ABSTRACT

The impact of Pneumocystis pneumonia (PcP) on morbidity and mortality remains substantial for immunocompromised individuals, including those afflicted by HIV infection, organ transplantation, cancer, autoimmune diseases, or subject to chemotherapy or corticosteroid-based therapies. Previous work from our group has shown that repurposing antimalarial compounds for PcP holds promise for treatment of this opportunistic infection. Following our previous discovery of chloroquine analogues with dual-stage antimalarial action both in vitro and in vivo, we now report the potent action of these compounds on Pneumocystis carinii in vitro. Identification of chloroquine analogues as anti-PcP leads is an unprecedented finding.

INTRODUCTION

Pneumocystis jirovecii pneumonia (PcP) is a potentially fatal opportunistic infection for immunocompromised individuals, such as those infected with HIV, or receiving immunosuppressive therapies for organ transplantation, cancer, or autoimmune diseases (13). Although fatality rates due to PcP in HIV-infected people have significantly dropped due to highly active antiretroviral therapies, PcP remains a major cause of death or of chronic lung dysfunction among people affected by diseases as diverse as lymphoma (4), rheumatoid arthritis (5), lupus erythematosus (6), or renal dysfunction due to IgA nephropathy (7). Moreover, atypical radiological presentation of PcP sometimes occurs, leading to delayed diagnosis and intervention, with frequently discouraging results (1, 7). Though considered an innocuous infection in immunocompetent people, P. jirovecii has been suggested as a morbidity factor in sudden infant death syndrome, given its high incidence in healthy infants (8).
P. jirovecii, formerly known as “P. carinii f. sp. hominis,” is the causative agent of PcP or pneumocystosis in humans and was originally identified as the pathogen responsible for the epidemics of interstitial plasma cell pneumonitis that infected premature infants in Europe during World War II (2, 9). Pneumocystis spp. are unicellular eukaryotic organisms now included in the fungal kingdom due to their high genetic homology with other fungi (2, 10). These fungi are thought to be host specific and infect a wide variety of mammals (11). P. jirovecii infects humans, while P. carinii and P. murina infect rats and mice, respectively. Preclinical drug screening is conducted using the latter two species, as a culture system to properly propagate P. jirovecii in vitro was developed only recently, and still requires validation (12).
Pneumocystis spp. have several features that are atypical among fungi, such as the lack of efficacy of common antifungal agents such as amphotericin B and the ketoconazoles, likely due to the lack of ergosterol as the major sterol in their membranes (13). Initially, these microorganisms were thought to be protozoans, since they were susceptible to antiprotozoals such as primaquine, trimethoprim, sulfa drugs, pentamidine, or atovaquone, among others; these are still present in anti-PcP therapies, where the recommended first-line regimen for PcP prophylaxis and treatment is the well-known trimethoprim-sulfamethoxazole (TMP-SMX) combination, used for more than 50 years as a wide-spectrum antibiotic, and also employed against malaria (13, 14).
Some reports documenting P. jirovecii gene mutations in the target of the sulfamethoxazole component, dihydropteroate synthase (15, 16), may indicate resistance of this pathogen to TMP-SMX, supporting earlier reports (1719). Alternative options for salvage therapy include intravenous pentamidine, the adverse effects of which are usually worse than those of TMP-SMX, or primaquine-clindamycin combinations (20). Clinical use of the latter still lacks approval in several countries, partly because primaquine (PQ; compound 1) (Fig. 1) is hemotoxic, in particular for people with 6-glucose-phosphate dehydrogenase (21, 22).
Since hemotoxicity and low oral bioavailability are major downsides of PQ's use in clinical practice, we have been working on its chemical modification over the past decade in an attempt to improve its action on malaria (2328), leishmaniasis (29, 30), and pneumocystosis (24, 25), with overall promising results. These successes provided the rationale to extend such “chemical recycling” efforts to other antimalarials, as chloroquine (CQ; compound 2 in Fig. 1), (31, 32) and mepacrine (MPR; compound 3 in Fig. 1) (32). This effort delivered N-acyl chloroquine analogues (compounds 2a to 2j in Fig. 1) as relevant antiparasitic leads, active against liver- and blood-stage Plasmodium parasites (31, 32), as well as Leishmania spp. (28), while being generally noncytotoxic (31, 32). Moreover, one of such leads, compound 2c, displayed remarkable in vivo performance in immunocompetent mice when encapsulated in conveniently functionalized liposomes specifically targeting Plasmodium-infected erythrocytes (33).
FIG 1
FIG 1 Classical antimalarial drugs and respective analogues, here under scrutiny as potential anti-Pneumocystis agents.
In view of the above, we investigated the anti-Pneumocystis activity of N-acyl chloroquine derivatives compounds 2a to 2j (Fig. 1) using a standardized in vitro assay with P. carinii (34). The study included N-acyl primaquine and N-acyl mepacrine analogues, compounds 1a/1b and 3a, respectively, as well as all relevant parent antimalarials, for comparison (see Fig. 1). The effects of replacing the covalent amide bond between the antimalarial pharmacophore and the cinnamoyl group in PQ derivatives 1a,b and CQ derivatives 2a,d by an ionic ammonium carboxylate bond, respectively producing PQ-derived ionic liquids 1′a and 1′b (26) and CQ-derived ionic liquids 2′a and 2′d (Fig. 1), were also assessed.

RESULTS

Chemical synthesis.

The test compounds were prepared with a minimum 95% purity as described elsewhere and presented structural data consistent with previous reports (27, 31, 32). Novel ionic liquids 2′a and 2′d were successfully synthesized and presented adequate purity and spectral data, as described in detail in Materials and Methods.

Anti-Pneumocystis activity and cytotoxicity assays.

All the test compounds (including parent drugs compounds 1 to 3) were first screened for their anti-Pneumocystis activity at 100 μg/ml (data not shown); remarkably, with the exception of PQ derivatives 1a, 1b, and 1′b and CQ derivative 2h, all compounds reduced P. carinii viability (ATP content) by at least 94% after 24 h and reached nearly 100% reduction after 72 h. Such potent anti-Pneumocystis activity of most compounds was confirmed by subsequent determination of their respective 50% inhibitory concentrations (IC50s) at 72 h, as shown in Table 1, which includes the compounds' toxicity for two mammalian cell lines (equally expressed as IC50 at 72 h), namely, A549 human lung adenocarcinoma and L2 rat pulmonary epithelial cells.
TABLE 1
TABLE 1 MICs at 50% (IC50) of test compounds at 72 h against P. carinii, A549 human lung adenocarcinoma cells, and L2 rat pulmonary epithelial cells
CompoundIC50 ± SD/μM (IC50 ± SD/μg/ml)aSIb
P. carinii*A549 cells†L2 cells†SI1SI2
14.92 ± 0.85 (1.28 ± 0.221)110.04 ± 49.93 (28.5 ± 13.0)>200 (>100)22.4>41
1a43.58 ± 3.14 (16.97 ± 1.22)>200 (>100)>200 (>100)>5>5
1b35.42 ± 7.52 (14.86 ± 3.16)>200 (>100)>200(>100)>6>6
1′a5.17 ± 4.46 (2.11 ± 1.82)120.49 ± 43.56 (49.1 ± 17.8)>200 (>100)23.3>39
1′b5.09 ± 1.11 (2.23 ± 0.486)100.15 ± 41.53 (43 ± 18.2)>200 (>100)19.7>39
23.13 ± 2.14 (1.00 ± 0.685)61.55 ± 6.38 (19.7 ± 2.04)>200 (>100)19.7>64
2a4.34 ± 1.34 (1.65 ± 0.509)19.01 ± 14.29 (7.22 ± 5.43)82.84 ± 7.27 (31.5 ± 2.76)4.384.36
2b1.85 ± 2.75 (0.730 ± 1.08)>200 (>100)>200 (>100)>100>100
2c2.75 ± 2.30 (1.16 ± 0.971)12.94 ± 6.99 (5.46 ± 2.95)75.01 ± 16.35 (31.6 ± 6.90)4.715.80
2d1.45 ± 0.06 (0.593 ± 0.023)80.02 ± 46.11 (32.8 ± 18.9)128.59 ± 72.75 (52.7 ± 29.8)55.31.61
2e5.81 ± 2.19 (2.41 ± 0.906)17.79 ± 12.41 (7.37 ± 5.14)48.13 ± 23.48 (19.9 ± 9.73)3.062.71
2f4.09 ± 3.21 (1.55 ± 1.22)41.04 ± 14.85 (15.6 ± 5.64)139.62 ± 17.98 (53.0 ± 6.83)10.03.40
2g2.61 ± 0.80 (1.10 ± 0.340)51.68 ± 30.19 (21.9 ± 12.8)79.26 ± 50.24 (33.6 ± 21.3)19.81.53
2h>200 (>100)    
2i7.00 ± 1.06 (2.70 ± 0.411)>200 (>100)>200 (>100)>29>29
2j8.45 ± 2.58 (2.79 ± 0.850)166.60 ± 53.27 (55.0 ± 17.6)>200 (>100)19.7>24
2′a4.99 ± 1.64 (2.34 ± 0.769)65.74 ± 7.84 (30.8 ± 3.67)>200 (>100)13.2>40
2′d4.60 ± 2.39 (2.29 ± 1.19)59.23 ± 22.19 (29.5 ± 11.0)>200 (>100)12.9>43
30.90 ± 0.21 (0.358 ± 0.083)17.58 ± 1.50 (7.03 ± 0.600)124.19 ± 11.45 (49.7 ± 4.58)19.67.07
3a2.27 ± 0.50 (1.11 ± 0.245)72.04 ± 60.20 (35.3 ± 29.5)>200 (>100)31.7>88
a
IC50s are expressed in both μM and μg/ml units (the latter, within parentheses). *, activity scale: high, <0.010 μg/ml; very marked, 0.011 to 0.099 μg/ml; marked, 0.10 to 0.99 μg/ml; moderate, 1.0 to 9.99 μg/ml; slight, 10.0 to 49.9 μg/ml; none, ≥50 μg/ml (24, 25). †, cytotoxicity scale: high, <1 μg/ml; moderate, 1.1 to 10 μg/ml; mild, 10.1 to 99.9 μg/ml; none, ≥100 μg/ml (24, 25).
b
SI = selectivity index, where SI1 = IC50(A549)/IC50(P. carinii) and SI2 = IC50(L2)/IC50(P. carinii).
Interestingly, despite the fact that, in most cases, activity differences are not markedly significant within experimental error, a number of trends for structure-activity relationships (SAR) were apparent. (i) The anti-Pneumocystis activity of PQ (compound 1) is markedly decreased upon N-acylation of the drug's primary amine with a cinnamoyl group, as in compounds 1a and 1b; in turn, when the amide bond between PQ and the cinnamic acid building blocks is replaced by a noncovalent ionic bond, as in compounds 1′a and 1′b, the activity of the parent drug is restored. (ii) In contrast to the previous situation, the moderate anti-Pneumocystis activity of CQ (compound 2) is preserved or even improved in the respective N-acyl analogues, except when a flexible alkyl spacer between the 4-aminoquinoline core and the N-acyl group is absent, as in compound 2h. Moreover, replacement of the amide bond, as in compounds 2a and 2d, by its ammonium carboxylate ionic counterpart, as in compounds 2′a and 2′d, does not affect activity expressively. (iii) Within the subset of CQ analogues 2a to 2g, compounds 2b (R = Me) and 2d (R = OMe) are markedly more active than the others, with compound 2b being nontoxic to both mammalian cell lines tested. The IC50s for this subset suggest that (a) the ideal length spacer between the 4-aminoquinoline core and the cinnamoyl group is butyl (n = 4), since activity slightly drops when this length is either decreased (compound 2b versus compound 2f) or increased (compound 2d versus compound 2g); notably, this was equally observed regarding anti-malarial activity (31); (b) there seems to be no obvious correlation between compounds' lipophilicity (clogP values, not shown) and anti-Pneumocystis activity, whereas former observations regarding the antimalarial activity of these compounds indicated that more lipophilic ones seemed preferred (31, 35); and (c) the influence of the stereoelectronic properties of the p-substituent R on the anti-Pneumocystis activity of compounds 2a to 2e was also surveyed by searching possible correlations between IC50s and classical descriptors, namely, the Hammett constant σpara, the inductive sigma constant σI, Charton's sterical constant σn, the Taft sterical parameter Es, the hydrophobicity parameter π, and molar refractivity (36). Interestingly, as shown in Fig. 2, a linear correlation (r2 = 0.96) seems to exist with the Hammett's constant σpara, where the positive slope of the linear fit suggests that electrodonating R groups favor anti-Pneumocystis activity. (iv) Replacement of the aryl group (compound 2a) by a nonaromatic cyclohexyl (compound 2i) or propyl (compound 2j) seems to slightly decrease activity, while improving selectivity compared to the rest of the compound 2a to 2g series, except when it comes to the best overall performer (activity plus selectivity), compound 2b. MPR (compound 3) and its N-acyl analogue 3a were included in this study only for comparison, since MPR was earlier reported as having low in vivo efficacy against Pneumocystis (37). Indeed, despite its marked activity, MPR was too toxic, whereas compound 3a was not, but it still did not outshine the activity and safety performance of its CQ counterparts compounds 2b and 2d.
FIG 2
FIG 2 Correlation between in vitro anti-Pneumocystis activity (IC50 at 72 h/μM) of compounds 2a to 2e and Hammett σpara constants for the aryl substituent group R.
Finally, all covalent compounds here reported were run through the following publicly available filters for Pan Assay Interference Compounds (PAINS) (38), namely, ZINC (http://zinc15.docking.org/patterns/home), (39) PAINS Remover (http://www.cbligand.org/PAINS/) (40), and Aggregator Advisor (http://advisor.docking.org) (41) to identify potential PAINS and aggregators. With the exception of compounds 3 and 3a, whose aminoacridine moiety triggered a PAINS alert (39, 40), and compound 2h, whose structural similarity to reported aggregators has been identified (39, 41), all compounds passed all filters used. In the case of organic salts 1′a, 1′b, 2′a, and 2′d, only ZINC was apparently able to conveniently process their ionic structures, returning no PAINS or aggregator alerts.

DISCUSSION

Globally, IC50s against P. carinii (Table 1) confirm that, with exception of compounds 1a and 1b (slightly active) and of compound 2h (inactive), all test compounds range from moderately to markedly active, particularly in the cases of CQ analogues 2b and 2d and of MPR (compound 3) and its analogue compound 3a. Moreover, the vast majority of the compounds displayed only mild or no cytotoxic activity against both mammalian cell lines used, the only exceptions being compounds 2a, 2c, 2e, and 3, which displayed IC50s below 10 μg/ml against A549 human lung carcinoma cells. Remarkably, one of the most active compounds against P. carinii, compound 2b, was also the most selective.
Altogether, these observations show that, in contrast to PQ, whose well-known anti-Pneumocystis activity is practically lost upon acylation of the drug's aliphatic amine, N-acyl chloroquine analogues, especially those embedding a cinnamoyl group, are quite promising anti-PcP leads. Interestingly, this is not the first time that N-cinnamoyl chloroquine analogues such as compounds 2a to 2g have performed surprisingly well against pathogens, e.g., P. carinii as here described, or against specific pathogen developmental stages, e.g., liver-stage plasmodia, as previously reported by us (31), for which PQ, not CQ, was one of the gold standards.
The significant activity of the test compounds against P. carinii may be different against P. jirovecii or P. murina, given the biological divergence among these species. Still, the predictive value of the in vitro methods applied here has been previously validated in vivo in a mouse model of Pneumocystis pneumonia (4244).
In conclusion, N-cinnamoyl chloroquine analogues such as compounds 2a to 2g are a notable family of anti-infective compounds, in particular because they have displayed remarkable activity with pathogens against which CQ, or other related compounds, is not currently considered useful. In the case of PcP, these compounds hold great promise, and the best performers, compounds 2a and 2b, will be further assessed in in vivo assays. To the best of our knowledge, unveiling CQ analogues as potent anti-PcP leads is unprecedented.
Ongoing work is addressing evaluation of the potential activity of these CQ analogues against another concerning class of lung pathogens, mycobacteria (45), since CQ itself has been reported as potentially useful in the fight against these bacteria (46). Results from these studies should be available soon.

MATERIALS AND METHODS

Chemical synthesis. (i) Chemicals and instrumentation.

Primaquine and chloroquine biphosphates, as well as mepacrine hydrochloride, were obtained from Sigma-Aldrich. The cinnamic acids were all from Acros Organics. The coupling agent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) was from Bachem, and all other chemicals were from Sigma-Aldrich. All solvents were of analytical grade and were purchased from VWR International. Nuclear magnetic resonance analyses were carried out on a Bruker Avance III 400 MHz spectrometer, and samples were prepared in either deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide [(CD3)2SO] with tetramethylsilane as an internal reference. Chemical shifts are reported downfield in parts per million (ppm), and the multiplicity of proton signals is indicated as s (singlet), d (doublet), dd (double doublet), t (triplet), or m (unresolved multiplet). High-resolution mass spectra were obtained on an LTQ Orbitrap XL/LTQ Tune Plus 2.5.5 spectrometer and processed using 2.1.0 Xcalibur software (Thermo Scientific). Compound purity was based on peak areas obtained through high-pressure liquid chromatography analyses that were run under the following conditions: 0 to 100% of solvent B in solvent A (A = H2O with 0.05% of trifluoroacetic acid; B = acetonitrile) in 30 min with a flow rate of 1 ml/min on a Merck-Hitachi Lachrom Elite instrument equipped with a diode array detector and thermostated (Peltier effect) autosampler, using a Purospher STAR RP-18e column (150 mm by 4.0 mm; particle size, 5 μm).

(ii) Synthesis of primaquine derivatives 1a, 1b, 1′a, and 1′b.

N-Cinnamoyl-primaquine derivatives 1a and 1b were prepared by previously described methods, and their analytical and structural data were consistent with those formerly reported (27). Similarly, the synthesis of the ionic counterparts of compounds 1a and 1b (ionic liquids 1′a and 1′b, respectively) was carried out as reported earlier, and the compounds' structural and analytical data concurred with those previously published (28).

(iii) Synthesis of N-acyl-chloroquine (compounds 2a to 2j) and of N-acyl-mepacrine (compound 3a) analogues.

The chemical synthesis procedures for compounds 2a to 2j and compound 3a were similar and are in accord with previously reported procedures, yielding the expected compounds, whose structural and analytical data agreed with those earlier described (31, 32, 35, 36, 47).

(iv) Synthesis of the new chloroquine/cinnamic acid-derived ionic liquids 2′a and 2′d.

Organic salts 2′a and 2′d, which are representative of the ionic counterparts of compounds 2a to 2g, were prepared as follows. Chloroquine was first obtained as a free base, by adding triethylamine (0.25 ml) to a suspension of chloroquine biphosphate (0.1298 g; 0.285 mol) in dichloromethane (4 ml). The mixture was stirred for 30 min in ice and in the dark, and then the organic layer was washed with water (three times using 10 ml each time), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Free chloroquine base, quantitatively obtained as a pale yellow oil, was next dissolved in methanol and titrated with an equimolar amount of the relevant cinnamic acid upon dropwise addition of a methanolic solution of the acid. The neutralization reaction proceeded in the dark at room temperature for 30 min; the methanol was removed by evaporation at reduced pressure, and the target organic salts 2′a and 2′d were obtained as chromatographically homogeneous colorless viscous liquids whose spectral data were in agreement with the target structures, as follows.

2-((7-Chloroquinolin-4-yl)amino)-N,N-diethylpropan-1-aminium cinnamate (compound 2′a).

Colorless oil (51 mg, 81%) δH (400 MHz, DMSO-d6) 8.37 (s, 1H), 8.35 (d, 1H, J = 4.4 Hz), 7.76 (d, 1H, J = 2.2Hz), 7.66 (m, 2H), 7.55 (d, 1H, J = 16.0 Hz), 7.42 (m, 5H), 6.90 (d, 1H, J = 7.9 Hz), 6.52 (m, 2H), 2.42 (m, 6H), 1.69 (m, 1H), 1.50 (m, 4H),1.23 (d, 4H, J = 6.3 Hz), 0.91 (t, 6H, J = 7.1 Hz); δC (100 MHz, DMSO-d6) 167.8, 151.8, 149.5, 149.2, 143.2, 134.4, 133.3, 130.0, 128.8, 128.0, 127.4, 124.3, 123.8, 120.1, 117.5, 98.8, 51.9, 47.5, 46.1, 33.3, 23.0, 19.8, 11.2; (EI+) m/z calculated for C18H27ClN3+: 320.19, found 320.33; (EI) m/z calculated for C9H7O2 147.05, found 147.33.

2-((7-Chloroquinolin-4-yl)amino)-N,N-diethylpropan-1-aminium p-methoxycinnamate (compound 2′d).

Colorless oil (52 mg, 87%) δH (400 MHz, DMSO-d6) 8.37 (s, 1H), 8.34 (d, 1H, J = 4.4Hz), 7.76 (d, 1H, J = 2.2Hz), 7.62 (d, 2H, J = 8.8 Hz), 7.51 (d, 1H, J = 15.9 Hz), 7.42 (dd, 1H, J = 9.0 Hz, J = 2.2 Hz), 6.96 (d, 2H, J = 8.8Hz), 6.89 (d, 1H, J = 8.0 Hz), 6.50 (d, 1H, J = 5.6 Hz), 6.36 (d, 1H, J = 15.9 Hz), 3.79 (s, 3H), 2.40 (m, 6H) 1.69 (m, 1H), 1.50 (m, 4H), 1.22 (m, 4H), 0.91 (t, 6H, J = 7.1 Hz); δC (100 MHz, DMSO-d6) 167.9, 160.8, 151.9, 149.5, 149.3, 143.4, 133.3, 129.8, 127.4, 126.9, 124.3, 123.7, 117.5, 116.9, 114.3, 98.8, 55.3, 52.0, 47.5, 46.1, 33.3, 23.3, 19.8, 11.5; (EI+) m/z calculated for C18H27ClN3+: 320.19, found 320.35; (EI) m/z calculated for C10H9O3 177.06, found 177.05.

In vitro assays. (i) Compound preparation.

Test compounds were stored at −20°C without exposure to light until use. Immediately prior to the first screening, compounds were solubilized in 100% dimethyl sulfoxide at a concentration of 20 mg/ml. For testing, the compounds were diluted directly into culture medium at dilutions of 100, 10, 1, and 0.1 μg/ml. Culture medium consisted of RPMI 1640 containing 20% horse serum, 1% minimal essential medium (MEM) vitamin solution, 1% MEM nonessential amino acid solution (NEAA), 200 U/ml penicillin, 0.2 mg/ml streptomycin, and 50 μg/ml vancomycin. Negative controls were medium alone and medium with 10 μg/ml ampicillin. The positive control included 1 μg/ml pentamidine isethionate. Medium with vehicle alone (dimethyl sulfoxide) was tested at the highest dilution to identify any associated toxicity.

(ii) P. carinii ATP assay.

P. carinii viability was assessed through a nonspecific ATP assay previously validated for this purpose (4244, 48, 49). Briefly, cryopreserved and characterized P. carinii isolated from rat lung tissue was distributed into triplicate wells of 48-well plates with a final volume of 500 μl and final concentrations of 5 × 107 P. carinii nuclei/ml. Controls and compound dilutions were added, followed by incubation at 36°C and 5% CO2. At 24, 48, and 72 h, 10% of the well volume was removed, and the ATP content was measured using a Perkin-Elmer ATP-liteM luciferin-luciferase assay. The bioluminescence generated by the ATP content of the samples was measured with a BMG POLARstar OPTIMA spectrophotometer and expressed as relative light units. A sample of each group was examined microscopically on the final assay day to rule out the presence of bacteria or other fungi. A quench control assay to determine compound interference in the luciferin/luciferase revealed quenching with six compounds at the highest (100 μg/ml) concentration. No quench was observed at 50 μg/ml. Test concentrations of these compounds were lowered accordingly.

(iii) L2 and A549 toxicity assay.

For mammalian cell toxicity testing, A549 cells were cultured in Dulbecco's modified Eagle medium and L2 cells with F-12 medium. Both were supplemented with 10% fetal calf sera, 1× MEM vitamins, and 1× NEAA. Cultured cells were plated at 2 × 105/ml and grown to confluent monolayers. The medium was removed and replaced with fresh medium containing controls and test compound at appropriate dilutions. Assays of three time points (24, 48, and 72 h) with triplicate wells were sampled. The medium was aspirated from the wells. Adherent cells were stained with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent (thiazolyl blue), followed by incubation for 1 h. The MTT reagent was aspirated from the cells, and MTT solvent was added to the stained cells, followed by rocking at room temperature until the crystals were dissolved. The plate was read in absorbance mode on a BMG POLARstar Optima spectrophotometer.

(iv) Calculations.

For the P. carinii ATP assay, the background bioluminescence was subtracted, and triplicate well readings were averaged. Using EXCEL software, the percent reduction in ATP for all groups was calculated (vehicle control − experimental/vehicle control × 100). The results of triplicate assays were averaged, and the standard deviations were determined. For the cell toxicity assay, triplicate well readings were averaged. For each day's readings, the percent reduction in absorbance for all groups was calculated as follows: medium control − experimental/medium control × 100. In both assays, the IC50 was calculated using the InSTAT linear regression program.

ACKNOWLEDGMENTS

We thank Fundação para a Ciência e Tecnologia (FCT, Portugal) for funding Research Unit LAQV-REQUIMTE (UID/QUI/50006/2013) and for a doctoral grant to A.G. (PD/BD/135073/2017). We also thank the Comissão de Coordenação e Desenvolvimento do Norte (CCDR-N, Porto, Portugal) for funding project DesignBiotechHealth (Norte-01-0145-FEDER-000024). Studies performed in Cincinnati were supported by the Cincinnati Veterans Affairs Medical Center and the Cincinnati Education and Research for Veterans Foundation. M.T.C. received a VA Research Career Scientist Award.

REFERENCES

1.
Matos O, Tomás A, Antunes F. 2017. Pneumocystis jirovecii and pneumocystosis, p 215–254. In Montes HMM, Lopes-Bezerra LM (ed), Current progress in medical mycology. Springer, New York, NY.
2.
Sokulska M, Kicia M, Wesołowska M, Hendrich AB. 2015. Pneumocystis jirovecii—from a commensal to a pathogen: clinical and diagnostic review. Parasitol Res 114:3577–3585.
3.
White PL, Backx M, Barnes RA. 2017. Diagnosis and management of Pneumocystis jirovecii infection. Expert Rev Anti Infect Ther 15:435–447.
4.
Jiang X, Mei X, Feng D, Wang X. 2015. Prophylaxis and treatment of Pneumocystis jirovecii pneumonia in lymphoma patients subjected to rituximab-contained therapy: a systemic review and meta-analysis. PLoS One 10:e0122171.
5.
Mori S, Sugimoto M. 2012. Pneumocystis jirovecii infection: an emerging threat to patients with rheumatoid arthritis. Rheumatology 51:2120–2130.
6.
Chia H-H, Lee M-C, Lee C-H, Huang S-F, Wu Y-K. 2012. Pneumocystis jirovecii pneumonia manifesting as a lung abscess in a woman with systemic lupus erythematosus. Tzu Chi Med J 24:88–89.
7.
Li H, Huang H, He H. 2016. Successful treatment of severe Pneumocystis pneumonia in an immunosuppressed patient using caspofungin combined with clindamycin: a case report and literature review. BMC Pulm Med 16:144.
8.
Vargas SL, Ponce C, Hughes WT. 1999. Association of primary Pneumocystis carinii infection and sudden infant death syndrome (SIDS). Clin Infect Dis 29:1489–1493.
9.
Gerrard JW, Moore DF. 1957. Interstitial plasma cellular pneumonia due to Pneumocystis carinii. Can Med Assoc J 76:299–302.
10.
Chabé M, Aliouat-Denis C-M, Delhaes L, El Moukhtar A, Viscogliosi E, Dei-Cas E. 2011. Pneumocystis: from a doubtful unique entity to a group of highly diversified fungal species. FEMS Yeast Res 11:2–17.
11.
Cushion MT, Stringer JR. 2010. Stealth and opportunism: alternative lifestyles of species in the fungal genus Pneumocystis. Annu Rev Microbiol 64:431–452.
12.
Schildgen V, Mai S, Khalfaoui S, Lüsebrink E, Pieper M, Tillmann RL, Brockmann M, Schildgen O. 2014. Pneumocystis jirovecii can be productively cultured in differentiated CuFi-8 airway cells. mBio 5:e01186–e01114.
13.
Porollo A, Meller J, Joshi Y, Jaiswal V, Smulian AG, Cushion MT. 2012. Analysis of current antifungal agents and their targets within the Pneumocystis carinii genome. Curr Drug Targets 13:1575–1585.
14.
Benfield T, Atzori C, Miller RF, Helweg-Larsen J. 2008. Second-line salvage treatment of AIDS-associated Pneumocystis jirovecii pneumonia: a case series and systematic review. J Acquir Immune Defic Syndr 48:63–67.
15.
Huang YS, Yang JJ, Lee NY, Chen GJ, Ko WC, Sun HY, Hung CC. 2017. Treatment of Pneumocystis jirovecii pneumonia in HIV-infected patients: a review. Expert Rev Anti Infect Ther 15:873–892.
16.
Lee SM, Cho YK, Sung YM, Chung DH, Jeong SH, Park J-W, Lee SP. 2015. A case of pneumonia caused by Pneumocystis jirovecii resistant to trimethoprim-sulfamethoxazole. Korean J Parasitol 53:321–327.
17.
Martin JN, Rose DA, Hadley WK, Perdreau-Remington F, Lam PK, Gerberding JL. 1999. Emergence of trimethoprim-sulfamethoxazole resistance in the AIDS era. J Infect Dis 180:1809–1818.
18.
Huovinen P. 2001. Resistance to trimethoprim-sulfamethoxazole. Clin Infect Dis 32:1608–1614.
19.
Huang L, Crothers K, Atzori C, Benfield T, Miller R, Rabodonirina M, Helweg-Larsen J. 2004. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance. Emerg Infect Dis 10:1721–1728.
20.
Kim T, Kim SH, Park K-H, Cho OH, Sung H, Kim M-N, Choi S-H, Jeong J-Y, Woo JH, Kim YS, Lee S-O. 2009. Clindamycin-primaquine versus pentamidine for the second-line treatment of Pneumocystis pneumonia. J Infect Chemother 15:343–346.
21.
Vale N, Moreira R, Gomes P. 2009. Primaquine revisited, six decades after its discovery. Eur J Med Chem 44:937–953.
22.
Teixeira C, Vale N, Pérez B, Gomes A, Gomes JRB, Gomes P. 2014. Recycling classical drugs for malaria. Chem Rev 114:11164–11220.
23.
Araújo MJ, Bom J, Capela R, Casimiro C, Chambel P, Gomes P, Iley J, Lopes F, Morais J, Moreira R, de Oliveira E, do Rosário V, Vale N. 2005. Imidazolidin-4-ones of primaquine as novel transmission-blocking antimalarials. J Med Chem 48:888–892.
24.
Vale N, Collins MS, Gut J, Ferraz R, Rosenthal PJ, Cushion MT, Moreira R, Gomes P. 2008. Anti-Pneumocystis carinii and antiplasmodial activities of primaquine-derived imidazolidin-4-ones. Bioorg Med Chem Lett 18:485–488.
25.
Vale N, Prudêncio M, Marques CA, Collins MS, Gut J, Nogueira F, Matos J, Rosenthal PJ, Cushion MT, do Rosário VE, Mota MM, Moreira R, Gomes P. 2009. Imidazoquines as antimalarial and anti-Pneumocystis agents. J Med Chem 52:7800–7807.
26.
Matos J, da Cruz FP, Cabrita E, Gut J, Nogueira F, do Rosário VE, Rosenthal PJ, Moreira R, Prudêncio M, Gomes P. 2012. Novel potent metallocenes against liver-stage malaria. Antimicrob Agents Chemother 56:1564–1570.
27.
Pérez B, Teixeira C, Albuquerque IS, Gut J, Rosenthal PJ, Prudêncio M, Gomes P. 2012. PRIMACINS, N-cinnamoyl-primaquine conjugates with improved liver-stage antimalarial activity. MedChemComm 3:1170–1172.
28.
Ferraz R, Noronha J, Murtinheira F, Nogueira F, Machado M, Prudêncio M, Parapini S, D'Alessandro S, Teixeira C, Gomes A, Prudêncio C, Gomes P. 2016. Primaquine-based ionic liquids as a novel class of antimalarial hits. RSC Adv 6:56134–56138.
29.
Vale-Costa S, Vale N, Tomás A, Moreira R, Gomes P, Gomes MS. 2012. Evaluation of dipeptide and peptidomimetic derivatives of primaquine against Leishmania infantum. Antimicrob Agents Chemother 56:5774–5781.
30.
Vale-Costa S, Gouveia J, Pérez B, Silva T, Teixeira C, Gomes P, Gomes P. 2013. N-cinnamoylated aminoquinolines as promising anti-leishmanial agents. Antimicrob Agents Chemother 57:5112–5115.
31.
Pérez B, Teixeira C, Albuquerque IS, Gut J, Rosenthal PJ, Gomes JRB, Prudêncio M, Gomes P. 2013. N-Cinnamoylated chloroquine analogues as dual-stage antimalarial leads. J Med Chem 56:556–567.
32.
Gomes A, Machado M, Lobo L, Nogueira F, Prudêncio M, Teixeira C, Gomes P. 2015. N-Cinnamoylation of antimalarial classics: effects of using acyl groups other than cinnamoyl towards dual-stage antimalarials. ChemMedChem 10:1344–1349.
33.
Moles E, Galiano S, Gomes A, Quiliano M, Teixeira C, Aldana I, Gomes P, Fernández-Busquets X. 2017. ImmunoPEGliposomes for the targeted delivery of novel lipophilic drugs to red blood cells in a falciparum malaria murine model. Biomaterials 145:178–191.
34.
Cushion MT, Walzer PD, Ashbaugh A, Rebholz S, Brubaker R, Vanden Eynde JJ, Mayence A, Huang TL. 2006. In vitro selection and in vivo efficacy of piperazine- and alkanediamide-linked bisbenzamidines against Pneumocystis pneumonia in mice. Antimicrob Agents Chemother 50:2337–2343.
35.
Pérez B, Teixeira C, Gut J, Rosenthal PJ, Gomes JRB, Gomes P. 2012. New cinnamic acid/chloroquinoline conjugates as potent agents against chloroquine-resistant Plasmodium falciparum malaria parasites. ChemMedChem 7:1537–1540.
36.
Hansch C, Leo A, Taft RW. 1991. A survey of Hammett substituent constants and resonance and field parameters. Chem Rev 91:165–195.
37.
Walzer PD, Kim CK, Foy J, Linke MJ, Cushion MT. 1988. Cationic antitrypanosomal and other antimicrobial agents in the therapy of experimental Pneumocystis carinii pneumonia. Antimicrob Agents Chemother 32:896–905.
38.
Aldrich C, Bertozzi C, Georg GI, Kiessling L, Lindsley C, Liotta D, Merz KM, Jr, Schepartz A, Wang S. 2017. The ecstasy and agony of assay interference compounds. J Med Chem 60:2165–2168.
39.
Sterling T, Irwin JJ. 2015. ZINC 15: ligand discovery for everyone. J Chem Infect Model 55:2324–2337.
40.
Baell JB, Holloway GA. 2010. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 53:2719–2740.
41.
Irwin JJ, Duan D, Torosyan H, Doak AK, Ziebart KT, Sterling T, Tumanian G, Shoichet BK. 2015. An aggregation advisor for ligand discovery. J Med Chem 58:7076–7087.
42.
Cushion MT, Collins MS. 2011. Susceptibility of Pneumocystis to echinocandins in suspension and biofilm cultures. Antimicrob Agents Chemother 55:4513–4518.
43.
Walzer PD, Ashbaugh A, Collins M, Cushion MT. 2001. In vitro and in vivo effects of quinupristin-dalfopristin against Pneumocystis carinii. Antimicrob Agents Chemother 45:3234–3237.
44.
Walzer PD, Ashbaugh A, Collins M, Cushion MT. 2001. Anti-human immunodeficiency virus drugs are ineffective against Pneumocystis carinii in vitro and in vivo. J Infect Dis 184:1355–1357.
45.
Rahman SA, Singh Y, Kohli S, Ahmad J, Ehtesham NZ, Tyagi AK, Hasnainb SE. 2014. Comparative analyses of nonpathogenic, opportunistic, and totally pathogenic mycobacteria reveal genomic and biochemical variabilities and highlight the survival attributes of Mycobacterium tuberculosis. mBio 5:e02020-14.
46.
Matt U, Selchow P, Molin MD, Strommer S, Sharif O, Schilcher K, Andreoni F, Stenzinger A, Zinkernagel AS, Zeitlinger M, Sander P, Nemeth J. 2017. Chloroquine enhances the antimycobacterial activity of isoniazid and pyrazinamide by reversing inflammation-induced macrophage efflux. Int J Antimicrob Agents 50:55–62.
47.
Gomes A, Pérez B, Albuquerque I, Machado M, Nogueira F, Prudêncio M, Teixeira C, Gomes P. 2014. N-cinnamoylation of antimalarial classics: new quinacrine analogues with reduced toxicity and dual-stage activity. ChemMedChem 9:305–310.
48.
Maciejewska D, Zabinski J, Kaźmierczak P, Rezler M, Krassowska-Świebocka B, Collins MS, Cushion MT. 2012. Analogs of pentamidine as potential anti-Pneumocystis chemotherapeutics. Eur J Med Chem 48:164–173.
49.
Cushion MT, Walzer PD. 2009. Preclinical drug discovery for new anti-Pneumocystis compounds. Curr Med Chem 16:2514–2530.

Information & Contributors

Information

Published In

cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 62Number 11November 2018
eLocator: 10.1128/aac.00983-18

History

Received: 11 May 2018
Returned for modification: 12 July 2018
Accepted: 19 August 2018
Published online: 24 October 2018

Permissions

Request permissions for this article.

Keywords

  1. Pneumocystis
  2. chloroquine
  3. cinnamic acids
  4. immunosuppressed
  5. lung infection
  6. malaria
  7. pneumonia
  8. primaquine

Contributors

Authors

Ana Gomes
LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal
Ricardo Ferraz
LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal
Ciências Químicas e das Biomoléculas, Escola Superior de Saúde, Politécnico do Porto, Porto, Portugal
Lauren Ficker
Cincinnati Veterans Affairs Medical Center, Cincinnati Education and Research for Veterans Foundation, Cincinnati, Ohio, USA
Margaret S. Collins
Cincinnati Veterans Affairs Medical Center, Cincinnati Education and Research for Veterans Foundation, Cincinnati, Ohio, USA
Cristina Prudêncio
Ciências Químicas e das Biomoléculas, Escola Superior de Saúde, Politécnico do Porto, Porto, Portugal
i3S–Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
Cincinnati Veterans Affairs Medical Center, Cincinnati Education and Research for Veterans Foundation, Cincinnati, Ohio, USA
Cátia Teixeira
LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal
LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal

Notes

Address correspondence to Paula Gomes, [email protected].

Metrics & Citations

Metrics

Note:

  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

View Options

Figures and Media

Figures

Media

Tables

Share

Share

Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy