Efflux pumps are transmembrane transporter proteins through which bacteria extrude toxic materials, including antibiotics, into the extracellular or periplasmic space. Drug efflux as a result of efflux pump activity is a significant concern in antimicrobial resistance, and the expression of efflux pumps has been observed to be increased in drug-resistant bacteria. Furthermore, certain transporters can extrude multiple types of drug, contributing to the development of multidrug resistance (Rahman et al., 2017). Currently known bacterial efflux pumps can be divided into six families – adenosine triphosphate-binding cassette (ABC) family, small multidrug resistance (SMR) family, major facilitator superfamily (MPS), and multidrug and toxic compound extrusion (MATE) family, resistance nodulation division (RND) family, and the recently characterised proteobacterial antimicrobial compound efflux (PACE) family (see figure 1) (Nishino et al., 2021; Thakur et al., 2021). The RND family in particular is considered to be of clinical significance in the multidrug resistance of gram negative bacteria, with well characterised examples including the AcrAB-TolC multidrug efflux pump in E. coli and the MexAB OprM multidrug efflux pump in P. aeruginosa (Thakur et al., 2021).
Observations of the role of efflux in drug resistance have led to the hypothesis that pharmacological inhibition of efflux pump activity during antibiotic treatment could increase the concentration of the antibiotic within bacteria, thereby improving its efficacy and reducing resistance. Additionally, efflux pump inhibition has been demonstrated to disrupt biofilm formation, which is another contributing factor in the development of drug resistance (Reza et al., 2019). The inhibition of drug efflux can be achieved through multiple mechanisms, with members of the efflux pump inhibitor (EPI) drug class doing so typically via the direct binding of the inhibitor to the efflux pump or indirect inhibition of pump activation by interfering with its energy source (Sharma et al., 2019; Thakur et al., 2021). Finding molecules capable of inhibiting the activity of efflux pumps has not proved to be a great challenge, and in the past few decades a variety of inhibitor molecules from various sources have been described in literature, including synthetic, plant-, and even microbially-derived EPIs (Tambat et al., 2022; Thakur et al., 2021). Furthermore, many currently existing and FDA-approved drugs for other indications have been observed to also exhibit inhibitory activity against efflux pumps, such as the antihypertensive calcium channel blocker verapamil which acts as an inhibitor at MATE-type efflux pumps, present, for example, in N. gonorrhoea and S. pneumonia (Thakur et al., 2021).
However, despite promising advances and years of research, no EPI has made it to clinical use, and several as well as economic hurdles must be overcome for EPIs to become clinically viable. For one, bacteria express multiple types of efflux pump capable of extruding antibiotics, which means that inhibiting a single type of efflux pump is likely not sufficient to curb resistance (Nishino et al., 2021; Rahman et al., 2017). This is the case with the EPI ABI-PP, which inhibits MexB but not MexY efflux pumps, both of which contribute to drug resistance in P. aeruginosa (Nishino et al., 2021). On the other hand, less specific EPIs capable of binding to a variety of targets are more likely to be accompanied with undesired off-target effects when used therapeutically, such as the inhibition of eukaryotic efflux pumps (Sharma et al., 2019). Another challenge is that because the proposed therapeutic benefit of EPIs is dependent on enhancing the action of antibiotics, any inhibitors and antibiotics being administered together would need to have a similar pharmacokinetic profile, which can be difficult to achieve and is not commonly taken into consideration in the early stages of EPI discovery (Nishino et al., 2021). Furthermore, some EPIs, particularly those in use for other indications, may not be safe to use simultaneously with certain antibiotics. For example, clarithromycin, an antibiotic used in the treatment of pneumonia, interferes with the metabolism of verapamil, an EPI that inhibits efflux pumps in S. pneumonia, making their simultaneous use dangerous and clinically unviable (Thakur et al., 2021). Finally, because no EPI has been previously approved for clinical use, any EPI aiming for approval by regulatory authorities would have to fulfil the stringent regulatory requirements of a New Chemical Entity (NCE), increasing the costs associated with EPI development and reducing the incentive for pharmaceutical companies to take on the challenge (Sharma et al., 2019).
Ultimately, although efflux pump inhibition is a promising strategy for overcoming antimicrobial resistance without the development of new antimicrobial agents, the considerable challenges associated with making EPIs suitable for clinical use indicate that they are unlikely to play a significant role in tackling global antimicrobial resistance in the near future. However, continued research interest in efflux pumps and their role in drug resistance means that the targeting of drug efflux as a therapeutic strategy, whether via EPIs or alternative methods, remains a possibility.
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