N-Acylated derivatives of sulfamethoxazole and sulfafurazole inhibit intracellular growth of chlamydia trachomatis

Article abstract of DOI:10.1128/AAC.02015-13

Antibacterial compounds with novel modes of action are needed for management of bacterial infections. Here we describe a high-content screen of 9,800 compounds identifying acylated sulfonamides as novel growth inhibitors of the sexually transmitted pathogen Chlamydia trachomatis. The effect was bactericidal and distinct from that of sulfonamide antibiotics, as para-Aminobenzoic acid did not reduce efficacy. Chemical inhibitors play an important role in Chlamydia research as probes of potential targets and as drug development starting points. Copyright

Full text of DOI:10.1128/AAC.02015-13

N-Acylated Derivatives of Sulfamethoxazole and Sulfafurazole Inhibit
Intracellular Growth of Chlamydia trachomatis
Sania Marwaha,a Hanna Uvell,b Olli Salin,a,c Anders E. G. Lindgren,a Jim Silver,d Mikael Elofsson,a,e,f Åsa Gylfec,e,f
Department of Chemistry, Umeå University, Umeå, Swedena; Laboratories for Chemical Biology Umeå, Chemical Biology Consortium Sweden, Umeå University, Umeå,
Swedenb; Department of Clinical Microbiology, Umeå University, Umeå, Swedenc; Department of Molecular Biology, Umeå University, Umeå, Swedend; Umeå Centre for
Microbial Research (UCMR), Umeå University, Umeå, Swedene; Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Swedenf
Antibacterial compounds with novel modes of action are needed for management of bacterial infections. Here we describe a
high-content screen of 9,800 compounds identifying acylated sulfonamides as novel growth inhibitors of the sexually transmit-
ted pathogen Chlamydia trachomatis. The effect was bactericidal and distinct from that of sulfonamide antibiotics, as para-ami-
nobenzoic acid did not reduce efficacy. Chemical inhibitors play an important role in Chlamydia research as probes of potential
targets and as drug development starting points.
hlamydia trachomatis causes sexually transmitted disease that analogs of compound 2 were identified (data available upon re-
C
can lead to infertility (1) and increased susceptibility to other
quest), while analogs of compound 1 had a range of MIC values
(Table 1). Basic structure-activity relationships for these acylated
sulfonamides demonstrated that the 5-methyl-3-isoxazolyl group
is preferred to the 3,4-dimethyl-5-isoxazolyl group (cf. com-
pounds 1 and 19, 3 and 21, and 17 and 18). Truncation as in the
case of compounds 7 and 10 or introduction of furane as in the
case of compounds 8, 9, 22, and 25 was not beneficial. Compound
18 with a benzothiophene-2-carboxamide group was the most
potent inhibitor (MIC, 6 ␮M). The antichlamydial effect was not
caused by general toxicity to the host cells. HeLa cell viability after
24 to 48 h at 50 ␮M was Ͼ70% for most compounds as deter-
mined using an XTT cell proliferation assay kit (ATCC) and un-
colored Dulbecco’s modified Eagle’s medium (DMEM) (Table 2).
A parahalogenated aryl ring in place of the isoxazole ring (com-
pounds 26 to 29) was associated with cytotoxicity.
