Article pubs.acs.org/jmc
Antimalarial Activities of 6‑Iodouridine and Its Prodrugs and Potential for Combination Therapy Ian E. Crandall,† Ewa Wasilewski,‡ Angelica M. Bello,†,‡ Asif Mohmmed,§ Pawan Malhotra,§ Emil F. Pai,∥,⊥ Kevin C. Kain,#,∞ and Lakshmi P. Kotra*,†,‡,# †
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, M5S 3M2, Canada ‡ Center for Molecular Design and Preformulations, Toronto General Research Institute, University Health Network, 5-356 TMDT/MaRS, 101 College Street, Toronto, Ontario, M5G 1L7, Canada § International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India ∥ Ontario Cancer Institute, Campbell Family Cancer Research Institute, Toronto Medical Discoveries Tower, 101 College Street, Toronto, Ontario, M5G 1L7, Canada ⊥ Departments of Medical Biophysics, Biochemistry, and Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, Ontario, M5S 1A8, Canada # McLaughlin Center for Molecular Medicine and Department of Medicine, University of Toronto, Toronto, Ontario, Canada ∞ McLaughlin-Rotman Center for Global Health, Toronto General and Western Hospital Foundation, Toronto Medical Discoveries Tower, 101 College Street, Toronto, Ontario, M5G 1L7, Canada S Supporting Information *
ABSTRACT: Resistance by Plasmodium falciparum to almost all clinically used antimalarial drugs requires the development of new classes of antimalarials. 6-Iodouridine (15), a novel and potent inhibitor of orotidine 5′-monophosphate decarboxylase (ODCase), exhibited efficacy in a mouse model infected by P. chabaudi chabaudi. Compound 15 exhibited promising antimalarial activity against P. falciparum, including drug-resistant isolates, and no rapid drug-resistant populations of the parasite were observed when challenged with 15. Uridine provided options to overcome any toxicity in the host but still suppressing the parasite load when treated with 15. In drug combination studies, compound 15 showed good efficacy in vivo with artemisinin and azithromycin. The propionyl ester of 15 exhibited superior antimalarial efficacy. Antimalarial activities of 15 and its prodrugs and potential for combination therapy are discussed in the context of novel strategies.
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INTRODUCTION Malaria is a parasitic infection in humans caused primarily by Plasmodium falciparum or P. vivax, with a minor fraction (95%. 6-Iodouridine (15). Compound 15 was synthesized following the method previously described.25 1H NMR (CD3OD) δ 6.42 (s, H5), 5.94 (d, H1′), 4.69 (dd, H2′), 4.31 (t, H3′), 3.87 (dt, H4′), 3.79 (dd, H5′), 3.66 (dd, H5″); 13C NMR (DMSO-d6) δ 161.92 (C4), 147.83 (C2), 118.37 (C6), 115.93 (C-5), 102.25 (C1′), 85.36 (C4′), 71.83 (C2′), 70.28 (C3′), 62.58 (C5′). UV (λmax, MeOH) 267.0 nm. ESI (+ve) m/z = 370.88 [M + H]+, m/z = 392.88 [M + Na]+. 5′-Propionyl-6-iodouridine (16). A solution of 6-iodouridine 15 (100 mg, 0.27 mmol) in anhydrous dimethylformamide (1 mL) was cooled to 0 °C and treated with anhydrous pyridine (33 μL, 0.41 mmol). After being stirred for 15 min, the mixture was treated with propionyl chloride (25 mg, 0.27 mmol) and stirred for an additional 1 h. The reaction mixture was then diluted with ethyl acetate (15 mL), washed with water (8 mL), brine (8 mL), and dried over anhydrous sodium sulfate. The organic layers were concentrated and the crude product was purified by silica gel column chromatography (0−8% MeOH in CH2Cl2) to yield 16 (45 mg, 39%). 1H NMR (CDCl3) δ 6.47 (s, H-5), 5.93 (d, H-1′), 4.67 (dd, H-1′), 4.46 (m, H-3′ and H5″), 4.25 (dd, H-5″), 4.04 (dt, H-4′), 2.34 (q, CH3-CH2-CO-), 1.14 (t, CH3-CH2-CO); 13C NMR (DMSO-d6) δ 173.97 (C6′, CO ester), 161.90 (C4), 147.70 (C2), 118.10 (C6), 115.93 (C5), 102.60 (C1′), 81.77 (C4′), 72.07 (C2′), 70.34 (C3′), 64.43 (C5′), 27.11 (CH2-CH3, C-7′), 9.38 (-CH3, C8′). UV (λmax, MeOH) 267.4 nm. ESI (+ve) calculated for C12H15N2O7I [M + H]+ 427.0002, found 426.9375. 5′-Butyryl-6-iodouridine (17). A solution of 6-iodouridine 15 (100 mg, 0.27 mmol) in anhydrous acetonitrile (1 mL) was cooled to 0 °C and treated with anhydrous pyridine (20 μL, 0.27 mmol). After 15 min of being stirred, the reaction mixture was treated with butyryl chloride (25 mg, 0.27 mmol) and then stirred at 4 °C for 18 h. The reaction mixture was concentrated, and the crude product was purified by column chromatography (0−5% MeOH in CH2Cl2) to yield 17 (37 mg, 31%). 