Chemical scaffolds with structural similarities to siderophores of nonribosomal peptide-polyketide origin as novel antimicrobials against Mycobacterium tuberculosis and Yersinia pestis

Abstract

Mycobacterium tuberculosis (Mtb) and Yersinia pestis (Yp) produce siderophores with scaffolds of nonribosomal peptide-polyketide origin. Compounds with structural similarities to these siderophores were synthesized and evaluated as antimicrobials against Mtb and Yp under iron-limiting conditions mimicking the iron scarcity these pathogens encounter in the host and under standard iron-rich conditions. Several new antimicrobials were identified, including some with increased potency in the iron-limiting condition. Our study illustrates the possibility of screening compound libraries in both iron-rich and iron-limiting conditions to identify antimicrobials that may selectively target iron scarcity-adapted bacteria and highlights the usefulness of building combinatorial libraries of compounds having scaffolds with structural similarities to siderophores to feed into antimicrobial screening programs.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, and Yersinia pestis (Yp), the etiologic agent of plague, are bacterial pathogens with serious impacts on global public health. Multidrug-resistant (MDR) tuberculosis is an emerging pandemic, whereas the more recent emergence of extensively drug-resistant (XDR) tuberculosis poses a new global threat.¹ Plague is a re-emerging disease for which the documented occurrence of MDR Yp strains and self-transferable Yp plasmids conferring antibiotic resistance raises concerns about future Plague control.² These grim scenarios underscore the need for expanding the anti-tuberculosis and anti-plague drug armamentarium.

Many bacteria utilize secreted, small (<1,000 Da) Fe³⁺-chelating compounds (K_d <10⁻²⁵ M) called siderophores to scavenge Fe³⁺ from their microenvironments and transport it into the cell.³ The Mtb siderophore (mycobactin/carboxymycobactin) and the Yp siderophore (yersiniabactin) are based on substituted scaffolds of nonribosomal peptide-polyketide origin (Figure 1).⁴ Studies in cellular and animal models of infection have established the relevance of the mycobactin/carboxymycobactin and yersiniabactin siderophore systems in these pathogens.⁵ The siderophores are believed to facilitate iron scavenging inside the host, where free iron is scarce (10⁻²⁵-10⁻¹⁵ M) and the pathogens experience and must adapt to iron-limiting conditions.⁶ These observations suggest that the Mtb and Yp siderophore systems represent potential in vivo conditionally essential target candidates for the development of alternative therapeutics against tuberculosis and plague.⁷

Figure 1.

Structures of M. tuberculosis and Y. pestis siderophores.

We hypothesize that screening compounds with structural features resembling Mtb and Yp siderophores for growth inhibitory activity against these pathogens may lead to the discovery of novel antimicrobial scaffolds. Such novel antimicrobials could illuminate alternative paths to drug development and/or be useful as small-molecule tools to assist in the elucidation of new target candidates for drug development. Compounds with structural features resembling Mtb and Yp siderophores may impair the siderophore systems (e.g., by inhibiting siderophore biosynthesis or transport) and halt bacterial growth in the host’s iron-limiting environments. Alternatively, these compounds might gain access to the intracellular environment using siderophore transport systems and inhibit essential functions unrelated to iron acquisition. Consistent with these views, we have recently demonstrated potent antimicrobial activity against Mtb and Yp for novel diaryl-carbothioamide-pyrazoline derivatives with structural features resembling the hydroxyphenyl-oxazoline/thiazoline-containing half of Mtb and Yp siderophores.⁸

In an effort to identify additional novel inhibitors of Mtb and Yp growth, we synthesized and evaluated the antimicrobial activity of new 3,5-diaryl-substituted pyrazoline (DAP) derivatives (cmpds 1–22,^9a Table 1). In addition, we synthesized and tested the activity of a group of (2E)-2-benzylidene-N-hydroxyhydrazine carbo(ox/thio/oximid)-amide (BHHC) derivatives (cmpds 23–32,^9b Table 2) with hydroxyphenyl-cap functionalities resembling that of the siderophores. The compounds were tested for growth inhibitory activity against Mtb and Yp in iron-limiting media, which mimic the iron-scarcity condition that the pathogens encounter in the host, and in standard iron-rich media.^10a We also assessed selected compounds for mode of action (bactericidal or bacteriostatic) in iron-limiting media and for cytotoxicity toward mammalian cells.^10b

Table 1.

