Abstract
Drugs inhibiting the iron scarcity-induced, siderophore-mediated iron scavenging systems of Mycobacterium tuberculosis (Mtb) and Yersinia pestis (Yp) may provide new therapeutic lines of defense. Compounds with structural similarities to siderophores were synthesized and evaluated as antimicrobials against Yp and Mtb under iron limiting conditions, which mimic the iron scarcity these pathogens encounter and must adapt to in the host, and under standard iron rich conditions for comparison. New antimicrobials were identified, some of which warrant exploration as initial leads against potentially novel targets and small-molecule tools to assist in the elucidation of targets specific to iron-scarcity adapted Yp and Mtb.
Keywords: Siderophore, Antimicrobial, drug target, Mycobacterium tuberculosis, Yersinia pestis, aryl-carbothioamide-pyrazolines
Mtb, the etiologic agent of tuberculosis, and Yp, the causative agent of plague and a potential agent of biowarfare and bioterrorism, are pathogens with serious impacts on global public health. MDR tuberculosis is an emerging pandemic and the surfacing of extensive drug-resistant (XDR) tuberculosis poses a new global threat.1,2 Plague is a re-emerging disease and the occurrence of MDR Yp strains and self-transferable Yp plasmids conferring antibiotic resistance raises concerns about future plague control.3,4 These scenarios underscore the need for expanding the anti-tuberculosis and anti-plague drug repertoires. Anti-infective drugs against in vivo conditionally essential targets may offer novel therapeutic possibilities, help the fight against MDR/XDR strains and the prevention of their selection and dissemination, and increase biodefense preparedness.5
Anti-infective drugs inhibiting the siderophore-mediated, iron scavenging systems of Mtb and Yp may provide lines of defense against tuberculosis and plague, respectively. The Mtb siderophores (mycobactins and carboxymycobactins) and the Yp siderophore (yersiniabactin) (Fig. 1) have high affinity for Fe3+ (Kd<10−25 M), their production is induced under iron scarcity, and they are believed to be required for scavenging iron inside the host, where free iron is scarce (10−25–10−15 M) and pathogens experience iron-limiting conditions.6,7 The Mtb siderophore-deficient mutant is impaired for growth in macrophages and iron-limiting culture medium.8 The Mtb mutant lacking the IrtAB ferri-siderophore uptake system is impaired for multiplication in macrophages, mouse lung, and iron-limiting medium.9 Siderophore system-deficient Yp strains are avirulent in mice infected subcutaneously (a route imitating the fleabite transmission of Yp) and unable to multiply in iron-limiting medium.10,11 Siderophore system-deficient mutants of enteropathogenic Yersinia spp. are also attenuated in mice.12–14
Figure 1.
Structures of M. tuberculosis and Y. pestis siderophores.
We recently developed the first antibacterial targeting siderophore biosynthesis, a salicyl-AMP biosynthetic intermediate analog called salicyl-AMS (Fig. 1S, supplementary data).15 Salicyl-AMS is a potent inhibitor of salicylic acid adenylation domains involved in biosynthesis of salicylate-derived siderophores, blocks production of Mtb and Yp siderophores, and inhibits Mtb and Yp growth with greater potency in iron-limiting media, where siderophores are crucial for uptake of essential iron.15 More recently, others have independently reported the activity of salicyl-AMS.16 Continuing the line of our previous work, we hypothesized that compounds with structural features resembling Mtb and Yp siderophores may also impair the siderophore system by, for example, inhibiting biosynthetic enzymes or (ferri-)siderophore transport systems, and halt bacterial growth under iron-limiting conditions. To begin testing this hypothesis, we synthesized a 32-member pilot library of 3,5-diaryl-1-carbothioamide-pyrazoline derivatives (compounds 1–32, Fig. 2) with structural features resembling the hydroxyphenyl-oxazoline/thiazoline containing half of the siderophores and tested these compounds as Mtb and Yp growth inhibitors in iron-limiting media, which mimic the iron scarcity condition that the pathogens encounter in the host, and in standard iron-rich media for comparison.17 We also assessed whether selected compounds were bactericidal or bacteriostatic in iron-limiting media18. The ability of the compounds to inhibit YbtE (the Yp salicylation enzyme that is the intended target of salicyl-AMS15) in vitro was also examined19. Lastly, cytotoxicity towards mammalian cells was evaluated using a HeLa cell-based assay.20
Figure 2.
Structures of compounds 1 to 32. In 6 and 7, 2-thiophenyl and 2-furyl groups replace, respectively, the R1-3-bearing phenyl group. In 32, H replaces the R5-6-bearing phenyl group.
Compounds 1–32 were synthesized from 2′-hydroxy chalcone derivatives21 (Supplementary material, Scheme 1). Hydroxy chalcones were prepared through Claisen-Schmidt Condensation. 2′-hydroxy acetophenone derived chalcones were prepared by adding 60% aqueous solution of sodium hydroxide or potassium hydroxide to the mixture of ketone and aldehydes in methanol at 0 °C and stirring the reaction mixture for 4 h. Adjusting the pH of the reaction mixture to 2 using 6N hydrochloric acid precipitated hydroxy chalcones. 2′,4′-dihydroxy acetophenone derived chalcones required two days with occasional stirring.22 Pyrazoline derivatives were obtained by condensing 2′-hydroxy chalcones with 85% hydrazine hydrate in ethanol.23 Hydrazine hydrate was used in excess and after 3 h of reflux, pyrazolines were precipitated out upon cooling. 2′,4′-dihydroxy chalcone derived pyrazolines were extracted using chloroform from the concentrated reaction mixture. The final products (1–31) were obtained by the reaction of pyrazoline derivatives with phenyl isothiocyanates.23 Most of the thiocarboxamide derivatives precipitated out while the reaction mixture was hot, few upon cooling, and the rest upon concentration. Compound 32 was obtained by the reaction of chalcone with thiosemicarbazide in alkaline medium.24 After 8 h of reflux, the reaction mixture was diluted with cooled water and acidified to precipitate it out. All the intermediates were characterized by IR spectroscopic analysis and elemental analysis for CHNS. In the elemental analysis, the observed values were within ±0.4% of the calculated values. Final compounds were characterized by 1H NMR and FAB-MS.21
Testing against Mtb revealed that 15 compounds had IC50s and MICs (3–500 μM range) within the concentration series tested in the iron-limiting medium, GAST-D (Table 1). Nine of these compounds also had determinable IC50s and MICs (4–500 μM range) in the iron rich medium, GAST-D-Fe. Interestingly, 10, 12, 16, 25, 26, and 32 were ≥3-fold more potent against Mtb cultured under iron scarcity (Table 1). Compound 32, with 16-fold higher potency in GAST-D, stood out in this group. This compound, along with 13 and 16, were the only bactericidal compounds (>99% inoculum killing) among those examined for mode of action against Mtb (10, 12 and 16, tested at 2×MICGAST-D; 13 and 32, tested at 5×MICGAST-D). These bactericidal compounds had CD50s against eukaryotic cells in the 21–398 μM range and were among those with the lowest cytotoxicity in the library, in which compounds had 10–3980-fold lower cytotoxicity than the reference cytotoxic compound cycloheximide25 (CD50 = 0.1 μM) (Table 3). Compound 13, with the highest activity against Mtb (IC50 = 3–4 μM, MIC = 12–13 μM; Table 1) and the second best selectivity index relative to Mtb (SIMtb = 14; Table 3), and 16 were 420- and 210-fold less cytotoxic than cycloheximide, respectively, but 5- and 9-fold more cytotoxic, respectively, than the reference anti-tuberculosis drug rifampicin used in our assay. Rifampicin had a CD50 of 190 μM (1,900-fold lower cytotoxicity than cycloheximide) and the same antimicrobial potency in GAST-D and GAST-D-Fe (MIC = 8 nM). Notably, 32, with one of the strongest antitubercular activity (Table 1), was 2-fold and 3980-fold less cytotoxic than the rifampicin and cycloheximide references, respectively, and had the best SIMtb (Table 3).
Table 1.
