Understanding the mechanisms of action and resistance to antibiotics and promising lead compounds is key to their prioritisation, rational improvement and to the development of strategies and molecules that can potentially bypass resistance, shorten treatment and be less toxic.
We apply modern metabolomic, proteomic, transcriptomic and chemical biological methods to define both pharmacodynamics and (intrabacterial) pharmacokinetics of antibiotics and antimycobacterial agents. Knowledge gained by these studies will rationally guide drug discovery and development. One of the drugs we have focused on is the antibiotic D-cycloserine (DCS), a second-line drug used as cornerstone for the treatment of multidrug-resistant tuberculosis.
Our studies to date indicate that: (1) DCS inhibits both alanine racemase (Alr) and D-Ala:D-Ala ligase (Ddl) in vitro, as expected; however, unlike has been observed in other species (2) DCS is a slow-onset inhibitor of M. tuberculosis Ddl. This is likely due to formation of a O-phospho-DCS inhibitor, in the active site of Ddl; (3) metabolomics and labelling experiments revealed that DCS successfully decreases the levels of D-Ala:D-Ala in M. tuberculosis without a full blockage of the synthesis of D-Ala, indicating suboptimal inhibition of Alr; and (4) that in M. tuberculosis, recovery of the pool size of D-Ala:D-Ala following DCS treatment is delayed, indicating a post-antibiotic effect for DCS which is consistent with time-dependent inhibition of Ddl observed in vitro.
Taken together, these results indicate that DCS kills M. tuberculosis chiefly by inhibiting D-Ala-D-Ala synthesis instead of Ala racemisation (Figure 2). These results are of great utility for the efficient design/development of novel inhibitors of the D-Ala branch of peptidoglycan biosynthesis in M. tuberculosis and other bacteria.