Sudapyridine (WX-081) antibacterial activity against Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium chelonae in vitro and in vivo

ABSTRACT Sudapyridine (WX-081) is a structural analog of bedaquiline (BDQ), which shows anti-tuberculosis and non-tuberculous mycobacteria (NTM) activities but, unlike BDQ, did not prolong QT interval in animal model studies. This study evaluated the antibacterial activity of this novel compound against Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium chelonae in vitro and in vivo. The minimum inhibitory concentration (MIC) of WX-081 against three kinds of non-tuberculous mycobacteria (NTM) clinical strains was determined using microplate-based alamarBlue assay (MABA), and the antibacterial activity of WX-081 against NTM in J774A.1 cells and mice was evaluated. MIC ranges of WX-081 against clinical strains of M. avium and M. abscessus were 0.05–0.94 μg/mL, 0.88–7.22 μg/mL (M. abscessus subsp. abscessus), and 0.22–8.67 μg/mL (M. abscessus subsp. massiliense), respectively, which were slightly higher than those of BDQ. For M. avium, M. abscessus, and M. chelonae, WX-081 can reduce the intracellular bacterial load by 0.13–1.18, 0.18–1.50, and 0.17–1.03 log10 colony forming units (CFU)/mL, respectively, in a concentration-dependent manner. WX-081 has bactericidal activity against three NTM species in mice. WX-081 exhibited anti-NTM activity to the same extent as BDQ both in vivo and in vitro. WX-081 is a promising clinical candidate and should be studied further in clinical trials. IMPORTANCE Due to the rapidly increased cases globally, non-tuberculous mycobacteria (NTM) disease has become a significant public health problem. NTM accounted for 11.57% of all mycobacterial isolates in China, with a high detection rate of Mycobacterium abscessus, Mycobacterium avium, and Mycobacterium chelonae during 2000–2019. Treatment of NTM infection is often challenging, as natural resistance to most antibiotics is quite common among different NTM species. Hence, identifying highly active anti-NTM agents is a priority for potent regimen establishment. The pursuit of new drugs to treat multidrug-resistant tuberculosis may also identify some agents with strong activity against NTM. Sudapyridine (WX-081) is a structural analog of bedaquiline (BDQ), which was developed to retain the anti-tuberculosis efficacy but eliminates the severe side effects of BDQ. This study initially evaluated the antimicrobial activity of this novel compound against M. avium, M. abscessus, and M. chelonae in vitro, in macrophages and mice, respectively.

is more difficult to treat than tuberculosis.NTM disease has the characteristics of a long treatment period, multidrug combination therapy, and limited active drugs, which suggests that we need to supplement the anti-NTM drug pipeline as soon as possible.
It has been reported that bedaquiline (BDQ) had good activity against NTM in vitro (2).Although BDQ is not explicitly listed in the guidelines as a treatment for NTM disease, it has been successfully used clinically in the empiric therapy of M. avium, M. abscessus, and Mycobacterium fortuitum infections (3)(4)(5).Sudapyridine (WX-081) is a new compound obtained by reasonable structural modification and optimization of BDQ.It retains the pharmacological scaffold of BDQ (composed of a quinoline heterocyclic nucleus and side chains of tertiary alcohol and tertiary amine groups), replacing the quinoline group with the pyridine group (6).Compared with BDQ, WX-081 had a higher exposure to target organs in the lung after oral administration and did not prolong the QT interval in animal model studies (6).In vitro, the antibacterial activity of WX-081 was comparable to that of BDQ against both susceptible and resistant Mycobacterium tuberculosis and different NTMs (7), and WX-081 and BDQ had similar efficacy in acute and chronic mouse tuberculosis models infected with small doses of aerosol (6).
Therefore, we evaluated the antibacterial activity of WX-081 against M. avium, M. abscessus, and M. chelonae in vitro, in macrophages and, firstly, in mice, respectively, in order to provide new drug candidates for the treatment of NTM disease.

In vitro antimycobacterial activity
The in vitro antibacterial activity of WX-081 and BDQ against 31 clinical strains of NTM (9 strains of M. avium, 13 strains of M. abscessus subsp.abscessus, and 9 strains of M. abscessus subsp.massiliense) was tested by microplate-based alamarBlue assay (MABA).The experiment was repeated three times.The minimum inhibitory concentration (MIC) of these drugs against the corresponding NTM standard strains was determined simultaneously in each experiment as a control.

