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. 2015 Apr;59(4):2256-64.
doi: 10.1128/AAC.05114-14. Epub 2015 Feb 2.

A novel antimycobacterial compound acts as an intracellular iron chelator

Marte S Dragset  1 , Giovanna Poce  2 , Salvatore Alfonso  2 , Teresita Padilla-Benavides  3 , Thomas R Ioerger  4 , Takushi Kaneko  5 , James C Sacchettini  6 , Mariangela Biava  7 , Tanya Parish  8 , José M Argüello  3 , Magnus Steigedal  9 , Eric J Rubin  10
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A novel antimycobacterial compound acts as an intracellular iron chelator

Marte S Dragset et al. Antimicrob Agents Chemother. 2015 Apr.

Abstract (V体育官网)

Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis. Mycobacterial iron uptake and metabolism are therefore attractive targets for antitubercular drug development. Resistance mutations against a novel pyrazolopyrimidinone compound (PZP) that is active against M. tuberculosis have been identified within the gene cluster encoding the ESX-3 type VII secretion system. ESX-3 is required for mycobacterial iron acquisition through the mycobactin siderophore pathway, which could indicate that PZP restricts mycobacterial growth by targeting ESX-3 and thus iron uptake. Surprisingly, we show that ESX-3 is not the cellular target of the compound VSports手机版. We demonstrate that PZP indeed targets iron metabolism; however, we found that instead of inhibiting uptake of iron, PZP acts as an iron chelator, and we present evidence that the compound restricts mycobacterial growth by chelating intrabacterial iron. Thus, we have unraveled the unexpected mechanism of a novel antimycobacterial compound. .

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Figures

FIG 1
FIG 1
Clones of M. tuberculosis resistant to PZP have mutations in the eccB3 gene of ESX-3. (A) Chemical structure of PZP. (B) The esx-3 gene cluster is composed of 11 genes stretching from MSMEG_0615 to MSMEG_0626 in M. smegmatis (SmegmaList, http://mycobrowser.epfl.ch/smegmalist.html) and rv0282 to rv0292 in M. tuberculosis (TubercuList, http://tuberculist.epfl.ch/index.html). (C) Amino acid sequence of the N-terminal end of EccB3. Black highlighting indicates conserved residues in mycobacteria. Orange, green, and red circles indicate the locations of mutated residues in PZP-resistant M. tuberculosis (R14L, N24H, and ΔN26 mutations). (D) EccB3 is a transmembrane protein with a predicted small (the first 71 amino acids) cytosolic tail on the N terminus and a large (amino acid 95 to 538) extracellular C-terminal domain. The mutations R14L, N24H, and ΔN26 are localized to the cytosolic tail, as indicated.
FIG 2
FIG 2
M. smegmatis is susceptible to PZP in an iron-dependent, ESX-3-independent manner. (A) Growth of M. smegmatis WT and ΔfxbA was monitored by OD600 measurements for 80 h under iron-replete (right) and iron-deplete (left) conditions, in the presence or absence of 25 μM PZP. M. smegmatis Δesx-3 ΔfxbA was included as a control. Results are representative of two independent experiments. (B) Growth of M. smegmatis WT and Δesx-3 was monitored for 70 h in iron-replete (right) and iron-deplete (left) medium, in the presence or absence of 25 μM PZP. (C) Growth of M. smegmatis WT (left) and Δesx-3 (right) was monitored for 70 h in iron-deplete medium in the presence of increasing concentrations of PZP. Results shown in panels B and C represent the averages of three independent biological replicates.
FIG 3
FIG 3
Treatment with PZP leads to iron accumulation and upregulation of iron uptake genes in M. smegmatis. (A) Whole-cell iron content was measured by atomic absorption spectroscopy in M. smegmatis WT and eccB3(ΔN26) strains grown to the late exponential phase in iron-deplete or iron-replete medium for 24 h in the presence (+) or absence (−) of 25 μM PZP. Error bars represent standard errors of at least five biological replicates. (B) M. smegmatis WT was grown in iron-deplete or iron-replete medium for 24 h in the presence (+) or absence (−) of 25 μM PZP. The relative transcript levels of three iron-responsive genes (mbtB, mbtL, and irtA) were measured by quantitative RT-PCR. Error bars represent the calculated maximum (RQmax) and minimum (RQmin) as determined from the standard errors of the cycle threshold changes (ΔCT values). (C and D) M. smegmatis WT and eccB3(ΔN26) mutant strains were grown under iron-depleted conditions with increasing concentrations of PZP, and the OD600 was monitored every second hour.
FIG 4
FIG 4
PZP chelates Fe2+. (A) Electrospray mass spectra of 10 μM PZP. (B) Electrospray mass spectra of a solution of 10 μM PZP with 10 μM Fe2+. The inset shows the isotopic pattern of the peaks at 551.92 and 1078.00 nm. All the reagents were dissolved in water-methanol (1:1, vol/vol).
FIG 5
FIG 5
PZP competes with ferrozine by binding to iron in a 3:1 stoichiometric manner. (A) Spectroscopic titration of 10 μM PZP with Fe2+ (increments up to 10 μM were reached). (Inset) Absorbance of the PZP-Fe2+ complex at 400 nm as a function of Fe2+. (B) Chemical structure of (PZP)3-Fe2+ complex. (C) Competition experiment between ferrozine and PZP for Fe2+. Curves: (a, black line), 60 μM ferrozine in 20 mM potassium phosphate buffer, pH 7.2 only; (b, red line) addition of 20 μM Fe2+; (c, green line) addition of 60 μM PZP; (d to f, yellow, purple, and blue lines, respectively) addition of 60 µM PZP for 60 min (d), 90 min (e), or 120 min (f).
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