High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against nontuberculous mycobacteria in vitro and in vivo

The F1FO-ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem–avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307–tebipenem–avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

The F 1 F O -ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions.Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the F O domain of the engine and preventing rotation and proton translocation.TBAJ-5307 is active at low nanomolar concentrations against fast-and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP.As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity.Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation.Furthermore, the TBAJ-5307-tebipenemavibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.
NTM infections are increasing exponentially in their global prevalence, morbidity, and mortality.Cell wall impermeability, along with biofilm formation, contributes to resistance against antimicrobials, high temperatures, and disinfectants.NTMs like Mab can exhibit either a smooth (S) or rough (R) morphotype as a consequence of the presence or the absence, respectively, of bacterial surface glycopeptidolipids (3).Importantly, S-R morphotypes show differences in virulence (2,3).These morphological differences are also associated with important physiological differences.In addition, NTM harbor intrinsic resistance mechanisms, quickly acquire resistance, and display drug tolerance, which renders most antimicrobial classes ineffective.Cure rates are globally poor, comparable to or worse than those of multidrug-resistant tuberculosis (4).
Importantly, most NTM are able to enter a metabolically quiescent state for instance under low oxygen tension (hypoxia), where the pathogens phenotypically become more tolerant to drug challenge with existing drugs (4).Being obligate aerobes, NTM need to regulate under such hypoxia conditions the essential metabolic processes of oxidation and recycling of the electron carriers (NADH 2 and FADH 2 ), the generation of a proton motive force, and the formation of sufficient amounts of ATP.The latter is driven by the oxidative phosphorylation pathway, including the F 1 F O -ATP synthase catalyzing ATP formation (5).Since the ATP pool is reduced in such persister cells, they become exquisitely sensitive to any further ATP depletion and susceptible to drugs targeting the maintenance of an optimal ATP:ADP ratio.This observation suggests that inhibitors targeting the essential NTM F 1 F O -ATP synthase (α 3 :β 3 :γ:ε:b-δ:b':a:c 9 ; Fig. 1A) could overcome the problem of NTM drug tolerance and shortening the duration of therapy for drug-resistant NTM diseases.This working model is supported by the antituberculosis therapeutic bedaquiline (BDQ), consisting of a quinoline-, a phenyl-, a naphthyl ring, and a dimethyl amino tether (Fig. 1B), and targeting the Mab F-ATP synthase (6).While BDQ is used as salvage therapy against Mab lung diseases (7), it has been overshadowed by its pharmacokinetic properties and tolerability profiles as well as its inhibition of the mitochondrial F-ATP synthase in human embryonic kidney 293S cell mitoplasts (8).Recently, BDQ's 3,5-dialkoxypyridine analog TBAJ-876 with improved pharmacokinetic and tolerability profiles has shown to be active in vitro and in vivo against the Mab complex (9).
However, in contrast to Mycobacterium tuberculosis, both inhibitors are not bactericidal and have 100 times lower growth-inhibitory potency in Mab, proposed to be correlated in part with crossresistance in Mab efflux pumps (6).In addition, the cryo-EM structure of Mycobacterium smegmatis F 1 F O -ATP synthase, a surrogate of the M. tuberculosis enzyme, showed BDQ binding to three sites of the F O domain, namely the leading sites and lagging sites and the c-ring, which binds five BDQ molecules (Fig. 1A) (10).The leading site involves a c subunit that has interacted with subunit a and picked up a proton from the periplasm, whereas the lagging site involves a c subunit poised to interact with subunit a to deposit a proton into the cytoplasm (10).The subunit a amino acid sequences of the three Mab subspecies differ from that of M. smegmatis, including the residues at the leading sites and lagging sites (Fig. S1).
Furthermore, the BDQ analogs TBAJ-5307, TBAJ-5316, and TBAJ-5366 with improved potency and physiochemical properties have been described (11).We showed that the racemates of (±)-5307, (±)-5316, and (±)-5366 (Cambridge Crystallographic Data Centre deposition numbers : 1967685, 1967683, 1967684, respectively; Fig. S2) exhibited improved growth and ATP synthesis inhibition against M. smegmatis, when compared with BDQ (12).Here, we demonstrate anti-Mab activity of the racemates of (±)-5307, (±)-5316, and (±)-5366 with (±)-5307 being the most potent one.Its enantiomer TBAJ-5307 displays high nanomolar efficacy against representatives of the fast and slow NTM growers, making it an attractive broad spectrum anti-NTM inhibitor.Its potency in cell growth and ATP synthesis inhibition is significantly higher compared with BDQ and TBAJ-876.Its mechanism of action is addressed by molecular dynamics (MD) simulations and binding free-energy calculations.TBAJ-5307 is efficacious ex vivo and in vivo without altering biofilm formation or being toxic and potentiates the activity of the clinical Mab antibiotics Potent anti-NTM inhibitor targeting ATP formation clofazimine (CFZ), amikacin, rifabutin (RFB), and the oral pair tebipenem (TBP) and avibactam (AVI).These features make TBAJ-5307 an ideal candidate to tackle the issue of NTM drug tolerance, high nanomolar anti-NTM potency, toxicity, pronounced combinatory efficacy with anti-Mab antibiotics to overcome intrinsic and acquired resistance as well as to design an oral cocktail in future, which reduces the expense and risks of intravenous therapies of today's anti-NTM drugs.
To determine whether TBAJ-5307 is bactericidal against Mab, we measured the survival of the strain upon drug exposure.TBAJ-5307 was bacteriostatic against M. subsp.abscessus at 30-fold its MIC 50 in 7H9 medium as BDQ (Fig. S3F) and as described for TBAJ-876 (9).
To determine whether Mab growth inhibition by TBAJ-5307 is related to depletion of ATP, intracellular ATP synthesis was measured.As shown in Figure 2C and Fig. S3G, TBAJ-5307 depleted ATP synthesis within the bacilli with an half-maximal inhibitory concentration (IC 50 ) of 7 ± 0.3 nM and 15 ± 1.4 nM in the S and R variants of M. subsp.abscessus, respectively.In comparison, lower ATP synthesis inhibition of the S and R variants was observed in the presence of BDQ (60 ± 12.5 nM and 148 ± 9.6 nM) or TBAJ-876 (106 ± 20 nM and 193 ± 35 nM).These results underline that the improved growth inhibition by TBAJ-5307 is consistent with higher intracellular ATP depletion when compared with BDQ or TBAJ-876.Since oxygen consumption within the respiratory chain of M. subsp.abscessus is unaffected at 125 nM and 250 nM of TBAJ-5307 (Fig. S3H), the data also indicate that the inhibitor targets the Mab F-ATP synthase.

