Alpibectir–Ethionamide combination (AlpE) for the treatment of tuberculosis

Domain: neuroscience
Published: 2026-04-07
Editorial outcome: minor_revision

Abstract
Ethionamide (Eto) and prothionamide (Pto) are second-line antibiotics used for tuberculosis (TB) treatment. Both are prodrugs whose antibacterial activity depends on bioactivation by oxidases in Mycobacterium tuberculosis, including the Baeyer-Villiger monooxygenase MymA. Through biophysical, genetic, and cellular assays, we show that the clinical candidate alpibectir (Alp, BVL-GSK098) binds the transcriptional regulator VirS, increasing MymA expression and potentiating Eto and Pto activity. Alpibectir also boosts the activity of the corresponding host-derived sulfoxide metabolites. We additionally show that alpibectir exhibits intrinsic antibacterial activity via overexpression of the mymA operon. The alpibectir/Eto (AlpE) combination is rapidly bactericidal in vitro and in mice, lowers the frequency of spontaneous resistance of Eto, and remains active on Eto- and isoniazid-resistant strains, including isolates with inhA promoter mutations. Alpibectir was safe in a Phase 1 human clinical trial. Together with the potentiation data presented here, these findings highlight its potential to optimize TB chemotherapy by reducing Eto/Pto doses, which can minimize dose-related side effects, enhancing adherence.

Introduction
Tuberculosis (TB) is one of the leading causes of mortality worldwide1. Its causative agent is the bacterial pathogen Mycobacterium tuberculosis (M. tuberculosis). The impact of the COVID-19 pandemic has been profound, resulting in a setback in diagnosis and treatment, reversing years of hard-won progress in reducing TB fatalities and underscoring the critical need to strengthen research and medical efforts to combat this persistent scourge. The growing threat of drug-resistant TB exacerbates this challenge, necessitating the use of less efficient and less safe second-line drugs2,3, thereby prolonging the treatment duration and diminishing its success.

Thioamides, including ethionamide (Eto) and prothionamide (Pto), are recommended by the World Health Organization (WHO) as second-line agents for treating drug-resistant pulmonary TB3 and as first line treatment for TB meningitis4. Despite their efficacy, these drugs pose a hurdle to treatment adherence due to dose-dependent adverse events5,6. Eto and Pto are prodrugs, so their antibacterial activity is linked to their level of bioactivation. The main bioactivating enzyme is the mycobacterial monooxygenase EthA, which initiates a series of catalytic events leading to the formation of an Eto/Pto-NAD adduct targeting InhA, an enoyl-reductase playing a pivotal role in the synthesis of mycolic acid in the mycobacterial cell envelope7,8. The expression of EthA is repressed by the transcriptional regulator EthR9. Inhibition of EthR by small molecules was shown to enhance EthA expression10–17. The most potent EthR inhibitor, BDM41906, demonstrated increased biotransformation of Eto and its major metabolite ethionamide-sulfoxide (Eto-SO) by EthA, and increased the susceptibility of M. tuberculosis to both Eto and Eto-SO in vitro 18 and to Eto in vivo13,18,19. Although this was an exciting proof of concept, the therapeutic potential was constrained. The continuous use of Eto and Pto over the last 60 years has led to the selection of resistant clinical isolates mutated in ethA, rendering these strains resistant to Eto/Pto, even in combination with BDM41906.

An unexpected rebound of optimism in this strategy emerged with our discovery of compounds capable of stimulating the expression of alternative Eto/Pto activation pathways20,21. Specifically, SMARt751 was shown to interact with VirS and induce the overexpression of the mymA operon, which was concomitantly identified for its ability to activate Eto. Importantly, by stimulating Eto activation through the upregulation of MymA, SMARt751 not only enhanced Eto efficacy but also circumvented resistance to Eto in clinical strains mutated in ethA21. However, despite these promising effects, SMARt751 also triggered activation of peroxisome proliferator-activated receptors alpha (PPAR\alpha) and the constitutive androstane receptor (CAR) at all tested doses in vivo. Due to these potential drug metabolism and pharmacokinetics (DMPK) and safety concerns, further development of SMARt751 was not pursued. Nevertheless, a multi-parametric lead optimization program led to the design of alpibectir, which demonstrated microbiological efficacy, pharmacokinetic properties, and safety profiles adequate for clinical development (manuscript in preparation).

Here, we elucidated the complex mode of action of alpibectir, linked to its molecular interaction with VirS. The ability of alpibectir to stimulate the mymA operon leads to a triple effect on the bacteria. First, overexpression of mymA improves the bio-activation of both Eto and its major metabolite Eto-SO. Importantly, alpibectir overcomes acquired Eto resistance by circumventing EthA-dependent Eto bioactivation. Finally, alpibectir has its own growth inhibitory effect, intriguingly, also subordinated to the overexpression of the mymA operon. The confluence of these effects allows alpibectir to potentiate the antibacterial activity of Eto in vivo in a 4-week acute murine model of TB and makes the combination a powerful anti-TB medication candidate.

Alpibectir was safe and well tolerated in a Phase I clinical trial22 and the results from a Phase 2a study (NCT05473195) in patients with pulmonary TB will be reported soon. It is envisaged for future use in combination with Eto and Pto in patients with isoniazid (INH) mono-resistant or multidrug-resistant (MDR) TB, including TB meningitis.

Results
Alpibectir enhances Eto activity against M. tuberculosis.
The antibacterial activity of the alpibectir/Eto (AlpE) combination on M. tuberculosis was first assessed using a checkerboard microtiter assay (Fig.1). Bacterial growth was monitored by quantifying fluorescence intensity of a GFP-expressing M. tuberculosis H37Rv strain after 5 days of incubation. Concentration-response curves of Eto were plotted for each individual concentration of alpibectir tested. IC50 and IC90 extrapolated from each curve refer to the concentrations of Eto required to inhibit 50% and 90% of bacterial fluorescence, respectively. The IC50 and IC90 values of Eto alone were found to be 1.0 mg/L and 3.7 mg/L. In this assay, a concentration of 0.03 mg/L (100 nM) of alpibectir was found to reduce the IC90 value of Eto by more than 100-fold (down to 0.03 mg/L). Maximum potentiation of Eto activity (almost 200-fold reduction in the IC90 of Eto) was observed at a concentration of 0.1 mg/L (300 nM) of alpibectir (Fig.1E).

Strikingly, we also observed concentration-dependent growth inhibition by alpibectir in the absence of Eto (Fig.1C). We confirmed through optical density measurements that the decrease in fluorescence observed upon treatment with alpibectir alone was due to bacterial growth inhibition and not to dose-dependent quenching of bacterial fluorescence by alpibectir (SI Fig.1). These results reveal that alpibectir not only potentiates Eto but can also contribute by itself to the inhibition of M. tuberculosis growth. Interestingly, close structural analogues of alpibectir that lack Eto-boosting activity also lack intrinsic growth inhibition, consistent with a narrow structural requirement shared between both activities (SI Fig.2).

Alpibectir boosts Eto activity in infected macrophages.
In the context of infection, M. tuberculosis primarily resides within host cells23. Therefore, penetration of host cells and the effectiveness of anti-TB drugs within the intracellular environment are crucial for treatment success. The antibacterial activity of Eto in the presence of alpibectir was assessed on luminescent M. tuberculosis H37Rv in infected THP-1 cells by measuring the luminescence of intracellular bacteria. As a control, macrophages were confirmed to be viable irrespective of drug treatment (SI Fig.3). The maximum intrinsic activity and boosting effect of alpibectir was observed at a concentration of \le 0.03 mg/L, reducing the IC90 of Eto from 0.1 mg/L (in the absence of alpibectir) to 0.01 mg/L (Fig.1F).

These experiments demonstrate that alpibectir is highly potent in increasing the antibacterial activity of Eto in infected macrophages. A comparable boost of Eto activity was observed in an independent macrophage survival assay that quantifies protection from infection-induced macrophage lysis (SI Fig.4). In this assay, however, alpibectir used alone showed no detectable intrinsic activity, suggesting that while it can inhibit intracellular bacterial growth, the effect is insufficient on its own to prevent macrophage lysis (Fig.1F).

Alpibectir preferentially activates the VirS/MymA pathway.
We then assessed the impact of alpibectir on the transcriptome and proteome of M. tuberculosis. When M. tuberculosis was exposed to a concentration of 0.1 mg/L of alpibectir, RNA-seq identified 37 genes that were upregulated by more than two-fold (Fig.2). Notably, the most prominently overexpressed mRNAs corresponded to the virS-mymA regulon (rv3082c to rv3089), a profile reminiscent of what was previously observed with SMARt75121. The transcription of rv0077c, the gene encoding the oxidoreductase EthA221, was also increased, although to a lesser extent. The addition of Eto did not change the transcriptomics profile induced by alpibectir, indicating that Eto does not interfere with the alpibectir-mediated induction of the mymA operon (SI Table 1).

Next, we quantified modifications of the M. tuberculosis global proteome upon treatment with alpibectir (SI Table 1). We compared the total protein content of an M. tuberculosis culture treated with alpibectir to that of a culture treated with DMSO using isobaric labelling-based mass spectrometry24. Out of the approximately 2,200 proteins identified, 9 proteins were overproduced upon alpibectir treatment. Eight of them correspond to the proteins of the virS-mymA regulon and the ninth is the EthA2 protein.

These findings confirm that the observed rise in transcription of the virS-mymA and ethR2-ethA2 regulons induced by alpibectir treatment leads to an increased production of their corresponding proteins. Altogether, this underscores the exceptional functional selectivity of alpibectir, which has virtually no other effect than predominantly stimulating the mymA operon, and to a lesser extent the ethA2 gene. Notably, while a tenfold reduction in EthA protein production was observed, no significant change in ethA expression (less than a 2-fold difference) was detected.

Alpibectir modifies VirS activity through direct interactions.
Transcriptomic data suggest that alpibectir may interact with the transcriptional regulator VirS to stimulate the expression of the virS/mymA operon. Here we wanted to elucidate whether this stimulation involves direct or indirect interactions between alpibectir and VirS. First, we developed a synthetic gene circuit in mammalian cells that quantitatively reports the expression of the secreted alkaline phosphatase (SEAP) gene, designed to sense interactions between the bacterial protein VirS and its cognate DNA operator region in a controlled biological context devoid of other bacterial proteins. We observed a strong concentration-dependent reduction of SEAP production upon alpibectir treatment (IC50 = 0.001 mg/L) (Fig.3A). Conversely, we showed that synthetic gene circuits designed to sense interactions between EthR2 or EthR and their respective cognate DNA operator were modestly impacted by alpibectir (IC50 = 0.27 mg/L) or not affected at all, respectively (SI Fig.5). These findings are consistent with the low change in transcriptomic level for ethA2 and ethA.

The direct binding between alpibectir and VirS was confirmed using differential scanning fluorimetry, a method that assesses the impact of a ligand on the temperature at which a protein unfolds25. The assay revealed a concentration-dependent thermostabilizing effect of alpibectir on VirS (Fig.3B-C). Notably, the most pronounced stabilization, resulting in a 10°C increase in the melting temperature of VirS, was achieved in the presence of an equimolar concentration of alpibectir (Fig.3C).

To improve our understanding of the direct interaction between VirS and alpibectir, we co-crystallized their complex and refined the crystal structure at a resolution of 1.49 Å (PDB ID 8RCX; SI Table 2). Alpibectir resides within a fully enclosed and predominantly hydrophobic cavity of VirS. The carbonyl group of alpibectir establishes 2 hydrogen bonds with the N\delta2 side chain amides of Asn11 and Asn149 (Fig.3D and Fig.3E). Its piperidine moiety is anchored on one side through the Ile80 side chain and on the opposite side by the Tyr129 and Ser146 side chains. The isoxazoline ring engages prominently with the Met153 side chain, while Gly142 provides the necessary spatial accommodation for the trifluoromethyl group adjacent to the CH2-CH2 aliphatic chain of the compound. This trifluoromethyl group is further stabilized by the side chains of Ile7, Leu49, Phe52, Val53, and Ser146. The second trifluoromethyl group is anchored by the side chains of Ile101, Leu114, Met153, and Phe197.

Conclusively, the integration of the transcriptomic and the structural data presented herein indicates that the upregulation of the mymA operon is caused by the direct physical interaction of alpibectir with the putative ligand-binding domain of VirS.

To assess whether the specificity of alpibectir for VirS also translated in bacterio, we tested its impact on a virS mutated strain of M. tuberculosis (strain 8F with mutation Cys278Arg in VirS). Consistent with published data, the loss of function mutation in virS resulted in a slight decrease in susceptibility to Eto21,26 (Fig.4). Alpibectir did not restore Eto activity in strain 8F, confirming the specificity of its activity through VirS, and highlighting the central role of this transcriptional regulator in controlling the boost of the bioactivation of Eto mediated by alpibectir.

Strikingly, the mutation in virS (strain 8F) not only abrogated the Eto-boosting activity of alpibectir, but it also suppressed the intrinsic antibacterial activity of alpibectir, leading to the conclusion that both the boosting and the intrinsic activities of alpibectir are under the control of VirS.

Alpibectir has distinct intrinsic and ethionamide boosting actions.
In the objective to delineate the intrinsic and the Eto-boosting activities of alpibectir, we investigated additional mutants in the virS-mymA regulon. Insertion of a transposon into the mymA open reading frame (Rv3083::Tn), which abrogates the expression of the entire operon without affecting the expression of VirS, resulted in the loss of both the Eto-boosting and intrinsic antibacterial activity of alpibectir (SI Table 3 and SI Fig.7). These findings rule out the possibility that the intrinsic antibacterial activity of alpibectir results from the expression of any VirS-regulated gene outside the virS-mymA regulon. They align with the RNAseq experiments, which demonstrate that alpibectir predominantly modulates the expression level of the virS-mymA regulon. This suggests that both the Eto-boosting effect and the intrinsic antibacterial activity of alpibectir arise from upregulation of the mymA operon. Remarkably, the inactivation of adhD, the fourth gene in the operon, by homologous recombination (which, in addition, might impact the alpibectir-mediated overexpression of the genes downstream of adhD), resulted in a complete loss of the intrinsic antibacterial activity of alpibectir, without affecting its Eto-boosting effect (Fig.5). Future studies aimed at generating clean, non-polar deletions of genes within the mymA operon and performing gene complementation experiments will be necessary to fully elucidate the genetic basis of the intrinsic activity of alpibectir.

In conclusion, by stimulating the expression of the mymA operon, alpibectir leads to two distinct outcomes: on one hand, the overproduction of the MymA protein potentiates the bioactivation of Eto; on the other hand, the overexpression of adhD and/or of its downstream genes directly or indirectly affects the growth of M. tuberculosis in vitro.

AlpE is bactericidal against M. tuberculosis.
Next, we assessed the speed of the anti-TB activity of AlpE using time-kill curves. At a concentration near its MIC (2.5 mg/L), Eto alone exhibited a constant yet relatively slow bactericidal activity in vitro, resulting in an approximate 4-log reduction of CFU in 7 days, never reaching the limit of detection of 102 CFU/mL. After this 7-day period, rapid regrowth was observed in the culture at day 21 (Fig.6A). At a tenfold higher Eto concentration (25 mg/L), a similar initial rate of CFU reduction was observed, reaching the detection limit after 14 days. Nonetheless, the onset of regrowth was observed by 21 days (Fig.6A).

Conversely, combining Eto 2.5 mg/L with alpibectir resulted in rapid bactericidal activity, driving bacterial counts down to the detection limit (10² CFU/mL) within 7 days, with no regrowth observed up to 21 days. In comparison, as well-documented, INH at 10 times its MIC (2.5 mg/L) failed to prevent bacterial regrowth.

In comparison to the intrinsic growth-inhibitory activity shown in Fig. 5, no bactericidal activity was observed when alpibectir was used alone at concentrations up to 0.3 mg/L suggesting that it affects bacterial fitness rather than viability. Similarly, AlpE was equally effective in killing the H37Rv strain mutated in adhD, indicating that the intrinsic effect of alpibectir does not contribute to the killing effect of the combination nor does it accelerate it (Fig.6B).

Alpibectir reduces the frequency of spontaneous resistance to Eto.
We then evaluated whether the enhanced killing effect of AlpE impacts the frequency of resistance (FoR) of M. tuberculosis H37Rv compared to Eto alone. INH and moxifloxacin (Mox) were used as references. The FoR to INH and Mox (at 10-fold and 4-fold agar MIC, respectively) were consistent with literature data27,28, with values around 10-6 and 10-8, respectively. Eto at 2.5 mg/L (agar MIC) resulted in a FoR of 5x10-7. Remarkably, the combination of 0.012 mg/L alpibectir with 2.5 mg/L Eto prevented the emergence of resistant colonies, indicating a FoR below the detection limit of 10-9 (Table 1).

In the presence of alpibectir, resistant mutants were only obtained at lower Eto concentrations, ranging from 0.375 to 1 mg/L (SI Table 4). Detailed analysis of these low-resistance clones revealed mutations in genes previously associated with Eto resistance, namely ethA, mymA, or virS. Four clones had simultaneous mutations in both ethA and mymA (SI Table 5). Subsequently, the susceptibility of 14 resistant clones to various anti-TB drugs was tested. All clones were sensitive to moxifloxacin, linezolid, ethambutol, bedaquiline, rifampicin, and pretomanid, indicating the absence of a global or specific mechanism of cross-resistance between AlpE and other TB drugs (SI Fig.8).

Alpibectir restores Eto and Pto activity in ethA mutants.
Pto is the propyl thioamide analogue of Eto (SI Fig.9). Both antibiotics are considered interchangeable for multidrug-resistant tuberculosis treatment regimens and for treatment of tuberculous meningitis in adults and children5,29.

Since the efficacy of the prodrugs depends on their bioactivation level, it is crucial to assess whether the overexpression of mymA mediated by alpibectir potentiates Pto and Eto to the same extent. The antibacterial activities of AlpE (alpibectir/ethionamide) and alpibectir/prothionamide were compared against the H37Rv strain and various isogenic clones with mutations in the ethR-ethA regulon, the virS-mymA regulon, or both simultaneously (SI Fig.10). The concentration-response curves for Eto alone (IC50 = 1.18 mg/L) and Pto alone (IC50 = 0.31 mg/L) were similar against H37Rv, with Pto being slightly more potent. The inclusion of alpibectir similarly enhanced the efficacy of Eto and Pto, shifting the IC50 values to 0.03 mg/L for both (SI Table 3). Consistent with published data, the potency of Eto and Pto was compromised by the inactivation of ethA (Rv3854::Tn) or mymA (Rv3083::Tn)26. Complete resistance to both thioamides was observed in the double mutant strain CD6R1 (ethA-/mymA-), which lacks both bioactivation pathways26. Remarkably, alpibectir restored the sensitivity of both Eto and Pto in the ethA-deficient strain at levels equivalent to those observed in H37Rv. This suggests that the enhanced expression of the mymA operon induced by alpibectir achieves maximum activation of both prodrugs, irrespective of the ethA expression level. As expected, alpibectir did not enhance Pto potency in the mymA-deficient strain (Rv3083::Tn). This set of results collectively demonstrates that alpibectir uniformly boosts the activity of both thioamides through stimulation of the virS-mymA regulon, even in strains that lack EthA production.

Alpibectir boosts the potency of Eto and Pto sulfoxide metabolites.
Upon oral administration, human flavin-containing monooxygenases (FMO) convert some of the circulating Eto or Pto into sulfoxide derivatives Eto-SO29–32 and Pto-SO (manuscript in preparation), respectively. Interestingly, these intermediates are as active as their corresponding parental prodrug. As already suggested by Vannelli and colleagues33 and confirmed by us 18,33, both Eto-SO and Pto-SO still require M. tuberculosis FMOs such as EthA to be further transformed and become effective. Interestingly, addition of alpibectir to Eto-SO and Pto-SO boosted their respective potency against H37Rv in a similar amplitude to the one observed with the respective parental compounds (SI Table 3), showing that MymA, like EthA, is able to finalize the transformation of the sulfoxide derivatives into active compounds. Finally, the reduced potency of Eto-SO and Pto-SO observed against an EthA-deficient mutant was fully restored by the addition of alpibectir. This observation implies that both the parental prodrugs and their respective SO derivatives, whether they are produced by the liver or the lung FMOs, will be boosted by alpibectir.

Alpibectir boosts Eto in recombinant strains overexpressing inhA.
Although the intracellular activation of Eto and INH occurs through distinct and independent enzymatic pathways, it results in the formation of analogous covalent adducts between each drug and nicotinamide adenine dinucleotide (NAD). Both adducts subsequently inhibit the enoyl reductase InhA, a key enzyme in mycolic acid biosynthesis30,31,32. Here, we assessed the antibacterial activity of Eto alone or in combination with alpibectir on M. tuberculosis overexpressing inhA (SI Fig.11). When inhA is expressed at basal level, alpibectir decreased the IC50 of Eto from 0.07 to 0.01 mg/L, consistent with the observed effect on the H37Rv strain (Fig.1). Overexpression of inhA increased the IC50 of Eto from 0.07 to 1.4 mg/L. Notably, treatment of the inhA-overexpressing strain with AlpE decreased the IC50 of Eto from 1.4 to 0.3 mg/L. This shows that alpibectir is potent enough to enhance the efficacy of Eto, even when its target, InhA, is significantly overproduced. This also suggests that the bioactivation of ethionamide by MymA results in the formation of an NAD-adduct, similar to the process observed with EthA. Addition of alpibectir did not impact the MIC of INH, confirming that although InhA is targeted by both INH and Eto, alpibectir is specific for the activation of Eto (SI Fig.12).

AlpE inhibits growth of MDR-TB, including ethA and inhA mutants.
The in vitro susceptibility of M. tuberculosis to Eto alone or the combination (AlpE) was assessed using liquid cultures (BACTEC MGIT 960). The activity is expressed as MIC to suppress the growth of the strains. First, the activity was assessed on the standard laboratory strain H37Rv. The MIC was 1 mg/L for Eto alone. In the presence of alpibectir (tested at 0.017, 0.05, or 0.1 mg/L), the MIC of Eto decreased significantly to \le 0.031 mg/L, once more demonstrating the strong Eto potentiation by the booster (SI Table 6). The presence of alpibectir alone did not inhibit the growth of the bacteria at any of the 3 concentrations tested. Next, we looked at a panel of INH-resistant M. tuberculosis clinical strains (MIC > 0.1 mg/L) that also exhibited cross-resistance to Eto (MIC > 5 mg/L). Among the 21 strains selected, 12 had mutations in the inhA promoter and 16 had a mutation in katG (S315T). Most strains (18/21) were also resistant to rifampicin (Table 2). The ability of alpibectir to potentiate Eto activity was evaluated on these strains using alpibectir at 0.017 mg/L or 0.05 mg/L, combined with Eto at 0.8 mg/L. This concentration corresponds approximately to 1/6th of the clinical breakpoint (5 mg/L) above which strains are classified as resistant to Eto34. This sub-inhibitory Eto concentration was chosen to allow detection of changes in drug susceptibility of the strains to Eto in the presence of alpibectir. The two alpibectir concentrations enable the detection of a dose-dependent effect for certain strains with the higher concentration capturing the full extent of Eto potentiation.

Remarkably, 11 (92%) of the 12 INHR/EtoR strains mutated in the inhA promoter were re-sensitized to Eto 0.8 mg/L in the presence of 0.05 mg/L alpibectir and 9 strains (75%) were re-sensitized with 0.017 mg/L alpibectir. All 9 INHR/EtoR clinical isolates without mutation in the promoter of inhA were sensitive to 0.8 mg/L Eto in the presence of 0.017 mg/L alpibectir.

Alpibectir alone did not inhibit the growth of any of the strains up to the highest tested concentration of 0.05 mg/L. Overall, 95% of INH/Eto cross-resistant isolates demonstrated sensitivity to the combination of Eto (0.8 mg/L) and alpibectir (0.05 mg/L). Only one isolate (13M2489, lineage 2) was unresponsive to the alpibectir/Eto combination. Whole genome sequencing of this strain revealed a large deletion in the virS/mymA regulon, consistent with the proposed mode of action of alpibectir. This mutation is extremely rare among lineage 2 strains. In the CRyPTIC database, which includes data from 12,287 M. tuberculosis isolates, 4,295 strains belong to lineage 2, and none of these exhibits a complete deletion of the virS-mymA regulon. It is worth noting, however, that M. tuberculosis sublineage 4.8 naturally harbors a similar large deletion encompassing the virS-mymA regulon35, rendering these strains intrinsically unresponsive to alpibectir. Accordingly, particular caution will be necessary in regions where this sublineage is prevalent, to avoid lowering the Eto dose to sub-MIC levels in patients infected with such strains.

AlpE is active in vivo in a 4-week acute murine model of TB.
The ability of alpibectir to potentiate the activity of Eto was evaluated in an acute mouse model of TB36,37. Mice were intravenously (IV) infected with M. tuberculosis H37Rv and then treated for 28 days with various doses of Eto, alone or in combination with alpibectir. In untreated controls, lung bacillary load increased by +3.5 log10 CFU (p<0.0001) relative to the initial load of 5.4 log10 CFU at pre-treatment. Oral administration of INH (25 mg/kg q24h, reflecting the human equivalent dose38) reduced the bacillary burden to 4.4 log10 at D28 (p=0.06), equivalent to a 1 log10 reduction from the initial load (Fig.7, SI Table 7A).

