Novel Antibacterial Activity of Febuxostat, an FDA-Approved Antigout Drug against Mycobacterium tuberculosis Infection

ABSTRACT

Accumulating evidence suggests that drug repurposing has drawn attention as an anticipative strategy for controlling tuberculosis (TB), considering the dwindling drug discovery and development pipeline. In this study, we explored the antigout drug febuxostat and evaluated its antibacterial activity against Mycobacterium species. Based on MIC evaluation, we found that febuxostat treatment significantly inhibited mycobacterial growth, especially that of Mycobacterium tuberculosis (Mtb) and its phylogenetically close neighbors, M. bovis, M. kansasii, and M. shinjukuense, but these microorganisms were not affected by allopurinol and topiroxostat, which belong to a similar category of antigout drugs. Febuxostat concentration-dependently affected Mtb and durably mediated inhibitory functions (duration, 10 weeks maximum), as evidenced by resazurin microtiter assay, time-kill curve analysis, phenotypic susceptibility test, and the Bactec MGIT 960 system. Based on these results, we determined whether the drug shows antimycobacterial activity against Mtb inside murine bone marrow-derived macrophages (BMDMs). Notably, febuxostat markedly suppressed the intracellular growth of Mtb in a dose-dependent manner without affecting the viability of BMDMs. Moreover, orally administered febuxostat was efficacious in a murine model of TB with reduced bacterial loads in both the lung and spleen without the exacerbation of lung inflammation, which highlights the drug potency. Taken together, unexpectedly, our data demonstrated that febuxostat has the potential for treating TB.

INTRODUCTION

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is among the top 10 leading causes of death worldwide. The global TB incidence rate is declining, but not fast enough to reach the first milestone of the End TB Strategy, and approximately a quarter of the world’s population remains infected with Mtb (1). Most Mtb-infected individuals develop a persistent but asymptomatic TB infection, rather than sterilizing immunity, following exposure. These individuals are at risk of reactivation during their lifetime because of comorbidities such as HIV or diabetes (2). Furthermore, the emergence of drug resistance threatens TB control worldwide and hinders effective treatment (3).

Regarding TB control, treatment with first-line drugs (2 months of isoniazid [INH], rifampin [RIF], pyrazinamide [PZA], and ethambutol [EMB], followed by 4 to 7 months of INH and RIF) is indicated as the standardized protocol. Although this regimen has >95% efficacy, several TB-attributable deaths occur each year (4), and drug-resistant TB hinders complete eradication of the epidemic (5). Specifically, treatment for multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB strains is much more expensive and requires up to 24 months of continuous drug therapy, with several side effects (6). Consequently, researchers have been using a high-throughput screening technique to discover new anti-Mtb agents and have attempted to find new drugs against TB (7). Several newly developed candidates are showing improved efficacy in clinical trials. However, the major challenge is the time and financial demands for preclinical studies and safety concerns, respectively (8, 9).

Without a prospective strategy for new anti-TB drugs, the research focus has mostly shifted toward drug repurposing, as is employed in other diseases (10). This new approach could be either host-directed or pathogen-directed. Similar to host-directed therapeutics (HDTs), which regulate host immune systems, pathogen-directed therapeutics (PDTs) have also been regarded as an alternative option, as they target the essential virulence factors of pathogens in vivo (11, 12). For instance, verapamil, a licensed drug to treat hypertension, displays inhibitory activity against the intracellular growth of Mtb, accompanied by inhibition of efflux pumps (13). Multidrug efflux pumps are associated with drug resistance, as increased activity in the efflux system lowers the level of antibiotics in the bacterial cytoplasm, which can eventually lead to drug resistance (14). Due to the inhibitory effect of expelling antibiotics on the bacterial efflux pumps, verapamil reduced the possibility of development of antimicrobial resistance (13) and further displayed anti-TB activity, in combination with first-line anti-TB drugs, in a murine model infected with the Mtb MDR strain (15). More recently, the bactericidal activity of verapamil has also been reported through a direct effect on the membrane potential (16). Similarly, a gastric proton-pump inhibitor, lansoprazole, for treating excessive gastric acid-associated diseases shows intracellular bactericidal activities against drug-sensitive Mtb and MDR clinical isolates by targeting the cytochrome bc₁ complex (17). Moreover, the incidence of TB among the users of lansoprazole was much lower than that among nonusers. Besides, there have been no reports of its antagonistic interaction with anti-TB drugs or of serious adverse effects, which indicates that it could be a promising treatment against TB (18, 19).

For many years, one of the under-investigated aspects of TB has been the hyperuricemia and acute gouty arthritis associated with the use of first-line anti-TB drugs, such as EMB and PZA (20, 21). PZA is a strong urate-retention agent and causes a more than 80% reduction in renal clearance of uric acid at a 300-mg therapeutic daily dose (22). Pyrazinoic acid, a major metabolite of PZA, is oxidized by xanthine oxidase and likely causes the hyperuricemic effect. Hyperuricemia was reported in 42% to 66% of patients treated with EMB (21) and 43% to 100% of patients treated with PZA (alone or combined with other drugs) (20).

Hyperuricemia, defined as a serum urate (SUA) concentration of >6.8 mg/dL, can occur because of either overproduction or underexcretion of uric acid in the body. If hyperuricemia is induced during anti-TB medications, antigout drugs such as allopurinol and febuxostat might be co-used with anti-TB drugs (23) to control EMB- and PZA-induced hyperuricemia. However, the direct effects of those drugs against Mtb infection have not been elucidated. Besides, uric acid use in Mycobacterium intracellulare and Mycobacterium scrofulaceum isolates has been demonstrated (24). Moreover, ammonia, a catabolic metabolite of uric acid and asparagine hydrolysis, was exploited to maintain phagosomal pH by inducing the Mtb asparagine transporters AnsA, AnsP1, and AnsP2 (25). The uric acid is a purine base and can be degraded by the purine metabolic/salvage pathway (26). Purine metabolism is conserved throughout eukaryote and prokaryote organisms, including Mtb. Unlike other intracellular pathogens such as Toxoplasma gondii, Plasmodium falciparum, and Leishmania donovani, which exploit purines for their life cycle (26, 27), the purine de novo and salvage pathway in Mtb have not been extensively elucidated.

Here, we hypothesized that the antigout drug may influence the life cycle of Mtb by affecting purine metabolism. Thus, we examined the antibacterial activity of the FDA-approved antigout drug febuxostat against Mycobacterium species, including in vivo efficacy in an established mouse model for TB, for its use as a potential anti-TB drug candidate.

RESULTS

In vitro antibacterial effect of antigout drug against Mtb.

Xanthine oxidase-related antigout drugs, including allopurinol, febuxostat, and topiroxostat, were evaluated for their antibacterial effects against Mtb. MIC tests against Mtb H37Rv strain showed that febuxostat inhibited bacterial growth at 100 μg/mL, compared to allopurinol (Fig. 1A). Furthermore, we compared the inhibitory effect of febuxostat with that of its next-generation candidate, topiroxostat. Although allopurinol is a xanthine oxidase inhibitor, it can function as purine nucleoside phosphorylase and orotidine 5′-monophosphate decarboxylase (28, 29). Considering this, a specific xanthine oxidase inhibitor, topiroxostat, which is superior to febuxostat, was selected (30). Interestingly, only febuxostat displayed inhibitory effects on both the Mtb H37Rv reference strain and Mtb Korean Beijing (K) strain (which caused a high-school TB outbreak in South Korea) (31) (Fig. 1B).

FIG 1.

Differential inhibitory effects of antigout drugs on mycobacterial growth. (A) Mtb H37Rv ATCC 27294 strain was incubated in 7H9 medium supplemented with 10% OADC for 14 days at 37°C. Then, the bacteria were treated with resazurin and subsequently incubated for 24 h at 37°C. Allopurinol and febuxostat were applied at a range of concentrations, beginning with 200 μg/mL and diluted serially by one-half. (B) Mtb H37Rv and K strains were treated with resazurin and incubated likewise. Febuxostat and Topiroxostat were administered at a range of concentrations: 75 to 200 μg/mL and 12.5 to 200 μg/mL, respectively. The red arrow indicates the MICs of drugs against each Mtb strain tested in this study. NC, negative control; PC, positive control.

Antibacterial activity spectrum of febuxostat against Mycobacterium species.

After observing the inhibitory potential of febuxostat against Mtb, the antimycobacterial activity spectrum of febuxostat was determined by screening against Mycobacterium species. MIC results against each Mycobacterium species are summarized in Table 1. Growth of Mtb H37Rv ATCC 27294, H37Ra ATCC 25177, CDC1551, and K strains was inhibited at 100 μg/mL of febuxostat. Febuxostat (50 μg/mL) also inhibited the growth of Mtb Erdman and HN878 strains (Fig. 2A). Additionally, febuxostat showed inhibitory effects against Mtb clinical strains at 50 or 100 μg/mL (See Fig. S1A in the supplemental material). Therefore, we attempted to determine the MIC against the Mtb complex (MTBC), comprising M. bovis and M. microti. Unsurprisingly, febuxostat displayed inhibitory activity on the growth of M. bovis AN5, BCG Pasteur 1173P2, BCG Danish-1331, and Tokyo-172 and clinical isolates #3, #4, #5, #6, and #10 at concentrations of 100 or 200 μg/mL (Fig. 2B; see also Fig. S1B). Additionally, the growth of M. microti was inhibited by febuxostat (Table 1). Considering that the inhibitory effect of febuxostat was intact against Mtb and MTBC, we performed MIC tests on other Mycobacterium species, such as nontuberculosis mycobacteria (NTM), to evaluate whether febuxostat exhibits broad-spectrum inhibition. Although most NTM was not inhibited, M. kansasii ATCC 12478 and clinical strain SMC #1 were inhibited by febuxostat at 100 μg/mL and 200 μg/mL, respectively (Fig. 2C; see also Fig. S1C). Surprisingly, M. kansasii is one of the nearest neighbors to MTBC according to multilocus sequence analysis-based phylogeny (32). Furthermore, in a more recent study, there have been other nontuberculous mycobacteria that were reported as genetically more closely related to Mtb than M. kansasii (33, 34). Among these nontuberculous mycobacteria, M. shinjukuense JCM 14233 and M. lacus JCM 15657 were employed and subsequently evaluated for their MIC against febuxostat treatment. Only M. shinjukuense JCM 14233 was inhibited by febuxostat at 200 μg/mL (Fig. 2D). These results demonstrated that the inhibitory effect of febuxostat on bacterial growth was relatively specific to Mtb and some of its closely related neighbors.

