Repurposing bioactive aporphine alkaloids as efflux pump inhibitors

Abstract

Extrusion of drugs or drug-like compounds through bacterial efflux pumps is a serious health issue that leads to loss in drug efficacy. Combinatorial therapies of low-efficacy drugs with efflux pump inhibitors may help to restore the activities of such drugs. In this quest, natural products are attractive molecules, since in addition to their wide range of bioactivities they may inhibit efflux pumps. The current work repurposed the bioactive alkaloid roemerine as a potential efflux pump inhibitor. In Bacillus subtilis, both Bmr and BmrA, belonging to the major facilitator and the ATP-binding cassette superfamilies, respectively, were found to be inhibited by roemerine. Scanning electron microscopy and RNA-Seq analyses showed that it potentiated the effect of berberine. Growth rates and checkerboard assays confirmed the synergy of roemerine and berberine and that roemerine prevented berberine efflux by inhibiting Bmr. Transport assays with inverted membrane vesicles prepared from Escherichia coli overexpressing BmrA showed that increasing roemerine concentration decreased the transport of doxorubicin, the BmrA substrate, confirming that roemerine may also be considered as an inhibitor of BmrA. Thus, these findings suggest that conjugation of roemerine to substrates of efflux pumps, Bmr and BmrA, may help to potentiate the activity of their drug substrates.

Evaluation of silibinin as an efflux pump inhibitor in Bacillus subtilis

“…Growth curves obtained with increasing silibinin concentrations have shown that 25 µgmL -1 silibinin is suitable for testing its EPI feature since it did not alter growth. The antimicrobial berberine is widely known to be a substrate of a number of drug efflux pumps therefore its combination with different natural pump inhibitors may offer a way to enhance its efficacy (Avci et al, 2019;Stermitz et al, 2000). In order to evaluate the EPI property of silibinin, its combination with berberine has been tested on B. subtilis cells.…”

“…In order to evaluate the EPI property of silibinin, its combination with berberine has been tested on B. subtilis cells. Berberine working concentration was determined based on previous work (Avci et al, 2019). In our previous work, 75 μgmL -1 berberine has been shown to only slightly affect B. subtilis 168 growth.…”

“…Isolation and identification of new EPIs will bring antimicrobials with lost efficacies back into the clinic. To date, many plant-based EPIs have been identified as reserpine (Gibbons et al, 2003;Neyfakh et al, 1991), piperine (Kumar et al, 2008), roemerine (Avci et al, 2019), baicalein (Chan et al, 2011), 5'-methoxy-hydnocarpin (Stermitz et al, 2000), and catechin gallates (Gibbons et al, 2004).…”

“…In the present study, the ability of silibinin to inhibit the efflux pumps of Bacillus subtilis was evaluated using minimum inhibitory concentration determination, growth studies, and ethidium bromide accumulation assay. Berberine was used in growth studies since it is the substrate of many efflux pumps and its activity is weakened due to the activity of efflux pumps (Avci et al, 2019). The clinical importance of B. subtilis is limited, it constitutes one of the model organisms for low G+C Gram-positives with a significant genomic abundance of multidrug transporters.…”

Naturally-Occurring Alkaloids of Plant Origin as Potential Antimicrobials against Antibiotic-Resistant Infections

“…Several assays showed that roemerine potentiated the effect of berberine with MIC values reduced from 256 and 64 μg/mL and from 64 to 16 μg/mL, respectively, by inhibiting the Bmr efflux pump. In addition, transport assays conducted using E. coli inverted membrane vesicles overexpressing BmrA confirmed that increasing concentrations of roemerine inhibited the transport of the BmrA substrate, doxorubicin and through this pump [ 111 ].…”