Characterization of Plasmodium falciparum Adenylyl Cyclase-β and Its Role in Erythrocytic Stage Parasites

The most severe form of human malaria is caused by the parasite Plasmodium falciparum. The second messenger cAMP has been shown to be important for the parasite’s ability to infect the host’s liver, but its role during parasite growth inside erythrocytes, the stage responsible for symptomatic malaria, is less clear. The P. falciparum genome encodes two adenylyl cyclases, the enzymes that synthesize cAMP, PfACα and PfACβ. We now show that one of these, PfACβ, plays an important role during the erythrocytic stage of the P. falciparum life cycle. Biochemical characterization of PfACβ revealed a marked pH dependence, and sensitivity to a number of small molecule inhibitors. These inhibitors kill parasites growing inside red blood cells. One particular inhibitor is selective for PfACβ relative to its human ortholog, soluble adenylyl cyclase (sAC); thus, PfACβ represents a potential target for development of safe and effective antimalarial therapeutics.


Introduction
Malaria remains a major burden in the developing world, causing approximately 1 million deaths per year. It is a vectorborne disease caused by protozoan parasites of the genus Plasmodium, the most lethal of which is Plasmodium falciparum. A diverse array of protozoal, fungal, and bacterial pathogens, including Plasmodium spp., depend upon the ubiquitous second messenger cyclic adenosine monophosphate (cAMP) for survival and environmental sensing [1]. In fact, two stages of the Plasmodium life cycle appear to depend upon cAMP: Sporozoites require cAMP generation for host cell invasion [2], and previous reports suggest that cAMP effectors play an important role in the asexual red blood cell stage of the life cycle. Specifically, inhibition of cAMP-catabolizing phosphodiesterases (PDEs) or addition of membrane-permeable cAMP analogs increase the percentage of schizonts in asynchronous, erythrocytic cultures of P. falciparum [3], and treatment of erythrocytic stage cultures with either pharmacological or genetic inhibitors of the main effector of cAMP, Protein Kinase A (PKA), inhibit growth [4,5]. While these data reveal that the cAMP pathway is required for progression through the erythrocytic, asexual stage of the life cycle, the stage of the life cycle that causes symptomatic malaria, it remains unclear how cAMP levels are controlled during this period. cAMP is synthesized by adenylyl cyclases (AC), and the P. falciparum genome encodes two such enzymes, PfACa and PfACb. Both enzymes contain class IIIB catalytic domains similar to mammalian soluble adenylyl cyclase (sAC) [6]. Mammalian sAC is structurally, molecularly, and biochemically distinct from other mammalian adenylyl cyclases, which are transmembrane proteins regulated by heterotrimeric G proteins (tmACs). Unlike tmACs, mammalian sAC is directly regulated by bicarbonate. In physiological systems, bicarbonate is in nearly instantaneous equilibrium with CO 2 and intracellular pH (pHi) due to the action of carbonic anhydrases [7]; thus, mammalian sAC serves as a physiological CO 2 /HCO 3 2 /pHi sensor [8,9], with specific roles in sperm activation [10,11], ciliary beat frequency in bronchii [12], pH homeostasis in epididymis [13], kidney [14,15], and shark gill [16], metabolism [17], and aqueous humor formation in the eye [18].
PfACa and PfACb differ in their modular architecture. PfACa . contains six predicted transmembrane domains and a single carboxy-terminal catalytic domain homologous to sAC-like ACs. The motifs required for metal cofactor binding, substrate binding, and catalysis are contained within this single catalytic domain, suggesting that this enzyme functions as a homodimer [19]. In contrast, PfACb has no predicted transmembrane regions and possesses two sAC-like AC catalytic domains. PfACb and ACb orthologs from other Plasmodium spp. possess all the motifs required for catalytic activity, but they are spread across the two presumptive catalytic domains suggesting that catalysis requires intramolecular heterodimerization, similar to mammalian sAC [20]. In addition, these ACs possess a threonine residue which is thought to be predictive for bicarbonate regulation in sAC-like . Samples were collected in triplicate. Luciferase activity is elevated between 4-16 hours due to increased promoter activity during primary round of infection. The peak of luciferase activity seen at ,44 hr under normal culture conditions, but absent in the absence of CO 2 /HCO 3 2 or presence of KH7, reflects reinvasion into RBCs. The graph was prepared with Prism software; error bars represent s.e.m of triplicate wells in the representative experiment. (D) Microscopic evaluation of Giemsa-stained parasites at 44 hr reveals parasites (P) maintained in normal culture completed mitosis and newly released merozoites are poised to reinvade new RBCs. Parasites treated with KH7 (E) or grown in low CO 2 /HCO 3 2 conditions (F) never form schizonts. doi:10.1371/journal.pone.0039769.g001 ACs [21]. Unlike other adenylyl cyclases including ACb orthologs from other Plasmodium spp., each catalytic domain of PfACb is interrupted by blocks of highly charged stretches of amino acids, which are encoded by low complexity regions of unknown function prevalent throughout the P. falciparum genome.
PfACa has been studied both in vivo and in vitro. PfACa is a predicted bifunctional protein comprising both a K + channel and an AC that is conserved in alveolata protozoans [22]. PfACa transcripts are abundant in sexual stage gametocytes [19], suggesting a possible role during sexual stages. Additionally, ACa proteins in Plasmodium spp. appear to play a role during the liver sporozoite stage. Specifically, P. berghei sporozoites deficient in ACa were shown to have reduced infectivity of cultured hepatocytes and reduced liver infectivity in a mouse model, but they were viable and exhibited normal growth during asexual, erythrocytic growth [2]. In contrast, PfACb has not yet been heterologously expressed or biochemically characterized, and attempts to generate PfACb-deficient parasites using protocols that demand growth of the haploid mutant parasite in erythrocyte cultures were repeatedly unsuccessful [2]. Interestingly, its mRNA is highly expressed during the erythrocyte stage; PfACb transcript levels begin to increase in the trophozoite stage and peak during schizogeny [23,24].
We took advantage of a number of small molecule inhibitors of sAC-like adenylyl cyclases to identify the essential source of cAMP during erythrocytic growth. Three distinct AC inhibitors blocked growth of P. falciparum inside red blood cells. We established conditions for in vitro characterization of PfACb . and we tested sensitivity of these three inhibitors against the in vitro AC activities of both PfACa and PfACb. Consistent with the differential expression patterns of the two cyclases, only PfACb proved to be sensitive to all three, providing strong evidence that it is the source of cAMP essential during erythrocytic growth. Interestingly, one of the three inhibitors was also selective for PfACb relative to mammalian sAC demonstrating that small molecules can distinguish between the parasite and host enzymes. These data define PfACb as a target for development of novel antimalarial therapeutics.

Results and Discussion
We have identified two, structurally distinct inhibitors of sAClike ACs; catechol derivatives of estrogen and KH7 ( Figure S1). Catechol estrogens (CEs), such as 2-hydroxyestradiol (2-CE), inhibit Class III ACs, including mammalian and bacterial sAC-like ACs, by chelating the catalytic magnesium ion in the active site [25]. The second structurally unrelated inhibitor, KH7, was identified as a potent, specific inhibitor of mammalian sAC [11,26,27] in a small molecule screen [11] and was subsequently found to inhibit a number of bicarbonate-sensitive ACs [16,28].
To determine the effect of these compounds on parasite growth and viability inside red blood cells, we measured the luminescence of the wild-type NF54 P. falciparum strain transfected with the pHLIDH plasmid, which constitutively expresses firefly luciferase [29]. The luminescence of this parasite strain directly corresponds to the measures of viability determined with the widely-used tritiated hypoxanthine-uptake assay [30] ( Figure S2). Both KH7 and 2-CE killed rapidly ( Figure 1A,B) [LD 50 = 8.5 mM (95% C.I. = 7.8-9.2 mM) for KH7 and 60 mM (95% C.I. = 43-90 mM) for 2CE] with death observed within a single replicative cycle (48 hours) of synchronized parasites ( Figure 1C). Giemsa-stained slides prepared from parasites treated with KH7 revealed condensed, pyknotic parasites ( Figure 1E), confirming that these compounds lead to rapid parasite death rather than simply inhibiting proliferation or reporter activity.