TABLE 1.

Summary of MIC results for Mycobacterium species

Mycobacterium species	MIC results (μg/mL)
Mycobacterium tuberculosis Complex
M. tuberculosis
H37Rv ATCC 27294	75–100
H37Ra ATCC 25177	100
HN878	50
Erdman	50
CDC1551	100
Clinical isolates (#2, #7, #10, #11, #20, #23)	50–100
M. bovis
AN5	200
BCG Pasteur 1173P2	200
BCG Tokyo 172	100
BCG Danish 1331	100
Clinical isolates (#3, #4, #5, #6, #10)	200
M. microti	200
NTM
M. kansasii ATCC 12478	100
M. kansasii clinical isolate (SMC #1)	200
M. shinjukuense JCM 14233	200
M. lacus JCM 15657	>200
M. abscessus ATCC 19977	>200
M. avium subsp. avium ATCC 25291	>200
M. avium subsp. avium 104	>200
M. intracellulare ATCC 13950	>200
M. fortuitum ATCC 49404	>200
M. massiliense CIP 108297	>200
M. smegmatis ATCC 700084	>200

FIG 2.

Evaluation of antibacterial spectrum of febuxostat on Mycobacterium species. (A) Mtb H37Rv ATCC 27294, H37Ra ATCC 25177, HN878, CDC1551, and Erdman strains were treated with resazurin and incubated for over 24 h at 37°C. (B) M. bovis AN5, BCG Pasteur 1173P2, BCG Danish-1331, and BCG Tokyo-172 strains were treated with resazurin and incubated for over 24 h at 37°C. (C) M. abscessus ATCC 19977, M. avium ATCC 25291, M. avium 104, M. intracellulare ATCC 13950, M. fortuitum ATCC 49404, M. massiliense CIP 108297, M. smegmatis ATCC 700084, and M. kansasii ATCC 12478 strains were treated with resazurin and incubated for over 24 h at 37°C. (D) M. shinjukuense JCM 14233 and M. lacus JCM 15657 were treated with resazurin and incubated for over 24 h at 37°C. The red arrow indicates MICs of febuxostat against each strain tested in this study. NC, negative control; PC, positive control.

Long-lasting inhibitory effect of febuxostat on Mtb growth.

To evaluate the effect of febuxostat over time, 5 × 10⁶ CFU Mtb H37Rv were initially inoculated and a time-kill curve analysis, which can monitor bacterial growth and death over an array of antimicrobial concentrations, was conducted (35). The onset of the bactericidal activity was dependent on the concentration of febuxostat against Mtb H37Rv. Specifically, 200 μg/mL of febuxostat-treated Mtb H37Rv experienced the most rapid death during the analysis, resulting in no growth in the 7H9 broth (Fig. 3A). In addition, scanning electron microscopy revealed significant structural changes in febuxostat-treated Mtb, featuring wrinkled or shrunken morphologies compared to those of either non- or solvent-treated Mtb (see Fig. S2). Furthermore, the inhibitory effect of febuxostat was investigated via phenotypic testing during an extra 8-week incubation period using low CFU inoculation (10³ CFU) of Mtb strains (Mtb H37Rv, K, HN878, and CDC1551) to 7H10 agar medium containing solvent or drug. The results showed that the growth of tested Mtb strains was significantly inhibited by febuxostat at 50 to 400 μg/mL compared to nontreated and solvent-treated groups (Fig. 3B; see also Fig. S3). To further explore the accurate growth time of drug-treated Mtb strains, Bactec MGIT analysis was performed for 10 weeks. MGIT analysis is a fully automated system that exploits the fluorescence of an oxygen sensor to accurately detect mycobacterial growth in culture (36). The MGIT data are summarized in Table 2. Following febuxostat treatment at 100 μg/mL, the inoculated H37Rv strain (at 10⁴ and 10⁵ CFU) was detected by the MGIT system at 23 and 17 days of incubation, respectively, while the solvent-treated group was detected at 9.5 and 5 days, respectively, in each group (Fig. 4A). The Erdman strain was gradually detected at 50 μg/mL of febuxostat compared to the solvent-treated group, and the 100 μg/mL-treated Erdman strain was undetected during analysis (Fig. 4B). Likewise, delayed detection was observed in febuxostat-treated HN878 (Fig. 4C). Additionally, a different set of MGIT analyses was performed for comparing the antimycobacterial effect of febuxostat with first-line anti-TB drugs, rifampin (RIF) and isoniazid (INH). Following febuxostat treatment at 100 μg/mL, the inoculated H37Rv strain (at 10⁵ CFU) was detected by the MGIT system at 16.5 days of incubation, while no drug-treated growth control group (at 10³ and 10⁵ CFU) was detected at 8.3 and 4.8 days, respectively, suggesting that the inhibitory activity of febuxostat (at 100 μg/mL) was achieved with more delayed detection than that of 100-fold diluted growth control. Notably, 200 μg/mL of febuxostat-treated Mtb H37Rv was not detected during analysis as observed in the RIF- and INH-treated group (Table 3). These results indicate that febuxostat durably mediated the inhibitory effect, contributing to the delayed detection of growth, resulting in relatively longer repopulation time for Mtb, and further 200 μg/mL of febuxostat can be needed for bactericidal activity.

FIG 3.

Direct growth inhibition of Mtb by febuxostat. (A) Mtb H37Rv ATCC 27294 strains (5 × 10⁶ CFU) were inoculated and treated with each febuxostat concentration. Then, they were incubated in a shaking incubator for 28 days at 140 rpm and 36°C. Simultaneously, CFU spotting was performed on 7H10 agar at indicated time points. The data are expressed as the mean ± SD (n = 4). Statistically significant differences between the groups were determined using an unpaired Student's t test. n.s., not significant; **, P < 0.01; ***, P < 0.001 comparing nontreated and febuxostat-treated groups. (B) Mtb H37Rv ATCC 27294, K, HN878, and CDC1551 strains were used. Bacterial stocks were diluted to 10³-fold with PBS and spotted on 7H10 agar containing 0.2% DMSO and 50, 100, 200, and 400 μg/mL of febuxostat. Then, all plates were incubated for 8 weeks at 37°C.

TABLE 2.

Bactec MGIT 960 time to detection (TTD) value^a

Bacterial strain	Standard equation	R square	Standard (104/105)	Positive control (104/105)	Solvent control (104/105)	50 μg/mL (104/105)	100 μg/mL (104/105)
H37Rv ATCC 27294 (99% inhibited at 105 CFU, 100 μg/mL)	y = −0.4443x + 8.487	0.9740	9.3 day/8 day	9.7 day/7.9 day	9.5 day/7.6 day	12.7 day/8.9 day	23.2 day/17.4 day
HN878 (96% inhibited at 105 CFU, 100 μg/mL)	y = −0.4118x + 8.122	0.9738	9.5 day/7.3 day	9.5 day/7.5 day	9.2 day/7 day	11.2 day/8.3 day	15.1 day/11.1 day
Erdman (93% inhibited at 105 CFU, 50 μg/mL)	y = −0.5551x + 9.485	0.9893	9.4 day/8 day	10.8 day/8 day	9.6 day/7.4 day	16.7 day/10.2 day	Not detected

^a400 μg/mL- and 200 μg/mL-treated groups in all tested Mtb strains were not detected over 10 weeks.

FIG 4.

Evaluation of Mtb growth in the Bactec MGIT 960 system. All bacteria were preincubated for 4 h in a 37°C incubator. For standard, each Mtb inoculum ranging from 10⁷ to 10¹ CFU were placed without drug treatment during analysis. Then, a positive control, solvent control, and 400 μg/mL, 200 μg/mL, 100 μg/mL, and 50 μg/mL febuxostat-treated groups were placed in the BD Bactec MGIT 960 system. Then, analysis against (A) Mtb H37Rv ATCC 27294, (B) Erdman, and (C) HN878 strains were performed.

TABLE 3.

Time to detection of growth in Bactec MGIT 960 upon drug treatment after inoculation of Mtb H37Rv

Bacterial strain	Inoculation (CFUs)	Drug treatment (μg/mL)	Time to detection (days)
H37Rv ATCC 27294	105	None	4.8 day
	103a	None	8.3 day
	105	RIF 0.25	Not detected over 30 days
	105	INH 0.1	Not detected over 30 days
	105	FBX 50	5.9 day
	105	FBX 100	16.5 day
	105	FBX 200	Not detected over 30 days

^a100-fold diluted growth control.

Intracellular inhibitory effect of febuxostat in Mtb-infected macrophages.

We assessed febuxostat activity against mycobacterial growth inside BMDMs. Before evaluating the intracellular inhibitory effect, viability assays were performed. Febuxostat did not influence cell viability when applied at the maximum concentration of 200 μg/mL, indicating that febuxostat does not interfere with cell viability (Fig. 5A). Subsequently, BMDMs were infected with Mtb H37Rv at multiplicity of infection (MOI) = 1 and incubated in the absence or presence of the drug. Importantly, febuxostat was active against the Mtb H37Rv by concentration-dependently inhibiting growth inside macrophages (Fig. 5B).

FIG 5.

Inhibition of intracellular Mtb growth by febuxostat. (A) Febuxostat-derived cell cytotoxicity against BMDMs was analyzed. Lipopolysaccharide stimulation was used as a positive control. Statistically significant differences between the groups were determined using an unpaired Student's t test. n.s., not significant comparing nontreated and febuxostat-treated group. (B) This experiment was conducted independently three times, and representative data were presented. BMDMs were infected with Mtb H37Rv ATCC 27294 at an MOI = 1 and then treated with febuxostat at concentrations ranging from 200 μg/mL to 25 μg/mL. After a 3-day infection, bacterial CFU was analyzed. The data are expressed as the mean ± SD (n = 8). Statistically significant differences between the groups were determined using an unpaired Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 comparing nontreated and febuxostat-treated groups.

In vivo efficacy of febuxostat in a murine model of tuberculosis.

Based on its potential as an antimycobacterial drug candidate, we further evaluated febuxostat in an established TB murine model. After Mtb H37Rv infection, febuxostat was administered orally for 26 days. Then, bacterial load in the lungs and spleens, and lung inflammation, of infected mice was investigated at 4 weeks postinfection (Fig. 6A). Compared to the vehicle-administered group, pulmonary inflammation tended to be ameliorated in the febuxostat-administered group, although there was no significant difference between groups (P = 0.0635; vehicle-treated versus febuxostat-treated) (Fig. 6B). Nevertheless, the significant bacterial reduction was observed in both organs in the febuxostat-treated group compared to the vehicle-treated group (Fig. 6C). Because of its inhibitory effects, especially on bacterial replications, in mice, febuxostat might be repurposed for use against Mtb infection.

FIG 6.

In vivo evaluation of the antibacterial activity of febuxostat in an Mtb-infected murine model. (A) Experimental scheme for measuring the efficacy of febuxostat against Mtb. (B) The superior lobes of the right lung of each subset of mice were analyzed using hematoxylin and eosin (H&E) staining, and representative lung lobes were depicted as gross images at 28 days postinfection (10×: scale bar = 2.0 mm; 40×: scale bar = 500.0 μm). The experimental results indicate the percentage of inflamed area in the lung and are presented by box and whisker plots. (C) CFU in the lungs and spleens of each group at 4 weeks postinfection were analyzed by enumerating the viable bacteria. Mann-Whitney rank tests were used to compare groups. n.s., not significant; *, P < 0.05; **, P < 0.01.

DISCUSSION

We evaluated the antibacterial activity of febuxostat against Mycobacterium species. Compared to other antigout drugs used in this study, such as allopurinol and topiroxostat, febuxostat displayed antibacterial activity against Mtb (Fig. 1). Moreover, febuxostat specifically targeted Mtb strains and Mtb-close strains such as MTBC, M. kansasii with their clinical isolates, and M. shinjukuense (Fig. 2; see also Fig. S1). Besides its direct effect on Mtb growth, intracellular growth inside BMDMs was also inhibited, and improved protection was further established in both lungs and spleens of the febuxostat-administered group (Fig. 3 to 6). Although febuxostat has already been exploited in Mtb infection to ameliorate hyperuricemia (23), this is the first study to evaluate febuxostat as an antimycobacterial agent via drug repurposing.

Drug repurposing is a novel strategy that is gaining interest because it facilitates the discovery of efficient and new therapies and reduces the time required to develop new anti-TB drugs (8). Based on this approach, we had reported the novel function of colchicine, an FDA-approved anti-gout drug as an anti-Mtb agent via regulation of the host immune system (37). Subsequently, we explored alternative agents to combat TB and focused on hyperuricemia following the use of PZA and EMB, which are first-line TB drugs. Uric acid is involved in the purine metabolic pathway and is required for reproduction in intracellular pathogens (27). Additionally, uric acid can be used as a nitrogen source, to synthesize proteins and enzymes for bacterial growth. Nitrogen is also used as a protein/enzyme source in Mtb (38). Therefore, we hypothesized that targeting uric acid production with antigout drugs, which are currently being used by patients with hyperuricemia during anti-TB treatments, might be an efficient way of combating TB. Based on that, we evaluated antigout drugs such as allopurinol, febuxostat, and topiroxostat for their antibacterial effects against Mycobacterium species. Among the tested drugs, only febuxostat displayed significant antimycobacterial activities (Fig. 1). Furthermore, febuxostat was also effective in controlling the growth of M. kansasii, which harbors intrinsic PZA resistance (39). Collectively, these results demonstrate that a distinct structural characteristic of febuxostat may contribute to this function rather than its role as an antigout drug, by reducing the production of uric acid, which furtherly weakened the gout connection.

Febuxostat exhibited specific antibacterial activity against Mtb and MTBC. However, except for M. kansasii and M. shinjukuense, NTM strains were not affected by febuxostat (Fig. 2). Interestingly, clinical features of pulmonary M. kansasii infection were almost identical to those of Mtb in terms of sharing a similar treatment regimen and showing similar clinical manifestations such as chest pain, cough, hemoptysis, fever, night sweats, malaise, and weight loss (40). In addition, M. shinjukuense, newly identified nontuberculous mycobacteria in 2011, displays high homology in the gene sequence of 16S rRNA compared to that of Mtb H37Rv, although the clinical features of pulmonary M. shinjukuense infection have been insufficiently accumulated (41, 42). Furthermore, based on the phylogeny study, these two species were closely related to Mtb complex among NTM species, including M. lacus not affected by febuxostat (32 to 34).