Evaluation of Eto alone (5–200 mg/kg) showed a dose-dependent improvement in treatment efficacy; however, only the 200 mg/kg dose led to a statistically significant 1 log10 CFU reduction compared with pre-treatment (SI Table 7B).

In mice treated solely with alpibectir at 0.1, 0.5, or 1.6 mg/kg, lung CFU counts at Day 28 did not fall below the initial load. Nevertheless, at 1.6 mg/kg, alpibectir produced lower CFU counts than at 0.5 or 0.1 mg/kg, and was 2.1 log10 below the untreated control, consistent with the intrinsic growth-inhibitory effect of alpibectir observed in vitro.

Combinations of alpibectir and Eto resulted in greater bacterial load reduction: whereas Eto at 5 mg/kg alone was only modestly active (7.8 log10 CFU), adding alpibectir at 0.1, 0.5, or 1.6 mg/kg reduced the CFU counts by an additional 1.5 (p=0.0043), 2.8 (p<0.0001), and 2.3 (p<0.0001) log10, respectively (Fig.7). Moreover, Eto doses of 15 or 50 mg/kg combined with 0.5 or 1.6 mg/kg alpibectir decreased the CFU counts by more than 1 log10 relative to pre-treatment levels. In combination with alpibectir, Eto at 15 mg/kg achieved maximal bactericidal activity (Emax). Increasing the ethionamide dose to 50 mg/kg provided no additional benefit, suggesting that the pharmacological target, InhA, is already saturated at 15 mg/kg. In contrast, the lowest tested dose of Eto (5 mg/kg) failed to reach this level of activity, indicating that the minimal effective Eto dose in the presence of alpibectir lies between 5 and 15 mg/kg in this murine model.

A sigmoidal dose-effect analysis (SI Fig.13) indicated that an Eto dose of \pm90 mg/kg was required to achieve a 1 log10 CFU reduction compared with the initial bacillary load, matching the effect of 25 mg/kg INH. In contrast, in the presence of alpibectir at 0.1, 0.5, or 1.6 mg/kg, the Eto dose needed for this same reduction fell to 33.0, 7.5, and 8.5 mg/kg, respectively. Notably, alpibectir at doses \ge 0.5 mg/kg lowered the required Eto dose by tenfold (SI Fig.13). Strikingly, alpibectir alone at 1.6 mg/kg prevented mortality (p=0.0001), consistent with its impact on lung CFU counts (SI Fig.14B) and with its intrinsic growth-inhibitory activity observed in vitro. Although alpibectir at 0.1 and 0.5 mg/kg also had a significant yet moderate impact on survival (17%), combining alpibectir with as little as 5 mg/kg Eto completely prevented mortality (SI Fig.14C).

Methods
Ethical statement
The in vivo experimental project was favorably evaluated by the Charles Darwin Ethics Committee no. 005, located at Pitié-Salpêtrière Hospital (France), and clearance was given by the French Ministry of Education and Research under the APAFIS no. 12380-2017112809414820 v3. The animal facility was authorized to conduct animal experiments (license number C-75-13-08). The individuals involved in the animal experiments underwent specific training recognized by the French Ministry of Education and Research.

Reagents and consumables
Alpibectir batch RL01L234A0 was provided by BioVersys AG. Details of the synthesis of alpibectir and its analogue will be described elsewhere (manuscript in preparation). Eto (E6005), Pto (SMB00387), INH (I0500000), and DMSO (D8418) were purchased from Sigma.

Chemical synthesis of Eto-SO and Pto-SO
Eto-SO and Pto-SO were synthesized as follows. Firstly, meta-chloroperoxybenzoic acid (2.97 g, 12.0 mmol for Eto-SO or 2.74 g, 11.1 mmol for Pto-SO) was added to a solution of Eto (2 g, 12.0 mmol) or Pto (2 g, 11.1 mmol) in MeOH (30 mL) at 0°C. After 4 h of agitation at 0°C, the solvent was removed in vacuo, and the crude product was purified by column chromatography (mobile phase: ethyl acetate/MeOH 90/10). The desired product was isolated as yellow microcrystals (yield: 0.65 g for Eto-SO and 1.41 g for Pto-SO). Eto-SO 1H NMR (300 MHz, DMSO-d6) \delta (ppm) 9.38 (s, 1H), 8.56 (dd, J = 5.2, 0.5 Hz, 1H), 8.48 (s, 1H), 7.39 (s, 1H), 7.31 (dd, J = 5.2, 1.7 Hz, 1H), 2.78 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). Pto-SO 1H NMR (300 MHz, DMSO-d6) \delta (ppm) 9.39 (brs, 1H), 8.57 (dd, J = 5.2, 0.6 Hz, 1H), 8.50 (brs, 1H), 7.38 (s, 1H), 7.32 (dd, J = 5.2, 1.7 Hz, 1H), 2.74 (t, J = 7.5 Hz, 2H), 1.70 (s, J = 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H).

Bacterial strains
All strains were cultured from frozen stocks in Middlebrook 7H9 medium (BD Difco) supplemented with glycerol 0.2% (Euromedex), Tween 80 0.05% (Sigma-Aldrich) and oleic acid-albumin-dextrose-catalase 10% (OADC, BD Difco) except if otherwise indicated.

All strains used are briefly described in Table 3. Recombinant M. tuberculosis strains expressing an enhanced green fluorescent protein (GFP) were obtained by transformation with an integrative plasmid containing the Aequoria Victoria egfp gene52. Luciferase-expressing M. tuberculosis H37Rv was obtained by transformation with the pEG200 plasmid53. Except for strain E1, which is a derivative of the W4 Beijing strain, all other strains are derived from H37Rv20. Strains 8F and CD6R1 are spontaneous mutants selected in vitro and Rv3854::Tn and Rv3083::Tn are transposon mutants26. CD6R1, Rv3854::Tn, and Rv3083::Tn were kindly gifted by Dr. Deborah Hung (Broad Institute, USA)26. M. tuberculosis H37Rv overexpressing InhA was obtained by transformation with plasmid pMV261\OmegainhA54. As a control, H37Rv was also transformed with the empty pMV261 vector.

H37Rv \DeltaadhD was constructed as previously described with some modifications55. The DNA substrate for allelic replacement of adhD (Rv3086) constituted of the 500 bp upstream and downstream regions of adhD on either side of a cassette composed of hygR (hygromycin resistance gene) under its own promoter and the sigA promoter in frame with Rv3087 in order to restore the expression of the last genes of the operon. The construct was synthesized and cloned into a pUC57 vector between NdeI and HindIII restriction sites (GeneCust). E. coli DH5\alpha strain transformed with the plasmid was grown in LB medium supplemented with 100 \mu g/mL ampicillin at 37°C. The pUC\DeltaadhD plasmid was isolated (QILAprep Spin Miniprep kit, Qiagen), digested with NdeI and HindIII (FastDigest, Thermo Fisher Scientific), and gel purified (QIAquick gel extraction, Qiagen). M. tuberculosis H37Rv pJV53 was cultured in 20 mL 7H9 supplemented with 0.2% succinate and 20 \mu g/mL kanamycin in 25 cm² tissue-culture flasks. Cultures were incubated at 37°C until mid-log phase and induced with 0.2% acetamide. Electrocompetent M. tuberculosis (200 \mu L) was transformed with 100 ng of salt-free dsDNA. Electroporated bacteria were resuspended in 6 mL 7H9 and following an incubation at 37°C for 72 h, harvested by centrifugation at 3,000 x g for 5 min and plated on 7H10 with 50 \mu g/mL hygromycin. Allelic exchange was confirmed by sequencing.

Extracellular antibacterial activity
Stock solutions of Eto, Pto, and INH were prepared in DMSO at 10 mg/mL, while alpibectir and its analogue were prepared at 10 mM. Aliquots were stored at -20°C. To set up the assay plates, all compounds were initially transferred to a 384-well low-volume polypropylene plate (Corning 3672).

For checkerboard assays, ten 3-fold serial dilutions of Eto and alpibectir (or its analogue) were distributed vertically and horizontally, respectively, in black Greiner 384-well clear bottom polystyrene plates (Greiner 781091) using an Echo 550 liquid handler (Labcyte). The assay was conducted with two technical duplicates per biological replicate, with a total of three biological replicates. DMSO was added to relevant wells to ensure a final concentration of 0.6% across all wells. The assay plates were inoculated with 50 \mu L/well of M. tuberculosis H37Rv cultures expressing GFP at an optical density (OD600 nm) of 0.02 and incubated at 37°C for 5 days. GFP fluorescence (\lambdaex = 485 nm/\lambdaem = 535 nm) was read using the Ensight Multimode plate reader (PerkinElmer, USA). For OD measurement of non-GFP expressing strains, an inoculum of OD600 nm 0.002 was used and plates were incubated at 37°C for 7 days before OD measurement at 600 nm using the Ensight multimode plate reader. IC50 and IC90 values were calculated by fitting fluorescence values for each concentration on GraphPad Prism version 10.2.2 (GraphPad Software, Inc., San Diego, CA).

For assays evaluating the antibacterial activity of Eto, Eto-SO, Pto, Pto-SO, or INH in combination with a fixed concentration of alpibectir, ten 3-fold serial dilutions of Eto, Eto-SO, Pto, Pto-SO, or INH were performed in duplicates into black Greiner 384-well clear bottom polystyrene plates (Greiner 781091) using an Echo 550 liquid handler (Labcyte). DMSO was added to relevant wells to ensure a concentration of 0.3% across all wells. To evaluate the effect of alpibectir on the antibacterial activity of the compounds, alpibectir was added at the indicated concentrations using the Echo 550 liquid handler. The assay plates were inoculated and incubated as mentioned above. GFP fluorescence or OD were measured and analyzed as detailed above.

Intracellular antibacterial activity
THP-1 cells were cultured at a density of 2x105 to 1x106 cells/mL in RPMI medium (Thermo Fisher 61870036) supplemented with 10% fetal bovine serum (FBS, Gibco 10500) at 37°C, 5% CO2.

For the intracellular assay in Fig.1F, which measures luminescence of intracellular M. tuberculosis: A single-cell bacterial suspension of M. tuberculosis H37Rv pEG200 (expressing a luciferase operon) was prepared for infection. Briefly, an exponential phase culture was centrifuged at 3,200 x g for 5 min and the pellet resuspended in RPMI-FBS before centrifugation at 100 x g to remove clumps. The supernatant was passed 5 times through a 0.9 mm diameter needle and the OD was measured. THP-1 cells (5 mL of 5x106 cells) were simultaneously infected by adding the bacterial suspension at a multiplicity of infection of 10 and differentiated with 40 ng/mL phorbol 12-myristate-13-acetate (Sigma P1585). The cells were incubated for 4 h at 37°C, 5% CO2 and the adherent cell monolayer was detached with a cell scraper. The cells were washed 4 times by centrifugation at 300 x g and resuspended in 25 mL of RPMI-FBS. Opaque white 384-well assay plates were prepared as described above for the extracellular assay. Plates were inoculated with 50 \mu L of the cell suspension (1x104 cells/well) and incubated for 5 days at 37°C, 5% CO2. The antibacterial activity of the compounds was determined by measuring luminescence of intracellular bacteria. The experiment was performed in triplicate. For this assay, THP-1 cells transfected with Incucyte® CytoLight Red Lentivirus (EF-1 Alpha promoter, puromycin selection, Sartorius 4482) were used to enable measurement of their viability in the presence of drug treatment. The infected THP-1 cells (as described above) were distributed to black 384-well assay plates and incubated for 5 days. Images (3 fields per well) were acquired at 20X using the GE IN Cell 6500HS (GE Healthcare Life Sciences) automated confocal microscope with a 561 nm laser and 600/54 nm emission filters for detection of THP-1 cells. Images were processed using the Columbus image analysis software version 2.9.1 (PerkinElmer).