1H NMR (CDCl3) δ 6.48 (s, H-5), 5.99 (d, H-1′), 4.68 (dd, H-2′), 4.54 (t, H-3′), 4.47 (dd, H-5′), 4.20 (dd, H-5″), 4.04 (dt, H-4′), 2.33 (t, CH3-CH2-CH2-CO-), 1.64 (m, CH3-CH2-CH2-CO-), 0.93 (t, CH3- CH2-CH2-CO-); 13C NMR (DMSO-d6) δ 173.10 (CO ester, C6′), 161.90 (C4), 147.68 (C2), 118.36 (C6), 115.92 (C5), 102.58 (C1′), 81.73 (C4′), 72.05 (C2′), 70.35 (C3′), 64.33 (C5′), 35.63 (CO-CH2-, C7′), 18.29 (CH2-CH3, C8′), 13.85 (-CH3, C9′). UV (λmax, MeOH) 267.4 nm. HRMS (ESI, +ve) calculated for C13H18N2O7I [M + H]+ 441.0154, found 440.9897. P. falciparum Cultures and CHO Cell Assays. The effects of compounds 15−17 on P. falciparum cultures were determined using a lactate dehydrogenase (LDH) assay that specifically detects parasite viability.27 Cultures of the laboratory line ItG were maintained following the method of Trager and Jensen using RPMI 1640 supplemented with 10% human serum, 25 mM HEPES, gentamicin, and 50 μM hypoxanthine (RPMI-A). All chemicals used in viability assays were purchased from Sigma Aldrich Canada (Oakville, Ontario, Canada). Briefly, compounds were dissolved in RPMI-1640 medium at 10 mg/mL and then filter-sterilized. To produce a serial dilution of compounds across a 96-well assay plate, an amount of 50 μL of RPMIA was added to each well. RPMI-A (40 μL) and a solution of an inhibitor (10 μL) were added to the wells in the first column, and the contents of the wells were mixed. Fifty microliters were removed from the wells of the first column and added to the next well in the series, and the process was repeated until the next-to-last well was reached.
Scheme 1. Synthesis of Compounds 16 and 17
Compounds 7, 8, and 10 are competitive inhibitors, and compounds 9 and 11 are potent covalent inhibitors of ODCase.22 Compound 11 was the first known covalent inhibitor of ODCase and has nanomolar potency.23 6Iodouridine 5′-monophosphate (11) inhibits the activity of ODCase from P. falciparum (Pf) irreversibly at a low concentration range of 2−5 times that of the enzyme. The second-order rate constant (kobs/[I]) for 11 was 6.8 × 105 M−1 s−1 against Pf ODCase. The high-resolution X-ray crystal structures of the complexes of compound 11 with ODCases from P. falciparum,22 Methanobacterium thermoautotrophicum,23 and Homo sapiens19 (PDB codes 2QCN and 3BGJ) all confirmed that there is indeed a covalent bond between Nε of Lys72 and C6 of the inhibitor. Compounds 8 and 10 were competitive inhibitors.22 Compound 8 was a nanomolar inhibitor with a Ki in the range of 800−840 nM, whereas the 6-methyl derivative 10 exhibited a micromolar inhibition constant against ODCase.22 Nucleoside derivatives of compounds 7−11 (i.e., their corresponding unphosphorylated derivatives) were evaluated for their antimalarial activities.24 Compound 15 (6-iodouridine, Chart 1) exhibited potent antiplasmodial activities in antiparasitic activity assays in vitro, with IC50 values of 1.2 ± 0.2 μM and 27.0 ± 3.9 μM against P. falciparum ItG and 3D7 isolates, respectively.25 These findings prompted us to further investigate the potential of 15 and its analogues as effective antimalarial agents. We synthesized two prodrugs 16 and 17 with small aliphatic groups and compared them to 15 for their antimalarial activities. Uridine was evaluated for its effects on the inhibitors of ODCase, specifically 15. Compound 15 was also evaluated for its additive and synergistic effects when administered together with azithromycin (18) and artemisinin (19). Here, we report the results of in vitro and in vivo investigations using 15, its prodrugs, and drug combination studies. 2350
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Table 1. Inhibitory Activities of Compound 15 against Plasmodium sp. Isolates in Vitroa species P. falciparum
P. berghei (mouse) P. chabaudi chabaudi (mouse) chloroquine P. falciparum a
r
isolate
IC50 (μM)
3D7 (lab strain) ItG (CQr) KC98 358 (Thailand, CQ/MQr) KC98 (Thailand, CQ/MQr) MB (West Africa, CQ/MDR) SB (India, CQ/MDR) FCR3 (The Gambia)
27.0 ± 3.9 1.2 ± 0.2 1.4 ± 0.4 2.3 ± 0.4 18.7 ± 4.7 29.1 ± 1.9 56.5 ± 14.0 303.0 ± 39.0 300.0 ± 34.0
ItG (CQr)
0.2 ± 0.08
r
CQ , chloroquine-resistant; MQ , mefloquine-resistant; MDR, multidrug resistant.
