Understanding Chloroquine Action at the Molecular Level in

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Understanding Chloroquine Action at the Molecular Level in Antimalarial Therapy: X-ray Absorption Studies in Dimethyl Sulfoxide Solution Monika S. Walczak,*,† Krystyna Lawniczak-Jablonska,† Anna Wolska,† Andrzej Sienkiewicz,†,‡ Liliana Suarez,§ Aaron J. Kosar,§ and D. Scott Bohle§ †

Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, PL-02-668 Warsaw, Poland Institute of Condensed Matter Physics, Ecole Polytechnique Federale de Lausanne, Lausanne, CH-1015, Switzerland § Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal QC H3A 2K6, Canada ‡

ABSTRACT: X-ray absorption spectroscopy is used to determine the local atomic structure around the iron atom from a soluble synthetic analogue of malaria pigment (hemozoin), cf. ferrimesoporphyrin IX of mesohematin anhydride, in the absence or presence of chloroquine (CQ) in dimethyl sulfoxide (DMSO). Of particular note are the CQ-induced changes in the structure of mesohematin anhydride, which might confirm the formation of CQ-ferrimesoporphyrin IX complex. Examination of solutions of mesohematin anhydride dissolved in DMSO reveals preservation of the dimerlike structure with the central iron atoms of the ferric porphyrin IX reciprocally linked by propionate side chains, which is typical for hematin anhydride (β-hematin). In the presence of CQ, additional light atoms, such as nitrogen, carbon, and oxygen, were detected surrounding the iron in a distance ranging from 2.48 to 3.77 Å. The changes introduced by CQ in DMSO are different from that observed in the acetic acid solution.

1. INTRODUCTION Malaria remains a severe infectious disease which is difficult to eliminate due to the spread of drug resistant strains.1 Alarmingly, there is no malaria vaccine approved for human use. With approximately 243 million cases and 863 000 attributed deaths, malaria affects more than 40% of the world's population, primarily in the most disadvantaged regions.2 Therapy relying on the synergistic or additive potential of two or more drugs with independent modes of action is recently recommended since sole use of chloroquine and artemisinin is less effective or ineffective against the parasite.3 It has been customarily accepted that quinoline-based antimalarial drugs work at the intraerythrocytic stage of parasite development.4,5 Despite many efforts, however, the exact mechanisms of chloroquine (CQ) action remains elusive. Inside the parasite's food vacuole, human hemoglobin undergoes hydrolysis to amino acids and free heme groups (Fe2þ-protoporphyrin IX), which in turn undergo a one-electron oxidation to produce ferriprotoporphyrin (Fe3þ-protoporphyrin IX). Since free heme groups are toxic and are thought to modify the parasite's vacuole and cell membranes, the parasite detoxifies heme into a neutral and insoluble microcrystalline material called hemozoin.6 Quinoline-based drugs probably exert their antimalarial effect by preventing the crystallization of hemozoin in the parasite's digestive vacuole. The drug may bind to free heme or adsorbed onto the hemozoin crystal. A detailed structure of this heme-drug complex remains the object of numerous theoretical modeling attempts.7-10 But there is an absence of hard physical data to evaluate the meaning of these in silico results. X-ray r 2010 American Chemical Society

absorption spectroscopy is an excellent method to solve this type of problem experimentally at least in the local absorbing atom environment at the distance up to 4 Å. Recent reports11 have examined the mechanism of heme-drug interaction and were focused on resolving the local structure around the central iron atom in the same synthetic analogue of malaria pigment but dissolved in acetic acid. No strong evidence has been found to indicate a complex formed upon addition of CQ in vivo. The existence of a CQ-ferriprotoporphyrin IX complex has, however, been suggested based on in vitro UV absorption spectroscopy.12,13 The π-π character of a complex of the components was claimed on the basis of nuclear magnetic resonance (NMR),14-17 and Raman spectroscopy18,19 studies. Herein, we report the results of the investigation of the local atomic structure of the iron atom in mesohematin anhydride, in the presence or absence of CQ dissolved in dimethyl sulfoxide (DMSO). It is shown that the change of the solvent influences the way of chloroquine action on the mesohematin anhydride, thus proving the importance of liquid environments on the mechanism of the drug action at the molecular level.