For the intracellular assay in Supplementary Fig.4 of the Supplementary Information, which measures macrophage survival: Prior to infection, 5 mL of 5x106 THP-1 cells (1x106 cells/mL) were differentiated with 40 ng/mL phorbol 12-myristate-13-acetate (Sigma P1585) and incubated for 3 days. A single-cell bacterial suspension was prepared for infection. Briefly, an exponential phase M. tuberculosis H37Rv culture was centrifuged at 3,200 x g for 5 min and the pellet resuspended in RPMI-FBS before centrifugation at 100 x g to remove clumps. The supernatant was passed 5 times through a 0.9 mm diameter needle and the OD was measured. The medium of the adherent THP-1 was renewed, and the bacterial suspension was added at a multiplicity of infection of 10. The cells were incubated for 2 h and then washed 3 times with RPMI. They were incubated with 50 mg/L amikacin (Sigma A1774) for 1 h to kill extracellular bacteria. The cell monolayer was washed 3 times with phosphate buffered saline (PBS, Sigma #D8537) and detached with trypsin-EDTA 0.25% (Gibco 25200056). The cell suspension was centrifuged at 100 x g for 5 min and the pellet resuspended in 25 mL of RPMI-FBS. 384-well assay plates were prepared as described above for the extracellular assay. Plates were inoculated with 50 \mu L of the cell suspension (1x104 cells/well) and incubated for 5 days at 37°C, 5% CO2. The antibacterial activity of the compounds was determined by measuring macrophage survival. On day 4, 5 \mu L of 0.05% resazurin was added to each well and resorufin fluorescence was read on day 5 after an incubation of 16 h at 37°C, 5% CO2. The experiment was performed in triplicate.

Time-kill assay
Serial ten-fold dilutions of Eto, INH, and alpibectir were prepared in DMSO. Appropriate volumes for each treatment were added to non-ventilated 25 cm² tissue-culture flasks. DMSO volume was compensated so that the concentration of DMSO in all flasks was equal to 0.25%.

The flasks were inoculated with 10 mL of M. tuberculosis H37Rv WT or \DeltaadhD cultures diluted to an OD600 nm of 0.005 (theoretically corresponding to 5 x 105 CFU/mL) and incubated at 37°C without shaking.

Cultures from each flask were plated on square plates containing Middlebrook 7H10 solid medium (BD) supplemented with glycerol 0.2% (Euromedex), Tween 80 0.05% (Sigma-Aldrich) and oleic acid-albumin-dextrose-catalase 10% (OADC, BD Difco) on days 0, 2, 4, 7, 14, and 21. On day 0, only 3 cultures were plated whereas on other days, all cultures were plated. Before plating, cultures were 10-fold serially diluted (up to 10-7) and 10 \mu L of each serial dilution and of the non-diluted culture were plated. CFUs were counted after incubating the plates for 3 weeks at 37°C. The lowest possible number of countable CFUs (limit of detection) is 1 when plating 10 \mu L of a non-diluted culture, which amounts to a log10 CFU/mL of 2.

Three biological replicates were performed for H37Rv WT and three replicates for H37Rv \DeltaadhD.

Transcriptomics
M. tuberculosis H37Rv cultures were diluted to an OD600 nm of 0.2 from a fresh exponentially growing culture and were incubated at 37°C without shaking until an OD600 nm of 0.7 was reached. To study the effect of alpibectir alone on gene expression levels, 25 mL cultures were treated with 0.1 mg/L of alpibectir for 24 h in triplicate. To study the effect of Eto combined with alpibectir, cultures were first treated for 20 h with 0.1 mg/L alpibectir followed by an additional 4 h with 2 mg/L Eto. The final DMSO concentration in all cultures was equal to 0.1%. Cultures were treated only with 0.1% DMSO as controls (in triplicate for comparison with alpibectir alone or in a single replicate for comparison with alpibectir and Eto treatment). RNA extraction and transcriptomics analysis were performed as previously described21. Briefly, after the treatment duration, 10 mL of the cultures were harvested by centrifugation at 3,000 x g for 5 min, resuspended in 1 mL of RNAproTM (FastRNA Pro Blue Kit, MP biomedicals) and homogenized in impact-resistant 2 mL tubes containing 0.1 mm silica spheres (Lysing Matrix B, MP biomedicals) using a FastPrep FP120 cell disrupter (Thermo Fisher Scientific) at 6.0 Hz for 40 s. The ribolysed cells were centrifuged at 12,000 x g to remove cellular debris and RNA was purified following the manufacturer's instructions. Ribosomal RNA (rRNA) depletion was performed using QIAseq FastSelect – 5S/16S/23S Kits; Qiagen). Libraries for Illumina sequencing were prepared with the TruSeq RNA sample preparation kit version 2.0 rev. A (Illumina Inc.). All cDNA libraries were uniquely indexed. cDNA libraries were sequenced using an Illumina NextSeq 500 system (Illumina Inc.) in high-output mode. All samples were multiplexed on one lane of the flow cell and sequenced in single-read sequencing mode with read lengths of 150 bp. Raw RNA-seq reads were processed with Illumina quality control tools using default settings. Sequences shorter than 50 bp and/or that contained any ‘Ns’ and/or with a mean quality score lower than 30 were removed using PRINSEQ (http://prinseq.sourceforge.net/index.html). Next, rRNA-specific reads were filtered out by mapping all the reads on M. tuberculosis rRNA sequences using Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Analysis of the RNA sequencing data was conducted using the SPARTA open-source software package with default parameters (https://sparta.readthedocs.io/en/latest/). A total of 4036 genes could be analyzed. Raw and processed RNA-seq reads were deposited in the ArrayExpress repository (accession code E-MTAB-16253).

Proteomics
Protein extraction and proteomics analysis were performed as previously described21. M. tuberculosis H37Rv was cultured in 7H9 without glycerol and supplemented with 2% (w/v) glucose and 0.025% (v/v) tyloxapol at 37°C for about 7 days until an OD600 nm of 0.8. The culture was diluted to an OD600 nm of 0.2 and split in 4 subcultures. Two of them were further incubated for 72 h in the presence of 1 \mu M (0.33 mg/L) alpibectir and the others were similarly incubated with DMSO. Protein extracts were prepared by centrifuging the culture and washing the pellet with PBS. The pellet was resuspended in 1 mL lysis buffer (50 mM Tris-HCl, pH 7.4, 0.8 (v/v) % NP-40, 1.5 mM MgCl2, 5% glycerol, 150 mM NaCl, 25 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol (DTT) and one Complete EDTA-free protease inhibitor tablet (Roche)) and disrupted using a TissueLyser II (Qiagen) for three cycles at full amplitude for 5 min in refrigerated supports. Bacterial lysates were centrifuged at 14,000 x g for 30 min and the supernatant filtered using Millex LG (PTFE) 0.2 \mu m/13 mm diameter. Filtered supernatants were further ultracentrifuged at 4°C for 60 min (140,000 x g, 55,000 rpm). Samples were prepared in quadruplicate.

SDS sample buffer (2% SDS, 10% glycerol, 0.005% bromophenol blue, 100 mM Tris-HCl, 125 mM Tris Base) was added to 37.5 \mu L of the M. tuberculosis protein extract. Proteins were digested according to a modified single pot solid-phase sample preparation (SP3) protocol56,57. Briefly, proteins were bound to paramagnetic beads (SeraMag Speed beads, GE Healthcare, CAT#45152105050250, CAT#651521050502) in 50% ethanol (final concentration). Beads were washed 4 times with 70% ethanol and proteins digested by resuspending in 0.1 mM HEPES (pH 8.5) containing TCEP, chloroacetamide, trypsin, and LysC followed by overnight incubation.

Peptides were labeled with isobaric mass tags (TMT10, Thermo Fisher Scientific, Waltham, MA) using the 10-plex TMT reagents, enabling relative quantification of 10 conditions in a single experiment57,58. The labeling reaction was performed in 40 mM triethylammoniumbicarbonate, pH 8.5 at 22°C and quenched with glycine. Labeled peptide extracts were combined into a single sample per experiment, lyophilized and subjected to LC-MS analysis.

Samples were pre-fractionated by reversed-phase chromatography at high pH to 16 samples of which 10 were measured59.

After lyophilization, dried samples were resuspended in 0.05 % trifluoroacetic acid in water. Half of the sample was injected into an Ultimate3000 nanoRLSC (Dionex) coupled to a Q-Exactive (Thermo Fisher Scientific). Peptides were separated into custom-made 35 cm x 100 \mu m (ID) reversed-phase columns (Reprosil) at 55°C, gradient elution was performed from 3.5% acetonitrile to 29% acetonitrile in 0.1% formic acid and 3.5% DMSO over 120 min.

The Q Exactive Plus operated with data-dependent top 10 method. MS spectra were acquired using 70.000 resolution and an ion target of 3E6. Higher energy collisional dissociation (HCD) scans were performed with 35% NCE at 35.000 resolution (at m/z 200), and ion target settings was set to 2E5 so as to avoid coalescence57. The instruments were operated with Tune 2.4 and Xcalibur 3.0 build 63.

Mascot 2.4 (Matrix Science, Boston, MA) was used for protein identification by using a 10 parts per million mass tolerance for peptide precursors and 20 mD (HCD) mass tolerance for fragment ions.

To create the fasta file for mascot searching, all proteins corresponding to the taxonomy ‘Mycobacterium tuberculosis H37Rv’ were downloaded from Uniprot (release 20170621) and supplemented with common contaminant protein sequences of bovine serum albumin, porcine trypsin and mouse, rat, sheep and dog keratins. To assess the false discovery rate (FDR), “decoy” proteins (reverse of all protein sequences) were created and added to the database, resulting in a database containing a total of 7,994 protein sequences, 50% forward, 50% reverse.

Unless stated otherwise, we accepted protein identifications as follows: (i) For single spectrum to sequence assignments, we required this assignment to be the best match and a minimum Mascot score of 31 and a 10x difference of this assignment over the next best assignment. Based on these criteria, the decoy search results indicated <1% false discovery rate (FDR). (ii) For multiple spectra to sequence assignments and using the same parameters, the decoy search results indicated <0.1% FDR. Quantified proteins were required to contain at least 2 unique peptide matches. FDR for quantified proteins was <0.1%.

Protein abundance ratios were calculated by IsobarQuant60. Foldchange ratios were calculated from sum ion areas of compound-treated samples and compared to one vehicle control that was set to 1. The complete list of analyzed proteins is provided as Supplementary Data 1.

In-cell transcriptional regulator assay
The in-mammalian cell bacterial regulator reporter assay was performed as previously described20,21. To construct chimeric bacterial-mammalian transcriptional regulators, the coding sequence of EthR, EthR2, or VirS was fused to the Herpes simplex derived transactivator protein VP16. The sequence was optimized for expression in human/mouse, synthesized (Genscript) and introduced into pSEAP2-control (Clontech) using EcoRI/XbaI to generate expression vectors PSV40-EthR-VP16-HA-pA (pCK310), PSV40-EthR2-VP16-HA-pA (pVT20), and PSV40-VirS-VP16-HA-pA (pCK461), respectively. To enable transcriptional control by the chimeric transcriptional regulators constructed above, the promoter region recognized by EthR, EthR2, or VirS, i.e., the intergenic region between ethA and ethR, between ethA2 and ethR2, or between virS and mymA, was cloned upstream of a minimal variant of the human cytomegalovirus-derived promoter (PhCMVmin) into pSEAP2-basic (Clontech) using XhoI/EcoRI to generate OEthR-PhCMVmin-SEAP-pA (pCK311), OEthR2-PhCMVmin-SEAP-pA (pVT23), OVirS-PhCMVmin-SEAP-pA (pCK462), respectively.

Baby hamster kidney cells (BHK-21, American Type Culture Collection CCL-10) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco 41966) supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) penicillin/streptomycin (Gibco 15140) in a humidified atmosphere with 5% CO2 at 37°C. Prior to transfection, cells from a confluent grown culture dish were split 1:2 to a new petri dish and grown for 16 hours. For transfections with the EthR and EthR2 regulatory systems, 10 \mu g of total plasmid DNA (8 \mu g pCK310 and 2 \mu g pCK311 or 5 \mu g pVT20 and 5 \mu g pVT23) were added to 1 mL Opti-MEM reduced serum medium (Gibco 11058021), mixed with 30 \mu L MegaTran 1.0 transfection reagent (Origene TT200003) and incubated for 15 min at room temperature. For transfections with the VirS regulatory system, 10 \mu g of total plasmid DNA (5 \mu g pCK461 and 5 \mu g pCK462, ratio 1:1) were mixed with 0.5 mL 1x Jetprime buffer and 10 \mu L of jetPRIME transfection reagent (Poly Transfection 114-15), vortexed for 10 sec, and incubated for 10 min at room temperature. The DNA mixes were each transferred to the cells and incubated for 6 h for plasmid uptake. The medium was then removed, the cells washed twice with PBS and detached by adding 1 mL of 0.05% trypsin-EDTA (Gibco 25300). The cells were resuspended in 8 mL DMEM to neutralize the trypsin, counted, and diluted to 500,000 cells/mL using fresh culture medium before 100 \mu L of the suspension was distributed per well in a 96-well plate. Compound dilutions were prepared by adding 4.8 \mu L of alpibectir (10 mM stock solution in DMSO) to 600 \mu L culture medium. For EthR and EthR2, 100 \mu L of a two-fold dilution of the compound solution were added to the cells to reach final compound concentrations of 20 nM to 40 \mu M (=13.3 mg/L). For VirS, 100 \mu L of a five-fold dilution of the compound solution were added to the cells to reach final compound concentrations of 0.0008 nM to 40 \mu M (=13.3 mg/L). After incubation for 48 hours, SEAP expression was quantified from cell culture supernatants using the p-nitrophenyl phosphate-based method58.