Antimicrobial activity against M. tuberculosis.
| Comp. | IC50 (μM)a |
Ratio | MIC90 (μM)b |
Ratio | Mode of action | ||
|---|---|---|---|---|---|---|---|
| GAST-D-Fe | GAST-D | GAST-D-Fe | GAST-D | ||||
| 1–3, 5, 9, 14, 15, 17–20, 22, 31 | ≥500 | ≥500 | nd | ≥500 | ≥500 | nd | nd |
| 4 | >250 | >250 | nd | >250 | >250 | nd | nd |
| 6 | 500 | 250 | 2 | >500 | >500 | nd | nd |
| 7 | 72 | 73 | 1 | 208 | 250 | 1 | nd |
| 8 | 65 | 29 | 2 | 100 | 83 | 1 | nd |
| 10 | 167 | 28 | 6 | >250 | 63 | >4 | BS |
| 11 | 42 | 28 | 2 | >500 | 104 | >5 | nd |
| 12 | 208 | 27 | 8 | >250 | 63 | >4 | BS |
| 13 | 4 | 3 | 1 | 12 | 13 | 1 | BC |
| 16 | 83 | 29 | 3 | 417 | 63 | 7 | BC |
| 21 | 96 | 101 | 1 | >500 | 417 | >1 | nd |
| 23 | >250 | 167 | >2 | >250 | >250 | nd | nd |
| 24 | 156 | 80 | 2 | 250 | 125 | 2 | nd |
| 25 | 250 | 63 | 4 | >500 | 167 | >3 | nd |
| 26 | 333 | 84 | 4 | >500 | 125 | >4 | nd |
| 27 | 55 | 51 | 1 | 125 | 125 | 1 | nd |
| 28 | 167 | 125 | 1 | >250 | >250 | nd | nd |
| 29 | 105 | 83 | 1 | 250 | 125 | 2 | nd |
| 30 | 42 | 24 | 2 | 167 | 125 | 1 | nd |
| 32 | 125 | 8 | 16 | 333 | 21 | 16 | BC |
| RIF | nd | nd | nd | 0.008 | 0.008 | 1 | nd |
IC50s were calculated from sigmoidal curves fitted to triplicate sets of dose-response data.
MIC90s are means of triplicates. All values were rounded to the nearest non-fractional number. RIF, rifampicin; nd, not determined; BS, bacteriostatic; BC, bactericidal.
Table 3.
Cytotoxicity and selectivity assessment
| Comp. | CD50 (μM)a | SIMtb (CD50/IC50GAST-D) |
SIYp (CD50/IC50PMH-D) |
|---|---|---|---|
| 1 | 3 | <0.006 | <0.3 |
| 2 | 5 | <0.01 | <1 |
| 3 | 5 | <0.01 | 10 |
| 4 | 6 | <0.02 | 15 |
| 5 | 5 | <0.01 | 7 |
| 6 | 3 | 0.01 | 300 |
| 7 | 3 | 0.04 | 3 |
| 8 | 3 | 0.1 | 4 |
| 9 | 15 | <0.03 | 8 |
| 10 | 1 | 0.04 | 1 |
| 11 | 17 | 0.6 | 9 |
| 12 | 11 | 0.4 | 14 |
| 13 | 42 | 14 | <2 |
| 14 | 1 | 0.002 | 0.5 |
| 15 | 4 | <0.008 | 6 |
| 16 | 21 | 0.7 | 7 |
| 17 | 3 | 0.007 | 3 |
| 18 | 13 | <0.03 | 7 |
| 19 | 11 | 0.02 | 11 |
| 20 | 25 | <0.05 | 42 |
| 21 | 15 | 0.1 | 5 |
| 22 | 16 | <0.03 | 16 |
| 23 | 63 | 0.4 | 63 |
| 24 | 23 | 0.3 | 33 |
| 25 | 62 | 1.0 | 155 |
| 26 | 30 | 0.4 | 50 |
| 27 | 26 | 0.5 | 2 |
| 28 | 31 | 0.2 | 155 |
| 29 | 248 | 3 | <3 |
| 30 | 19 | 0.8 | <1 |
| 31 | 29 | 0.06 | <0.8 |
| 32 | 398 | 50 | <10 |
| RIF | 190 | nd | nd |
| STR | >500 | nd | nd |
| CHX | 0.1 | nd | nd |
CD50s were calculated from sigmoidal curves fitted to triplicate sets of dose-response data. SIMtb and SIYp, selectivity index relative to activity against M. tuberculosis and Y. pestis, respectively. CD50 and SI values <1 and values >1 were rounded to one significant digit and to the nearest non-fractional number, respectively. CHX, cycloheximide. Other abbreviations as in Table 1.
Most compounds were more active against Yp than against Mtb, which has a thick, waxy cell envelope that makes penetration of many drugs difficult. Twenty-five compounds had considerable activity against Yp in the iron limiting medium, PMH-D (IC50s = 0.01–17 μM range) (Table 2). Notably, 17 compounds had both IC50s and MICs that were at least >3-fold higher in PMH-D-Fe (iron rich medium) than in PMH-D (Table 2). In this group were 18, 20, 23, 24, 25 and 26, each of which had >30-fold more potent IC50s and MICs against Yp cultured under iron scarcity. These compounds were 130–630-fold less cytotoxic than cycloheximide, but have over 250–1250-fold greater cytotoxicity than the reference anti-plague drug streptomycin. Streptomycin had a CD50 >500 μM (>5,000-fold less cytotoxic than cycloheximide) and the same antimicrobial activity in PMH-D and PMH-D-Fe (MIC = 0.2 μM). Compound 25, along with 6 and 28, had the best selectivity indexes relative to Yp (SIYp) (Table 3). Twenty-two compounds evaluated for mode of action against Yp were bacteriostatic when tested at up to the maximum multiple of the MICPMH-D permitted by solubility, which ranged from 1× to 38×MICPMH-D (Table 2).
Table 2.
Antimicrobial activity against Y. pestis.
| Comp. | IC50 (μM)a |
Ratio | MIC90 (μM)a |
Ratio | Mode of action | ||
|---|---|---|---|---|---|---|---|
| PMH -D-Fe | PMH -D | PMH -D-Fe | PMH -D | ||||
| 1 | >9 | >9 | nd | >9 | >9 | nd | nd |
| 2 | >5 | >5 | nd | >5 | >5 | nd | nd |
| 3 | >5 | 0.5 | >10 | >5 | 1 | >5 | BS |
| 4 | >5 | 0.4 | >13 | >5 | >5 | nd | nd |
| 5 | >19 | 0.7 | >27 | >19 | >19 | nd | nd |
| 6 | >9 | 0.01 | >900 | >9 | >9 | nd | nd |
| 7 | >9 | 1 | >9 | >9 | 2 | >5 | BS |
| 8 | >19 | 0.7 | >27 | >19 | 2 | >10 | BS |
| 9 | >5 | 2 | >3 | >5 | 5 | >1 | BS |
| 10 | >2 | 1 | >2 | >2 | 3 | >0.7 | BS |
| 11 | >19 | 2 | >10 | >19 | 5 | >4 | BS |
| 12 | >9 | 0.8 | >11 | >9 | 2 | >5 | BS |
| 13 | >19 | >19 | nd | >19 | >19 | nd | nd |
| 14 | >19 | 2 | >10 | >19 | 6 | >3 | BS |
| 15 | >19 | 0.7 | >27 | >19 | 2 | >10 | BS |
| 16 | >19 | 3 | >6 | >19 | 5 | >4 | BS |
| 17 | >9 | 0.9 | >10 | >9 | 3 | >3 | BS |
| 18 | >150 | 2 | >75 | >150 | 5 | >30 | BS |
| 19 | >9 | 1 | >9 | >9 | 6 | >2 | BS |
| 20 | >150 | 0.6 | >250 | >150 | 2 | >75 | BS |
| 21 | >19 | 3 | >6 | >19 | 9 | >2 | BS |
| 22 | >19 | 1 | >19 | >19 | 2 | >10 | BS |
| 23 | >150 | 1 | >150 | >150 | 2 | >75 | BS |
| 24 | >150 | 0.7 | >214 | >150 | 3 | >50 | BS |
| 25 | >150 | 0.4 | >375 | >150 | 5 | >30 | BS |
| 26 | >150 | 0.6 | >375 | >150 | 2 | >75 | BS |
| 27 | >38 | 17 | >2 | >38 | >38 | nd | BS |
| 28 | >9 | 0.2 | >45 | >9 | 1 | >9 | BS |
| 29 | >75 | >75 | nd | >75 | >75 | nd | nd |
| 30 | >19 | >19 | nd | >19 | >19 | nd | nd |
| 31 | >38 | >38 | nd | >38 | >38 | nd | nd |
| 32 | >38 | >38 | nd | >38 | >38 | nd | nd |
| STR | nd | nd | nd | 0.2 | 0.2 | 1 | nd |
STR, streptomycin. All values <1 and values >1 were rounded to one significant digit and to the nearest non-fractional number, respectively. Other footnotes and abbreviations as in Table 1
Compounds 1–32 were not specifically designed to inhibit a particular enzyme in the siderophore biosynthetic pathways. However, the compounds were tested as inhibitors of YbtE, which is the intended target of salicyl-AMS15. None of the compounds was as potent as the bona fide inhibitor salicyl-AMS (Table 1S, supplementary data). The compounds had IC50s in the 0.2- to >12.5-μM range and were 29- to >1786-fold less potent than salicyl-AMS (IC50s = 0.007 μM). Moreover, no clear structural-activity relationships emerged from these data. Interestingly, however, the three library compounds (29, 30, and 31) lacking the hydroxyl ortho to the 5-membered ring as seen in the siderophores were among the four compounds with drastically increased IC50s (>12.5 μM; Table 1S) compared with salicyl-AMS. No meaningful correlation trend between the IC50s in the YbtE assay and the IC50s in the Yp growth assay was observed. Considering that the compounds active against Yp had IC50s in the high nanomolar-low micromolar range in the cellular assay under the iron scarcity condition (Table 2) and that the activity of these compounds is likely to be drastically reduced in the cellular assay (due to factors such as penetration/efflux, intracellular stability, and off-target binding) compared with the enzymatic assay, it is unlikely that YbtE inhibition plays a major role in the antimicrobial activity of these compounds.
In sum, 30 compounds of our pilot library had detectable antimicrobial activity. To our knowledge, these are novel scaffolds not previously shown to have this property. In line with our aforementioned hypothesis, several compounds had higher potency under iron-limiting conditions. Under this condition bacteria depend on siderophores for iron scavenging and engage an adaptive response to tailor their physiology to iron scarcity, thus exposing novel potential in vivo conditional targets.5 Some of these antimicrobials may impair siderophore system functioning as discussed above, a property that would result in bacteriostatic activity conditional to environmental iron scarcity (e.g., as seen with 25 against Yp). Others may inhibit functions conditionally essential to the iron scarcity-associated physiology, which is expected to be adopted by the pathogens in the iron-limiting environments of the host, and could have bactericidal activity (e.g., as seen with 32 against Mtb). Compounds with antimicrobial activity that was independent from the iron content of the media were also identified (e.g., 13 against Mtb). These antimicrobials may target essential bacterial functions required under both low and high iron conditions.
This study provides proof-of-principle for the effectiveness of screening compound libraries in iron limiting conditions to identify antimicrobials that may selectively target iron scarcity-adapted bacteria. Our screening approach allowed us to identify compounds with antimicrobial activity that is conditional to iron scarcity and would not have been revealed in conventional screens, which are performed under iron-rich conditions. Some of the identified antimicrobials warrant exploration as initial leads against potential in vivo conditionally essential targets and small-molecule tools to assist in the elucidation of targets and pathways critical to iron-scarcity adaptation in Yp and Mtb. Studies are underway to elucidate the molecular mechanisms of action of selected library compounds, a process that may lead to the discovery of novel mechanisms of antimicrobial activity and drug target candidates.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:
Acknowledgments
This work was supported in part by NIH grants AI063384 and AI056293-01, and the New England Regional Center of Excellence in Biodefense and Emerging Infectious Disease. The Department of Microbiology and Immunology acknowledges support from the W. R. Hearst Foundation. LQ is a Niarchos Scholar. We are grateful to the Sophisticated Analytical Instrument Facility (CDRI, Lucknow, India) for providing spectral data, to Dr. D. S. Tan’s laboratory (Memorial Sloan-Kettering Cancer Center) for providing salicyl-AMS for this study, and to R. Moy, G. Sadhanandan and Dr. W. He (Cornell) for assistance with antimicrobial testing and helpful discussions.
Footnotes
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References and notes
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Supplementary Materials
Supplementary data associated with this article can be found, in the online version, at doi: