Phenotypic, chemical and functional characterization of cyclic nucleotide phosphodiesterase 4 (PDE4) as a potential anthelmintic drug target

Background Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer’s disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). Methodology/Principal findings A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite’s genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target. Conclusions/Significance The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A’s contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets.

Introduction Schistosomiasis, also known as bilharzia, is a 'neglected' tropical disease caused by the Schistosoma flatworm parasite that resides in the bloodstream. The disease is chronic and morbid, and affects more than 240 million people worldwide [1][2][3]. For over 35 years, treatment and control has relied on just one drug, praziquantel (PZQ) [4][5][6]. There are a number of ongoing international efforts that aim to increase the distribution of PZQ for mass drug administration [7,8]. Consequently, there is concern regarding the possible emergence and establishment of drug resistance [5,[9][10][11]. Furthermore, PZQ has a number of pharmacological problems that encourage the search for alternate anti-schistosome therapies. The drug has diminished or no also, tavaborole derivatives have been developed as anti-bacterial candidates [72]. In addition, crisaborole, which inhibits human (hu)PDE4 [73], has completed clinical trials for treatment of atopic dermatitis [74,75]. Finally, SCYX-7158 has completed Phase I clinical testing as an oral, single dose cure of Human African Trypanosomiasis [76]. A phase II/III trial is now underway with recruitment of patients in the Democratic Republic of Congo [77].
Based on data from the phenotypic screen, we followed up by recombinantly expressing the orthologous S. mansoni PDE4 enzyme, SmPDE4A, and uncovered an association between enzyme inhibition and anti-parasite activity. Given the challenges of genetically manipulating the schistosome [78], we employed Caenorhabditis elegans to understand whether the parasite gene could functionally replace the nematode's endogenous pde4 gene, and whether that transgene is the target of the anti-schistosomal benzoxaborole inhibitors.

Results
Screening of S. mansoni somules with a benzoxaborole library identifies a subset of huPDE4 inhibitors that induce hypermotility and degeneration A 5 μM single concentration screen of S. mansoni somules (schistosomula) over 6 days with a collection of 1,085 benzoxaboroles identified three phenotype response groups as judged by microscopical observation (Fig 1): (i) 104 compounds eliciting an early and sustained hypermotile phenotype, of which, 30% was associated with a progressive degeneration of the parasite; (ii) 94 compounds that yielded a range of phenotypic responses (e.g., rounding, darkening), including hypermotility, which was either transient (noted at 24 h only) or appeared later in the incubation period (on or after day 3), and (iii) 887 compounds that yielded no phenotype.
Of the 1,085 benzoxaboroles phenotypically screened, 174 also had associated IC 50 data for inhibition of huPDE4B2 (Fig 1) that were distributed as 77, 82 and 15 compounds across the sustained hypermotile, no phenotype and transient hypermotile groups, respectively. Of the 77 compounds in the sustained hypermotile group, 65 had IC 50 values for inhibition of huP-DE4B2 of < 1 μM. In contrast, for the 82 compounds with no phenotype, only 16 had IC 50 values of < 1 μM. The association between the sustained hypermotile phenotype and submicromolar inhibition of huPDE4 was highly significant with a Fishers exact p-value of <0.0001. Also, the 5-(3-cyanopyridyl-6-oxy) benzoxaborole scaffold, which is known to preferentially inhibit huPDE4 over other PDEs [73], was enriched in compounds that caused the sustained hypermotile phenotype (Fig 1). In sum, therefore, the phenotypic and associated biochemical data focused our attention on identifying a schistosome PDE4 and understanding whether engagement of that enzyme was associated with the hypermotility (and degeneracy) recorded.
Among huPDEs, PDE4 has unique sequence features upstream of the catalytic domain, namely Upstream Conserved Regions (UCR)1 and 2, each of which is succeeded by a Linker Region (LR1 and 2; Fig 2). The presence or part absence of these UCRs characterizes the three principal huPDE4 variants. Thus, PDE4 'long isoforms' contain both UCRs; 'short isoforms' lack the UCR1 and 'super short isoforms' contain an N-terminally-truncated UCR2 [34, [79][80][81]. As a general rule, long isoforms act as dimers whereas short forms are monomers [82]. Dimerization is facilitated via both UCRs [28,83] and in an engineered construct of huP-DE4B2, the dimerization domain comprises the C-terminus of UCR1 and the N-terminus of UCR2 which form an antiparallel helix pair [79].
Based on the sequence alignment (Fig 2), huPDE4B2, SmPDE4A-C and the C. elegans ortholog share obvious homology with the human enzyme in the last one-third of UCR1: SmPDE4D has no UCR1. Downstream of LR1, UCR2 is better conserved across all of the sequences except for SmPDE4C which is missing approximately the C-terminal half of the region as well as LR2 and approximately 60 amino acids at the N-terminus of the catalytic domain. The catalytic domain itself is well-conserved across all of the sequences, except, as just indicated for SmPDE4C; also, SmPDE4D has a 16 amino acid insert at position 892 (Fig 2). SmPDE4B stands out in possessing the longest N-terminal sequence (~315 amino acids) The screen involving 1,085 benzoxaboroles was performed at 5 μM for 6 days with observations taken every day using a constrained nomenclature, as noted in the text. Three main phenotype response groups could be adjudicated by microscopical observation: (i) 104 compounds eliciting an early and sustained hypermotile phenotype, of which, 30% was also associated with a parasite degeneration; (ii) 94 compounds that yielded a range of phenotypic responses (e.g., rounding, darkening), including hypermotility, which were either transient (noted at 24 h only) or appeared later (on or after day 3 of the incubation), and (iii) 887 compounds that produced no phenotype. Of the 1,085 benzoxaboroles screened, 174 also had IC 50 data for inhibition of huPDE4B2 that were distributed as 77, 82 and 15 compounds across the sustained hypermotile, no phenotype and transient hypermotile groups, respectively. Sixty-five of 77 compounds in the sustained hypermotile group inhibited huPDE4B2 with IC 50 values of < 1 μM. In contrast, for the no phenotype group, only 16 of 82 compounds had IC 50 values of < 1 μM. The association between the sustained hypermotile phenotype and sub-micromolar inhibition of huPDE4 was highly significant with a Fishers exact p-value of <0.0001. The 5-(3-cyanopyridyl-6-oxy) benzoxaborole scaffold, which is known to preferentially inhibit huPDE4 over other PDEs [73], was well represented in Group 1.  [79], C. elegans (NP_495601.1) and S. mansoni Smp_134140 (PDE4A), Smp_141980 (PDE4B), Smp_129270 (PDE4C) and Smp_044060 (PDE4D). Upstream Conserved Regions (UCR) 1 and 2, and the catalytic domain are shaded in blue, green and pink, respectively. Demarcations of these domains are according to [80]. Linker regions (LR) 1 and 2 are indicated by blue horizontal bars. If present, the PKA and ERK phosphorylation sites, in the UCR1 and the catalytic domain, respectively, are indicated by the red and blue typeface, respectively. The conserved PDE signature motif HNX 2 HNX N E/D/QX 10 HDX 2 HX 25 E is indicated with blue circles and those residues that coordinate directly with the catalytic zinc in the substrate binding pocket are also indicated by red circles [33,93]. The residues in UCR1 and upstream of UCR1 and a large insert (~156 amino acids) between UCR2 and the catalytic domain.
For huPDE4, UCR1 regulates phosphohydrolase activity via a R-R-E-S variant of the R-X-X-S/T phosphorylation consensus motif for protein kinase A (PKA; Fig 2) which increases PDE4 activity and results in enhanced cAMP degradation [86][87][88]. This site is present in SmPDE4A and B but absent in the other helminth orthologs (Fig 2). Absent in all of the helminth sequences is a P-X-S/T-P consensus motif for ERK phosphorylation near the C-terminus of the catalytic domain [89][90][91]. The functional consequences of ERK phosphorylation are dependent on the presence of UCR1 and UCR2 such that long isoforms are catalytically inhibited whereas short isoforms have increased activity, and super-short isoforms are again weakly inhibited [33,91]. The absence of PKA and ERK phosphorylation sites in some or all the helminth orthologs suggest differences in how the respective proteins are regulated relative to mammalian PDE4s. SmPDE4A was chosen for recombinant expression and subsequent enzyme activity/inhibition studies as it was the least divergent in its protein sequence and domain organization from huPDE4, which has been successfully expressed by Anacor for its own drug development programs [92].
SmPDE4 genes are expressed in various developmental stages of S. mansoni with some orthologs being present in the genomes of Schistosoma haematobium and Schistosoma japonicum Querying the GeneDB database reveals that all four SmPDE4 enzymes are expressed in a number of different developmental stages of S. mansoni relevant to infection in humans (cercariae, somules, and adult male and/or female worms; S1 Table). NCBI BLAST analysis of the genomes of S. haematobium [94] and S. japonicum [95], indicates that orthologs of SmPDE4A, B and D (not C) are present in adult S. haematobium, and that orthologs of SmPDE4A and B (not C or D) are found in the adult male and somules of S. japonicum, respectively (S1 Table and S1-S3 Figs for alignments).