Binding interaction between alpibectir and VirS
The recombinant protein VirS was produced as previously described59. Briefly, BL21(DE3) cells transformed with pET28a\OmegaVirS were cultured in LB medium containing 50 \mu g/mL kanamycin and protein expression was induced with 1 mM isopropyl-\beta-D-thiogalactoside overnight at 28°C. Harvested bacteria were suspended in lysis buffer (1X PBS, 100 mM L-arginine, 5 mM imidazole) containing a tablet of cOmplete EDTA-free protease inhibitor cocktail (Roche) and lysed by Emulsiflex-C3 (Avestin). The lysate was centrifuged (46,000 x g, 4°C) and the supernatant incubated with HisPur Cobalt-NTA affinity resin (Thermo Fisher Scientific) at 4°C for 1 h. VirS was eluted in 1X PBS, 100 mM L-arginine, 1 M NaCl, 500 mM imidazole, and 10% glycerol. The binding interaction between alpibectir and VirS was evaluated by thermal shift assay (TSA), conducted in an iCycler RT-PCR system (Bio-Rad, Hercules, CA). SYPRO Orange (Thermo Fisher Scientific) was used as extrinsic fluorescent dye at a final concentration of 5X and VirS was used at a final concentration of 8.17 mg/L. Alpibectir was added at a concentration comprised between 0.08 and 33.3 mg/L. Sample solutions were dispensed into a 96-well optical reaction plate (MicroAmp™ Optical, Thermo Fisher Scientific) and the plate was sealed with optical PCR plate sheet (Thermo Fisher Scientific). The final DMSO concentration was equal to 0.5% in all tested conditions. The plate was heated from 20 to 95°C with a heating rate of 0.7°C/min. The fluorescence intensity was recorded with wavelength ranges of 450-490 and 560-580 nm for excitation and emission, respectively. Each condition was measured at least in quintuplicate. Data were analyzed with GraphPad Prism 10 (GraphPad Software, Inc., San Diego, CA).

Crystallization and structure determination of the alpibectir/VirS complex was performed as previously described59. Briefly, a VirS construct (6HIS-SEET3a-VirS) integrated in the pET28a plasmid was expressed recombinantly in E. coli and purified to homogeneity using cobalt-NTA affinity resin. Crystals of alpibectir/VirS complex were obtained through an in situ proteolysis approach. The method involved incubating a mixed solution of VirS and alpibectir (at molar ratio of 1:1) on ice for 20 min, followed by the addition of subtilisin (final concentration of 0.1 mg/mL). Subsequently, sitting-drop vapor diffusion crystallization experiments were conducted at 20°C using a reagent solution containing 0.3 M sodium acetate trihydrate, 0.1 M Tris pH 7.5, 8% (w/v) PEG20,000, 8% (v/v) PEG500-MME. The crystal structure was solved by the molecular replacement method using the MOLREP program60 and the VirS AlphaFold2 model61 as search template. Data collection and refinement statistics are provided in SI Table 2. An electron density omit map was generated by excluding the alpibectir molecule from the structure, then randomly modifying the remaining atom coordinates within a range of 0-0.3 Å, followed by updating the corresponding structure factors and computing a residual map. The analysis of the interactions between alpibectir and VirS in the crystal structure was facilitated using the LigPlot+ program62.

Binding interaction between alpibectir and \alpha-latalbumin
As a negative control, the binding interaction between alpibectir and the \alpha-latalbumin protein (Sigma, L-5385) was evaluated by thermal shift assay (TSA), conducted in a CFX-Opus 96 Dx RT-PCR detection system (Bio-Rad CFX-Opus 96, Hercules, CA). SYPRO Orange (Thermo Fisher Scientific) was used as an extrinsic fluorescent dye at a final concentration of 5X and \alpha-latalbumin was used at a final concentration of 5 \mu M. Concentrations of alpibectir ranged from 0.250 to 100 \mu M. Sample solutions were dispensed into a 96-well optical reaction plate (MicroAmp™ Optical, Thermo Fisher Scientific) and the plate was sealed with optical PCR plate sheet (Thermo Fisher Scientific). The final DMSO concentration was kept at 0.5% in all wells. The plate was heated from 20 to 85°C with a heating rate of 1.8°C/min. The fluorescence intensity was recorded with a range of excitation and emission wavelengths of 450-490 and 560-580 nm, respectively. Each condition was measured at least in quintuplicate. Data was analyzed with Prism 10 (GraphPad Software Inc.).

Determination of the FoR to alpibectir/Eto
Resistant mutants were selected on 7H11 plates containing various drug concentrations. Plates were prepared in triplicate to spread 1 mL of culture containing 107, 108, 109 bacteria. The inoculum was prepared by sonicating an exponentially growing culture of M. tuberculosis H37Rv using a LabSonic M (Sartorius) sonicator equipped with a Microtip MS1 of 1 mm diameter for 1 min at 80% amplitude and a 0.8 cycle. Colonies were counted after an incubation of 21 days at 37°C.

To test for cross-resistance, 14 clones selected on agar plates containing alpibectir/Eto combinations (and cultured with selective pressure) were streaked on plates containing either no antibiotic or moxifloxacin (1 mg/L; GSK978567A), linezolid (1 mg/L; GSK GW602383X), ethambutol (4 mg/L; GSK GF133424A), bedaquiline (0.5 mg/L; GSK3377910A), pretomanid (PA824, 2 mg/L; GSK3184596A), or rifampicin (0.5 mg/L; Sigma R3501). These concentrations correspond to 2x the MIC or, whenever available, 2x the epidemiological cut-off (ECOFF; representing measures of a drug MIC distribution that separate bacterial populations into those representative of a WT population and those with acquired or mutational resistance to the drug).

Determination of MIC values by BactecTM MGITTM 960
Minimal inhibitory concentration (MIC) values of compounds against M. tuberculosis H37Rv or clinical strains were determined in BactecTM MGIT960TM culture tubes supplemented with 10% OADC and inoculated with an exponential phase preculture of the corresponding bacterial strain. Tubes were continuously monitored and analyzed using the EpiCenter software, thus enabling a precise assessment of bacterial growth. The minimal inhibitory concentration is defined as “no growth detected” at the time a 100-fold diluted control culture (Growth control 1%) reaches 400 Growth Unit. An extended incubation period of a further 7 days following the positivity of the “Growth control 1%” allows determination of the drug concentration at which no bacterial “late growth” is observed.

Eto was tested at 2, 5 and 10 mg/L and in a few instances also at 20 mg/L. Alpibectir was tested alone at 0.05 mg/L. Alpibectir at 0.017 mg/L and in a few cases 0.05 mg/L was tested in the presence of 0.8 mg/L Eto. Growth was measured by detection of oxygen consumption in the BD BACTECTM MGIT960TM automated system in all tubes when the control tube showed growth.

Mouse efficacy study
To assess the bactericidal activity of boosted Eto, we used a 4-week treatment model of TB. A total of 175 4-week-old female Balb/c mice were intravenously infected into the tail vein with 0.5 mL of a calibrated bacterial suspension and an injected bacillary load of 106 CFU at D063.

Eto, INH, and alpibectir were suspended in 1% methylcellulose (MC) (400cPs) in water. Alpibectir/Eto combinations were formulated together into 1% MC. Mice were randomly allocated to 20 groups. A pretreatment group consisted of 12 mice that were sacrificed at D1. A negative control group consisted of 12 infected mice that were not treated. Treatment was started the day after infection and given orally on 6 out of 7 days for 4 weeks (D28). The treatment groups consisted of 6 or 12 mice each; for the latter, 6 mice were sacrificed after 2 weeks (D14) of treatment. A positive control group was treated with INH alone at the human equivalent dose of 25 mg/kg64. Eto (5, 15, 50, 100, or 200 mg/kg) and alpibectir (0.1, 0.5, or 1.6 mg/kg) were either given as monotherapy or Eto (5, 15, or 50 mg/kg) was combined with alpibectir (0.1, 0.5, or 1.6 mg/kg). Treatment efficacy was assessed in survival rate at D28 and bacillary load reduction at D14 and D28 compared to pretreatment (D1).

Mice were euthanized by cervical elongation. Lungs were removed, homogenized, serially diluted, and spotted onto drug-free Lowenstein Jensen for the determination of colony forming units (CFUs).

CFU numbers were converted to CFU/lung by multiplying the number of CFUs by the volume of the lung homogenate spotted and the dilution at which the colonies were counted. CFU data was then transformed into log10 CFU/lung for calculation of means and standard deviations (SD). The changes in the log10 CFU/lung at the endpoint compared to the pretreatment (D1) were calculated.

Dose-effect curves were fitted to the log10 CFU data by using a sigmoidal maximum-effect (Emax) model (GraphPad Prims, version 10.1.2, GraphPad Software Inc, San Diego, CA, USA) to determine the effect of Eto or of alpibectir/Eto combinations on the change in CFUs. The total daily dose (TDD) needed for 1-log10 growth, stasis and 1-log10-kill were determined. Mean CFU counts were compared using one-way analysis of variance (One-Way ANOVA, no matching or paring, assuming Gaussian distribution and equal SDs, correction for multiple comparisons with Dunnet test (recommended by Prism); F (19, 103) = 35.59, p<0.0001, R2 = 0.8678). The exact p-values are mentioned in the text. Data from D14 and D28 were analyzed separately. All experiments were performed in female mice; sex was not considered as a variable in the study design. All animals were grown in an animal facility under filtered air conditions (20-24°C), with a 12-hour light/dark cycle and relative humidity ranging from 45 to 85%, in plastic cages using sterilized wood shavings as bedding.