2. EXPERIMENTAL METHODS Investigated Materials. Iron(III) mesoporphyrin IX anhydride, chemical formula C68H70N8O8Fe2 ((CH3)2SO), called Received: July 21, 2010 Revised: October 27, 2010 Published: December 29, 2010 1145

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Figure 1. Comparison of the experimentally measured EXAFS spectra (a) and their Fourier transforms (b) for microcrystalline powder phase of mesohematin anhydride (solid line), mesohematin anhydride dissolved in DMSO (MDDMSO, open circles), and mesohematin anhydride and CQ dissolved in DMSO (MDDMSOQ, filled squares).

mesohematin anhydride, and chloroquine, chemical formula C18H26N3Cl, called CQ, were synthesized as it was described elsewhere.11 The solvent dimethyl sulfoxide, chemical formula (CH3)2SO), called DMSO, of 99.9% purity, was purchased from Alfa Aesar (Ward Hill, MA) and used as received. Two solution samples were prepared: a solution of mesohematin anhydride in DMSO called MDDMSO and a solution of mesohematin anhydride and CQ in DMSO called MDDMSOQ. The solutions were prepared in the same way as described previously11 and measured under the same conditions. The iron K-edge extended X-ray absorption fine structure (EXAFS) spectra were measured at the ID26 undulator station at the European Synchrotron Radiation Facility (ESRF) in Grenoble. The EXAFS oscillations χ(E) were extracted from the experimental raw data using VIPER software.20 The χ(E) refining procedure, taking into account single as well as multiple scattering up to fourth order, was performed with Excurve program21 starting from structural model based on the crystallographic data.22,23 The fast spherical wave approximation was applied in calculations of the scattered wave amplitude and phases.24-26

3. RESULTS The structure of the condensed phase of mesohematin anhydride was chosen as a starting model for EXAFS spectra

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analysis. A three-dimensional model was built based on unpublished XRD results, and atomic distance values were corrected according to EXAFS refinement.23 The structural model for local iron environment was then modified during EXAFS refinement taking into account the shift of atom position and its presence. Experimental EXAFS spectra of mesohematin anhydride in DMSO solutions measured with and without chloroquine, along with their Fourier transforms, are presented in Figure 1a,b. These are compared with the spectrum of mesohematin anhydride measured as a powder. Significant differences in the shape of the EXAFS oscillation spectra (Figure 1a) of MDDMSO and MDDMSOQ are visible in the following ranges of the wave vectors: 5-6 and 6.5-8 Å-1. In the ranges of 4-6 and 7-9 Å-1, both solutions spectra are clearly different from the EXAFS function shape for a powder sample of mesohematin anhydride. The oscillation function of MDDMSO within the range of 6.57 Å-1 is similarly located as the one for powder state mesohematin anhydride, whereas the one for MDDMSOQ is clearly shifted in the direction of increasing values of the k wave vector. Fourier transforms of χ(E) EXAFS oscillations of both solutions MDDMSO and MDDMSOQ and mesohematin anhydride powder are markedly different for each of the resolved atomic coordination spheres (Figure 1b). In the preceding paper,23 we attributed to the first peak the scattering contributions from the axial oxygen (Oax), and four pyrrolic nitrogens atoms (N1234) which are present in the range from 1.4 to 2.5 Å. Within the 2.5-4.0 Å range, the 8 CR and 4 Cmeso carbons are emphasized in their scattering input (second peak) and next in the distance up to 4.3 Å a significant contribution is made by 8 Cβ carbon atoms (third peak). An additional feature is turning up in the range 2.4-2.9 Å of Fourier transform of χ(k) oscillation function of the MDDMSOQ solution with chloroquine, suggesting the presence of additional atoms at this distance from the central absorber. To verify this suggestion, quantitative analysis of the spectra was performed. Parameters obtained during the refining process of the EXAFS χ(k) oscillations of MDDMSO and MDDMSOQ solutions are presented in the Table 1 and compared to the EXAFS study of powder mesohematin anhydride.23 In the case of the MDDMSO sample, its EXAFS spectrum could be satisfactorily fitted using a structural model of powder mesohematin anhydride. Nevertheless, the considerable shortening (0.12 Å) in axial oxygen (Oax) ligand distance was detected and the tendency for Cmeso carbons sphere to be located further from Fe atom was observed. The disorder parameters for atoms belonging to the ferric mesoporphyrin IX ring in solution are increasing as expected, contrary to the disorder for oxygen ligand where its DebyeWaller parameter value diminishes by 0.005 Å2. It is difficult to claim the differences in distance values of others than oxygen ligand atoms forming the propionate group (marked as a.a.) because the observed changes are within the estimated error. To construct the proper model for MDDMSOQ spectrum analysis, it appeared to be necessary to include in the model some additional atoms (marked as o.r.) at a distance from 2.48 to 3.77 Å from central absorber. The substantial changes of parameters within the ferrimesoporphyrin IX ring structure after adding the quinoline drug mainly relates to the shortening of the distance of four pyrrolic nitrogens by 0.023 Å and shortening the distance to the sphere of Cmeso carbons by 0.11 Å. Coordination numbers of the axial oxygen and other atoms bound to the oxygen moiety do not significantly differ for both solutions. The same concerns the values of the distance parameter except for carbon C2 a.a. of 1146

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Table 1. Structural Parameters Obtained for Selected Atom Spheres from the Refining Procedure of the Structural Model of Mesohematin Anhydride Molecule Dissolved in DMSO in the Presence (MDDMSOQ) or Absence (MDDMSO) of CQa MDDMSO

MDDMSOQ

mesohematin anhydride

R

51.88

53.69

27.85

E0

-3.0(7) 2

-3.3(7)

N(X)

r(Fe-X) (Å)

2σ (X) (Å )

N(X)

r(Fe-X) (Å)

2σ (X) (Å )

N(X)

r(Fe-X) (Å)

2σ2(X) (Å2)

O1 ax N1234

1 4

1.78(1) 2.083(6)

0.007(3) 0.010(2)

0.96(4) 4

1.80(2) 2.06(1)

0.012(4) 0.013(3)

1 4

1.90(2) 2.07(averaged)

0.012(2) 0.0073(5)

CR

8

3.09(1)

0.011(3)

8

3.08(2)

0.011(4)

8

3.09(averaged)

0.007(1)

Cmeso

4

3.51(5)

0.013(9)

4

3.40(6)

0.015(6)

4

3.46(1)

0.009(2)

Cβ

8

4.28(4)

0.02(1)

8

4.30(7)

0.018(8)

8

4.28(averaged)

0.021(6) 0.007(1)

X

a

-3.0(7) 2

2

2

C1 a.a.

1

3.1(2)

0.011(3)

0.96(4)

3.2(1)

0.011(4)

1

3.06(7)

C2 a.a.

1

3.5(3)

0.010(8)

0.96(4)

4.0(4)

0.018(8)

1

3.72(7)

0.010(6)

O2 a.a.

1

3.90(9)

0.010(8)

0.96(4)

4.0(2)

0.018(8)

1

4.05(3)

0.010(6)

N1 o.r. C1 o.r.

-

1.10(5) 1.01(8)

2.48(6) 2.88(6)

0.002(1) 0.002(1)

-

-

-

C2 o.r.

-

0.98(8)

2.89(6)

0.002(1)

-

-

-

C3 o.r.

-

6.3(1)

3.60(7)

0.014(3)

-

-

-

O1/N2 o.r.

-

2.9(1)

3.77(9)

0.004(2)

-

-

-

The parameters obtained for the powder state mesohematin anhydride are also shown for comparison. Abbreviations used: ax = axial oxygen ligand; o.r. = atoms outside the ring of ferrimesoporphyrin IX; a.a. = axial atoms from the propionate group.

which the distance from the absorber is increased by 0.5 Å. The value of the Debye-Waller factor for axial oxygen ligand is pointing out to the greater structural disorder after chloroquine supplementation. Analysis of the spectra of MDDMSOQ solution with chloroquine identified some additional atoms which do not belong to ferric porphyrin IX ring structure. Among these are an atom of nitrogen at 2.48 Å distance, and two carbon atoms in the distance of around 2.88 Å, and additional light atoms in the distance above 3.5 Å. At that distance it is difficult to distinguish between N and O. Graphical representations of the experimental and fitted χ(k) functions, and their Fourier transforms are shown in the Figure 2a,b for both MDDMSO (1) and MDDMSOQ (2) solutions.

4. DISCUSSION The EXAFS oscillations refined for both MDDMSO and MDDMSOQ solutions point to the fact that the coordination number of the axial oxygen ligand remains very close to unity, with a reasonable value of structural disorder parameter. Therefore, study of the local atomic structure of mesohematin anhydride dissolved in DMSO without and with the chloroquine provides a clear indication of the stability of the dimer structure in solution. This is contrary to the result of the study of mesohematin anhydride dissolved in acetic acid.11 The lack of the second axial ligand bound to the absorber as determined by EXAFS analysis supports the maintenance of the dimer structure of mesohematin anhydride molecule after dissolvation in DMSO. Moreover, no evidence for substitution of the propionate group with solvent molecule was found. To check this, several additional models were considered. The models with additional sulfur or oxygen atom at a distance of up to 4.0 Å did not refine with acceptable quality. A search of the Cambridge Crystallographic Database (CCDB) for oxygen bound ferric DMSO complexes resulted in 15 different complexes with 22 different iron—sulfur separations within a

range of 3.074-3.28 Å. This gives an average value of 3.17 Å. Searching within this set for possible iron-oxygen distances, we found values within a range of 1.928-2.237 Å (average 2.056 Å). From the EXAFS analysis the best possible distances for the additional sulfur and oxygen atoms are 2.64(2) and 2.47(3) Å, respectively, and by comparison to that found in other compounds seem to be physically unreasonable. Moreover, the resulting fits do not match well to the data. The fit quality parameter R for the model with additional single sulfur atom in MDDMSOQ solution is equal to 66.97 (curve 5, Figure 2a,b), and for additional single oxygen atom to 67.03 (curve 4, Figure 2a,b). These values are significantly higher than the R value (53.69) for the model with all additional atoms (o.r.) listed in Table 1. An alternative model with a chlorine atom originating from the chloroquine located close to iron was also checked, Figure 2a,b, curve 3. However, the best fit of the model with chlorine at a distance of 2.62(3) Å turned out to be less probable (with R factor equal to 67.37) than that with the light atoms proposed in Table 1. The next model, with a μ-oxo dimer between two ferrimesoporphyrin IX molecules, was also considered.12,28 This model requires the presence of second strongly scattering Fe atom in the distance about 3.6-4.0 Å. EXAFS analysis unambiguously excludes the possibility of the presence of a μ-oxo dimer in DMSO solution, before or after chloroquine addition. The EXAFS oscillation function which results after excluding from the model all additional atoms marked as o.r. but including Fe at the distance 3.8 Å is shown in Figure 2a,b, curve 6. This is again contrasted with the experimental EXAFS spectrum of dissolved mesohematin anhydride in the presence of chloroquine. The R factor for this structural model with a second iron atom rises to 68.20. Another form of ferric porphyrin IX dimer consists of two hematin molecules interacting noncovalently by their unligated 1147

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Figure 2. Experimental EXAFS and refined oscillation χ(k) functions (a) and their Fourier transforms (b) for the synthetic analogue of hemozoin, mesohematin anhydride, dissolved in DMSO (MDDMSO) (1), and mesohematin anhydride dissolved in DMSO with the presence of chloroquine (MDDMSOQ)—model with additional o.r. atoms as proposed from EXAFS analysis (Table 1) (2), MDDMSOQ—model with additional Cl atom (3), additional O atom (4), additional S atom (5), and additional Fe atom at the distance 3.8 Å (6) instead of o.r. atoms (details in the text).

faces was recently proposed by de Villiers et al.29 Taking into account the parameters we have found for a coordinated oxygen ligand, this model does not seem to be plausible under these conditions for mesohematin anhydride both dissolved with and without chloroquine. Furthermore, we expect that such outer facial ligands would be labile and susceptible to facile exchange and substitution which would lead to a significant reduction of the coordination number as in case of hematin.23 Therefore, the addition of all atoms listed in Table 1 marked o.r. in a ferric mesoporphyrin IX structural model for MDDMSOQ solution with chloroquine leads to a considerable improvement in the agreement of the data with the model as indicated by the parameter R describing the quality of the fit. This parameter improves by a factor of 14.88 (from R = 68.57 for a model without any additional atoms to R = 53.69). In the Figure 3 the models with subsequent addition of consecutive shells of scattering of the additional atoms listed in Table 1 are presented. There is a significant improvement of the agreement of the model with the experimental data with a systematic stepwise addition of scattering atoms as follows: after adding N1 o.r. by value 1.74, after adding C1 o.r. by 3.74, after adding C2 o.r. by 1.68, and after adding C3 o.r. and O1/N2 o.r. by 7.72. It is known that multiple scattering contributions which originate from pyrrolic rings have significant influence on spectra. In all refinement procedures the fourth order of multiple scattering was included. The inclusion of fifth order multiple scattering improves the fit quality value only by 0.44 (Figure 3a,b, curve 6). Reduction of the multiple scattering to third order deteriorates the fit quality value R by 5.87 and influences the refinement in the low range k, 4-6 Å-1 (Figure 3a, curve 7) or agreement with model above 3 Å in Fourier transform of EXAFS (Figure 3b, curve 7).

The detection of additional atoms of N, C, and O within iron's atomic neighborhood might be interpreted as being due to the presence of the chloroquine molecule close to the ferrimesoporphyrin IX ring, and therefore the formation of a complex with the mesohematin anhydride. Looking for structural evidence to support the presence of a nitrogen atom found in the solution with chloroquine in the distance 2.48 Å, we again searched the PDB and CCDB databases. The systems with a ferric porphyrin structure, and having as a ligand a nitrogen atom built into the aromatic ring, e.g., histidine as in hemoglobin, were found but their structures did not reveal any configuration with iron-nitrogen distance beyond the limit of 2.4 Å. Moreover, generally the length of iron-nitrogen ligand bond does not exceed 2.37 Å.27 Therefore, the interaction of the nitrogen and iron cannot be reliably distinguished between either a direct coordination bond or a weak London forces π-adduct. Considering the possible origin of all additional o.r. atoms, the location of the iron atom from ferrimesoporphyrin IX molecule toward CQ structure is proposed (Figure 4) in agreement with the distance that resulted from EXAFS analysis. The optimal location of Fe is found to be the intersection of three spheres with the centers located in the position of nitrogen and two closest to it carbon atoms, all originating from the CQ ring structure. The sphere radiuses are 2.48, 2.88, and 2.89 Å, respectively. Although CQ has three different nitrogens which at first glance might interact with the iron, the EXAFS data suggest that neither the diethylamino nor the secondary amino nitrogens is closely interacting with the iron. In both cases the number of carbon atoms at distance around 3.6 Å, which might be considered as part of C3 o.r. sphere, is lower than inside the proposed model structure where four carbon atoms can be found at the following 1148

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Figure 3. Experimental EXAFS and calculated oscillation χ(k) functions (a) and their Fourier transforms (b) for MDDMSOQ solution after adding shell by shell all additional scattering atoms marked in Table 1 as o.r. Curve (1), all o.r. atoms rejected as in model without chloroquine; curve (2), after addition of N1 o.r.; curve (3), after addition of C1 o.r.; curve (4), after addition of C2 o.r.; curve (5), after addition of both C3 o.r. and O1/N2 o.r.; in all above models 4th-order multiple scattering were included; curve (6), with 5th-order multiple scattering; and curve (7), with 3rd-order multiple scattering.

of CQ and even strong covalent π-backbonding would not cause much alteration in its geometry. The further atoms may originate from no aromatic part of CQ and solvent.

Figure 4. Possible location of iron, from ferrimesoporphyrin IX molecule in relation to CQ structure, determined based on the refined Fe-N1 o.r, Fe-C1 o.r., and Fe-C2 o.r. atoms distances (black lines). Carbon atoms which might be C3 o.r. atoms in the refined model for MDDMSOQ solution are marked with squares.

distances from iron: 3.45, 3.56, 3.64, 3.93 Å. We emphasize that in all of this discussion the CQ relative atoms positions are idealized and neither CQ structure nor CQ behavior in DMSO solution was refined nor experimentally determined in this research. This is not likely to have an important bearing on our conclusions as there is considerable rigidity to the aromatic ring

5. CONCLUSION The local iron environment of mesohematin anhydride— soluble synthetic analogue of malarial pigment—in the solutions of dimethyl sulfoxide before and after treatment with chloroquine was studied by means of X-ray absorption spectroscopy. It is shown that the dissolution of mesohematin anhydride in dimethyl sulfoxide does not affect the dimer character of synthetic hemozoin analogue molecule independently on the presence of chloroquine. However, the strong influence (fit quality factor reduction of 14.88) of antimalarial drug is observed at a distance of 2.5 to 3.8 Å from the absorbing atom. According to EXAFS study, the complex of ferric porphyrin with additional light atoms (carbon, oxygen, and nitrogen) which come from chloroquine molecule, placed within considerable distance range, is most probable. EXAFS method is not able to determine the character of bonding between these atoms and ferric porphyrin ring. Nevertheless, from the refined distances of additional atoms it is possible to conclude that neither of them forms ionic or covalent bonds to the central iron atom. Moreover, the EXAFS results for mesohematin anhydride and CQ in DMSO solutions are clearly different from those results obtained for the same system in aqueous solutions of acetic acid, where monomer-like ferric porphyrin IX structure and modifications around axial oxygen ligand were detected.11 This points to the fact that the formation and structure of the CQ-ferrimesoporphyrin IX complex depends strongly upon the environmental conditions. 1149

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The Journal of Physical Chemistry B Clearly, the most credible information should be obtained in an aqueous milieu mimicking the parasite food vacuole matrix.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: (þ48-22) 843 60 34. Phone: (þ48-22) 843 66 01 ext. 3201.

’ ACKNOWLEDGMENT This work was supported by research grant N20205332/1197, special project ESRF/73/2006 from the Ministry of Science and High Education grants to M.S.W., and by NSERC and CRC grants to D.S.B. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we thank M. Sikora, T. C. Weng, and J. Swarbrick for their assistance in using beamline ID26. We thank P. W. Stephens of the Department of Physics and Astronomy, State University of New York at Stony Brook, NY, for providing the crystallographic data of mesohematin anhydride.

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