Figure 1. (A) Dose−response curves for the treatment of CHO cells with compound 15 in the presence (●) and absence (○) of uridine. The fit to range range + background , and eq 2, y = the experimental data was generated in Grafit 5.0 using eq 1, y = . The effect of compound 15 in ⎛ ⎞s ⎛ ⎞s x
⎟ 1+⎜ ⎝ IC50 ⎠
x
⎟ 1+⎜ ⎝ IC50 ⎠
the absence of uridine was divided into two sets and fit individually. The first set of data was fit to eq 1 to account for the nonzero data points (the middle plateau). The second set of data was fit to eq 2 (from the middle plateau to the last point). (B) Dose−response curves for the treatment of P. falciparum cells with compound 15 in the presence (●) and absence (○) of uridine. The fit to the experimental data obtained in the presence of uridine was generated in Grafit 5.0 using eq 2. . This produced a plate with a series of 2-fold dilutions across it except for the last well in the series, which contained RPMI-A alone. Parasite culture (2% hematocrit, 2% parasitemia, 50 μL), with or without 1 mM uridine supplementation, was added to each well, and the plates were then incubated at 37 °C in 95% N2, 3% CO2, and 2% O2 for 72 h. The viability of the parasites in each well was then determined by measuring the LDH enzyme activity of the parasites in individual wells as described previously.27 Experiments to determine the effects of uridine supplementation were performed in parallel with those without uridine supplementation, i.e., on the same days using the same cells and culture conditions, to eliminate possible effects due to variations in the contents of the culture medium or environment. For cell toxicity assays, CHO cells (ATCC, Manassas, VA) were grown in RPMI-1640 supplemented with 10% fetal calf serum (Sigma, St. Louis, MO), 25 mM HEPES, and gentamicin (RPMI-10). Cells were seeded in 96-well plates and grown to 20% confluency in 100 μL of RPMI-10 per well prior to the addition of test compounds. Prior to the introduction of a gradient of the test compound, the RPMI-A in each well was removed and replaced either with 100 μL of RPMI-A or RPMI-A supplemented with 500 μM uridine. A gradient of the test compound was prepared by adding 90 μL of RPMI-10 mixed with 10 μL of compound solution to the first well in the series, mixing, transferring 100 μL to the next well, and repeating until the next-tolast well was reached. After 48 h, the viability of the cells was determined by measuring the ability of the cells to reduce 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO) to its corresponding colored formazan. The medium was discarded from the wells, followed by the addition of 100 μL of 10
mg/mL of MTT in RPMI-10, incubating the plates for an additional 30 min, then removing the medium, adding 100 μL of DMSO, and reading the absorbance at 650 nm.28 The IC50 values of individual compounds were determined by applying a nonlinear regression analysis of the dose−response curve using the computer program Sigma Plot (Jandel Scientific). In Vivo Efficacy Studies. Animals were housed at the Department of Comparative Medicine at the University of Toronto, Canada. Animals were allowed to feed and drink ad libitum. In vivo evaluation of efficacy for the test compounds was performed by infecting 6- to 8week-old female Balb/C mice with 1 × 106 erythrocytes parasitized with either P. chabaudi chabaudi or P. berghei that were obtained from a passage mouse that had previously been infected with cryopreserved parasites. Mice were monitored daily for the presence of parasites by examining a Giemsa-stained peripheral blood film. Once the presence of parasites was confirmed, mice were infused once daily by an intraperitoneal injection with compound or saline alone. Parasitemia was determined by light microscopy, examining a minimum of 400 erythrocytes per sample. Animals were weighed daily and profiled to monitor their well being. The relative weights of the mice were determined by dividing the weight of each mouse on individual days by the mouse’s weight on day 0. Chloroquine, azithromycin, uridine, and artemisinin were purchased from Sigma Aldrich Canada. Compound 20 is an inhibitor of parasite invasion into RBCs and was reported earlier.26 During the experiments animals that were judged to be terminally ill were promptly euthanized. 2351
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RESULTS AND DISCUSSION ODCase is an attractive antimalarial target to investigate because of the essential role of de novo pyrimidine biosynthesis Table 2. Inhibition of Plasmodia Cultures and CHO Cells upon Treatment with Compound 15 in the Presence and Absence of 500 μM Uridine Supplementationa IC50 (μM) CHO P. falciparum therapeutic index
with uridine
without uridine
3.4 ± 3.6 (IC50A) 391.0 ± 9.0 (IC50B) 11.2 ± 0.8 35
491.0 ± 56.0 13.2 ± 1.0 38
a
Two inhibition constants in CHO cells in the presence of uridine indicate the two points of inflection (see also Figure 1). Figure 3. In vivo efficacy studies using 15 and its prodrugs 16 and 17. Mice were infected with 106 P. chabaudi chabaudi parasites on day 0 and were then infused with either saline or 15−17 on days 4, 5, and 6. Data points represent mean parasitemias of 4 animals per group with standard error indicated by bars. Groups are the following: untreated control (●); 6 mg per day of 15 (○); 6.9 mg of 16 (▼); 7.1 mg per day of 17 (▽).