Statistics & Reproducibility
All experiments were conducted with at least 2 independent biological replicates as detailed in figure legends and “Methods”. No statistical methods were used to predetermine sample sizes, which are considered as standard. No data were excluded from experiments except the in vivo study for which some mice were excluded from analysis due to culture contamination as indicated in Supplementary Table 7A. Experiments were not randomized except the in vivo study for which mice were randomly assigned to treatment and control groups. The investigators were not blinded to allocation during experiment and outcome assessment. Statistical analyses were carried out using GraphPad Prism version 10.2.2 (GraphPad Software Inc.). Data are presented as mean \pm SD when applicable. For the in vivo study, statistical significance was analyzed using one-way ANOVA.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presents interesting findings on the potential of alpibectir to enhance the antibacterial activity of ethionamide (Eto) and prothionamide (Pto) through its interaction with the transcriptional regulator VirS. The study is well-designed, combining biophysical, genetic, and cellular assays to demonstrate the mechanism of action and therapeutic potential of alpibectir. However, some minor revisions are needed to strengthen the manuscript.\n1. In Line 285, The author mentioned “In contrast to the intrinsic growth-inhibitory activity shown in Fig.4, no bactericidal effect was observed when alpibectir was used alone at concentrations up to 1 mg/mL” and attribute the results to the impact on bacterial adaptability. However, the animal assay of alpibectir showed a decrease of Mtb CFU when treat with 1.6 mg/mL of alpibectir. Can the author provide a more detailed discussion on this results?\n2. In Line 258-262, the author suggested polar effect may occur during KO of adhD. The gene complementation experiment needs to be conducted.\n3. In transcriptomic/proteomic analysis, the author only showed sharp LFC of selected genes. However, it is necessary to declared the statistically significance. Also, a volcano plot with annotation of these differentially expressed genes would also be more intuitive.\n4. It would be beneficial to include an INH + alpibectir treatment group as an additional control in Fig. 6. This would help to distinguish whether the observed effects are unique to the Eto+alpibectir combination or not.\n5. The authors presented a virS/mymA-inactivated Beijing strain in Table 3. Is this mutation frequently observed in clinical strains? This would determine the effectiveness of the combination therapy.", "reviewer_2": "The manuscript by Edoo et al. reports work using a combination of orthogonal approaches to show that Alpibectir, an activator of VirS with bactericidal activity against Mycobacterium tuberculosis (Mtb), boosts the activation of the oxidoreductase MymA, promoting ethA-independent conversion of ethionamide into its active form to potentiate inhibition of InhA. The authors show that ethionamide administered in combination with Alpibectir led to lower bacterial loads in Mtb-infected mice. The work presented in the manuscript is well executed, robust and cogent. However, some aspects of the work should be clarified and discussed in more detail. Specific comments are as follows:\n- It is unclear whether Alpibectir alone can kill intracellular Mtb. In Figure 1F, Alpibactir at concentrations ranging from 0.050-0.333mg/L does not prevent replication of intracellular Mtb at concentrations at which ethionamide is ineffective against Mtb, suggesting that Alpibectir is not effective against intracellular Mtb. This should be clearly discussed. In addition, the authors use a rather unusual murine model of tuberculosis wherein the drug is administered 24 hours post infection. Rationale for using this model should be included. Did the lack of effect of Alpibectir against intracellular Mtb motivate the use of this model?. Does the short time lapse between infection and treatment amplify the effect of Alpibectir alone because the drug helps reduce the extracellular Mtb load?\n- Figure 2, panel B. Although the data are compelling, some relevant controls are missing in the thermal denaturation experiment. The authors should either repeat it with inclusion of another protein that does not bind Alpibectir, or ideally with inclusion of a variant of VirS that cannot bind Alpibectir. The structural data should help in designing such a variant.\n- Figure 4. There is no attempt to complement the ∆adhD mutant. It would be useful to complement the ∆adhD mutant with adhD and each of the three downstream genes individually first, and then in combination if unsuccessful, to define which gene(s) are responsible for the intrinsic activity of Alpibectir.\n- Figure 5. The molecular mechanisms underlying ethionamide potentiation by Alpibectir remain unclear. Ethionamide is primarily converted into its active form by EthA in the absence of Alpibectir, the addition of which favours bioconversion of ethionamide by MymA. If EthA and MymA activate ethionamide into the same bioactive form (the NAD adduct), do they do so at the same rate? For the same concentration of ethionamide, how do levels of the bioconverted form compare in H37Rv versus H37Rv treated with Alpibectir? Does activation by MymA allow for larger accumulation of the bioconverted, active form compared to activation by EthA? This could explain why the combination of Alpibectir/ethionamide is better able to kill drug-recalcitrant Mtb subpopulations, and why the combination is effective at killing strains overexpressing InhA (lines 368-373). These measurements should be done; at the very least, these questions should be discussed.\n- p12, line 282: the use of “only” might be misleading as ethionamide alone at 2.5 or 25 mg/L leads to statistically similar levels of bacterial clearance at day 7.\n- The grounds on which the authors conclude that InhA is expressed at a basal level or overexpressed are unclear.\n- How do the authors explain the absence of dose effect between administration of 15 and 50mg/kg of ethionamide?\n- Please check the policy of Nature Communications regarding the use of “data not shown”.", "reviewer_3": "The objective of discovering new treatments for tuberculosis is an undeniable priority, especially for those areas and regions and which tuberculosis have a high incidence for example in sub-Saharan Africa and in south-east Asia. The reporting of novel drugs or drug combinations are therefore not only important but necessary.\nIn this study Edoo and colleagues report on the novel drug combination that involves Alpibectir and ethionamide. This study reports on it to effects in studies that were directly conducted against M. tuberculosis bacilli, against bacilli harboured in macrophages as well as its effects tested in mice.\nGeneral Comments:\nThe study design is well constructed with logical flow in the experimental steps. The findings that alpibactir potentiates Eto activity and has antibacterial activity independent of Eto is highly promising.\nIn Fig.1A the effects of alpibactir is generally evaluated against the effect of Eto in its absence. While this is a necessary control, the inclusion of a compound in the same class of amino piperidines that does not potentiate Eto is an additional control required to demonstrate specificity.\nIn the next phase of the study, the effects of Alp/E was tested against intracellular bacilli (Fig 1F). The authors report efficacy, however whether macrophages remain intact and viable for the duration of the assay is not presented. Should macrophages be lysed, the test would essentially evaluate efficacy against extracellular bacilli. The inclusion of a data that demonstrate macrophage viability for the duration of the experiment must be included to validate the conclusion of efficacy against intracellular bacilli. Again, while the absence of alpibactir serves as a control in Fig 1F, a control to demonstrate drug specificity is absent.\nIn line 169, the authors refer to the “…exceptional specificity of alpibactir…”. The inclusion of a non-specific control for alpibactir would enhance this claim. The authors should address why this was not done.\nThe graphical lines and points in Fig 5A and 5B is difficult to distinguish. The resolution of the data points should be improved. In lines 274-277 the authors states the following: “At a concentration near its MIC (2.5 mg/L), Eto alone exhibited a constant yet relatively slow bactericidal activity in vitro, resulting in an approximate 4-log10 reduction of CFU in 7 days, never reaching the limit of detection of 102 CFU/mL.” This is very difficult to verify. From Fig 5A it appears that 102 is indeed reached prior to regrowth, specifically at 25mg/ml and is sustained until D21. The authors should comment on this.\nWhile, Alp/E is shown to be more effective than Eto alone, measurements terminate at 21 days. Has measurement been done for longer periods? What happens with regrowth after D21?\nWhy is there a limit of detection of 102 CFU’s? For example, it is possible to obtain absolute pulmonary bacterial burdens (<100) if the whole lung volume is homogenised and plated. This would give an indication of whether treatment induces levels of sterility.\nIt appears that the addition of alpibactir to Eto beyond 15mg/ml has no added benefit in terms of reducing pulmonary bacilli burdens, at least at the 4 week time point reported in Fig 6. It may be that the optimal Eto concentration is less than 15 (between 5-15mg/ml). The authors may want to comment on this. The addition of alpibactir to Eto at a concentration at 50mg/ml has no added potentiation. The authors should comment on this observation.\nMinor comments/questions:\nBlue to Yellow scale need to be indicated in Fig 1A.\nLine 194, should be Fig 2B-2C, rather than Fig 2A-C\nWhy was the concentration of 0.33mg/ml for alpibactir selected for the study in Fig 3?\nthe authors should provide context for the selection of concentrations used, for both Eto and alpibactir in the study section under the heading “Alpibectir/Eto inhibits growth of MDR-TB, including ethA and inhA mutated strains” page lines 304-404. What is the relevance of 1/6th of the clinical breakpoint (line 387)?"}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe manuscript presents interesting findings on the potential of alpibectir to enhance the antibacterial\nactivity of ethionamide (Eto) and prothionamide (Pto) through its interaction with the transcriptional\nregulator VirS. The study is well-designed, combining biophysical, genetic, and cellular assays to\ndemonstrate the mechanism of action and therapeutic potential of alpibectir. However, some minor\nrevisions are needed to strengthen the manuscript.\n1. In line 285, the author mentioned “In contrast to the intrinsic growth-inhibitory activity shown in\nFig.4, no bactericidal effect was observed when alpibectir was used alone at concentrations\nup to 1 mg/mL” and attribute the results to the impact on bacterial adaptability. However, the\nanimal assay of alpibectir showed a decrease of Mtb CFU, when treat with 1.6 mg/mL of\nalpibectir. Can the author provide a more detailed discussion on these results?\nThank you for highlighting this important point and for noting the inconsistencies in the original\nparagraph. First, as shown in Fig. 6, the maximum concentration of alpibectir used in the in vitro time-\nkill assay was 0.3 mg/L and not 1 mg/L as we had previously stated. The corresponding sentence (line\n287) has now been corrected to: “In comparison to the intrinsic growth-inhibitory activity shown in Fig.\n5, no bactericidal activity was observed when alpibectir was used alone at concentrations up to 0.3\nmg/L”.\nIt is important to emphasize that the checkerboard assay shown in Fig. 5 measures the evolution of\nbacterial density over a five-day period. In this assay, even at 1 mg/L alpibectir, the bacterial population\nat day 5 remains higher than at day 0 (noting that OD at day 0 is below the detection limit). The OD\ndifference between the 0 mg/L and 1 mg/L conditions is approximately 0.08 on day 5, supporting our\nconclusion that alpibectir limits bacterial growth but does not exert a bactericidal effect. A similarly low,\nstatistically non-significant inhibitory trend was observed in the kill-curve assay, which lacks the\nsensitivity required to detect such modest growth-inhibitory effects.\nAs rightly noted by the reviewer, the in vivo experiment shows a more pronounced growth-inhibitory\neffect, with a 2.05-log CFU reduction (down to 6.84 log CFU) at 1.6 mg/kg alpibectir compared to the\n10 10\nuntreated control on day 28 (8.89 log CFU). This effect was not detectable at the lower tested doses\n10\n(0.5 mg/kg and 0.1 mg/kg). This confirms that alpibectir, consistent with the time-kill results, can reduce\nbacterial multiplication in vivo but does not achieve bactericidal activity, which would require CFU levels\nto fall below 5.36 log .\ncorresponding sentence in the in vivo section (line 441) as follows: “Strikingly, alpibectir alone at 1.6\nmg/kg prevented mortality (p=0.0001), consistent with its impact on lung CFU counts (SI Fig.14B) and\nwith its intrinsic growth-inhibitory activity observed in vitro”.\n2. In line 258-262, the author suggested polar effect may occur during KO of adhD. The gene\ncomplementation experiment needs to be conducted.\nResponse: Indeed, since we used homologous recombination to replace the adhD gene with a\nhygromycin-resistance cassette, we acknowledge the possibility of a polar effect on downstream genes.\nAs noted in the manuscript, the loss of intrinsic activity of alpibectir observed in the ΔadhD strain may\ntherefore not be solely attributable to the disruption of adhD itself.\nTo determine whether one or more downstream genes contribute to intrinsic activity of alpibectir, it would\nbe necessary to generate clean, non-polar deletions of each gene in the mymA operon, followed by\nsystematic complementation analyses. We have begun to initiate such an approach, and we now realize\nthat this represents a substantial amount of work that will require many additional months.\nWhile we agree that a detailed understanding of the contribution of each gene of the mymA operon is\nan important future objective, the focus of the present manuscript is to reveal the dual mode of action of\nalpibectir. We believe that an in-depth genetic dissection would refine mechanistic insight but would not\nalter the central conclusion regarding the involvement of the mymA operon.\nTo address the reviewer’s comment, we propose adding a clarifying sentence at line 264 indicating that\nidentifying the specific genetic determinants underlying the intrinsic activity of alpibectir will be pursued\nin a future manuscript.\n3. In transcriptomic/proteomic analysis, the author only showed sharp LFC of selected genes.\nHowever, it is necessary to declared the statistically significance. Also, a volcano plot with\nannotation of these differentially expressed genes would also be more intuitive.\nResponse: We thank the reviewer for this valuable suggestion. In response, we have repeated the\ntranscriptomics experiment to include 3 biological replicates, which now allows us to provide statistical\nsignificance for the differentially expressed genes. We have added a volcano plot summarizing these\nresults (new Figure 2), with annotations of key genes discussed in the manuscript.\nSince the proteomics analysis (n=2) corroborates the transcriptomics data for the most highly expressed\ngenes previously shown, we have moved the proteomics results to the Supplementary Information (SI\nTable 1). We also provide, in the supplementary section, the complete list of proteins detected in this\nexperiment (Supplementary Data 1).\n4. It would be beneficial to include an INH + alpibectir treatment group as an additional control in\nFig.6. This would help to distinguish whether the observed effects are unique to the\nEto+alpibectir combination or not.\nResponse: To address this comment, we performed an additional in vitro experiment as the question\ncan be resolved without requiring a new animal study.\nWe evaluated the effect of alpibectir in combination with INH and observed that the addition of alpibectir\ndid not alter the MIC of INH in a wild-type strain. This confirms that alpibectir selectively boosts Eto but\nnot INH. The corresponding data have been included in the Supplementary Information (SI Fig. 12) and\na sentence describing this result has been included in the main text (line 367).\n5. The authors presented a virS/mymA-inactivated Beijing strain in Table 3. Is this mutation\nfrequently observed in clinical strains? This would determine the effectiveness of the\ncombination therapy.\nResponse: The mutation observed in 13MY2489 is very rare among lineage 2 strains. In the CRyPTIC\ndatabase, which includes data from 12,287 M. tuberculosis isolates, 4,295 strains belong to lineage 2.\nAmong these 4,295 strains, none displays a complete deletion of the virS-mymA regulon; only two\nstrains contain a partial deletion of mymA and two additional strains contain a complete deletion of\nmymA. In contrast, as previously reported in our study on the SMARt751 booster of Eto, strains\nbelonging to sublineage 4.8 are characterized by a large genomic deletion encompassing the virS–\nmymA regulon. As a result, this sublineage does not respond to the boosting effect of alpibectir. We\nhave added the following sentence in the corresponding paragraph (line 401) to reiterate this known\nlimitation of the approach: “It is worth noting that M. tuberculosis sublineage 4.8 naturally harbors a\nsimilar large deletion encompassing the virS-mymA regulon36, rendering these strains intrinsically\nunresponsive to alpibectir. Accordingly, particular caution will be necessary in regions where this\nsublineage is prevalent to avoid reducing the Eto dose to sub-MIC levels in patients infected with such\nstrains.”\nReviewer #2 (Remarks to the Author):\nThe manuscript by Edoo et al. reports work using a combination of orthogonal approaches to show\nthat Alpibectir, an activator of VirS with bactericidal activity against Mycobacterium tuberculosis (Mtb),\nboosts the activation of the oxidoreductase MymA, promoting ethA-independent conversion of\nethionamide into its active form to potentiate inhibition of InhA. The authors show that ethionamide\nadministered in combination with Alpibectir led to lower bacterial loads in Mtb-infected mice. The work\npresented in the manuscript is well executed, robust and cogent. However, some aspects of the work\nshould be clarified and discussed in more detail. Specific comments are as follows:\n1. It is unclear whether Alpibectir alone can kill intracellular Mtb. In Figure 1F, Alpibactir at\nconcentrations ranging from 0.050-0.333mg/L does not prevent replication of intracellular Mtb\nat concentrations at which ethionamide is ineffective against Mtb, suggesting that Alpibectir is\nnot effective against intracellular Mtb. This should be clearly discussed.\nResponse: We thank the reviewer for giving us the opportunity to clarify this aspect. In our in vitro\nexperiments, we observed that alpibectir exhibits modest intrinsic growth-inhibitory activity when used\nalone but displays a strong boosting effect on the activity of ethionamide.\nIn the intracellular assay originally presented in Fig. 1F in the first version of the manuscript (now SI Fig.\n4), differentiated THP-1 macrophages were infected with a high multiplicity of Mtb. Under these\nconditions, macrophage survival relies on antibiotic treatment capable of preventing infection-induced\nlysis. In this stringent setting, we were able to detect the boosting effect of alpibectir on ethionamide,\nbut not its intrinsic activity. We reasoned that the intrinsic activity of alpibectir alone is insufficient to\nprevent macrophage lysis in this model.\nTo directly assess the intrinsic intracellular activity of alpibectir, we have performed an additional assay\nin which THP-1 cells were simultaneously differentiated and infected with luciferase-expressing Mtb,\nfollowed by 5 days of incubation with antibiotics. In this setup, macrophages remain viable regardless\nof treatment (new figure added in Supplementary Information, SI Fig. 3), allowing direct quantification of\nintracellular bacterial luminescence. This new experiment (new Fig. 1F) shows both the intrinsic activity\nof alpibectir and its boosting effect on ethionamide. Importantly, the magnitude of the boosting effect\n(ca. 10-fold decrease of the IC of ethionamide in the presence of alpibectir) is consistent across both\n90\nintracellular assays.\nAccordingly, we have moved the initial experiment to the Supplementary Information (SI Fig. 4) and\nadded a clarifying sentence in the main text (line 134) to reflect these updates.\n2. In addition, the authors use a rather unusual murine model of tuberculosis wherein the drug is\nadministered 24 hours post infection. Rationale for using this model should be included. Did\nthe lack of effect of Alpibectir against intracellular Mtb motivate the use of this model?. Does\nthe short time lapse between infection and treatment amplify the effect of Alpibectir alone\nbecause the drug helps reduce the extracellular Mtb load?\nResponse: We used the preventive murine model, in which treatment is initiated one day after infection,\nwhen the bacterial load remains limited (105 to 10⁶ CFU). The model can be applied to BALB/c or\nC57BL/6 mice, with a treatment duration of 4 weeks. This model is particularly suited for comparing the\nactivity of different compounds used alone, which was the rationale for its use in our study. Importantly,\nit parallels human early bactericidal activity (EBA) studies by reproducing the early, rapidly replicating\nphase of infection and allowing the measurement of short-term reductions in bacterial burden. Our\nteam, along with others, including the expert group of Eric Nuermberger, uses this model for the\npreclinical evaluation of novel antitubercular agents. We have added the supplementary reference below\nto our text (line 411) to reflect this.\nNuermberger EL et al. GSK2556286 Is a Novel Antitubercular Drug Candidate Effective In Vivo with the\nPotential To Shorten Tuberculosis Treatment. Antimicrob Agents Chemother. 2022 Jun\n21;66(6):e0013222. doi: 10.1128/aac.00132-22.\n3. Figure 2, panel B. Although the data are compelling, some relevant controls are missing in the\nthermal denaturation experiment. The authors should either repeat it with inclusion of another\nprotein that does not bind Alpibectir, or ideally with inclusion of a variant of VirS that cannot\nbind Alpibectir. The structural data should help in designing such a variant.\nResponse: We thank the reviewer for this valuable suggestion. In accordance with the comment, we\nhave included α-latalbumin as a negative control in the thermal denaturation experiment. No effect of\nalpibectir on the thermal denaturation profile of α-lactalbumin was observed, indicating the absence of\nbinding. This additional control experiment has been included in the Supplementary Information (SI Fig.\n6) and mentioned in the main text (line 206).\n4. Figure 4. There is no attempt to complement the ∆adhD mutant. It would be useful to\ncomplement the ∆adhD mutant with adhD and each of the three downstream genes\nindividually first, and then in combination if unsuccessful, to define which gene(s) are\nresponsible for the intrinsic activity of Alpibectir.\nResponse: We thank the reviewer for this comment. Please refer to our response to Comment 2 from\nReviewer 1, which addresses this point in detail.\n5. Figure 5. The molecular mechanisms underlying ethionamide potentiation by Alpibectir remain\nunclear. Ethionamide is primarily converted into its active form by EthA in the absence of\nAlpibectir, the addition of which favours bioconversion of ethionamide by MymA. If EthA and\nMymA activate ethionamide into the same bioactive form (the NAD adduct), do they do so at\nthe same rate? For the same concentration of ethionamide, how do levels of the bioconverted\nform compare in H37Rv versus H37Rv treated with Alpibectir? Does activation by MymA allow\nfor larger accumulation of the bioconverted, active form compared to activation by EthA?\nThis could explain why the combination of Alpibectir/ethionamide is better able to kill drug-\nrecalcitrant Mtb subpopulations, and why the combination is effective at killing strains\noverexpressing InhA (lines 368-373). These measurements should be done; at the very least,\nthese questions should be discussed.\nWe thank the reviewer for these insightful questions. The mechanisms underlying ethionamide activation\nby EthA and MymA are not completely understood despite substantial efforts from several groups. To\ndate, the relative rates of ethionamide activation by EthA and MymA have not been determined and\ntherefore cannot be directly compared. Measuring the rate of ethionamide bioconversion has proven\ntechnically challenging for many groups, including ours, as the pathway proceeds through the\nethionamide S-oxide (Eto-SO) intermediate, likely involves radical species, and ultimately results in the\nformation of the ethionamide-NAD adduct (see Johnston et al. 1967). Importantly, this Eto-NAD adduct\nhas never been directly quantified in M. tuberculosis; its existence is demonstrated solely through\ncrystallographic structure of InhA in complex with this adduct. Consequently, to the best of our\nknowledge, no reliable method currently exists to precisely quantify either the rate of ethionamide\nactivation or the intracellular accumulation of its bioactive species.\nWhat we have demonstrated, however, is that alpibectir-induced overexpression of mymA (as evidenced\nby our transcriptomic and proteomic analyses) confers ethionamide hypersensitivity with MIC shifts\nequivalent to those observed upon ethA de-repression by EthR inhibitors (BDM31343, BDM41906; see\nWilland et al. 2009 and Flipo et al. 2011).\nThis functional equivalence strongly suggests that both conditions achieve comparable steady-state\nlevels of the bioactive Eto-NAD species, despite utilizing different monooxygenases.\nMoreover, overexpression of inhA reduces the efficacy of both alpibectir (SI Fig. 11) and BDM41906 to\ncomparable extents, confirming that: (i) both monooxygenases generate the same InhA-targeting active\nspecies (Eto-NAD) and (ii) the boosted monooxygenase activities under alpibectir (MymA-mediated)\nand BDM41906 (EthA-mediated) treatment are functionally equivalent in terms of ethionamide\nbioactivation capacity.\nPer the reviewer’s request, we have now discussed this aspect at line 496 of the main text.\nReferences:\n- Johnston JP et al. The metabolism of ethionamide and its sulfoxide. J Pharm Pharmacol.\n1967Jan;19(1):1-9. doi: 10.1111/j.2042-7158.1967.tb07986.x.\n- Willand N et al. Synthetic EthR inhibitors boost antituberculous activity of ethionamide. Nat Med. 2009\nMay;15(5):537-44. doi: 10.1038/nm.1950.\n- Flipo M et al. Ethionamide boosters. 2. Combining bioisosteric replacement and structure-based drug\ndesign to solve pharmacokinetic issues in a series of potent 1,2,4-oxadiazole EthR inhibitors. J Med\nChem. 2012 Jan 12;55(1):68-83. doi: 10.1021/jm200825u.\n- DeBarber AE et al. Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium\ntuberculosis. Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9677-82. doi: 10.1073/pnas.97.17.9677.\n- Wang F et al. Mechanism of thioamide drug action against tuberculosis and leprosy. J Exp Med 2007\nJan 22;204(1):73-8. doi: 10.1084/jem.20062100.\n- Ushtanit A et al. Molecular Determinants of Ethionamide Resistance in Mycobacterium tuberculosis.\nInt J Mol Sci 2022 Jan 20;11(2):133. doi: 10.3390/antibiotics11020133.\n- Laborde J et al. Ethionamide biomimetic activation and an unprecedented mechanism for its\nconversion into active and non-active metabolites. Org Biomol Chem 2016 Sep 21;14(37):8848-8858.\ndoi: 10.1039/c6ob01561a.\n6. - p12, line 282: the use of “only” might be misleading as ethionamide alone at 2.5 or 25 mg/L\nleads to statistically similar levels of bacterial clearance at day 7.\nResponse: The reviewer is correct. We have removed the word “only” from line 284 (originally line 282).\n7. - The grounds on which the authors conclude that InhA is expressed at a basal level or\noverexpressed are unclear.\nResponse: We thank the reviewer for highlighting this important point. To provide evidence showing\noverexpression of inhA, we have now included additional experimental validation. We repeated our\nexperiments using a well-characterized and previously published inhA overexpressing strain (see\nreference below). This strain exhibits a 30-fold increase in INH MIC compared to H37Rv, confirming\nfunctional inhA overexpression as previously described.\nWe have updated SI Fig. 11 in the Supplementary Information accordingly.\nHartkoorn RC, Sala C, Neres J, Pojer F, Magnet S, Mukherjee R, Uplekar S, Boy-Röttger S, Altmann\nKH, Cole ST. Towards a new tuberculosis drug: pyridomycin - nature's isoniazid. EMBO Mol Med.\n8. - How do the authors explain the absence of dose effect between administration of 15 and\n50mg/kg of ethionamide?\nResponse: We thank the reviewer for inviting us to clarify this observation. When administered as a\nmonotherapy, ethionamide displays a clear dose-dependent bactericidal effect between 5 and 50 mg/kg\n(exceeding a 3-log reduction), followed by a plateau corresponding to target saturation between 50\n10\nand 200 mg/kg. In the presence of alpibectir (0.5 or 1.6 mg/kg), the ethionamide dose-response curve\nis shifted leftward, with the maximal antibacterial effect, slightly surpassing that of 200 mg/kg\nethionamide alone, achieved at 15 mg/kg. These findings indicate that the pharmacodynamic saturation\npoint (InhA inhibition), which is only reached at ≥50 mg/kg under ethionamide monotherapy, is attained\nat 15 mg/kg when alpibectir is co-administered. Consequently, increasing the ethionamide dose to 50\nmg/kg in the presence of alpibectir does not confer any additional efficacy, as the pharmacological target\nis already saturated at 15 mg/kg.