TBAJ-5307's target specificity and mechanism of action
To confirm that the observed whole-cell ATP depletion correlates with the inhibition of the Mab F 1 F O -ATP synthase only, ATP formation of inverted-membrane vesicles of M. subsp.abscessus R variant was measured, resulting in an IC 50 of 1.4 ± 0.5 nM (Fig. 2D), which is comparable to the IC 50 observed for the whole cell assay described previously (7 ± 0.3 nM; Table 1).This indicates that TBAJ-5307's high potency of Mab growth inhibition correlates with the inhibition of Mab's F-ATP synthase.
To explore the inhibitory potency at an energetic level and to understand the mechanism of action, we performed MD simulations and free-energy calculations as successfully done previously for BDQ and TBAJ-876 binding to the M. smegmatis (14) and BDQ binding to the Mab F O domains (15).First, the structure of the Mab F O domain was modeled based on the cryo-EM structure of the M. smegmatis F O part (10), and TBAJ-5307 was fitted on to BDQ, which was resolved in the M. smegmatis structure (Protein Data Bank ID: 7JGC) (10).While the c-ring residues are conserved between M. smegmatis and the three Mab subspecies, differences in subunit a at the leading and lagging sites can be observed (Fig. S1), which in part may explain the 100 times lower potency of BDQ and TBAJ-876 against Mab when compared with M. smegmatis (6,9).The MD simulations (see supporting information: Computational procedures and data) revealed the binding site of TBAJ-5307 with emphasis on the lagging and leading sites, since they contribute mainly to the proposed inhibitory mechanism when compared with the c-ring-only site (10).TBAJ-5307 interacts with the Mab c-ring residues cA25, cV58, cG59, cL60, cE62, cA63, cA64, cY65, cF66, cI67, and cL69 and subunit a residues aV174, aF177, aI178, and aI181 for the lagging site (Fig. 3), whereas in the leading site, residues cA25, cV58, cG59, cL60, cE62, cA63, cA64, cY65, cF66, cI67, and cL69 and residues aA218, aP219, aI222, aW223, and aF226 interact with the inhibitor (Fig. S4 and Table S1).Interestingly, the Mab-specific residues aF177 and aI181 of the lagging site (aP172 and aV176 in M. smegmatis) form hydrophobic interactions with the 2,3-dimethoxy-pyridin-4-yl moiety of TBAJ-5307 (Fig. 3) and contribute to the strong binding of the agent, which hinders rotation of the cring turbine and movement of the proton to residue aR193 for further translocation via the second half-channel to the cytoplasmic side, a requirement for ATP formation.In comparison, the 2,6-dimethoxy-pyridin-4-yl moiety exhibits hydrophobic contacts with the Mab-specific amino acids aA218 and aI222 (aF213 and aV217 in M. smegmatis) in the leading site (Fig. S4).
Free energy calculations resulted in a binding free energy of −9.4 ± 1.3 kcal/mol compared with −12.0 kcal/mol derived from experimental data (Fig. 2D) for the lagging site; the offset is similar to that derived from our previous work for strong BDQ binding to M. smegmatis (14).The free-energy calculations are Potent anti-NTM inhibitor targeting ATP formation thus in reasonable agreement with the experimental data, thereby confirming the accuracy of the modeled complex.The offset in free energy likely results from the modeled neutral state of the compound and the key cE62; the alternative charged form would lead to artifactual membrane solvation, as discussed previously (16).For the leading site, we obtained a binding free energy of −9.8 ± 3.8 kcal/mol and thus predict a comparable inhibition of the rotation for the leading site.
To further confirm TBAJ-5307's target specificity and to demonstrate that the molecule does not affect biofilm formation or planktonic bacteria, the compound was assayed against Pseudomonas aeruginosa PAO1 or Escherichia coli UTI 189.As revealed in Figure 4, no killing of planktonic cells or biofilm cells was observed upon addition of 9× and 26× MIC 50 (40 and 120 nM) of TBAJ-5307 to batch biofilms of P. aeruginosa PAO1 or E. coli UTI 189 under both nutrient-limited conditions, which was simulated by treatment in PBS, and under growth conditions, which is simulated by addition of fresh growth media.These results confirm TBAJ-5307's target specificity and underlines that the compound is not a broadspectrum antibiotic.

Ex vivo anti-Mab potency of TBAJ-5307
To explore anti-Mab potency of the compound in macrophages, a THP-1 infection model was used.Macrophages were infected with M. subsp.abscessus S and R variants and treated with TBAJ-5307 in a range of 5 nM to 2500 nM. Figure 5A demonstrates that TBAJ-5307 was active against both morphotypes, reflected in decrease in viable bacterial count (colony-forming unit/ml) when compared with initial inoculum at 250 and 2500 nM.Drug-treated macrophages did not show any visual alteration of the membrane, cell form, or size as tested microscopically (Fig. 5B).Immunofluorescence imaging also showed reduced numbers of infected macrophages and cords (for Mab R) during treatment with TBAJ-5307 (Fig. 5B), as reported earlier with RFB (16,17), in agreement with the reduced intracellular bacterial burden (Fig. 5A).

In vivo potency and nontoxicity of TBAJ-5307
In vivo potency and toxicity of TBAJ-5307 has been studied in the zebrafish model of infection, which have arisen as alternative because of the advantages of rapid development, Potent anti-NTM inhibitor targeting ATP formation easy and fast chemical administration, visualizing, highthroughput capabilities, and the possibility to study the dynamics of the infection in the presence of the active compound (6).By analogy with the protocol developed for BDQ (6), the activity of TBAJ-5307 against Mab infection in zebrafish larvae was tested using red fluorescent tdTomato-expressing M. subsp.abscessus (R variant), which was injected in the caudal vein of embryos at 30 h postfertilization and transferred to 24-well plates.TBAJ-5307 was then directly added at 1-day postinfection (dpi) to the water containing the infected zebrafish, and the TBAJ-5307 supplemented water was then changed daily for 4 days.Noninfected embryos were first exposed to increasing concentrations of TBAJ-5307 and observed under a microscope.No signs of toxicity-induced killing or developmental abnormalities were recorded in the range of concentrations tested (Fig. 6A).When infected embryos were exposed for 4 days to 2500 nM TBAJ-5307, a significant increased survival rate (80%) was observed compared with the untreated group of embryos (Fig. 6B).We next monitored the kinetics of the bacterial burden upon intravenous infection of embryos by quantification of the total area of the red fluorescent signal (fluorescent pixel count) at 3 and 5 dpi in the presence or the absence of TBAJ-5307.The addition of 2500 nM TBAJ-5307 for 4 days in fish water was accompanied by a significant reduction in the fluorescent pixel count at both time points (Fig. 6C).Importantly, our results indicate that at both 3 and 5 dpi, treatment with TBAJ-5307 coincided with a significant reduction in the number of infection foci (Fig. 6D).Whole embryo imaging at 3 and 5 dpi clearly highlighted the effect of the compound, with limited infection, particularly evident in the presence of 2500 nM TBAJ-5307 (Fig. 6E), consistent with bacterial burden quantification (Fig. 6C).This pronounced effect of the compound in reducing the bacterial burden and infection foci is likely to explain the protective efficacy of TBAJ-5307 on embryo survival.Abscess formation can be linked to loss of infection control, typically occurring following cord formation and expansion and representing a marker of disease severity (18-20), associated with Potent anti-NTM inhibitor targeting ATP formation cell debris, tissue destruction, and acute infection.We note that treatment with TBAJ-5307 was associated with a decrease in abscess formation and cording (Figs.6E and S5).This indicates that TBAJ-5307 is very efficient in vivo against infection and protects the zebrafish from killing by the pathogen M. subsp.abscessus.

TBAJ-5307 enhances potency of existing anti-Mab drug candidates
Since leveraging the potential of synergistic drug interactions in multidrug treatment regimens to accelerate durable cure is a growing field and the treatment of Mab infections requires drug combinations, the growth inhibition activity of TBAJ-5307 along with clinical Mab antibiotics CFZ, amikacin, and the RNA polymerase inhibitor, RFB, was measured using a checker-board titration assay.Drastic reduction of cell growth was observed with all three drugs tested (Fig. 7, A-C).The fractional inhibitory concentration index was calculated to characterize the interaction between TBAJ-5307 and each of the test drugs (21).Fig. S6, A-C shows an additive growth reduction triggered by CFZ or RFB with TBAJ-5307, achieving a fractional inhibitory concentration    S6D) (22)(23)(24).Here, we tested whether inhibiting the fundamental cell processes of peptidoglycan synthesis in cell wall formation and silencing of the essential mycobacterial enzyme F-ATP synthase by TBAJ-5307 provide a potent combination.As shown in Figure 7D, combining the TBP-AVI pair with TBAJ-5307 enhanced growth inhibition.The efficacy of TBAJ-5307 increased by a factor of 14 (MIC 50 of 0.7 nM) in combination of TBP-AVI.Importantly, TBAJ-5307 does not exert any observable antagonism in killing activity with bactericidal TBP-AVI (Fig. S6E), suggesting that TBAJ-5307 could be coadministered with the dual drug.
The data demonstrate that TBAJ-5307 is a highly potent anti-NTM agent covering the broad range of fast and slow NTM growers by targeting the F 1 F O -ATP synthase, which converts ADP and Pi to the biological currency ATP.The data also show, for the first time, that TBAJ-876, reported to be active against the M. abscessus complex (9), displays attractive in vitro activity against M. mucogenicum, M. fortuitum, M. avium, and M. intracellulare, although with lower potency compared with TBAJ-5307.

Conclusion
NTM infection presents a growing global health problem, complicated by ubiquitous exposure to the organisms, incomplete understanding of the immune susceptibility to disease, increasing numbers of immune-compromised patients, and costly multidrug treatment regimens that often fail to cure.Therefore, there is a pressing need to identify inhibitors with high efficacy and enzyme targets being essential for NTM under various metabolic conditions, being nontoxic and with attractive combinatory potency.Here, we identified TBAJ-5307 as a novel anti-Mab inhibitor targeting the F-ATP synthase, essential for ATP synthesis, regulation of ATP homeostasis, and proton motive force under multiple growth conditions.Based on our MD simulations and free-energy calculations, the inhibitor interacts with the F O domain via the c-ring as well as the subunits a-c lagging and leading sites, including the Mab-specific residues aF177, aI181, aA218, and aI222 (Fig. 3).TBAJ-5307's binding to the a-c interfaces inhibits proton translocation via the F O domain half-channels by preventing rotation of the c-ring relative to subunit a.The high potency of TBAJ-5307 in growth inhibition correlates with whole-cell ATP depletion, reduction in ATP formation of Mab inverted-membrane vesicles, and free-energy calculations (Fig. 2).The compound displays higher efficacy compared with BDQ and TBAJ-876.TBAJ-5307 exhibits anti-Mab potency in macrophages (Fig. 5) and zebrafish embryos without being toxic (Fig. 6), which is essential for further clinical developments.The compound potentiates the anti-Mab activity of the NADH dehydrogenase inhibitor CFZ, the 16S rRNA targeting amikacin, the RNA polymerase-targeting antibiotic RFB, and the oral pair TBP-AVI, with TBP inhibiting peptidoglycan synthesis, respectively (Fig. 7).In addition, the TBAJ-5307-TBP-AVI cocktail is bactericidal (Fig. S6E).The presented combinations of the low nanomolar ATP synthesis inhibitor TBAJ-5307 with antibiotics targeting transcription, protein synthesis, or cell wall formation would be a step forward (i) to silence major cell processes of the pathogen under different metabolic states, (ii) to lower the emergence of drug resistance, (iii) to reduce the required concentrations and thus dosing of these existing antibiotics because of the enhanced inhibitory activity of each within the cocktail, leading to reduced toxicity and side effects, and (iv) in case of an TBAJ-5307-TBP-AVI to aim for an oral drug combination, which would reduce costs and risks compared with intravenous therapies.Importantly, TBAJ-5307 displays pronounced nanomolar anti-NTM activity, which correlates with wholecell ATP depletion, underscoring binding of TBAJ-5307 to the NTM F 1 F O -ATP synthase (Fig. 8).Its improved potency compared with BDQ and TBAJ-876 makes the compound an attractive candidate to eradicate a broad range of NTM infectious disease in future.