charged monophosphate group; thus, nucleoside derivatives are used in the cell based assays as well as in in vivo studies. Nucleoside derivatives such as 15 are expected to enter the cells where cellular nucleoside kinases convert them into the corresponding monophosphate forms. The results in Table 1 indicate that parasites infecting different species display highly variable susceptibilities toward the same drug. P. falciparum isolates produced IC50 values that varied from 1.2 ± 0.2 to 56.5 ± 14.0 μM with no apparent correlation with their sensitivity to mefloquine or chloroquine susceptibility. Compound 15 inhibited drug-resistant isolates ItG and KC98 at low micromolar concentrations, while multidrug resistant (MDR) isolates such as MB and SB were less susceptible to compound 15.31 This finding is not surprising, as chloroquine and mefloquine act in the food vacuole of the parasite, while 15 acts in the cytosol. A much bigger difference in sensitivity is seen between P. falciparum and the rodent parasites P. berghei and P. chabaudi chabaudi because ODCase enzyme in mice may be less sensitive to 15. It is already known that a variety of ligands to ODCases from different species (such as human, bacterial, and plasmodial) could exhibit a range of inhibitory constants, differing by almost 3 orders of magnitude.32 The observation that mouse parasites are as much as 25- to 40-fold less sensitive to compound 15 compared to P. falciparum suggests that 15 would be more effective in humans and may be a desirable component of future combination therapies. Effect of Compound 15 and Uridine on Cultured Cells. Mammalian CHO cells and P. falciparum cells in culture were both treated with compound 15 and monitored using a dose− response assay (Figure 1). The effect of compound 15 on CHO cells was complex and was dependent on the presence of uridine (Figure 1A and Table 2). When there was no uridine supplement, two distinct inflection points were seen in the dose−response profile with compound 15. At high levels of 15 (>250 μM), parasite growth was completely inhibited, while at lower levels of 15, i.e., concentrations ranging from 5 to 250 μM, reduced growth was observed for the CHO cells. When
Figure 2. Effect of exposure of ring and mature stage parasites to compound 15. Parasite cultures were synchronized to either the ring or mature stage of development and were then exposed to 4 μM 15 for the duration of the experiment (continuous treatment) and for a period of 3 h after which the compound was removed (3 h treatment) or were not exposed to 15 (untreated). The cultures were then allowed to incubate for a further 48 h before culture viability was determined by adding SYBR-Green I. Relative viability was determined by dividing the relative fluorescence by the values obtained for untreated parasites.
in Plasmodium. The absence of a pyrimidine salvage pathway in the Plasmodium genome indicates that these parasites have evolved to rely on de novo synthesis alone. When this mechanism is crippled, the parasite will not survive,6,29 validating this enzyme as a focus of antimalarial drug discovery.30 Identification of compound 11 as a potent inhibitor of ODCase catalytic activity and the finding that its nucleoside analogue 15 is a potent in vitro inhibitor of Plasmodium growth led to further investigations of these compounds as potential antimalarial agents.22,23 Typically, mononucleotide derivatives such as 11 cannot pass through the cell wall because of the 2352
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Figure 4. Effect of compound 19 on the in vivo activity of 15. Mice infected with 106 P. berghei on day 0 were treated on days 5, 6, and 7. Mice were given ip injections of saline (control mice, ●), 3 mg per dose of 15 (○), 3 mg per dose of 15 combined with 200 μg of 19 (▼), or 200 μg of 19 (▽) daily for a period of 3 days, and parasitemia (A) and the relative weight loss (B) were monitored. Results represent the average for 5 mice in each group.
Figure 5. Effect of compound 18 on the in vivo activity of 15. Mice infected with P. berghei were treated on days 4, 5, 6, and 7. The parasitemia was determined by examining blood films (A), and the relative weights of the mice were determined (B) by dividing the weight of the mice on individual days by the mouse’s weight on day 0. Results represent the average for 8 mice in each group. Mice were given ip injections of saline (control mice, ●), 8 mg per dose of 15 (○), 8 mg per dose of 15 combined with 50 μg of 18 (▼), or 50 μg of 18 (▽) daily for a period of 4 days.
Figure 6. Effect of compound 20 on the in vivo activity of 15. Mice infected with P. berghei on day 0 were treated on days 5, 6, and 7. Results represent the average for 5 mice in each group. Mice were given ip injections of saline (control mice, ●), 3 mg per dose of 15 (○), 3 mg per dose of 15 combined with 75 μg of compound 20 (▼), or 75 μg of compound 20 (▽) daily for a period of 3 days, and parasitemia (A) and the relative weight loss (B) were monitored.
the culture medium was supplemented with 500 μM uridine, a single transition point at >250 μM was observed suggesting that the presence of uridine permitted unrestricted growth of the
CHO cells to occur. Cell viability was observed at its maximum when supplemented with uridine at the same concentration range that showed the phase transition in the biphasic profile 2353
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and absence of uridine along with compound 15 (Figure 1A). Above 250 μM 15, complete disruption of the human erythrocytes was observed, which may result from effects other than ODCase inhibition. However at intermediate values of 5−250 μM, the intracellular parasites display decreased vitality. The observations that partial inhibition of the CHO cell culture occurs at a similar concentration to where P. falciparum cultures are completely inhibited (Figure 1B) and that the partial inhibition can be reversed by uridine supplementation suggest that salvage of the uridine is the factor limiting the CHO cultures at intermediate concentrations. This finding reveals that if compound 15 were used to suppress a Plasmodium infection in a mammalian host, uridine supplementation could increase the efficacy of the compound, an advantage that rarely exists for other antimalarial compounds. Effects of Covalent Inhibition of ODCase on Parasite Growth. On the basis of various in vitro experiments, it can be observed that a 48 h exposure to 15 effectively kills P. falciparum cultures,23 and in these in vitro assays a constant compound concentration of the drug is present during the experiment challenging the parasites. However, in the in vivo experiments, a constant concentration of the drug would not be presented to the parasites. Thus, we were interested in timecourse exposure of the parasites to 15 to determine if ODCase activity in P. falciparum could be restored rapidly after exposure to this drug. Parasite cultures were treated with 15 for a period of 3 h. Then the parasites were transferred into a culture medium without the drug and the cultures were allowed to grow for a further 48 h. Malaria parasites have two phases to their asexual growth cycle: (i) the ring stage, in which relatively little metabolic activity occurs, and (ii) the mature stage, which is characterized by high metabolism and rapid protein and nucleic acid synthesis. The viability of ring stage parasites exposed to 15 for 3 h was found to be intermediate in comparison to the continuously treated and untreated controls (Figure 2). This suggested that ring stage parasites are sensitive to ODCase loss, showing affects on their growth. Mature stage parasites, however, did not have a detectable loss of viability after a transient treatment with 15, indicating that functional levels of ODCase activity was restored after the removal of the compound. Suppression of parasites in the mature but not the ring stage of development with a transient dose of chloroquine is consistent with the mechanism of action of chloroquine whereby heme is liberated in large amounts by mature parasites only. This finding suggests that in a mouse model if serum levels of 15 drop below efficacious concentrations, then mature parasites seem to be able to regenerate their ODCase activity and continue to propagate. Effects of Prodrugs 16 and 17. We synthesized two prodrugs to compound 15 by incorporating a small hydrophobic group at the O5′ position. Compound 16, containing a propionyl ester, and compound 17, containing a butyryl ester were synthesized by reacting compound 15 with the appropriate acid chloride (Scheme 1). When 15−17 were assayed for activity in P. falciparum FCR-3 cultures, IC50 values of 81 ± 3, 85 ± 12, and 166 ± 18 μM, respectively, were obtained. The compounds were evaluated in vivo using mice infected with P. chabaudi chabaudi. Compound 15 produced results similar to those of the untreated group, while treatment with 17 resulted in higher parasitemias initially, while 16 appeared to suppress the parasitemia in the later days of the
Figure 7. Effect of compound 15 on the outcome of a P. chabaudi chabaudi infection. Infected mice were infused with either compound 15 alone or compound 15 supplemented with uridine on days 4, 5, and 6. (A) Data points represent mean parasitemias of 4 animals per group with standard error indicated by bars. The groups included untreated control (▲), 6 mg per day of compound 15 (○), and 6 mg of compound 15 combined with 5 mg of uridine (●). (B) Mouse survival for the three groups was plotted: untreated control group represented as a dotted line, compound 15 alone as a solid line, and compound 15 in combination with uridine as a broken line.
when treated with 15 alone. Similar results were obtained when C32 almelanotic melanoma cells were used in the assay (data not shown). Compound 15 inhibited the P. falciparum cultures with an IC50 of 13.2 ± 1.0 μM in the absence uridine and of 11.2 ± 0.8 μM in the presence of 500 μM uridine, indicating little effect of uridine on compound 15’s inhibitory effect on plasmodia (Figure 1B). This observation underscores that mammalian cells (CHO and C32 melanoma) utilize the pyrimidine salvage pathway to take up uridine into the cells, and thus, any growth restriction due to a limited supply of pyrimidine nucleotides caused by compound 15 can be overcome (Figure 1A). The inability of P. falciparum to utilize the salvage pathway to access uridine is clearly demonstrated by the similarity between the response and IC50 values for compound 15 in the presence and absence of uridine (Figure 1B). Both the de novo and salvage pathways for pyrimidine nucleotides synthesis are present in the CHO (mammalian) cells, as exemplified by the two different outcomes in the culture assays resulting from the presence 2354
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Figure 8. Effect of uridine supplementation on the in vivo activity of compound 15. Female Balb/c mice infected with P. berghei were treated with 3 mg/dose (A, B), 6 mg/dose (C, D), or 12 mg/dose (E, F) of compound 15. Mice were then given ip injections of saline (control mice, ●), compound 15 (▼), compound 15 combined with 5 mg of uridine (□), or chloroquine (○) on days 5, 6, and 7. Relative weight was calculated as the weight of individual mice divided by the same mouse’s weight on day 5 of the experiment.
