Non-tuberculous mycobacterial disease: progress and advances in the development of novel candidate and repurposed drugs

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens that can infect all body tissues and organs. In particular, the lungs are the most commonly involved organ, with NTM pulmonary diseases causing serious health issues in patients with underlying lung disease. Moreover, NTM infections have been steadily increasing worldwide in recent years. NTM are also naturally resistant to many antibiotics, specifically anti-tuberculosis (anti-TB) drugs. The lack of drugs targeting NTM infections and the increasing drug resistance of NTM have further made treating these mycobacterial diseases extremely difficult. The currently recommended NTM treatments rely on the extended indications of existing drugs, which underlines the difficulties of new antibiotic discovery against NTM. Another challenge is determining which drug combinations are most effective against NTM infection. To a certain extent, anti-NTM drug development depends on using already available antibiotics and compounds. Here, we aimed to review new antibiotics or compounds with good antibacterial activity against NTM, focusing on their mechanisms of action, in vitro and in vivo antibacterial activities.


Introduction
Non-tuberculous mycobacteria (NTM) refer to mycobacteria other than Mycobacterium leprae and Mycobacterium tuberculosis complex (MTC).NTM prevalence has demonstrated an increasing global trend in the last few decades (Yu et al., 2016;Brode et al., 2017;Lin et al., 2018;Santin et al., 2018;Cowman et al., 2019).In China, the proportion of NTM isolates among the specimen cultures positive for Mycobacterium species rose from 4.3% in 1979 to 22.9% in 2010 (Zhou et al., 2020).NTM are present in soil and water and are widely dispersed in the natural world.Most of the more than 200 isolated NTM species are non-pathogenic, whereas approximately 30 are clinically relevant.Although the nosocomial spread of M. abscessus (Mab) in cystic fibrosis patients was reported, the precise source of the remaining NTM species infection has no conclusive proof of transmission between people, which is assumed to be acquired via environmental exposure (Bryant et al., 2016;Wu et al., 2018).
The resistance of NTM to existing treatments is increasingly becoming an internationally recognized problem (Griffith et al., 2007;Johansen et al., 2020;Griffith and Daley, 2022).These include the most common clinical cases of Mab and Mycobacterium avium complex (MAC), which account for >90% of all documented NTM pulmonary disease (NTM-PD) cases (Yu et al., 2016).The regimens for Mab infections contain macrolide antibiotics, including clarithromycin (CLA) and azithromycin.However, most patients respond poorly to this class of antibiotics due to the inducible resistance phenotype that occurs during therapy, which is driven by the macrolide-inducible ribosomal methylase encoded by erm (41) (Richard et al., 2020).Mab has recently developed increased resistance to CLA, with reported resistance rates of 14%-38% (Broda et al., 2013;Zhuo et al., 2013;Lee et al., 2014).In addition, Mab also exhibits resistance to azithromycin (resistance rate of 10%), which is relatively lower than that of CLA (Pasipanodya et al., 2017;Hirama et al., 2020).A meta-analysis shows that the estimated pooled treatment success rate for patients with MAC disease was 39% (Xu et al., 2014), similar to the treatment outcomes of extensively drug-resistant tuberculosis (XDR-TB).Macrolides have also been used as the key medication in MAC therapy regimens, with a meta analysis indicating a treatment success rate of 60% (95% confidence interval [CI], 55.1%-64.8%)for macrolide-containing regimens in MAC-PDs (Kwak et al., 2017).Despite the combination strategy of fluoroquinolones, aminoglycosides, and surgical resection, macrolideresistant MAC lung disease has a poor treatment outcome, resulting in a 1-year all-cause mortality rate of 10% (Park et al., 2019).
However, no drugs have been specifically developed for the increasingly prevalence of NTM infection worldwide.All pharmaceuticals currently recommended by the American Thoracic Society for NTM therapy regimens are derived from the expanded indications of currently available medications.Over the last 50 years of tuberculosis (TB) research, only bedaquiline (BDQ), delamanid (DLM), and pretomanid are the new drugs that have been approved for commercialization by the US Food and Drug Administration (FDA) due to the challenges in developing novel medications.Therefore, maximizing the anti-NTM activity of presently accessible drugs or antibiotics may be useful for developing anti-NTM medications.In this study, we elaborate on new antibiotics or compounds with potent antimycobacterial activity against NTM.Reported drug type, targets, range of concentration, side effects, and structure are summarized in Table 1 and Figure 1.

Oxazolidinone
In vitro and in vivo investigations have shown that oxazolidinones are extremely effective at eliminating M. tuberculosis (Mtb) (Pstragowski et al., 2017;Guo et al., 2021).Their mechanism of action involves attaching to the 23S ribosome, composed of 5S and 23S rRNAs and 36 riboproteins (L1-L36), and blocking the tRNA at the peptidyl transferase center on the ribosomal subunit, thereby preventing microbial protein synthesis (Kadura et al., 2020).The first approved oxazolidinone drug was linezolid (LZD) in 2000.LZD has a wide spectrum of activity against gram-positive bacteria, including methicillin-resistant staphylococci, penicillin-resistant pneumococci, and vancomycin-resistant Enterococcus faecalis, as well as drug-resistant TB (Clemett and Markham, 2000).Subsequently, tedizolid (TZD) was approved by the FDA in 2014 as a novel, effective oxazolidinone precursor drug (Zhanel et al., 2015).Another promising drug is sutezolid (SZD, PNU-100480), a thiomorpholine analog of LZD, showing preliminary evidence of superior efficacy against Mtb and readily detectable bactericidal activity in the sputum and blood (Wallis et al., 2014).Similarly, delpazolid (DZD, LCB01-0371) is a novel oxazolidinone analog that exhibits broad-spectrum anti-gram-positive activity in vitro and in animal infection models as well as effective protection against Mab (Kim et al., 2017).
Among these four oxazolidinones, LZD and TZD have been used for the therapy of NTM infection, including MAC, M. abscessus, M. fortuitum and M. kansasii (Table 2).Consistent with in vitro antimycobacterial activity, two of 3 (67%) patients with M. chelonae and M. abscessus were cured or clinically cured with LZD-containing regimens.Seven of 12 (58%) patients with M. chelonae and Mab were cured or clinically cured with TZDcontaining regimens (Poon et al., 2021).Potential therapeutics of LZD and TZD and the detailed information of case reports using these two drugs are presented in Table 2 and Table S1.

Tetracycline
Tetracyclines have served as cornerstone antibacterial drugs for over 70 years.This class of drugs blocks protein synthesis by attaching to the 30S ribosomal subunit of the mRNA translation complex to inhibit the binding of aminyl tRNA to the mRNAribosomal complex (Yadav et al., 2023).Within this tetracycline group, tigecycline (TGC) is the first and only clinically available glycylcycline (Ng and Ngeow, 2022).Additionally, several new antibiotics from the tetracycline class were recently created to address the drawbacks of TGC, i.e., high rate of adverse gastrointestinal effects and mortality (Heaney et al., 2019).Among them, eravacycline (ERC), a synthetic fluorocycline, was authorized by the FDA in 2018.ERC is injected intravenously to treat difficult intra-abdominal infections caused by antibioticresistant bacteria (Anonymous, 2018a).In 2018, the FDA approved another drug called omadacycline (OMC) for managing acute bacterial skin and skin structure infections (ABSSI) and community-acquired bacterial pneumonia (CABP).This antibiotic can be administered orally (PO) or intravenously (IV) once per day (Anonymous, 2018b).Sarecycline (SAC), another oral drug, represents the first narrow-spectrum tetracycline-class Modes of actions of drugs or compounds against NTM.
Previous studies have demonstrated strong in vitro antimycobacterial activity of TGC against RGM and have recommended using TGC for treating Mab infections according to the current guidelines (Wallace et al., 2002;Lerat et al., 2014;Wallace et al., 2014;Haworth et al., 2017;Daley et al., 2020).Except for SAC, the other tetracyclines (i.e., TGC, OMC, and ERC) had MICs of ≤0.5 mg/ml against 27 RGM reference strains.In particular, ERC generally presented the lowest MICs, with MIC 90 values of 0.25 mg/mL, 0.25 mg/mL, and 0.06 mg/mL against the clinical isolates of Mab subsp.abscessus, Mab subsp.massiliense, and M. fortuitum, respectively (Shoen et al., 2019).In the case of TGC and OMC, equivalent in vitro inhibitory activities were found against these isolates, in which the TGC showed MIC 90 values that were lower or equal to those of OMC for Mab subsp.abscessus, Mab subsp.massiliense, and M. fortuitum (1 mg/mL, 1 mg/mL, and 0.25 mg/ mL versus 1 mg/mL, 2 mg/mL, and 2 mg/mL) (Zhang et al., 2023).
Furthermore, the in vitro antimycobacterial activity of ERC was similar to or better than that of CLA (MIC: 0.0625-2 mg/mL for susceptible strains), with CLA being the core antimycobacterial in the Mab treatment regimen (Fujiwara et al., 2021).Case reported by Frizzell M et al. showed that a patient with M. chelonae infection receiving OMC containing regimen was considered clinically improved (Frizzell et al., 2020).Notably, 44 of 95 (46%) patients with Mab-PD had 1 or more negative cultures, with 17 of 95 (18%) achieving culture conversion (Mingora et al., 2023).Furthermore, the adverse drug effects (ADEs) were relatively mild, 35 patients (29.9%) experienced direct ADEs, nausea/emesis occurring in 21.4% of patients (Table S1).Oral administration, high antimycobacterial activity and relatively low adverse effects make O M C a p r o m i s i n g d r u g f o r M a b a n d M .c h e l o n a e infection (Table 2).

Diarylquinoline
Diarylquinoline antibiotics act by inhibiting ATP synthase (subunit c encoded by atpE) required for oxidative phosphorylation (Kim et al., 2023).BDQ was the first diarylquinoline approved by the FDA in 2012 for treating pulmonary multidrug-resistant TB (MDR-TB) (Cox and Laessig, 2014).Recently, an innovative diarylpyridinated drug called sudapyridine (WX-081), which is formed by substituting the bromoquinoline of BDQ with a 5-phenylpyridine, was applied at the clinical development stage in 2018 and revealed better safety on QTc intervals.This drug has been included in phase III clinical trials as a TB treatment (JYP0081M301) in China since 2022 (Huang et al., 2022).

Pfaeffle et al., 2021
There are several studies utilizing BDQ containing regimens for NTM infection (Philley et al., 2015;Erber et al., 2020;Gil et al., 2021), including Mab, MAC and M. fortuitum (Table S1).A small preliminary report indicated the potential clinical and microbiological activity of BDQ in patients with advanced MAC (n = 6) or Mab (n = 4) lung disease, with 60% of patients (six of 10) demonstrating a microbiologic response (Philley et al., 2015).Due to strong antimycobacterial activity of BDQ, it seems to be used for all pathogenic NTM, especially for refractory Mab and MAC infection (Table 2).

Nitroimidazole
Nitroimidazole is a class of novel antimycobacterial agents that eradicate active Mtb by inhibiting mycolic acid biosynthesis and blocking cell wall production (Lewis and Sloan, 2015;Zhang et al., 2019).DLM, a bicyclic nitroimidazole, was initially approved by the European Medicines Agency in 2014 for pulmonary MDR-TB in adult.Pretomanid was the second bicyclic nitroimidazole drug that received its approval in the USA in 2019 for treating adults with pulmonary XDR-TB or treatment-tolerant or non-responsive MDR-TB (Gils et al., 2022).
Our earlier research demonstrated that DLM had highly variable antimycobacterial activity against 19 tested SGM species, with MICs of <0.25 mg/ml in 11 species.In contrast, DLM displayed no significant inhibitory activity against most tested RGM species, with 28 of the 33 tested strains having MICs >32 mg/ml (Yu et al., 2019).Except for a few strains with MICs ≤1 mg/mL, DLM did not possess a strong inhibitory effect against M. intracellulare and M. abscessus.Similarly, Philley JV et al. found that DLM had extremely high MIC 90 (>16 mg/ml) values against MAC and Mab complex.Compared to the MICs of DLM for those NTM, relatively low MIC 50 (0.25 mg/mL) and MIC 90 (1 mg/ml) values were observed against M. kansasii (Kim et al., 2019).Similar to DLM, pretomanid expressed high in vitro antimycobacterial activity against M. kansasii (MIC = 1.71 mg/mL) and moderate antimycobacterial activity against M. xenopi, with an MIC of 3.84 mg/mL (Zheng et al., 2023).Although high inhibitory potency was detected in DLM and pretomanid against M. kansasii in vitro, DLM or pretomanid containing regimens has not been used for the therapy of NTM infection in patients to date.Thus, DLM or pretomanid may be a potential efficacy drug for drug resistant M. kansasii (Table 2).

CFZ
CFZ, a traditional hydrophobic riminophenazine, has been prescribed for leprosy management since the 1950s.Although the exact mechanism of CFZ-mediated antimycobacterial activity remains undeciphered, the cell membrane may be the primary target (Cholo et al., 2017;Mirnejad et al., 2018;Stadler et al., 2023).The antimycobacterial activity of this riminophenazine covers a wide range, extending from anti-leprosy to NTM efficacy.Our prior investigation showed that CFZ had good activity against reference strains and clinical isolates of varied SGM species, with MICs well below 1 mg/ml for most strains (Luo et al., 2018).Furthermore, most clinical isolates of Mab and M. fortuitum had MICs >2 mg/ml.However, the ECOFF values for M. kansasii, M. avium and M. intracellulare were defined as 0.5 mg/ml, 1 mg/mL, and 2 mg/ml, respectively, based on their MIC distributions (Luo et al., 2018).In a study of the Mab clinical isolates obtained from patients with cystic fibrosis, 70% of the isolates presented with an MIC of ≤1.5mg/mL (Banaschewski et al., 2019).
7 Compound drugs

