Lysyl-tRNA synthetase, a target for urgently needed M. tuberculosis drugs

Abstract

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.

Fig. 1. Chemical evolution of DDD2049209 (8) from the initial hit 1.

Potencies are shown against: in vitro enzymes LysRS & KARS1; M. tuberculosis phenotypic growth in culture (MIC), M. tuberculosis growth within macrophages, and cytotoxicity against human HepG2 cells. Data shown are the geometric mean of at least two independent replicates. Source data are provided as a Source Data file.

Fig. 2. Compounds bound in the LysRS catalytic binding site.

a 2 is shown in pink carbon atoms, and surrounding protein residues are shown with gray carbon atoms. Oxygen atoms are shown in red, nitrogen atoms in dark blue, and fluorine in pale blue. Key interactions are indicated with dashes (PDB 7qhn). b 8 is shown with gold carbon atoms, other atoms as in a (PDB 7qi8).

Fig. 3. In vivo efficacy of LysRS inhibitors in murine models of M. tuberculosis infection.

a Compounds 5 and 8 were tested in an acute model of infection in C57BL/6 mice. Dosing started 1 day after infection and lasted for 8 days; each group consisted of one mouse/dose. The effect on the number of colony-forming units (CFU) in mouse lungs is shown. b Compound 8 was tested in a chronic model of infection in C57BL/6 mice. Dosing started 6 weeks after infection and lasted for 8 weeks (7 days/week), each group consisted of two mice/dose. c Compound 5 (K: 100 mg/kg bid) was evaluated in a subacute model combination study in BALB/c mice along with bedaquiline (B: 25 mg/kg qd), pretomanid (Pa: 25 mg/kg bid), and linezolid (L: 50 mg/kg bid). Dosing (8 weeks: 5 days/week) started 14 days post infection (D0), five animals/group were used. Source data are provided as a Source Data file.

Fig. 4. Studies to explore the cellular mechanism of action.

a A resistant mutant was isolated against 7, resistance was confirmed by looking at sensitivity over a concentration range compared to parental H37Rv for both 7 and the unrelated drug isoniazid. b Impact of TetON overexpression of either wild-type (WT) LysRS or mutant LysRS.491 on sensitivity to 8 ± anhydrotetracycline (ATc), for comparison the impact of 8 on H37Rv is also shown. For a, b, graphs are representative data from one of two independent experiments each run in triplicate presented as mean values ± standard deviation. c Metabolomic analysis of cells treated with three active and one inactive members of the LysRS inhibitor series (5× MIC) for 18 h. Metabolites related to the lysine biosynthetic pathway are shown. Drug concentrations were evaluated in triplicate per experiment, and the results are representative of at least two independent experiments. Data are depicted on a log₂ transformed ratio of ion intensity of metabolite abundance in treated vs untreated M. tuberculosis and displayed using the image generation program treeview (http://jtreeview.sourceforge.net/). Source data are provided as a Source Data file.