compounds in combination (Figure 4). A combination of 15 and 19 (3 mg and 200 μg, respectively) led to a drop in parasitemia when treatment commenced, and a sharp “rebound” in the parasitemia was observed when treatment was stopped. Weight loss of the mice was used as a surrogate marker for general mouse health,36 and the group treated with the combination of 15 and 19 showed relatively little weight loss during the course of the experiment. Further, the animals in the combination treatment group did not show a decrease in body weight during the experiment. Overall, combining 15 with 19 provided the greatest benefit. Compound 18 inhibits protein synthesis in bacteria and also inhibits the growth of Plasmodium.37 It was of interest to challenge malaria parasites with a nucleic acid synthesis inhibitor (such as 15) and a protein synthesis inhibitor (such as 18) to explore potential for synergy. When P. falciparum was exposed to either 15 or 18 continuously at suboptimal concentrations, no synergistic effects were observed, but an additive effect was observed (Supporting Information). A pilot study was initiated to determine the effect of the combination of 15 and 18 on Plasmodia. Mice were infected with P. berghei and were then treated with 15 (8 mg per dose) alone, 18 (50 μg per dose) alone, or a combination of 15 and 18 (Figure 5). Animals receiving 18 alone showed a progression of parasitemia
experiment (Figure 3). It is not known whether the observed effects resulted because of an altered interaction with the parasite or the host. Mice treated with 17 developed higher parasitemias than the other mouse groups, suggesting that the presence of the butyryl ester was shifting the balance in the parasite’s favor while 16 may have been slightly more efficacious than 15. The addition of esters did not have a dramatic effect; therefore, we turned our attention to combination therapies that may enhance the positive pharmacodynamics of 15. Drug Combination Studies. We examined the effect of combining compound 15 with compounds 18 and 19. Compound 19 is an epoxide-containing compound that rapidly and covalently modifies a spectrum of biomolecules when it is present in the parasite’s cytosol.33 It is frequently used as part of a combination therapy for malaria because it is fast acting; however, it possesses the disadvantage of a short half-life in serum.34 Recent experiments with artemisinin derivatives with longer half-lives are yielding good results.35 When combinations of 15 and 19 were evaluated in the parasite culture, it was observed that compound 15 exhibited an additive effect in combination with 19 (Supporting Information). To determine if 19 enhanced the effect of 15 in vivo, mice were infected with P. berghei and were treated with 19 alone, 15 alone, or the two 2355
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compound 15 alone. There was no significant difference in parasitemia reduction in the 3 mg/dose arm whether coadministered with uridine (5 mg/dose) or not. In parallel, when the weights of the animals were monitored as a surrogate marker for animal health, uridine supplementation made no significant difference at the 3 and 12 mg doses; however, it significantly improved the weight loss at the 6 mg dose. An early onset of weight loss in the 12 mg group was observed, although it was only moderate and further investigation is necessary in this direction. Chloroquine was used as a positive control in these experiments for comparison purposes, and P. berghei parasitemia could be cured by chloroquine because of its high sensitivity to this drug. Drug Resistance to Compound 15. The possible development of parasites resistant to treatments was one of our concerns with our ODCase inhibitors, since it is a problem with current medications. In separate experiments, we evaluated compound 15 for its potential to generate rapid drug resistant populations of the parasite in vitro. To determine if resistance to ODCase inhibitors, in particular to compound 15, would develop rapidly in culture, we implemented a stepwise resistance selection method.41 Briefly, parasite cultures were exposed for 10 days to concentrations of compound 15 that represented 0.5 times the IC50 value, followed by exposure to 3× the IC50 value for 15 days and then followed by 5× the IC50 value for 15 days. In three separate attempts the parasite cultures were observed to grow in the first selection step; however, no viable parasites were observed at 3 days into the second selection step at the concentration of 3× the IC50 concentration. A determination of the IC50 values of unselected cultures and cultures that had been subjected to the first step of selection indicated that no change in the IC50 value had occurred (t test, P = 0.66). Inability to select for parasite populations that were fully or partially resistant to compound 15 suggests that ODCase variants do not already exist, nor are they rapidly generated, in our parasite cultures. Further, the continued suppression of murine parasites in vitro during treatment suggests that rapid selection of resistant parasites is not occurring in this model. This is an important factor for the selection of antimalarial drugs for development in order to avoid the existing drug resistance problem. Although more studies are required, the potential for compound 15 or analogues to provide significant treatment for malaria, perhaps in combination with an uridine adjuvant, seems promising, especially in light of the fact that P. berghei is about 2 orders of magnitude less sensitive than P. falciparum to compound 15. The data obtained from both in vitro cultures and in vivo mouse experiments