Figures
Abstract
Chloroquine (CQ) is a cost effective antimalarial drug with a relatively good safety profile (or therapeutic index). However, CQ is no longer used alone to treat patients with Plasmodium falciparum due to the emergence and spread of CQ-resistant strains, also reported for P. vivax. Despite CQ resistance, novel drug candidates based on the structure of CQ continue to be considered, as in the present work. One CQ analog was synthesized as monoquinoline (MAQ) and compared with a previously synthesized bisquinoline (BAQ), both tested against P. falciparum in vitro and against P. berghei in mice, then evaluated in vitro for their cytotoxicity and ability to inhibit hemozoin formation. Their interactions with residues present in the NADH binding site of P falciparum lactate dehydrogenase were evaluated using docking analysis software. Both compounds were active in the nanomolar range evaluated through the HRPII and hypoxanthine tests. MAQ and BAQ derivatives were not toxic, and both compounds significantly inhibited hemozoin formation, in a dose-dependent manner. MAQ had a higher selectivity index than BAQ and both compounds were weak PfLDH inhibitors, a result previously reported also for CQ. Taken together, the two CQ analogues represent promising molecules which seem to act in a crucial point for the parasite, inhibiting hemozoin formation.
Citation: Aguiar ACC, Santos RdM, Figueiredo FJB, Cortopassi WA, Pimentel AS, França TCC, et al. (2012) Antimalarial Activity and Mechanisms of Action of Two Novel 4-Aminoquinolines against Chloroquine-Resistant Parasites. PLoS ONE 7(5): e37259. https://doi.org/10.1371/journal.pone.0037259
Editor: Anne Charlotte Gruner, Museum National d'Histoire Naturelle, France
Received: September 29, 2011; Accepted: April 19, 2012; Published: May 23, 2012
Copyright: © 2012 Aguiar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Work supported from the Brazilian funding agencies MCT/CNPq/CT-Saúde/MS/SCTIE/DECIT, FIOCRUZ-PAPES, FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais), FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro) and by CNPq (Conselho Nacional de Pesquisa) fellowships to AUK, ACCA, FJBF and WAC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Malaria is one of the most prevalent parasitic diseases in the world. It affects approximately 500 million individuals throughout the tropical and subtropical areas of developing countries and causes considerable morbidity and mortality with about 800,000 deaths worldwide each year [1]. P. falciparum infection in non-immune adults and children is often associated with severe cerebral malaria. The global importance of this disease, current limitations of vector control and the absence of an effective vaccine, makes the development of therapeutic antimalarial drugs the main strategy of malaria control [2].
CQ is a cost effective antimalarial drug with a relatively good safety profile [3]. However, CQ is no longer used alone due to the emergence and spread of P. falciparum CQ-resistant strains and, more recently, of P. vivax [4]–[6]. Artemisinin-based combination therapy (ACT) is the first line treatment in P. falciparum malaria (WHO, 2009). However, the limited availability of ACT and the decreased susceptibility of P. falciparum to artemisinin derivatives [7]–[8] have required the development of novel antimalarial drugs [9]–[11]. Previous studies have described the discovery of novel antimalarial drugs through analysis of medicinal plants [12] and through novel drug synthesis protocols [13]–[14], however, no new active compound has been shown to be as effective as CQ.
Despite the resistance of P. falciparum to CQ, novel drug candidates based on the structure of CQ continue to be considered [15]–[18]. In the present work, CQ analogs were synthesized as mono- and bisquinoline-based derivatives, referred as MAQ and BAQ, respectively. The main structural aspects considered included the maintenance of the 4-aminoquinoline pharmacophore group and the presence of proton-accepting sites to increase drug bioavailability in the digestive vacuole of the parasite. The compounds were tested: (i) as blood schizonticides against P. falciparum in vitro and against P. berghei malaria in mice; (ii) for their in vitro cytotoxicity; (iii) for their ability to inhibit hemozoin formation; and (iv) for their binding mode to lactate dehydrogenase and dimeric hematin in silico.
Results
Synthesis and characterization of CQ analogs
The structure of the two aminoquinoline derivatives, MAQ and BAQ, and their synthesis strategy are depicted in Scheme S1. Both CQ analogs possess the conserved 4-aminoquinoline moiety (a crucial pharmacophore group), and have a chlorine substituent at the 7 position on the quinoline ring, and, both have amino groups on the alkyl chain [19]. MAQ, a new monoquinoline molecule, and BAQ, a previously synthesized bisquinoline already tested against P. falciparum and proven to be active [20], were prepared from 4,7-dichloroquinoline and diethylenetriamine. These reactions occurred via a SNAr synthesis step, which eliminated the use of solvents [21]. MAQ and BAQ were obtained by controlling the stoichiometric relationship between these reagents. The synthesis protocol for the bisquinoline compound BAQ was described previously [20].
