Novel Inhibitors of the Two-Component System VanRS Re-Sensitize Enterococcus Faecalis to Vancomycin

Two-component systems (TCS) are the main signalling system in bacteria and are involved in adaptation, virulence, antimicrobial resistance, etc. TCS are formed by a sensor Histidine Kinase (HK) that detects a specific stimuli and undergoes an autophosphorylation via an ATP bound to the catalytic domain (CA). The phosphate is transferred to the Response Regulator (RR) that then changes gene expression accordingly to the detected stimuli. TCS have been proposed as good targets for antibiotics due to their presence in all bacteria, each bacteria possess multiple TCS, their catalytic domains are conserved and they are absent in mammalian cells. In this research we used a fragment-based drug design approach to develop inhibitors that compete with ATP for the ATP-binding pocket on the CA domain of TCS.

Following a fragment-based drug design approach, a series of fragments were screened in silico for binding to the ATP-pocket of different HK. Promising fragments were synthesized and protein binding was confirmed via X-crystallography. The most promising fragment CT14 was rationally and virtually grown into a compound library. This library was synthesized, and screened for in vitro binding to the HKs CheA from Thermotoga maritima and PhoR from Staphylococcus aureus. Best compounds bound with Kds in the low μM range. The library was then screened for inhibition of VanRS TCS mediated vancomycin resistance in E. faecalis and E. faecium. Surprisingly, compound 752 completely re-sensitized E. faecalis vanA and vanB to vancomycin. Expression analysis showed a significant downregulation of vanA induction from vancomycin treatment when 752 was present. Even though 752 failed to re-sensitize E. faecium vancomycin, it led to a similar reduction in vanA induction.

In summary, TCS inhibitor 752 re-sensitizes E. faecalis to vancomycin treatment by, at least partially, inhibiting VanRS TCS. Compound 752 also reduces VanRS related pathways in E. faecium but this inhibition is not enough to re-sensitize it to vancomycin. However, optimization for complete inhibition of VanRS could serve to also re-sensitize E. faecium to vancomycin, with great implications in nosocomial infections.