Identification of Potent Phosphodiesterase Inhibitors that

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Identification of Potent Phosphodiesterase Inhibitors that Demonstrate Cyclic Nucleotide-Dependent Functions in Apicomplexan Parasites. Brittany L Howard, Katherine L Harvey, Rebecca Stewart, Mauro F Azevedo, Brendan S. Crabb, Ian G Jennings, Paul R Sanders, David T. Manallack, Philip E. Thompson, Christopher J Tonkin, and Paul R Gilson ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb501004q • Publication Date (Web): 02 Jan 2015 Downloaded from http://pubs.acs.org on January 4, 2015

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ACS Chemical Biology

Identification of Potent Phosphodiesterase Inhibitors that Demonstrate Cyclic Nucleotide-Dependent Functions in Apicomplexan Parasites. Brittany L. Howard1*, Katherine L. Harvey2,4,*, Rebecca Stewart3,5, *, Mauro F. Azevedo2, Brendan S. Crabb1,2,4, Ian G. Jennings1, Paul R. Sanders2, David T. Manallack1, Philip E. Thompson1 +, Christopher J. Tonkin3,5 + and Paul R. Gilson1,2 +

1. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia 2. Macfarlane Burnet Institute, Melbourne, Victoria, Australia 3. The Walter & Eliza Hall Institute, Melbourne, Victoria, Australia 4. University of Melbourne, Victoria, Australia 5. Department of Medical Biology, The University of Melbourne

* These authors contributed equally to this work. + Corresponding Authors

Philip Thompson Medicinal Chemistry Monash Institute of Pharmaceutical Sciences, 381 Royal Pde, Parkville, Melbourne, Victoria, Australia Email: [email protected]

Chris Tonkin The Division of Infection and Immunity

ACS Paragon Plus Environment

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The Walter & Eliza Hall Institute of Medical Research 1G Royal Pde, Parkville, Melbourne, Victoria, Australia. Email: [email protected]

Paul Gilson Macfarlane Burnet Institute, 85 Commercial Rd, Melbourne, Victoria, Australia email: [email protected]


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ABSTRACT Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds of host cell invasion, replication and egress (exit), destroying host cell tissue in the process. Previous work has identified important roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress and invasion, yet little is understood about the regulation of cyclic nucleotides, cGMP and cAMP, that activate these enzymes. To address this we have focused upon the development of inhibitors of 3’,5’-cyclic nucleotide phosphodiesterases (PDEs) to block the breakdown of cyclic nucleotides. This was done by repurposing hPDE inhibitors noting various similarities of the human and apicomplexan PDE binding sites. The most potent inhibitors blocked the in vitro proliferation of P. falciparum and T. gondii more potently than the benchmark compound, Zaprinast. 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) was found to be a potent inhibitor of recombinant PfPDEα and activated PKGdependent egress of T. gondii and P. falciparum likely by promoting the exocytosis of micronemes, an activity that was reversed by a specific Protein Kinase G inhibitor. BIPPO also promotes cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion suggesting that the compound inhibits single or multiple PDE isoforms that regulate both cGMP and cAMP levels. BIPPO is therefore a useful tool for the dissection of signal transduction pathways in apicomplexan parasites.


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INTRODUCTION The phylum Apicomplexa comprises a large group of obligate intracellular parasites of wide social, economic and medical significance. Plasmodium falciparum, the causative agent of the most severe form of malaria, remains one of the biggest scourges to human health infecting 207 million people a year and killing 627,000 1, 2. Toxoplasma gondii on the other hand is chronically carried by 30-80% of human populations and whilst it causes few problems in healthy individuals, acute infection in the developing foetus or people with compromised immune systems (HIV, transplant recipients) can cause massive tissue destruction. This can lead to a range of disease manifestations including hydrocephalus, epilepsy, mental retardation when passed to the unborn foetus or blindness and neurological deficits in the immuno-compromised. Both P. falciparum and T. gondii are obligate intracellular pathogens and thus rely on the invasion of host cells for survival and proliferation. During their asexual amplification stage P. falciparum exclusively targets human erythrocytes whereas T. gondii can infect almost any cell in most vertebrates. After replication inside a host cell, both T. gondii and P. falciparum must activate a cellular program, which elicits egress (exit) from one cell and reinvasion of the next. Molecular processes that are required for invasion and egress are of significant interest given their potential as vaccine and drug targets for the treatment and prevention of toxoplasmosis and malaria. Recently, it has been determined that three second messenger signalling pathways control parasite egress and reinvasion of host cells 3-6. An increase in cytoplasmic concentration of the secondary messengers cyclic-AMP (cAMP), cyclic-GMP (cGMP) and calcium ions (Ca2+), typically cause changes in the cellular program by activating kinases through their direct or indirect binding. This results in phosphorylation of specific substrates, which ultimately leads to a change in cellular function. cAMP for example is a known activator of


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Protein Kinase A (PKA) and this enzyme has been implicated in the phosphorylation of the cytoplasmic tail of the parasite adhesin Apical Membrane Antigen 1 (AMA1), which is essential for P. falciparum erythrocyte invasion 5. Protein Kinase G (PKG) on the other hand is directly activated upon a rise in cGMP concentration and in apicomplexan parasites this enzyme appears to occupy an important nexus during multiple transition stages within P. falciparum 4, 7-9 and during egress and motility of Toxoplasma 6, 10, 11. Given that PKA and PKG require a rise in intracellular concentration of cAMP and cGMP, enzymes that control the production and degradation of these cyclic nucleotides likely play a critical role in the activation of egress and invasion. 3’-5’ cyclic nucleotide phosphodiesterases (PDEs) hydrolyse cAMP and cGMP into AMP and GMP respectively thus attenuating their cellular accumulation. Indeed, physiological PDE inhibition (eg, by regulatory domain phosphorylation) has been described as a mechanism to increase cAMP and cGMP levels thus activating downstream events 12. Small molecule inhibitors of parasite PDEs could therefore assist in the determination of events that activate egress and invasion in apicomplexan parasites. The hPDE5 inhibitor, Zaprinast (1) (Figure 1) was the first compound identified as an inhibitor of parasite PDEs 13. Specifically, it was shown to inhibit P. falciparum proliferation (IC50 35 µM), inhibit recombinant P. falciparum PDEα activity and cause elevation of intracellular cGMP levels 14. Zaprinast has also been used in the study of parasite egress where it was shown to induce premature egress from erythrocytes, which can be blocked by the PKG inhibitor, Compound 1 (Cmpd1) 4. Indeed, Zaprinast can also induce PKGdependent egress in T. gondii 2. While Zaprinast has been useful to determine the role of PDEs in infection, and as a tool for activating parasite egress, it is only a moderately potent inhibitor and therefore has limited use. Several other PDE inhibitors were evaluated in the 2009 National Institute of Health


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(NIH) malaria screen 15, including the non-isoform selective PDE inhibitor, Dipyridamole and the hPDE3 inhibitor Cilostazol with EC50 values against the 3D7 strain of approximately 10µM. In addition, Beghyn and co-workers originally explored the repurposing of the human PDE5 inhibitor, tadalafil (2) (Figure 1) as a potential anti-plasmodium compound. The most potent tadalafil analogue (3) (Figure 1) had an EC50 of 0.5 µM 16. Recently, we reasoned that more potent PfPDE inhibitors might be identified by examining the homology at the catalytic site of P. falciparum PDE’s (PfPDE) as compared to human PDEs (hPDE) 17 and using this we hypothesised that hPDE9 and hPDE1 isoforms were most like PfPDE, thus providing a mechanism to identify new apicomplexan PDE inhibitors. Here we have extended our examination of this topic now to the apicomplexan parasite T. gondii PDEs (TgPDE). Furthermore, we show that a series of compounds that inhibit hPDE9/PDE1 elicit much more potent cell responses than Zaprinast on both P. falciparum and T. gondii. We provide evidence as to the mode-of-action of these new inhibitors showing that they potently inhibit recombinant PfPDEα, induce host cell egress in both P. falciparum and T. gondii, which can be blocked by inhibiting PKG and this is likely due to their ability to potently activate microneme secretion. Interestingly, we also show that our new PDE inhibitors act to induce an accumulation of cAMP during AMA1 tail phosphorylation, thus suggesting that the PDE target(s) of these compounds break down both cAMP and cGMP during egress, motility and invasion in apicomplexan parasites. Together, this data describes potent new compounds that can be used to study cyclic nucleotide signalling across the Apicomplexa, which in time could offer new insights for future disease control therapies.


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RESULTS AND DISCUSSION Homology of PfPDEs and TgPDE. Previous work demonstrating that Zaprinast and other compounds are active against P. falciparum and T. gondii suggested that these parasites rely on one or more PDEs for critical cellular processes 2, 4. The P. falciparum genome encodes four highly similar PfPDEs of which only two, PfPDEα and PfPDEβ, appear to be expressed in asexual blood stages. It is this stage that produces symptomatic disease and is therefore the stage that most PDE inhibition studies have targeted. Collins et al suggested PfPDEβ as a good candidate for Zaprinast inhibition 4, but the PfPDEs are highly homologous to one another at the active site and it is conceivable that it inhibits multiple isoforms. The PfPDEs are also homologous to the human targets of Zaprinast hPDE1, 5 and 9 17. Yuasa et al showed that Zaprinast inhibits PfPDEα with an IC50 of 3.8µM and in their studies of PfPDEδ knockout gametocytes Taylor et al showed that PfPDEδ is relatively insensitive to Zaprinast whereas the residual activity is zaprinast-sensitive 13, 18. To extend this work we wished to determine the PDE target(s) of Zaprinast in T. gondii. We retrieved 18 putative PDE sequences from ToxoDB, the T. gondii genome database and reviewed the homology to human and Plasmodium sequences. When aligned to the Pf- and hPDE sequences TgME49_202540 showed good homology to PfPDEβ (27%) and hPDE1B (25%) and in particular showed 72% homology at the cyclic nucleotide binding site with PfPDEβ (Table 1), and matched hPDE1/9 nucleotide-binding residues as well at a number of conserved amino acid positions. Other T. gondii sequences also showed sufficient homology to the hPDE and PfPDE sequences to be considered candidate PDEs and include TgME49_266920 [PfPDEβ (26%) and hPDE1 (28%)] and TgME49_228500 [PfPDEβ (29%) and hPDE1 (33%)] (data not shown).