\nThis clarification has been added to the revised manuscript at line 429.\n9. - Please check the policy of Nature Communications regarding the use of “data not shown”.\nResponse: We initially used “data not shown” on 2 instances.\nFor the first instance (originally line 186; now line 193), we have replaced the phrase by adding the\ncorresponding data as a new panel in the Supplementary Information (SI Fig. 5) which shows the\nsynthetic gene circuit experiments assessing the interaction of EthR2 and EthR with alpibectir.\nFor the second instance (originally line 286; now line 287), we have modified our sentence to only\nmention the results that are shown in the survival kinetics analysis (Fig. 6).\nReviewer #3 (Remarks to the Author):\nThe objective of discovering new treatments for tuberculosis is an undeniable priority, especially\nfor those areas and regions and which tuberculosis have a high incidence for example in sub-\nSaharan Africa and in south-east Asia. The reporting of novel drugs or drug combinations are\ntherefore not only important but necessary.\nIn this study Edoo and colleagues report on the novel drug combination that involves Alpibectir\nand ethionamide. This study reports on it to effects in studies that were directly conducted against\nM. tuberculosis bacilli, against bacilli harboured in macrophages as well as its effects tested in\nmice.\nGeneral Comments:\nThe study design is well constructed with logical flow in the experimental steps. The findings that\nalpibactir potentiates Eto activity and has antibacterial activity independent of Eto is highly\npromising.\n1. In Fig.1A the effects of alpibactir is generally evaluated against the effect of Eto in its absence.\nWhile this is a necessary control, the inclusion of a compound in the same class of amino\npiperidines that does not potentiate Eto is an additional control required to demonstrate\nspecificity.\nResponse: We thank the reviewer for this comment. As requested by the reviewer, we performed an\nadditional experiment to evaluate the effect of a close analogue of alpibectir on the growth inhibitory\nactivity of ethionamide. The hydrophobic electron-withdrawing trifluoromethyl group was replaced by a\nmore polar, electron-donating group (aminomethyl) on the spiroisoxazoline motif. With this analogue,\nwe observed no boosting effect on ethionamide activity, nor intrinsic antibacterial activity, clearly\ndemonstrating that it does not bind VirS and does not induce expression of the mymA operon. These\nresults are fully consistent with the VirS Alpibectir X-ray co-structure, in which the trifluoromethyl group\nengages hydrophobic contacts with Phe197 and Ile101, interactions that the aminomethyl group is\nunlikely to support. In addition, the bend imposed by the nitrogen atom may introduce steric clashes that\nfurther prevent binding. We have added a figure in the Supplementary Information (SI Fig. 2), as well as\na corresponding sentence in the main text at line 121.\n2. In the next phase of the study, the effects of Alp/E was tested against intracellular bacilli (Fig\n1F). The authors report efficacy, however whether macrophages remain intact and viable for\nthe duration of the assay is not presented. Should macrophages be lysed, the test would\nessentially evaluate efficacy against extracellular bacilli. The inclusion of a data that\ndemonstrate macrophage viability for the duration of the experiment must be included to\nvalidate the conclusion of efficacy against intracellular bacilli. Again, while the absence of\nalpibactir serves as a control in Fig 1F, a control to demonstrate drug specificity is absent.\nResponse: In the intracellular assay originally presented in Fig.1F (now moved to the Supplementary\nInformation as SI Fig. 4), THP-1 cells were first differentiated and then infected with M. tuberculosis at\na multiplicity of infection of 10, a condition that leads to macrophage lysis in the absence of antibiotic\ntreatment, as evidenced by increased resazurin fluorescence in wells containing the lowest antibiotic\nconcentrations. This experiment enabled us to determine the concentration of AlpE required to prevent\nmacrophage lysis caused by M. tuberculosis infection.\nIn response to comment 1 from Reviewer 2, we have performed an additional intracellular assay in\nwhich THP-1 cells were simultaneously differentiated and infected with luciferase-expressing Mtb. In\nthis new setup, we have used THP1-cells stably expressing a red fluorescence protein, enabling\nmonitoring of macrophage viability over the 5-day infection period (See the following references\ndescribing the model). Macrophages remained viable across all treatment groups. These new data have\nbeen added to the Supplementary Information (SI Fig. 3), and a clarifying sentence has been\nincorporated in the main text (line 134) to reflect these updates.\nReferences:\n- Zheng X, Av-Gay Y. 2017. System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting\nIntracellular Mycobacterium tuberculosis. J. Vis. Exp., e55273, doi: 10.3791/55273.\n- Sorrentino F, Gonzalez del Rio R, Zheng X, Presa Matilla J, Torres Gomez P, Martinez Hoyos M,\nPerez Harran ME, Mendoza Losana A, Av-Gay Y. 2016. Development of an intracellular screen for new\ncompounds able to inhibit Mycobacterium tuberculosis growth in human macrophages. Antimicrob\nAgents Chemother 60:640–645. doi: 10.1128/AAC.01920-15.\n- Kavanagh ME, McLean KJ, Gilbert SH, Amadi CN, Snee M, Tunnicliffe RB, Arora K, Boshoff HIM,\nFanourakis A, Rebollo-Lopez MJ, Ortega F, Levy CW, Munro AW, Leys D, Abell C, Coyne AG. 2025. J\nMed Chem 68 (14), 14416-14441. doi: 10.1021/acs.jmedchem.5c00478\n3. In line 169, the authors refer to the “…exceptional specificity of alpibactir…”. The inclusion of a\nnon-specific control for alpibactir would enhance this claim. The authors should address why\nthis was not done.\nResponse: We apologize for the confusing sentence. By using the word “specificity” in line 169 (now\nline 177), we were not referencing the structural determinants of alpibectir but rather the functional\nselectivity of alpibectir towards the mymA operon and to a lesser extent, towards EthA2. We have\nreplaced “specificity” in line 177 by “functional selectivity” to make the context explicit.\n4. The graphical lines and points in Fig 5A and 5B is difficult to distinguish. The resolution of the\ndata points should be improved. In lines 274-277 the authors states the following: “At a\nconcentration near its MIC (2.5 mg/L), Eto alone exhibited a constant yet relatively slow\nbactericidal activity in vitro, resulting in an approximate 4-log10 reduction of CFU in 7 days,\nnever reaching the limit of detection of 102 CFU/mL.”\nThis is very difficult to verify. From Fig 5A it appears that 102 is indeed reached prior to\nregrowth, specifically at 25mg/ml and is sustained until D21. The authors should comment on\nthis.\nResponse: We agree that the original presentation of Fig. 5A and 5B (now Fig. 6A and 6B) made the\ndata difficult to interpret. Two issues contributed to this:\n1. the near-complete overlap of five series of data points at day 7 and\n2. the difficulty distinguishing cultures that exhibited regrowth at days 14 and 21 from those that did not.\nTo address this, we have separated the data of each strain into two independent graphs, one showing\ncultures treated with compounds alone and the other showing cultures treated with Eto + alpibectir. This\npresentation clearly highlights the absence of bacterial rebound in the presence of alpibectir, in contrast\nto the conditions where Eto is used alone.\nRegarding the reviewer’s specific point on the time-kill curves with Eto monotherapy: at 2.5 mg/L, the\nlimit of detection was not reached at day 7; the data point lies slightly above the 10² CFU/mL threshold\n(please refer to the source data of Fig. 6 for the exact values). In contrast, for Eto at 25 mg/L, the limit\nof detection was indeed reached at day 14. However, the regrowth observed at day 21 demonstrates\nthat reaching the detection limit does not equate complete sterilization under these conditions.\n5. While, Alp/E is shown to be more effective than Eto alone, measurements terminate at 21\ndays. Has measurement been done for longer periods? What happens with regrowth after\nD21?\nResponse: No measurements were done beyond 21 days; therefore, we do not claim complete\nsterilization of the cultures. However, the rebound observed at day 21 in the condition where bacteria\nwere undetectable at 14 days (Eto 25 mg/L) indicates that, if surviving bacilli remain, they are present\nat extremely low concentration. While their persistence cannot be fully excluded, this observation must\nbe interpreted in the context of clinical polytherapeutic regimens. In patients, AlpE would not constitute\nthe sole active antimicrobial component, and any rare surviving bacilli would be expected to be\neliminated by the additional drugs present in the treatment combination.\n6. Why is there a limit of detection of 102 CFU’s? For example, it is possible to obtain absolute\npulmonary bacterial burdens (<100) if the whole lung volume is homogenised and plated. This\nwould give an indication of whether treatment induces levels of sterility.\nResponse: The limit of detection in the in vivo experiment was 1.24 log CFU, and this has now been\n10\nadded to Fig. 7 for clarity. Importantly, none of the lung samples reached this detection threshold.\nIf the reviewer is instead referring to the in vitro time-kill curves, the LOD is determined by the\nexperimental setup: 10 µL of culture were plated at each time point, corresponding to a detection limit\nof 2 log CFU/mL. By definition, time-kill assays involve sampling and plating only a small, fixed volume\n10\nof the culture at each time point, which intrinsically imposes a higher LOD than would be achieved by\nplating the entire culture volume.\n7. It appears that the addition of alpibactir to Eto beyond 15mg/ml has no added benefit in terms\nof reducing pulmonary bacilli burdens, at least at the 4 week time point reported in Fig 6. It\nmay be that the optimal Eto concentration is less than 15 (between 5-15mg/ml). The authors\nmay want to comment on this. The addition of alpibactir to Eto at a concentration at 50mg/ml\nhas no added potentiation. The authors should comment on this observation.\nResponse to first part of comment: The reviewer is correct. The opportunity offered by alpibectir lies in\nachieving ethionamide maximal activity (i.e. Emax) with lower ethionamide doses. We agree that the\noptimal ethionamide dose lies between 5 mg/kg (which, even in combination with alpibectir, does not\nreach Emax) and 15 mg/kg (which achieves Emax when combined with either 0.5 or 1.6 mg/kg of\nalpibectir). We added a sentence to mention this at line 429.\nResponse to second part of comment: Please see below our response to reviewer 2 who basically raised\nthe same comment.\nReviewer #2 : How do the authors explain the absence of dose effect between administration\nof 15 and 50mg/kg of ethionamide?\nResponse: We thank the reviewer for inviting us to clarify this observation. When administered as a\nmonotherapy, ethionamide displays a clear dose-dependent bactericidal effect between 5 and 50 mg/kg\n(exceeding a 3-log reduction), followed by a plateau corresponding to target saturation between 50\n10\nand 200 mg/kg. In the presence of alpibectir (0.5 or 1.6 mg/kg), the ethionamide dose-response curve\nis shifted leftward, with the maximal antibacterial effect, slightly surpassing that of 200 mg/kg\nethionamide alone, achieved at 15 mg/kg. These findings indicate that the pharmacodynamic saturation\npoint (InhA inhibition), which is only reached at ≥50 mg/kg under ethionamide monotherapy, is attained\nat 15 mg/kg when alpibectir is co-administered. Consequently, increasing the ethionamide dose to 50\nmg/kg in the presence of alpibectir does not confer any additional efficacy, as the pharmacological target\nis already saturated at 15 mg/kg. This clarification has been added to the revised manuscript at line 429.\n8. Minor comments/questions:\nBlue to Yellow scale need to be indicated in Fig 1A.\nResponse: We have modified Fig. 1A accordingly.\n9. Line 194, should be Fig 2B-2C, rather than Fig 2A-C\nResponse: We have corrected this oversight.\n10. Why was the concentration of 0.33mg/ml for alpibactir selected for the study in Fig 3?\nResponse: With 0.33 mg/L of alpibectir, we are able to obtain maximum boosting effect of ethionamide\nand maximum intrinsic activity of alpibectir (as shown in Fig. 1C and 1D). This has prompted us to use\nthis concentration of alpibectir for several experiments.\nThe authors should provide context for the selection of concentrations used, for both Eto and alpibactir\nin the study section under the heading “Alpibectir/Eto inhibits growth of MDR-TB, including ethA and\ninhA mutated strains” page lines 304-404. What is the relevance of 1/6th of the clinical breakpoint\n(line387)?\nResponse: We thank the reviewer for pointing out the absence of an explanation for the concentrations\nwe used in this experiment. The ethionamide concentration used in the MGIT assay was set at one sixth\nof the clinical breakpoint, a sub-inhibitory level chosen to permit detection of sensitization effects. At this\nconcentration, growth of M. tuberculosis is only partially inhibited, allowing quantification of changes in\ndrug susceptibility by alpibectir. We selected two concentrations of alpibectir to capture both the plateau\nand the ascending phase of the potentiation curve of alpibectir. The maximal concentration (0.05 mg/L)\nconfirms the full extent of the effect, while the lower concentration (0.017 mg/L) demonstrates that the\nresponse is dose-dependent and not a non-specific consequence of high compound exposure. We have\nadded a sentence to explain the concentrations chosen at line 384."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The manuscript is well-delineated, cogent and an exemplary illustration of the use of multidisciplinary and orthogonal approaches to tackle scientific questions. The authors addressed most comments. This reviewer has two additional, pedantic ones: the acronym DMPK, line 85, should be defined and there is a problem with color labels between the graphs and the legend in Figure 6 that must be fixed (“Alpibactir alone” is certainly wrongly labeled)."}, "author_response": "We thank the reviewer for their thoughtful comment and for pointing out these oversights. We have\nduly amended the manuscript."}]

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