Figure 1 .
Figure 1.Binding-sites of BDQ in the mycobacterial engine and the key chemical groups of BDQ and TBAJ-5307.A, Mycobacterium smegmatis F-ATP synthase cryo-EM structure with bound BDQ (Protein Data Bank ID: 7JGA).The hexamer forming α and β subunits is shown in green and orange color, respectively.The γ (yellow), ε (red), bδ (blue), b' (cyan), and a (magenta) subunits and c-ring (gray) are shown in surface representation.The seven BDQ molecules that bind in c-ring are also shown in surface representation.BDQ binds five c-only sites (yellow), a leading site (green), and a lagging site (cyan).For clarity, the c-ring along with subunit a are shown in cartoon representation in two views.In the zoomed-in view, the residues, which are interacting with BDQ in the leading site and lagging site, are shown.B, BDQ with its key pharmacophore features (a quinoline, a phenyl (Ar 1 ), a naphthyl (Ar 2 ) ring, and a dimethyl amino [DMA] tether).In comparison, TBAJ-5307 has a 2,3-dimethoxy-pyridin-4-yl moiety as Ar 1 group and a 2,6-dimethoxy-pyridin-4-yl moiety as Ar 2 group.BDQ, bedaquiline.

Figure 2 .
Figure 2. Anti-Mab testing of BDQ analogs and racemates.A, growth inhibition dose-response curve of Mab subsp.abscessus ATCC 19977 cells by the racemates (±)-5307, (±)-5316, and (±)-5366.Two biological replicates were carried out each in three technical replicates.Data represent the average of all the experiments.Bacterial growth is expressed as percentage of control/untreated sample.B, growth inhibition dose-response curve of smooth morphotype strains of Mab subsp.abscessus ATCC 19977 by BDQ, TBAJ-876, and TBAJ-5307.Two biological replicates were carried out each in three technical replicates.Data represent the average of all the experiments.Bacterial growth is expressed as percentage of control/untreated sample.C and D, intracellular ATP synthesis inhibition of smooth (C) morphotype strains of Mab and (D) Mab IMVs.The ATP content of the cells was measured by adding BacTiter-Glo (Promega) to the cells.The total ATP content is directly proportional to relative luminescence units (RLUs).Two biological replicates were carried out each in three technical replicates.Data represent the average of all the experiments.****p < 0.0001, statistical analysis was carried out using two-way ANOVA test for all the experiments presented.ATCC, American Type Culture Collection; IMV, inverted-membrane vesicle; Mab, Mycobacterium abscessus.

Figure 3 .
Figure 3. Predicted binding of TBAJ-5307 to the Mab F O -lagging site.Position of TBAJ-5307 bound to the F O domain in the side view (top left) and top view (bottom left), respectively.Zoom into the binding site (top right) and the LigPlot+ representation (27) (bottom right) are shown.Residues of the c-ring are displayed in gray, and residues of subunit a are shown in purple (same residues as in Mycobacterium smegmatis) and orange (different residues than in M. smegmatis).Figures representing molecular representations were produced with VMD (visual molecular dynamics) (28).Mab, Mycobacterium abscessus.