Intracellular antimycobacterial activity
To evaluate the antibacterial activity of WX-081 and BDQ against intracellular NTMs in J774A.1 cells, the optimal multiplicity of infection (MOI) and infection time of M. abscessus to J774A.1 cells were determined.M. abscessus bacterial solution (diluted with Dulbecco's modified Eagle medium [DMEM] medium containing 10% fetal bovine serum [FBS]) was added to J774A.1 cells at MOI of 1, 3, and 5, respectively.After co-incubation for 6, 12, and 24 hours, the cell growth status was observed, and then the cells were lysed to count the colony forming units (CFU) of intracellular mycobacteria.The experiment was repeated twice.The results are shown in Table 3.
The results showed that all combinations of MOI and infection time allowed M. abscessus to infect macrophages to a great extent.However, J774A.1 cells were observed to aggregate and lose refraction at 24 hours after infection, and some cells were suspended in the culture medium.Aggregated macrophages have insufficient adherent capacity, and washing with 1× phosphate buffer saline(PBS) prior to lysis may cause them to fall off the plate, ultimately resulting in CFU counts less than the cells infected for 12 or 6 hours.After 12 hours of infection, no macrophages died, but some cells gathered  into clusters, and the bacterial count was slightly lower than that of cells infected for 6 hours.The J774A.1 cells infected for 6 hours grew well, all cells did not form clumps or die, and the intracellular bacteria could reach more than 6 log 10 CFU/mL.After 6 hours post-infection, at MOI of 1, 3, and 5, while there was no cell death or aggregation, higher MOI resulted in increased susceptibility to cell damage, thereby affecting the evaluation of intracellular antibacterial activity of BDQ and WX-081.To keep the vector cells in an optimal state to cope with the toxicity of the compound, we finally determined that the optimal MOI for M. abscessus infection of the J774A.1 cells was 1, and the optimal infection time was 6 hours.However, the intracellular bacterial load was only ~4 log 10 CFU/mL 6 hours after infection with M. avium with MOI = 1 (data not shown).Such results demonstrated that the optimal MOI for different NTM-infected macrophages was different.In this study, the J774A.1 cells were infected with M. avium and M. chelonae, respectively.The macrophages cultured in 24-well plates were added to mycobacteria solution with MOI 1, 3, and 5, respectively.The cell growth was observed, and the number of mycobacteria in the cells was counted 6 hours after infection.The experiment was repeated twice, and the results are shown in Table 4.
The results showed that after 6 hours of incubation, both M. avium and M. chelonae could enter macrophages in a large proportion.At 6 hours after infection, the intracellu lar bacterial counts of M. avium were 5.87 ± 0.05, 6.63 ± 0.10, and 6.54 ± 0.07 log 10 CFU/mL at MOI of 1, 3, and 5, respectively, whereas the intracellular bacterial counts of M. chelonae were 6.24 ± 0.08, 6.95 ± 0.04, and 6.74 ± 0.02 log 10 CFU/mL at MOI of 1, 3, and 5, respectively.Therefore, it can be preliminarily determined from the results that the optimal MOI of M. avium was 3, and the optimal MOI of M. chelonae was 1.
Therefore, under the conditions of optimal MOI and optimal infection time, the antibacterial activities of BDQ and WX-081 against M. avium and M. chelonae in the J774A.1 cells were tested at 1× MIC, 5× MIC, and 10× MIC, respectively.Since the concentration of WX-081 at 10× MIC for M. chelonae was greater than the 50% inhibitory concentration (IC 50 ) for J774A.1 cell, the intracellular antibacterial activity of WX-081 at this concentration was not determined.The experiment was repeated twice, and the specific results are shown in Table 5 and Fig. 1.BDQ and WX-081 showed similar antibacterial activity against mycobacteria within the J774A.1 cells.BDQ reduced intracellular bacterial load by 0.03-1.19,0.11-1.54,and 0.27-1.34log 10 CFU/mL for M. avium, M. abscessus, and M. chelonae, respectively.WX-081 reduced the intracellular bacterial load by 0.13-1.18,0.18-1.50,and 0.17-1.03log 10 CFU/mL, respectively.The activities of the two compounds were concentration dependent.