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In developing homology models of the PfPDE isoforms 17, we identified some key features that we believed would dictate potency of inhibitors of those PDEs and these were found to be conserved in the T. gondii orthologues (Table 1). In particular, the residue adjacent to the conserved “purine-scanning” glutamine is small in hPDE9 (A452) , hPDE1 PfPDEβ (S283, Figure 2) and Tg49_202540 , compared to a bulky M in most human isoforms (Table 1 and Figure 2). We had also identified that a residue in the core pocket (N405 in PDE9) as H or N in the various parasite sequences (H235 in PfPDEβ, Figure 2) as well as hPDE1, -4, -7, -8 and -9, but A in PDE5. The larger H residue in PfPDEs may explain why the hPDE5 inhibitor sildenafil, is a much less potent inhibitor in P. falciparum proliferation assays relative to Zaprinast. Finally we identified a common hydrophobic pocket formed by three residues (L421, Y424 and F441 of hPDE9 ; L251, F254 and L271 in PfPDEβ, Figure 2). Overall, the similarity of key nucleotide binding residues of hPDE1 or hPDE9 with the parasite PDEs supported inhibitors of human hPDE1 and/or 9 as potential starting points from which to develop parasite PDE inhibitors. We speculated that a series of reported 1H-pyrazolo[4,3d]pyrimidin-7(6H)-one PDE1/9 inhibitors described by De Ninno et al displayed the requisite binding pharmacophore (Figure 2) 19. Subsequently an analogous 1H-pyrazolo[3,4d]pyrimidin-4(5H)-one compound in co-crystal with hPDE9 was reported adopting the pose as modelled by us 19, 20.

Discovery of 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (6): Given that both P. falciparum and T. gondii most closely resemble hPDE1/9 we prepared a focused library of compounds based upon a series of the already reported inhibitors of this enzyme 19. The synthesis of all the compounds derive from the key precursor 4 prepared by adaptation of literature methods (Scheme 1). Condensation of 4 with various carboxylic acids


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using PyBroP under microwave conditions yielded intermediate amides 5, which underwent base catalysed cyclisation to yield the desired pyrazolopyrimidinones (6-23). An N-methyl derivative 24 was prepared by direct methylation of 6 (see Supporting Information). The compounds were first tested for their growth inhibitory activity in cultured asexual blood stage P. falciparum parasites by measuring the activity of the parasite enzyme lactate dehydrogenase, after 72 hrs of parasite cultivation with the inhibitors (Scheme 1 and Table 2). Compounds 6-11 showed the most potent growth inhibitory activity in that order. Compound 6 or 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one which we abbreviated to ‘BIPPO’ and with no substitution on the phenyl ring was the most effective compound with an IC50 of 0.4µM in P. falciparum. The potency of BIPPO was followed by compounds 7 and 8, indicating the respective para-chloro and para-fluoro substitutions were tolerated (Scheme 1 and Table 2). The presence of other substituents, shorter or longer links to the aryl substituent reduced potency as did, most notably, methylation at the 7-positon (24) consistent with the proposed pharmacophore. Proliferation inhibitory studies were performed also with T. gondii tachyzoites growing in human fibroblasts. Growth potential was qualitatively measured by the number and size of zones of host cell clearance (plaques) created by the lytic growth of T. gondii. We decided to take the three best compounds as determined by P. falciparum growth inhibition studies (above) and determine their ability to block T. gondii growth. BIPPO showed effective inhibition at concentrations as low as 2µM and was at least 30 times more effective than Zaprinast at reducing plaque formation (Figure 3). Just as for P. falciparum, compounds 7 and 8 were the next most potent at reducing tachyzoite proliferation and all were much more effective than Zaprinast (Figure 3). Note that the IC50 cannot be calculated using this plaque assay as a readout.