3.5-Diarvl-substituted pyrazoline (DAP) derivatives (1–22)

Compound	R	R’	R
1	–H	–OH	–H
2	–OH	–H	–H
3	–H	–OH
4	–OH	–H
5	–H	–OH
6	–OH	–H
7	–H	–OH
8	–OH	–H
9	–H	–OH
10	–H	–CH3
11	–H	–OH
12	–OH	–H
13	–H	–OH
14	–OH	–H
15	–H	–OH
16	–OH	–H
17	–H	–OH
18	–OH	–H
19	–H	–OH
20	–OH	–H
21	–H	–OH
22	–OH	–H

Table 2.

(2E)-2-Benzylidene-N-hydroxyhydrazine carbo(ox/thio/oximid)-amide (BHHC) derivatives (23–32)

Compound	R	R1	R2	R3
23	–H	–OH	–H	=O
24	–OH	–H	–H	=O
25	–H	–OH	–CH3	=O
26	–OH	–H	–CH3	=O
27	–H	–OH	–H	=S
28	–OH	–H	–H	=S
29	–H	–OH	–CH3	=S
30	–H	–OH	–H	–NH
31	–OH	–H	–H	–NH
32

Testing against Mtb revealed that 17 compounds (1, 2, 5, 8–15, 24, 27–31) had IC₅₀s and MICs (3–222 μM range, Table 3) within the concentration series tested in the iron-limiting medium, GASTD. Of these 17 compounds, 15 (1, 2, 5, 9-12, 14, 15, 24, 27–31) also had determinable IC₅₀s and MICs (2–132 μM range) in the iron-rich medium, GASTD+Fe. Examination of IC_50GASTD+Fe/IC_50GASTD and MIC_GASTD+Fe/MIC_GASTD ratios revealed that the inhibitors had no noteworthy increased potency in the iron-limiting medium within the concentration series tested. This suggests that interference with iron acquisition, or any other bacterial process differentially required for growth under the iron-limiting condition, is not a property that significantly contributes to the compounds’ antimicrobial activity against Mtb. Interestingly, 14 is 5-fold more potent against Mtb cultured in GASTD+Fe, as judged by MIC values. This phenomenon might suggest that the mechanism(s) of action of 14 against Mtb might be potentiated by an elevated production of cytotoxic hydroxyl radicals originated through increased levels of Fenton reaction in the iron-rich medium. Such a potentiation would be in line with recent findings of Collins and coworkers regarding antibiotic-induced cell death.¹⁶ Alternatively, 14 might inhibit an oxidative stress protection function(s) more critically needed in the iron-rich medium. Testing for cytotoxicity at the MIC against Mtb (125 μM) revealed that 14 had no significant cytotoxicity at short cell-cmpd contact time (4 hr), yet cell viability was reduced by 70% relative to untreated controls after prolonged contact time (24 hr) (Supplementary Data, Fig. S1).

Table 3.

Antimicrobial activity against M. tuberculosis

Compound	IC50a (μM)		Ratio	MIC90b (μM)		Ratio	Mode of actionc
	GASTD+Fe	GASTD		GASTD+Fe	GASTD
DAP series
1	18	25	1	59	66	1	BC (2 × MIC)
2	37	46	1	125	125	1	BC (I × MIC)
3	>16	>31	nd	>16	>32	nd	nd
4	>8	>8	nd	>8	>8	nd	nd
5	64	77	1	104	208	0.5	BC (2 × MIC)
6	>32	>63	nd	>63	>63	nd	nd
7	>32	54	nd	>31	>63	nd	nd
8	40	42	1	>31	125	nd	BC (1 × MIC)
9	50	53	1	125	125	1	BC (2 × MIC)
10	7	7	1	16	16	1	BC (2 × MIC)
11	23	44	1	66	66	1	BC (2 × MIC)d
12	26	31	1	76	125	1	BC (2 × MIC)d
13	32	37	1	>125	125	>l	BC (2 × MIC)
14	22	47	0.5	31	125	0.2	BC (2 × MIC)
15	8	8	1	16	16	1	BC (2 × MIC)
16	>4	>8	nd	>4	>8	nd	nd
17	98	84	1	>500	>500	nd	nd
18	>16	>63	nd	>63	>63	nd	nd
19	>16	>63	nd	>31	>63	nd	nd
20	>31	>63	nd	>31	>63	nd	nd
21	>31	>63	nd	>31	>63	nd	nd
22	>31	>63	nd	>31	>63	nd	nd
BHHC series
23	>16	>16	nd	>16	>16	nd	nd
24	40	97	0.4	97	146	1	BC (2 × MIC)
25	>8	>8	nd	>8	>8	nd	nd
26	>500	>500	nd	>500	>500	nd	nd
27	2	3	1	6	7	1	BC (4 × MIC)
28	33	31	1	59	59	1	BC (2 × MIC)
29	4	4	1	9	10	1	BC (2 × MIC)
30	15	11	1	26	222	1	BC (2 × MIC)
31	43	43	1	132	222	1	BC (2 × MIC)
32	>8	20	nd	>8	>31	nd	nd
Isoniazid	0.09	0.07	1	0.2	0.2	1	BC (2 × MIC)

^aIC₅₀ values were calculated from sigmoidal curves fitted to triplicate sets of dose–response data.

