Antibiotics, among the most important medical discoveries of the 20th century, remain the main anti-infective drugs and have saved countless lives, while improving quality of life for humanity broadly (Cook and Wright, 2022). Despite continuous development and advances in medical science, genetic changes and the widespread and irrational use of antibiotics in healthcare, animal husbandry, and agriculture have led to the emergence and rapid spread of antimicrobial resistance (AMR) and multidrug-resistant (MDR) bacteria, with disastrous consequences for health and the economy. According to global data, 4.95 million deaths in 2019 were linked to bacterial AMR, of which 1.27 million deaths were directly attributable to bacterial AMR (Murray et al., 2022). It is projected that the annual number of deaths from bacterial AMR by 2050 (10 million) will even exceed that attributable to cancer (8.2 million) (Laxminarayan et al., 2013). Although the number of newly approved antimicrobial drugs has slightly increased over the past five years, it remains insufficient to address the growing problem of AMR. The primary impediment to progress is that identifying new drug targets and novel antibacterial compounds through whole-cell phenotypic, target-based, or gene identification studies is lengthy, costly, and not commercially profitable, which has led to gradual withdrawal of large pharmaceutical companies from the antibiotic market (Koh Jing Jie et al., 2022; Vila et al., 2020). The search for antibiotic adjuvants to existing antimicrobial drugs offers a productive and valuable approach to this problem. In most cases, adjuvants do not kill bacteria directly. However, when combined with existing antibiotics, they can increase the antibiotic’s antimicrobial efficiency by increasing the accumulation of the antibiotic in the bacterial cell or interfering with the bacterial defense system. Therefore, it is essential to tap into novel antibiotic adjuvants that can enhance the activity of existing antibiotics and extend their lifespan.

Drug combinations usually include two active compounds or one antibiotic and one non-antibiotic adjuvant molecule (Meric-Bernstam et al., 2023), where the latter can overcome the redundancy of safe but ineffective or obsolete antibiotics. An example of a clinically approved combination therapy is β-lactam with a β-lactamase inhibitor, which has played a significant role in treating infections with drug-resistant strains of bacteria. Combination therapies are common and critical in many other areas of medicine. Examples include cancer treatment (Prager et al., 2023; van der Heijden et al., 2023) or artemisinin-based combination therapy for malaria (Sutanto et al., 2023; Yeka et al., 2005). An ideal drug combination should simultaneously fulfill the following three conditions: (1) synergistic effect, mutually enhancing drug efficacy and therapeutic effect can be achieved using the lowest dose of the drug; (2) reduction of bacterial mutation rate and slowing down the development of drug resistance; and (3) even at high concentrations, no toxicity to the host cell (Sharma et al., 2021).

In March 1990, mefloquine (MFL) was recommended by the Centers for Disease Control for malaria chemoprophylaxis in areas where Plasmodium falciparum is endemic and was first marketed in U.S. pharmacies in May 1990 by Roche under the trade name Lariam (Kozarsky and Eaton, 1993). In recent years, MFL has increasingly been studied as a potential adjuvant for antibiotics against MDR bacteria. In this paper, we provide the first overview of the synergistic effects and potential mechanisms of MFL activity when used in combination with conventional antibiotics for treatment of MDR bacteria. Further, we summarize the antibacterial potential of MFL derivatives, as well as possible MFL applications in other areas of medical research. Finally, we discuss the favorable pharmacological properties of MFL, in terms of bacterial inhibition, and the associated shortcomings and challenges. The aim of this review is to provide new ideas to inform future deployment of synergistic combinations of MFL and antibiotics to address the antibiotic resistance crisis.

MFL is a synthetic 4-quinoline-methanol derivative that is structurally very similar to the first potent antimalarial drug, quinine (Kucharski et al., 2022). MFL is effective against all strains of malaria known to infect humans and has an essential role in prevention and in unimmunized individuals, as well as in treating malaria caused by MDR Plasmodium falciparum (Mairet-Khedim et al., 2023). In November 2020, the World Health Organization (WHO) released a 10-year (2000–2019) surveillance report on antimalarial drug efficacy, resistance, and response (World Health Organization, 2020). The report recommends six artemisinin-based combination therapies (ACTs) as first- and second-line treatments for Plasmodium falciparum, including artemisinin derivatives (artesunate, artemether, or dihydroartemisinin); artesunate-MFL is used as one of these ACTs.

Recently, an increasing number of studies have shown that MFL is an effective antibiotic adjuvant that enhances the susceptibility of various drug-resistant bacteria to a wide range of antibiotics. Additionally, research into its medical use in other areas has revealed potential for clinical applications ( Table 1 ).

Lutz et al. first documented MFL synthesis in the 1970s (Lutz et al., 1971), and it was also briefly summarized by Kucharski et al. (2022) ( Figure 1 ). MFL is a chiral compound with two asymmetric centers and exists in two racemic forms (erythro and threo), each of which comprises a pair of optical isomers; i.e., (±)-erythro-enantiomers and (±)-threo-epimers. When the term MFL is used in clinical settings, it is often referring to the erythro enantiomer (erythro-isomer racemic mixture) (Kucharski et al., 2022).

After the entry of MFL into the body, approximately 75%–80% is absorbed, with peak drug concentrations occurring between 7 and 24 h after administration (Karbwang and White, 1990). In the plasma, MFL is primarily bound to proteins in large amounts and has a high affinity for lipids, allowing it to reach very high intracellular concentrations (Wernsdorfer et al., 2013). MFL has a long half-life, and studies in mice have shown that it can be administered every three days to treat disseminated Mycobacterium avium infections (Bermudez et al., 2003) and weekly for treating malaria in humans (Schlagenhauf et al., 2010). MFL is extensively metabolized, primarily in the liver, by CYP3A4, to produce the pharmacologically inactive form, carboxy MFL, which is finally excreted from the body in the bile via the enterohepatic circulation (Piedade et al., 2015).

The main disadvantage of MFL is the occurrence of dose-related neuropsychiatric adverse effects, including abnormal dreaming, anxiety, nausea, and dizziness (the last two being the most common symptoms), with women and individuals with a lower body mass index (BMI) being more affected by the side effects (Toovey, 2009). Due to the side effects of MFL, a doctor’s prescription is required in countries such as Australia (Administration (TGA), T.G, 2022), Canada (Government of Canada, 2013) and the United Kingdom (Medicines and Healthcare Products Regulatory Agency, 2024). The United States Food and Drug Administration (2017) and the European Medicines Agency (Schlagenhauf et al., 2015) are also cautious about the use of MFL because its safety is not fully proven, and studies on the mechanism of action of MFL neurotoxicity still need to be better understood. A limited number of studies have shown that enantiomer (-)-erythro- MFL binds to adenosine receptors and cholinesterase in the central nervous system, regulates neurotransmitter release, disrupts intracellular homeostasis to produce oxidative stress, and impairs the function of voltage-dependent calcium channels and gap junctional intercellular communication are thought to be responsible for neuropsychiatric symptoms, whereas (+)- erythro- MFL enantiomer does not bind tightly to adenosine receptors in the brain (Schmidt et al., 2012). Therefore, selective synthesis and use of (+)-erythro- MFL rather than racemates could result in a better risk-benefit assessment. Müller et al. (2013) applied residual dipolar coupling (RDC)-enhanced NMR spectroscopy in combination with optical rotatory dispersion (ORD) and circular dichroism(CD) spectroscopy, determined the (+)-erythro- MFL absolute configuration to be (11S, 12R), and the absolute configuration of (-)-erythro- MFL to be (11R, 12S). Selective synthesis of (+)-erythro- MFL enantiomers could provide safer antimalarial drugs. Although MFL has the side effects of neuropsychiatric adverse effects, considering the wide range of potential applications of MFL in antibacterial, antitumor, and antiviral ( Table 1 ), it is worthwhile for researchers to conduct neurotoxic mechanism of action studies, such as new tools based on transcriptomic and proteomic analysis to gain new insights into MFL-induced neurotoxicity and to identify and provide potential early biomarkers or molecular targets that may be involved in its adverse neurological effects for further investigation.

Antibiotic adjuvants can include compounds or herbal products with known pharmacology or toxicology that do not have direct bactericidal activity, but may enhance the antibacterial efficacy of an antibiotic through various mechanisms (e.g., blocking resistance, enhancing intracellular antibiotic accumulation, complementing the bactericidal pathway, inhibiting signaling and modulation pathways, or augmenting the host response to bacterial infection) (Sharma et al., 2012). Synergism is commonly measured using the fractional inhibitory concentration index (FICI); a criterion used to assess synergism, additivity, or antagonism between two drugs (Coates et al., 2020). The FICI is calculated using the checkerboard method, which involves generating 96 different concentrations containing gradient dilutions of combinations of two drugs and observing their effects on the growth of microorganisms, to determine the optimal concentration that produces the most potent interaction.

The formula for calculating FICI is as follows:

Where MIC indicates ‘minimum inhibitory concentration’; and FICI values are interpreted as follows: FICI ≤ 0.5, synergistic; FICI > 0.5–4.0, additive; FICI > 4.0, antagonistic.

We used the above approach to evaluate the effectiveness of MFL as an adjuvant in combination with other antibiotics for the treatment of clinically prevalent MDR bacteria, including gram-negative bacteria (e.g., Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae), gram-positive bacteria [e.g., methicillin-resistant Staphylococcus aureus), and mycobacteria (Mycobacterium tuberculosis and M. avium complexes (MACs)].

Several MFL derivatives with potent antitubercular activity have been described ( Table 3 ). Silva et al. (2022) used MFL hydrochloride ([MFLH][Cl]) as a raw material and complexed it with several sodium salts of organic acids to obtain eight MFL organic salts, all of which showed higher bioavailability than the raw materials. Among them, the compound [MFLH][TsO] ( Figure 2 ) obtained via tosylate (TsO) was the most promising, in terms of antituberculosis effects, showing the highest activity against H37RV, with an MIC of 12.5 μg/mL, which is 1.3 times higher than that of [MFLH][Cl] (9.6 μg/mL), and was not cytotoxic to macrophages at the MIC.

da Silva Araújo et al. (2019) obtained 11 new compounds by a series of reactions centered on MFL. Among them, 3C ( Figure 3A ), 7 ( Figure 3B ), and 9 ( Figure 3C ) showed the highest activity against H37RV, with MIC values of 12.5, 25, and 25 μg/mL, respectively. MIC values against the resistant strain, SR 2571/0215 (resistant to rifampicin and isoniazid), were 12.5, 51, and 25 μg/mL, respectively. The most potent inhibitory effects against resistant and sensitive M. tuberculosis were observed with compound 3 C.

Gonçalves et al. (2010, 2012) synthesized a series of MFL-oxazolidine derivatives in 2010 and 2012, and these new compounds were not cytotoxic, but exhibited potent antitubercular activity. Among them, compounds 1j ( Figure 4A ), 1e ( Figure 4B ), and 2a ( Figure 4C ) had MIC values of 2.7, 2.7, and 2.8 μg/mL against H37RV, which were approximately 2.6-fold higher than that of MFL, and their antimicrobial activities were even better than that of the first-line antituberculosis drug, ethambutol. Compounds 1e and 2a were also assayed for MIC against T113, and the same MIC values as those for H37RV were observed. Moreover, these compounds were not cytotoxic to mouse macrophages at concentrations close to their MIC values.

Rodrigues-Junior et al. (2016) synthesized the MFL-oxazolidine derivative, compound 1E ( Figure 5 ), which showed MIC values of 0.5–4 μg/mL against 11 clinical isolates of quinolone-resistant M. tuberculosis, which were lower than the MIC of MFL (8 μg/mL). In addition, 1E was effective in reducing H37Rv bacterial load in RAW 264.7 macrophages, and in vivo experiments demonstrated that 1E could effectively reduce the bacterial load in the lungs and spleen of an H37Rv mouse infection model.

In 2009 and 2010, Mao et al. (2009, 2010) synthesized a series of MFL-isoxazole carboxylates with good metabolic stability in vitro and in vivo, based on pharmacokinetic data and rational drug design principles, including compounds 3 ( Figure 6A ) and 9d ( Figure 6B ). Compound 9d had an MIC value of 0.06 μg/mL against H37Rv, which was only two-fold higher than that of RFP, and was active against H37Rv (MIC = 3.68 μg/mL) under acidic conditions (pH 6.8). Compound 3 showed good activity against both replicating and nonreplicating M. tuberculosis, with MIC values of 0.4 and 5.3 μg/mL, respectively. Furthermore, the corresponding acid of compound 3 showed increased antituberculosis activity in an acidic environment, and significantly reduced the number of bacteria in a macrophage infection model, suggesting that compound 3 may be active in the acidic environment produced by inflammation in the lungs of patients with tuberculosis.

Invasive fungi can cause acute CNS infections (Giuliano et al., 2023). MFL and its derivatives cross the blood-brain barrier, facilitating sufficiently high penetration into the CNS (Shin et al., 2014), which makes them attractive candidates for treatment of fungal meningitis.

Compared with MFL, MFL derivatives exhibit more efficient antifungal activity. MFL shows poor or no antifungal activity of against Candida albicans, Cryptococcus neoformans, or Aspergillus fumigatus, with MIC values of ≥ 128, 32, and > 64 μg/mL, respectively (Kunin and Ellis, 2000); however, MFL structural analogs showed efficient antimicrobial activity against these fungi.

Montoya et al. (2020) screened four MFL derivatives with antifungal activity from the National Cancer Institute chemical library, including 2450 ( Figure 7A ), 4377 ( Figure 7B ), 13480 ( Figure 7C ), and 305758 ( Figure 7D ), of which 4377 exhibited the strongest antimicrobial activity, with MIC values of 1–4 μg/mL against all tested organisms (including C. albicans, Candida glabrata, S. cerevisiae, C. neoformans, and A. fumigatus), and the MIC values of the other three derivatives were also in the range of 2–8 μg/mL. In addition to direct antifungal activity, subinhibitory concentrations of MFL derivatives could reduce the expression of virulence traits, including filamentation in C. albicans and capsule formation/melanization in C. neoformans (Montoya et al., 2020).