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BIPPO, 6 with its robust activity in both growth inhibition assays appeared to be the compound to investigate further to establish parasite PDE inhibition as the mechanism of action. We first attempted to confirm that BIPPO was a potent inhibitor of PfPDEs by testing it against recombinant enzymes. We expressed PfPDEα with a glutathione S transferase (GST) tag in Escherichia coli, and obtained a partially purified protein (Supplementary Figure s1) that showed robust hydrolysis of cGMP. While full characterisation of the enzyme and inhibitors was not possible due to its poor stability, it was found that BIPPO inhibited this activity in a dose dependent manner, with an IC50 of approximately 150 nM, while zaprinast was much less potent 5 µM affording only approximately 40% inhibition, (consistent with the report of Yuasa et al 13) (Supplementary Figure s2). We also observed that the enzyme showed modest hydrolytic activity against cAMP, (Supplementary Figure s3) and when both cGMP and cAMP were in the same reaction, hydrolysis of cGMP was virtually complete while significant cAMP remained (Supplementary Figure s4). We also attempted to prepare recombinant PfPDEβ and while observing some activity against cGMP the enzyme was even less stable than PfPDEα making reproducible measurements problematic and the data will not be presented here. BIPPO was also screened against recombinant hPDE isoforms and confirmed potent hPDE9 activity (IC50 = 30nM), with significant inhibition at 1 µM against hPDE1 (42%), hPDE5 (66%) and hPDE6 (64%). Attempts to directly measure the increase in levels of these cyclic nucleotides in cultured blood stage P. falciparum, using commercial microplate kits were unsuccessful since nucleotide levels were too low to reliably measure (data not shown).

BIPPO potently activates parasite egress in P. falciparum and T. gondii. We assessed the activity of BIPPO in comparison to Zaprinast in a number of cyclic nucleotide dependent functional assays in both P. falciparum and T. gondii. To quantitatively


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measure egress in P. falciparum cultures we created an egress reporter parasite line by transfecting the 3D7 strain with a highly active luciferase called Nanoluc fused to a signal peptide sequence at its N-terminus 21, 22. The signal peptide promotes secretion of Nanoluc into the parasitophorous vacuole (PV) space that surrounds the intracellular parasite. Just prior to egress, the parasite breaks down the PV membrane and the plasma membrane of the erythrocyte host and the extent to which this occurs can be ascertained by measuring the levels of luciferase released into the supernatant. After 20 minutes incubation, BIPPO induced greater egress at substantially lower concentrations than Zaprinast and induced a 2.5 fold higher egress peak at saturating levels of the compounds (Figure 4A).

To determine if the egress response seen in P. falciparum due to PDE inhibition was also seen in T. gondii, we monitored egress response to a titration of our panel of compounds and compared their effectiveness to Zaprinast. Egress was measured as a function of host cell lactate dehydrogenase release (LDH) release, which occurs due to damage created by egressing tachyzoites 2. In comparison to Zaprinast, T. gondii egress was more sensitive to five compounds (BIPPO, 7, 8, 10 and 12, Scheme 1 and Table 2), one drug (18) showed a similar response to Zaprinast (17) and one compound was ineffective at inducing tachyzoite egress at all (15) (Figure 4b-h). The data largely correlates with the activity shown by the compounds in the T. gondii proliferation (plaque) assay (Figure 3).

To understand further the effect that PDE inhibition has on parasite egress we undertook live cell imaging of both P. falciparum and T. gondii. Zaprinast has been recently shown to induce premature egress in asexual, late stage blood cultures of P. falciparum with an EC50 25µM 4 and also activate tachyzoite egress from host cells in T. gondii 2. To confirm that BIPPO was actually inducing the breakdown of the PV membrane and the plasma membrane


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of infected erythrocytes, time-lapse imaging was performed on late stage parasites. Over a 30 minute period 2µM BIPPO appeared to induce the breakdown of the erythrocyte and vacuole membranes around fully formed merozoites but they could not invade the surrounding erythrocytes presumably because they either were not developmentally mature or because BIPPO also inhibited the invasion process (Figure 5A, left, Movie 1). Immature parasite forms undergoing cytokinesis were also released highlighting the fact that BIPPO was accelerating breakdown of the host before the merozoites were mature, presumably through an increase in cGMP levels (Figure 5A, right, Movie 1). Zaprinast induced egress was not done since it has been performed previously 4.

To investigate the response of T. gondii to these new inhibitors, BIPPO and Zaprinast were compared for egress induction via live microscopy (Figure 5B and Movie 2). Tachyzoites were allowed to invade and replicate for 30hrs to produce vacuoles with >16 parasites. Tachyzoite egress was seen rapidly after addition of the compounds (Cf =55µM) with BIPPO stimulating egress at 1:30 mins faster than Zaprinast (5:30 mins). Compounds 7 and 8 were also tested and they too induced egress with rapidity consistent with the drug curves (Supplementary Figure s5). It is also worth noting that T. gondii and P. falciparum undergo intracellular replication in a different manner. T. gondii undergoes binary division and is always ready to egress, thus premature egress is not possible.

PKG inhibitor can block BIPPO indicating specificity for a cGMP-dependent PDE. In other systems cGMP can function by activating ion channels and promoting phosphorylation via PKG. If BIPPO and the other PDE inhibitors exert their egresspromoting effects in a PKG-dependent processes, then inhibition of this kinase should block


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the egress-promoting effects of BIPPO. Compound 1 (Cmpd1) is a specific inhibitor of apicomplexan PKG 11 and it has been shown that Cmpd1 blocks the effects of Zaprinast in both P. falciparum and T. gondii 2, 4, 23. To determine if Cmpd1 could therefore block the proegress effects of BIPPO the P. falciparum Nanoluc reporter parasites were incubated +/- 2.5 µM Cmpd1 along with the approximate IC50 levels of BIPPO (0.7µM) and Zaprinast (40µM) for 0, 10, 20 and 40 minutes (Figure 6A). Under these conditions Cmpd1 efficiently blocked the egress-promoting effects of Zaprinast and BIPPO confirming the latter is also a cGMP PDE inhibitor (Figure 6A).

We performed parallel experiments in T. gondii, which is susceptible to Zaprinast in a Cmpd1-dependent manner 2. After pretreatment of intracellular tachyzoites with a titration of Cmpd-1, parasites were stimulated to egress using a fixed concentration of our compounds (BIPPO, 7, 8 and Zaprinast) (Figure 6b-d), while increasing the concentration of Cmpd1. The most potent PDE inhibitor, BIPPO required greatest concentrations of Cmpd1 to inhibit egress than 7, 8 and Zaprinast. Furthermore, as visualized by live video microscopy, Cmpd1 pretreatment completely ablated tachyzoite response to our compounds, showing a complete lack of response even after 10 minutes of stimulation (Supplementary Figure s5, Movie 3).

PDE inhibitors stimulate the release of secretory organelles in Toxoplasma Host cell egress and motility in apicomplexan parasites relies on the secretion of adhesins and perforin-like molecules from the microneme organelles, which is controlled by intracellular calcium and PKG signaling events 6, 11, 24. Given that our new PDE inhibitors rapidly stimulate host cell egress in both T. gondii and P. falciparum we hypothesized that this was due to the activation of microneme secretion. To test this we applied the inhibitors to a wellestablished semi-quantitative microneme secretion assay in T. gondii 6, 24 (this assay is not


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well developed for P. falciparum and hence could not used here). Here the amount of proteolytically cleaved Mic2 (a major Toxoplasma adhesin) is measured in the supernatant of extracellular tachyzoites after stimulation and is used as a surrogate for total microneme release. To do this we treated extracellular tachyzoites for 20 minutes with 500µM of Zaprinast or 55µM of BIPPO, 7 and 8. The supernatant (containing any excreted and cleaved MIC2 in response to stimulation) and pellet fraction was collected by centrifugation and then fractionated by SDS-PAGE. Western blot was then used to determine the amount of MIC2 present in the supernatant. All our new PDE inhibitors induced the secretion of MIC2 into the supernatant and appear more potent over the given timeframe than Zaprinast (Figure 7). Overall, this provides further evidence that our new compounds are much more potent PDE inhibitors than Zaprinast and act, at least in part, to induce parasite egress through the stimulation of microneme secretion.

BIPPO enhances PKA/cAMP-dependent AMA1 tail phosphorylation Given the restricted expression of PDE isoforms through the blood stage of the parasite in P. falciparum we hypothesized that the target(s) of BIPPO may also break down cAMP. We have shown previously in P. falciparum (not studied at all in T. gondii) that the cytoplasmic tail of the merozoite invasion ligand apical membrane antigen 1 (AMA1), is phosphorylated by the cAMP-dependent protein kinase A, at serine 610 5. To measure phosphorylation here, an in vitro reaction was performed on recombinant GST-AMA1 tail in the presence of parasite lysate, [γ-32P] ATP and 2µM cAMP. To determine if BIPPO could block cAMP hydrolysis we added 6µM to a series of in vitro phosphorylation reactions containing 2, 1, 0.5, 0.25 and 0µM cAMP (Figure 8 and Supporting Information). Without BIPPO the levels of AMA1 tail phosphorylation declined very rapidly with decreasing cAMP concentrations suggesting the nucleotide was being rapidly hydrolyzed. The rate of phosphorylation decline


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however, could be substantially inhibited by BIPPO, suggesting it acts against a cAMPdegrading PfPDE (Figure 8).

Given their greater potency and rapid action, BIPPO and its analogues have great potential as tools to understand signaling pathways mediating egress and invasion in apicomplexan parasites. Previously, use of Calcium Ionophores (Ionomycin or A23187) were standard for assessing active or defective biological responses such as egress, motility, microneme secretion and invasion 3, 6, 24-26. However, this approach is problematic as calcium ionophores induce a crude, global Ca2+ change in the intracellular environment by indiscriminately releasing calcium from intracellular stores and allowing Ca2+ to enter from external sources 27

. This completely ablates the intricacies and importance of calcium dynamics, such as

calcium flux, amplitude and periodicity. Furthermore, signal pathway induction by ionophores prohibits analysis of any events preceding calcium release. Indeed, cGMP signaling has been suggested to act before intracellular Ca2+ release and therefore argues that the use of BIPPO provides a superior alternative to current stimuli in a more biologically relevant environment 9. The combination of the use of BIPPO, Cmpd1 and Ca2+ ionophore may indeed help tease out the order of events that are required for apicomplexan host cell egress and help understand how a change in extracellular K+ activates egress or how the engagement of host cell receptors promotes invasion.

The consequences of inducing rapid and premature parasite egress in an infected host are not absolutely clear and so the therapeutic potential of PDE inhibition remains to be examined. On the other hand, dysregulation of key events in host cell invasion such as AMA1 phosphorylation, that rely upon cAMP dependent PfPKA phosphorylation for efficiency, may


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provide one mechanistic pathway to a therapeutically useful application of PDE inhibition or render parasites more susceptible to other drugs.

In conclusion, the broad homology between the mammalian and parasitic PDE active site residues 14 suggested that it should be possible to repurpose hPDE inhibitors for use in studying parasite signaling. Our approach to use homology modelling to select the most homologous of the human enzymes for parasite orthologues led us to develop BIPPO which is much more potent than the previously utilized tool compound, Zaprinast at inhibiting growth and inducing premature parasite egress in a PKG-dependent manner. While BIPPO shows potent inhibition of PfPDEα consistent with its observed cell-based activity, the specific PfPDE and TgPDE targets of BIPPO in parasites are still unknown and the effect on both cGMP and cAMP pathways suggest they may emerge as being a single dual specificity PDE, multiple PDEs or a cGMP or cAMP-PDE involve in PDE cross-talk and this is a key goal of future work. The greater activity of PfPDEα against cGMP over cAMP reflects the current order in which we think these nucleotides function ie, cGMP stimulates egress followed by cAMP dependent phosphorylation of AMA1 which enables invasion. Optimizing the pharmacological inhibitors of parasite PDE functions may provide novel compounds for the study of signal transduction processes governing apicomplexan host cell egress and invasion and identify new intervention points for therapy in these diseases.


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METHODS Plasmodium falciparum strains and transfections. P. falciparum (strain 3D7) asexual blood stage parasites were cultured as per 28 in RPMI-HEPES media supplemented with Lglutamine (Sigma) and Albumax® II (Invitrogen). To express a luciferase enzyme that could be secreted into the parasitophorous vacuole of infected red blood cells, DNA sequence corresponding to 23 amino acid endoplasmic reticulum signal sequence of merozoite surface protein 1 was appended onto the 5’ end of the Promega NanoLuc sequence. 3D7 parasites were transfected with the NanoLuc construct by culturing in erythrocytes that had been electroporated with 100µg of the DNA 29, 30 and selected on 5 µg/mL blasticidin S. Toxoplasma culture. Toxoplasma was grown in Human Foreskin Fibroblasts (HFFs) and passaged as required. Here HFFs were grown to confluency in DME supplemented with 10% Cosmic Calf Serum (Hyclone) and just before inoculation with T. gondii media was refreshed and serum levels dropped to 1% fetal calf serum. All culture and assay conditions performed at 37°C, 10% CO2. Plasmodium growth assay. Asexual blood stage parasites 12-24 hour post infection (hpi), were grown for 72 hrs in compounds serially diluted in DMSO (0.2% v/v in culture media). Parasite growth was assessed by measuring the activity of parasite lactate dehydrogenase using the Malstat assay 31. Toxoplasma Plaque Assay. Growth was determined by the ability of tachyzoites to form zones of clearance (plaques) in host cells through repeated cycles of invasion, replication and egress. This was done by adding tachyzoites to confluent HFFs (Cf 100cells/ml) and treated with indicated concentrations of drug for the entirety of the assay. Plates were left undisturbed for 7 days at 37°C, fixed with 70% methanol, stained with Crystal Violet and imaged.


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Plasmodium Egress Assays. 3D7_NanoLuc parasites22 were cultured until 44h post-invasion at 5% parasitemia. The culture was washed in RPMI medium to remove any NanoLuc from the supernatant and resuspended at 2% hematocrit in RPMI medium. Compound 1 was added to half of the culture at 2.5µM and 0.2µL of Zaprinast or BIPPO dilutions in DMSO were added to 100µL of Compound 1-treated and -untreated culture in duplicate. Samples were allowed to warm to 37oC for 5min and then placed on ice after a 0, 10, 20 or 40min incubation period. Cells were pelleted at 3000g for 5 mins then 10µL culture supernatant was mixed with 10µL NanoLuc assay buffer (2x Promega cell lysis buffer and 1µL Nano Glo substrate per mL) in a luminometer plate and the activity was measured with a FluoStar Optima instrument (BMG Labtech). Relative light units (RLU) were plotted as a function of inhibitor concentration or time in Prism (GraphPad), using non-linear regression analysis as a sigmoidal dose-response curve with variable slope. Samples were assayed in triplicate. Live cell imaging of P. falciparum parasites was performed as per 32 after addition of 2µM BIPPO. Toxoplasma LDH Egress assay. Egress was determined as a function of lactate dehydrogenase (LDH) released from the host cell as parasites egress 2. Tachyzoites (Cf 5x105cells/ml) were added to confluent HFFs in a 96 well plate format and grown for 32hrs. Wells were washed once with Ringers/5%FCS before 1:3 titration of PDE inhibitors and Zaprinast for 5 mins at 37°C (Ctop 500µM). For Cmpd1 inhibition, cells were pre-treated with a titration of Cmpd1 (Ctop 18µM) for 20mins at 37°C then stimulated with PDE inhibitors (55 µM) or Zaprinast (500µM) for 5 mins at 37°C. Supernatants were taken and LDH detected using Promega CytoTox LDH assay kit according to manufacturers instructions. Data was normalised to 100% egress response in each assay. IC50 and EC50 were determined using Prism (GraphPad) plotting normalised, log transformed (x axis), non-linear regression analysis as a sigmoidal dose-response curve with variable slope. Samples were assayed in


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triplicate for each assay, n=3. Treatments did not induced significant LDH release in absence of parasites (data not shown). Toxoplasma Live Egress. Tachyzoites were added to confluent HFFs in imaging chambers at 1x104 cells/ml and grown for 30hrs. In relevant instances, cells were pre-treated with 2µM Cmpd1 for 20mins at 37°C. Wells were rinsed and media was replaced with Ringers/5%FCS. Cells were imaged using a heated chamber at 37°C with PDE inhibitors (55µM) and Zaprinast (500µM) added at 00:30 time-points. Images were recorded for 10 minutes. Toxoplasma Microneme secretion assay. Fresh extracellular cells (2 x 108 cells/ml) were treated for 20 minutes at 37°C with PDE inhibitors (Cf 55µM) and zaprinast (Cf 500µM) in 3%FCS DME. Microneme secretion was detected using western blot, probing for micronemal protein MIC2 33.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org This paper contains enhanced objects

ACKNOWLEDGEMENT B.L.H, K.L.H and R.S. are recipients of Australian Postgraduate Awards and C.J.T is recipient of an Australian Future Fellowship (FT1200100164). This work was supported by NHMRC Project grants 1025598 and 603720 as well as the Victorian State Government Operational Infrastructure Support Program and NHMRC IRIISS. We are grateful to the


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Australian Red Cross for the supply of red blood cells. We also thank K. Rogers for help with microscopy, and T. Luc for technical assistance.


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TABLES

Residuea Regionb hPDE1 292 M H 293 M D 296 M H 322 M E 325 M H 402 M D 251 Q Y 405 Q H 413 Q H 420 Q+H L 423 Q E 453 Q Q 456 Q F 490 Q W 421 H M 424 H F 441 H L 301 L N 302 L N 303 L F 452 L S 455 L G 459 L F 406 P 417 T

hPDE5 H D H E H D Y A Q V E Q F W A F L N S Y M G A I A

hPDE9a PfPDEβa TgME49_202540 H292 H119 H D293 D120 D 296 123 H H H E322 E149 E 325 152 H H H D402 D232 D 251 78 F Y Y N405 H235 N A413 H243 H 420 L V250 L 423 253 E E E Q453 Q284 Q F287 F F456 490 Y W322 W L421 L251 S 424 254 Y F F F441 L271 L N301 N128 N L129 A T302 Y303 F130 L A452 S283 S 455 286 G T G F290 F F459 406 236 E G C V417 C247 C

a

Numbering based upon PDE9 x-ray structure pdb code: 3DYN and PfPDEβ model based on 17, bRegion guide M = metal binding, Q = core pocket, H = hydrophobic pocket, L = Lid region (see 34). Shaded residue positions are discussed in the text.

Table 1. Alignment of human, Pf and Tg PDE catalytic site residues based upon full sequence alignment.


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Compound IC50 (µM) 1 Zaprinast 35 ± 4.2 a 6 (BIPPO) 0.40 ± 0.14b 7 0.64 ± 0.21 8 0.73 ± 0.30 9 0.74 ± 0.23 10 1.2 ± 0.24 11 2.4 ± 0.6 12 2.9 ± 0.6 13 3.6 ± 0.85 14 4.7 ± 1.9 15 7.8 c 16 5.6 17 10 18 11.5 ± 3.7 19 25 20 35 ± 6.7 21 40 22 >100 23 >100 24 70

a

Reference 13; b IC50 ± SEM (n = 3 or 4); c IC50 (n = 2).

Table 2. Anti proliferative activity of the pyrazolopyrimidinones against the asexual blood stage of Plasmodium falciparum 3D7 strain.


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Scheme 1. Synthesis of compounds 6-24 (top) and structures of R- groups (bottom).

FIGURES

Figure 1 Structures zaprinast (1), tadalafil (2) and tadalafil analogue (3).

Figure 2 Homology model of PfPDEβ catalytic site modelled with BIPPO (6) by analogy to hPDE9 co-crystal (PDB: 3JSI). Residues are highlighted which may dictate selective interactions between inhibitors and binding site including Q284 conserved glutamine (Q453 in PDE9), H243 (A413), H235 (N405), S283 (A452), L251 (L421), F254 (Y424), L271 (F441) and H277 (V447).