In vivo efficacy in mice
Our previous experiments showed that the administration of dexamethasone to mice before and after infection increased the burden of mycobacteria in the organs of mice and made the effect of antibiotics more pronounced (8).So, in this study, we used the same method to establish the mouse models of M. avium, M. abscessus, and M. chelonae infection, and investigated the anti-NTM activity of BDQ and WX-081 in vivo (Fig. 2).
After 4 weeks of treatment, there were no differences in lung index and spleen index between the BDQ and WX-081 groups (Table 6).The results showed that BDQ and WX-081 showed similar bactericidal activity against M. avium, M. abscessus, and M. chelonae in mice after 4 weeks of monotherapy; that is, they could reduce the organ

DISCUSSION
With the rising incidence of NTM disease worldwide, how to treat NTM lung disease safely and effectively has become the focus of scientists.Because most of the gen eral antibiotics are ineffective, the pulmonary infection caused by NTM is one of the internationally recognized refractory chronic infectious diseases, and it is urgent to find new and effective treatment drugs.
BDQ is a diarylquinoline antibiotic, which can interfere with the proton motive force (PMF) of mycobacteria and inhibit ATP synthase of mycobacteria (9)(10)(11).Research has shown that the survival rate of zebrafish embryos infected with M. abscessus was significantly higher than that of untreated zebrafish when exposed to the lowest concentration of BDQ (1 µg/mL), which was comparable to that of imipenem 360 µg/mL, and the antibacterial activity was concentration dependent (12).Le Moigne et al. ( 13)  reported that mice given 30 mg/kg BDQ had significantly fewer M. abscessus in the lungs and spleen compared with mice given 150 mg/kg amikacin (AMK).In the nonobese diabetic/severe combined immunodeficient (NOD SCID) mouse model, 10 mg/kg of BDQ had similar anti-M.abscessus activity as 250 mg/kg of clarithromycin (CLR) (14).In a small clinical trial conducted by Philley et al. (15), 9 of 10 (90%) treatment-unsuc cessful patients infected with M. abscessus or M. avium experienced symptom relief after 2 months of treatment with a BDQ-containing regimen.At 6 months of treatment, sputum culture results improved in 60% of patients.WX-081 has not been used in clinical treatment of NTM-infected patients.
Ruth et al. ( 2) reported a BDQ MIC ranging from 0.25 to 0.5 µg/mL for rapid-growing mycobacteria (RGM) and 0.03 to 0.25 µg/mL for slow-growing mycobacteria (SGM) in 7H9 liquid medium.Our study also showed that both compounds had good antibacterial activity against clinical strains of M. avium and M. abscessus in vitro.In addition, BDQ showed excellent bactericidal activity against M. abscessus within the human leukemia monocytic cell line THP-1 in a concentration-dependent manner, which was consistent with our experimental results.However, 1× MIC and 5× MIC BDQ or WX-081 had little inhibitory effect on M. avium within the J774A.1 cells.Kilinç et al. ( 16) also reported that BDQ had only partial inhibitory activity against M. avium strain 101 at 1.74 µg/mL in primary human macrophages (difference not statistically significant).However, when the concentration of BDQ or WX-081 was increased to 10× MIC, the antibacterial activity of the two compounds against M. avium increased significantly.Therefore, BDQ or WX-081 may be a potential drug for the treatment of NTM disease.
As there is no study on the anti-NTM effect of WX-081 in macrophage and mice, our group conducted the first evaluation of the anti-NTM activity of WX-081 at both macrophage and animal levels.In our established mouse model, WX-081 exhibited bactericidal effects on M. avium, M. abscessus and M. chelonae.Therefore, WX-081 and BDQ have similar antibacterial activity against NTMs both in vitro and in vivo.
Our study also has some limitations.First, the number of clinical strains tested was small, and all of the tested isolates were obtained from Beijing Chest Hospital.Conse quently, some isolates could be related epidemiologically.Second, there was a substan tial variation in MIC values against M. abscessus.Only one reference strain was tested in macrophages and in vivo.In our future studies, more species of M. abscessus will be used in macrophages and in vivo studies.Nonetheless, our study provides a comprehensive description of the antibacterial activity of WX-081 against M. avium, M. abscessus, and M. chelonae in vitro and in vivo.

Conclusion
Sudapyridine (WX-081) effectively inhibited the growth of M. avium, M. abscessus, and M. chelonae in vitro and in vivo, and its activity was equivalent to that of BDQ.As such, Sudapyridine (WX-081) represents a potential clinical candidate for incorporation into novel therapeutic anti-NTM regimens.

MIC measurements
The MICs of CLR, BDQ, and WX-081 against M. avium and M. abscessus reference strains and clinical strains (no clinical strains of M. chelonae were collected) are tested using the MABA.Specific experimental methods refer to previous studies (17).