Each of the SmPDE4 genes shares greatest homology with its respective ortholog in S. haematobium over the full sequence (83-90%; S2 Table) or the catalytic domain (87-98%; S3 Table). For SmPDE4A and B, the corresponding ortholog identities in S. japonicum are generally lower, 63 and 34% for the full sequence, and 89 and 35% for the catalytic domain. For either the full length sequence or that of the catalytic domain, the percentage identities with huPDE4B2 are approximately 60% for SmPDE4A and SmPDE4B, 50% for SmPDE4C and 34% for SmPDE4D. Similar data were obtained for the same comparisons between the SmPDE4 and the C. elegans sequences. approximately 55 g bacterial paste from a 6 L culture, 8.2 mg of purified His 6 -tagged SmPDE4A was obtained. Phosphohydrolase activity was measured using [ 3 H]-cAMP as described [96]. The recombinant enzyme displayed Michaelis-Menten kinetics with a Michaelis constant (K m ) of 3.0 μM and a maximum velocity (V max ) of 32.6 pmol/min ( Fig 3B). The K m value is similar to values of 0.98, 2.25 and 7.81 μM reported previously for huPDE4A, B and D, respectively [96].

SmPDE4 inhibition potency is associated with anti-schistosomal activity
The phenotypic screen of 1,085 benzoxaboroles with S. mansoni somules had identified a cluster of compounds that induced sustained hypermotility and, in 30% of those cases, degeneracy. For 77 of these compounds for which huPDE4B2 inhibition data were also available to Anacor, the majority (65) had sub-micromolar IC 50 values. The underlying implication was, therefore, that a schistosome PDE4 may be associated with the phenotypes observed. Accordingly, we selected benzoxaboroles (compounds 1-7) with various peripheral substitutions to understand whether an association between enzyme inhibition and anti-parasite activity could be measured (Fig 4). Compounds were selected on the bases of (i) availability, (ii) existence of IC 50 values for huPDE4 and (iii) absence of IP-constraints to reveal structures. The analysis also included two catechol drugs that inhibit huPDE4, namely, rolipram and roflumilast [41].
IC 50 values for the selected benzoxaboroles and catechols were determined for the recombinant SmPDE4A and compared to Anacor's in-house data for huPDE4B2 (Fig 4); see Fig 5 for representative IC 50 curves). For SmPDE4A, the most potent benzoxaborole inhibitors (1-5) containing p-cyano and 2-oxy substitutions yielded IC 50 values of < 50nM. Compounds 6 and 7 with 3-sulfone and 2-hydroxy substitutions were less effective (415 and 123 nM, respectively). The catechol, roflumilast, was an effective inhibitor (IC 50 = 18 and 11 nM), whereas rolipram was ineffective (IC 50 >10 μM). For huPDE4B2, a similar trend was noted: compounds 1-4 yielded IC 50 values of 30nM, whereas the values for 5-7 ranged between 19 and 69 nM. The catechol, roflumilast, was a potent inhibitor (IC 50 = 0.65 nM) and rolipram much less so (IC 50 = 540 nM) but still at least 19-fold more effective than against SmPDE4A (Fig 4). Inhibition values obtained for these two catechols against huPDE4 are consistent with those previously reported [79,97,98]. For the nine compounds, bioactivity as a function of time against both somules (at 5 μM) and adult S. mansoni (at 10 μM) was recorded observationally using our constrained nomenclature [99][100][101]: for adults, we also used Wormassay [102] to measure motility (Fig 4). Those most potent inhibitors of SmPDE4A were also the most bioactive against the parasite irrespective of developmental stage. Thus, by the first time point of 24 h for somules and 1 h for adults, compounds 1-4 induced intense hypermotility, which by day 6 for somules and day 3 for adults had progressed to include severe degenerative changes (Fig 4). For both somules and adults, degeneration appeared to occur throughout the worm body (not localized to a particular region or feature) and was irreversible upon removal of the inhibitors after the respective   In support of the intense hypermotility observed for adults after 1 h in the presence of compounds 1-4, motility as measured by Wormassay was 6-10-fold greater than that of the DMSO control. For the less potent inhibitors (5-7) of SmPDE4A, bioactivity, if observed at all, was restricted to mild and/or transient increase in motility (for adults a maximum 3.9-fold over DMSO controls by Wormassay) without any associated degeneration. Finally, the catechols were inactive against somules and only induced transient hypermotility in adults (maximum 3.2-fold by Wormassay for roflumilast) without major degenerative changes. Overall, therefore, there appears to be a reasonable association between the potency of inhibition of SmPDE4A and the degree of hypermotility of the parasite, which at its most extreme, is associated with degenerative changes.
Functional phenotypic rescue of pde4-deficient mutant of C. elegans by expression of SmPDE4A cDNA In the model nematode, C. elegans, a single PDE4 gene is responsible for maintaining normal motility such that disruption of that gene (ce268 mutation) results in hypermotility [103]. Hypermotility of this C. elegans mutant is thought to be due to excessive cAMP accumulation and consequent hyper-activation of signaling pathways that promote motility [103]. To investigate whether the SmPDE4A can functionally substitute for the C. elegans pde4, we generated transgenic C. elegans that express full-length SmPDE4A under a pan-neuronally expressed promoter. In two independently generated transgenic lines of C. elegans that express smpde4a in the C. elegans pde4(ce268) mutant background, smpde4a(a) and smpde4a(b), we found that the S. mansoni transgene restored normal motility rates (Fig 6). This was not simply due to nonspecific motility-reducing effects of these transgenes as the same two transgenes did not affect the motility rates of otherwise wild type (WT; N2 Bristol strain) animals ( Fig 6). Thus, the function of the endogenous C. elegans pde4 gene can be complemented by the smpde4a transgene.
PDE4 inhibitors act via the endogenous pde4 or the smpde4a transgene to induce hypermotility in C. elegans We first asked whether exemplar PDE4 inhibitors (compounds 2 and 4) could induce hypermotility in WT C. elegans. Exposure of WT C. elegans to rolipram, roflumilast and compound 2 increased motility relative to the DMSO control whereas compound 4 did not ( Fig 7A). Consistent with the notion that these compounds cause hypermotility through inhibition of PDE4, the already elevated motility of pde4 mutant C. elegans was not further modulated by exposure to any of the PDE4 inhibitors ( Fig 7B).
Exposure of the two mutant C. elegans lines carrying the smpde4a transgene to each of the PDE4 inhibitors induced hypermotility (Fig 7C and 7D) indicating that the compounds act via the schistosome transgene. The differential effects of compound 4 on WT and transgenic C. elegans may indicate differences between the susceptibilities of the C. elegans and S. mansoni PDE4 target enzymes to inhibition by this compound. Interestingly, rolipram, which was a weak inhibitor of recombinant SmPDE4A (Fig 4), increased motility significantly in both mutant C. elegans lines, albeit less so than the hypermotility induced by compounds 2 and 4 (Fig 7C and 7D).
Differences in the inhibition of huPDE4B and SmPDE4A by catechols is associated with particular residues surrounding the binding site To interpret these data, molecular models of each enzyme in complex with rolipram and roflumilast were built using ICM-pro and huPDE4B1 as a template (PDB ID: 4X0F) [79]. The ligand-protein interaction diagrams of rolipram and roflumilast are shown in Fig 8. The ligand-binding residues are highly conserved between both enzymes with the exception of two and three differences for binding to rolipram and roflumilast, respectively. Specifically, for rolipram-binding, I953 and M954 in huPDE4 are replaced by L and I in SmPDE4A, respectively (Fig 8, left panel). For binding to roflumilast, the same residues are changed in the same manner with the addition of a S!T809 substitution (Fig 8, right panel). Notably, and common for both inhibitors, the switch from I953 to L953 would make ligand binding unfavorable as shown by the high positive changes of binding free energies (14.06 kcal/mol and 15.77 kcal/ mol for rolipram and roflumilast, respectively). This change helps explain the weaker inhibition measured for SmPDE4A with rolipram and roflumilast compared to the human enzyme.

Discussion
A number of benzoxaboroles have been successfully brought through to the clinic and/or market for a variety of molecular drug targets, including aminoacyl-tRNA synthetase [70] and huPDE4 [73], and disease conditions [74,75], such as Human African Trypanosomiasis [76]. Accordingly, we leveraged a benzoxaborole library from Anacor Pharmaceuticals to identify new drug development opportunities for schistosomiasis, a disease for which treatment currently relies on just one drug, praziquantel. We first employed a phenotypic screen of 1,085 benzoxaboroles using S. mansoni somules to identify phenotypes of interest. We resolved three phenotype response groups: (i) 104 compounds eliciting an early and sustained hypermotile phenotype, including 30% that also induced degenerative changes; (ii) 94 compounds that yielded a range of phenotypic responses and (iii) 887 compounds that yielded no phenotype. The possibility that a PDE4 may be a target of interest arose via a statistically significant association for compounds that induced PDE4 as a potential anthelmintic drug target sustained hypermotility in the parasite and were sub-micromolar inhibitors of huPDE4. This notion was strengthened by a previous report that a mutation (ce268) of the single pde4 gene in C. elegans causes hypermotility [103]. Given the circumstantial evidence, therefore, we searched for and identified four PDE4-like gene sequences in the S. mansoni genome, which we termed SmPDE4A through SmPDE4D.
Of the four putative PDE4 proteins identified in the S. mansoni genome, SmPDE4A is the most similar in protein sequence and inferred domain architecture to huPDE4 whereas the other three are more divergent in various respects such as possessing an extended Nterminal domain (SmPDE4B), sequence inserts (SmPDE4B and D) or sequence truncations (SmPDE4C). At this time, the possible functional significance of the sequence variations is unclear, however, based on the conservation of key amino acids directly involved in catalysis, all four gene products may be active. For each of the four protein sequences, corresponding expression products were identified in various developmental stages of S. mansoni relevant to infection in humans and orthologs of three of the SmPDE4 sequences (A, B and D) were identified in adults and/or somules of S. haematobium and S. japonicum.
Because of SmPDE4A's greater similarity in sequence and domain organization to huPDE4, which had already been functionally expressed in bacteria during a campaign to develop benzoxaborole inhibitors [74], we chose to recombinantly express this enzyme and determine whether, for exemplar benzoxaboroles, an association between enzyme inhibition and phenotypic effects on the parasite existed. After expression of the SmPDE4A catalytic domain in E. coli and chromatographic purification, an enzyme that was catalytically active against the relevant cAMP substrate was obtained.
For seven exemplar benzoxaboroles, the potency of inhibition of SmPDE4A trended with the severity of parasite hypermotility, either recorded observationally in somules and adults, or using Wormassay, an image-based method to measure motility in adults [102]. For the most potent benzoxaborole inhibitors (1-4) of SmPDE4A, somules and adults underwent  degenerative changes in addition to, and perhaps, as a consequence of, the extreme hypermotility recorded. Neither the sustained hypermotility nor degeneracy was noted for the weaker inhibitors of SmPDE4A. Although we cannot discount the possibility that the inhibitors tested interact with one or more of the other three putative PDE4s identified in S. mansoni, or indeed, other phosphodiesterases, the trends uncovered for the benzoxaboroles tested would indicate that the induction of parasite hypermotility and degeneracy is, at the least, mediated via inhibition of SmPDE4A, an interpretation that is consistent with the data for C. elegans transfected with a cDNA for SmPDE4A (discussed below). Relevant in this context is that the 5-(3-cyanopyridyl-6-oxy) benzoxaborole scaffold represented in the most potent SmPDE4A inhibitors (Fig 4) provides between 4 and >2,000-fold better potency (IC 50 values) for huPDE4 over other huPDE enzymes [73].
The catechol, roflumilast, although a potent inhibitor of SmPDE4A, produced only marginal and transient phenotypic effects on the parasite. The reason(s) for this is unclear, but may be due to a lack of penetrance or rapid metabolism of the catechol by the parasite. The second catechol tested, rolipram, was inactive against SmPDE4 and, again, marginally bioactive. Both catechols are considerably weaker inhibitors of SmPDE4A than huPDE4 (approximately 20-fold). Molecular modeling revealed that there are up to three amino acid residue differences in the ligand-binding sites between SmPDE4A and huPDE4, but that the change at one position in particular, namely I!L953, would generate an unfavorable binding free energy value that would contribute to the weaker inhibition values measured for SmPDE4A with the catechols. Given how similar the ligand binding sites are otherwise, the differences noted could be important in a future campaign to derive more parasite-specific inhibitors and decrease potential off-target interactions with host PDE4.
To determine whether SmPDE4A can operate as a bona fide PDE4 in a heterologous biological system, we generated two C. elegans transgenic lines for full-length smpde4a on the ce268 background, which lacks a functional pde4 gene and is, consequently, hypermotile relative to WT worms [103]. The decision to use C. elegans as a functional read-out was motivated by the fact that genetic manipulation of S. mansoni, in spite of progress in this area [78], is still not a standardized undertaking. Both smpde4a transgene lines depressed hypermotility in ce268 worms to the levels measured for WT-an original demonstration that a platyhelminth gene can compensate for gene functionality in this nematode model. The finding opens the possibility of using the smpde4a transgenic C. elegans as a research tool to perform mutational/mechanistic studies on enzyme function.
To understand whether the transgenic C. elegans system responded to PDE4 inhibitors, we tested the two catechols, rolipram and roflumilast, and two benzoxaboroles, compounds 2 and 4, with the pde4(ce268);smpde4 lines. Encouragingly, all of the inhibitors tested increased worm motility demonstrating that they engage and inhibit the SmPDE4A transgene. This raises the interesting possibility that the current transgenic model could be a useful tool in the further development of more specific inhibitors (see below), especially considering that the bacterial expression of 'long isoform' huPDE4, i.e., including both UCR1 and UCR2, is associated with difficulties relating to activity and the aggregation of different molar forms [79].
The finding that rolipram produced a modest, yet statistically significant, increase in the motility of transgenic C. elegans was initially surprising given the compound's apparent lack of inhibition of the recombinant catalytic domain of SmPDE4A (IC 50 >10 μM). One possible explanation may lie in the recent demonstration (consistent with earlier reports [82,83,104,105]) that huPDE4B dimerizes via certain residues in UCR1 and UCR2, and that the UCR2 from one monomer contributes to the topography of the active site of the other monomer [79] (Fig 2). This UCR2-mediated alteration of the active site increases the rolipram-binding contacts and accounts for the existence of a high-affinity inhibition of huPDE4 by rolipram versus a low-affinity inhibition that involves the catalytic domain only [97,98,106]. In support of this explanation, the residues in UCR1 and UCR2 that contribute to the dimerization interface in huPDE4 are strongly conserved in SmPDE4A (and isoforms B and C, but not D; Fig 2). Also, the rolipram-facing residue in the UCR2 of huPDE4B (Y471 in Fig 2 designated Y274 in [79]) that enhances the binding potential of rolipram is conserved in SmPDE4A (and isoform B, but not C and D). Thus, it is conceivable that the UCR2 region present in the full length SmPDE4A cDNA that was transfected into the C. elegans pde4(ce268) mutant provides the additional necessary contacts for rolipram's enhanced binding and induction of hypermotility. This interaction would, however, imply adjustments in the pose and contacts made by rolipram in the SmPDE4A binding site given the unfavorable binding presence of L953 compared to I953 in huPDE4. Unfortunately, our attempts to perform the corollary experiment of transfecting C. elegans with a truncated form of SmPDE4, i.e., minus the UCR2 domain, and provide support for UCR2's contribution to rolipram's enhanced binding were unsuccessful. If confirmed, however, then the extra specificity determinants present in the UCR2-augmented binding site (in addition to other control elements in the full-length enzyme [79,107]) could be exploited in a program to improve inhibitor specificity especially given the strong similarities between the ligand binding sites of SmPDE4A and huPDE4 noted above.
To conclude, a phenotypic screen of a benzoxaborole collection with S. mansoni identified a particular phenotype-chemotype association that suggested an underlying PDE4 molecular target. An association between inhibition of the recombinant SmPDE4A, and parasite hypermotility and degeneration was noted. Employing C. elegans as a transgene expression system, we confirmed SmPDE4A's contribution to modulating worm motility and its relevance as the molecular target for benzoxaborole inhibitors. The applicability of C. elegans as screening platform for small molecules to flatworm (schistosome) molecular targets, coupled with the differences noted between the human and schistosome PDE4s could support a structure-based approach to optimize inhibitor specificity, bioavailability and safety.

Ethics statement
Maintenance and handling of vertebrate animals were carried out in accordance with a protocol (AN107779) approved by the Institutional Animal Care and Use Committee at the University of California San Francisco. UCSF-IACUC derives its authority from the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Animal Welfare Act and Regulations.

S. mansoni life-cycle
We employ a Puerto Rican isolate of S. mansoni that is cycled between Biomphalaria glabrata snails and female Golden Syrian hamsters (infected at 4-6 weeks of age) as intermediate and definitive hosts, respectively. The acquisition, preparation and in vitro maintenance of mechanically transformed somules (derived from infective stage cercariae) and adults have been described [99,108].

Cloning and expression of SmPDE4
The catalytic domain of SmPDE4A (Smp_134140; XM_002573613; residues 668-1,060 in Fig  2) was synthesized (Genscript) with codons optimized for Escherichia coli expression (including a translation-start methionine codon) and cloned into the pET15b vector to yield an N-terminally His 6 -tagged protein. The protein was produced in E. coli BL21(DE3) cells grown in Terrific Broth medium supplemented with 0.1 mM zinc acetate and 50 μg/ml carbenicillin. For the large scale expression of recombinant SmPDE4A, cells were grown at 37˚C to an OD 600 approaching 0.5, the temperature was then dropped to 15˚C, and the cells induced for 24 h with 0.1 mM isopropyl β-D-1-thiogalactopyranoside. Cells were collected by centrifugation at 4˚C, flash frozen in liquid nitrogen and stored at -80˚C.
For purification, frozen cells were suspended (1 g/ 5 ml) in 20 mM TRIS-HCl, pH 7.2, 250 mM NaCl, 10 mM imidazole, 1 mM phenyl methane sulfonyl fluoride (PMSF), and once fully homogenous, were lysed by microfluidization. Cellular debris was centrifuged at 4˚C for 1 h at 12,500 g. The resulting lysate was then purified by metal-ion affinity chromatography using a His-TRAP FF column (GE Healthcare). Prior to purification, the column was washed with 10 column volumes of elution buffer (20 mM TRIS-HCl, pH 7.2, 250 mM NaCl, 500 mM imidazole) and equilibrated with 10 column volumes of binding buffer (20 mM TRIS-HCl, pH 7.2, 250 mM NaCl, 10 mM imidazole). The protein eluted at~27.5% elution buffer. Major fractions containing the protein of interest were combined, concentrated and treated with an equal volume of 20 mM TRIS-HCl, pH 8.0, 2 M (NH 4 ) 2 SO 4 . This was done in order to prepare the protein sample for hydrophobic interaction chromatography using a HiTRAP Butyl HP column (GE Healthcare). The column was equilibrated with binding buffer (20 mM TRIS-HCl, pH 8.0, 1 M (NH 4 ) 2 SO 4 ), followed by loading of SmPDE4A and elution with a linear gradient of 20 mM TRIS-HCl, pH 8.0. The protein eluted at~50% elution buffer. Fractions containing the protein of interest were pooled, concentrated and buffer exchanged to decrease the concentration of (NH 4 ) 2 SO 4 to below 5 mM. As a final step, the protein was purified by ion-exchange chromatography using a Mono Q column (GE Healthcare). The column was equilibrated with binding buffer (20 mM TRIS-HCl, pH 8.0) followed by loading of SmPDE4A and elution with a linear gradient of 20 mM TRIS-HCl, pH 8.0, 1 M NaCl. The protein eluted at~65% elution buffer. Fractions containing the protein of interest were pooled and concentrated, and the purity assessed by SDS-PAGE. The concentration of recombinant SmPDE4A was estimated by the Bradford Assay (BioRad) using bovine serum albumin (BSA) as a standard.

PDE4 inhibition assay
Assay of PDE4 enzymatic activity was as described [96]: huPDE4B2 was purchased from Proteros Biostructures, GmbH, Martinsried, Germany. The reaction contained 0.15 μM [ 3 H]-cAMP (10 uCi/ml; Perkin Elmer, Waltham, MA) and activity was measured by ZnSO 4 /Ba (OH) 2 precipitation of the AMP product after reaction quenching. The precipitate was collected by filtration onto Multi-Screen HTS FB plates (Millipore, Billerica, MA), washed and then dried for quantitation of radioactivity. For tests with PDE4 inhibitors, fifty percent inhibitory concentration (IC 50 ) values were calculated based on a four-parameter logistic equation: the means and number of replicates are reported in Fig 4. Racemate rolipram was purchased from Sigma (Cat. no. R6520) and roflumilast was from Selleckchem (cat. no. S2131).
Adult schistosome screens were performed in 24-well plates (Corning Inc., cat. # 3544) using five worm pairs per well in a final volume of 2 ml of the above Basch medium. Compound was added in a volume of 1 μl DMSO such that the final concentration was 10 μM. Incubations were maintained for 3 days at 37˚C under 5% CO 2 .
Parasite responses to chemical insult were adjudicated visually every 24 h (also at the 1 h time point for adults) using an inverted microscope and employed a constrained nomenclature of phenotype descriptors (e.g., rounding, degeneration, overactivity and slowed motility) as described [99][100][101]. For adult parasites, in addition to observation-based annotations, we employed Wormassay [102] to measure worm motility. Briefly, Wormassay comprises a commodity digital movie camera connected to an Apple personal computer that operates an open source software application to automatically process multiple wells (in 6-, 12-or 24-well plate formats). The application detects worm-induced changes in the occupation and vacancy of pixels between frames (outputted as an average ± S.D.). Worm motion was quantified using the "Consensus Voting Luminance Difference" option.

Sequence-analysis and expression-profiling of SmPDE4 and its orthologs
To determine in which developmental stages the SmPDE4 genes are expressed, the GeneDB (http://www.genedb.org/Homepage) Gene IDs for SmPDE4A (Smp_134140), SmPDE4B (Smp_141980), SmPDE4C (Smp_129270) and SmPDE4D (Smp_044060) were each used as key words to search for the respective sequences. The "Transcript Expression" file was selected for each sequence to view transcriptomic expression data [65,66]. To determine whether the SmPDE4 genes are expressed differentially in adult male and female parasites, the amino acid sequences were queried via tBLASTn in NCBI (http://ncbi.nlm.nih.gov/) against the EST (Expressed Sequence Tag) database and constraining the organism ID to "Schistosoma" (taxid: 6181). Only the information associated with returned sequences that shared ! 97% identity with the query sequence was scrutinized.
To identify orthologous sequences in the genomes of the human, C. elegans, S. haematobium [94] and S. japonicum [95], each SmPDE4 protein sequence was analyzed via tBLASTn in NCBI, again constraining for the taxid ID of 6181. The returned sequences that shared an identity of 30% or more were subsequently analyzed via BLASTp (at NCBI and GeneDB) to (i) obtain the full length sequence (ii) confirm the accession (gene ID) numbers and (iii) determine the sequence identity. Then, a sequence alignment was generated using the PRABI (Pôle Rhône-Alpes de Bioinformatique) MULTALIN tool (https://npsa-prabi.ibcp.fr/) to define the relative positions of the various PDE4 domains (UCR1, UCR2 and the catalytic domain). The catalytic domains were then used as queries via BLASTp in NCBI to determine sequence identities with the other orthologs.