Drugs can show synergistic relevance in various ways. The two most accepted synergy models are (1) the parallel pathway inhibition model and (2) the bioavailability model (Jia et al., 2009). In the parallel pathway inhibition model, two drugs are considered synergistic if they can simultaneously target two proteins in parallel pathways that are phenotypically critical for inhibition (Yeh et al., 2009). In the bioavailability model, two drugs are considered synergistic if one acts in a way that can contribute to increased accumulation of the concentration and availability of the other in target cells (Cokol et al., 2011). A typical example of a synergistic mechanism via the bioavailability model is when one drug enhances the intracellular concentration of another by destabilizing a drug transport barrier or inhibiting drug efflux. Although MFL may exhibit more than one mode of activity, the bioavailability model appears to be more consistent with its mechanism of action, based on the results observed when MFL acts against various bacteria with different antibiotics.

Pathogen mutation and the spread of drug resistance are recognized as major public health problems affecting human health and food quality. In Europe and the United States alone, at least 50,000 people die annually from microbial infections (European Antimicrobial Resistance Collaborators, 2022), and the number of deaths is much higher in other parts of the world. In this context, reintroducing approved unconventional antimicrobials, or applying them as antibiotic adjuvants in combination therapies, is becoming increasingly important.

As a potential antibiotic adjuvant, MFL has little or no antimicrobial activity but can enhance the antimicrobial effect of antibiotics. Studies have shown that MFL has an antibacterial effect on S. aureus only at high concentrations and has no antibacterial activity against P. aeruginosa, E. coli, and K. pneumonia. Combined with MFL, it can reduce the resistance of many known MDR bacteria to specific antibiotics and even reverse their resistance phenotype. The antimicrobial effects of a wide range of antibiotics, including colistin, β-lactams (oxacillin), antituberculosis drugs (isoniazid, pyrazinamide and ethambutol), quinolones (gatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, levofloxacin, ofloxacin, and sparfloxacin), and oxazolidinones (linezolid), can be enhanced in combination with MFL. These effects provide new opportunities for the treatment of common clinically drug-resistant gram-negative bacteria (P. aeruginosa, E. coli, and K. pneumoniae), gram-positive bacteria (MRSA and MSSA), and mycobacteria (M. tuberculosis and MACs). The mechanisms by which MFL enhances the bactericidal activity of antibiotics include attenuating antibiotic efflux, disrupting bacterial cell wall integrity, and inhibiting biofilm formation or eradicating pre-formed mature biofilms. Despite the long-term use of MFL for treating malaria, the exact mechanism of action underlying its antimalarial activity remains unclear, and the mechanism through which MFL exerts its antimicrobial activity has not been fully elucidated. Hence, further research is warranted to provide deeper understanding of the mechanism of action of MFL, optimize its use in the clinic and infectious disease, and ensure that it can be safely and effectively applied.

In addition, various MFL derivatives with strong bactericidal effects against M. tuberculosis and activities against some fungi have been successfully synthesized. The known favorable pharmacological properties and novel multitarget mechanism of action of MFL provide strong support for the development and optimization of MFL scaffolds and their derivatives for the treatment of Mycobacterial and fungal infections. First, MFL has a good affinity for lipids and can reach high concentrations in serum and tissue cells after oral administration, a feature that is attractive for the treatment of intracellular M. tuberculosis. Second, MFL and its derivatives can penetrate the blood-brain barrier, making them promising candidates for treating infectious diseases of the CNS. Third, it has become established practice to use prophylactic strategies in patients at high risk for infectious diseases, and MFL has a long half-life and can be used in dosing regimens to prevent infection. Notably, the side effects of MFL on the CNS limit its potential use in clinical settings. However, this issue should be carefully addressed when considering the treatment of infectious diseases with MFL, as it provides a new strategy for tackling multidrug-resistant bacterial infections in the context of limited antibiotic development and a continuing rise in drug resistance. There are still limitations in the research on MFL as an antibiotic adjuvant, such as the incomplete and unsystematic design of combined antimicrobial susceptibility experiments and limited specimen size. This has hindered a comprehensive evaluation of the synergistic effect of MFL with commonly used antibiotics on susceptible, resistant, MDR, and XDR bacteria. The exact mechanism of MFL’s auxiliary function has not been fully elucidated through more profound studies, which has led to some obstacles to achieving synergistic effects in vivo, and solving this dilemma requires consideration of complex pharmacology and in vivo drug metabolism, as well as thorough toxicological evaluation, and the study and characterization of its targets in bacteria, in order to clarify the exact mechanism of MFL antibiotic adjuvant activity and better promote its clinical application.