BAQ and MAQ were isolated as white solids, which underwent a satisfactory elemental analysis and were fully characterized by NMR and IR spectroscopy. On the 1H NMR spectrum, MAQ showed five of the expected signals of the aromatic region (between 8.32 and 6.50 ppm) and four signals related to the methylenic groups (between 3.47 and 2.78 ppm). The hydrogen signals of the amino groups were either not present or had an integration level lower than the predicted value due to the fast H/D exchange with the deuterated solvent. In the 13C NMR spectrum, it was possible to identify nine signals related to the aromatic carbons (between 152 and 97 ppm) and the four signals associated with the methylenic carbons (50 to 40 ppm). In the infrared spectrum of MAQ, the typical absorption bands for this kind of chemical structure were observed and the 1H NMR spectrum was consistent with the five typical aromatic signals (between 8.25 and 6.46 ppm). However, only two signals related to the methylenic groups were observed due to the symmetry of the molecule (3.45 and 2.95 ppm). In the 13C NMR spectrum of BAQ, the expected nine signals related to the aromatic carbons (between 152 and 97 ppm) and the two possible signals of the methylenic carbons (46.8 and 41.8 ppm) were observed. The infrared spectrum of BAQ exhibited the absorption bands predicted for this structure.
BAQ and MAQ activities against P. falciparum in vitro
The 4-aminoquinoline derivatives MAQ and BAQ were compared with CQ for their antiplasmodial activities against the W2 clone of P. falciparum (CQ-resistant and mefloquine-sensitive) and against a CQ-sensitive strain 3D7. All compounds showed activity in the nanomolar range in the HRPII and hypoxanthine tests (Table 1). The IC50 values were similar in both assays, although somewhat lower for BAQ in the hypoxanthine test with W2 parasites. As expected, the IC50 values of BAQ and MAQ were lower with the CQ- sensitive 3D7 strain than with W2 CQ- resistant in most tests.
Neither MAQ or BAQ were toxic against hepatoma (HepG2) or kidney (BGM) cell lines, and important, the new CQ analog MAQ was less toxic than BAQ, based on MTT assay (Table 2) or neutral red method (data not shown). Additionally, the CQ analogs were not lytic to human erythrocytes. Even at doses 100 times higher than the IC50 values no hemolysis was detected.
The selectivity index- IS (the ratio between MDL50 and IC50) was determined and both compounds had an IS greater than 90. The IS was particularly high for MAQ, 384 and 1038, against BGM and HepG2 cells respectively (Table 2). Nevertheless, neither compound was better then CQ (IS>3083)
BAQ and MAQ were also active in vivo against rodent malaria
The antimalarial activity of the new CQ analogs was evaluated in mice with P. berghei and is summarized in Table 3. MAQ and BAQ were active in vivo at a dose of 50 mg/kg on days 5, 8 and 10 days post-infection, at 25 mg/kg only MAQ was active. MAQ caused a 95% reduction of parasitemia on day 5. However, only CQ caused 100% suppression of parasitemia and increased the animal survival, but the survival differences were not significant from the other groups.
The antimalarial activity of BAQ and MAQ involves inhibition of hemozoin formation
Whether anti P. falciparum activity of BAQ and MAQ involves inhibition of hemozoin formation was evaluated using an indirect in vitro test. Both compounds significantly inhibited hemozoin formation in vitro (Figure 1) in a dose-dependent reaction. The inhibition was more evident with MAQ than with BAQ or CQ (Figure 1A).
Statistical differences as compared to drug-free controls are indicated in each graph by an asterisk (p<0.05). The p and r values represent the statistical correlation analyses.
Similarly, docking studies with MAQ and BAQ showed that the MolDock Score energies between the antimalarials and the dimeric hematin (Table 4) were approximately –100.000 kcal mol–1. Both aminoquinoline derivatives, similar to CQ, were able to interact with dimeric hematin to form a complex. The best conformations (Figure 2) obtained for the complexes showed that the aromatic rings of both compounds were parallel to the hematin ferriprotoporphyrin group (Figure 3). Weak hydrogen bonds (approximately −5.0 kcal mol–1) corroborate previous studies demonstrating that the hydrophobic interactions were the main contributors for the binding between CQ and the dimer [22]–[23].
(A) Protonated CQ, (B) MAQ-1, (C) MAQ-2, (D) MAQ-3, (E) BAQ-1, (F) BAQ-2.
The antimalarial action of BAQ and MAQ involves binding to lactate dehydrogenase
Table 5 presents the H-bond energy and MolDock Scores of the residues involved in H-bonds with NADH as well as the protonated forms of chloroquine, BAQ and MAQ docked to PfLDH. Figure 4 shows that these compounds presented the most stable energy conformations in the binding site of NADH. This suggests that they are NADH competitors. The H-bond energy between the protonated chloroquine and the enzyme was −3.8 kcal mol−1, a high value compared with that of NADH H-bond energy (−35.3 kcal mol−1). The energy of interaction between the enzyme and the protonated forms of MAQ were approximately −250.00 kcal mol−1. Furthermore, MAQ like the protonated chloroquine, bound to the active site of PfLDH; BAQ-1 and BAQ-2 presented the lowest energies, which were extremely large when compared to NADH. These findings suggest that all these compounds are weak inhibitors of PfLDH, as previously reported for chloroquine [24].
(A) Protonated chloroquine, (B) MAQ-1, (C) MAQ-2, (D) MAQ-3, (E) BAQ-1, (F) BAQ-2.
Discussion
A large series of bisquinoline derivatives containing proton-accepting amino groups on the aliphatic chain moiety has already been described [19] and considered a promising class of compounds for treating drug-resistant malaria parasites. Two CQ analogs MAQ and BAQ were obtained as low-cost antimalarial prototypes, which is important considering that disease transmission is concentrated in developing countries and is most frequent among the impoverished classes of several endemic countries [9]. Comparisons between their antimalarial activities aimed to evaluate the molecular structural-activity relationship between mono- and bisquinoline compounds.
We showed that MAQ and BAQ are active in vitro against CQ-resistant and CQ-sensitive P. falciparum parasites at nanomolar doses in the hypoxanthine incorporation test and in an enzyme-linked immunosorbent assay (ELISA), using antibodies against the histidine-rich protein 2 (HRP-II), a parasite protein.Our results show that MAQ, a new monoquinoline, has a higher IS, in consonance with the tendency of a decreased toxicity when comparing mono- to bisquinoline analogs. Such in vitro activity was confirmed in vivo in mice infected with P. berghei; MAQ was more effective as proven by parasitemia reduction in response to drug treatment.
Further in vitro analysis showed that the compounds interacted with dimeric hematin and inhibited heme polymerization. Although the mechanism of action of CQ is not well understood, several experimental studies describe the interaction between CQ and the dimeric form of heme [25], [26], [27], [28]. This interaction seems responsible for the inhibition of hemozoin formation, a hypothesis further supported by docking studies, which also suggest that all protonated forms of MAQ and BAQ interact with the rings parallel to the heme group, as described previously for CQ [26], [27], [28], [29].
The presence of two quinoline moieties in BAQ increases the probability of hydrophobic interactions when compared with MAQ and also results in lower docking energies for all protonation states of the amino groups. The results of this study suggest that the protonated BAQ at the central nitrogen (BAQ-1) should be more effective at inhibiting heme polymerization.
Experimental and modeling studies have shown that chloroquine binds to PfLDH, suggesting that CQ possesses another mechanism of drug action or a mechanism complementary to the one already proposed [24], [29]. Although PfLDH is not a direct chloroquine target, experimental data have shown its weak binding to chloroquine [30]. The existence of this shared target-binding site suggests that PfLDH is related to drug effectiveness. The docking data (Figure 4) suggest that the protonated forms of BAQ and MAQ present the most stable energetic conformations at the binding site of NADH inside PfLDH. The H-bond energy and MolDock Scores, the residues involved in the H-bonds with NADH and the protonated forms of chloroquine as well as BAQ and MAQ binding to PfLDH (Table 5) suggest that these compounds are NADH competitors. The H-bond energy between protonated chloroquine and the enzyme is −3.8 kcal mol−1, a high value compared with the H-bond energy of NADH (−35.3 kcal mol−1). The energies of interaction between the enzyme and the protonated forms of MAQ were approximately –250.0 kcal mol−1. Furthermore, MAQ compounds showed a similar behavior at the active site of PfLDH compared with protonated chloroquine. BAQ-1 and BAQ-2 possessed the lowest energies, which were extremely large compared with NADH. Overall, these findings suggest that all of these compounds are weak inhibitors of PfLDH, as previously reported for chloroquine [22]–[23].
One of the recent strategies to develop effective antimalarial agents is based on the development of metal-drugs [31], [32], [33]. Thus, MAQ and BAQ were associated with metals readily available to be tested for their antimalarial activity, in promising ongoing tests (data not shown). Metallocomplexed drugs, considered first-line anticancer drugs [34], [35], are increasingly used to develop anti-parasitic agents [36], [37], [38].
Materials and Methods
Reagents and drug synthesis
All reactions for drug synthesis were performed under a 100% argon atmosphere using a dual vacuum/argon line and standard Schlenk techniques. Reagents and solvents were purchased from Sigma Aldrich and used without further purification. The IR spectra were recorded on a Varian 640-IR with an ATR device and the elementary analyses were conducted at the analytical center, University of São Paulo. The 1H NMR spectra were recorded at 400.130 MHz and the 13C NMR spectra at 100.613 MHz on a Bruker instrument (Bruker Avance 400) and were externally referenced to the tetramethylsilane (TMS). Chemical shifts (δ) and coupling constants (J) were expressed in ppm and Hz, respectively. The melting or decomposition points of the structures were determined on a MQAPF 302 apparatus.