The 50% inhibitory concentration (IC50) in C. trachomatis and
C. pneumoniae was determined for well-tolerated compounds
with MIC Ͻ 25 ␮M (Table 2). C. pneumoniae T45 (17) was grown
in HEp-2 cells (CCL-23; ATCC) for 70 h in the presence of 0.5
␮g/ml cycloheximide (14). Inclusion counts were logarithmized
and normalized. Nonlinear regression was used, and the IC50s
were derived from fitted curves (18). Dose-dependent activity was
demonstrated for C. trachomatis (see Fig. S2 and S3 in the supple-
mental material) and less prominent with C. pneumoniae, indicat-
ing lower specificity in C. pneumoniae and differences in the mo-
lecular targets. Compounds listed in Table 2 were bacterial, as
formation of infectious progeny was completely inhibited. No in-
clusions were detected by immunostaining after passage of C. tra-
sexually transmitted pathogens such as HIV (2). Chlamydophila
pneumoniae is a respiratory pathogen that can cause pneumonia
(1). These common infections are treated with broad-spectrum
antibiotics such as doxycycline and azithromycin which can select
for resistant strains (3). Specific treatments would affect the nor-
mal bacterial flora less and reduce the use of these important an-
tibiotics. High-content screening (HCS) uses cell-based assays,
automated microscopy, and image analysis to collect complex in-
formation (4) and is well suited to screen for compounds for use
against the obligate intracellular bacterium Chlamydia (5). Inhib-
itors of Chlamydia have been used to investigate the Chlamydia
life cycle (6–11) and have been suggested for prevention of trans-
mission (12–14).
HCS of 9,800 compounds gave 12 hits that inhibited C. tra-
chomatis growth without visual changes in morphology of HeLa
cell nuclei. The compound collection (Chembridge Corporation,
San Diego, CA) was selected based on chemical diversity and drug
similarities. In 96-well plates, 104 HeLa 229 cells (CCL-2.1;
ATCC) were infected with 3,000 CFU C. trachomatis serovar L2
(VR-902B; ATCC) in 30 ␮l Hanks balanced salt solution (HBSS)
(14). After 1 h, HBSS was replaced with 100 ␮l RPMI cell culture
medium with 50 ␮M test compounds or 1% dimethyl sulfoxide
(DMSO) and incubated for 18 h. DAPI (4=,6-diamidino-2-phenylin-
dole) was used for staining together with fluorescein isothiocyanate
(FITC) (Molecular Probes, Eugene, OR)-conjugated purified serum
IgG (Melon Gel IgG Spin Purification kit; Thermo Scientific) from
rabbit (Agrisera AB, Vännäs, Sweden) immunized with formalin-
fixed C. trachomatis L2 elementary bodies (15). Photomicrographs
were generated (20ϫ objective), and the number and area of Chla-
mydia inclusions were determined (spot detection method) using
an ArrayScan VTi HCA Reader (Thermo Fisher Scientific, Pitts-
burgh, PA). Artifacts and cellular toxicity were judged visually. Six
hits were excluded due to the lack of a dose response or visible
toxicity to the host cells. Two potent hits, compounds 1 and 2,
were selected for further investigation (Table 1).
Received 16 September 2013 Returned for modification 16 October 2013
Accepted 17 February 2014
Published ahead of print 24 February 2014
Address correspondence to Mikael Elofsson, mikael.elofsson@chem.umu.se, or
Åsa Gylfe, asa.gylfe@climi.umu.se.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.02015-13.
Statistical molecular design (16), cherry-picking, and chemical
synthesis (see the supplemental material) were used to select 28
and 44 analogs of compounds 1 and 2, respectively. MICs for C.
trachomatis were determined for all compounds (14). No potent
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.02015-13
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Novel Chlamydia Growth Inhibitor
TABLE 1 Structures and MICs against C. trachomatis for hit 1 and hit 2
TABLE 1 (Continued)
and for the analogs of hit 1, compounds 3 to 30
MIC
MIC
(␮M)
Compound
13
Structure
(␮M)
Compound
1
Structure
Ͼ50
Ͼ50
25
14
15
2
3
50
50
50
25
50
4
12
16
17
5
6
50
Ͼ50
18
19
6
7
8
Ͼ50
Ͼ50
12
20
21
22
23
24
12
9
Ͼ50
Ͼ50
25
10
Ͼ50
12
11
12
25
25
12
(Continued on following page)
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Marwaha et al.
TABLE 1 (Continued)
MIC
Compound
25
Structure
(␮M)
Ͼ50
12
26
27
28
29
25
25
25
30
25
chomatis grown 44 h with 25 ␮M compound and harvested by
freeze-thawing in sterile water.
All compounds follow Lipinski’s rules of five (19) with minor
exceptions for compounds 22 and 25. Even though the aqueous sol-
ubility was low, good oral absorption was predicted in silico (20)
mainly due to favorable cellular permeability (see Table S2 in the
supplemental material). The predicted numbers of metabolites (0 to
5) are comparable to those of average drugs (21). Acylated sulfon-
amides share the structural core with the orally bioavailable sulfon-
amide antibiotics sulfafurazole and sulfamethoxazole. The acylated
sulfonamides are, however, more lipophilic and might concentrate
more readily in chlamydial inclusions.
Sulfonamide antibiotics inhibit folate synthesis by competing
with the substrate para-aminobenzoic acid (PABA) (22). Chla-
mydiae are capable of synthesizing folate, and levels of suscepti-
bility to sulfonamide antibiotics have been reported to differ
among species and strains (23–25). Compounds 1, 18, and 23 had
identical MICs in DMEM with or without 10 ␮M PABA (50, 12.5,
and 25 ␮M, respectively), while sulfamethoxazole inhibited C.
trachomatis growth only in PABA-free DMEM (25% inhibition at
100 ␮M). Our compounds did not inhibit growth of Escherichia
coli and Straphylococcus aureus (in-house strains), except com-
pound 14, which inhibited E. coli 80% at 100 ␮M. Overnight
growth curves were recorded in Mueller-Hinton broth with 100 to
1.56 ␮M test compounds or 1% DMSO (600 nm absorbance)
(Tecan Safire microplate reader; Tecan, Männerdorf, Switzer-
land), and sulfamethoxazole IC50s were 12 and 6 ␮M for E. coli
and S. aureus, respectively. These data suggest that acylated sul-
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Novel Chlamydia Growth Inhibitor
9. Sandoz KM, Eriksen SG, Jeffrey BM, Suchland RJ, Putman TE, Hruby
DE, Jordan R, Rockey DD. 2012. Resistance to a novel antichlamydial
compound is mediated through mutations in Chlamydia trachomatis
secY. Antimicrob. Agents Chemother. 56:4296–4302. http://dx.doi.org
/10.1128/AAC.00356-12.
fonamides do not affect folate synthesis. The different mecha-
nisms of action are likely due to the fact that acylated derivatives
lack the free amine of sulfonamide antibiotics that fits the active
site in dihydropteroate synthetase (22).
Acylated and benzylidene sulfonamides with structural simi-
larity to compound 10 in this study have recently been reported to
inhibit mycobacteria and staphylococci (26). However, 10 com-
pletely lacked antichlamydial and antistaphylococcal activities.
Except for compound 14, our compounds did not inhibit growth
of representative extracellular bacteria, and further investigation
is needed to determine if the antimicrobial spectrum is narrow.
The compounds presented here are promising starting points
for development of novel antichlamydial drugs. Specific treat-
ments for these common infections would reduce the risks of dis-
turbing the normal flora or spreading antibiotic resistance. The
mode of action is unknown but may be identified by selection for
resistant mutants and subsequent whole-genome sequencing (9,
27). Novel antichlamydial compounds may also be of importance
as probes to validate potential drug targets and may thereby reveal
new insights into Chlamydia biology.
10. Nguyen BD, Cunningham D, Liang X, Chen X, Toone EJ, Raetz CR,
Zhou P, Valdivia RH. 2011. Lipooligosaccharide is required for the gen-
eration of infectious elementary bodies in Chlamydia trachomatis. Proc.
Natl. Acad. Sci. U. S. A. 108:10284–10289. http://dx.doi.org/10.1073/pnas
.1107478108.
11. Lei L, Li Z, Zhong G. 2012. Rottlerin-mediated inhibition of Chlamydia
trachomatis growth and uptake of sphingolipids is independent of p38-
regulated/activated protein kinase (PRAK). PLoS One 7:e44733. http://dx
.doi.org/10.1371/journal.pone.0044733.
12. Slepenkin A, Chu H, Elofsson M, Keyser P, Peterson EM. 2011. Pro-
tection of mice from a Chlamydia trachomatis vaginal infection using a
salicylidene acylhydrazide, a potential microbicide. J. Infect. Dis. 204:
1313–1320. http://dx.doi.org/10.1093/infdis/jir552.
13. Chu H, Slepenkin A, Elofsson M, Keyser P, de la Maza LM, Peterson
EM. 2010. Candidate vaginal microbicides with activity against Chla-
mydia trachomatis and Neisseria gonorrhoeae. Int. J. Antimicrob. Agents
36:145–150. http://dx.doi.org/10.1016/j.ijantimicag.2010.03.018.
14. Ur-Rehman T, Slepenkin A, Chu H, Blomgren A, Dahlgren MK, Zetter-
ström CE, Peterson EM, Elofsson M, Gylfe Å. 2012. Pre-clinical phar-
macokinetics and anti-chlamydial activity of salicylidene acylhydrazide
inhibitors of bacterial type III secretion. J. Antibiot. 65:397–404. http://dx
.doi.org/10.1038/ja.2012.43.
ACKNOWLEDGMENTS
David Andersson is acknowledged for computational assistance.
This work was supported by Laboratories for Chemical Biology Umeå,
Chemical Biology Consortium Sweden, the Swedish Government Fund
for Clinical Research (ALF), the Scandinavian Society for Antimicrobial
Chemotherapy foundation (for Å.G.), and the Swedish Research Council,
the Swedish Governmental Agency for Innovation Systems (VINNOVA),
the Knut & Alice Wallenberg foundation, and the Carl Trygger founda-
tion (for M.E.).
15. Caldwell HD, Kromhout J, Schachter J. 1981. Purification and partial
characterization of the major outer membrane protein of Chlamydia tra-
chomatis. Infect. Immun. 31:1161–1176.
16. Linusson A, Elofsson M, Andersson IE, Dahlgren MK. 2010. Statis-
tical molecular design of balanced compound libraries for QSAR mod-
eling. Curr. Med. Chem. 17:2001–2016. http://dx.doi.org/10.2174
/092986710791233661.
17. Kuoppa Y, Boman J, Scott L, Kumlin U, Eriksson I, Allard A. 2002.
Quantitative detection of respiratory Chlamydia pneumoniae infection by
real-time PCR. J. Clin. Microbiol. 40:2273–2274. http://dx.doi.org/10
.1128/JCM.40.6.2273-2274.2002.
REFERENCES
1. Batteiger BE. 2012. Chlamydia infection and epidemiology, p 1–26. In 18. GraphPad Software Inc. 2012. GraphPad Prism, 6.0b ed. GraphPad Soft-
Tan M, Bavoil P (ed), Intracellular pathogens I: Chlamydiales. ASM Press,
Washington, DC.
2. Johnson LF, Lewis DA. 2008. The effect of genital tract infections on
HIV-1 shedding in the genital tract: a systematic review and meta-
analysis. Sex. Transm. Dis. 35:946–959. http://dx.doi.org/10.1097
/OLQ.0b013e3181812d15.
ware Inc., La Jolla, CA.
19. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. 2001. Experimental
and computational approaches to estimate solubility and permeability in
drug discovery and development settings. Adv. Drug Deliv. Rev. 46:3–26.
http://dx.doi.org/10.1016/S0169-409X(00)00129-0.
20. Schrödinger Inc. 2011. Qikprop, 3.3 ed. Schrödinger Inc., New York, NY.
3. Bhengraj AR, Srivastava P, Mittal A. 2011. Lack of mutation in macro- 21. Wenlock MC, Barton P. 2013. In silico physicochemical parameter predictions.
lide resistance genes in Chlamydia trachomatis clinical isolates with de- Mol. Pharm. 10:1224–1235. http://dx.doi.org/10.1021/mp300537k.
creased susceptibility to azithromycin. Int. J. Antimicrob. Agents 38:178– 22. Yun MK, Wu Y, Li Z, Zhao Y, Waddell MB, Ferreira AM, Lee RE,
179. http://dx.doi.org/10.1016/j.ijantimicag.2011.03.015.
Bashford D, White SW. 2012. Catalysis and sulfa drug resistance in
dihydropteroate synthase. Science 335:1110–1114. http://dx.doi.org/10
.1126/science.1214641.
4. Abraham VC, Taylor DL, Haskins JR. 2004. High content screening
applied to large-scale cell biology. Trends Biotechnol. 22:15–22. http://dx
.doi.org/10.1016/j.tibtech.2003.10.012.
5. Osaka I, Hills JM, Kieweg SL, Shinogle HE, Moore DS, Hefty PS. 2012.
An automated image-based method for rapid analysis of Chlamydia in-
fection as a tool for screening antichlamydial agents. Antimicrob. Agents
Chemother. 56:4184–4188. http://dx.doi.org/10.1128/AAC.00427-12.
6. Wolf K, Betts HJ, Chellas-Gery B, Hower S, Linton CN, Fields KA.
2006. Treatment of Chlamydia trachomatis with a small molecule inhibi-
23. Kuo CC, Wang SP, Grayston T. 1977. Antimicrobial activity of several
antibiotics and a sulfonamide against Chlamydia trachomatis organisms
in cell culture. Antimicrob. Agents Chemother. 12:80–83. http://dx.doi
.org/10.1128/AAC.12.1.80.
24. Hammerschlag MR. 1982. Activity of trimethoprim-sulfamethoxazole
against Chlamydia trachomatis in vitro. Rev. Infect. Dis. 4:500–505. http:
//dx.doi.org/10.1093/clinids/4.2.500.
tor of the Yersinia type III secretion system disrupts progression of the 25. Hammerschlag MR. 1994. Antimicrobial susceptibility and therapy of
chlamydial developmental cycle. Mol. Microbiol. 61:1543–1555. http://dx
infections caused by Chlamydia pneumoniae. Antimicrob. Agents Che-
.doi.org/10.1111/j.1365-2958.2006.05347.x.
mother. 38:1873–1878. http://dx.doi.org/10.1128/AAC.38.9.1873.
7. Bailey L, Gylfe Å, Sundin C, Muschiol S, Elofsson M, Nordström P, 26. Krátký M, Vinšová J, Volková M, Buchta V, Trejtnar F, Stolarˇíková
Henriques-Normark B, Lugert R, Waldenström A, Wolf-Watz H, Berg-
ström S. 2007. Small molecule inhibitors of type III secretion in Yersinia
block the Chlamydia pneumoniae infection cycle. FEBS Lett. 581:587–595.
http://dx.doi.org/10.1016/j.febslet.2007.01.013.
J. 2012. Antimicrobial activity of sulfonamides containing 5-chloro-2-
hydroxybenzaldehyde and 5-chloro-2-hydroxybenzoic acid scaffold.
Eur. J. Med. Chem. 50:433–440. http://dx.doi.org/10.1016/j.ejmech
.2012.01.060.
8. Muschiol S, Bailey L, Gylfe Å, Sundin C, Hultenby K, Bergström S, 27. Engström P, Nguyen BD, Normark J, Nilsson I, Bastidas RJ, Gylfe Å,
Elofsson M, Wolf-Watz H, Normark S, Henriques-Normark B. 2006.
A small-molecule inhibitor of type III secretion inhibits different
stages of the infectious cycle of Chlamydia trachomatis. Proc. Natl.
Acad. Sci. U. S. A. 103:14566–14571. http://dx.doi.org/10.1073/pnas
.0606412103.
Elofsson M, Fields KA, Valdivia RH, Wolf-Watz H, Bergström S. 2013.
Mutations in hemG mediate resistance to salicylidene acylhydrazides,
demonstrating a novel link between protoporphyrinogen oxidase
(HemG) and Chlamydia trachomatis infectivity. J. Bacteriol. 195:4221–
4230. http://dx.doi.org/10.1128/JB.00506-13.
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