More than 100 years after being first described, Chagas disease remains endemic in 21 Latin American countries and has spread to other continents. Indeed, this disease, which is caused by the protozoan parasite Trypanosoma cruzi, is no longer just a problem for the American continents but has become a global health threat.
KEYWORDS: Chagas disease, drug discovery, imidazole
ABSTRACT
More than 100 years after being first described, Chagas disease remains endemic in 21 Latin American countries and has spread to other continents. Indeed, this disease, which is caused by the protozoan parasite Trypanosoma cruzi, is no longer just a problem for the American continents but has become a global health threat. Current therapies, i.e., nifurtimox and benznidazole (Bz), are far from being adequate, due to their undesirable effects and their lack of efficacy in the chronic phases of the disease. In this work, we present an in-depth phenotypic evaluation in T. cruzi of a new class of imidazole compounds, which were discovered in a previous phenotypic screen against different trypanosomatids and were designed as potential inhibitors of cAMP phosphodiesterases (PDEs). The confirmation of several activities similar or superior to that of Bz prompted a synthesis program of hit optimization and extended structure-activity relationship aimed at improving drug-like properties such as aqueous solubility, which resulted in additional hits with 50% inhibitory concentration (IC50) values similar to that of Bz. The cellular effects of one representative hit, compound 9, on bloodstream trypomastigotes were further investigated. Transmission electron microscopy revealed cellular changes, after just 2 h of incubation with the IC50 concentration, that were consistent with induced autophagy and osmotic stress, mechanisms previously linked to cAMP signaling. Compound 9 induced highly significant increases in both cellular and medium cAMP levels, confirming that inhibition of T. cruzi PDE(s) is part of its mechanism of action. The potent and selective activity of this imidazole-based PDE inhibitor class against T. cruzi constitutes a successful repurposing of research into inhibitors of mammalian PDEs.
INTRODUCTION
American trypanosomiasis, also known as Chagas disease (CD), was described in 1909 by the Brazilian researcher Carlos Chagas (1). More than 100 years later, this neglected tropical disease, caused by the protozoan parasite Trypanosoma cruzi, remains endemic in 21 countries in Latin America, according to the WHO (2). Moreover, although the disease was confined to the region of the Americas, over the past century it has spread to other continents, mainly due to human migration (3).
The disease has an acute phase that occurs immediately after infection and is usually asymptomatic or oligosymptomatic. The extent of parasite proliferation is controlled by a competent host immune response, although the patient remains chronically infected if untreated. Many patients could remain asymptomatic, but 30% to 40% develop a severe stage that is characterized mainly by cardiac and/or gastrointestinal pathologies (4). Chemotherapy is highly recommended for all asymptomatic patients with acute or chronic infection, but the current drugs, nifurtimox and benznidazole (Bz), are far from adequate due to side effects and their poor efficacy in all clinical phases (especially inactive against the chronic phase), besides the occurrence of naturally resistant parasite strains belonging to some different discrete typing units (DTUs) (5).
For these reasons, the search for effective, well-tolerated, and affordable CD drugs is both urgent and of genuine importance. Despite significant advances in the discovery and development of new effective drugs for this tropical disease, this remains an unmet clinical need (6, 7), and for many patients there is no effective treatment. This paper describes the therapeutic potential of a class of imidazole compounds discovered to have antiprotozoan activity in a previous screen of different human phosphodiesterase (PDE) inhibitors from our in-house chemical library against different trypanosomatids (8). Compounds from this chemical class showed potential utility in Parkinson’s disease due to the ability to inhibit mammalian cyclic nucleotide PDE10A (9). Results from the previous phenotypic screen of a focused library of 69 imidazole derivatives against a panel of three pathogenic trypanosomatids, Trypanosoma brucei, T. cruzi, and Leishmania infantum, highlighted these imidazoles as promising hits for the development of anti-Leishmania therapies (8). Interestingly, 12 members of this library (compounds 1 to 12) also showed promising activities against the intracellular forms of T. cruzi (Tulahuen β-galactosidase-transfected parasites; DTU VI), some with potency quite similar to that of Bz (50% inhibitory concentration [IC50] = 3.18 μM) (see Table S1 in the supplemental material), justifying further analysis of their potential against T. cruzi, including chemical optimization. The work presented here includes an evaluation of the mechanism of action of this compound class and initial efforts toward hit optimization and the development of a structure-activity relationship (SAR).
RESULTS
Optimization of imidazole hit compounds.
The first priority of the present work was to improve the drug-like properties of the imidazoles with the most interesting anti-T. cruzi activities. In order to improve aqueous solubility, we decided to increase the polarity, designing a new series of imidazole-related compounds with a reduced number of aromatic rings as substituents on the heterocyclic core and introducing a polar group such as the urea moiety.
The first synthetic approach to obtain these newly designed compounds used different aminothiazole and aminoimidazole derivatives as the starting material, together with different isocyanates, to easily synthesize the corresponding urea in one step by microwave irradiation in tetrahydrofuran (THF), with low to moderate yields (Fig. 1). The second approach was focused on a 4-phenyl-2-amino imidazole scaffold and employed a two-step synthesis based on the use of 1,1ʹ-carbonyldiimidazole (CDI) as a reagent to convert the amino group into urea. After the addition of CDI to the aminoimidazole, the amine was activated and a carbonyl group was added. The addition of the corresponding substituted amine then allowed the formation of the final urea derivative, with moderate yields (Fig. 2).
FIG 1.
Synthesis of substituted N-(thiazole-2-yl)urea derivatives (compounds 13 to 17) and N-(1H-imidazol-2-yl)urea derivatives (compounds 18 to 23) using isocyanates. MW, microwave.
FIG 2.
Synthesis of substituted N-(1H-imidazol-2-yl)urea derivatives (compounds 24 to 38) using CDI. rt, room temperature.
Thus, 26 new imidazole-related compounds bearing a urea motif were synthesized and evaluated against the standard panel (9) of T. brucei, T. cruzi, and L. infantum, and their cytotoxicity was evaluated on human lung fibroblasts (MRC-5) and primary cultures of peritoneal mouse macrophages (PMMs) (Table 1). Among the newly synthesized compounds, thiazole derivatives (compounds 13 to 17) were not active at all, while the most promising compounds against intracellular β-galactosidase-transfected T. cruzi (Tulahuen strain; DTU VI) were the ureaimidazoles (compounds 23, 31, 33, 37, and 38). Remarkably, fluoride substituents appeared likely to be favorable for activity against T. cruzi (trifluoromethyl at position 4 of the R2 substituent in compounds 33, 37, and 38 and trifluoromethoxy at position 4 of one of the phenyl substituents in compound 23). Moreover, the activity of most of these compounds was specific to T. cruzi, although some activity was observed against the other trypanosomatids, particularly the closely related species T. brucei; crucially, the compounds displayed little or no toxicity to the mammalian cell lines. The finding that the introduction of the polar urea group was well tolerated and in some cases actually enhanced the selective anti-T. cruzi activity was very encouraging for further exploration in this chemical space.
TABLE 1.
In vitro antiparasitic activities of new imidazole derivatives (compounds 13 to 38)
| Compound | IC50 (μM)a |
||||
|---|---|---|---|---|---|
| MRC-5 | T. cruzi | L. infantum | T. brucei | PMMs | |
| 13 | >64.0 | 50.5 | 53.5 | >64.0 | >64.0 |
| 14 | >64.0 | >64.0 | 57.4 | >64.0 | >64.0 |
| 15 | >64.0 | >64.0 | 49.7 | >64.0 | >64.0 |
| 16 | >64.0 | >64.0 | 48.2 | >64.0 | 48.0 |
| 17 | >64.0 | >64.0 | 36.0 | >64.0 | 36.0 |
| 18 | 19.5 | 24.5 | 32.3 | 32.3 | 48.0 |
| 19 | >64.0 | >64.0 | 37.8 | >64.0 | 48.0 |
| 20 | 20.9 | 26.0 | 19.9 | 8.2 | 48.0 |
| 21 | 12.8 | 26.5 | 26.5 | >64.0 | >64.0 |
| 22 | >64.0 | >64.0 | 32.5 | >64.0 | 32.0 |
| 23 | >64.0 | 7.1 | 11.7 | 6.8 | >64.0 |
| 24 | >64.0 | >64.0 | >64.0 | >64.0 | >64.0 |
| 25 | >64.0 | >64.0 | >64.0 | >64.0 | >64.0 |
| 26 | >64.0 | >64.0 | 53.5 | >64.0 | >64.0 |
| 27 | >64.0 | >64.0 | 53.5 | >64.0 | >64.0 |
| 28 | >64.0 | >64.0 | 19.0 | >64.0 | 32.0 |
| 29 | >64.0 | 37.0 | 15.7 | 1.8 | >64.0 |
| 30 | >64.0 | >64.0 | 35.1 | >64.0 | 45.2 |
| 31 | 16.9 | 3.0 | 30.0 | 2.1 | 33.4 |
| 32 | >64.0 | 13.5 | 32.5 | >64.0 | 32.0 |
| 33 | >64.0 | 2.2 | 7.1 | 6.0 | 8.0 |
| 34 | >64.0 | 54.6 | >64.0 | >64.0 | >64.0 |
| 35 | >64.0 | 49.5 | >64.0 | >64.0 | >64.0 |
| 36 | >64.0 | >64.0 | 49.4 | >64.0 | 54.6 |
| 37 | >64.0 | 1.7 | 29.3 | 7.1 | 34.0 |
| 38 | >64.0 | 8.1 | 39.0 | 19.3 | 45.2 |
IC50 values for inhibition of the growth of T. cruzi, L. infantum, and T. brucei or for cytotoxicity toward human lung fibroblasts (MRC-5 cells) and primary cell cultures of PMMs are indicated. Each value is the mean of two independent determinations.
Activity against bloodstream trypomastigotes.
In order to verify the therapeutic potential of this new compound class, we phenotypically assayed the original imidazole hits (compounds 1 to 12) (see Table S1 in the supplemental material) and the most promising of the new 4-phenyl 2-ureaimidazoles (compounds 23, 31, 33, 37, and 38, i.e., all of the derivatives with IC50 values below 10 μM) against the other parasite form relevant for mammalian infections (bloodstream trypomastigotes [BTs]). To address the possibility that the compounds might not be effective against other T. cruzi DTUs, we also assayed another strain from a different DTU (Y strain; DTU II) in this assay. We found that 5 of the 17 compounds tested showed greater activity than Bz (IC50 = 12.9 ± 1.9 μM) against Y strain BTs, with IC50 values ranging from 1.2 to 11.5 μM (Table 2). Compound 9 stood out as the most active against this life cycle stage (IC50 = 1.2 ± 0.3 μM). Because heart muscle is an important target for T. cruzi infection and inflammation, we also investigated the potential toxicity of these compounds toward primary cultures of mouse cardiac cells (CCs). As shown in Table 2, most compounds were not cardiotoxic, exhibiting 50% lethal concentration (LC50) values ranging from 65 to >200 μM. Selectivity index (SI) values were calculated, showing compound 9 to be the most selective (SI of >85), followed by compound 33 (IC50 = 4.6 ± 0.1 μM, with SI of >44).
TABLE 2.
Activity of selected imidazole derivatives against Y strain BTs, toxicity profiles against CC cultures, and respective SI values after 24 h of incubation
| Compound | IC50 for Y strain BTs (μM)a | LC50 for CC cultures (μM) | SI |
|---|---|---|---|
| 1 | >50 | >200 | NDb |
| 2 | 11.5 ± 0.2 | >200 | >17 |
| 3 | >50 | >200 | ND |
| 4 | 29.7 ± 7.3 | >200 | >7 |
| 5 | 27.4 ± 4.7 | >200 | >7 |
| 6 | 16.8 ± 2.7 | >200 | >12 |
| 7 | >50 | >200 | ND |
| 8 | 11.1 ± 0.2 | >200 | >18 |
| 9 | 1.2 ± 0.3 | >100 | >85 |
| 10 | >50 | >200 | ND |
| 11 | 35.7 ± 2.2 | >200 | >6 |
| 12 | 22.1 ± 0 | >100 | >4 |
| 23 | 13.9 ± 1.8 | 65.6 | 5 |
| 31 | 4.1 ± 1.8 | 68.6 | 17 |
| 33 | 4.6 ± 0.1 | >200 | >44 |
| 37 | >50 | >200 | ND |
| 38 | 15.7 ± 5.8 | >200 | >13 |
| Bz | 12.9 ± 1.9 | >1,000 | >77 |
IC50 results are presented as means ± standard deviations.
ND, not determined.
Cellular effects of compound 9.
Imidazole 9 was chosen for further analysis of the cellular effects of the imidazole compound class. BTs of Y strain were incubated for 2 h with 1 times the 50% effective concentration (EC50) of compound 9, and the effects on their ultrastructure were analyzed using transmission electron microscopy (Fig. 3). Treated parasites exhibited severe features, including flagellar pocket dilation, disruption of the Golgi apparatus, and extensive blebs and shedding events in the plasma membrane, in addition to a large number of myelin figures and membranous profiles surrounding cytoplasmic organelles, an apparent sign of autophagy.
FIG 3.
Transmission electron microscopy of Y strain BTs that were untreated (A) or treated with compound 9 for 2 h (B to K). The treated parasites exhibited important features that included flagellar pocket dilation (B, hash mark), disruption of the Golgi apparatus (C, asterisk), and extensive blebs (E and G, angle brackets) and shedding events (J, double asterisks) in the plasma membrane, as well as a large number of myelin figures (K) and membranous profiles surrounding cytoplasmic organelles (D and H) and complete disorganization of cytoplasmic structures (I).
Because related imidazoles were found to be able to increase cAMP levels in Leishmania promastigote cultures (8), here we tested whether compound 9 was similarly able to increase the levels of this cyclic nucleotide in T. cruzi. With this aim, Y strain BTs were incubated with either 2×EC50 or 5×EC50 of this compound for 2.5 h, and both the cellular cAMP content and the released cAMP in the medium were determined. Cultures incubated in parallel without a test compound and with known T. brucei PDE inhibitors (NPD-001 [9] and NPD-008 [10]) served as negative and positive controls, respectively.
Our data demonstrated that incubation with compound 9 dose-dependently increased the intracellular content of cAMP (1.8 times the untreated control [P < 0.05] and 2.5 times the untreated control [P < 0.01] at 2×EC50 and 5×EC50, respectively) (Fig. 4A). cAMP was also released from the cells and could be measured in the medium; NPD-001, NPD-008, and compound 9 all induced highly significant increases in the extracellular cAMP concentration, relative to the untreated control (P < 0.001) (Fig. 4B).
FIG 4.
Intracellular (A) and extracellular (B) cAMP levels after incubation of BTs with compound 9 or positive controls (NPD-001 and NPD-008). All bars represent the mean and standard error of the mean of three independent experiments, each conducted in duplicate. P values were calculated using Student t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
DISCUSSION
Today, CD no longer is a problem only for Latin America but has become a global public health threat. Current therapeutic options rely on only two related (nitrofuran and nitroimidazole) drugs, nifurtimox and Bz, which, although they are active in the acute phase of the disease, suffer from a significant decrease of efficacy in the later chronic stage. Moreover, it has been found that some clinical isolates have acquired resistance to the nitro-heterocyclic compounds and other strains are innately resistant to these drugs (11). This unsatisfactory situation has led to sustained efforts, including clinical trials with posaconazole, E1224, and fexinidazole (12–14), although today no new drugs for CD are on the horizon (5).
Our aim in this work was to study previously described antikinetoplastid hits with an imidazole ring against T. cruzi, including improvements in their pharmaceutical profiles through increased polarity. Of an initial set of 69 imidazole compounds, 12 derivatives showed promising activities against intracellular T. cruzi amastigotes, with acceptable selectivity, and thus deserve future studies (see Table S1 in the supplemental material) (8). However, these imidazoles, all with multiple phenyl substitutions, suffer from suboptimal solubility. With the aim of decreasing their lipophilicity, we developed a medicinal chemistry strategy that, while retaining the core five-membered ring (imidazole or thiazole) with different substituents, introduced a polar 2-urea bridge to the other part of the molecule, which contains either aromatic or aliphatic tails with different substituents. A series of 26 new imidazoles, closely related to the original hits but containing fewer aromatic rings and containing a urea moiety, were synthesized in one-step (Fig. 1) or two-step (Fig. 2) procedures.
The evaluation of the 26 new compounds in a primary in vitro screen against a standardized panel of T. brucei, T. cruzi, L. infantum, and mammalian cell lines showed that 5 compounds (compounds 23, 31, 33, 37, and 38) displayed promising effects against intracellular T. cruzi forms (Table 1). These findings motivated advanced studies with T. cruzi with the most potent derivatives (IC50 values of <10 μM). In a first step, the activity against another parasite form relevant for human infection, the BTs, was evaluated. For this assay, another parasite DTU (Y strain, belonging to DTU II) was used as well. Of the 17 compounds evaluated (Table 2), 5 compounds (compounds 2, 8, 9, 31, and 33) showed better activity than Bz against the bloodstream forms in vitro, and we selected imidazole 9 for preliminary cellular studies. Electron microscopy data demonstrated that the major early ultrastructural insults included flagellar pocket dilatation, disruption of the Golgi apparatus, and extensive blebs and shedding events in the plasma membrane. These events might be linked to defects in osmoregulation, which is thought to be one of the functions of cAMP signaling in T. cruzi (15). In addition, large numbers of myelin figures were observed, as well as membranous enclosures surrounding cytoplasmic organelles, which are the morphological characteristics of autophagy, as reported for other trypanocidal compounds (16). Autophagy is a process involved in life cycle progression and differentiation, and cAMP signaling in Leishmania has been directly linked to autophagy and differentiation (17). Similarly, cAMP signaling has been implicated in life cycle progression and differentiation in T. cruzi (18–20). Thus, our ultrastructural observations are consistent with the phenyl-substituted imidazoles acting through the inhibition of cAMP PDEs, as shown previously in Leishmania (8).
Validation measurements of the intracellular and extracellular cAMP contents after incubation of BTs with compound 9 were therefore conducted. These experiments were conducted with trypomastigotes, because it is impossible to separate the cAMP of the much larger host cells from the cAMP inside amastigotes. Lacking an established positive control for the stimulation of cAMP in T. cruzi, we utilized two well-characterized inhibitors of T. brucei PDE-B1/B2, namely, NPD-001 and NPD-008 (9, 10), relying on the very high level of structural conservation in PDEs among kinetoplastids (21, 22). As expected, both compounds induced clear and highly significant increases in the cellular cAMP concentrations at concentrations just 2 times or 5 times their EC50 values. Imidazole 9 similarly induced cAMP increases, i.e., 246% of the untreated control at 5×EC50 after just 2.5 h of incubation and despite a highly significant increase in cAMP efflux. Interestingly, all three PDE inhibitors also stimulated the efflux of cAMP from the trypomastigotes, significantly increasing the cAMP concentration in the medium over that of the control (P < 0.001). The phenomenon of cAMP efflux has been described for mammalian cells, where it is mediated by ABC class transporters (23), but to the best of our knowledge it has not been reported for protozoan cells, although we previously observed it in T. brucei and Leishmania species (J. C. Munday, T. Kalejaiye, and H. de Koning, unpublished results). The increased efflux from the trypomastigotes clearly shows that this mechanism in part compensates for the inhibition of PDE activities in the cells; however, as the cellular levels still increased significantly, we propose that the efflux mechanism was saturated upon treatment with the PDE inhibitors, leading to toxic cAMP levels and cell death, as demonstrated previously for T. brucei (9, 10). Our findings demonstrate promising in vitro activity of a series of phenyl-substituted imidazole derivatives, with several being very active against the different parasite forms and strains, especially compound 9, which merits further studies in order to contribute to the identification of novel therapies for CD.
MATERIALS AND METHODS
Compounds studied.
Imidazoles 1 to 12 (see Table S1 in the supplemental material) were prepared following previously described procedures (24) and had purities of ≥95% by high-performance liquid chromatography (HPLC). Detailed synthetic procedures and full characterization of compounds 13 to 38 (Fig. 1 and 2) are given in the supplemental material.
In vitro parasite growth inhibition assays.
An integrated screening was used to define the activity profiles of the test compounds from Table 2 using standard assay protocols, as described previously (25). A brief description of each model is given. For Leishmania infantum, amastigotes harvested from the spleens of infected donor hamsters were used for infection. PMMs were obtained after intraperitoneal stimulation with 2% starch in water for 24 to 48 h and were plated in 96-well microplates at 104 cells/well. After the addition of 105 amastigotes per well and 5 days of incubation, parasite burdens were microscopically assessed after Giemsa staining. For Trypanosoma brucei brucei, bloodstream forms of a drug-sensitive T. brucei brucei strain were axenically grown in Hirumi-9 medium at 37°C under an atmosphere of 5% CO2. Assays were performed in 96-well tissue culture plates, with each well containing 104 parasites. After 4 days of incubation, parasite growth was assessed by adding resazurin (product no. R7017; Sigma) and recording fluorimetric readings after 4 h at 37°C. For Trypanosoma cruzi, the nifurtimox-sensitive Tulahuen strain (LacZ transfected) of T. cruzi was maintained on MRC-5 cells. Assays were performed in 96-well tissue culture plates, with each well containing the compound dilutions together with 3 × 103 MRC-5 cells and 3 × 104 trypomastigotes. After 7 days of incubation, colorimetric readings were performed after the addition of chlorophenol red β-d-galactopyranoside (CPRG) (product no. 10884308001; Sigma) as substrate. For cytotoxicity assessments, MRC-5 cells were cultured in minimal essential medium supplemented with 20 mM l-glutamine, 16.5 mM NaHCO3, and 5% fetal calf serum. Assays were performed at 37°C in 5% CO2 in 96-well tissue culture plates with confluent monolayers. After 7 days of incubation, cell proliferation and viability were assessed after the addition of resazurin, with fluorescence readings.
Stock solutions of selected compounds.
Stock solutions (20 mM) of selected compounds from Table S1 and Table 1 were prepared in pure dimethyl sulfoxide (DMSO) (maximum final concentration of 1% in assays). Bz [N-benzyl-2-(2-nitroimidazol-1-yl)acetamide] (Laboratório Farmacêutico do Estado de Pernambuco, Brazil) was used as a reference drug (26).
Mammalian cells.
Primary CC cultures were obtained from mice embryos and plated onto 0.01% gelatin-coated coverslips in 96-well plates (27). PMMs were purified as reported previously (25).
Parasites.
BTs of the Y strain of T. cruzi were obtained by cardiac puncture of infected Swiss Webster mice, at the parasitemia peak (27, 28).
Cytotoxicity assays in CCs.
Noninfected CCs were incubated at 37°C for 24 h with increasing concentrations of each compound (12.5 to 200 μM; 1:2 serial dilutions) diluted in supplemented Dulbecco’s modified Eagle’s medium (DMEM). CC morphology was evaluated by light microscopy, and the cellular viability of the CCs was determined with a standardized PrestoBlue test. The results were expressed as the difference in reduction between treated and nontreated cells, according to the manufacturer’s instructions, and the LC50 (minimum concentration that reduces cellular viability by 50%) values were determined by nonlinear regression using a sigmoid curve with a variable slope (29).
Trypanocidal activity.
Y strain BTs (5 × 106/ml) were incubated for 24 h at 37°C in RPMI 1640 medium, in the presence or absence of 1:3 serial dilutions of the compounds (0.2 to 50 μM), for determination of parasite death rates through direct quantification of live parasites by light microscopy. The IC50 (compound concentration that reduces the number of live parasites by 50%) values were calculated by nonlinear regression (30). The SI is expressed as the ratio between the LC50 (toxicity for mammalian cells) and the IC50 (activity against the parasite).
Transmission electron microscopy.
Y strain BTs (5 × 106 parasites/ml) were treated with compound 9 for 2 h at the concentration corresponding to its 24-h IC50 value. The parasites were fixed at room temperature with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 40 min and postfixed with a solution of 1% OsO4, 0.8% potassium ferricyanide, and 2.5 mM CaCl2 in the same buffer for 30 min. The samples were washed in phosphate-buffered saline (PBS), dehydrated in an ascending acetone series, and embedded in epoxy resin. Ultrathin sections (Leica Ultracut; Leica, Vienna, Austria) were stained with 2% uranyl acetate and lead citrate and examined using an EM10C Zeiss microscope (Zeiss, Oberkochen, Germany) (31).
cAMP measurements.
Y strain BTs (15 × 106 parasites/ml) were treated with compound 9, at the concentrations of 2 times and 5 times the IC50, for 2.5 h at 37°C. After the incubation, the samples were centrifuged at 5,000 rpm for 2 min and the supernatant was collected. The pellet was resuspended in 100 μl of 0.1 M HCl and incubated at 4°C for 20 min, followed by centrifugation at 12,000 rpm for 10 min and collection of the supernatant. Samples were stored at −80°C and then analyzed using a cAMP enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemicals, Ann Arbor, MI, USA), according to the manufacturer’s instructions (9). Each experiment was performed independently at least three times, and all samples were assayed in duplicate.
Supplementary Material
ACKNOWLEDGMENTS
Funding from the EC 7th Framework Program (PDE4NPD, grant 602666), Red de Investigación Cooperativa en Enfermedades Tropicales (grant RD16/0027/0010), Fondo Europeo de Desarrollo Regional (FEDER), and Ministerio de Educación, Cultura y Deportes (grant FPU15/1465 to V.S.-P.) is acknowledged. M.N.C.S. is a Cientista do Nosso Estado (CNE) from the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) researcher. The Fundação Oswaldo Cruz (Fiocruz), CNPq, and FAPERJ are acknowledged.
This paper is dedicated to the memory of our friend and colleague, Mercedes González.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02156-18.
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