Synthesis of the 4-aminoquinolines, MAQ and BAQ
MAQ, N1-(2-aminoethyl)-N2-(7-chloroquinol-4-yl)ethane-1,2-diamine, was prepared according to the synthesis procedures described by Musonda and collaborators [39]. Briefly, a mixture of 4,7-dichloroquinoline (0.98 g, 4.9 mmol) and diethylenetriamine (2.7 mL, 25 mmol) was heated at 80°C for 1 h without stirring, following by heating at 135°C for 3 h with stirring. The mixture was cooled to room temperature, alkalized with an aqueous solution of 10% NaOH (30 mL), extracted with hot EtOAc (3×50 mL) and dried over Na2SO4. The solvent was removed under vacuum and the residue was crystallized with CH2Cl2/hexane, thereby leading to a white solid. Chemical and spectral analyses revealed the following: Yield: 0.52 g, 40%. M.F: C13H17ClN4; M.M.: 264.75 g/mol. Elem. Anal., theoretical: C13H17ClN4, C, 58.98; H, 6.47; N, 21.16, experimental: C13H17ClN4.H2O, C, 55.23; H, 6.54; N, 19.22. M.P.: 99–100°C; I.R. (νmáx cm−1): 3256 (Ar- NH); 3065 (CH quinoline ring); 2929 and 2839 (CHas/s and CH2); 1607, 1577 and 1528 (C = C quinoline ring); 1449 (CH2); 1280 (Ar-C-N); 1133 (C-N); 874, 795 and 761 (δ = C-H out-of-plane aromatic ring). RMN 1H (400 MHz, CD3OD): δ 1.93 (s, NH amine), 2.78 (t, J = 5.76 Hz, 2H, CH2-16), 2.85 (t, J = 5.76 Hz, 2H, CH2-15), 2.95 (t, J = 6.38 Hz, 2H, CH2-13), 3.47 (t, J = 6.38 Hz, 2H, CH2-12), 3.70 (ls, NH aniline), 6.50 (d, J = 5.71 Hz, 1H, Ar-H C-3), 7.35 (dd, J = 9.01 Hz, J = 2.14 Hz, 1H, Ar-H C-6), 7.73 (d, J = 2.14 Hz, 1H, Ar-H C-8), 8.07 (d, J = 9.01 Hz, 1H, Ar-H C-5), 8.32 (d, J = 5.62 Hz, 1H, Ar-H C-2). RMN 13C (100 MHz, CD3OD): δ 151.1 (C-2), 98.28 (C-3), 151.4 (C-4), 124.6 (C-5), 123.08 (C-6), 134.9 (C-7), 126.2 (C-8), 148.2 (C-9), 117.32 (C-10), 50.03 (C-12), 42.08 (C-13), 39.9 (C-15), 38.46 (C-16).
BAQ, N1-(7-chloroquinol-4-yl)-N2-{2-[(7-chloroquinol-4-il)amino]ethyl}ethane-1,2-diamine, was synthesized according to the synthesis procedures described by Zhang and collaborators [40]. Briefly, a mixture of 4,7-dichloroquinoline (1.20 g, 6.00 mmol) and diethylenetriamine (0.22 mL, 2.00 mmol) was heated at 80°C for 6 h without stirring and then at 130°C for 3 h with stirring. The reaction mixture was cooled to room temperature and the resulting solid that formed was washed with an aqueous solution of 10% NaOH (30 mL) and recrystallized with MeOH/water. The solid was recuperated by filtration and dried under vacuum, which lead to a white solid. Chemical and spectral analyses revealed the following: Yield: 0.43 g, 50%. M.F: C22H21Cl2N5; M.M.: 426.34 g/mol. Elem. Anal., theoretical: C22H21Cl2N5, C, 61.98; H, 4.96; N, 16.43; experimental: C22H21Cl2N5.4H2O, C, 53.03; H, 4.91; N, 14.07. M.P.: 212–215°C; IR. (νmax in cm−1): 3272 (Ar- NH); 3071 (CH quinoline ring); 2901 and 2843 (CH2 as/s); 1608, 1576 and 1531 (C = C quinoline ring); 1441 (CH2); 1328 (Ar-CN); 1136 (C-N); 865 and 798 (δ out-of-plane aromatic ring). 1H RMN (400 MHz, CD3OD): δ 2.97 (t, J = 6.22 Hz, 2H, CH2-13), 3.45 (t, J = 6.24 Hz, 2H, CH2-12), 6.45 (d, J = 5.69 Hz, 1H, Ar-H C-3), 7.20 (dd, J = 9.01 Hz, J = 2.14 Hz, 1H, Ar-H C-6), 7.67 (d, J = 2.12 Hz, 1H, Ar-H C-8), 7.87 (d, J = 8.97 Hz, Ar-H C-5), 8.24 (d, J = 5.61 Hz, 1H, Ar-H C-2). RMN 13C (100 MHz, MeOD): δ 150.9 (C-2), 98.3 (C-3), 151.2 (C-4), 124.6 (C-5), 122.6 (C-6), 134.9 (C-7), 126.2 (C-8), 148.14 (C-9), 117.3 (C-10), 46.8 (C-12), 41.8 (C-13).
Continuous cultures of P. falciparum
P. falciparum blood parasites (W2 clone, CQ-resistant and mefloquine-sensitive) were maintained in continuous culture as previously described [41] with slight modifications [42]. The 3D7 strain, CQ- sensitive, originally received from the New York University Medical School, was also used. Briefly, the parasites were kept at 37°C in human erythrocytes (A+) with complete medium (RPMI 1640 supplemented with 10% human serum blood group A+, 2% glutamine and 7.5% NaHCO3) in Petri dishes in a candle jar or in a 25-cm culture flask in a defined atmosphere (with 3% O2, 5% CO2 and 91% N2). Human erythrocytes (A+) and serum blood group A+ were kindly donated by Center of Hemotherapy and Hemathology of Minas Gerais (HEMOMINAS). Immediately before the tests, ring stage-parasites were synchronized in sorbitol [43]. Synchronized parasites were stained with orange green in pre-dried coverslips [44] then immediately examined under a fluorescent microscope. The suspension was adjusted for parasitemia and hematocrit as described below for each test. The infected red blood cells were distributed in a 96-well microtiter plate (Corning, Santa Clara, CA, EUA) at 180 µL/well, in which 20 µL of the test drugs and controls were previously diluted at different concentrations (400-0.625 ng/mL) in 0.005% DMSO (v/v) (Sigma, USA).
In vitro assays with P. falciparum infected erythrocytes
The effects of BAQ and MAQ on the W2 and 3D7 P. falciparum blood cultures were assayed through the incorporation of 3H hypoxanthine (PerkinElmer, Waltham, MA, EUA) [45] and with monoclonal antibodies to the parasite histidine and alanine-rich protein (HRPII) (MPFM ICLLAB-55A®, MPFG55P ICLLAB®, USA) (Noedl 2002). The [3H]-hypoxanthine assay was performed at 1% parasitemia and 1% hematocrit and the level of isotope incorporation was read in a beta-counter (PerkinElmer, Waltham, MA, EUA). The anti-HRPII test was performed at 0.05% parasitemia and 1.5% hematocrit and the HRPII quantification was read at 450 nm in a spectrophotometer (SpectraMax340PC384, Molecular Devices). The results of three of these drug activity assays were expressed as the mean of the half-maximal inhibitory dose (IC50) and compared with the drug-free controls. Curve-fitting was performed using Origin 8.0 software (OriginLab Corporation, Northampton, MA, USA) [46].
Inhibition of hemozoin assay
The hemozoin inhibition assay was performed as described by Ncokazi et al., (2002) [47] with some modifications. Briefly, 10.1 µL of the aminoquinoline derivatives at different concentrations (40-0.62 mg/mL) and 101.2 µL of the hematin stock solution (1.68 mM in 0.1 M NaOH) were combined in triplicate in a flat-bottomed 96-well plate. The solutions were further mixed and 58.7 µL of acetate solution (12.9 M, pH 5.0) then added to each well. After incubation at 60°C, 80 µL of 30% (v/v) pyridine solution in 20 mM HEPES, pH 7.5, were added at room temperature. The solids formed were resuspended and allowed to settle for 15 min at room temperature. Then, 38 µL of the supernatant were transferred to a new plate and diluted to 250 µL with a 30% (v/v) pyridine solution (20 mM HEPES, pH 7.5). The color reaction was read in a spectophometer at 405 nm and the results were expressed as the concentration of free heme in each well, which was calculated from the standard absorbance curve of a known hemin solution. CQ was used as a positive control and the cut off was determined by the amount of free heme in the drug-free control. Each compound was evaluated in two different experiments.
Antimalarial tests against P. berghei in mice
A suppressive parasite growth test was used with P. berghei, NK65 strain (originally received from the New York University Medical School) infected mice as described previously [48] with some modifications [49]. Briefly, adult Swiss outbred mice (20±2 g weigh) were inoculated with 1×105 red blood cells infected with P. berghei, by intraperitoneal route. The infected mice were maintained together for at least 2 h and then divided randomly into groups of 3–5 animals per cage, which were subsequently treated with 25 and 50 mg/kg of each compound diluted in DMSO 3% (v/v) given daily by oral gavage for three consecutive days. Two control groups were used in parallel, one treated with CQ (20 mg/kg) and one with the vehicle. Blood smears from mouse tails were prepared on days 5, 8 and 10 post-infection and then methanol-fixed, stained with Giemsa and examined microscopically. The parasitemia was evaluated and the percent inhibition of parasite growth calculated in relation to the untreated control group (considered 100% growth) using the following equation: [(C - T)/C] ×100; where C is the parasitemia in the control group and T is the parasitemia in the treated group.
The use of laboratory animals was approved by the Ethics Committee for Animal Use of the Oswaldo Cruz Foundation - Fiocruz (CEUA L-0046/08).
Cell cultures and cytotoxicity tests
The human hepatoma cell line (HepG2) was originally received from the New University of Lisbon, and the monkey kidney cell line (BGM) from the Federal University of Minas Gerais. These cells were cultured in 75 cm2 plates with RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 40 mg/L gentamicine in a 5% CO2 atmosphere at 37°C. For the in vitro tests, a confluent cell monolayer was trypsinized, washed with culture medium, distributed in a flat-bottomed 96-well plate (5×103 cells/well) and incubated for 18 h at 37°C to ensure cell adherence.
The cytotoxicity was evaluated with the MTT assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [50]. Briefly, the HepG2 and BGM cell lines were incubated with 20 µL of the compounds at different concentrations (1000-1 µg/mL) for 24 h in a 5% CO2 and air atmosphere at 37°C. For the MTT assay, which evaluates mitochondrial viability, 20 µL of MTT solution (5 mg/mL) were added and the plates were incubated for a further 3 h. After incubation, the supernatant was carefully removed from the wells, followed by the addition of 100 µL DMSO with thorough mixing. The optical density at 570 and 630 nm (background) was determined on an ELISA reader (SpectraMax340PC384, Molecular Devices).
