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 Gylfe c,e,f Department of Chemistry, Umeå University, Umeå, Sweden a ; Laboratories for Chemical Biology Umeå, Chemical Biology Consortium Sweden, Umeå University, Umeå, Sweden b ; Department of Clinical Microbiology, Umeå University, Umeå, Sweden c ; Department of Molecular Biology, Umeå University, Umeå, Sweden d ; Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, Sweden e ; Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden f 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. C hlamydia trachomatis causes sexually transmitted disease that can lead to infertility (1) and increased susceptibility to other 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, 10 4 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 (20 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). 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 analogs of compound 2 were identified (data available upon re- 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 70% 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 (IC 50 ) in C. trachomatis and C. pneumoniae was determined for well-tolerated compounds with MIC  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 IC 50 s 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- 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. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.02015-13 May 2014 Volume 58 Number 5 Antimicrobial Agents and Chemotherapy p. 000 aac.asm.org 1 AQ: au AQ: aff T1/AQ:A AQ:B/T2 zac00514/zac2825d14z xppws S=1 2/27/14 21:49 ArtID: 02015-13 NLM: brief-report CE: jtc Editor: Section: Designation: Shlaes Susceptibility S