Thus, we investigated the genes conserved as xanthine dehydrogenase with the comparative genomic approach for all Mycobacterium species used in this study. Specifically, we verified a putative operon sequence, including the rv0370c, rv0371c, and rv0372c genes, which are known as a hypothetical oxidoreductase, molybdenum cofactor, and xanthine dehydrogenase accessory factor, respectively, only in the NTM species closely related to Mtb. Although, compared with M. kansasii, M. lacus generally has a closer genetic similarity to Mtb (33, 34), a putative operon sequence of M. kansasii showed closer genetic similarity than M. lacus to Mtb (see Fig. S4). Such genetic variations in a putative operon sequence may differentially influence the antimycobacterial function of febuxostat against M. lacus and M. kansasii. Furthermore, the efficacy of new compounds against clinically relevant Mtb strains should be evaluated, because of differential treatment outcomes between clinical isolates and laboratory-adapted strains (43, 44). That the antimycobacterial function of febuxostat remained intact even against clinical isolates of Mtb (see Fig. S1A) demonstrated that febuxostat could be a prospective anti-TB drug. Although we did not test the antimycobacterial function of febuxostat against Mtb complex-related neighbors such as M. decipiens and M. riyadhense (33, 34), based on multiple alignments and growth inhibition observed in M. kansasii, M. shinjukuense, and M. lacus (Fig. 2; see also Fig. S4), it is possible that febuxostat functions by targeting Mycobacterium species that harbor the putative operon sequence containing rv0370c to rv0372c similarity, rather than by targeting all close neighbors of the Mtb complex.

Next, we validated the antibacterial role of febuxostat by Bactec MGIT 960 analysis and MIC test for resazurin. Febuxostat concentration-dependently exhibited antimicrobial activity with sustained effects (Fig. 3 and 4). Thus, the role of febuxostat against Mtb was further supported with these assays by especially displaying comparable bactericidal activity at 200 μg/mL of febuxostat as observed in RIF- and INH-treated groups, respectively (Table 3).

Although the exact means of ensuring intracellular Mtb survival remain debatable (45, 46), potential new anti-TB drugs should exhibit activity against Mtb inside host cells. For instance, in phase 2 clinical trial, Q203 potently blocked Mtb growth in vitro and in macrophages, and these features made Q203 a promising candidate for TB treatment (47). Therefore, we evaluated whether febuxostat may target Mtb inside host cells. During infections, macrophages act as reservoirs of replicating mycobacteria. Thus, anti-TB drugs need to be functional inside the host cell. Thus, similar to in vitro dormancy models, Mtb-infected macrophages are a beneficial tool for developing improved TB drugs (48). Notably, the growth of Mtb inside BMDMs was significantly inhibited by febuxostat in a dose-dependent manner not interfering with cell viability (Fig. 5). Next, the in vivo efficacy of febuxostat in the Mtb-infected murine model was tested. After 4 weeks postinfection, both the lungs and spleens from febuxostat-treated mice displayed markedly reduced bacterial loads compared to those from vehicle-treated mice (Fig. 6).

Our current study has certain limitations; hence, it deserves further consideration. First, despite showing anti-Mtb infection ability in vitro, in vivo, and in macrophages, febuxostat may not be implemented in the direct treatment of Mtb infections in clinical settings because of the high concentration levels required for anti-Mtb activity compared to that of anti-Mtb drugs such as RIF and INH (Table 3). Given that the standard regimen for treating TB lasts at least 6 months, the use of febuxostat in clinical settings should be carefully approached in terms of determining concentration, the precise length of treatment, and adverse drug–drug interaction because febuxostat is currently used only in hyperuricemia during anti-TB medications. For the treatment of hyperuricemia and gouty arthritis in humans, 40 or 80 mg/day oral dosage is generally recommended. However, a dosage of 120 mg/day could be administered according to the symptoms and countries (49). With regard to the body weight, the recommended oral dosage in humans weighing on an average 70 kg is at least >3-fold diluted concentration to that employed in our in vivo study (5 mg/kg). Although further evaluations on whether oral dosing would be expected to reach the concentrations that demonstrate in vitro anti-Mtb activity of febuxostat were required to optimize the regimen, febuxostat significantly reduced the bacterial loads, accompanied by a tendency to ameliorate lung inflammation (Fig. 6). Pharmacokinetic, pharmacodynamic, and safety profiles of febuxostat in mice should be determined for securing its toxicity profile during Mtb infection. In addition, febuxostat should be investigated for its tolerance at higher doses and prolonged exposures without any clinical signs. Evaluation of the drug localization or concentration, particularly in lungs, is also one of the desirable properties for an anti-Mtb drug (47, 50). Along with its desirable safety and pharmacokinetic/pharmacodynamic profiles, the use of febuxostat against TB should be further warranted. The anti-Mtb activity of febuxostat was not evaluated in combination with anti-Mtb drugs such as INH, RIF, PZA, and EMB in the murine TB model to clarify the beneficial effects of combination therapy, considering that febuxostat is being used clinically for ameliorating hyperuricemia during anti-TB medications. However, febuxostat-induced disruptions in bacterial membrane structure may contribute to a synergistic effect, in combination with first-line anti-Mtb drugs, similar to that elicited by verapamil (16). Finally, further research is required to explore and validate whether the putative operon sequence is a precise target of febuxostat within Mtb components, using an analytical approach such as RNA sequencing. In addition, it might be also noteworthy whether the derivatives of febuxostat, made based on its structure, could play antimycobacterial function at a comparable concentration as RIF and INH did (Table 3). Thus, further elucidation is required to steer the development of a new class of drugs.

Nevertheless, our results indicate that febuxostat is a potent inhibitor of Mtb growth both in vitro and in vivo. Therefore, this study is a credible example of drug repurposing, and antimycobacterial mechanisms of febuxostat warrant further investigation.

MATERIALS AND METHODS

Mycobacterial strains and culture conditions.

The following strains and isolates were purchased: Mtb H37Rv ATCC 27294, Mtb H37Ra ATCC 25177, M. intracellulare ATCC 13950, M. avium ATCC 25291, M. abscessus ATCC 19977, M. kansasii ATCC 12478, M. fortuitum ATCC 49403, M. fortuitum ATCC 49404, and M. smegmatis ATCC 700084 from the American Type Culture Collection (ATCC, Manassas, VA, USA); Mtb K strain from the Korean Institute of Tuberculosis (KIT, Osong, Chungcheongbuk-do, South Korea); Mtb HN878 and CDC1551 strains from the strain collection of the International Tuberculosis Research Center (ITRC; Changwon, Gyeongsangnam-do, South Korea); M. shinjukuense JCM 14233 and M. lacus JCM 15657 strains from the Korean Institute of Tuberculosis (KIT; Cheongju, Chungcheongbuk-do, South Korea); Mtb clinical isolates (#2, #7, #10, #11, #20, and #23) from the strain collection bank of the ITRC; M. massiliense CIP 108297 from the Collection of Institute Pasteur; M. kansasii clinical isolate SMC #1 from the Samsung Medical Center (SMC; Seoul, South Korea); M. microti from the strain collection of the ITRC; and M. bovis AN5, BCG Pasteur 1173P2, Tokyo-172, and Danish-1331 strains from the strain collection of the ITRC. M. bovis clinical isolates #3, #4, #5, #6, and #10 were provided by Sungmo Je of Yonsei University (Seoul, South Korea) (51). Strains were cultured in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.02% glycerol and 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC; Becton, Dickinson, Sparks, MD) for 28 days at 37°C. Single-cell suspensions of each strain were prepared as previously described (31).

Drugs and chemicals.

Febuxostat (Sigma-Aldrich, St. Louis, MO, USA), allopurinol (Sigma-Aldrich, St. Louis, MO, USA), and topiroxostat (Cayman Chemical, Ann Arbor, MI, USA) were diluted in dimethyl sulfoxide (DMSO), 1 M NaOH, and DMSO, respectively. All drugs and chemicals were stored in the dark at 4°C until use.

In vitro antimycobacterial susceptibility testing of antigout drugs.

Bacteria were incubated in 96-well cell culture plates (SPL Life Science, Pocheon, Gyeonggi-do, South Korea) at a density of 1.5 × 10⁵ CFU/well in a 200-μL Middlebrook 7H9 broth supplemented with 10% OADC. The highest concentration of the drugs, including febuxostat, allopurinol, and topiroxostat, was 200 μg/mL; the drugs were 2-fold serially diluted to 12.5 μg/mL and incubated at 37°C. After incubation, the bacteria were treated with resazurin (Sigma-Aldrich) for 24 h and the inhibitory concentration was confirmed (52).

Time-kill curve analysis.

The Mtb H37Rv ATCC 27294 strain was seeded in 20-mL 7H9 Middlebrook broths supplemented with OADC. Febuxostat was added at the above-indicated concentrations and placed in a 50-mL conical tube (SPL Life Science, Pocheon, Gyeonggi-do, South Korea). Then, the lid was sealed with parafilm (Sigma-Aldrich, St. Louis, MO, USA), and the tubes were incubated in a shaking incubator maintained at 140 rpm and 36°C for 10 days. Additionally, for the previously collected bacteria in the conical flask, CFU were spotted on 7H10 agar plates daily for 28 days. Then, the plates were kept in an incubator at 37°C. CFU were counted 12 to 14 days after incubation.

Scanning electron microscopy.

Mtb H37Rv was observed with scanning electron microscopy 2 days after febuxostat treatment. Briefly, specimens were fixed in Karnovsky fixative (2% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4; all reagents were purchased from Sigma-Aldrich) for 24 h and washed twice for 30 min in 0.1 M phosphate buffer. They were then fixed with 1% OsO₄ (Polysciences, Warrington, PA, USA) for 2 h, dehydrated in an ascending series (50% to 100%) of graduated ethanol (Sigma-Aldrich), and dried using a Critical Point Dryer (Leica EM CPD300). Samples were coated with platinum using an ion sputter (Leica EM ACE600) and observed with a field emission scanning electron microscope (Zeiss Merlin).

Phenotypic assay of antimycobacterial susceptibility.

The prepared 7H10 Middlebrook agar medium contained OADC, 400 μg/mL, 200 μg/mL, 100 μg/mL, and 50 μg/mL of febuxostat, and 0.2% (vol/vol) DMSO. Additionally, the Mtb H37Rv ATCC 27294, HN878, and Erdman K strains were prepared by serial dilution from 10⁸ CFU. Then, the bacterial culture in the PBS was serially diluted 10⁴ and 105-fold, spotted on the 7H10 agar plates, and incubated at 37°C for 8 weeks to visualize 30 to 300 CFU.

Bactec MGIT 960-automated mycobacterial detection analysis.

Three Mtb strains, H37Rv ATCC 27294, HN878, and Erdman K, were preincubated in Middlebrook 7H9 broth supplemented with OADC for 4 h at 37°C. Next, 0.1 mL 7H9 broth containing bacteria was mixed with 0.8 mL BBL MGIT PANTA mixed solution containing polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin. The resultant mixture was then mixed with 10.5 mL of BBL MGIT OADC enrichment supplemented with OADC and 0.1 mL of 7H9 broth containing 400 μg/mL, 200 μg/mL, 100 μg/mL, and 50 μg/mL of febuxostat, respectively. Then, the final reaction mixture was placed in an MGIT Bactec 12B vial (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and analyzed using Bactec MGIT 960 (BD Biosciences, USA) according to the manufacturer’s instructions. In another set of experiments, 0.8 mL of OADC supplement was added to each of the MGIT tubes. After 0.1 mL of the anti-TB agents (RIF, 0.25 μg/mL; INH, 0.1 μg/mL) and febuxostat (50, 100, and 200 μg/mL) was added respectively to each of the MGIT tubes, 0.5 mL of Mtb H37Rv ATCC 27294 inoculum (10⁵ CFU) was well mixed. For the growth control, 100-fold diluted inoculum (10³ CFU) without drug treatment was used. Then, the final reaction mixture was analyzed using Bactec MGIT 960 (BD Biosciences, USA) according to the manufacturer’s instructions.

Mice.

All animal studies were carried out in accordance with the guidelines of the Korean Food and Drug Administration (KFDA). The experimental protocols used in this study were reviewed and approved by the Ethics Committee and Institutional Animal Care and Use Committee (permit number: 2018-0229) of the Laboratory Animal Research Center at Yonsei University College of Medicine (Seoul, South Korea). Specific pathogen-free female C57BL/6J (6 to 7 weeks old) mice were purchased from Japan SLC, Inc. (Shizuoka, Japan) and maintained under barrier conditions in an ABSL-3 biohazard animal facility at the Yonsei University Medical Research Center. The animals were fed a sterile commercial mouse diet and provided with water ad libitum. The mice were monitored daily, and none showed any clinical symptoms or illness.

Murine bone marrow-derived macrophage culture.

Isolated mouse bone marrow cells were differentiated into BMDMs using L929-conditioned medium. Cells were maintained in L929-conditioned Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 100 units/mL of penicillin/streptomycin and incubated with 5% CO₂ at 37°C.

Cell cytotoxicity test.

BMDMs were plated at 5 × 10⁴ cells/well along with the drug in a 96-well cell culture plate. The highest drug concentration was 400 μg/mL, and this was 2-fold serially diluted to 25 μg/mL, added to the CCK-8 cell cytotoxicity test kit agent (Dojindo Laboratories, Kumamoto, Japan), and incubated with 5% CO₂ for 3 h at 37°C. An Epoch microplate spectrophotometer was used (BioTek Instruments, VT, USA) to analyze samples at 450 nm.

Measuring intracellular bacterial growth in macrophages.

After BMDMs were generated, they were incubated in a 48-well cell culture plate (SPL Life Sciences) for 24 h. Next, BMDMs were infected with Mtb H37Rv ATCC 27294 for 4 h at a multiplicity of infection of 1 (MOI). BMDMs were washed with PBS (Biowest Inc., Nuaillé, France) to remove extracellular bacteria and added to fresh DMEM. Then, they were grown in the incubator for 72 h with 5% CO₂ at 37°C. After incubation, the cells were lysed with 0.05% Triton X-100, and 10-fold serial dilutions were plated onto a 7H10 agar medium (Becton, Dickinson) to count the viable bacteria. Colonies were enumerated after incubation for 4 weeks at 37°C.

In vivo treatment efficacy of febuxostat against Mtb infection in a murine model.

Mice were exposed to Mtb H37Rv strain in the calibrated inhalation chamber of an airborne infection apparatus for 60 min at a predetermined dose (Glas-Col, Terre Haute, IN, USA); approximately 400 viable bacteria were delivered. For treatment, mice were administered febuxostat (5 mg/kg) diluted in 0.5% carboxymethyl cellulose (Sigma-Aldrich) orally from postinfection day 1. For bacterial enumeration, mice were euthanized with CO₂, and the lungs and spleens were homogenized. The number of viable bacteria was determined by plating serial dilutions of the organ homogenates onto Middlebrook 7H10 agar (Difco, USA) supplemented with 10% OADC (Difco, USA) and amphotericin B (Sigma-Aldrich, USA). Colonies were counted after a 4-week incubation at 37°C.

Histopathology.

For histopathological analysis, the right superior lobes of the lungs were preserved in 10% neutral buffered formalin overnight and embedded in paraffin. Then, the lungs were vertically or horizontally sectioned and stained with hematoxylin and eosin (H&E). The level of inflammation in the lungs was evaluated using the ImageJ software (National Institutes of Health, Bethesda, MD), as previously described (53).

Statistical analysis.

Data for all experiments are presented as the mean ± standard deviation (SD). Significant differences between samples in the two selected groups were assessed using an unpaired t test, and the Mann-Whitney rank test was used to compare the differences between two different groups. GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, California, USA, www.graphpad.com) was used for statistical analysis. A P of <0.05 was considered statistically significant.

Data availability.

The data sets generated and analyzed in this study are available from the corresponding author upon reasonable request.