Figure 3 PDE inhibitors prevent T. gondii growth. Parasites incubated with confluent HFF cells for 7 days with indicated concentrations (µM) of PDE inhibitors. Each zone of clearance (white plaques) represents one tachyzoite undergoing repeated rounds of invasion, replication and egress. T. gondii is more sensitive to new PDE inhibitors than zaprinast with plaques prevented at concentrations upwards of 55µM in PDE inhibitors tested. Scale bar = 5mm.

Figure 4 BIPPO (Compound 6) induces parasite egress. a) In P. falciparum blood stages BIPPO more efficiently induces egress than zaprinast through being more active at lower concentration and achieving higher peak activity after 20 minutes incubation. Egress was quantified by measuring luciferase activity as relative light units (RLU) in the growth media of parasites induced to breakdown their enveloping vacuole and erythrocyte membranes


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thereby releasing the luciferase. The data points represent mean ±SD, n=3. b-f) Tachyzoite egress following 5 min treatment with different concentrations of PDE inhibitors was measured as a function of lactate dehydrogenase release from host cells, and normalised to maximal egress. Each plot displays indicated drug treatment compared with zaprinast. Mean +/- S.E.M, n=3. Plots indicate that compounds BIPPO (b), C7 (c), C8 (d), C12 (e) and C10 (f) out perform zaprinast as an egress stimulant while C18 (g) is comparable to zaprinast and C15 (h) is ineffective.

Figure 5 Movie stills showing rapid egress of Plasmodium falciparum and Toxoplasma gondii induced by BIPPO (added at black arrow). a, left) Sequential video images showing P. falciparum merozoite egress (red box) beginning 10:30 min after addition of 2µM BIPPO. (Right) After 15:30 min, BIPPO induces breakdown of membranes surrounding doubly infected erythrocyte before the schizonts have undergone complete cytokinesis. Time stamp is minutes:seconds. Scale bar =5µm. b) Intracellular T. gondii tachyzoite vacuoles (outlined by dashed white line) were exposed to either zaprinast (500µM) or BIPPO (55µM) at 30 secs (arrow). BIPPO-treated T. gondii respond much more rapidly and at lower concentration than zaprinast treatment. Scale bar = 20µm.

Figure 6 The cGMP specific protein kinase G (PKG) inhibitor compound 1 (Cmpd1), can block egress-promoting effects of BIPPO and other putative PDE inhibitors confirming the inhibitors are targeting parasite cGMP-specific PDEs . a) Cmpd 1 at 2.5µM was able to block the pro-egress effects of 0.7µM BIPPO and 40µM zaprinast upon Nanoluc expressing P. falciparum blood stage parasites. Egress was measured via luciferase present in the supernatant after host cell lysis. RLU was plotted as a function of time for the various treatments (mean +/- SD, n=3). b-d) Inhibition of tachyzoite egress following 20min pre-


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incubation with different concentrations of Cmpd1 (Ctop 18µM) and 5 min treatment with a fixed concentration of PDE inhibitors (Cf 55µM). Egress was measured as a function of lactate dehydrogenase release from host cells, and normalised to maximal egress. Each plot displays indicated drug treatment compared with Zaprinast. mean +/- S.E.M, n=3. Plots demonstrate egress response was inhibited by Cmpd1 at a comparable rate to Zaprinast.

Figure 7 PDE inhibitors stimulate microneme secretion. Extracellular tachyzoites treated with Zaprinast (Cf 500µM) or PDE inhibitors (Cf 55µM). Microneme secretion was determined by Western Bot detection of secreted and cleaved micronemal protein MIC2 (cMic2) relative to non-secreted, full length Mic2 (fMic2). Supn (supernatant).

Figure 8 BIPPO blocks the decline of cAMP-dependent phosphorylation by inhibiting degradation of cAMP by PDEs in parasite extracts. a) Diagram of recombinant GSTAMA1S610only tail fusion protein that has had all its known phosphorylation sites mutated to alanine (red) except for the S610 PKA site (blue). b) An autoradiograph of [γ32P] labelled GST-AMA1S610only tail proteins that have been phosphorylated by PfPKA in lysates from P. falciparum blood stage parasites. At lower levels of cAMP, phosphorylation declines rapidly presumably due to degradation by native PDEs, which could be reversed by BIPPO. c) Densitometry plots as a function of cAMP concentration (mean +/- SD, n=3).


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Movies Movie 1 Treatment of Plasmodium falciparum blood stage parasites with 2µM BIPPO results in premature egress. Movie 2 Treatment of Toxoplasma gondii infected human fibroblasts with 55µM BIPPO triggers rapid parasite egress from their host cells. Movie 3 Pre-treatment of Toxoplasma gondii infected human fibroblasts with 2µM Compound 1 blocks the egress normally triggered by BIPPO.


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Identification of Potent Phosphodiesterase Inhibitors that

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