Final Report Summary - SETTREND (Schistosoma Epigenetics - Targets, Regulation, New Drugs)
Executive summary:
4.1.1 Executive summary
the overall aim of the SETTREND project was to develop new drug leads for the treatment of schistosomiasis. This disease, caused by blood-dwelling flatworm parasites of the genus Schistosoma, affects more than 200 million people worldwide, causing upwards of 200,000 deaths annually and its control currently depends on the use of the only available drug, praziquantel. The current use of this drug in mass-treatment campaigns in sub-Saharan Africa increases the probability of the development of resistance by the parasite. The development of new drugs is therefore imperative and our approach is to target enzymes involved in the epigenetic modification of the chromatin, the histone modifying enzymes (HME). In particular, we chose to focus our approach on enzymes catalyzing histone acetylation/deacetylation and methylation/demethylation. These enzymes are increasingly targeted in the therapy of several pathologies and in particular cancer, allowing us to benefit from the expertise and inhibitors already developed. The enzymes targeted in the project are the histone deacetylases (HDAC), histone acetyltransferases (HAT), histone methyltransferases (HMT) and histone demethylases (HDM) of Schistosoma mansoni the species responsible for intestinal schistosomiasis in Africa and South America.
the objective of the project was to identify novel inhibitors of HMEs that will be drug leads. To do this we used a holistic approach that enabled us to progress from the cloned target protein, via structural analysis, in silico and high-throughput screening, to the identification of new lead compounds validated by their toxicity for the parasite in vitro and in vivo.
the main achievements of the SETTREND strategy are as follows:
- The identification of the HMEs encoded in the schistosome genomes. Molecular cloning of the coding sequences of selected HMEs and the investigation of their expression during the parasite life-cycle.
- The elucidation of gene expression signatures of HDAC inhibition in parasite larvae, leading to the identification of key pathways and proteins.
- The implementation of a reverse chemical genetics strategy showing that inhibitors of all HME families induced apoptosis and death of schistosome larvae in culture.
- The validation of an HDAC (SmHDAC8) and two other HMEs (SmPRMT3 and SmKDM1) as therapeutic targets by RNAi.
- The production of SmHDAC8 as an enzymatically active recombinant protein and the solution of the structures of the apo-form of the protein, the protein bound to pan-HDAC inhibitors and to selective inhibitors generated during the project. Two other recombinant HME proteins, SmSirt2 and SmSirt6 were also produced.
- In silico screening of SmHDAC8 based on the solved crystal structure identified compounds with novel scaffolds and warheads with greater selectivity than reference inhibitors and that provoked apoptosis and death of schistosomula in culture. Treatment of schistosome-infected mice with one of these inhibitors, J1075, led to reduced worm burdens and egg loads.
- High-throughput inhibitor library screening of SmHDAC8 and bioguided synthetic chemistry optimization produced lead inhibitors that were extremely active against both schistosome larvae and adult worms in culture. High-throughput inhibitor screening was also carried out for SmSirt2.
project Context and Objectives:
4.1.2 Project context and objectives
the need for new drugs to combat schistosomiasis
schistosomiasis is a neglected tropical parasitic disease of major public health importance [1] caused by blood flukes of the Schistosoma spp. (class Trematoda, family Schistosomatidae). S. haematobium, S. japonicum and S. mansoni are the three main species parasitizing more than 200 million people worldwide, with 780 million at risk of infection [2]. S. mansoni, the agent of human intestinal schistosomiasis, is endemic in 54 countries, mostly in Africa, South America and the Middle-East [3]. There is currently no effective vaccine against schistosomiasis [4] and the following control strategies are recommended in a recent WHO report: preventive chemotherapy, intensified case management, vector control and provision of safe water, sanitation and hygiene. Unfortunately interruption of the transmission cycle through chemical or biological vector (snail) control and health education have generally proved insufficient on their own as control methods 5]. The treatment of the disease relies on a single drug, praziquantel (PZQ) [6]. In Africa, where the annual mortality rate is estimated to be as high as 280,000 [7], several control programs, such as those conducted by the Schistosome Control Initiative (SCI, see http://www3.imperial.ac.uk/schisto online), the United States Agency for International Development (USAID) and the Department for International Development (DFID), distribute PZQ on a large scale because PZQ is safe, cheap and effective against adult worms. Another recent input into the fight against schistosomiasis is Merck's commitment to a tenfold increase, from 25 million to 250 million, of its annual donation of PZQ tablets (see http://www.4-traders.com/MERCK-KGAA-436395/news/Merck-KGaA-Merck-to-Raise-Donation-Tenfold-to-Eliminate-Worm-Disease-13991828/ online). In Africa, PZQ is distributed generally to the whole population where overall prevalence is greater than 50% or to school-aged children in endemic areas. Treatment results in an immediate reduction in the prevalence and intensity of infection but because immunity to infection is weak, reinfection often brings the prevalence back to pre-treatment levels within 2 years [8].
epigenetics, the histone code and transcriptional regulation
epigenetic modifications, particularly those affecting DNA methylation or posttranslational modifications of histones (including acetylation and methylation of lysine or arginine residues), are known to be aberrant in a number of diseases, including cancer, diabetes, autoimmune disorders, asthma and neurological disorders (see [10] for review). In the case of cancer a global hypomethylation of DNA, leading to genetic instability, is accompanied by localized hypermethylation, in particular of the promoters of tumour suppressor genes. Additionally, histone hypoacetylation in malignant compared to the corresponding normal tissue has been found in lymphomas and colon cancers [11] characterized by the loss of acetylation at Lys16 of H4 (H4K16). This was also accompanied by the trimethylation of H4K20. Histone acetylation is generally associated with the activation of gene transcription via a relaxation of the chromatin structure, whereas deacetylation has the opposite effect. Methylation on the other hand can be associated both with transcriptional activation (H3K4 methylation) or repression (H3K9 or H3K27 for example) with nuanced effects depending on the degree (mono-, di- or tri-) of methylation. These modifications of the charged histone "tail" that extends from the nucleosome, along with others including phosphorylation, SUMOylation and ubiquitinylation, make up the "histone code" [12]. The different combinations of histone modifications enable the storage of a large amount of information that has a central role in the regulation of transcription, or its dysregulation in pathology. The enzymes that effect these modifications are consequently privileged targets for pharmacological intervention.
histone modifying enzymes (HME)
the enzyme families involved in epigenetic modification of histones are conserved in eukaryotes and the most studied as drug targets are those catalyzing acetylation/deacetylation and methylation/demethylation. Acetylation of lysine residues in histone tails and of other proteins is carried out by a large number of proteins with histone acetyltransferase (HAT) activity of which it is possible to distinguish at least six families [13]: the GNAT superfamily (PCAF/GCN5), the MYST family, the CBP/p300 family, the nuclear receptor coactivator family, the TAFII 250 family and the GNAT-related family (TFIIIC).
HME inhibitors as drug candidates
inhibitors of epigenetic targets have been under active investigation for decades, particularly as potential cancer drugs [reviewed in 20]. Although the two HME inhibitors already approved for use, as well as 17 others currently in clinical trials [20], are all inhibitors of HDACs or sirtuins, other enzyme classes are also being targeted, such as the lysine methyltransferase EZH2 for which inhibitors have been shown to have potential in the treatment of lymphoma [21].
strategy and objectives of the SETTREND project
we adopted a holistic approach to drug discovery with schistosome HMEs as the targets. The targeting of histone acetylation and methylation (to the exclusion of other types of modification) was deliberate since they are the two crucial modifications that impact directly on gene transcription and are the subject of intense investigation, both in the context of cancer research and, increasingly, in the search for parasiticidal drugs.
the main elements in our strategy were as follows:
- The identification of the members of HDAC classes I, II and III (sirtuins) HAT, HMT and HDM encoded in the genome of S. mansoni. Protein motifs and hidden Markov models of conserved, mainly catalytic, domains were used to search the databases.
- Molecular cloning of the coding sequences of selected HMEs in order to allow production of the corresponding recombinant proteins. The biological roles of selected HMEs were investigated in order to give insights into the potential action of inhibitors.
- A reverse chemical genetics approach using generic inhibitors of HME subclasses available within the consortium in cultures of schistosome larvae was employed to identify those classes that are bona fide drug targets. This allowed the earliest indications about crucial epigenetic targets in Schistosoma.
- Validation of these enzymes as therapeutic targets individually or collectively using RNA interference (RNAi) to knockdown the transcription of the corresponding genes. Individual HMEs, chosen using the criteria of structural divergence from human orthologues and/or the phenotypic effects of inhibitors of HME classes, were validated as targets using RNAi
- Production of enzymatically active, recombinant HME proteins and structural analysis by X-ray crystallography in the presence or absence of inhibitors. HMEs selected due to their divergence (catalytic domain) from the corresponding human orthologues and/or shown to be vital for worm survival in the phenotypic screen will be produced as recombinant proteins. Selected proteins were to be crystallized for structural analysis by X-ray diffraction.
- Target based synthesis and enzyme testing. Enzyme assays were to be optimized for the recombinant S. mansoni HMEs. Target based synthesis of inhibitors was initiated with HDAC inhibitors. In vitro testing of compounds identified by virtual screening (supported by structural studies) and synthetic bioguided optimization of HDAC inhibitors and inhibitors of other targets that originate from virtual screening were also performed.
- In silico screening for potential inhibitors (HDACi, HATi, HMTi, HDMi) was carried out by docking to the modelled catalytic domains (based on the crystal structures) of the enzymes. Bioguided optimization of inhibitors was also performed.
- High-throughput screening, hit to lead optimisation, synthetic optimisation of lead structures. After initial screening of a large set of compounds for their ability to inhibit the activity of the corresponding recombinant proteins in high-throughput assays, collections of analogues were tested both in biochemical assays and phenotypic assays on the parasite. Bioguided optimization of inhibitors was performed.
- The establishment of gene expression profiles corresponding to inhibition using drug candidates in cultured larval stages (schistosomula) was carried out to enable the determination of metabolic and signalling pathways affected.
- In vivo testing of the best lead candidates was done in infected mice. In this way, during the study period we aimed to develop a series of candidate molecules that could progress to clinical trials.
project Results:
4.1.3 Main S/T Results/Foregrounds
identification and cloning of S. mansoni HMEs
the predicted proteome of S. mansoni was screened using protein motif searches and Hidden Markov Models (by beneficiary 7) and a total of 55 HMEs were identified, as well as a number of alternatively spliced products. Molecular phylogeny shows that all are orthologues of human proteins. The most abundant group are the histone methyltransferases (23 representatives) that include both lysine and arginine methyltransferases. The data generated during this search was used to update the database SchistoDB. Moreover, the corresponding sequences and gene identifications were used to devise strategies for cloning the cDNAs encoding these proteins. In addition, since the sequence data for the Schistosoma japonicum and the Schistosoma haematobium genomes became available after the start of the project, the same methods were applied to determine the complement of HMEs in the genomes of the related parasites. Although the fragmentary nature of the S. japonicum genome meant that fewer HMEs were identified, the sequences obtained allow us in particular to compare those of proteins of interest, such as HDAC8. The genome of S. haematobium was much more comparable to that of S. mansoni. Importantly, no further HMEs were detected in addition to those already identified in the S. mansoni genome. This study will be instrumental in determining whether HME inhibitors can be developed that will be active against both species and in this context it should be noted that the catalytic domain of S. japonicum HDAC8 possesses the same modifications in relation to the human orthologue as does SmHDAC8. This indicates that inhibitor specificity should be similar for the two helminth enzymes and hence that broadband anti-schistosomal drugs could be envisaged.
schistosome HMEs as drug targets: phenotypic screening and RNAi
A reverse chemical genetics approach was used in order to identify HMEs or HME classes that are potential therapeutic targets. For this we used an in vitro assay in which schistosomula larvae or adult worms were incubated in the presence of different concentrations of HME inhibitors. For schistosomula, mortality was assessed by appearance (dead larvae are opaque) and motility and the induction of apoptosis was measured using the TUNEL assay, and in some cases the measurement of caspase 3/7 activity and Annexin V staining. For adult worms, the separation of worm pairs and in vitro egg laying were measured and in some cases worm morphology, particularly of the genital apparatus, was examined.
A focused library of HME inhibitors (including 21 novel compounds supplied by beneficiary 6) of all classes of HME (class I and II histone deacetylases, sirtuins, histone acetyltransferases, histone methyltransferases (protein arginine methyltransferases) and histone demethylases) were tested by beneficiary 1. All induced TUNEL positivity and larval death to varying degrees in the assay. One (LH 19N, a PRMT inhibitor) showed a very high level of activity (85% TUNEL positivity) at a concentration of only 5μM, whereas CS13, a sirtuin inhibitor, gave only 14% positivity at 100μM. Results of these assays showed notably that inhibitors of HDACs and in particular potential inhibitors of SmHDAC8 (e.g. Pe 11/5) efficiently induce parasite death via apoptosis.
gene expression signatures
another means of determining the roles of HME in the parasite is to identify with microarrays the Gene Expression Signatures (GES) of S. mansoni treated with candidate HME inhibitors in order to elucidate the downstream molecular pathways engaged to mediate their effects. Work was carried out by beneficiary 10 with the pan-HDAC inhibitor TSA with the aim of identifying such pathways and gene networks that might reveal new therapeutic targets.
total RNA was extracted from schistosomula (LE strain) treated for 12, 24, 48 and 72 hours either with 1 μM of Trichostatin A (TSA) or with ethanol (vehicle of the drug) as a control. The integrity of RNA samples was evaluated using microfluidic electrophoresis in the Bioanalyser equipment (Agilent Technologies). The 4x180k oligoarray platform used in this work was previously designed by beneficiary 10, which has single-strand oligonucleotide probes representing essentially all known S. mansoni transcripts [29], and it was extensively re-annotated according to the newly available S. mansoni genome sequence [30]. Each sample (biological and technical replicates) was labeled with either Cy3 or Cy5 and hybridized to the oligoarrays. For data analysis a Log2 ratio was calculated between gene intensities of TSA Treated/Control, and Significance Analysis of Microarray (SAM) was used as the statistical test to identify significantly (p-value = 0.05) differentially expressed genes. For further functional analysis, only significantly affected genes with a minimal fold change cut-off of 2 were used, allowing for a more stringent analysis of expression change. Ingenuity Pathway Analysis software (IPA) was used to identify significantly enriched gene networks among the differentially expressed S. mansoni genes.
in order to identify the gene expression signature of schistosomula treated with TSA, microarray experiments were performed in three biological replicates (each one with a technical replicate). Gene expression analyses show a set of genes regulated by inhibition of histone deacetylases, either induced or repressed by the effect of TSA over time.
recombinant protein production and X-ray crystallography
while active inhibitors can be discovered using phenotypic screening, for a rational optimization of drugs towards parasite selectivity it was vital to be able to produce recombinant HME proteins to allow structural analyses, enzyme assay development, comparative studies with human enzymes and high-throughput inhibitor screening. The first target chosen was SmHDAC8, based on its dissimilarity to and phylogenetic distance from its human orthologue and initial results from the phenotypic assay showing that inhibitors of human HDAC8 induced apoptosis and death of schistosomula in culture.
the cDNA encoding SmHDAC8 was cloned into E. coli expression vectors by beneficiary 2 and although initially the expression of soluble protein was difficult to obtain, subsequent efforts were highly successful and high yields (1.5 mg per litre of culture) of pure protein were obtained. The enzyme has been supplied (almost gram quantities throughout the duration of the project) for high-throughput inhibitor screens, tests of its catalytic activity and used for the generation of specific antibodies, as well as being crystallized for structural studies (see below).
of the 58 HMEs identified by genome data-mining, many were eliminated as candidates for drug screening since they were too conserved (e.g. HDAC1 and HDAC3), too large to permit efficient protein production (a number of the lysine methyltransferases) or enzymatically active only when in complexes with partner proteins. Eight cDNA clones encoding HMEs (Sirtuins 1, 2 and 6, PRMT 1 and 4, the HATs Tip60 and GCN5 and the HMT SET8) were cloned into expression vectors and produced in E. coli. Only two (Sirt2 and Sirt6) could be expressed as soluble proteins in sufficient quantity and although the clones producing insoluble proteins were also tested in the baculovirus expression system, this has so far proved unsuccessful. An initial protocol was set up which yielded large amounts of these proteins (more than 5 mgs per litre of culture). The protocol was further modified to prevent degradation of the purified proteins, notably in the case of SmSirt2. During the project, tens of milligrams of SmSirt2 and smSirt6 have been provided to beneficiaries 6 and 9 for their use in inhibitor assays and high-throughput screening, respectively. Sirt6 has not shown activity in standard sirtuin assay systems up to now. The setup of the purification of SmSirt2 has been carried out successfully and the target has undergone inhibitor screening. However, all attempts to obtain well-diffracting crystals of these two sirtuins have failed so far.
as the major initial target of inhibitor screening in the project, the crystallization of the SmHDAC8 protein was initiated by beneficiary 2 as a priority. Crystals were successfully obtained and the structure of the apo form (the first time for an HDAC8) was solved at 1.8 Å resolution. Interestingly, comparison of this structure to the inhibitor-bound structure of human (h) HDAC8 revealed only few gross structural changes between both proteins. Major changes concern the different insertions that are observed at the sequence level in the SmHDAC8 enzyme. Structural comparison shows that these insertions form loops at the surface of the protein, possibly providing S. mansoni specific protein/protein interaction surfaces.
compound screening and molecular modelling
the collection of a focused library of about 300 HME inhibitors led to the supply of subsets for phenotypic screening (see above) and in vitro screening assays were successfully developed for the two HMEs so far produced as recombinant proteins, SmHDAC8 and SmSirt2. The reference inhibitors of SmHDAC8 were further supplied for the phenotypic assay. These have been used to screen the candidates from the focused library, and also from the virtual screening strategy.
assay development
the development of biochemical assays for the targeted HMEs was necessary for the filters downstream of virtual screening. Furthermore, acquired knowledge was shared with beneficiary 9 who also carried out assay development for high-throughput screening. As SmHDAC8 was chosen as the initial target, assays were first developed for this parasite deacetylase subtype. Besides this, an in vitro assay for sirtuin 2 from S. mansoni (SmSirt2) was also set up.
it was already known that small acetylated substrates are not well accepted by human HDAC8 (hHDAC8), which we found out to be true for smHDAC8 as well. Thus, the well-established deacetylase assay that was developed by beneficiary 6 was adapted by using a small peptidic substrate with a trifluoroacetyl moiety. In cooperation with beneficiary 9, also a tripeptidic substrate carrying a trifluoracetyl group was used in the same assay. It could be shown that both substrates were converted by SmHDAC8 to a comparable degree. Therefore, our small substrate was used for further testing.
being aware of the fact that using a trifluoroacetyl group instead of the endogenously converted acetyl moiety might lead to false negatives or the discovery of false positives, a commercially available assay setup was adapted that uses an acetylated substrate derived from a sequence of the deacetylase substrate p53.