consistently demonstrate that ODCase is the site of action for compound 15. The in vitro experiments suggest that mammalian cells remain viable but that their replication is constrained by the amount of uridine in their environment, when their ODCase activity is inhibited. Consistent with the hypothesis that compound 15 primarily inhibits ODCase activity, part of the de novo pathway within the cell, and the only way for these parasites to obtain uridine, we observed that parasite cultures are not responsive to changes in the uridine levels in the culture medium. In a culture environment, we expect the levels of compound 15 to remain relatively constant; however, this is not the case in an animal model. Administration of compound 15 to an infected mouse should have two opposing effects: (i) It should inhibit the ODCase enzyme of the parasite, and unless the enzyme can be replaced in a timely manner, this will be fatal to the parasite. (ii)
that was similar to that of untreated animals. The group that received 15 alone displayed a significantly reduced progression of parasitemia, and the animals that received the combination displayed the slowest progression of parasitemia seen in the experiment. Similar results were seen when relative weight loss was determined suggesting that the combination of 15 and 18 provided superior control of the infection compared to each compound used alone. The antimalarial effects of azithromycin are primarily exerted through the apicoplast in the parasite;38 however, the exact function of this organelle in the parasite remains unclear.39 The apicoplast is a chloroplast-like organelle that is required for normal parasite replication; however, as is observed in mitochondria, it contains DNA for its own replication. Although further experiments are required for a better understanding of the mechanism,39 the results above suggest that the combination of 15 and 18 is significantly better than either treatment alone (Figure 5). Further, the combination treatment group lost noticeably less weight than the other groups, supporting the hypothesis that there was a degree of positive interaction between the effects of the two compounds. Azithromycin is notable for having a long half-life in serum;40 therefore, no “rebound effect” was observed in the groups that received this treatment. Finally, the effect of 15 in combination with the novel bistriazolium compound 20 was investigated. Compound 20 inhibits P. falciparum cultures with an IC50 of 8 ± 2 nM and was able to suppress a P. berghei infection in mice. Compound 20 inhibits the interaction of a parasite ligand necessary for erythrocyte recognition with a receptor domain in the erythrocyte membrane protein AE1 and thereby prevents merozoite entry. Compounds 15 and 20 thus also target two unrelated pathways. Treatment with compound 20 alone resulted in a suppression of the parasitemia, as was reported earlier (Figure 6).26 Treatment with the combination of compounds 15 and 20 produced partial parasitemia that was less than that seen with either compound alone; however, a dramatic increase in parasitemia was observed on day 12. Loss of body weight was most notable in the group of mice receiving the combination of 15 and 20, and it was concluded that the combination of 15 and the bis-triazolium compound 20 may not be suitable as a combination therapy. In Vivo Studies with Uridine Supplementation. To determine whether compound 15 itself could be effective in vivo and also to examine the potential effect of including uridine as a supplement during compound 15 dosing, we again set up a study using a mouse malaria model. The study we performed examined the time course of parasitemia in Balb/c mice infected with P. chabaudi chabaudi, treated with either compound 15 on its own or compound 15 plus supplementary uridine (Figure 7A). Treatment with compound 15 and uridine together resulted in parasite suppression during the critical period of days 10−12. Treatment with compound 15 alone enhanced survival (Figure 7B) as expected, but treatment with the combination of compound 15 and uridine further enhanced survival times and rates. Further exploration into the effect of uridine supplementation on the in vivo activity of compound 15 was undertaken using three doses (3, 6, and 12 mg/dose) of compound 15 (Figure 8). Mice were administered compound 15 ip in combination with uridine or without uridine (5 mg/dose) after the development of parasitemia in mice. When uridine was given as a supplement with compound 15 at the 6 and 12 mg/ doses, parasitemia was further reduced in comparison to 2356
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(2) Alonso, P. L.; Brown, G.; Arevalo-Herrera, M.; Binka, F.; Chitnis, C.; Collins, F.; Doumbo, O. K.; Greenwood, B.; Hall, B. F.; Levine, M. M.; et al. A research agenda to underpin malaria eradication. PLoS Med. 2011, 8, e1000406 (DOI: 10.1371/journal.pmed.1000406). (3) Dondorp, A.; Nosten, F.; Yi, P.; Das, D.; Phyo, A.; Tarning, J.; Lwin, K.; Ariey, F.; Hanpithakpong, W.; Lee, S.; et al. Artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 2009, 361, 455−467. (4) Dhingra, N.; Jha, P.; Sharma, V. P.; Cohen, A. A.; Jotkar, R. M.; Rodriguez, P. S.; Bassani, D. G.; Suraweera, W.; Laxminarayan, R.; Peto, R. Adult and child malaria mortality in India: a nationally representative survey. Lancet 2010, 376, 1768−1774. (5) The malERA Consultative Group on Drugs.. A research agenda for malaria eradication: drugs. PLoS Med. 2011, 8, e1000402 (DOI: 10.1371/journal.pmed.1000402). (6) Gero, A. M.; O’Sullivan, W. J. Purines and pyrimidines in malarial parasites. Blood Cells 1990, 49, 253−279. (7) Jones, M. E. Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis. Annu. Rev. Biochem. 1980, 49, 253−279. (8) Miller, B. G.; Wolfenden, R. Catalytic proficiency: the unusual case of OMP decarboxylase. Annu. Rev. Biochem. 2002, 71, 847−885. (9) Radzicka, A.; Wolfenden, R. A proficient enzyme. Science 1995, 267, 90−93. (10) Sievers, A.; Wolfenden, R. Equilibrium of formation of the 6carbanion of UMP, a potential intermediate in the action of OMP decarboxylase. J. Am. Chem. Soc. 2002, 124, 13986−13987. (11) Snider, M. J.; Wolfenden, R. The rate of spontaneous decarboxylation of amino acids. J. Am. Chem. Soc. 2000, 122, 11507−11508. (12) Warshel, A.; Florian, J. Computer simulations of enzyme catalysis: finding out what has been optimized by evolution. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5950−5955. (13) Donovan, W. P.; Kushnerm, S. R. Purification and characterization of orotidine-5′-monophosphate decarboxylase from Escherichia coli K-12. J. Bacteriol. 1983, 156, 620−624. (14) Pragobpol, S.; Gero, A. M.; Lee, C. S.; O’Sullivan, W. J. Orotate phosphoribosyltransferase and orotidylate decarboxylase from Crithidia luciliae: subcellular location of the enzymes and a study of substrate channeling. Arch. Biochem. Biophys. 1984, 230, 285−293. (15) Kotra, L. P.; Pai, E. F.; Bello, A. M.; Fujihashi, M. Inhibitors of Orotidine Monophosphate Decarboxylase (ODCase) Activity. U.S. Patent 8,067,391 B2, 2011. (16) Christopherson, R. I.; Lyons, S. D.; Wilson, P. K. Inhibitors of de novo nucleotide biosynthesis as drugs. Acc. Chem. Res. 2002, 35, 961−971. (17) Levine, H. L.; Brody, R. S.; Westheimer, F. H. Inhibition of orotidine-5′-phosphate decarboxylase by 1-(5′-phospho-beta-Dribofuranosyl)barbituric acid, 6-azauridine-5′-phosphate, and uridine5′-phosphate. Biochemistry 1980, 19, 4993−4999. (18) Scott, H. V.; Gero, A. M.; O’Sullivan, W. J. In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo. Mol. Biochem. Parasitol. 1986, 18, 3−15. (19) Wittmann, J. G.; Heinrich, D.; Gasow, K.; Frey, A.; Diederichsen, U.; Rudolph, M. G. Structures of the human orotidine-5′-monophosphate decarboxylase support a covalent mechanism and provide a frame work for drug design. Structure 1980, 16, 82−92. (20) Krungkrai, J.; Krungkrai, S. R.; Phakanont, K. Antimalarial activity of orotate analogs that inhibit dihydroorotase and dihydroorotate dehydrogenase. Biochem. Pharmacol. 1992, 43, 1295−1301. (21) Seymour, K. K.; Lyons, S. D.; Phillips, L.; Rieckmann, K. H.; Christopherson, R. I. Cytotoxic effects of inhibitors of de novo pyrimidine biosynthesis upon Plasmodium falciparum. Biochemistry 1994, 33, 5268−5274. (22) Bello, A. M.; Poduch, E.; Liu, Y.; Wei, L.; Crandall, I.; Wang, X.; Dyanand, C.; Kain, K. C.; Pai, E. F.; Kotra, L. P. Structure−activity relationships of C6-uridine derivatives targeting plasmodia orotidine monophosphate decarboxylase. J. Med. Chem. 2008, 51, 439−448.
It will lead to a temporary loss of ODCase activity in the host which may lead to impairment of many functions including the immune regulation of the parasite infection.42 The second effect is proposed as a possibility for the mice study where higher in vivo doses of 15 were needed in order to reach the effective doses for P. chabaudi chabaudi that are 20−25 times higher than that of P. falciparum. We hypothesized that adjunct uridine therapy should improve the outcome of mice treated with compound 15. We investigated the effects of uridine supplementation in combination with 15 on Plasmodium, on the mammalian cells, and in an in vivo malaria model, since the presence of uridine should only affect mammalian cells. There is evidence that this hypothesis is correct, since we observed that uridine administered with compound 15 (6 mg dose) did result in an improved outcome. However, this was not observed when compound 15 was administered at 3 and 12 mg doses. The lowest dose at 3 mg did not suppress parasitemia completely, and the mice were sick. At the higher dose of compound 15 at 12 mg dose, there was no observable improvement in the suppression of parasitemia in comparison to the group receiving 6 mg dose, and early weight loss was observed. The combined experimental results also support the hypothesis that general toxicity due to ODCase inhibitors used in malaria treatment can be reversed by the administration of uridine. The data from the in vivo mouse studies using compound 15 are consistent with those obtained from the in vitro studies against P. falciparum, confirming that ODCase is the most significant target in both systems. The results also suggest ways to improve the efficacy and outcomes of compound 15 in vivo. Coadministration of uridine with compound 15 reduced host toxicity at higher doses of 15 while maintaining the efficacy.
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ASSOCIATED CONTENT
* Supporting Information S
Purity data for compounds 16 and 17; isobolograms and experimental details for the combination studies of 15 with 18 and 19. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Address: University Health Network/TGRI, 5-356 TMDT/ MaRS, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: (416) 581-7601. Fax: (416) 581-7621. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant from the ISTPCanada (L.P.K.), Department of Biotechnology, Government of India (A.M., P.M.), Genome Canada (K.C.K.), Canadian Genetics Disease Network (K.C.K.), and PSI (K.C.K). E.F.P. and K.C.K. acknowledge support through the Canada Research Chairs program. This research was funded in part by the Ontario Ministry of Health and Long Term Care. The views expressed do not necessarily reflect those of the OMOHLTC.
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