The neutral red assay was used to evaluate cell viability by the accumulation of dye in the viable cell lysosome [51]. Briefly, the cells were incubated for 2 h with 100 µL of a neutral red solution (100 µg/mL). Then, the supernatants were discard and 200 µL of a formaldehyde (0.5% v/v) and CaCl2 (1%) solution were added, which was followed by another 5 min incubation. Finally, the supernatant was removed, then 100 µL of an alcohol-acid solution added to the sediment. The absorbance was read at 540 nm on an ELISA reader (SpectraMax340PC384, Molecular Devices). Cell viability was expressed as the percentage of control absorbance obtained in untreated cells after subtracting the absorbance of the appropriate background. Lastly, the minimum lethal dose for 50% of the cells (MLD50) was determined as previously described [52].
Hemolysis assay
The test and control compounds were diluted in a 0.2% (v/v) DMSO solution and incubated (0–50 µg/mL) with a human erythrocytes suspension (1% hematocrit) at 37°C for 30 min in a shaking water bath. The mixtures were centrifuged at 1000 g for 10 min and the absorbance of the supernatants was measured at 540 nm in an ELISA reader (SpectraMax340PC384, Molecular Devices). The hemolytic rate was calculated in relation to the hemolysis of erythrocytes in 0.05% saponin, which was considered to be 100% [53].
Docking energy calculations
The 3D optimized structure of dimeric hematin was taken from our previous studies [29]. The structures of CQ and its analogs were built using the Gaussian View software and optimized using Gaussian 03 [54]. The structural analysis of CQ analogs (BAQ and MAQ) was performed and all possible protonation forms in the biological medium were considered (Figure 2). The geometry optimizations and partial atomic charge distributions were performed using the B3LYP/3-21G level of theory. The calculations for the docking energies were performed with Molegro Virtual Docker (MVD) software [55] as described previously [25]. The algorithm used in the docking studies was the MolDock Score, which is an adaptation of the Differential Evolution (DE) algorithm [53]. The MolDock Score was used because it yields a higher docking accuracy than other state-of-the-art docking algorithms (MVD: 87%, Glide: 82%, Surflex: 75%, FlexX: 58%) [46].
The 3D structure of PfLDH complexed with NADH and the substrate oxamate used in the present work was obtained from the Protein Data Bank (PDB) file 1LDG [56], [57]. We performed flexible docking studies (considering the presence of water molecules in the crystal) with the protonated forms of BAQ and MAQ to compare these interactions with protonated chloroquine at the active site of PfLDH.
Acknowledgments
We are grateful to Dr. Marcus Fernandes de Oliveira and Juliana B. R. Corrêa Soares for helping with the hemozoin assay.
Author Contributions
Conceived and designed the experiments: MRM TCCF AUK. Performed the experiments: ACCA FJBF WAC RMS ASP. Analyzed the data: ACCA FJBF WAC RMS ASP MRM TCCF AUK. Contributed reagents/materials/analysis tools: MRM TCCF AUK. Wrote the paper: ACCA WAC ASP MRM TCCF AUK.
References
- 1.
WHO (2010) World Malaria Report 2010. Available at: http://www.who.int/malaria/world_malaria_report_2010/worldmalariareport2010.pdf. Accessed: march/2011.
- 2. de Ridder S, van der Kooy F, Verpoorte R (2008) Artemisia annua as a self-reliant treatment for malaria in developing countries. J Ethnopharmacol 120: 302–314.
- 3. Greenwood BM, Bojang K, Whitty CJ, Targett GA (2005) Malaria. Lancet 365: 1487–1498.
- 4. Baird JK (2004) Chloroquine resistance in Plasmodium vivax. Antimicrob Agents Chemother 48: 4075–4083.
- 5. de Santana Filho FS, Arcanjo AR, Chehuan YM, Costa MR, Martinez-Espinosa FE, et al. (2007) Chloroquine-resistant Plasmodium vivax, Brazilian Amazon. Emerg Infect Dis. 13: 1125–1126.
- 6. Tjitra E, Anstey NM, Sugiarto P, Warikar N, Kenangalem E, et al. (2008) Multidrug-resistant Plasmodium vivax associated with severe and fatal malaria: a prospective study in Papua, Indonesia. PLoS Med 5: e128.
- 7. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, et al. (2009) Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 361: 455–467.
- 8. Lin JT, Juliano JJ, Wongsrichanalai C (2010) Drug-Resistant Malaria: The Era of ACT. Curr Infect Dis Rep 12: 165–173.
- 9. Rosenthal PJ (2003) Antimalarial drug discovery: old and new approaches. J Exp Biol 206: 3735–3744.
- 10. Krettli AU, Adebayo JO, Krettli LG (2009) Testing of natural products and synthetic molecules aiming at new antimalarials. Curr Drug Targets 10: 261–270.
- 11. Enserink M (2010) If artemisinin drugs fail, what's plan B? Science 328: 846.
- 12. Krettli , AU (2009) Antimalarial drug discovery: screening of Brazilian medicinal plants and purifi ed compounds. Expert Opin. Drug Discov 4: 95–108.