Figure 4 .
Figure 4. Activity of 40 or 120 nM of TBAJ-5307 against 3 h preformed biofilm of Pseudomonas aeruginosa.PAO1 WT (A and C) and Escherichia coli UTI 189 (B and D).Treatment was carried out in 1× PBS media (A and B) or M9 glucose media (C and D).Both planktonic and biofilm cells were collected from the same sample well, with planktonic cells (black circles) referring to suspended cells present in treatment buffer prior to washing and collection of biofilm cells (open blue triangles).No significant differences were observed between TBAJ-5307 and untreated controls.At least two independent experiments were carried out, each with two technical replicates.Each data point represents averaged data of two independent experiments with three technical replicates, and the line represents the means of all data.Colony-forming unit (CFU) counts were analyzed using GraphPad Prism, version 9.3.0 using two-way ANOVA and multiple comparison of column effect (concentration of compound against untreated control) within each row (planktonic versus biofilm samples).

Figure 6 .
Figure 6.Therapeutic activity of TBAJ-5307 against Mab subsp.abscessus (R variant) in an embryonic zebrafish infection model.A, groups of around 20 uninfected embryos were immersed in water containing increasing concentrations of TBAJ-5307 (ranging from 50 to 2500 nM) for 4 days.The magenta bar indicates the duration of treatment.The graph shows the survival of the TBAJ-5307-treated and -untreated embryos over an 11-day period.B, zebrafish embryos (around 35 per group) at 30 h postfertilization were infected with 250 colony-forming unit (CFU) of Mab subsp.abscessus expressing red fluorescent TdTomato via caudal vein injection.A standard PBS injection control (uninfected) was included.At 1 day postinfection, embryos were exposed to increasing concentrations of TBAJ-5307 (250 or 2500 nM) in fish water.Drugs were renewed at a daily basis for 4 days (magenta bar) after which embryos were washed twice in fresh embryo water and maintained in fish water.Survival was monitored daily over 11-day period.Each treatment group was compared against the untreated control group with significant differences determined using the log rank (Mantel-Cox) statistical test for survival curves: ****p < 0.0001; ns, nonsignificant.Three experiments were performed.C, Mab infection burden was quantified by fluorescent pixel count (FPC) determination using the ImageJ software after 2 (3 dpi) or 4 (5 dpi) days of exposure to TBAJ-5307 (2500 nM).The data are from three experiments (containing around 35 embryos per group), with each data point representing one infected zebrafish larva.The error bar represents the mean and standard deviations of the dataset.Statistical comparison of the difference using a Mann-Whitney's t test: *p < 0.05, ****p < 0.0001.C and D, the 2500 nM values are normalized to the values of the untreated control at each time point.D, Mab infection foci in embryos after 2 (3 dpi) or 4 (5 dpi) days of exposure to TBAJ-5307 (2500 nM) by FPC determination using the ImageJ software.The error bar represents the mean and standard deviations of the dataset.Statistical comparison was done using a Mann-Whitney's t test: ***p < 0.001 and ****p < 0.0001.E, representative embryos from the untreated group (upper panel) and from the group treated with 250 or 2500 nM TBAJ-5307 at 3 dpi (left panels) or 5 dpi (right panels).Scale bars represents 300 μm.* highlights autofluorescence.dpi, day postinfection; Mab, Mycobacterium abscessus.
risks of intravenous therapies, more potent oral combinations would provide huge advantages.Recently, the combination of the oral β-lactam TBP, targeting the L,D-transpeptidases LdtMab1 and LdtMab2 and D,D-transpeptidases PonA1, PonA2, and PbpA, which are essential for peptidoglycan synthesis, with the Mab β-lactamase inhibitor AVI has shown an attractive, low micromolar, and strong cidal activity (Fig.

Figure 8 .
Figure 8. Anti-M.avium potency of TBAJ-5307.A, growth inhibition dose-response curve of Mycobacterium avium by TBAJ-5307 in comparison to BDQ and TBAJ-876.B, TBAJ-5307 inhibits oxidative phosphorylation in a whole-cell ATP synthesis assay better than BDQ or TBAJ-876.Three biological replicates were carried out each in three technical replicates.Data represent the average of all the experiments.****p < 0.0001, statistical analysis was carried out using two-way ANOVA test for all the experiments presented.BDQ, bedaquiline.

Table 1
Growth-inhibitory and ATP synthesis potency of BDQ, TBAJ-876, and TBAJ-5307 against NTM strains in 7H9 broth medium n.d.n.d.The experiments were carried out three times independently.Abbreviation: n.d., not determined.