Intracellular activity assay
The J774A.1 cells were grown in cell culture dishes in DMEM containing 10% FBS.The cells were detached with trypsin digestion and were resuspended to a final concentration of 4 × 10 5 cells/mL.Aliquots (1 mL) of cell suspension were distributed into 24-well plates, and the plates were incubated at 37°C in a 5% CO 2 incubator for 12 hours (18).
The MOI was the ratio of mycobacterial concentration to cell concentration when NTM was used to infect cells.The best MOI and the best time of infection were defined as the MOI and the time of infection when the cells infected with mycobacterium had the most amount of bacteria and the most complete cell morphology.The NTMs were the reference strains of M. abscessus, M. avium, and M. chelonae, respectively.The culture medium of DMEM (containing 10% FBS) was used to mix the M. abscessus in a logarith mic growth period into 4 × 10 5 CFU/mL (MOI = 1), 1.2 × 10 6 CFU/mL (MOI = 3), and 2 × 10 6 CFU/mL (MOI = 5).Infection was carried out for 6, 12, and 24 hours, followed by three times of washing with 1× PBS to remove the extracellular mycobacteria.Monolayers were visually inspected under the microscope to ensure they remained intact, and then the medium was removed and the macrophages were lysed with 200 µL of 0.1% sodium dodecyl sulfate.Then the lysates were diluted with fresh media and plated onto 7H10 plates supplemented with 10% OADC to measure the CFU (19).The optimal MOI values of M. avium and M. chelonae were determined by the same method after the optimal infection time was determined.
The J774A.1 cells were co-incubated with DMEM medium containing 10% FBS and different concentrations of BDQ and WX-081 (1, 5, and 10 times the MICs of the compounds against the standard strains) for 48 hours (20), and the cells were lysed at 48 hours and CFU counts were performed.Cell lysis and CFU counts were then performed as in our previous study.Control wells received drug-free medium (19).

Establishment of infection in mice
A total of 75 BALB/c mice were given dexamethasone (5 mg/kg) by gavage from 7 days before infection to 6 days after infection, once a day, 0.2 mL each time, and no drug was given on the day of infection.Normal saline was used to dilute the M. avium, M. abscessus, and M. chelonae bacterial solution at the logarithmic growth stage to 1 × 10 7 CFU/mL.These 75 mice were randomly divided into four groups and infected with three NTMs by tail vein injection (0.2 mL each) (8), respectively.
Five mice infected with different NTMs were randomly grabbed and sacrificed 1 day after infection (D-6) and on the day of treatment initiation (D0).Serial dilutions of tissue homogenates were inoculated on 7H10 solid medium, and the initial number of mycobacteria implanted in lungs and spleens was determined.

Experimental chemotherapy trials
Fifteen mice infected with each NTM were randomly divided into three groups.One group was treated with CMC as a control group, and the other two groups were treated with BDQ and WX-081 on day 7 post-infection (D0).Both BDQ and WX-081 were suspended in CMC solution as single agents and were given for 28 days (seven times weekly, 0.2 mL each time) by gavage.The dose of BDQ was 25 mg/kg.Since WX-081 is a structural analog of BDQ and has similar antibacterial activity against NTMs in vitro and in macrophages to BDQ, we chose the same dose as BDQ.

Statistical analysis
Figures were plotted using GraphPad prism 8.0 (GraphPad Inc., USA) software, and the data were statistically analyzed using SPSS 26.0.Measurement data are expressed as mean ± standard deviation (x± s).Organ CFU counts were log-transformed before analysis, and the mean CFU counts were compared by one-way analysis of variance with Dunnett's post hoc test to control for multiple comparisons.The Mann-Whitney test was used to test for significance on non-normally distributed CFU data.A P value of 0.05 was considered significant.

TABLE 2
MICs of BDQ and WX-081 against clinical strains of M. abscessus a a The experiment was repeated three times.MICs are the mean of three experiments.

Mycobacterial species MOI Infection time (hours) CFU count (log 10 CFU/mL，x̄ ± s)
a The experiment was repeated two times.The CFU count is the mean ± standard deviation of the results of six experimental holes (n = 6).

TABLE 4
Results of determination of optimal MOI of J774A.1 cells infected with M. avium and M. chelonae (n = 6)

TABLE 6
Weight and organ index of mice infected with NTMs after drug treatment a a "D28" represents the blank control groups when therapy ends ； "BDQ and WX-081" represent the 28-day treatment with BDQ and WX-081 (25 mg/kg).