Antimalarials inhibit hematin crystallization by unique SEE COMMENTARY drug–surface site interactions

Katy N. Olafsona, Tam Q. Nguyena, Jeffrey D. Rimera,b,1, and Peter G. Vekilova,b,1

aDepartment of Chemical and Biomolecular Engineering, University of Houston, Houston, TX 77204-4004; and bDepartment of Chemistry, University of Houston, Houston, TX 77204-5003

Edited by Patricia M. Dove, Virginia Tech, Blacksburg, VA, and approved May 9, 2017 (received for review January 3, 2017) In pathophysiology, divergent hypotheses on the inhibition assisted by a protein complex that catalyzes hematin dimerization of hematin crystallization posit that drugs act either by the (45). In a previous paper, we reconciled these seemingly opposite sequestration of soluble hematin or their interaction with crystal viewpoints by suggesting that β-hematin crystals grow from a thin surfaces. We use physiologically relevant, time-resolved in situ shroud of that coats their surface (46), a mechanism that surface observations and show that quinoline antimalarials inhibit is qualitatively consistent with experimental observations (47). β-hematin crystal surfaces by three distinct modes of action: step Driven by the evidence favoring neutral as a preferred en- pinning, kink blocking, and step bunch induction. Detailed experi- vironment for hematin crystallization in vivo (46), we use a sol- mental evidence of kink blocking validates classical theory and dem- vent comprising octanol saturated with citric buffer (CBSO) at onstrates that this mechanism is not the most effective inhibition pH 4.8 as a growth medium. pathway. Quinolines also form various complexes with soluble he- Recent observations of hematin crystallization from a bio- matin, but complexation is insufficient to suppress detoxifica- mimetic organic medium demonstrated that it strictly follows a tion and is a poor indicator of drug specificity. Collectively, our classical mechanism of growth (48), where new layers are gener- findings reveal the significance of drug–crystal interactions and ated by 2D nucleation and advance by the attachment of solute open avenues for rationally designing antimalarial compounds. molecules. This finding allowed identification of the rate of nu- cleation of new layers, J2D, and the velocity of advancing steps v as malaria | P. falciparum | crystallization inhibition | hematin crystals | heme the main quantitative measures of β-hematin crystal growth and detoxification suggested criteria to differentiate the specificity and efficacy of growth inhibitors that bind to β-hematin crystal surfaces (48). The any pathological conditions are understood within the physiological relevance of these conclusions is reinforced by the Mrealms of molecular biology and biochemistry. An impor- observation that the morphology of the grown crystals is identical tant class of diseases, whose pathology involves the formation of to biological hemozoin (28), and their growth rate is similar to solid or liquid condensate, stands to benefit from the introduction those observed in parasites (42, 48). of concepts and mechanisms from physics and materials science To establish whether drug–crystal interactions are the dominant (1–3). A prominent example is the formation of crystalline mechanism of hematin growth inhibition, we monitor and quantify hemozoin, a vital component of malaria parasite physiology (4–7). the processes of hematin crystallization by time-resolved in situ Malaria infection starts with a bite introducing Plas- atomic force microscopy (AFM). We use CBSO as the solvent (48). modium sporozoites, which invade the host’s liver cells (Fig. 1). To this growth solution, we introduce six common antimalarial Within 2 wk, merozoite-stage parasites invade the erythrocytes, drugs: (QN), (CQ), pyronaridine (PY), amo- where they catabolize and release Fe(II) heme (5). diaquine (AQ), mefloquine (MQ), and artemisinin (ART). We find The released heme rapidly oxidizes to Fe(III) hematin, which is that in the presence of any of the tested drugs, the basal {100} toxic to the parasite in its free state (6). The main mechanism of CHEMISTRY heme detoxification (8) implemented by the parasite is seques- Significance tration as nontoxic hemozoin crystals (9–12) (Fig. 1C). Hematin crystallization (the synthetic analog of hemozoin has been referred Approximately 3.2 billion people are at risk for malaria. The re- to as β-hematin) (7) has been the most successful molecular target sistance of malaria parasites to current advanced multidrug treat- for antimalarial drugs (13, 14). Malaria parasite resistance to ments, recently recorded to spread outofSoutheastAsia,hasraised current drugs (15–18), including the most advanced line of anti- concerns of dire public health consequences. We demonstrate that malarial defense, artemisinin (19–24), has renewed the impetus to antimalarial drugs suppress heme detoxification in the malaria elucidate the pathways of parasite suppression (25–27). parasites in a manner that is counter to the prevailing hypothesis in It is generally accepted that the quinoline class of antimalarials the field. We find that quinoline-class drugs work by specific in- (quinine, chloroquine, amodiaquine, and others) work by sup- teractions with β-hematin crystals, which are the by-product of pressing hematin crystallization (8, 13, 21, 25, 26, 28, 29). The heme detoxification within the digestive vacuole of the parasites. prevailing hypothesis is that drugs decrease the activity of soluble We also identify specific drug adsorption sites on crystal surfaces. hematin by forming noncrystallizable complexes (13, 30). An These insights may potentially spur development of antimalarial alternative inhibition mechanism, involving drug–crystal in- – drugsthatovercomeparasiteresistancethrougharationalap- teraction, has been suggested by experimental evidence (29, 31 proach, far superior to currently used combinatorial methods. 38) and theoretical models (39, 40). A controversy surrounds the environment of hematin crystal- Author contributions: J.D.R. and P.G.V. designed research; K.N.O. and T.Q.N. performed lization in the parasite digestive vacuole (DV). A group of au- research; K.N.O., T.Q.N., and P.G.V. analyzed data; and J.D.R. and P.G.V. wrote the paper. thors has argued that several neutral lipids are present in the DV The authors declare no conflict of interest. as mesoscopic phases that accumulate hematin and serve as This article is a PNAS Direct Submission. – crystallization medium (41 43); this scenario has been supported See Commentary on page 7483. by numerical modeling (44). High-resolution electron micros- 1To whom correspondence may be addressed. Email: [email protected] or vekilov@ copy and X-ray tomography, however, have failed to detect lipid uh.edu. phases larger than 25 nm, interpreted in favor of growth from the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. aqueous environment (10–12), where crystallization may be 1073/pnas.1700125114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1700125114 PNAS | July 18, 2017 | vol. 114 | no. 29 | 7531–7536 Downloaded by guest on October 1, 2021 developed means to reduce CQ concentration within the DV to levels that permit effective heme detoxification. Besides step pinning, which suppresses J2D and slows down v, the drug PY appears to follow an additional mechanism* of action. AFM observations reveal a decrease in v along the b crystallo- graphic direction in response to increasing PY concentration that * is more pronounced than along the c direction. Correspondingly, the islands in Fig. 2E exhibit exaggerated ellipticity, with a c/b ratio ∼ of *8, compared with 2.7 in pure solutions (51). The sensitivity of b the -oriented step growth to PY* concentration suggests that this drug preferentially attaches to b steps, blocking the growth sites available for hematin incorporation. An idealized schematic* of this interaction is presented in Fig. 2G. The hindered b steps form strips of high-density step bunches, Fig. 2E and SI Appendix, Fig. S3 (52–54), which amplifies the PY inhibition due to competitive supply of solute to closely spaced steps (51). vðcDÞ profiles similar to those in Fig. 2 B and C are compatible with an alternative mechanism of growth inhibition, whereby inhibitors incorporate in the crystals lattice. The resulting lattice strain increases the chemical potential of the crystal, which raises

Fig. 1. Lifecycle of malaria parasites and hemozoin formation. (A) In human hosts, the lifecycle of malaria parasites consists of liver and red cell (RBC) stages. The characteristic times after RBC invasion for parasite growth and division, RBC rupture, and transfer of infection are displayed. (B) Sche- matic rendering of RBCs. (C) Parasite inhabiting an RBC with a nucleus (N) and a DV containing hemozoin crystals. (D) SEM image of a hematin crystal grown in vitro. (Scale bar, 5 μm.)

β-hematin faces grow via a classical layer mechanism, analogous to the one observed in pure CBSO solutions (48). Results and Discussion Molecular Mechanisms of Drug Action. Three of the tested drugs, QN, CQ, and PY, depicted in Fig. 2A, cause a sharp decay of J2D (Fig. 2B) accompanied by a monotonic decrease in v relative to its value v0 in solutions of pure hematin with concentration cH = 0.28 mM, at supersaturation σ = lnðcH =ceÞ ≅ 0.56, where ce = 0.16 mM is the solubility at 28 °C, the temperature in the AFM liquid cell (48) (Fig. 2C). AFM observations, presented in Fig. 2D and SI Appendix, Figs. S1–S3, reveal protrusions along ad- vancing steps in the presence of drugs. These corrugated step edges are in contrast to observations in pure solutions (48), in- dicating a specificity of the drugs for {100} faces; the {100} faces are the largest of the crystal, implying that they are the slowest growing. The concomitant reduction of J2D and v with increasing drug concentration cD suggests that the drugs inhibit 2D nucle- ation of new layers and step motion by a step-pinning mechanism (49), illustrated in Fig. 2F. This mechanism assumes that inhib- itors preferentially bind to terraces with a surface coverage governed by the dynamics of adsorption. If the separation be- Fig. 2. Step-pinning action of three common antimalarials. (A) The struc- tween a pair of adsorbed inhibitors Δx is less than the diameter tures of QN, CQ, and PY. (B) A decrease in the rate of 2D nucleation of new of the critical layer nucleus 2Rcrit, the adsorbates enforce a cur- layers J2D relative to that in the absence of any drug, J2D,0, with increasing vature at which the advancing step is undersaturated. The growth drug concentration. (C) A decrease in step velocity v relative to that in the absence of any drug, v0, with increasing drug concentration. In the presence * * of existing steps is arrested (49), whereas newly formed 2D is- of QN and CQ, v in the c and b directions maintains a constant ratio of 2.7 * lands are prevented from reaching the critical size and are forced (here, only v in the c direction is shown). For PY, the ratio increases to ∼8. to dissipate. At intermediate inhibitor concentrations, step pin- (D and E) AFM images of islands and steps on a (100) face growing in a ning produces corrugated steps (Fig. 2D) and suppresses 2D 0.27 mM hematin solution in CBSO in the presence of QN and PY at concen- layer nucleation. The significance of this mechanism is reflected trations shown in the respective images. The gold arrow in E indicates an area ∼ × μ 2 in the sensitivity of the crystallization rate to the drug concen- of step bunching. The imaged face is 1 13 m .(F) Schematic of the step- pinning mechanism, where Δx is the separation between drug molecules tration (Fig. 2 B and C), which may be a critical factor underlying adsorbed on flat crystal terraces, shown in yellow, and Rcrit is the critical radius the increased resistance of falciparum parasite to of the 2D nucleus. (G) Schematic of step bunching induced by the putative * CQ (50). For instance, resistant parasite strains may have interaction between PY molecules, shown in purple, and b direction steps.

7532 | www.pnas.org/cgi/doi/10.1073/pnas.1700125114 Olafson et al. Downloaded by guest on October 1, 2021 incorporation mechanism remains a feasible pathway of β-hematin growth inhibition by CQ, QN, and PY. SEE COMMENTARY Trends of J2D and v suggest that the drugs AQ and MQ (Fig. 3A) exhibit mechanisms that differ from those of QN, CQ, and PY (SI Appendix, Fig. S7). Both AQ and MQ suppress v by about half (Fig. 3C), but only AQ inhibits the nucleation of new layers (Fig. 3B). The gradual decrease of v at increasing inhibitor concentration is consistent with a mechanism wherein inhibitor molecules adsorb on kinks at the steps and block hematin mol- ecules from incorporating, as seen with other materials (55, 58) and illustrated in Fig. 3F. To quantify the inhibition of step motion by partial blocking of kinks, we assume that v is pro- portional to the density of unoccupied kinks, v = v0 – ðv0 – v∞ÞnD, where v0 = v in pure solutions, v∞ is reached at the maximum surface inhibitor coverage, and nD is drug adsorption coverage −1 on the kinks, governed by a Langmuir isotherm, nD = cDðA + cDÞ ,

Fig. 3. Inhibition of hematin growth by kink blocking. (A) Structures of AQ,

MQ, and ART. (B) The rate of 2D nucleation of new layers J2D relative to that in the absence of any drug, J2D,0, with increasing drug concentration. (C) The step velocity v relative to that in the absence of any drug, v0, with increasing *

drug concentration for c direction steps. Dashed and dotted lines denote the CHEMISTRY reduction of v due to the sequestration of hematin in respective drug– hematin complexes (Fig. 4C). (D and E) The correlation between v and the

respective concentrations of AQ and MQ, cD, in modified reciprocal coordi- nates. (F) Schematic of drug molecules (shown in green) inhibiting step advancement by partial blocking of kinks. (G) AFM image of islands and steps growing in a 0.27 mM hematin solution in CBSO in the presence of 1.5 μM AQ. (H) Submolecular resolution image of a (100) hematin crystal surface. Blue spheres highlight carboxyl, methyl, and vinyl C atoms as seen in the hematin model (Inset). C atoms marked with yellow spheres belong to the same molecule. (I) High-resolution image of a step edge, highlighted with a white contour; higher terrace is on the right. (J) Monitoring the evolution of a step edge with disabled y-axis scans, beginning at time = 4s (yellow line) reveals temporal displacements of the step due to the attach- ment or detachment of solute molecules; higher terrace is on the right.

the solubility and lowers the driving force for growth (55–57). In Fig. 4. Drug effects on crystal size and morphology. (A) Illustration of β previous observations of this scenario, the solubility increase was -hematin crystal habit. (B) Growth in pure solutions preserves the crystal shape. (C and D) Variations of the average length l and length-to-width, l/w, significant if the inhibitor concentration in the solution was aspect ratio Asp of crystals grown in pure CBSO solutions and in the presence comparable to that of the solute (55–57). AFM images of he- of increasing concentrations of five drugs for 16 d at 23 °C. (E and F) Illus- matin crystals reveal protrusions along advancing steps in the trations of modifications to the crystal habit. Drug-induced suppression of presence of drug (Fig. 2D and SI Appendix, Figs. S1–S3), which crystal l and w by interaction of drugs with axial (E) and lateral (F) crystal seems to indicate that alternative pathways (e.g., step pinning) faces, respectively. (G) Tapering due to enhanced adsorption of drugs near the crystal edges. (H–L) Scanning electron micrographs of crystals grown in are the principal routes to inhibit step growth; however, in the the presence of drugs at concentrations listed in each panel. Tapering due to absence of data on the thermodynamics of drug incorpora- impurity action on the ð011Þ face is shown in K, where (K, I) and (K, II)are tion into β-hematin crystals and their elastic properties, the enlargements of respective dashed boxes.

Olafson et al. PNAS | July 18, 2017 | vol. 114 | no. 29 | 7533 Downloaded by guest on October 1, 2021 Fig. 5. Drug–hematin complexation and speciation. (A) UV-visible spectra of hematin at three concentrations cH in the presence of QN, CQ, AQ, and MQ where the inhibitor concentration increases in the direction of the arrows (note that PY and ART data revealing the lack of complexation are displayed in SI

Appendix, Figs. S10 and S11). (B) Relative decrease of the absorbance of a solution with cH = 0.3 mM at 594 nm as a function of the concentration of the respective drug. Symbols are absorbance data and lines are the complexation model fits (H, hematin; D, drug; HD and H2D, complexes). The relevant binding −1 −2 constants are listed; units of K1 and K2 for QN are mM , whereas the unit of K for the other drugs is mM .(C) Distribution of hematin, drug, and complex species as a function of total drug concentration at cH = 0.3 mM. Vertical dotted line marks the QN concentration at which the concentration of unliganded hematin ½H decreases below the solubility ce = 0.16 mM, marked with horizontal dashed lines in each plot. Purple shading highlights the concentration range of each drug tested in Figs. 1 and 2.

where A is a constant. We obtain, similar to Bliznakov (59), reveals that it shifts by about 0.55 nm, consistent with in- −1 −1 −1 −1 ðv0 – vÞ = ðv0 – v∞Þ + ½Aðv0 – v∞Þ cD . The scaling between corporation/removal of a single hematin molecule. The high −1 −1 v and the cD in the presence of AQ and MQ in Fig. 3 D and E kink density suggests that the tail of a drug molecule occupying and SI Appendix,Fig.S8A and B supports that action of a kink may overlap adjacent sites and thereby protect them this mechanism. from docking of other drug molecules, but may be insufficient The morphology of advancing layers in the presence of AQ to prevent hematin molecules from accessing kinks and thus and MQ (Fig. 3G and SI Appendix, Figs. S5 and S6) is similar to inhibit further step growth. that in pure solutions, suggesting that inhibitor adsorption on kinks is independent of step orientation. The independence of Drug Effects on Bulk Crystal Morphology. Owing to the crystal J2D of the MQ concentration suggests that MQ does not adsorb anisotropy, inhibitor molecules that bind to crystal surfaces on molecularly flat surfaces or dock to the edges of small, newly exhibit marked differences in inhibition of growth in different nucleated islands. Meanwhile, the addition of ART has no ap- crystallographic directions. To assess the anisotropic growth parent effect on the surface features and the kinetics of layer rates, as illustrated in Fig. 4 A and B, we perform bulk crys- nucleation and growth (Fig. 3 B and C and SI Appendix, Fig. S4). tallization studies to determine the macroscopic dimensions of This result is not surprising given that ART is believed to attain β-hematin crystals grown for 16 d at varying concentrations of activity after reduction of the endoperoxide bridge by freshly the six drugs (Fig. 4 C and D); these experiments were carried released Fe(II) heme (4, 22, 24, 60). out at cH = 0.24 mM and at supersaturation σ = lnðcH =ceÞ ≅ 0.7, We observe that drug action by kink blocking does not fully where ce = 0.12 mM is the solubility at 25 °C. The drug ART suppress step growth, but rather results in a finite step velocity does not affect the size and morphology of crystals grown in its v∞ at high drug concentrations (i.e., 0.65v0 for AQ and 0.4v0 for presence, indicating that ART does not specifically interact MQ in Fig. 3C). To elucidate this phenomenon, we image the with β-hematin surfaces (Fig. 4B), which is consistent with AFM structure of advancing steps at submolecular resolution, as data.InthepresenceofCQ,QN,PY,andAQ,averagecrystal shown in Fig. 3H. High-resolution AFM images of the step lengths l are shorter (Fig. 4C), indicating preferential inhibition  edges (Fig. 3I) indicate that kinks are separated by distances of of the axial faces, (001) and/or ð011Þ, as illustrated in Fig. 3E. ∼1 nm. Monitoring the evolution of a step edge (as in Fig. 3J) Changes in the average aspect ratio Asp of crystal length to

7534 | www.pnas.org/cgi/doi/10.1073/pnas.1700125114 Olafson et al. Downloaded by guest on October 1, 2021 width (Fig. 4C) is an indication that the drug selectively binds to values at or below its solubility ce. To compare the relative ef-

 SEE COMMENTARY either the lateral {010} faces or the axial (001) and ð011Þ faces ficacy of complexation and crystal surface interaction as in- (Fig. 4 E and F).ThedatainFig.4C indicate that CQ pref- hibition pathways and to test if complexation dictates the erentially suppresses growth of the axial faces, whereas QN, selection of the drug mode of action, we characterized drug PY, and AQ have identical effects on the lateral and axial (D)–hematin (H) binding equilibria in CBSO, using a method faces. Similar studies of MQ reveal it to be a weak inhibitor based on UV-visible spectroscopy, previously used for pre- of β-hematin crystal growth in both the lateral and axial dominantly aqueous solvents (65–68). The UV-visible spectra of directions. hematin in the presence of six drugs reveal that PY and ART do Fig. 4 C and D reveals that higher inhibitor concentrations are not form complexes with hematin in CBSO (SI Appendix,Figs. needed to suppress bulk growth than the nucleation and spreading of S10 and S11). Using the spectroscopic data obtained in the pres- layers on the {100} faces (Figs. 2 and 3). Discrepancies between the ence of QN, CQ, AQ, and MQ (Fig. 5A), we discriminate between inhibitor concentrations needed to arrest step growth observed by several different stoichiometries and complexation steps and eval- AFM and those that suppress the growth of crystals in a bulk crys- uate the respective binding constants (Fig. 5B and SI Appendix). tallization experiment are common in the crystallization literature Drug–hematin complexation lowers ½H below ce = 0.16 mM (56, 61, 62). Several processes may contribute to this disparity. Bulk (48) when the drug concentrations exceed 0.09 and 0.07 mM for crystallization experiments are carried out at σ = 0.70, whereas AFM QN and CQ, respectively. This is more than 20-fold higher than surface monitoring uses σ = 0.56. The rates of nucleation and growth the concentrations that induce complete growth cessation in of layers are significantly faster at the higher σ, leading to shorter AFM studies (∼1 μM in Fig. 2 C and D). These considerations times between subsequent layers and lower impurity adsorption. suggest that drug interaction with the β-hematin crystal surface is Furthermore, the crystal dimensions evolve owing to the growth of a significantly more efficient pathway to inhibit hematin crys- all faces in the crystal habit, whereas Fig. 4 C and D characterizes tallization than complexation of free hematin in the solution. ½  J v growth in the [010], [001], and 011 directions. 2D and on the Our findings indicate that the formation of complexes does not (100) face contribute to growth in the [100] direction. Growth ki- correlate with the selection of specific inhibition mechanism; netics and their response to inhibitors are anisotropic and may however, for drugs such as AQ and MQ, the combination of significantly differ for individual crystallographic directions. In relatively strong drug–hematin binding and the limited growth addition, it is feasible that in a bulk experiment, in which nu- suppression (Fig. 3 C, I, and J) increases the significance of drug merous crystals with significant surface area grow for 16 d, the complexation as a contributing inhibition pathway. inhibitor concentration decreases during this time owing to in- corporation into the growing crystals, whereas it was maintained at Conclusions a constant value during short-term AFM monitoring. Lastly, We have demonstrated that five antimalarials exhibit distinct during surface scans the iterative motion of the AFM probe and mechanisms of β-hematin crystal growth suppression based on cantilever stirs the solution and homogenizes the inhibitor con- their specific interactions with crystal terraces, step edges, kinks, centration throughout the solution volume. In contrast, a signifi- or newly nucleated islands. We show that drug–crystal interactions cant inhibitor concentration gradient may develop in a steady are a significantly more efficient pathway to inhibition of hematin solution during long-term bulk crystallization experiments, leading crystallization than sequestration of soluble hematin into drug– to significantly lower inhibitor concentration at the crystal surface hematin complexes; it is feasible that the species that absorbs on than in the solution bulk (63). a specific surface site is a drug–hematin complex. Through phys- H–L Observations of the crystal morphology (Fig. 4 ) reveal iologically relevant techniques, we find that kink blocking is sharp distinctions between the different drugs. Electron micro- implemented by two antimalarials. We find that drug–crystal in- H β graphs in Fig. 4 indicate that CQ renders -hematin crystals teractions do not correlate with the propensity of drugs to form short and wide whereas PY induces crystal tapering at both axial complexes with soluble hematin, but are predominantly driven by I J termini (Fig. 4 and ). Tapering has been attributed (64) to chemical recognition between drug molecules and the topological blocking of steps on an orthogonal face on approach to a shared features of β-hematin crystal surfaces. These findings may prove CHEMISTRY edge, induced by enhanced supply of inhibitors at the edge, as influential for the design of new antimalarials and demonstrate, in G I J illustrated in Fig. 4 . The symmetric tapering in Fig. 4 and the broader context of condensation diseases, that the application ð Þ indicates that PY interacts with both (001) and 011 faces. In of physical science approaches to crystallization in living organisms ð Þ contrast, QN and AQ induce tapering at a single axial face, 011 can provide valuable insight into parasite physiology. for QN in Fig. 4K, and (001) for AQ in Fig. 4L. ACKNOWLEDGMENTS. We thank David Sullivan, Sergey Kapishnikov, and How Important is the Contribution of Drug–Hematin Complexation? Leslie Leiserowitz for insightful discussions on hematin crystallization and Our findings refute the hypothesis that drug–hematin binding, drug–hematin interactions and Bryan G. Alamani for gold coating the SEM which sequesters hematin into soluble complexes, is the main sample. This work was supported by NIH through the Nanobiology Interdis- ciplinary Graduate Training Program of the Gulf Coast Consortia for Quan- mode of hemozoin growth inhibition (13). Sequestration would titative Biomedical Sciences (Grant T32EB009379) and directly (Grant completely suppress the growth of β-hematin crystals if the re- 1R21AI126215-01), NASA (NNX14AD68G and NNX14AE79G), and The Welch sidual concentration of unliganded hematin ½H is lowered to Foundation (Grant E-1794).

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