Drug repurposing for identification of new efflux inhibitors and/or antibiofilm agents against Staphylococcus aureus and Staphylococcus epidermidis

Background
Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are frequent pathogens
causing nosocomial infections. Efflux-mediated resistance and biofilm formation may
contribute to the emergence of resistance in these species and resilience of such infections to
chemotherapeutics. In this work, we applied an in silico drug repurposing strategy to identify
drugs that can target efflux and/or biofilm formation, to be then assessed in vitro for their
efflux inhibitory and/or antibiofilm activities.
Methods
A list of targets, corresponding to all SA and SE membrane transporters and biofilmassociated
proteins was used to interrogate the DrugBank database and generate a list of
approved drugs targeting these proteins or their homologues. Representative candidate drugs
were chosen for validation in an experimental model based on reference and isogenic strains
differing in norA expression (gene encoding the main staphylococcal efflux pump NorA). To
assess efflux inhibitory activity, the drugs MICs were determined by broth microdilution and
each drug (at ¼ MIC) tested for its ability to reduce MICs of selected antimicrobials (NorA
substrates and non-substrates). Drugs with significant effect (≥ four-fold MIC reduction)
were further tested for synergism with those antimicrobials by checkerboard assays and their
potential to inhibit biofilm formation by the crystal violet adhesion method.
Results
We identified over 200 drugs that potentially target SA and/or SE membrane transporters or
biofilm-associated proteins. Seven of these candidate drugs were tested: desipramine,
chloroquine, atovaquone, topiramate, amlodipine, tariquidar and sulpiride. All drugs showed
high MICs (>64 mg/L) against all staphylococcal strains. Tariquidar, amlodipine,
desipramine and chloroquine reduced the MICs of NorA substrates in norA-overexpressing
SA and SE strains, suggesting they possess efflux inhibitory activity. Moreover, amlodipine
and tariquidar presented significant synergism with some NorA substrates. Amlodipine was
the only drug able to abolish biofilm formation in SA, whereas desipramine and chloroquine
were more effective against SE biofilms.
Conclusions
This work reveals amlodipine, desipramine and chloroquine as new potential dual target
drugs and tariquidar as a potent efflux inhibitor in staphylococci. These drugs may, in the
future, be included in the fight against antimicrobial-resistant S. aureus and S. epidermidis
infections.
J. Neves and C. Antunes contributed equally to this work.

Acknowledgement of grants and fundings, word count: 30 words
Project DREBI, Ref. 2022.07931.PTDC funded by Fundação para a Ciência e a Tecnologia (FCT,
Portugal) and Project BIOSAFE funded by FEDER through the Programa Operacional Factores de
Competitividade - COMPETE and FCT (Portugal), Grant LISBOA-01-0145-FEDER-030713,
PTDC/CAL-EST/30713/2017. Further support by FCT to GHTM (UID/04413/2020).