Biomimetic Assay for Hematin Crystallization Inhibitors: A New ... - DOI

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Biomimetic Assay for Hematin Crystallization Inhibitors: A New Platform to Screen Antimalarial Drugs Megan A Ketchum, Andrea M Lee, Peter G. Vekilov, and Jeffrey D Rimer Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b01424 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

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Crystal Growth & Design

Biomimetic Assay for Hematin Crystallization Inhibitors: A New Platform to Screen Antimalarial Drugs Megan A. Ketchum1, Andrea M. Lee1,†, Peter G. Vekilov1,2,*, Jeffrey D. Rimer1,* 1

Department of Chemical and Biomolecular Engineering and

2

Department of Chemistry,

University of Houston, 4726 Calhoun Road, Houston, TX 77204, USA KEYWORDS growth inhibitor, malaria, high-throughput, pathological crystallization ABSTRACT Molecular inhibitors are commonly utilized in natural, synthetic, and biological crystallization as a means of regulating crystal size and habit. In pathological crystallization, growth inhibitors are commonly employed as therapies to decrease the rate of crystal growth, thereby reducing the incidence rate of diseases. When designing inhibitors for such purposes, it is often unknown a priori what properties (e.g., structure, functionality) are critical for drug efficacy and specificity. Identification of lead candidates is often accomplished through brute force screening without knowledge of the molecular-level driving force(s) governing favorable inhibitor-crystal interactions. Here, we present a biomimetic assay to characterize the crystallization of hematin, a critical component of Plasmodium parasite survival in malaria. Many antimalarial drugs have

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been shown to function as inhibitors that suppress hematin crystal growth. The increasing resistance of malaria parasites to current antimalarials has created an impetus to design alternative drugs; however, current approaches to identify lead candidates rely on combinatorial methods employing parasite assays to screen compounds, which are incapable of elucidating the effect(s) of inhibitors on hematin crystallization. In this study we use citric-buffer saturated octanol (CBSO) as a physiologically-relevant growth medium to develop a facile, robust method of screening compounds that inhibit hematin crystal growth. As benchmarks, we selected antimalarials and antibiotics that are commonly used in combination therapies. We assessed drug solubility in CBSO and designed a biomimetic assay to quantify the relative efficiency of hematin growth inhibitors. We demonstrate that this assay can be used as a high-throughput platform to screen libraries of compounds as a more streamlined approach to identify inhibitors of pathological crystallization.

1. INTRODUCTION Crystallization is a major part of the pathophysiology of several debilitating diseases, including kidney stones,1,2 atherosclerosis,3,4 gallstones,5,6 and malaria7,8. Correspondingly, suppression of crystal nucleation and growth is a common mechanism of action of the respective therapies. In the case of malaria, crystallization is essential for the survival of the implicated parasite, Plasmodium falciparum. Parasites infiltrate red blood cells and catabolize hemoglobin,9 releasing heme, which rapidly oxidizes to hematin. The latter is toxic to the parasite, but is sequestered into innocuous crystals called hemozoin.10 Many antimalarial drugs block hemozoin formation, inhibit the sequestration of heme, and preserve soluble hematin, thereby eliminating the parasite.11 The increased resistance of parasites to current antimalarial drugs, including the


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most advanced artemisinin combination therapies, is fueling the demand for the development of new drugs.12,13 Currently 40% of the world’s population is at risk for malaria transmission and the disease causes approximately 200 million clinical episodes and 600,000 deaths annually.14-17 The release of hematin and its sequestration into hemozoin occurs within the parasite’s digestive vacuole (DV), a complex environment comprised of membrane interfaces, an acidic aqueous solution with pH 4.8 – 5.5, and a subphase of neutral lipids.7 The latter are mostly mono- and diglycerides that result from the degradation of the membranes of the transport vesicles that supply hemoglobin into the DV. Attempts to replicate hematin crystallization in vitro have traditionally focused on the use of aqueous solvents.18-20 Recent in vivo and in vitro tests, however, suggest that the lipid subphase is the preferred crystallization medium. Notably, transmission electron micrographs21,22 and X-ray tomography images23 of parasites have shown hemozoin crystals seemingly immersed in lipid nanospheres and attached to DV membranes coated with lipid.21,23 While there is still debate regarding the growth medium of hemozoin (i.e., aqueous or organic), we have provided convincing evidence that the lipid environment is crucial for crystallization.24-27 The design of new antimalarial medications relies on screens of large libraries of potential drug molecules aimed to discern differences in inhibitor efficacy. Prior work has concentrated on bulk crystallization assays in aqueous solvents,28-31 which may be less physiologically relevant. Current methods to screen putative inhibitors involve time-intensive parasite assays.32,33 Lead candidates are identified by virtue of their impact on parasite survival without knowledge of their direct effect on hematin crystallization. However, the latter could help streamline the selection of compounds as a more rational and efficient approach to drug screening.


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Here, we present a physiologically-relevant assay based on crystallization from organic solvent to quantify the efficacy of hematin crystallization inhibitors. We performed bulk crystallization tests of the effect of drugs on the rate of crystal growth. This approach was adapted to a high-throughput multi-well configuration that simultaneously assesses numerous drug candidates in less than five hours, significantly faster than assays based on parasite suppression. The proposed assay is a commercially-viable platform for identifying novel antimalarial drugs. 2. EXPERIMENTAL METHODS Chemicals. The following reagents were purchased from Sigma-Aldrich (St. Louis, MO) and were used without further purification: porcine hematin, n-octanol (anhydrous, ≥99%), citric acid (anhydrous), sodium hydroxide (anhydrous reagent-grade pellets, ≥98%), chloroquine diphosphate salt (≥98%), quinine (anhydrous, ≥98%), sulfadoxine (≥98%), amodiaquine dihydrochloride dihydrate, doxycycline hyclate (≥98%), artemisinin (≥98%), mefloquine hydrochloride (≥98%), pyronaridine tetraphosphate (≥95%), porcine hemin (≥98%), NaHCO3 (≥99%), sodium dodecyl sulfate (SDS, ≥98.5%), Drierite (with indicator), and pyridine (anhydrous, ≥99%). Deionized (DI) water was produced by a Millipore reverse osmosis–ion exchange

system

(RiOs-8

Proguard

2–MilliQ

Q-guard).

Monopalmitoylglycerol,

monostearoylglycerol, dipalmitoylglycerol, dioleoylglycerol, and dilinoleoylglycerol were purchased from Nu-Chek Prep (Elysian, MN) and used without further purification. Preparation of Biomimetic Solvent. Citric buffer (pH 4.80) was prepared by dissolving citric acid at 50 mM in DI water and titrating with 0.10 M NaOH under continuous stirring to the desired pH. The buffer was stored with a maximum shelf life of 1 month. Before each


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experiment, the buffer pH was verified. To prepare citric buffer-saturated octanol (CBSO), noctanol (ca. 15 mL) was deposited over citric buffer stock solution (5 mL) held in a pre-cleaned 40 mL glass vial. The resulting two-phase solution was sealed and allowed to equilibrate without stirring at 23°C for 30 min. During this time, the organic layer becomes saturated with water, forming the CBSO solution. The top CBSO layer was carefully removed with a pipette far from the interface to avoid resuspension of the two phases. Fresh CBSO solution was prepared at the beginning of each crystallization experiment. Preparation of Seed Crystals. All glassware was thoroughly cleaned with 0.1 M NaOH to remove residual hematin. The glass containers were then washed with soap and rinsed with DI water and acetone. Hematin solutions for crystal growth experiments were prepared by placing 20 mL CBSO and an amount of hematin powder sufficient for a 6 mM solution in a 20 mL glass vial. The unsealed vial was kept at 100°C in the absence of direct light. Solution samples were periodically extracted from the solution by pipette. Each aliquot was filtered through a 0.2 µm polyvinylidene fluoride (PVDF) membrane, and the hematin concentration was measured spectrophotometrically using a Beckman Coulter DU 800 Spectrophotometer with Tungsten and D2 lamps. Solutions of antimalarials or antibiotics were prepared by placing solid drug powder in contact with 20 mL of CBSO in a clean glass vial sealed with a plastic cap. The solutions were incubated in the dark at 22.6 ± 0.2°C, and then were filtered with a 0.2 µm PVDF membrane. The exact drug concentration was determined spectrophotometrically using extinction coefficients determined from data in Figures S1 – S3 in the Supporting Information, SI. When necessary, the concentration was adjusted by dilution with CBSO.


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To prepare β-hematin seed crystals, a 7.7 mM hemin solution was first prepared by dissolving hemin in 0.1 M NaOH in a sealed glass container that was heated at 60°C in a water bath under continuous stirring at 150 rpm.34 Within 10 min, the solution was neutralized using 1.0 M HCl; we found that exposing hemin to NaOH for more than 20 minutes resulted in an amorphous product. We prepared 12.9 M acetate buffer at pH 4.5 by mixing sodium acetate trihydrate and glacial acetic acid, which was then stored at 60°C for an hour and subsequently incubated at 70°C for 22 hours. This buffer was added to the hematin solution at 1:1 ratio. The mixture was placed in a freezer at -18°C for 15 minutes, and the formed residue was filtered through a 0.20 µm Nylon filter and rinsed sequentially with 100 mL DI water, 100 mL of a 0.225 M NaHCO3 solution containing 0.105 M SDS, and 100 mL DI water. The filter cake was placed in a glass Petri dish over a layer of Drierite and dried for 48 hours in an incubator at 37°C. It was then removed and crushed into a fine powder using a mortar and pestle. This powder was stored for a maximum of three weeks in a sealed vial in the dark. Characterization. Powder X-ray diffraction (XRD) patterns of seed crystals were collected on a Siemens D5000 X-ray diffractometer with CuKα radiation (40 kV, 40 mA, λ = 1.54 Å). Crystal size and morphology were analyzed by scanning electron microscopy (SEM) using a FEI XL-30FEG microscope. Seed samples for microscopy imaging were mounted on metal specimen stands using adhesive carbon tape (Ted Pella Inc.) and coated with ca. 25 nm carbon using an AJA UHV six-source sputtering device. Thermogravimetric analysis (TGA) was carried out using a TA Instruments SDT-Q600 device with nitrogen flowing at a rate of 50 mL min-1. Determination of Drug Solubility. The concentration of antimalarials in CBSO and citric buffer was determined by UV-Visible spectrophotometry using the Beer-Lambert law A = εbc, where A is the absorbance, ε is the extinction coefficient, b is the optical path length, and c is the


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concentration of the attenuating species. To determine the solubility of antimalarial drugs in each solvent, we placed solid drug powder in contact with 5 mL of solvent at room temperature in a clean 6 mL vial. This procedure was repeated until the powder no longer dissolved, and the vial was then sealed with a plastic cap. A total of three vials were prepared for each drug-solvent combination for each temperature that was analyzed. The solutions were incubated at four temperatures: 5 (or 9), 25, 37, and 45°C. Every week, a small aliquot was removed from each vial and filtered (0.22 µm PES membrane) to determine the concentration using the same spectrophotometric procedure described above. The change in concentration was tracked as a function of time and the solubility was determined as the plateau across three time points. In the case of pyronaridine, its relatively high solubility in citric buffer enabled only one determination at 25°C due to a limited supply of reagent. Conversely, the low solubility of artemisinin required an alternative method involving the slow addition of solvent to a known mass of dry powder over the course of one month, until the entire artemisinin powder dissolved. Given the long timeframe of the latter measurement, this procedure was only performed at a temperature of 25°C. Biomimetic Crystallization Assay. A filtered solution of 0.60 mM hematin in CBSO was prepared using the method described above. Hematin seed crystals were added to this solution at a molar ratio of 1:4 seeds to hematin. Portions of the seeded growth solution (ca. 0.2 mL) were extracted by pipette while continuously stirring and transferred to polypropylene centrifuge tubes containing 0.2 mL of an antimalarial drug solution to yield a final hematin concentration of 0.3 mM. Each centrifuge tube was shaken to ensure proper mixing and incubated in the dark at 22.6 ± 0.2°C. To monitor the time evolution of the concentration of uncrystallized hematin, three tubes were removed and analyzed every hour over a five-hour period (for experimental statistics). We first added pyridine (21.1 L, 5% v/v) to re-solubilize the hematin precipitated as


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amorphous solid (pyridine does not induce crystal dissolution within the time scale of the determination). To separate the crystals, we centrifuged each tube at 4000 rpm and 20°C for 10 minutes. The concentration of hematin in the supernatant was determined spectrophotometrically by measuring the absorbance at λ = 525 nm (see Figure S4). The entire procedure is illustrated in Figure 1. For each trial, the rate of crystallization r (mM h-1) is determined from the slope of hematin concentration as a function of time. The percent inhibition of crystallization is calculated using the relation 1

100% ,

(1)

where ri is measured in the absence (i = control) and presence (i = inhibitor) of drug.

Figure 1. Schematic of the biomimetic assay to quantify the efficacy of potential antimalarial drugs as inhibitors of hematin crystallization. (I) Combination of solutions containing an antimalarial drug and crystal seeds suspended in supersaturated hematin; (II) crystal growth over a set period of time; (III) addition of pyridine to solubilize amorphous hematin; and (IV) centrifugation to isolate the supernatant and solids for further characterization. Benchmark studies in centrifuge tubes were adapted to a high-throughput configuration using multi-well plates.


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The above procedure was adapted to a high-throughput configuration (Figure 1) with only minor modification. The volume of growth solution was reduced to accommodate measurements in microwell plates. The concentration of filtered hematin solution in CBSO was increased to 0.60 mM while maintaining the same ratio of seed crystals to hematin. For this study, we used a V-bottom Corning polypropylene 96 well plate. A small volume of hematin/seed solution (ca. 100 L) was transferred by pipette into wells containing an equal volume of CBSO with the desired drug concentration. The plates were immediately shaken to ensure proper mixing and the procedure described above was used to determine the rate of crystallization and percent inhibition. To remove solids from each sample prior to UV-Vis spectroscopy measurements, the plate was centrifuged at 3,000 rpm and 20°C for 10 minutes, then 100 L of the supernatant in each well were transferred to a flat bottom Corning polypropylene 96 well plate. The absorbance at λ = 525 nm was measured for all samples using a Tecan Infinite200 Pro microplate reader (an entire 96-well plate requires 1.5 minutes of scanning time). 3. RESULTS AND DISCUSSION Identifying a Surrogate Lipid Phase. Prior findings21,22,35-39 suggest that the organic subphase within the digestive vacuole (DV) of malaria parasites is the preferred medium for hematin crystallization. We scrutinized the suggested growth environment to identify a solvent that best mimics physiological conditions. The lipid blend identified within the parasite DV is a mixture of five neutral lipids: monopalmitoyl glycerol, monostearoyl glycerol, dipalmitoyl glycerol, dioleoyl glycerol, and dilinoleoyl glycerol. These lipids are reportedly present in the DV in a molar ratio of 2:4:1:1:1, respectively.21 The chemical structure of each lipid is shown in Scheme 1. Only two of the five lipids, dioleoyl glycerol and dilinoleoyl glycerol, are liquid at 25°C.40 To test the phase of the lipid blend, we prepared a mixture of the five lipids in


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proportions according to their reported physiological molar ratio. At room temperature the mixture is solid; the melting point of the lipid blend is reported at approximately 37°C.41 Thus, it is feasible that this mixture is indeed present as a liquid subphase within the parasite DV. However, the lipid blend is not a practical solvent for in vitro tests of hematin crystallization inhibitors owing to the high cost of the constituents, the large quantity of lipids required for highthroughput characterization, and the inability to perform experiments at room temperature. Scheme 1. Chemical structures of five neutral lipids identified in the digestive vacuoles of malaria parasites.21

Given the challenges associated with using a lipid blend, we searched for an alternative organic solvent with properties similar to the lipid blend, but one that is inexpensive, available in large volume, and liquid at room temperature. To this end, we selected n-octanol as a surrogate organic solvent. Octanol possesses a long aliphatic carbon chain and a polar alcohol group that mimics the amphiphilicity of the lipids. Octanol is routinely used to model lipids during


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determinations of the lipophilicity of drugs. Another property of the surrogate solvent that may be significant for hematin crystallization is the presence of water. The organic phase in the parasite DV is in direct contact with an aqueous phase that maintains a pH between 4.8 and 5.5.42,43 To estimate the amount of dissolved water within the organic subphase, we prepared the lipid blend and placed it in intimate contact with water for one hour to generate a water-saturated organic phase. The latter was extracted and the solubility of water in the lipid blend was determined using TGA. We recorded the mass fraction of evaporated solvent as a function of temperature increasing from 25 to 120°C. Three different runs with ramp rates 0.1, 0.3, and 1.0 °C min-1 yielded identical results.

Figure 2. The water content in organic solvents. (A) Thermogravimetric analysis of the biomimetic blend of five lipids at a ramp rate of 0.3oC/min shows the mass loss of water as a function of temperature. (B) Comparison of the water solubility in n-octanol (reported by Segatin et al.)44 and the biomimetic lipid blend.


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As shown in Figure 2A, an increase in temperature from 25 to 85°C results in a loss of mass from the lipid blend, but no further loss was observed between 85 and 120°C. An identical study performed with an anhydrous lipid blend revealed no mass loss at temperatures lower than 120°C. These temperatures are significantly lower than the boiling points of the lipids; therefore, we conclude that the evaporated compound is water. The average amount of dissolved water in the lipid mixture is 8.5 ± 0.5% mass, corresponding to a solubility of 4.2 ± 0.3 mol kg-1. In comparison, the water solubility in noctanol is slightly lower (2.7 mol kg-1), as shown in Figure 2B.44 Previous results have indicated that anhydrous n-octanol does not support hematin crystallization.25 The factors that underlie the need for water in the crystallization solvent are likely related to the shared structure of both physiological and synthetic hematin crystals. In these crystals, hematin assembles into dimers bound by reciprocal COO–-Fe(III) coordination bonds, and connected through chains of COOH···HOOC- hydrogen bonds.45 The formation of hydrogen bonds requires the presence of water and hydrogen ions. Hence, as a biomimetic solvent (i.e., surrogate for the lipid blend) we use n-octanol saturated with citric buffer and refer to it as CBSO. Drug Solubility. An argument that is frequently presented in the literature in favor of hematin crystallization in an aqueous medium is the presumed low solubility of quinoline-class antimalarials in organic solvent, which would seemingly reduce their efficacy as inhibitors of crystal growth in the lipid phase. A low threshold of drug efficacy in hematin crystallization inhibition was identified for chloroquine (CQ) as ca. 1 µM.27 If the solubility of drug in the organic solvent exceeds this concentration, the partitioning of drug from an aqueous phase to the


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organic phase would not limit its efficacy. We determined the solubility of eight drugs listed in Scheme 2. A general approach to malaria treatment, recommended by The World Health Organization,14 involves combination therapies. In this approach, two or more drugs with different modes of action and biochemical targets are applied simultaneously to produce a highly effective treatment.46 For example, an antibiotic such as doxycycline (DC), which causes abnormal cell division, and an antimalarial such as quinine (QN), which inhibits crystallization of hematin, are administered together to eradicate the parasite Scheme 2. Molecular structures of six antimalarials (AQ, QN, CQ, MQ, PY, ART) and two antibiotics (SX and DC) that are commonly used in malaria combination therapies.

from the body. For these reasons, we selected six common antimalarials – amodiaquine (AQ), mefloquine (MQ), pyronaridine (PY), artemisinin (ART), CQ, and QN – and two antibiotics,


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sulfadoxine (SX) and DC. The solubility of each drug was measured in CBSO and citric buffer to assess the difference between organic and aqueous solvents, respectively. Drug solubility was determined spectrophotometrically by monitoring the evolution of soluble drug concentration over a 50-day period. A representative data set for CQ in CBSO (Figure 3A) shows that the soluble drug concentration reaches a plateau with prolonged time.

Figure 3. Drug solubility in CBSO and citric buffer. (A) Evolution of the concentration of chloroquine (CQ) dissolved in CBSO at four different temperatures: 5, 25, 37, and 45°C. (B) Comparison of solubility of six antimalarials and two antibiotics in CBSO (top) and pH 4.8 citrate buffer (bottom) at 25°C. (C and D) Solubility of antimalarials and antibiotics in CBSO (C, left column) and citric buffer (D, right column) as a function of temperature. Dashed lines are interpolations to guide the eye. (E and F) Solubility in CBSO and citric buffer plotted in van ’t Hoff coordinates. Solid lines are linear regression fits.


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We interpret this as thermodynamic equilibrium and the average concentration value at the plateau as solubility. The solubility for each drug at 25oC is listed in Figure 3B for CBSO (top plot) and citrate buffer (bottom plot). We found that the majority of drugs have a solubility that exceeds 100 µM in both solvents, with the exception of PY in CBSO and ART in citric buffer. When comparing the solubility of antimalarials, we observed that QN, MQ and ART have higher solubility in CBSO (that exceed 10 mM). The antibiotics SX and DC exhibit comparable solubility in both solvents, with slightly higher values in CBSO. Note that even the drugs that exhibit lower solubility in CBSO than in citric buffer are soluble at concentrations sufficient to inhibit hematin crystallization. The effect of temperature on drug solubility was quantified within the range 5 – 45°C, which encompasses the conditions selected for bulk crystallization assays in vitro (25°C) and the physiological environment of hemozoin formation in vivo (37°C). Notably, drug solubility within this temperature range does not fall below 1 µM, the approximate threshold concentration for effective inhibition of hematin crystal growth. The determinations in CBSO (Figure 3C) reveal that all tested drugs generally exhibit higher solubility at higher temperature. The solubility of AQ exhibits a 1.5-fold increase. The solubility of MQ and QN is nearly independent of temperature. For the other drugs, the solubility increases only marginally between 25 and 37°C. The effect of temperature on drug solubility in citric buffer (Figure 3D) reveals disparate effects. The solubility increases with temperature for the majority of drugs tested; however, the solubility of DC, and to a lesser extent MQ, decreases with increased temperature. The solubility of PY at 25°C is 223.3 ± 0.6 mM, which lies between that of AQ and CQ; however, limited PY availability prohibited an exhaustive analysis over the full temperature range.


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Van ’t Hoff plots of the temperature dependence of solubility (Figure 3E and F) yield the enthalpy ∆H and entropy ∆S of drug crystallization from the slope and intercept, respectively, of the linear relation between ln Ce and T-1: ∆%

!"# &'

∆( &

(2)

Equation 2 is based on the crystallization equilibrium constant Keq = Ce-1. The values of ∆H and ∆S for each drug extracted from data in Figure 3E and F are listed in the SI (Table S2). Screening Drug Efficacy as Hematin Crystallization Inhibitors. The most significant feature of the proposed assay is the use of CBSO as a solvent. To monitor the extent of hematin crystallization in CBSO we quantified crystalline hematin as the difference between initial hematin in the solution and the amount present during crystallization, as in previous studies.28,29 A problem encountered in this approach is the presence of amorphous hematin, which sequesters a fraction of hematin in a third phase and lowers the measurement of the concentration of uncrystallized hematin. To address this issue, Egan and coworkers47 proposed the addition of pyridine prior to concentration determination. Pyridine forms a relatively strong complex with hematin and dissolves potential amorphous residue.47 In the presence of excess pyridine, all noncrystalline hematin is complexed with pyridine. The amount of hematin-pyridine complex is used as a measure of uncrystallized hematin in solution when determining the rate of hematin crystal growth. Tests using varying concentrations of added pyridine, as discussed in the SI, suggested that a pyridine concentration of 5 % v/v is optimal for the assay. We employed seed crystals (Figure 4A) to bypass nucleation and reduce the time of crystallization for more efficient screening of inhibitors, similar to Chong and coworkers.29 Since


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β-hematin crystal surfaces oxidize in contact with air, seeds should not be stored for long periods of time. Each batch of seeds was analyzed by powder X-ray diffraction (XRD) to

Figure 4. (A) Scanning electron micrograph of hematin crystal seeds. (B) Powder XRD patterns of amorphous hematin (blue line) and crystalline β-hematin (green line). The latter is arbitrarily shifted in the y-axis for visual clarity. (C) Evolution of the concentration of soluble hematin in the absence of seeds and in the presence of amorphous and crystalline seeds. Dashed lines are guides for the eye.


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test their crystallinity. We found that published protocols for seed synthesis can yield either crystalline or amorphous product (Figure 4B). The lack of crystallinity may lead to irreproducible crystallization kinetics. For example, we performed a hematin crystallization assay using three different approaches: (i) absence of seeds, (ii) use of amorphous seeds, and (iii) use of crystalline seeds. The data in Figure 4C demonstrate that there is a marked difference in the induction period and crystallization rates between the three approaches. In the absence of seeds, there is a short induction period (ca. 1 day) followed by a rate of growth much slower compared to the crystalline seeded growth assays. The addition of either amorphous or crystalline seeds eliminates the induction period and leads to more rapid depletion of solute. The use of amorphous seeds can falsely give the appearance of crystallization where the apparent rate of growth (i.e., slope of curves in Figure 4C) lies between the values of non-seeded and crystalline seed assays. We could not determine whether the reduction in free hematin concentration was associated with heterogeneous nucleation and growth of new crystals or condensation of amorphous solid (i.e., the mass of products was insufficient for XRD analysis). This observation underscores the importance of verifying the crystallinity of seed batches. An important aspect of the proposed procedure is that hematin crystallization in the presence or absence of inhibitors can be assessed in a relatively short period of time (≤ 5 hours), which is practical for drug screening assays. Within the first five hours of crystallization the growth rate is approximately linear. Variations from one seed batch to another lead to disparities in the measured rate of crystallization. To account for this experimental uncertainty, we report the effect of antimalarials on hematin crystallization using the percent inhibition, defined in Eq. 1 as the ratio of growth rate in the presence of inhibitor relative to that of the control in the absence of drug. Using this relative change in growth rate minimizes variability between different seed


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batches. An example of hematin growth in the presence of the antibiotic DC is provided in Figure 5. The rate of crystal growth, measured as the slope of the linear correlation (solid lines), decreases monotonically with increasing drug concentration. From these plots we extract drug potency and its efficacy for inhibiting the growth of hematin crystals.

Figure 5. The evolution of free hematin concentration CH over five hours in the presence of varying concentration of doxycycline (DC). The percent inhibition is evaluated according to Eq. 1 using the slopes of CH(t) in the absence of inhibitor and in the presence of the inhibitor to evaluate the crystallization rates rcontrol and rinhibitor, respectively. Solid lines are linear regression fits.

In Figure 6A, we report the percent inhibition of hematin crystallization as a function of inhibitor concentration for four quinoline-class antimalarials: AQ, QN, CQ, and MQ. As shown in Scheme 2, each drug is comprised of different functional moieties, but they share a common quinoline base. With the exception of MQ, all of the tested antimalarials are capable of inhibiting growth by more than 80%. Our findings reveal that 0.1 mM concentration of AQ is sufficient to nearly suppress hematin crystallization. Few studies in literature report growth inhibitors capable of reaching such high efficacy in bulk crystallization assays without leading to the promotion of


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polymorphs or the precipitation of amorphous product. Reported values of percent inhibition for a wide range of crystalline materials tend to be around 60%,48 which is the approximate value we observed for hematin growth in the presence of MQ. The other two antimalarials tested here (CQ and QN) inhibit growth in the range 60 – 90%, which is similar to highly effective inhibitors reported in prior studies of inorganic crystals.49-52 The general trends in Figure 6A are consistent with a Langmuir-like behavior wherein the coverage of inhibitor on hematin crystal surfaces reaches a maximum (corresponding to the plateau in percent inhibition) beyond which any further addition of inhibitor has no appreciable effect on crystal growth.

Figure 6. Percent inhibition of hematin crystallization as function of drug concentration. (A) The effect of four antimalarials: amodiaquine (AQ), chloroquine (CQ), quinine (QN), and mefloquine (MQ). (B) The effect of artemisinin (ART) and two antibiotics: sulfadoxine (SX) and


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doxycycline (DC). The negative percent inhibition observed for SX indicates promotion of hematin crystallization. Each symbol is the average of 9 measurements over 3 separate experiments (with the exception of MQ data, which were collected from 2 separate experiments). Error bars equal two standard deviations and dashed lines are interpolations to guide the eye.

We also determined the effect of one additional antimalarial, ART, along with two antibiotics, DC and SX. The solubility of PY in CBSO (Figure 3) was not sufficient to generate a full inhibition curve. As shown in Figure 6B, quantification of hematin crystallization in the presence of ART reveals no apparent change in growth rate with drug concentration, which is not surprising considering prior studies suggest that an activated form of ART (with the endoperoxide bridge cleaved) is the effective suppressor of malaria parasites.53,54 Conversely, we find that DX is a weak inhibitor of hematin crystallization that is capable of achieving comparable percent inhibition as the antimalarials in Figure 6A, but at much higher drug concentration. Unexpectedly, measurements of SX revealed promotion of hematin crystal growth (observed as negative percent inhibition) at low drug concentration (< 0.06 mM), and no appreciable influence on crystal growth at higher concentrations. Presently, there are three proposed mechanisms for modifier promotion of crystal growth: (i) solute-promoter interactions increase the local supersaturation at crystal interfaces due to an enhanced attraction of solute molecules to the surface,55 (ii) reduction of the energy barrier(s) for solute attachment by disruption of the solvation layers at the crystal surface as a result of promoter-crystal interactions,56-58 and (iii) adsorbed promoters decrease the energy of advancing layers at step edges on the crystal surface.59 Drawing upon examples from literature for other types of crystalline systems, it is reported for calcite (CaCO3) crystallization that certain molecules act as a growth promoter at low concentration and switch to a growth inhibitor at higher concentration.56,60 Prior studies seem to indicate that the promotion-to-inhibition transition


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occurs when the adsorbate coverage on crystal surfaces is sufficient to induce step pinning. In other examples, such as calcium oxalate monohydrate crystallization, molecules have been observed to act strictly as a promoters, irrespective of their concentration.49 In this study of hematin crystallization, the exact mode of action governing SX promotion of growth is not well understood. Table 1. Comparison of antimalarial and antibiotic IC50 values. IC50 values Compound

Assay

Amodiaquine [AQ]

16 µM

Chloroquine [CQ]

25 µM

Quinine [QN]

32 µM

Mefloquine [MQ]

71 µM

Pyronaridine [PY]

--------

Doxycycline [DC]

110 µM

CQ-susceptible parasites (strain)a 7.8 nM (3D7)61 8.5 nM (HB3)61 3.0 nM (NF54)62 10.1 nM (NF54)63 14.0 nM (3D7)61 18.5 nM (HB3)61 13.0 nM (NF54)65 10.0 nM (NF54)62 12.9 nM (NF54)63 25 nM (3D7)66 41 nM (HB3)66 34.2 nM (3D7)61 36.8 nM (HB3)61 22.0 nM (NF54)68 132.0 nM (NF54)63 134 nM (3D7)66 109 nM (HB3)66 43.9 nM (NF54)63 51 nM (3D7)66 36 nM (HB3)66 1.9 nM (NF54)63 24 nM (3D7)66 16 nM (HB3)66 10.8 µM (3D7)66 12.1 µM (HB3)66

CQ-sensitive parasites (strain)a 9.3 nM (wild)64

29.0 nM (KH-266)67 76 nM (wild)64

178 nM (L3)69 90.1 nM (KH-266)67 150 nM (wild)64

11.4 nM (L3)69 67.7 nM (KH-266)67

11.3 µM (wild)64

No --------Sulfadoxine 23.7 µM (wild)70 inhibition [SX] a NF54 = African origin Plasmodium falciparum strain; 3D7 = parasite line cloned from NF54; HB3 = Plasmodium falciparum strain from Honduras; L3 = strain of Plasmodium falciparum from Ivory Coast; wild = strain from Gabon; KH-266 = Southeast Asia origin Plasmodium falciparum strain


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When comparing the relative effect of antimalarial drugs at 50% inhibition of hematin growth, we observed the following order of inhibitor potency (from lowest to highest drug concentration): AQ > CQ > QN > MQ >> DC >> SX. Interestingly, this trend is almost identical to the IC50 values of each drug reported in literature for parasite models using a variety of Plasmodium strains (see Table 1). The IC50 value refers to the inhibitory concentration (IC) of drug where the response (i.e., parasite population) is reduced by half. Prior studies reveal the following order of drug potency (from lowest to highest IC50): AQ > CQ > MQ > QN >> DC >> SX. Comparison of our assay with parasite models shows excellent agreement, with the exception of one outlier: the relative potency of MQ and QN in our assay does not match the parasite model. Caution should be taken when directly comparing the results of in vitro and in vivo studies. Nevertheless, the similarity in IC50 trends suggests that the bulk crystallization assay is an effective tool for identifying lead drug candidates and comparing relative efficacy. High-throughput Assay. The commercial relevance of drug-screening assays relies on their ability to identify lead candidates in a rapid timeframe. As a proof of concept, we adapted the biomimetic assay developed above to high-throughput screening of numerous prospective drugs in parallel. Instead of centrifuge tubes, we employed 96-well plates. In this high-throughput configuration, the solution volume in individual runs was reduced from 400 µL (centrifuge tubes) to 200 µL (plate wells). We tested the modified platform using the conditions described above. In the multi-well plates, hematin crystallizes at a much slower rate than in the centrifuge tubes. Slower rates of crystal growth impede the quantification of the percent inhibition of hematin crystallization as the differences between hematin growth in the absence and in the presence of inhibitor are commensurate with the error of UV-Vis measurements; and this


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similarity increases the likelihood of identifying false positives during inhibitor screening. To address this problem, we adjusted the starting hematin concentration CH to increase the supersaturation, which alters the rate of crystallization. As shown in Figure 7A, the growth rate increases monotonically as a function of CH. The data in Figure 7 illustrate the good reproducibility of the assay in multi-well plates, i.e., growth rates obtained using two separate crystal seed batches, each in duplicate, agree. For all measurements reported herein, we use a hematin concentration of 0.60 mM that induces growth at a rate observed for the 0.3 mM concentration in the centrifuge tubes.

Figure 7. (A) Effect of starting hematin concentration CH on the rate of growth using a 96-wellplate configuration. The growth rate of hematin crystallization, rcontrol, was measured in the absence of inhibitor. We performed experiments in duplicate using two crystal seed batches (labeled with circles and diamonds, respectively). Symbols represent the average value measured


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from eight wells, and error bars equal two standard deviations. The solid line is a linear regression fit. (B) Percent inhibition of hematin crystallization as a function of quinine concentration, CQN, in biomimetic assays employing centrifuge tubes (diamonds) and a 96-well plate (squares). Each data point for the centrifuge tubes is the average of three measurements, while data points for the well plates are averages of 16 measurements from two separate trials. Error bars equal two standard deviations and the dashed lines are interpolations to guide the eye.

In Figure 7B, we compare the percent inhibition of hematin measured in the multi-well plate and centrifuge tubes, selecting QN as a representative growth inhibitor. The general trend of the two curves is similar, which confirms that the high-throughput configuration can be used to screen inhibitors of hematin crystallization. For reasons that are not entirely clear, the percent inhibition measured in the multi-well plate is slightly higher. Potential reasons for this observation may be attributed to the fact that multi-well plates have smaller volume (which may slow down buoyancy-driven convection), the plates are made of different polymeric material (which may potentially alter crystal and/or inhibitor adsorption on container walls), and the multi-well plate requires higher supersaturation. Despite these small differences, this proof-ofconcept study indicates that the biomimetic assay proposed here has the potential to be applied as a screening platform for hematin crystallization in order to identify and characterize new generations of antimalarial drugs. 4. CONCLUSIONS In summary, we have assessed the effects of antimalarial and antibiotic drugs on bulk hematin crystallization in a biomimetic growth medium. Systematic analysis of drug-solvent thermodynamics confirmed that all drugs are sufficiently soluble in CBSO, resulting in concentrations that are high enough to inhibit hematin crystals. We showed that bulk crystallization in CBSO employing hematin crystal seeds is an effective method of quantifying


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inhibitor efficacy. Moreover, comparison of seeded growth assays for various drugs revealed their relative potency. Here we report several advantages of the biomimetic bulk crystallization assay over current parasite models. For instance, seeded growth in CBSO requires relatively short time (ca. ≤ 5 hours) to assess the efficacy of growth inhibitors. Crystallization-based assays also provide more information regarding the mechanism of drug action. Notably, crystalline product from drug screening assays can be analyzed by more rigorous techniques, such as electron or scanning probe microscopy, to examine the effects of inhibitors on crystal size, habit, and surface topography from the macroscopic to near molecular length scale. Identifying effective crystal growth inhibitors can also help develop structure-performance relationships wherein the properties of the drug (i.e., molecule structure and functionality) could potentially serve as heuristic guidelines to design molecular analogues for additional testing, thereby reducing the number of compounds that need to be screened prior to identifying a lead candidate(s). We also demonstrated a high-throughput configuration that is potentially viable for conducting bulk crystallization in parallel, thus offering an efficient method of screening antimalarial drug compounds. One aspect of this approach that is lacking is the ability to discern the bioavailability of these compounds. To this end, in vivo tests are essential, and could be combined with bulk crystallization assays wherein the latter would serve as a pre-screening stage to collect useful information on drug-crystal interactions for the design of new compound libraries, and improve the overall efficiency of drug screening. ASSOCIATED CONTENT


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Supporting Information. Addition details of the experimental methods, determinations of the extinction coefficients in various solvents, and thermodynamic data for antimalarial and antibiotic compounds are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *Email: [email protected] (J.D.R.), *Email: [email protected] (P.G.V.) Present Addresses †

The Village School, 13077 Westella Dr., Houston, TX 77077

AUTHOR CONTRIBUTIONS The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. NOTES The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank Katy Olafson for help in the experiments and for numerous helpful discussions. This work was supported by NIH (Grant 1R21AI126215-01), The Welch Foundation (Grant E-1794), the National Science Foundation (Grant 1207441), and NASA (Grants NNX14AD68G and NNX14AE79G).


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For Table of Contents Use Only:

Biomimetic Assay for Hematin Crystallization Inhibitors: A New Platform to Screen Antimalarial Drugs Megan A. Ketchum1, Andrea M. Lee1,†, Peter G. Vekilov1,2,*, Jeffrey D. Rimer1,* 1

Department of Chemical and Biomolecular Engineering and

2

Department of Chemistry,

University of Houston, 4726 Calhoun Road, Houston, TX 77204, USA

Hematin crystallization, an essential part of heme detoxification in malaria parasites, occurs in a mixed aqueous-organic(lipid) medium, contrary to suggestions in literature that crystal growth occurs in aqueous solution. Here we present a high-throughput screening assay that is capable of quantifying the efficacy and potency of antimalarials as crystal growth inhibitors in a biomimetic growth medium.


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Biomimetic Assay for Hematin Crystallization Inhibitors: A New ... - DOI

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