- 13. Cunico W, Cechinel C, Bonacorso H, Martins M, Zanatta N, et al. (2006) Antimalarial activity of 4-(5-trifluoromethyl-1H-pyrazol-1-yl)-chloroquine analogues. Bioorg Med Chem Lett 16: 649–653.
- 14. de Pilla Varotti F, Botelho A, Andrade A, de Paula R, Fagundes E, et al. (2008) Synthesis, antimalarial activity, and intracellular targets of MEFAS, a new hybrid compound derived from mefloquine and artesunate. Antimicrob Agents Chemother 52: 3868–3874.
- 15. Ryckebusch A, Debreu-Fontaine MA, Mouray E, Grellier P, Sergheraert C, et al. (2005) Synthesis and antimalarial evaluation of new N1-(7-chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine derivatives. Bioorg Med Chem Lett 15: 297–302.
- 16. Yearick K, Ekoue-Kovi K, Iwaniuk DP, Natarajan JK, Alumasa J, et al. (2008) Overcoming drug resistance to heme-targeted antimalarials by systematic side chain variation of 7-chloro-4-aminoquinolines. J Med Chem 51: 1995–1998.
- 17. Ekoue-Kovi K, Yearick K, Iwaniuk DP, Natarajan JK, Alumasa J, et al. (2009) Synthesis and antimalarial activity of new 4-amino-7-chloroquinolyl amides, sulfonamides, ureas and thioureas. Bioorg Med Chem 17: 270–283.
- 18. Andrews S, Burgess SJ, Skaalrud D, Kelly JX, Peyton DH (2010) Reversal agent and linker variants of reversed chloroquines: activities against Plasmodium falciparum. J Med Chem 53: 916–919.
- 19. Blackie MA, Yardley V, Chibale K (2010) Synthesis and evaluation of phenylequine for antimalarial activity in vitro and in vivo. Bioorg Med Chem Lett 20: 1078–1080.
- 20. Girault S, Grellier P, Berecibar A, Maes L, Lemière P, et al. (2001) Antiplasmodial activity and cytotoxicity of bis-, tris-, and tetraquinolines with linear or cyclic amino linkers. J Med Chem 44: 1658–1665.
- 21. Carey FA, Sundberg RJ (1990) Advanced Organic Chemistry Part B: Reactions and Synthesis, 3rd Ed, Plenum, New York pg 597:
- 22. Dorn A, Vippagunta S, Matile H, Jaquet C, Vennerstrom J, et al. (1998) An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials. Biochem Pharmacol 55: 727–736.
- 23. Portela C, Afonso CM, Pinto MM, Ramos MJ (2003) Receptor-drug association studies in the inhibition of the hematin aggregation process of malaria. FEBS Lett 547: 217–222.
- 24. Menting J, Tilley L, Deady L, Ng K, Simpson R, et al. (1997) The antimalarial drug, chloroquine, interacts with lactate dehydrogenase from Plasmodium falciparum. Mol Biochem Parasitol 88: 215–224.
- 25. Penna-Coutinho J, Cortopassi WA, Oliveira AA, França TC, Krettli AU (2011) Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies. PLoS One 6: e21237.
- 26. Dios AC, Casabianca , LB , Kosar A, Roepe PD (2004) Structure of the Amodiaquine-FFIX Oxo dimer solution complex at atomic resolution. Inorg Chem 43: 8078–8084.
- 27. Vippagunta SR, Dorn A, Matile H, Bhattacharjee AK, Karle JM, et al. (1999) Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth. J Med Chem 42: 4630–4639.
- 28. Leed A, Dubay K, Ursos LMB, Sears D, Dios AC, Roepe PD (2002) Solution structures of antimalarial drug-heme complexes. Biochemistry 41: 10245–10255.
- 29. Cortopassi WA, Oliveira AA, Guimarães AP, Rennó MN, Krettli AU, et al. (2011) Docking studies on the binding of quinoline derivatives and hematin to Plasmodium falciparum lactate dehydrogenase. J Biomol Struct Dyn 29: 207–218.
- 30. Read J, Wilkinson K, Tranter R, Sessions R, Brady R (1999) Chloroquine binds in the cofactor binding site of Plasmodium falciparum lactate dehydrogenase. J Biol Chem 274: 10213–10218.
- 31. Chellan P, Nasser S, Vivas L, Chibale K, Smith GS (2010) Cyclopalladated complexes containing tridentate thiosemicarbazone ligands of biological significance: Synthesis, structure and antimalarial activity. J.Organomet. Chem. 695: 2225–2232.
- 32. Sánchez-Delgado RA, Navarro M, Pérez H, Urbina JA (1996) Toward a novel metal-based chemotherapy against tropical diseases. 2. Synthesis and antimalarial activity in vitro and in vivo of new ruthenium- and rhodium-chloroquine complexes. J Med Chem 39: 1095–1099.
- 33. Navarro M, Pérez H, Sánchez-Delgado RA (1997) Toward a novel metal-based chemotherapy against tropical diseases. 3. Synthesis and antimalarial activity in vitro and in vivo of the new gold-chloroquine complex [Au(PPh3)(CQ)]PF6. J Med Chem 40: 1937–1939.
- 34. Gasser G, Ott I, Metzler-Nolte N (2011) Organometallic anticancer compounds. J Med Chem 54: 3–25.
- 35. Galanski M, Jakupec MA, Keppler BK (2005) Update of the preclinical situation of anticancer platinum complexes: novel design strategies and innovative analytical approaches. Curr Med Chem 12: 2075–2094.
- 36. Gabbiani C, Messori L, Cinellu MA, Casini A, Mura P, et al. (2009) Outstanding plasmodicidal properties within a small panel of metallic compounds: Hints for the development of new metal-based antimalarials. J Inorg Biochem 103: 310–312.
- 37. Navarro M, Vásquez F, Sánchez-Delgado RA, Pérez H, Sinou V, et al. (2004) Toward a novel metal-based chemotherapy against tropical diseases. 7. Synthesis and in vitro antimalarial activity of new gold-chloroquine complexes. J Med Chem 47: 5204–5209.
- 38. Ajibade P A, Kolawole GA, O'Brien P, Helliwell M (2006) Synthesis and characterization of Ni(II), Pd(II) and Pt(II) complexes of 2,4-diamino-5-(3, 4, 5-trimethoxybenzyl) pyrimidine complexes. J Coord Chem 59: 1621–1628.
- 39. Musonda CC, Gut J, Rosenthal PJ, Yardley V, Carvalho de Souza RC, et al. (2006) Application of multicomponent reactions to antimalarial drug discovery. Part 2: New antiplasmodial and antitrypanosomal 4-aminoquinoline gamma- and delta-lactams via a ‘catch and release’ protocol. Bioorg Med Chem 14: 5605–5615.
- 40. Zhang H, Solomon VR, Hu C, Ulibarri G, Lee H (2008) Synthesis and in vitro cytotoxicity evaluation of 4-aminoquinoline derivatives. Biomedicine & Pharmacotherapy 62: 65–69.
- 41. Trager W, Jensen J (1976) Human malaria parasites in continuous culture. Science 193: 673–675.
- 42. de Andrade-Neto V, Goulart M, da Silva Filho J, da Silva M, Pinto MC, et al. (2004) Antimalarial activity of phenazines from lapachol, beta-lapachone and its derivatives against Plasmodium falciparum in vitro and Plasmodium berghei in vivo. Bioorg Med Chem Lett 14: 1145–1149.
- 43. Lambros C, Vanderberg J (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418–420.
- 44. Xu L, Chaudhuri A (2005) Plasmodium yoelii: a differential fluorescent technique using Acridine Orange to identify infected erythrocytes and reticulocytes in Duffy knockout mouse. Exp Parasitol 110: 80–87.
- 45. Desjardins R, Canfield C, Haynes J, Chulay J (1979) Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16: 710–718.
- 46. Andrade-Neto V, Brandão M, Oliveira F, Casali V, Njaine B, et al. (2004) Antimalarial activity of Bidens pilosa L. (Asteraceae) ethanol extracts from wild plants collected in various localities or plants cultivated in humus soil. Phytother Res 18: 634–639.
- 47. Ncokazi KK, Egan TJ (2005) A colorimetric high-throughput beta-hematin inhibition screening assay for use in the search for antimalarial compounds. Anal Biochem 338: 306–319.
- 48. Peters W (1965) Drug resistance in Plasmodium berghei Vincke and Lips, 1948. I. Chloroquine resistance. Exp Parasitol 17: 80–89.
- 49. Carvalho L, Brandão M, Santos-Filho D, Lopes J, Krettli A (1991) Antimalarial activity of crude extracts from Brazilian plants studied in vivo in Plasmodium berghei-infected mice and in vitro against Plasmodium falciparum in culture. Braz J Med Biol Res 24: 1113–1123.
- 50. Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89: 271–277.
- 51. Borenfreunda E, Babicha H, Martin-Alguacila N (1987) Comparisons of two in vitro cytotoxicity assays: The neutral red (NR) and tetrazolium MTT tests. Toxicology in vitro 2: 1–6.
- 52. Madureira MC, Martins AP, Gomes M, Paiva J, Proença da Cunha A, et al. (2002) Antimalarial activity of medicinal plants used in traditional medicine in S Tomé and Príncipe islands. J. Ethnopharmacol 8: 23–29.
- 53. Wang C, Qin X, Huang B, He F, Zeng C (2010) Hemolysis of human erythrocytes induced by melamine-cyanurate complex. Biochem Biophys Res Commun 402: 773–777.
- 54.
Frisch MJ (2003) Gaussian 03, Revision C.02 Gaussian, Inc.
- 55. Thomsen R, Christensen M (2006) MolDock: a new technique for high-accuracy molecular docking. J Med Chem 49: 3315–3321.
- 56. Bernstein F, Koetzle T, Williams G, Meyer EJ, Brice M, et al. (1977) The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol 112: 535–542.
- 57. Berman H, Battistuz T, Bhat T, Bluhm W, Bourne P, et al. (2002) The Protein Data Bank. Acta Crystallogr D Biol Crystallogr 58: 899–907.