DprE1 inhibitor
In 2009, the decaprenylphosphoryl-beta-D-ribose oxidase (DprE1) enzyme was identified as a novel anti-TB drug target owing to its crucial role in mycobacteria and its location in the bacterial cell wall (Morrisette et al., 2021;Edwards and Field, 2022).Currently, several DprE1 inhibitors are enrolled in clinical trials, including BTZ-043, macozinone (MCZ, PBTZ169), OPC-167832 and TBA-7371 (Edwards and Field, 2022).There is no in vitro activity of BTZ-043 and TBA-7371 against NTM.PBTZ169 had poor activity against MAC and Mab isolates with MIC 90 of >32 mg/ mL (Shi et al., 2018).Surprisingly, after 4 weeks treatment in mice, PBTZ169 showed an average 3.33 and 2.29 log 10 CFU reductions in the lung against Mab and M. chelonae infection (Zheng et al., 2023).Among these DprE1 inhibitors, OPC-167832 displayed superior efficacy even at low doses in a mouse TB model using as monotherapy or combined treatment with other anti-TB drugs (Robertson et al., 2021;Tasneen et al., 2022).Additionally, other studies have revealed that OPC-167832 harbored substantial activities against Mab in vitro, with MICs ranging from 5.2 mM (2.37 mg/mL) to 15 mM (6.85 mg/mL) (Hariguchi et al., 2020;Sarathy et al., 2022), which made it a promising candidate for Mab infection.

SPR720
SPR720, is a prodrug that is converted to SPR719, is a novel aminobenzimidazole that inhibits the gyrase enzyme by targeting its ATPase subunits (Brown-Elliott et al., 2018;Pennings et al., 2021).Brown-Elliott BA et al. showed that SPR719 had MIC 50 values of 0.06-4 mg/mL for 93 RGM isolates, whereas the 41 MAC strains were associated with MIC 90 and MIC 50 values of ≤2mg/mL and ≤1mg/mL, respectively (Brown-Elliott et al., 2018).Another study of SPR719 demonstrated its activity against clinically relevant mycobacteria in mouse models of M. avium and Mab infections (Talley et al., 2021).Furthermore, a phase I trial showed that a once-daily oral administration of SPR720 (a phosphate prodrug of SPR719) could provide predicted therapeutic exposures of SPR719.Recently, SPR720 was granted the Investigational New Drug status by the FDA as a novel oral agent for pulmonary NTM infections and was recently enlisted in a phase IIa clinical trial for these infections.

GSK286
GSK286 is a Leucyl-tRNA synthetases (LeuRS) inhibitor with potent in vitro activity against Mtb and a Phase IIa clinical trials for systemic use against tuberculosis is underway (Bouz and Zitko, 2021).GSK286 showed potent antibacterial activity against Mab, with MICs of ≤0.25 mg/mL, yielding a MIC 90 of 0.063mg/mL.In contrast, it was not effective against MAC with the MIC 50 and MIC 90 values were >8mg/mL (Dong et al., 2020).

Indole-2-carboxamide derivatives
Mycolic acid transporter protein MmpL3 is inhibited by a wide range of structurally unrelated small molecules (Raynaud et al., 2020).Among these MmpL3 inhibitors, indole-2-carboxamides (ICs) block the export of alginate monomycolate to the outer membrane and can significantly inhibit bacterial growth.ICs have been identified as a novel chemical scaffold exhibiting good preclinical efficacy against Mtb and NTM pathogens.Several novel ICs with MIC values of 0.0039-8 µg/mL against NTM have been recently reported (Pandya et al., 2019).Of these, two lead IC compounds (compounds 5 and 25) showed effective bactericidal activity against Mab in vitro (MIC = 0.125 µg/mL) (Pandya et al., 2019).Considering these findings, the chemical inhibition of MmpL3 can be hypothesized to enhance the efficacy of other drugs owing to their crucial role in modulating cell wall structure and composition.
In summary, identifying novel anti-NTM drugs is of vital importance in the face of increasing global NTM infections.Anti-TB drugs, such as BDQ and CFZ have shown good in vitro anti-NTM activity and have been proposed for clinical use.Compounds, including OPC-167832, have also shown good in vitro antibacterial activity in clinical trials involving common pathogenic NTM.Therefore, these drugs or compounds have potential for in NTM treatment to improve patient outcomes.Although in vitro and preclinical trials have detected many promising compounds with potential therapeutic effects against NTM infection, clinical trials are urgently required to investigate their efficacy in NTM disease management.In addition, phage therapy for NTM infection also acquired favorable outcomes with exceptional safety profiles and no evidence of phage resistance was observed, which makes it a promising potential therapy.Thus, we believe that the increasing attention on NTM diseases should result in increased efforts on relevant drug discovery necessary to close the gaps in NTM treatment.

TABLE 1
Main characteristics of the described new drugs against NTM.

TABLE 2
Potential or existing therapeutics for different species of NTM.