MmpL3

Similar efficaciousness against MDR isolates of M is exhibited by AU1235 in conjunction to fluoroquinolones, ethambutol, and/or streptomycin. resistance to pyrazinamide, rifampicin, and isoniazid in tuberculosis patients. medicinal qualities. Although the minimum inhibitory concentration (MIC) of AU1235 (3.2 to 6.4 μg/ml) is considerably higher than that of Mycobacterium tuberculosis and Mycobacterium bovis BCG, it still inhibits Mycobacterium smegmatis and Mycobacterium fortuitum [2].

Selection of spontaneous AU1235-resistant mutants of M. tb [2]
AU1235-resistant mutants were selected at 37°C (M. tb H37Rv) or 30°C (M. tb H37Ra) on 7H11 plates supplemented with OADC and 0.2 to 0.4 μg ml−1 of the inhibitor (2 to 4 × MIC) and scored for resistance 3 to 7 weeks post-inoculation.

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Mycolyltransferase assays [2]
The TMM transesterification assay described in ref. 24 was used to measure the mycolyltransferase activity of the purified FbpA FbpB and FbpC proteins in the presence of cold TMM purified from M. tb, [U-14C]trehalose and different concentrations of AU1235 (see Supplementary Fig. 5). Purified FbpA and FbpB proteins were obtained through the NIH - TB Vaccine Testing and Research Materials Contract and NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: Ag85A, purified native protein from M. tb H37Rv (NR-14856); Ag85B, purified native protein from M. tb H37Rv (NR-14857). Purified recombinant FbpC (Ag85C) was kindly provided by Dr. K. Dobos.

Determination of MIC.[1]
MICs were determined on either solid or liquid media as described (31, 32). For solid medium, MICs were determined in 24-well plates inoculated with 5 μl of culture at 1 × 105 CFU/ml. Plates were incubated at 37°C for 4 weeks, and the MIC was defined as the minimum concentration that prevented growth. For liquid medium, assays were performed in 96-well plates inoculated with 35 μl of culture at an optical density at 590 nm (OD590) of 0.06 to 0.10; growth was measured by OD590 after 5 days at 37°C. The MIC90 (IC90) was defined as the concentration at which 90% of growth was inhibited. All compounds were dissolved in dimethyl sulfoxide (DMSO). SQ109 and AU1235 were synthesized according to published protocols (4). MIC determinations were performed in at least biological duplicates.

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Drug susceptibility testing[2]
MIC values of various antibiotics against Mycobacterium clinical isolates and recombinant strains were determined in 7H9-OADC-Tween 80 or 7H9-S-OADC-Tween 80 broth at 37°C in 96-well microtiter plates using the colorimetric resazurin microtiter assay and by visual readout for growth. Low-oxygen tension experiments were performed using the Rapid Anaerobic Dormancy (RAD) model as described. Control tubes contained methylene blue dye (1.5 μg ml−1) as an indicator of oxygen depletion. Different concentrations of AU1235 and control drugs (isoniazid, rifampicin, ethambutol and metronidazole) were injected through the septa of oxygen-depleted cultures and the cultures allowed to grow at 37o C with stirring for another 4 days, at which point the septa were removed and cultures serially diluted in saline and plated onto 7H11-OADC agar plates for enumeration of CFU.

[1]. McNeil MB, et al. Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors. mSphere. 2020;5(5):e00985-20. Published 2020 Oct 14.
[2]. Grzegorzewicz AE, et al. Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane. Nat Chem Biol. 2012;8(4):334-341. Published 2012 Feb 19.

