An engineered prodrug selectively suppresses ß-lactam resistant bacteria in a mixed microbial setting.

The rise of ß-lactam resistance necessitates new strategies to combat bacterial infections. We purposefully engineered the ß-lactam prodrug AcephPT to exploit ß-lactamase activity to selectively suppress resistant bacteria producing extended-spectrum-ß-lactamases (ESBLs). Selective targeting of resistant bacteria requires avoiding interaction with penicillin-binding proteins, the conventional targets of ß-lactam antibiotics, while maintaining recognition by ESBLs to activate AcephPT only in resistant cells. Computational approaches provide a rationale for structural modifications to the prodrug to achieve this biased activity. We show AcephPT selectively suppresses gram-negative ESBL-producing bacteria in clonal populations and in mixed microbial cultures, with effective selectivity for both lab strains and clinical isolates expressing ESBLs. Time-course NMR experiments confirm hydrolytic activation of AcephPT exclusively by ESBL-producing bacteria. In mixed microbial cultures, AcephPT suppresses proliferation of ESBL-producing strains while sustaining growth of ß-lactamase-non-producing bacteria, highlighting its potential to combat ß-lactam resistance while promoting antimicrobial stewardship.

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.