Molecular modeling of SmPDE4
Modeling was performed with the internal coordinate mechanics (ICM-pro) package for structure prediction, homology modeling and docking [112]. HuPDE4B1 in complex with (R)-(-)-rolipram (PDB code 4X0F; [79]) was used to build models incorporating the rolipram binding site. The residues surrounding the binding site of rolipram were mutated into the corresponding residues in SmPDE4A to build a SmPDE4A-rolipram model. The residue side chains around the binding site of rolipram in the human and parasite enzymes were then globally optimized using Biased Probability Monte Carlo (BPMS) sampling [112] with ICM energy functions in the context of rolipram. Similarly, the model of huPDE4B-roflumilast was built based on the PDB structures 4X0F [79] and 1XOQ [113]. A model of SmPDE4A-roflumilast was built by the mutating residues around the binding site of roflumilast and globally optimized with BPMS sampling in the context of roflumilast.

Ligand-residue interaction diagram and in silico mutation analysis
With the models for huPDE4B1 and SmPDE4A in complex with rolipram and roflumilast, 2D diagrams of the ligand-residue interactions were built using the requisite tool in ICM-pro. The hydrophobic interaction cutoff was 5.0 Å. Then, with the model of huPDE4B1 in complex with rolipram and roflumilast, the residues around the binding site of rolipram and roflumilast that distinguish SmPDE4A from the human ortholog were mutated. The differences in ligand binding free energies were calculated using following equations: where DG wt bind represents the binding free energy of protein and ligand in WT huPDE4B1; DG mut bind represents the binding free energy of protein and ligand in those residues mutated in huPDE4B1; E comp intra represents the internal energy of the protein-ligand complex and E parts intra represents the sum of the internal energy of protein and ligand. Similarly, DG comp solv represents the solvation energy of the protein-ligand complex and DG parts solv represents the sum of solvation energies of protein and ligand [114] C. elegans strains and culture methods All strains were cultured at 20˚C on nematode growth medium (NGM) plates seeded with E. coli strain OP50 [115]. N2 Bristol was used as the WT reference strain. The mutant strain used in this study, pde4(ce268), carries a D448N mutation relative to WT pde4 and is encoded by the C. elegans gene denoted as R153.1. The D448N change disrupts the catalytic domain by changing one of the four active residues that together chelate an active-site zinc atom. The consequence is a strong decrease in gene function [103].

Plasmid constructs and generation of C. elegans WT and pde4 transgenic mutants
Plasmids were constructed using Gateway Technology (Invitrogen) reagents as described [116]. The entire S. mansoni SmPDE4A sequence (Smp_134140; XM_002573613) was PCR amplified from the start codon to immediately preceding the stop codon (2-1880 bp) using mixed sex, adult S. mansoni cDNA. PCR primers contained the gateway attB recombining sequences (in lower case): SmPDE4Fw, 5'-ggggacaagtttgtacaaaaaagcaggctTGGAGTTAC GAACCGATAAAGTGATTTCATC-3' and SmPDE4Rv, 5'-ggggaccactttgtacaagaaagctggg TATGTGTTTCCTGAAGTTGTAGA. The PCR fragment was cloned into a donor vector pKA5 (pDONR-221) and the correct sequence confirmed. Then, the entry vector pKA5-SmP-DE4A was recombined with the 2 kb of sequence upstream of the start site of unc-119 promoter [117][118][119] into a Gateway destination vector containing GFP (pKA453) to obtain promoter::SmPDE4A::intercistronic::gfp polycistronic fusions as previously described [120]. This also allows for co-expression of GFP and SmPDE4A from the same transcript without modifying SmPDE4A and facilitates the selection of transgenic animals via the GFP tag.
To generate C. elegans that carry the SmPDE4A transgene, the plasmids described above were purified and microinjected into the gonads of both WT (N2) and pde4(ce268) mutant strains at a concentration of 50 ng/μl. The injected worms (P0) were transferred to individual freshly seeded bacteria plates and allowed to reproduce. F1 progeny were screened for evidence of transgene expression based on the GFP marker. Each transgenic F1 was then singled onto a new plate. This process was repeated until lines that stably transmit the transgene were established in WT or pde(ce268) animal backgrounds. To verify the consistency of GFP expression in the transgenic lines, 10-20 transgenic animals from each line were examined using a Zeiss Axioplan II stereoscope equipped with a FITC/GFP filter (emission 500-515 nm).

C. elegans motility assays
Compounds were directly added onto the OP50 food source at a 100 μM final concentration or the equivalent 0.2% DMSO as control. Previously synchronized early L4 stage larvae were cultivated on NGM plates with OP50 and compounds at 20˚C for 16 h. For each experimental condition and transgenic line, motility was measured for 10-20 animals. The animals were washed twice in S basal buffer, transferred onto a new NGM plate in the absence of bacteria. After a brief period of recovery from this manipulation, locomotion was measured by counting the number of body bends in 30 s intervals under a stereoscope.