacterial activity of oxadiazolone-core derivatives against Mycobacterium abscessus”. PLoS ONE 2020, 15(9), e0238178. DOI: doi.org/10.1371/journal.pone.0238178.

Tuberculosis (TB) caused by the pathogenic bacterium Mycobacterium tuberculosis (M.tb), is the deadliest infectious disease worldwide. Infection with M. tb leads to the formation of granulomas in the lung, where some of the infected macrophages accumulate lipids in lipid bodies (LB) giving the cells a foamy appearance. In such foamy macrophages (FM), bacilli can persist in a non-replicating state for decades but also be reactivated to cause acute disease. A better understanding of how bacilli persist inside lipid-rich FM is needed to find new ways for eradicating this disease.

To persist inside FM, M. tb hydrolyzes host lipids into fatty acids that are reused as lipid reserves within intracytoplasmic lipid inclusions (ILI). Recent results suggest a direct link between the presence of ILI in mycobacteria and their inability to divide. The latter may be of central importance for mycobacterial persistence within granulomas.

Over the past 20 years, lipolytic enzymes, which are responsible for the release of long-chain fatty acids, have become the focus of intense research. Indeed, these enzymes, strongly involved in the host-pathogen cross-talk, play several roles in the physiopathology of the disease during both the active and persistent phases of infection. Although their role in the control of host lipid breakdown and ILI consumption during infection is documented, the molecular mechanisms involved in these processes remain elusive. Recently, these enzymes have become real mycobacterial drug targets. Accordingly, finding ways to inhibit their activity could then pave the way for discovery of new modalities for the treatment of TB.
In this competitive and exciting context, we have recently discovered two new families of promising anti-TB agents with no cytotoxic effects towards host cells, and targeting enzymes involved in lipid metabolism.

Our project, involving 5 partners, aims to use this novel class of inhibitors to monitor mycobacterial infection in order to i) identify and validate in vitro & in vivo the mycobacterial enzymes impaired by our compounds leading to M. tb death during extracellular growth and in infected macrophages; and ii) use them as probes to decipher M. tb lipid metabolism during the phase of active replication, latency and reactivation in infected macrophages.

Based on preliminary data, we believe that exposure to lipase inhibitors would profoundly alter the outcome of the disease by strongly impairing mycobacterial growth within host cells as well as entry of actively replicating bacilli into the persistence phase and/or reactivation of dormant bacilli within granuloma FM.

The mutual complementation of scientific and technical skills between all contributing teams will be a major asset. We believe that this project will generate a large amount of results that are directly relevant for the pathogenesis of actively replicating and latent bacilli.

Above all, this project will provide significant achievements in the understanding of the mechanisms that operate during mycobacterial replication in non-FM as well as during persistence and/or reactivation in FM that are major issues for understanding the susceptibility and the general development of TB. This knowledge will contribute to background information for the development of new therapeutic strategies for elimination of either actively replicating or latent bacilli from infected individuals.