Piperidine-4-Carboxamides Target DNA Gyrase in Mycobacterium abscessus

ABSTRACT New, more-effective drugs for the treatment of lung disease caused by nontuberculous mycobacteria (NTM) are needed. Among NTM opportunistic pathogens, Mycobacterium abscessus is the most difficult to cure and intrinsically multidrug resistant. In a whole-cell screen of a compound collection active against Mycobacterium tuberculosis, we previously identified the piperidine-4-carboxamide (P4C) MMV688844 (844) as a hit against M. abscessus. Here, we identified a more potent analog of 844 and showed that both the parent and improved analog retain activity against strains representing all three subspecies of the M. abscessus complex. Furthermore, P4Cs showed bactericidal and antibiofilm activity. Spontaneous resistance against the P4Cs emerged at a frequency of 10−8/CFU and mapped to gyrA and gyrB encoding the subunits of DNA gyrase. Biochemical studies with recombinant M. abscessus DNA gyrase showed that P4Cs inhibit the wild-type enzyme but not the P4C-resistant mutant. P4C-resistant strains showed limited cross-resistance to the fluoroquinolone moxifloxacin, which is in clinical use for the treatment of macrolide-resistant M. abscessus disease, and no cross-resistance to the benzimidazole SPR719, a novel DNA gyrase inhibitor in clinical development for the treatment of mycobacterial diseases. Analyses of P4Cs in recA promoter-based DNA damage reporter strains showed induction of recA promoter activity in the wild type but not in the P4C-resistant mutant background. This indicates that P4Cs, similar to fluoroquinolones, cause DNA gyrase-mediated DNA damage. Together, our results show that P4Cs present a novel class of mycobacterial DNA gyrase inhibitors with attractive antimicrobial activities against the M. abscessus complex.

whole cell active for the treatment of M. abscessus disease, we measured its activity against reference strains representing the three subspecies of the M. abscessus complex as follows: M. abscessus subsp. abscessus ATCC 19977, M. abscessus subsp. bolletii CCUG 50184T, M. abscessus subsp. massiliense CCUG 48898T, and a collection of clinical isolates. 844 retained activity against the M. abscessus complex strains with MICs ranging from 6 to 14 mM (Table 1).
P4Cs are bactericidal and active against M. abscessus biofilm cultures. We previously reported reduced viability of M. abscessus subsp. abscessus Bamboo treated with 844 in broth culture, suggesting bactericidal activity of the compound (21). To confirm and characterize the bactericidal activity of the class, we determined time-concentration kill for 844 and 844-TFM against the reference strain M. abscessus subsp. abscessus ATCC 19977. Both compounds were bactericidal in planktonic cultures ( Fig. 2A) and against M. abscessus grown as biofilm (Fig. 2B).
Resistance against P4Cs is caused by missense mutations in M. abscessus DNA gyrase. Based on in silico analyses, 844 was proposed to act as an inhibitor of mycobacterial ABC transporters (23). To determine the mechanism of action of 844 experimentally, we isolated spontaneous resistant mutants in M. abscessus subsp. abscessus Bamboo on 844-containing agar. Resistant colonies emerged at a frequency of 10 28 / CFU. Three randomly selected resistant strains were further characterized, showing 2-to more than 8-fold increases in 844 MIC. Whole-genome sequencing, confirmed by targeted sequencing, revealed missense mutations in gyrA and gyrB, the genes encoding the subunits of DNA gyrase (Table 2). We repeated the mutant selection experiment for 844-TFM using M. abscessus subsp. abscessus ATCC 19977, again yielding resistant colonies at a frequency of 10 28 /CFU. Characterization of five randomly selected resistant colonies revealed 3-to more than 66-fold increased 844-TFM MICs and, again, missense mutations in gyrA and gyrB ( Table 2). MIC determinations of the 844-resistant strains for 844-TFM and of the 844-TFM-resistant strains for 844 revealed cross-resistance of all strains to both compounds (Table 2). To confirm that the observed polymorphisms are indeed causing resistance, one representative resistant strain (M. abscessus subsp. abscessus 19977 844-TFM r -1), harboring a D91N missense mutation in gyrA associated with high-level P4C resistance (Table 2), was complemented with a copy of M. abscessus subsp. abscessus ATCC 19977 wild-type (wt) gyrAB, which abscessus ATCC 19977 were treated with 1Â, 4Â, and 8Â MIC of 844 or 844-TFM, moxifloxacin (MXF), SPR719, or clarithromycin (CLR), and CFU were enumerated by plating samples on agar after 2 and 3 days. (B) Exponentially growing biofilm cultures were treated with 1Â, 4Â, and 8Â MIC of 844, 844-TFM, MXF, SPR719, or CLR, and surface-attached CFU were enumerated by suspending bacteria and plating on agar after 2 and 3 days. MXF, SPR719, and CLR are included for comparison (MXF, SPR719) or as control (CLR). Experiments in panels A and B were carried out three times independently, and the results are represented as mean values with the standard deviations displayed as error bars. A two-way ANOVA multiple comparison test was performed using GraphPad Prism 8 software to compare treated groups with untreated day 0 CFU. partially restored sensitivity to both 844 and 844-TFM (Fig. 3). These results suggest that missense mutations in M. abscessus DNA gyrase genes cause resistance to P4Cs and that the compounds target this enzyme.
P4Cs inhibit activity of recombinant M. abscessus wild-type DNA gyrase but not mutant enzyme harboring a P4C resistance mutation. To provide direct evidence that the P4Cs indeed target DNA gyrase, we tested whether the molecules inhibit the supercoiling activity of recombinant M. abscessus DNA gyrase. The two DNA gyrase inhibitors, moxifloxacin and SPR719, inhibited supercoiling activity as expected, whereas the negative control, the ribosome inhibitor clarithromycin, did not affect the activity of the enzyme (Fig. 4). Both P4Cs inhibited activity of DNA gyrase (Fig. 4). Consistent with its improved whole-cell activity, the 50% inhibitory concentration (IC 50 ) of 844-TFM (1.9 mM) was 2.4-fold lower than the IC 50 of the parental 844 (4.6 mM) (Fig.  4). To confirm the mechanism of resistance, we tested activity of the P4Cs against recombinant M. abscessus DNA gyrase harboring the resistance mutation D91N in gyrA (M. abscessus subsp. abscessus ATCC 19977 844-TFM r -1) ( Table 2; Fig. 3). The mutant version of DNA gyrase was not inhibited by P4Cs (Fig. 4).
P4C resistance causing DNA gyrase mutations show limited cross-resistance to moxifloxacin and no cross-resistance to SPR719. Taken together, the genetic and biochemical analyses indicate that P4Cs target M. abscessus DNA gyrase, an essential and clinically validated target in mycobacteria (24). The type IIA DNA topoisomerase is a GyrA 2 GyrB 2 heterotetrameric protein that regulates DNA topology (25). The unwinding of DNA during replication and transcription introduces positive supercoils that, left unaddressed, would affect DNA function. This problem is resolved by DNA gyrase, which introduces negative supercoils into DNA in an ATP-dependent fashion. To do this, the enzyme generates a DNA double-stranded break, passes a segment of doublestranded DNA through the break, and subsequently reseals the DNA molecule (25).
The DNA gyrase inhibitor moxifloxacin (Fig. 1) is a pillar of the treatment of multidrug-resistant tuberculosis (26) and is used less widely for the treatment of macrolide-resistant M. abscessus disease (27,28). The fluoroquinolone targets the catalytic core of the enzyme comprised of the C-terminal TOPRIM domains of two GyrB subunits and the N-terminal breakage-and-reunion domains of two GyrA subunits (29). Consequently, acquired fluoroquinolone resistance involves missense mutations within these domains (30). P4C resistance mutations ( Table 2) are also located in the TOPRIM and breakage-and-reunion domains. Interestingly, D91 missense mutations causing high-level P4C resistance have been reported to also confer resistance to moxifloxacin in M. tuberculosis (31). To determine whether the P4C resistance  (Table 2) was transformed with plasmid pMV262 not carrying an insert (pMV262-empty; control) or with pMV262 carrying wild-type gyrAB constitutively expressed from the hsp60 promoter. Cultures were either grown without drug (DF, drug free) or treated with MIC of 844 (8 mM) or 844-TFM (1.5 mM) for 2 days (D2), and growth was measured by OD 600 determination. Clarithromycin (CLR) treatment at MIC (2 mM) was used as control. The experiments were carried out three times independently, and the results are represented as mean values with the standard deviations displayed as error bars. A twoway ANOVA with Sidak's multiple comparison test was performed to compare the two groups using GraphPad Prism 8 software.
mutations in M. abscessus confer cross-resistance to moxifloxacin, we measured the MICs of moxifloxacin for the P4C-resistant strains. The strains harboring D91 missense mutations in gyrA showed low level cross-resistance with a 2-to 8-fold increase in MIC ( Table 2). None of the other P4C resistance mutations altered the fluoroquinolone MIC (Table 2).
A novel DNA gyrase inhibitor, SPR719 ( Fig. 1), is in clinical development for the treatment of mycobacterial lung diseases. This benzimidazole acts as an inhibitor of the ATPase activity of DNA gyrase located in the N-terminal domain of GyrB (32). SPR719-resistant mutants in M. tuberculosis have been mapped to the ATPase domain (32). As expected, P4C resistance mutations did not cause cross-resistance to SPR719 (Table 2). Interestingly, several P4C-resistant mutations conferred hypersusceptibility to SPR719. The mechanistic basis for this phenotype remains to be determined. Thus, P4C-resistant mutations caused limited or no cross-resistance to moxifloxacin or SPR719, suggesting a novel on-target mechanism of this new DNA gyrase inhibitor.
P4Cs trigger induction of recA DNA damage reporter in wild-type but not in P4C-resistant M. abscessus. Fluoroquinolones arrest DNA gyrase-DNA complexes in their double strand broken state. This mechanism of action results in DNA damage, which contributes to the bactericidal activity of the class (33). Similar to moxifloxacin, P4Cs are bactericidal and resistance mutations map to the catalytic core of DNA gyrase. To determine whether P4Cs also cause DNA damage, we constructed a DNA damage reporter strain by introducing the DNA damage inducible recA promoter controlling expression of the bioluminescence LuxCDABE operon into M. abscessus subsp. abscessus ATCC 19977. The positive control moxifloxacin strongly induced recA promoter-dependent reporter expression (Fig. 5), similar to what has been described for M. tuberculosis (34,35). SPR719, inhibiting gyrase's ATPase activity, caused only a weak increase, and treatment of M. abscessus with the protein synthesis inhibitor clarithromycin as negative control did not cause any increase in reporter expression (Fig. 5). Similar to moxifloxacin, treatment of reporter cultures with P4Cs strongly induced expression of the reporter gene (Fig. 5), suggesting that the compounds damage bacterial DNA. If P4C-mediated recA promoter induction is due to interaction of the compounds with DNA gyrase, induction should not occur in P4Cresistant gyrAB mutant background. To test this prediction, we introduced the recA reporter operon into the P4C-resistant M. abscessus subsp. abscessus ATCC 19977 844-TFM r -1 strain harboring the D91N allele of gyrA (Table 2; Fig. 3 and 4). In this background, induction of recA promoter activity was strongly reduced, suggesting that the increase of recA promoter activity in the wild-type background is DNA gyrase dependent (Fig. 5). Together, these results suggest that inhibition of DNA gyrase by P4Cs results in DNA damage.
In vivo and in vitro pharmacokinetic properties of 844-TFM. To identify the pharmacokinetic (PK) liabilities of 884-TMF, we determined its concentration-time profile in mice and measured basic PK properties in established in vitro assays. 884-TFM was not orally bioavailable in mice with a calculated bioavailability of 0.05 to 0.2% ( Fig. 6A and Table 3). To determine whether this was due to poor solubility, low permeability, or rapid metabolism, these properties were evaluated in standard in vitro PK assays ( Table 3). 844-TFM exhibited adequate solubility at both pH 2.0 (simulated gastric fluid) and pH 7.4 (standard physiological conditions). In the parallel artificial membrane permeability assay (PAMPA), 844-TFM showed modest permeability with a  logPe of 25.7 cm/s (logPe of 25.0 cm/s is considered the border between high and low permeability). In mouse liver microsomes, 844-TFM was highly unstable with a half-life of 5 min and a high rate of clearance (Table 3), nearly as high as the mouse hepatic blood flow of 90 ml/min/kg (36), predicting an extraction ratio of ;1, consistent with rapid first-pass liver metabolism and in line with poor oral bioavailability. To circumvent first-pass metabolism, we administered 844-TFM via the subcutaneous route to CD-1 mice, leading to improved exposure compared to that of the oral route (Fig. 6A). However, the compound was still rapidly eliminated, and bioavailability remained modest at 3%. Indeed, 844-TFM was rapidly degraded in mouse plasma (Fig. 6B). Identification of the two major breakdown products in mouse plasma revealed cleavage of the central amide bond of 844-TFM (see Fig. S1 and S2 in the supplemental material). Interestingly, the compound was markedly more stable in rabbit, monkey, and human plasma, with approximately 80% remaining after 24 h at 37°C (Fig. 6B). Taken together, characterization of the PK properties of 844-TFM revealed limited permeability and metabolic instability as the major liabilities of the lead compound in the mouse model. Conclusion. Fluoroquinolones are used successfully as second-line agents for the treatment of multidrug-resistant tuberculosis (37). Variable in vitro susceptibilities due to (unknown) intrinsic resistance mechanisms limit the therapeutic utility of this drug class against M. abscessus (5). Thus, moxifloxacin is used only rarely as a second-line drug for the treatment of macrolide-resistant infections, and there is no effective DNA gyrase inhibitor available for the treatment of M. abscessus disease. Recently, the benzimidazole SPR719, an inhibitor of ATPase activity of the mycobacterial DNA gyrase, entered early clinical development for tuberculosis and NTM lung diseases, bringing new hope for patients suffering from mycobacterial infections (38). Here, we have identified a novel class of mycobacterial DNA gyrase inhibitors with attractive bactericidal and antibiofilm activity against M. abscessus complex. We characterized the PK properties of the lead compound to enable medicinal chemistry programs. A novel DNA gyrase inhibitor would be a welcomed addition to the anti-M. abscessus drug pipeline. Mycobacterium abscessus was grown in complete Middlebrook 7H9 broth (271310; BD Difco, Spark, MD, USA) supplemented with 0.05% Tween 80, 0.2% glycerol, and 10% albumin-dextrose-catalase. 7H10 agar (262710; BD Difco, Sparks, MD, USA) was used as solid medium. 844 was obtained from the Medicines for Malaria Venture's (Geneva) compound archive. 844-TFM was synthesized as described below. Moxifloxacin (MXF, SML1581) and clarithromycin (CLR, C9742) were purchased from Sigma-Aldrich, USA. SPR719 (HY-12930) was purchased from MedChemExpress LLC, USA. MXF, SPR719, and CLR were dissolved in 100% dimethyl sulfoxide (DMSO) (MP Biomedicals, USA) at 10 mM. 844 and 844-TFM were dissolved in ethanol (BP2818100; Fisher Scientific, USA) at 5 mM and 10 mM, respectively. All compounds were stored in aliquots at 220°C until use.

MATERIALS AND METHODS
Chemicals and physical methods. Starting materials were purchased and used as received. Solvents were of reagent grade and distilled before use. N-(6-methoxy-1,5-naphthyridin-4-yl)-4-piperidinecarboxamide was prepared according to the literature (45,46). The melting point (uncorrected) was determined on a Boetius melting-point apparatus (VEB Kombinat NAGEMA, Dresden, GDR). 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded at room temperature on an Agilent Technologies VNMRS 500 NMR spectrometer. The residual solvent signals of methanol-d 4 (d 1H = 3.31 ppm; d 13C = 49.00 ppm) were used to reference the spectra (abbreviations, s = singlet, d = doublet, t = triplet, q = quartet, td = triplet of doublets, m = multiplet). The mass spectrum was recorded on a Q Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) using methanol as solvent.
Synthesis of 844-TFM. A mixture of N-(6-methoxy-1,5-naphthyridin-4-yl)-4-piperidinecarboxamide (50 mg, 0.17 mmol), triethylamine (35 ml, 0.26 mmol, 1.5 eq), and 1-(2-bromoethyl)-4-(trifluoromethyl) benzene (58 ml, 0.34 mmol, 2 eq) in dimethylformamide (DMF) (5 ml) was stirred at 50°C for 16 h. The reaction was quenched by the addition of water (20 ml). The mixture was extracted with ethyl acetate (3 Â 10 ml), and the combined organic phases were washed with water (10 ml) and brine (10 ml). The solution was dried over Na 2 SO 4 , filtered, and evaporated under reduced pressure. The residue was chromatographed on silica gel with methanol/ethyl acetate (0 to 5%) to afford the product as a white solid ( 174.7, 161.8, 148.3, 144.7, 144.7, 140.4, 140.3, 139.2, 132.0, 129.0 Determination of MICs. MIC was determined using the broth microdilution method in 96-well plates as described previously (47). Briefly, a 10-point 2-fold serial dilution of compounds was performed in 96-well plates (Costar 3370; Corning, USA) starting at twice the desired highest concentration. Exponentially growing M. abscessus cultures (optical density at 600 nm [OD 600 ] = 0.4 to 0.8) were adjusted to a density of OD 600 = 0.01 in Middlebrook 7H9 broth (Becton, Dickinson). One hundred microliters of the bacterial suspension was seeded onto the 96-well plates containing 100ml of the serially diluted compounds to give a final volume of 200ml in each well with the final OD 600 of 0.005. The plates were sealed with parafilm, placed onto wet paper towels in a lock-lock box and incubated for 3 days at 37°C with orbital shaking at 90 rpm. Absorbance at 600 nm was measured using a TECAN Infinite Pro 200 plate reader after resuspension. Absorbance values at day 3 were subtracted from the day 0 readout. Percent growth inhibition was calculated by dividing the absolute absorbance value of treated cells with untreated control and multiplying by 100. CLR was included in all MIC experiments as a positive control to monitor assay reproducibility. MIC was defined as 90% of growth inhibition relative to untreated controls.
Time To prevent compound carryover effects, we plated out serially diluted samples onto 7H10 agar supplemented with 0.4% activated charcoal (C9154; Sigma-Aldrich, USA) for all time points as described (48). CFU were enumerated after 4 days of incubation at 37°C. A two-way analysis of variance (ANOVA) multiple comparison test was performed using GraphPad Prism 8 software to compare treated groups with untreated day 0 CFU.
Determination of antibiofilm activity. The antibiofilm activity of 844 and 844-TMF was determined employing the MBEC 96-well Biofilm assay kit (19111; Innovotech, Edmonton, AB, Canada) as described previously (15) with minor modifications. Briefly, exponentially growing M. abscessus subsp. abscessus ATCC 19977 cultures were spun down at 3,200 Â g for 10 min and washed with 7H9 broth without Tween 80 ('7H9'). Bacteria were resuspended in '7H9' to an OD 600 of 0.0125, and 150 ml was dispensed into the wells of Innovotech 96-well plates. Pegs (protruding from the specialized lids of the Innovotech multiwell plates) were inserted into the bacterial suspensions in the wells, and the cultures were grown 24 h with shaking at 110 rpm at 37°C to allow attachment and growth of the bacteria on the surface of the pegs. Then, the lids with the pegs were transferred to a new multititer plate containing 150 ml fresh '7H9' broth (without bacteria; time zero) (Fig. 2B) with no drug or with 1Â, 4Â, or 8Â MIC of 844, 844-TFM, MXF, SPR719, or CLR and incubated for 0, 2, and 3 days. Biofilm growth on the pegs was measured by CFU determination. The pegs were washed in 200 ml '7H9' medium, removed, and placed in 1.7-ml microcentrifuge tubes (87003-294; VWR, Radnor, PA, USA) containing 500 ml PBS/Tween 80 (0.025%). The tubes were vortexed at 2,000 rpm for 90 s to detach and suspend the bacteria from the pegs before samples were serially diluted and plated onto 7H10 agar supplemented with 0.4% activated charcoal to determine CFU/peg. A two-way ANOVA multiple comparison test was performed using GraphPad Prism 8 software to compare treated groups with untreated day 0 CFU.