As a reference, the terminal phenotype of KH7-killed parasites was indistinguishable from that of parasites maintained in the absence of CO 2 /HCO 3 2 . Synchronized cultures grown in CO 2 / HCO 3 2 in the presence of the inhibitor KH7 or grown in the absence of CO 2 /HCO 3 2 lacked the burst of luciferase due to the reinvasion observed in normal cultures ( Figure 1C,D). Microscopic evaluation confirmed that the drug-treated parasites ( Figure 1E) resembled dead CO 2 /HCO 3 depleted parasites ( Figure 1F); neither formed merozoites, indicating they had not completed schizogeny. In addition, we tested KH7 against a chloroquineresistant P. falciparum strain (Dd2), and it was lethal, as determined microscopically, with similar efficacy as observed against the chloroquine-sensitive NF54 strain (data not shown).
In order to determine the temporal effect of KH7 on synchronized parasites, we added KH7 to synchronized cultures at different time points throughout the cell cycle ( Figure 2A). Addition of KH7 in the first 24 hours of the cell cycle led to complete cell cycle arrest. However, if KH7 was added to the culture at a point well into schizogeny (34 hours), parasites were able to complete the cell cycle and invade new erythrocytes. In a complementary experiment to determine a ''window of KH7sensitivity,'' synchronized cultures were incubated in the presence of KH7 for various times, at which point the drug was washed out and cultures were grown for the remainder of a 48-hour cell cycle. When KH7 was removed at 24 hours or before, cultures were able to progress through the cell cycle, reinvade erythrocytes, and enter G1 ( Figure 2B). If KH7 remained on cultures past 24 hours, parasites appeared unable to recover within the 48-hour culture period. These data demonstrate that parasites are most sensitive to KH7 at 24-31 hours post-invasion. This corresponds to the period in the cell cycle during which PfACb mRNA levels are beginning to rise dramatically ( Figure S3).
We next sought to determine whether the in vitro activities of PfACa and/or PfACb were sensitive to 2-CE and KH7. PfACa has been heterologously expressed and characterized previously [22], but the in vitro activity of PfACb has not yet been demonstrated. We expressed a synthetic gene encoding the catalytic domains of PfACb . AA 1-785) with mammalian codon usage as a fusion protein with a carboxy-terminal glutathione-Stransferase (GST) using a baculovirus (BV) expression system. GST-PfACb 1-785 was soluble, and we were able to purify it only under high salt conditions ( Figure S4). This high salt requirement for GST-PfACb 1-785 solubility may be due to the blocks of charged amino acids inserted into its catalytic domains. Similar to other sAC-like ACs [21,31,32,33,34], including PfACa [22], which exhibit much greater activity using Mn 2+ -ATP as a substrate relative to Mg 2+ -ATP, purified GST-PfACb 1-785 was active in the presence of Mn 2+ -ATP ( Figure 3A). We were unable to detect measurable activity in the presence of Mg 2+ -ATP ( Figure 3B). A similar Mn 2+ -ATPdependency was observed in assays of AC activity in erythrocytic stage P. falciparum lysates [35].
GST-PfACb 1-785 displayed Michaelis-Menten kinetics with a lack of cooperative binding of substrate at the active site ( Figure 3A). The enzyme has an apparent Michaelis constant (Km) for substrate ATP of ,0.6 mM using Mn 2+ as a cofactor with a maximum reaction velocity of ,265 nmol cAMP/min/mg. This Km value is similar to that obtained for human sAC (0.9 mM) [34]. The optimal ratio of divalent cation (Mn 2+ ) to substrate (ATP) was 4:1 ( Figure 3B), similar to mammalian sAC [34], and GST-PfACb 1-785 displayed minimal ability to produce cGMP when supplied with GTP as substrate (data not shown).
Mammalian sAC is directly regulated by bicarbonate [34,36] and calcium [34,37], and the threonine residue thought to be predictive of bicarbonate stimulation [21] is found in PfACb and ACb orthologs from other Plasmodium spp. However, because bicarbonate precipitates in the presence of Mn 2+ , and because we found bicarbonate and calcium activation to be unique to Mg 2+ -ATP-dependent activity in mammalian sAC, we were unable to explore bicarbonate-or calcium-responsiveness of BV-expressed GST-PfACb 1-785 . Instead, we explored the pH responsiveness of GST-PfACb 1-785 .
In contrast to mammalian sAC, GST-PfACb 1-785 exhibited a strong pH dependence ( Figure 3B) [36]. Varying the reaction pH from 7 through 9 revealed a pH optimum of 7.5, and activity decreased sharply at both higher and lower pH values. Thus, PfACb activity will be sensitive to changes in pHi, which, in physiological systems, is dependent upon the carbonic anhydrase-mediated equilibrium between CO 2 , bicarbonate, and protons. It is important to note that the pH dependence observed for PfACbis strikingly similar to the pH dependence of P. falciparum in culture. When pH of growth media is maintained between 7.1 and 7.5, parasitemias increase 20-30 fold after three days, with sharp reductions in yield outside of this pH range [38]. During the trophozoite stage, when PfACb mRNA is first expressed [23,24] (Figure S4), the intracellular pH (pHi) of parasites is approximately 7.3 [39,40,41]. Therefore, we speculate that PfACb functions as the parasite's pH sensor during growth inside red blood cells.
GST-PfACb 1-785 activity was inhibited by both KH7 and 2-CE with affinities that reflect their observed efficacies in culture. KH7 inhibited GST-PfACb 1-785 with an IC 50 of 5 mM, and 2-CE showed inhibition with an IC 50 of 8 mM ( Figure 4A,B). In contrast, although PfACa adenylyl cyclase activity was inhibited by 2-CE, it  was largely insensitive to KH7 ( Figure 4C). Thus, among adenylyl cyclases in P. falciparum, only PfACb is inhibited by the two structurally unrelated inhibitors which kill parasites in erythrocytic cultures.
While these data suggest PfACb may be a relevant target for killing malaria parasites inside red blood cells, both KH7 and 2-CE are also known to inhibit mammalian sAC, leaving open the possibility that host red blood cell sAC may be the relevant target of these compounds. To address this concern, we sought to identify a PfACb selective inhibitor. During our screen to identify KH7 as a mammalian sAC inhibitor, we tested numerous KH7-like compounds ( Figure S1). Most of the KH7-like compounds were ineffective against sAC-like cyclases, and these proved to have little effect on P. falciparum growth ( Figure S5). However, one KH7-like compound, KH7.15, which is inert against mammalian sAC [27], inhibited GST-PfACb with an IC 50 of 150 mM ( Figure 4D). KH7.15 killed parasites ( Figure 4E) with a similar efficacy [LD50 = 67 mM (95% C.I. 58-78 mM)] as it inhibited PfACbactivity in vitro. The fact that parasites were killed by two structurally unrelated inhibitors (2-CE and KH7) and by a third inhibitor (KH7.15) selective for PfACb relative to both PfACa and to the host sAC suggest that PfACb is the relevant target of these compounds and is essential for parasite growth inside red blood cells.
Our data include the first characterization of PfACb and suggest that PfACb is essential for erythrocytic-stage parasite viability. We have demonstrated PfACb is biochemically distinct from other Class IIIb adenylyl cyclases and exhibits significant pH-sensitivity. Additionally, we have shown that small molecule inhibitors can distinguish PfACb from mammalian sAC. Although the profile of KH7.15 is not ideal for clinical use, the data presented here provide proof-of-principle that PfACb can be selectively targeted, thereby identifying it as a therapeutic target for a new class of antimalarial drugs.
Although effective pharmacological therapies for malaria exist, the widespread and expanding resistance to these drugs demands new approaches to therapeutic intervention. The spread of multidrug resistant strains of P. falciparum threatens to increase the malaria burden, and novel therapeutics to combat malaria are desperately needed. This work is an initial step in attempts to address that need by defining PfACbas a novel, attractive therapeutic target.  Parasite Culture and Microscopy

Compounds
The parasite strains NF54 and NF54 transfected with pHLIDH were grown in 5% hematocrit in RPMI 1640 (Invitrogen/Life Technologies) supplemented with 0.5% Albumax II (Invitrogen/ Life Technologies), 0.25% sodium bicarbonate (standard media), and 0.01 mg/ml gentamycin. Human red blood cells for culture were obtained from human volunteers, cleared of leukocytes by passage through a Sepacell R-500 column (Baxter Health Care), and washed three times in RPMI 1640. Parasites were grown in sealed culture flasks under an atmosphere of 90% nitrogen, 5% oxygen, and 5% carbon dioxide. Parasitemias were maintained between 1 and 10%. Fixed parasites were stained with Giemsa to allow microscopic analysis of cultures using an Olympus BX40 compound microscope.
A synthetic gene with mammalian codon usage was used as the template. A 4-nucleotide addition was included in the FWD primer for directional topoisomerase-based cloning, and a stop codon was included in the REV primer. Following the PCR reaction, fragments were resolved on a 1% Agarose gel. Bands corresponding to the appropriate size were excised and fragments were gel-purified (Qiagen gel purification kit). After quantification by gel electrophoresis and comparison to a High Mass Ladder (Invitrogen), 10 ng of each fragment was used in a 2-hr topoisomerase-based cloning reaction with pENTR/TEV-D-TOPO (Invitrogen). Two microliters of the cloning reaction was transformed into TOP10 E. coli (Invitrogen). Colonies were screened by restriction digest, and positive clones were sequenced using M13 forward and M13 reverse primers and multiple genespecific primers. Clones found to be correct by sequencing were subsequently recombined into the ''destination'' vector pDEST20 (N-terminal GST tag) using a 1-hr LR Clonase II recombination reaction (Invitrogen). pDEST20-PfACb plasmid was transformed into DH10Bac E. coli (Invitrogen). Transformed bacteria were plated onto LB agar plates containing 50 mg/mL kanamycin (Sigma-Aldrich), 7 mg/ mL gentamicin (Sigma-Aldrich), 10 mg/mL tetracycline (Sigma-Aldrich), 100 mg/mL Bluo-gal (Invitrogen), and 40 mg/mL isopropyl-b-D-1-thiogalactopyranoside (Sigma-Aldrich). White colonies, indicative of successful bacmid recombination, were picked and streaked on fresh plates to confirm the phenotype. Blue colonies were streaked on a separate area of the same plate as a control. Confirmed white colonies were cultured in 500 mL of LB containing 50 mg/mL kanamycin, 7 mg/mL gentamicin, and 10 mg/mL tetracycline.
Subsequently, bacmid DNA was isolated from the cell pellet using the NucleoBond Bac 100 DNA isolation kit (Macherey-Nagel). Isolated bacmid DNA was immediately transfected into Sf9 cells plated at ,80% confluency on a 6-well plate (Becton-Dickenson) using Cellfectin reagent (Invitrogen). After transfection, successful recombination of bacmid DNA was confirmed by PCR analysis using M13 forward (Invitrogen), M13 reverse (Invitrogen), and the PfACb FWD primer indicated above. Four days post-transfection, cells showed significant signs of baculovirus infection. Cell media containing recombinant baculovirus was harvested and clarified by centrifugation at ,1,0006g. This P1 baculovirus stock was amplified first in a volume of 20 mL (400 mL P1 baculovirus was added) and subsequently in a volume of 500 mL (10 mL P2 baculovirus was added). For expression studies, 25 mL P3 baculovirus was added per liter of insect cells (either Sf9 or Hi-Five).

Heterologous Protein Expression
Insect cells are a proven system for expression and characterization of adenylyl cyclases [42]. Hi-Five cells at a density of 1610 6 cells/mL were infected with GST-PfACb 1-785 baculovirus at a concentration of 25 mL P3 baculovirus/L of culture. Infected cells were cultured for 40 hrs and harvested by centrifugation at ,10006g. Cells were frozen in liquid nitrogen and stored. Frozen pellets were resuspended in lysis buffer containing 50 mM Tris (pH 7.5), 5 mM DTT, 2 M NaCl, 10 mg/mL aprotinin/leupeptin, 1 mM PMSF, 1 mM benzamidine, 10 mM b-mercaptoethanol at a ratio of ,10 mL lysis buffer/100 mL of pelleted culture. This lysate was sonicated 5 times at 10-second intervals at 12 watts with a Misonix Microson cell disruptor. The sonicated lysate was clarified by centrifugation at 100,0006g using a Ti-75 rotor (Beckman). The resulting supernatant was passed over a Superdex G-25 column with a 5-mL bed volume for further clarification. The clarified lysate was incubated on ice with minor agitation for 1 hr with 1 mL (packed volume) of glutathione sepharose 4B (Amersham) per 100 mL of lysate. The lysate was allowed to flow through, and the resin was washed with 3610 bed volumes of lysis buffer. Finally, bound protein was eluted with 15 mM reduced glutathione in lysis buffer in 1 bed volume fractions. PfACb protein was detected by activity and anti-GST Western blot (data not shown). PfACa pressed as previously described [22].

Radioactivity-based Two-Column Adenylyl Cyclase Assay
Adenylyl cyclase assays with purified PfACb and PfACa were performed according to the method of Salomon [43]. Purified GST-PfACb . 50-500 ng) was incubated in 50 mM Tris, pH 7.5 (unless otherwise indicated), 1 mM DTT, 300 mM NaCl, 10 mM MnCl 2 and 2.5 mM ATP (unless otherwise indicated) with ,1,000,000 cpm [a-32 P]ATP (Perkin Elmer) and ,5,000 cpm [ 3 H]cAMP (Perkin Elmer). (Tris buffers were pH-adjusted at room temperature for use at 37uC.) Reactions were performed in 100 mL for 20 minutes at 37uC and stopped with 150 mL 1.5% SDS. Product [ 32 P]cAMP was separated from substrate [a-32 P]ATP by sequential column chromatography over dowex 50WX4-400 resin (Fluka) followed by aluminum oxide resin (Sigma). Product [ 32 P]cAMP was eluted from dowex, directly onto the alumina by water, and the cAMP was eluted from alumina by 0.1 M imidazole, pH = 7.3.

Viability Assays
The NF54 strain transfected with pHLIDH expresses the firefly luciferase gene under the control of the constitutively active Hrp3 promoter [44]. This strain of parasites was created by transfection and stable integration of the plasmid pHLIDH into the genome of the NF54 wildtype parasite line. pHLIDH is a derivative of the pHLH-1 plasmid [44], in which the drug selectable marker hdhfr was inserted under the control of the PcDT59 promoter [45]. Parasites were plated on day 0 at 1% parasitemia in 96-well plates in standard media in the presence of the indicated concentrations of DMSO (vehicle control), KH7, 2-CE, or KH7.15. Media plus compounds were replenished on day 1. On day 2, red blood cells were lysed with Bright-Glo Lysis Buffer (Promega), and luminescence was read using a luminometer (Molecular Devices) after injection with 10 ml Bright-Glo Luciferase Reagent (Promega) for a 2-sec integration time and a 15-sec read time. Data shown are normalized to the luminescence of vehicle-treated control parasites.

Parasite Synchronization
NF54 parasites were synchronized as described [46]. Briefly, parasites in cultured RBCs were centrifuged for 4 min at 4000 rpm. The pellet was layered atop a 40%/70% Percoll-Sorbitol gradient and centrifuged for 20 min at 10,000 rpm. The late-stage fraction at the interface of the gradient was collected, washed in media, and reconstituted with fresh RBCs and media. Following erythrocyte invasion, the synchronized culture was expanded into 6 20-mL cultures at 3% parasitemia. At each indicated time point, one 20-ml culture was centrifuged for 2 min at 4000 rpm. The pellet was resuspended in 500 ml phosphatebuffered saline (PBS), and RBCs were lysed with 10 ml 10% saponin and microcentrifuged for 2 min at 13,000 rpm. The supernatant was aspirated, and pellets were frozen at 280uC until all time points were collected.

Protection of Human Subjects
Blood was purchased from the New York City Blood Center or obtained from healthy human volunteers for use in parasite culture. A protocol for acquisition and use of human blood has been approved and is on file with the Internal Review board at Weill Medical College of Cornell University (Protocol #0010004662). For blood purchased from the New York City Blood Center (NYBC), contact of blood donors will not be attempted and is not necessary for the livelihood of the study. Informed consent is not required (other than NYBC in-house protocol). The blood will be used for research purposes onlysolely for in vitro culture of Plasmodium falciparumand not for transfusion into humans or animals. NYBC policy states that only surplus blood will be made available for research purposes, and thus this study will not compromise blood supplies. Blood will be used for research purposes only -solely for in vitro culture of Plasmodium falciparum -not for transfusion into humans or animals. The blood purchased from NYBC will only be used as a resource for propagation of malaria parasites and no data will be collected with regard to the blood itself. Therefore the inclusion of women, minorities or children is not applicable.

Ethics Statement
Blood used in parasite cultures was obtained under a protocol approved by and on file with the Internal Review board at Weill Medical College of Cornell University or at New York Blood Center. All donors gave prior written consent. Figure S1 Structures of compounds used in this study. Parasite viability with measured with the luciferase-based (yellow curves) or tritiated hypoxanthine-based viability assay (red curves) in the presence of increasing concentrations of chloroquine (A), quinine (B), mefloquine (C), and artemisinin (D). Best-fit curves are shown. Y-axis is percentage assay readout; X-axis is log 10 drug concentration. EC 50 s for each drug are shown below the figure.

Supporting Information
Best-fit curves are highly similar for each drug. (TIFF) Figure S3 Expression levels of PfACb in the red blood cell. RT-PCR using PfACb-specific primers confirms publicly available microarray data [23,24]. Both primer sets 1 (blue bars) and 2 (red bars) amplify high levels of PfACb mRNA in the late trophozoite and schizont stages of the parasite. Representative photos of Giemsa-stained parasites corresponding to the time of RNA extraction for the RT-PCR analysis are shown below the graph.
(TIFF) Figure S4 The solubility of His-tagged PfACb 1-785 is increased by high salt conditions. (Similar results were obtained with GST-PfACb 1-785 ). Hi-5 insect cells were infected with His-tagged PfACb 1-785 baculovirus and harvested after 42 hrs (determined to be the optimal time for maximal activity and expression of intact protein). Cell pellets were resuspended in a lysis buffer containing 50 mM Tris (pH = 7.5), 10 mg/mL aprotinin/leupetin, 1 mM PMSF, 1 mM benzamidine, 200 mM NaCl, and 1 mM DTT at ,10 mL lysis buffer/100 mL of pelleted culture. This lysate was sonicated five times at 10-second intervals at 12 watts with a Misonix Microson cell disruptor. Sonicated lysate was clarified by centrifugation at 100,0006g using a Ti-75 rotor (Beckman). The pellet fraction was resuspended in lysis buffer and adenylyl cyclase activity corresponding to PfACb 1-785 activity remained in the insoluble pellet fraction. The various additives indicated above were added to the resuspended pellet fraction, and the solution was again clarified by centrifugation. Soluble fractions were assayed for adenylyl cyclase activity. This was used as a measure of PfACb 1-785 amount. Only 2 M NaCl significantly solublized PfACb 1-785 . (TIFF) Figure S5 Effect of KH7-like compounds on parasite viability. P. falciparum cultures were maintained in a 96-well plate in the presence of 40 mM of the indicated compound. Luminescence was measured after 48 hrs. Reactions were performed in duplicate. (TIF)