The Mycobacterium tuberculosis protein MmpL3 plays a crucial role in cell wall synthesis because it influences the transmembrane transport of trehalose monomycin. Many structurally diverse pharmacophores have been identified as MmpL3 inhibitors, primarily based on the identification of resistant strains carrying MmpL3 mutations. For some compounds, different primary or secondary targets may exist. This paper investigates resistance to spiroamine compounds. Isolation and sequencing of resistant mutants revealed the presence of MmpL3 mutations in all mutants. We hypothesized that if other targets exist for this pharmacophore, then successive screening of resistant strains might uncover mutations at other sites. Since the compounds remained effective against resistant strains, albeit with reduced potency, we isolated resistant mutants at higher concentrations in this context. After a second round of screening with spiroamines, we discovered additional mutations in MmpL3. To increase the probability of discovering alternative targets, a third round of isolation was performed using a different molecular backbone (AU1235, an adamantyl urea). Unexpectedly, we obtained even more mutations in MmpL3. Multiple mutations in MmpL3 increased resistance levels and the spectrum of resistance to different pharmacophores without inducing an adaptive cost in vitro. These results support the hypothesis that MmpL3 is the primary resistance mechanism and a likely target of these pharmacophores. Importance: Mycobacterium tuberculosis is a leading human pathogen globally, urgently requiring new drugs and drug targets. Cell wall biosynthesis is a major target for current tuberculosis drugs and new drugs under development. Several novel molecules appear to share the same target, MmpL3, which is involved in the export and synthesis of mycobacterial cell walls. However, whether MmpL3 is the primary or sole target of these molecules remains controversial. We aimed to confirm the resistance mechanisms of a series of molecules. We identified mutations in the MmpL3 gene that resulted in resistance to the spiroamine series of compounds. Multiple mutations in the same protein (MmpL3) conferred high levels of resistance to these compounds as well as two other series of compounds. These mutations did not reduce growth rates in culture. These results support the hypothesis that MmpL3 is the primary resistance mechanism and a likely target of these pharmacophores. [1] Additional mutations in the MmpL3 gene broadened the resistance spectrum. In our previous two rounds of isolation of resistant mutants, we did not find any resistant strains other than those with the MmpL3 gene. Since the strains carrying the two mutations now exhibited relatively high resistance (>50 μM) to both of our initial compounds (Tables 1 and 2), we were unable to attempt further isolation of resistant strains for IDR-0334448 or IDR-0033216. However, the cross-resistance profiles of SQ109 and AU1235 differed (Table 2), which was consistent with the expectation that the compounds had unique interactions with MmpL3. Therefore, we used the observation that the strains were not completely resistant to these compounds to conduct a third round of isolation of resistant mutants. We screened two strains exhibiting varying degrees of resistance to SQ109 but no significant resistance to AU1235: (i) LP-0334448-RM102 carrying F255L and L567P mutations, demonstrating a 5.5-fold increase in resistance to SQ109; and (ii) LP-0334448-RM107 carrying F255L and V646M mutations, demonstrating a 3.3-fold increase in resistance to SQ109. These mutations were chosen because they provide resistance to other pharmacophores and are functionally important residues (5, 24, 25). Since the MIC value of AU1235 changed by less than 2-fold, we were able to isolate the resistant mutants at a 5-fold MIC concentration on solid medium, as before. We again hypothesized that if other target sites mutated, these isolated resistant mutants would not mutate in MmpL3. However, sequencing results of nine strains (three from LP-0334448-RM102 and six from LP-0334448-RM107) showed that all strains had additional mutations at the F644 site, namely F644L or F644I (Table 3), and exhibited high resistance to AU1235 (MIC values changed 16-fold and 100-fold, respectively). These strains also showed cross-resistance to SQ109, but to a lesser extent (5-fold and 6.7-fold, respectively). The F644L mutation did not lead to increased resistance to the structure-associated helical amine (i.e., IDR-0541243), while the F644I mutation increased resistance by 7-fold (Table 3). This is consistent with the predicted functional importance of the F644 site and the targeting of multiple pharmacophores to this site. [1] In summary, the accumulation of AU1235 was not altered in Mycobacterium tuberculosis cells expressing mmpL3-G253E, and the decreased expression of mmpL3 mimicked the phenotypic effect of treating mycobacteria with AU1235. These facts refute the hypothesis that MmpL3 is an adamantylurea efflux pump and confirm that this transporter is a direct target of our prototype inhibitor. [2]

C17H19F3N2O

324.3408

324.144

C, 62.95; H, 5.90; F, 17.57; N, 8.64; O, 4.93

1338780-86-1

941678-49-5;

54752297

Solid powder

3.9

2

4

2

23

445

0

FRRHMLGKNPFRKT-UHFFFAOYSA-N

InChI=1S/C17H19F3N2O/c18-12-1-2-13(15(20)14(12)19)21-17(23)22-16-10-4-8-3-9(6-10)7-11(16)5-8/h1-2,8-11,16H,3-7H2,(H2,21,22,23)

1-(2-Adamantyl)-3-(2,3,4-trifluorophenyl)urea

AU1235; AU-1235; AU1235; 1338780-86-1; 1-(1-adamantyl)-3-(2,3,4-trifluorophenyl)urea; CHEMBL1818385; SCHEMBL18423981; SCHEMBL18464994; SCHEMBL18464996; AU 1235

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol : ~65 mg/mL
DMSO : 10~25 mg/mL ( 30.83~77.08 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10% DMSO+90% (20% SBE-β-CD in Saline): ≥ 2.5 mg/mL (7.71 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)