In vitro DNA gyrase inhibition assay. The mycobacterial DNA gyrase inhibition assay was performed using the kit from Inspiralis Limited (MTS002; Norwich, UK) as described by the suppliers using the generated recombinant DNA gyrases from M. abscessus instead of the recombinant M. tuberculosis DNA gyrase supplied by the kit. The assay was carried out in a 30-ml final volume, containing 50 mM HEPES KOH (pH 7.9), 6 mM magnesium acetate, 4 mM dithiothreitol (DTT), 1 mM ATP, 100 mM potassium glutamate, 2 mM spermidine, 0.05 mg/ml bovine serum albumin, 40 nM either wild-type or GyrA D91N mutant DNA gyrase, and 0.5 ml (1mg/ml) of relaxed pBR322 DNA. The reaction mixture was incubated for 30 min at 37°C, and then stopped by adding 30 ml of chloroform/isoamyl alcohol (24:1) followed by 30 ml of STEB (40% sucrose, 100 mM Tris-HCl [pH 8.0], 100 mM EDTA, and 0.5 mg/ml bromophenol blue) solution. The tubes were briefly vortexed and centrifuged for 1 min, and 20 ml of the aqueous (upper blue) phase was loaded onto a 1% (wt/vol) agarose gel. The gel was run for 2 h at 75 V and stained with 1 mg/ml ethidium bromide in water. After destaining with water for 10 min, images were taken and analyzed using Invitrogen iBright FL1000 imaging system. To determine IC 50 values, the intensity of the bands was measured and compared to the drug-free reaction using iBright analysis software. The IC 50 values were determined using nonregression model fit of GraphPad Prism 8.0.1 software.
recA DNA damage report assay. To assess whether compounds cause DNA damage, luminescence of M. abscessus cultures harboring a LuxCDABE reporter expressed under the control of the recA promoter was measured. A recA-LuxCDABE reporter plasmid was constructed by replacing the NotI-EcoRI hsp60 promoter fragment of pMV306hsp60-LUX (54) (Addgene, 26159) with the M. abscessus subsp.
abscessus ATCC 19977 recA promoter (34). The M. abscessus recA promoter was amplified with primers Fw-PrecA (NotI, underlined) 59-gcgcggccgcGTTGGGGGAACCGCGTTAC-39 and Rv-PrecA (EcoRI, underlined) 59-ccggaattcGGTGTTCTCCGTTTCGTCG-39 using Phusion high-fidelity DNA polymerase (F530S; Fisher Scientific, USA). The resulting amplicon was digested with NotI (no. R31189L; New England BioLabs) and EcoRI (no. R3101S; New England BioLabs), gel purified (Qiagen, Hilden, Germany), and ligated into NotI-EcoRI-digested pMV306hsp60-LUX, resulting in the reporter plasmid pMV306recA-LUX (54). The ligation product was transformed into E. coli DH5a and plated on LB agar medium containing 25 mg/ml kanamycin. The plasmid was verified by PCR and transformed into wild-type M. abscessus subsp. abscessus ATCC 19977 and the P4C-resistant M. abscessus subsp. abscessus ATCC 19977 844-TFM r -1 strain harboring a D91N allele of gyrA (Table 2) via electroporation (52). To measure the effect of compounds on recA promoter activity, exponentially growing reporter cultures (OD 600 = 0.4 to 0.8) were adjusted to an OD 600 of 0.1 and treated with various concentrations of drugs. Luminescence intensity was measured using a TECAN Infinite Pro 200 plate reader at 250 ms integration time with automatic attenuation at time zero and after 4 h at 37°C. Data analysis was performed using GraphPad Prism 8 software after subtracting the luminescence at time zero.
In vitro pharmacokinetic analyses. Kinetic solubility, parallel artificial membrane permeability assay (PAMPA) permeability, and stability in the mouse liver microsome assay were performed by BioDuro (Shanghai, China) according to standard protocols.
In vivo mouse pharmacokinetics. All animal experiments were approved by the Center for Discovery and Innovation, Institutional Animal Care and Use Committee, and were conducted in compliance with their guidelines. Female CD-1 mice were weighed and received a single dose of 884-TFM via the intravenous (i.v.) (5 mg/kg of body weight), oral (p.o.) (25 mg/kg), or subcutaneous (s.c.) (25 mg/kg) dosing route. The compound was formulated as a solution in 5% N,N-dimethylacetamide (DMA)/95% Milli-Q water vehicle. Blood samples were serially collected via the tail snip from each individual mouse at 0.017, 0.25, 1, 3, 5, and 8 h postdose following i.v. dosing and at 0.5, 1, 3, and 5 h postdose following p.o. and s.c. dosing. Blood (50 ml) was collected in capillary Microvette K 2 EDTA tubes (16.444.100; Sarstedt, Inc.) and kept on ice prior to centrifugation at 1,500 Â g for 5 min. The supernatant (plasma) was transferred into a 96-well plate and stored at 280°C.
HPLC-MS analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on a Sciex Applied Biosystems Qtrap 65001 triple-quadrupole mass spectrometer coupled to a Shimadzu Nexera 2 high-pressure liquid chromatography (HPLC) system to quantify each drug in plasma. Neat 1 mg/ml DMSO stocks for 844-TFM were serial diluted in 50:50 acetonitrile (ACN)/water to create standard curves and quality control (QC) spiking solutions. Standards and QCs were created by adding 10 ml of spiking solutions to 90 ml of drug-free plasma (CD-1 K 2 EDTA Mouse; Bioreclamation IVT). Twenty microliters of control, standard, QC, or study sample were added to 200 ml of ACN/methanol (MeOH) 50:50 protein precipitation solvent containing internal standard (10 ng/ml verapamil). Extracts were vortexed for 5 min and centrifuged at 4,000 rpm for 5 min. One hundred microliters of supernatant was transferred for HPLC-MS/MS analysis and diluted with 100 ml of Milli-Q deionized water.
Chromatography was performed on an Agilent Zorbax SB-C 8 column (2.1 Â 30 mm; particle size, 3.5 mm) using a reverse-phase gradient. Milli-Q deionized water with 0.1% formic acid was used for the aqueous mobile phase and 0.1% formic acid in ACN for the organic mobile phase. Multiple-reaction monitoring of precursor/product transitions in electrospray positive-ionization mode was used to quantify the analytes. The following multiple reaction monitoring (MRM) transitions were used for 844-TFM (459.10/133.00) and verapamil (455.4/165.2). Sample analysis was accepted if the concentrations of the quality control samples were within 20% of the nominal concentration. Data processing was performed using Analyst software (version 1.6.2; Applied Biosystems Sciex).
Plasma stability analysis and metabolite identification. The plasma stability assays were carried out using plasma from female CD-1 mice, New Zealand white rabbits, rhesus monkeys, and humans containing K 2 EDTA anticoagulant (Bioreclamation). Stability samples consisted of 5 ml of stock compound solution in 50:50 ACN/water and 95 ml of plasma to a final concentration of 1 mg/ml. The samples were incubated at 37°C with shaking; 10 ml of plasma was removed at each time point and combined with 100 ml of ACN/methanol 50:50 protein precipitation solvent containing internal standard (10 ng/ml verapamil). Chromatography for metabolite identification was performed the same as specified for pharmacokinetic sample analysis. Full scan total ion chromatograms (TIC) of plasma extracts were acquired using a Q Exactive high-resolution mass spectrometer (QE-HRMS) at 70,000 mass resolution. Thermo Fisher Compound Discoverer software was used to assist in identifying the 844-TFM metabolites from the QE-HRMS TIC mass spectrum. Figure S2 in the supplemental material illustrates the extracted ion chromatograms (XIC) of 844-TFM and the metabolites using 5 ppm mass accuracy before and after 12 h of incubation in mouse plasma.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.5 MB.

ACKNOWLEDGMENTS
We are grateful to Wei Chang Huang (Taichung Veterans General Hospital, Taichung, Taiwan) for providing M. abscessus Bamboo, to Jeanette W. P. Teo (Department of Laboratory Medicine, National University Hospital, Singapore) for providing the