^bMIC₉₀ values are means of triplicates. Ratio, GASTD+Fe/GASTD. All values >l, <l, >0.5, and ≤0.5 were rounded to the nearest whole number, to 1, and to one significant digit, respectively.

^cMode of action was evaluated in GASTD in duplicate. The concentration at which each compound was tested for mode of action is indicated between parentheses.

^dSome bactericidal activity detected, yet below the 99% killing criterion set for defining bactericidal mode of action. BS, bacteriostatic; BC, bactericidal; nd, not determined.

Among the active DAP derivatives, 10 and 15 were the most potent against Mtb (IC₅₀ = 7–4 μM, MIC = 16 μM, Table 3). Of these two cmpds, only 10 displayed significant cytotoxicity at the MIC against Mtb (16 μM). Cmpd 10 had a modest impact on cell viability, which was reduced only by 24% after 24 hr of cell-cmpd contact (Supplementary Data, Fig. S1). Encouragingly, these and most other inhibitors in the DAP derivatives series examined for mode of action against Mtb were bactericidal (>99% killing relative to inoculum) at concentrations of 1-2 × MIC_GASTD (Table 1). This finding is significant since bactericidal activity is a desirable property in any early lead compound evaluated for antibacterial drug development programs. It is worth noting that the only two compounds (11 and 12) defined as bacteriostatic in Table 1 showed significant bactericidal activity, yet below the 99% killing criterion set in this study for defining bactericidal mode of action. Among the compounds of the BHHC derivatives series with defined IC₅₀ and MIC values, 27 and 29 were the most active against Mtb (IC₅₀ = 2–4 μM, MIC = 6–10 μM, Table 3). These two compounds displayed no significant cytotoxicity in mammalian cells at their respective MIC values determined against Mtb (Supplementary Data, Fig. S1). Gratifyingly, 27, 29 and other active cmpds in this series displayed bactericidal mode of action against Mtb at concentrations of 1.7-4 × MIC_GASTD (Table 3).

Testing against Yp revealed that 14 cmpds (1, 2, 7–9, 11, 13, 14, 19, 27–29, 31, 32; Table 1) reached IC₅₀s (0.04–181 μM range, Table 4) within the concentration series tested in the iron-limiting medium, PMHD. Nine of these compounds (1, 11, 13, 14, 27–29, 31, 32) also reached MICs (0.2–388 μM range) in PMHD. Only 29 and 30 had determinable activity in the iron-rich medium, PMHD+Fe (29: IC50 = 156 μM, MIC = 233 μM; 30: IC50 = 305 μM). Interestingly, examination of the IC_50PMHD+Fe/IC_50PMHD ratios revealed that a number of inhibitors (7, 11, 13, 14, 19, 27, 29, 31, 32) with increased potency (>3-fold) against Yp cultured under iron scarcity. Cmpd 32, with >100-fold and >20-fold higher potency in PMHD based on IC₅₀ and MIC values, respectively, stood out in this group. The higher potency of these compounds in the iron-limiting medium raises the possibility that interference with an iron acquisition function, or other function more critically required for growth under the iron-limiting condition, is a property that significantly contributes to the compounds’ antimicrobial activity against Yp. One of the possible mechanisms of action of these compounds could be related to iron-binding properties. An iron-binding ability strong enough to outcompete the powerful iron chelating capacity of the bacterial siderophore could lead to sequestration of the traces of iron in the iron-limiting medium, thus reducing further iron bioavailability and producing a stronger antimicrobial activity under the iron-scarcity condition. Alternatively, it is possible that these compounds gain intracellular access using the iron-scarcity-unregulated yersiniabactin’s uptake system,^5e therefore reducing IC₅₀ and MIC values in PMHD.

Table 4.

Antimicrobial activity against Y. pestis

Compound	IC50a (μM)		Ratio	MIC90b (μM)		Ratio	Mode of actionc
	PMHD+Fe	PMHD		PMHD+Fe	PMHD
DAP series
1	>8	9	nd	>8	31	nd	BC (1 × MIC)
2	>31	20	>2	>31	>31	nd	nd
3	>31	>31	nd	>31	>31	nd	nd
4	>16	>16	nd	>16	>16	nd	nd
5	>250	>250	nd	>250	>250	nd	ml
6	>31	>31	nd	>31	>31	nd	nd
7	>31	8	>4	>3l	>31	nd	nd
8	>31	13	>2	>31	>31	nd	nd
9	>63	181	nd	>63	>250	nd	nd
10	>31	>62	nd	>31	>63	nd	nd
11	>63	7	>9	>63	42	>l	BC (1 × MIC)
12	>63	>125	nd	>63	>125	nd	nd
13	>16	4	>4	>16	16	>1	BC (2 × MIC)
14	>63	5	>13	>63	21	>3	BC (2 × MIC)
15	>16	>16	nd	>16	>16	nd	nd
16	>8	>8	nd	>8	>8	nd	nd
17	>63	>31	nd	>63	>31	nd	nd
18	>16	>31	nd	>16	>3l	nd	nd
19	>31	8	>4	>31	>31	nd	nd
20	>31	>16	nd	>31	>16	nd	nd
21	>16	>31	nd	>16	>31	nd	nd
22	>16	>31	nd	>16	>31	nd	nd
BHHC series
23	>4	>16	nd	>4	>16	nd	nd
24	>250	>250	nd	>250	>250	nd	nd
25	>2	>4	nd	>2	>4	nd	nd
26	>500	>500	nd	>500	>500	nd	nd
27	>59	4	>15	>59	15	>4	BC (2 × MIC)
28	>233	137	>2	>233	388	nd	BC (1 × MIC)
29	156	61	>3	233	116	2	BC (2 × MIC)
30	305	70	4	>500	250	>2	BC (2 × MIC)
31	>500	>500	nd	>500	>500	nd	nd
32	>4	0.04	>100	>4	0.2	>20	BC (2 × MIC)
Streptomycin	1	1	1	2	1	2	BC (2 × MIC)

^aIC₅₀ values were calculated from sigmoidal curves fitted to triplicate sets of dose response data.

^bMIC₉₀ values are means of triplicates. Ratio, PMHD+Fe/PMHD. All values >1, <l, >0.5, and ≤0.5 were rounded to the nearest whole number, to 1 and to one significant digit, respectively.

^cMode of action was evaluated in PMHD in duplicate The concentration at which each compound was tested for mode of action is indicated between parentheses. nd, not determined; US, bacteriostatic; BC, bactericidal.

Compounds 13 (IC_50PMHD = 4 μM, MIC_PMHD = 16 μM) and 14 (IC_50PMHD = 5 μM, MIC_PMHD = 21 μM) were the most potent among the active DAP derivatives with detectable activity against Yp (Table 4). These two compounds displayed no significant cytotoxicity when they were evaluated at their respective MIC values determined against Yp (Supplementary Data, Fig. S1). Testing of these and two other active DAP derivatives (1, 11) for mode of action against Yp revealed that the four compounds were bacteriostatic at concentrations of 1-2 × MIC_PMHD (Table 4). Among the BHHC derivatives with defined IC₅₀ and MIC values in at least one condition (iron limiting or iron rich), 32 stood out due to its remarkable potency (IC_50PMHD = 0.04 μM, MIC_PMHD = 0.2 μM) against Yp cultured under iron scarcity. Encouragingly, 32 displayed no significant cytotoxicity when evaluated in cytotoxicity assays at the MIC determined against Yp (Supplementary Data, Fig. S1). Notably, the activity of 32 was significantly higher than that of streptomycin (IC₅₀ and MIC ~1 μM), a bactericidal drug used to treat plague and included herein as an anti-Yp activity reference. Five compounds (27-29, 31, 32) of the BHHC derivatives series were tested for mode of action against Yp in PMHD at concentrations of 1-2 × MIC_PMHD. Under the conditions tested, 29 displayed bactericidal activity, whereas 27, 28, 31 and 32 were bacteriostatic.

In sum, 20 of 32 compounds synthesized and evaluated herein have detectable antimicrobial against Mtb and/or Yp in at least one condition, iron scarcity or iron sufficient. To our knowledge, these are novel scaffolds not previously shown to have antimicrobial properties. Most active compounds identified herein have comparable potency in the low and high iron conditions. This finding suggests that their pharmacological targets are likely to be essential bacterial functions required under both these conditions. In line with our aforementioned hypothesis, however, some of our compounds have higher potency under the iron-limiting condition. Under this condition, bacteria depend on siderophores for efficient iron scavenging and engage an adaptive response to tailor their physiology to iron scarcity, thus exposing novel potential in vivo conditional target candidates.^7a 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 27 and 32 against Yp). Overall, our study illustrates the possibility of screening compound libraries in both iron-sufficient and iron-limiting conditions to identify antimicrobials that may selectively target iron scarcity-adapted bacteria and highlights the usefulness of building combinatorial libraries of compounds having scaffolds with structural similarities to siderophores to feed antimicrobial screening programs.