most of the compounds tested in both in vitro assay systems (trifluoroacetyl substrate and acetylated peptide) led to comparable results, but in some cases the resulting potency of the tested compounds depended on the assay (and substrate) that was used. Hence, it was decided that the use of one assay system would be sufficient for pre-screening of library compounds. Identified hits, however, were then countertested in the other assay setup. The influence of pH on activity (around 6-7) and also mutants provided by beneficiary 2 that highlight the importance of a cysteine near the catalytic site were also tested.
screening for inhibitors of SmHDAC8
virtual screening of inhibitors for SmHDAC8 was initially done by beneficiary 3 using 3D structural models based on known structures of human orthologues and used for docking of known HDAC inhibitors. This pointed out the similarity of the residues of the active site between the human and schistosome HDAC8 enzymes and suggested a similar interaction pattern with inhibitors, but also indicated a notable difference at the rim of the active site (Met/His substitution). The in silico screen for inhibitors was refined with the contribution of the 3D structure of SmHDAC8 determined by X-ray crystallography. A virtual database of ~2 million compounds was focused on compounds with zinc-chelating groups. Of 4300 compounds with such moieties, 3209 were docked into the SmHDAC8 active site. About 150 compounds selected for stability and availability were tested as inhibitors in the fluorescence-based assays. We identified as novel smHDAC8 inhibitor the internal hydroxamate J1036 from the Ibscreen compound library. This compound and analogues represent a class of compounds with a scaffold not previously described for HDAC inhibitors. Similarity-based screening and docking identified ten further analogues that were sent to beneficiary 6 for phenotypic screening. For J1036 beneficiary 2 obtained a low-resolution X-ray structure with SmHDAC8. Two compounds, J1036 and J1060 were identified as the most potent SmHDAC8 inhibitors from this series (IC50 values around 5 μM). The similarity-based screening identified also J1075 as an SmHDAC8 inhibitor active in the low micromolar range. Biological characterization was carried out and a crystal structure in complex with SmHDAC8 was obtained by beneficiary 2 that confirmed the docking results.
screening for inhibitors of SmSirt2
although it proved impossible to produce well-diffracting crystals of SmSirt2, a homology model was generated using the MODELLER approach and analyzed by means of molecular dynamics simulations using the AMBER software. The SmSirt2 model was compared with the human Sirt2 X-ray structure and was used for docking studies of in house sirtuin inhibitors. Superposition of SmSirt2 with hSirt2-inhibitor complex reveals that the residues in the active site are similar and therefore will have similar interaction pattern with inhibitor/substrate. However a notable difference was found at the peptide substrate binding site in which I169, I232, F234, and L239 in human Sirt2 are mutated into V132, V193, L195, and M200 in SmSirt2.
high-throughput inhibitor library screening
SmHDAC8
recombinant SmHDAC8 was first used by beneficiary 9 to develop a biochemical assay suitable for high-throughput screening (HTS). A carefully designed set of 80,000 diverse compounds was implemented, eliminating promiscuous compounds and plated. After screening, counterassays were performed to filter out false positives. The 66 compounds obtained in this screen were subjected to quality control and then clustered according to their structures, resulting in 9 hit series and 21 singletons. Three of the hit series and 3 singletons were then selected for analogue searches, resulting in the identification of 122 analogous compounds. Dose response testing allowed us to concentrate on 2 series that are most promising in terms of potency, but with a large spread in activity.
two novel SmHDAC8 lead series originating from the HTS campaign have been generated in the course of the project. The main series consists of greater than100 novel compounds with potencies in the biochemical assay ranging from 43 nM to inactive. The second (backup) series consists of 19 novel compounds with potencies in the biochemical assay ranging from 0.8 to 12 μM.
the main SmHDAC8 inhibitor series: Considerable efforts have been invested in establishing chemical routes for the synthesis of different types of these compounds. About 75 compounds have been synthesized and tested in the biochemical assay leading to the establishment of a clear structure/activity relation (SAR). The activities of five selected compounds were confirmed by beneficiary 6 using two alternative substrates. Testing of compounds against human HDAC8 has been performed and showed that there is no selectivity of the tested compounds against human HDAC8. Ligand-detected NMR binding experiments (transverse relaxation filtered experiments with equimolar amounts of protein and compound) have confirmed direct binding of representative compounds to SmHDAC8. Information on the binding mode of the main series of compounds was first obtained from a modelling/docking study (beneficiary 3) and later from the crystal structure determination of two highly potent compounds in complex with SmHDAC8 (beneficiary 2), including one of the selected lead compounds. The crystal structures have allowed a deeper understanding of the SAR and provided hypotheses on how to obtain selectivity against human HDAC8. Compounds from the main inhibitor series have been tested in the phenotypic assay at different times during the project. The first set of main series compounds tested did not show any significant effect on the schistosomula, only a compound from the backup series clearly induced apoptosis in the schistosome larvae. Since the main series compounds were potent SmHDAC8 inhibitors we hypothesized that the compounds did not pass the double tegument membrane of the larvae. Following this, synthesized compounds were tested in a membrane affinity assay in order to direct the chemistry towards compounds with higher membrane affinity and thus higher passive diffusion over a membrane. A second batch of compounds in the main series with higher membrane affinities did indeed induce apoptosis in the schistosome larvae. In order to further improve the permeability of selected lead compounds, a pro-drug strategy has been employed.
the backup SmHDAC8 inhibitor series: The chemical route for synthesis has been worked out and 19 novel compounds have been synthesized and quality controlled. The compounds have been tested in dose-response in the biochemical assay against both SmHDAC8 and hHDAC8. An early SAR is emerging but there is no clear selectivity against human HDAC8. Two compounds from this series has been tested in the in vitro schistosomula assay by beneficiary 1, both compounds showing toxicity against the schistosomula at both 5 and 20 μM compound concentration. Further, one of the two tested compounds also showed effects in the adult worm pairing assay.
three potent compounds (denoted compounds 1, 2 and 3 below) from the main inhibitor series were selected as leads; their IC50s in the biochemical assay were respectively 43, 45 and 350 nM. Most efforts have been invested in the two first compounds that are quite similar and the most potent in the biochemical assay. The crystal structure of SmHDAC8 in complex with the most potent compound shows a perfect fit of the lead compound in the active site and the optimization of the lead compounds have been focused on increasing their permeability.
the three lead compounds have been subjected to a pro-drug strategy in order to further improve and optimize the permeability of the compounds. The strategy takes advantage of the fact that the concentration of reducing agent (GSH) inside the worm (or cell) is on the order of 1,000 times higher compared to the outside. The pro-drugs are designed so that they stay intact outside of the worm (cell) and upon entering the worm (cell) are reduced to the active compound. A large variety of different pro-drugs have been synthesized starting from the lead compounds. The synthesized compounds have been subjected to QC analyses and tested in the biochemical assay in presence and absence of reducing agent to mimic the situation inside and outside the worm (cell), respectively. Also, a smaller number of pro-drugs have been synthesized starting from a few other parent compounds and tested in the biochemical assay and cell assays.
before sending compounds to be tested in the in vitro phenotypic assays, as a first filter a much larger number of SmHDAC8 inhibitors have been tested in a 48 hour cell toxicity assay. This has been done in order to test if the prodrug strategy is working on cells and thus to select compounds for the phenotypic assays with a higher probability to be active against the schistosomes. A high-throughput cellular assay in 384-well format has been developed which, apart from testing of compounds on cell lines, also allows the testing of compounds on primary cells due to the small volumes needed. Effects of active SmHDAC8 inhibitors have been tested in dose-response (11 concentrations from 20 μM down to 20 nM, in duplicate) on the standard HepG2 cell line as well as primary fibroblasts and a few cancer cell lines (osteosarcoma, neuroblastoma, pancreatic cancer). The lead compounds show low toxicity while most of the pro-drugs based on the lead compounds show a much higher cell toxicity demonstrating that the prodrug is indeed cleaved intracellularly and that the prodrug strategy is working. Further, the prodrug versions of the lead compounds are more toxic against the cancer cell lines (several compounds with low μM IC50s) compared to the primary fibroblasts and the HepG2 cell line (no IC50 values, i.e. IC50 greater than 20 μM).
ten compounds that showed a good effect in the cell assays were selected for the last round of testing in the in vitro phenotypic assays. The selected compounds were five prodrugs based on the lead compound 1, two prodrugs based on the lead compound 2, one prodrug based on lead compound 3, and two compounds from the backup series. All ten compounds induced mortality of schistosomula at 20 μM compound concentration, and a few of them a clear effect also at 5 μM. Further, an adult worm pairing assay was performed on the ten compounds. Four of them showed a clear effect by inducing separation of worm pairs at 20 μM compound concentration in this assay.
SmSirt2
recombinant SmSirt2 was supplied by beneficiary 2. A biochemical assay with fluorescence read-out suitable for high-throughput screening (HTS) of SmSirt2 was successfully developed and formatted by beneficiary 9. During the assay development several different substrates were tested, among them a substrate supplied in small amounts by beneficiary 6. This latter substrate was selected for the HTS and was synthesized in large amounts at iNovacia. In order to increase the sensitivity of the assay with respect to protein consumption, an extensive buffer optimisation was performed resulting in an assay that required reasonable amounts of protein and thus making an HTS possible to perform.
the HTS of a carefully designed screening set consisting of ~80,000 diverse compounds was performed at 10 μM compound concentration. 875 positives (hit rate 1.08%) were identified in the primary SmSirt2 HTS. 27 positives that have been identified as hits in 4 or more previous HTS campaigns were removed from the hit list, and dose-response curves (11 concentrations from 100 μM and down to 1.5 nM, in duplicate) were performed for 848 compounds. Dose-dependent SmSirt2 inhibitory activity was confirmed for 764 compounds. Of these 9 compounds showed an IC50 less than 0.1 μM; 111 compounds showed an IC50 less than 1 μM and 646 compounds showed an IC50 less than 10 μM. Several counterassays were employed to filter out compounds active in the biochemical assay by non-desired mechanisms. These assays included detection of compounds that interfere with the fluorescence readout (quenching the substrate fluorescence) and detection of compounds that are redox-active. Further, the activities of selected hits were determined in the biochemical assay also in the absence of buffer components such as reducing agent and detergent. The hits were subjected to quality control (QC, checked for identity, purity, chemical stability and solubility in buffer). The confirmed hits were then clustered with respect to their structures and the resulting 165 hit series and 159 singletons were evaluated by the beneficiary 9 medicinal chemistry group. The evaluation aimed at identifying the most promising hit series and singletons from a medicinal chemistry perspective (e.g. lead likeness, synthetic and library expansion tractability, emerging structure-activity relationship (SAR) and IP space). Six hit series and four singletons were given the highest prioritization and a further six series and nine singletons were selected as backups.
the six inhibitor series and four singletons with the highest prioritization were selected for analogue searching. 551 analogous compounds were identified and tested in dose-response followed by counterassays and compound QC. The analogue testing further increased the number of active compounds in the highest prioritized inhibitor series and six of these series contain a large number of active compounds. In addition to these ten series there are 159 series and 155 singletons containing active compounds that have been given a lower priority. The abundance of active compounds that have passed all counterassays and the emerging SAR in the highest prioritized inhibitor series constitutes a very good foundation for the further development of SmSirt2 inhibitors.
conclusions
the aim of the SETTREND project was to develop new drugs for use in treating schistosomiasis, the second most important parasitic disease after malaria and involves steps to bring drugs to the pre-clinical testing stage, determining their activity in vitro and in vivo in infected mice. The rationale for the project is the existence of only one effective drug against schistosomes, praziquantel, and the increasing likelihood of the development of strains of parasite resistant to this drug, given its intensive use, particularly in the context of the Schistosomiasis Control Initiative (see http://www3.imperial.ac.uk/schisto online). We have attempted to fulfil this aim using the most comprehensive strategy yet assembled to resolve such a problem. We have provided a number of lead compounds for development as drugs (or at least valuable tool compounds for cellular and animal studies) that could either proceed to clinical trials or be further optimized. Our strategy, combining high-throughput and rational approaches has proved its worth and novel compounds with potent activity against the parasite in vitro and in vivo have been generated by both. Therefore, the proof of concept of the strategy has already been delivered. Moreover, the comparison of the HME targets from S. mansoni and the related species Schistosoma japonicum, added to the project, shows that, in the case of schistosome HDAC8, drugs developed against the enzyme from the former species are likely to be active against the latter. As we stated in our submission, we do not consider that clinical trials (mentioned as a possible aim of the original call) represent a reasonable aim within the project duration. However, the development of optimized lead compounds will lay the foundations for such trials.
summary of main scientific achievements
- The identification of the HMEs encoded in the schistosome genomes. Molecular cloning of the coding sequences of selected HMEs and the investigation of their expression during the parasite life-cycle.
- The elucidation of gene expression signatures of HDAC inhibition in parasite larvae, leading to the identification of key pathways and proteins.
- The implementation of a reverse chemical genetics strategy showing that inhibitors of all HME families induced apoptosis and death of schistosome larvae in culture. This validated both the SETTREND approach and the targeted enzymes.
- The validation of an HDAC (SmHDAC8) and two other HMEs (SmPRMT3 and SmKDM1) as therapeutic targets by RNAi. This showed that single HMEs (and not just whole families) can be therapeutic targets and validated our target-based approach to develop selective inhibitors for individual HMEs.
- The production of SmHDAC8 as an enzymatically active recombinant protein and the solution of the structures of the apo-form of the protein, the protein bound to pan-HDAC inhibitors and to selective inhibitors generated during the project. Two other recombinant HME proteins, SmSirt2 and SmSirt6 were also produced.
- In silico screening of SmHDAC8 based on the solved crystal structure identified compounds with novel scaffolds and warheads with greater selectivity than reference inhibitors and that provoked apoptosis and death of schistosomula in culture. Treatment of schistosome-infected mice with one of these inhibitors, J1075, led to reduced worm burdens and egg loads.
- High-throughput inhibitor library screening of SmHDAC8 and bioguided synthetic chemistry optimization produced lead inhibitors that were extremely active against both schistosome larvae and adult worms in culture. High-throughput inhibitor screening was also carried out for SmSirt2.
- These results justify our strategy and suggests that further lead optimization will generate highly selective and potent compounds that could provide the basis for clinical trials.
Overall, the objectives set out for the project have been achieved with minor exceptions. Potential pitfalls in our strategy have been avoided and problems solved. No major changes in strategy have been necessary during the project. The main aim, to develop a series of candidate molecules with a future potential for clinical trials, has been achieved.
references
[1] Hotez PJ, Pecoul B. "Manifesto" for advancing the control and elimination of neglected tropical diseases. PLoS Neg Trop Dis 2010; 5: e718.
[2] Steinmann P, Keiser J, Bos R et al. Schistosomiasis and water resources development: systematic review, meta-analysis and estimates of people at risk. Lancet Infect Dis 2006; 6: 411-25.
[3] Crompton DW. How much human helminthiasis is there in the world? J Parasitol 1999; 85: 397-403.
[4] Siddiqui A A, Siddiqui B A, Ganley-Leal L. Schistosomiasis vaccines. Hum Vaccin 2011; 7: 1192-7.
[5] Fenwick A, Webster JP. Schistosomiasis: challenges for control, treatment and drug resisitance. Curr Opin Infect Dis 2006 ; 19 : 577-82.
[6] Doenhoff MJ, Hagan P et al. Praziquantel: its use in control of schistosomiasis in sub-Saharan Africa and current research needs. Parasitology 2009; 136: 1825-35.
[7] van der Werf MJ, de Vlas Sj, Brooker S et al. Quantification of clinical morbidity associated with schistosome infection in sub-Saharan Africa. Acta Trop 2003; 86: 125-39.
[8] McManus DP, Loukas A. Current status of vaccines for schistosomiasis Clin Microbiol Rev 2008; 21: 225-42.
[9] Doenhoff MJ, Pica-Mattoccia L. Praziquantel for the treatment of schistosomiasis: its use for control of areas with endemic disease and prospects for drug resistance. Expert Rev Anti Inf Ther 2006; 4: 199-210.
[10] Kelly TK, De Carvalho DD, Jones PA. Epigenetic modifications as therapeutic targets. Nat Biotechnol 2010; 28:1069-78.
[11] Fraga MF, Bellestar E, Villar-Garea A et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 2005; 37: 391-400.
[12] Jeunwein T, Allis CD. Translating the histone code. Science 2001; 293: 1074-80.
[13] Sterner DE, Berger SL. Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 2000; 64: 435-59.
[14] Gregoretti IV, Lee Y-M, Goodson HV. Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis. J Mol Biol 2004; 338: 17-31.
[15] Frye RA. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun 2000;273:793-8.
[16] Zhu AY, Zhou Y, Khan S et al. Plasmodium falciparum Sir2A preferentially hydrolyzes medium and long chain fatty acyl lysine. ACS Chem Biol 2012; 7: 155-9.
[17] Martin C, Zhang Y. The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 2005; 6: 838-49.
[18] Anand R, Marmorstein R. Structure and mechanism of lysine-specific demethylase enzymes. J Biol Chem 2007; 282: 35425-9.
[19] Andrews KT, Haque A, Jones MK. HDAC inhibitors in parasitic diseases. Immunol Cell Biol 2012; 90: 66-77
[20] Arrowsmith CH, Bountra C, Fish PV et al. Epigenetic protein families: a new frontier for drug discovery. Nat Rev Drug Disc 2012; 11: 384-400.
[21] McCabe MT, Ott HM, Ganji G et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 2012; 492: 108-12.
[22] Koppenol WH, Bounds PL, Dang CV. Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer 2011; 11:325-37.
[23] Dubois F, Caby S, Oger F et al. Histone deacetylase inhibitors induce apoptosis, histone hyperacetylation and up-regulation of gene transcription in Schistosoma mansoni. Mol Biochem Parasitol 2009; 168: 7-15.
[24] Ezhkova E, Lien WH, Stokes N, et al. EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev. 2011; 25: 485-98.
[25] Anderson L, Pierce RJ, Verjovski-Almeida S. Schistosoma mansoni histones : from transcription to chromatin regulation ; an in silico analysis. Mol Biochem Parasitol 2012; 183:105-14
[26] de Moraes Maciel R, de Silva Dutra DL, Rumjanek FD et al. Schistosoma mansoni histone acetyltransferase GCN5: linking histone acetylation to gene activation. Mol Biochem Parasitol 2004; 133: 131-5.
[27] Yamauchi Y, Boukari H, Banerjee I et al. Histone deacetylase 8 is required for centrosome cohesion and influenza A virus entry. PLoS Pathogens 2011; 7: e1002316
[28] Deardorff MA, Bando M, Nakato R et al. HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature 2012; 489: 313-7
[29] Verjovski-Almeida S, Venancio TM, Oliveira KC et al. Use of a 44k oligoarray to explore the transcriptome of Schistosoma mansoni adult worms. Exp Parasitol 2007; 117:236-45.
[30] Protasio AV, Tsai IJ, Babbage, A et al. A systematically improved hig-quality genome and transcriptome of the human blood fluke Schistosoma mansoni. PLoS Negl Trop Dis 2012; 6: e1455.
potential Impact:
4.1.4 Potential Impact
the impact of the work done is assessed in terms of the criteria set out in the original project application.
strategic Impact of the SETTREND project
development of new drugs for treating schistosomiasis
the strategy for drug development used in the SETTREND project was the most comprehensive yet assembled in anti-parasitic drug discovery. The combination of both in silico and high-throughput screening, both rigorously target-based, allowed the identification of a large number of inhibitors of HMEs, particularly of HDACs.
proof of concept was provided early on in the project with:
- Phenotypic screening showing that HME inhibitors were capable of inducing apoptosis and mortality in schistosomes.
- The validation of SmHDAC8 as a therapeutic target, the production of the active recombinant enzyme, structural studies and initial screening results identifying potent inhibitors
subsequent and parallel work showed that the approach could represent a new paradigm for drug discovery for parasitic diseases in general, but also for other pathologies.
- Optimized lead compounds that are nM range inhibitors of SmHDAC8 have been produced from both in silico and high-throughput screens that are also highly toxic for schistosomes in culture. In one case the compound was also effective in vivo on schistosome-infected mice.
- Potent inhibitors of another HME, SmSirt2 have also been produced by both screening methods.
- The screening approaches merge in the phase of lead optimization with the docking of compounds into the solved crystal structure of the target enzyme or after co-crystallization with the enzyme.
- Testing of compounds in phenotypic assays to judge the effects of compounds on the parasite is applied at each step of the hit to optimized lead process.
the candidate drugs effective against S. mansoni are likely to be effective against the two other main species of schistosome infecting humans Phylogenetic and structural studies confirm that candidate drugs effective against S. mansoni are very likely to be effective against the other two main species of schistosome infecting humans. For example, the catalytic site differences of SmHDAC8 compared to its human orthologue are conserved in both S. haematobium and S. japonicum HDAC8. This is very probably the case for other HMEs.
the candidate drugs developed against schistosomes will serve as lead compounds for the treatment of other helminth diseases. The lack of effective treatments for hydatid disease caused by Echinococcus granulosus (cystic hydatid disease) and E. multilocularis (alveolar hydatid disease) suggests that the exploration of the possible application of inhibitors developed against S. mansoni HMEs should be explored. As a first step the exploration of the HME content and orthologies in the E. multilocularis genome will be possible once the reference quality genome has been produced (see http://www.sanger.ac.uk/resources/downloads/helminths/echinococcus-multilocularis.html online).
candidate drugs developed against schistosomes could provide novel lead compounds for cancer therapy. Our strategy for drug development targeting histone modifying enzymes was directly inspired by emerging approaches to develop treatments for cancer. Moreover, two HDAC inhibitors are currently approved for the treatment of cutaneous T-cell lymphoma and many others are in clinical trials. Beneficiary 9 has tested compounds developed from hits obtained for SmHDAC8 on cancer cell lines and shown that some are highly toxic with low μM IC50 values. It is clear that a number of the novel inhibitor scaffolds identified by screening the schistosome HMEs will prove of interest as leads for the development of anti-cancer drugs.
european dimension of the project
the project required a combination of expertise and capacity not available at the national level in any of the beneficiary countries, whether in Europe or Brazil. Moreover, the project required the participation of beneficiaries working on aspects of parasite molecular biology and with specific expertise and access to material not available in Europe, but only in Brazil, a country endemic for schistosomiasis. Funding for collaborations between Europe and Brazil is not available on the necessary scale via any other mechanism. The financial support of the EC is essential.
one substantial impact of the project has been to develop and encourage scientific contacts between the Brazilian and European partners and between the different European partners. This has been reflected in the exchanges of students between these partners.
this collaboration has kept Europe at the forefront of a domain (the search for drugs against parasitic diseases) that is highly competitive, with a strong representation in the USA. The model character of our approach could be the seeding point for other similar campaigns aimed at other infectious diseases. This is in fact already the case, since the SETTREND project has formed the basis of a new project, A-PARADDISE, submitted under FP7, which will apply the same strategy to drug development against schistosomes and three protozoan parasites.
the SETTREND project has been the starting point to move toward the production of solid industrial standard-based candidate drugs that could be taken over by a larger European pharmaceutical company. Thus, on top of the humanitarian objective, the economics will drive a larger interest into these diseases by the medium and large European companies,
european competitiveness
impact on industrial activity within the project. The consortium included an industrial participant, the SME iNovacia (beneficiary 9), as well as a company involved in intellectual property management and dissemination (beneficiary 8). The project undoubtedly contributed to their credentials in the domain of drug development, although these are already well-established, and involvement in this collaborative project will have increased awareness of their expertise, within and outside Europe. Moreover, the SME iNovacia was a full partner in the project and had a major influence on the direction taken by the research. More specifically, for the SME in the project, working within the Life Science sector in a comparatively small country like Sweden, it is important to reach out and extend the customer base. Participation in an FP7 project could provide new contacts throughout Europe that may lead to business opportunities. However, this is an objective, not an achieved impact. Similarly, an impact on the biotech industry by a bottom-up approach to innovation and funding has not yet had any tangible effects, and the progression towards an industrial exploitation of the compounds developed during the SETTREND project via partnerships with pharmaceutical industry remains a future aim.
innovation impact
the scientific impact of the project within parasitology research has been considerable, both in terms of innovation and dissemination (see below). The major innovation impacts predicted in the submitted project have been achieved:
- The identification and characterization of schistosome HMEs has been completed. The improved annotation is available in SchistoDB and a manuscript describing the main conclusions is in preparation (beneficiary 7), in particular concerning the phylogenetic relationships of schistosome HMEs to their orthologues in other species and the implications for the evolution of these enzyme families in invertebrates. Very divergent HMEs were not found in S. mansoni or the two other schistosome species investigated, demonstrating the overall conservation of both these enzymes and the epigenetic marks they generate. In addition, a study of the schistosome histones was carried out making use of the newly available RNASeq data newly available (beneficiary 10) and which allowed the correction of some of the genomic assemblies and annotations in the databases. Moreover, it allowed the confirmation of the structures of schistosome histones and the conservation of the sites of epigenetic marks. The more detailed characterization of the functional properties of HMEs demonstrated the role of HATs in the transcriptional control of a gene encoding a major eggshell protein, confirming HAT inhibition as a therapeutic goal (beneficiary 4). Furthermore, the main HME target studied during the project, SmHDAC8, was found to interact with proteins involved in cell division (beneficiary 1). This underlines that many HMEs, in addition to functions in transcriptional control, are also involved in other processes and have substrates other than histones.
- Comparative structural studies were carried out between SmHDAC8 and its human orthologue (beneficiary 2). These studies (manuscript submitted for publication) were central to the demonstration of differences in catalytic pocket architecture that can be exploited to identify and synthesize inhibitors that are selective for the schistosome enzyme. The studies allowed precise modelling of inhibitor-enzyme interactions (beneficiary 3) and the identification of inhibitors that exploited the specific characteristics of the SmHDAC8 catalytic site. Although we focused on the crystal structure of SmHDAC8 and small molecule interactions, modelling studies of SmSirt2, for which it proved impossible so far to obtain crystals, also flagged up catalytic pocket differences with respect to the human orthologue which will again allow the identification of selective inhibitors.
- Gene expression signatures. The studies of gene expression signatures did not allow the definition of drug specificity via the comparison of expression profiles of parasites treated with a drug or knocked down for the transcription of the HME drug target by RNAi (for reasons elaborated in the second periodic report). However, the profiles generated by HDAC inhibition and the analysis of the pathways and interaction networks of the genes for which expression was modulated (beneficiary 10) have already allowed the identification of the lysine methyltransferase EZH2 as a therapeutic target, which was confirmed using a specific inhibitor.
- New HME inhibitors available for functional studies. In addition to the focused inhibitor library generated by beneficiary 6, which supplied inhibitors already used in functional studies of HATs and sirtuins, the project has generated several hundred new compounds that are hits from high-throughput and in silico screening of SmHDAC8 and SmSirt2, analogues or optimized leads. Many of these have novel scaffolds or warheads for HDAC or sirtuin inhibitors and promise to be extremely useful, not only for functional studies in schistosomes, but more particularly as lead compounds for other human pathologies. Moreover, these novel structures are candidates for IP protection (see below).
societal impact
schistosomiasis remains a neglected tropical disease (in terms of the financial resources devoted to its prevention and treatment) and is a leading cause of chronic disability and poverty, in addition to the direct mortality that can be attributed to it in the 75 endemic tropical and sub-tropical countries. The ultimate aim of the work contained in this project, to contribute to reducing the impact of schistosomiasis on the affected populations, will have a dual impact on both health and living standards. This future impact is predicated on the development of an effective drug that is relatively cheap to produce. The results and foreground generated by the SETTREND project show this outcome is attainable based on the strategy that we have employed.
training and technology transfer impact
direct training activities during the project concerned PhD students from beneficiaries 1, 4 and 7 who underwent training in beneficiary laboratories (1, 2, and 6) for recombinant protein production, enzyme inhibition assays, gene transcription techniques or RNAi (in a laboratory outside the consortium, expert in the domain).
in addition to these activities, technology transfer between beneficiaries has concerned:
- Exchanges and discussions between beneficiaries 1 and 7 concerning the invalidation of HME gene transcription by RNAi and the phenotypic screen using HME inhibitors on parasite larvae in vitro.
- Transfer of technology concerning the functional characterization of schistosome HMEs between beneficiaries 1 and 4, specifically the chromatin immunoprecipitation methodology.
- Development and standardisation of enzyme assay methods between beneficiaries 2, 6 and 9. This has specifically concerned the assays used for SmHDAC8 and SmSirt2.
- Molecular modelling and virtual screening approaches between beneficiaries 2 and 3.
plan for the use and dissemination of foreground
knowledge generated by the project has been made available to the public respecting the general rules laid down in the Consortium Agreement and according to the European Commission guidelines. Participants planning dissemination informed the consortium via e-mail and by posting the proposed communication or article on the consortium website (consortium intranet section) in order to obtain approval, or to allow for objections if it was felt that certain data required protection before dissemination. In practice, however, this latter proviso did not prove necessary.
dissemination to the general public and media
the aims and strategies of the project were disseminated in seven press releases or articles published in the popular press, as well as in the project brochure, which was widely distributed.
publications and communications
to date the project results have been published in nine full journal articles. This number is destined to rise significantly in the coming months. One reason for this is that the publication of a certain number of articles in preparation will be subsequent to that of the major article concerning the structural characterization of SmHDAC8 and the characterization of selective inhibitors, which will be submitted after finalization of the reporting procedure.
A large number of presentations (29) were given by project beneficiaries in international conferences and workshops. These included international conferences on parasitology, specifically on schistosomiasis or on epigenetics. In addition to these conferences, 19 communications and posters were also presented at such meetings. A further seven presentations were given to more select (single country) audiences.
databases
the results of the project concerning the identification of HMEs have permitted the improved annotation of the corresponding genes in SchistoDB (see http://schistodb.net/schisto/ online) which is part of the EuPathDB project (see http://eupathdb.org/eupathdb/ online). Furthermore, cloned cDNA copies of schistosome HMEs have led to the correction of some assemblies and the elucidation of splicing isoforms. Similarly, the results of RNASeq studies have led to the correction of histone gene predictions in SchistoDB. Fully characterized transcript sequences have also been submitted to the NCBI database (see http://www.ncbi.nlm.nih.gov/ online)
consortium website
the SETTREND website (see section 4.1.5) as well as being used for consortium business (posting proposed articles and abstracts for approval by consortium members) also posted news of activities of consortium members, in addition to basic information about the project.
exploitation of foreground
the plan is to develop the current foreground to pharmaceutical agents for treatment of schistosomiasis. Most of the compounds under consideration were identified first as inhibitors of SmHDAC8. Further research is necessary to optimize these compounds, primarily medicinal chemistry work and iterative testing of compounds in several different assays such as the primary biochemical assay, the in vitro assays on schistosome larvae and adult worms, ADMET assays, and finally schistosome-infected mice. Optimized compounds or candidate drugs are possible to exploit once such work has been done. Novel scaffolds and warheads of HDAC inhibitors were identified that are potentially patentable. However, further research by the SETTREND consortium members is necessary to validate the in vivo activity of a number of these. Moreover, some of the inhibitors still have to be tested on human enzymes.
some compounds have been tested against human HDACs and show activity. A number have also been tested on human cancer cell lines and show toxicity. These compounds could therefore potentially be developed into pharmaceutical agents for treatment of human cancers and other human diseases.
this is also true for the sirtuin inhibitors that it may be possible to develop some of them into pharmaceutical agents for treatment of human cancers, neurodegenerative diseases and other human diseases.
in addition to the novel HDAC8 inhibitors active on human and schistosome enzymes, the consortium has identified potential drugs that interfere with parasite sexual development.
this foreground can be exploited with further research as follows:
1. Cloning S. mansoni HMEs in mammalian vectors to study the molecular mechanisms involved in the action of these HMEs in parasite biology.
2. Screening new drugs to interfere with any of the signaling pathways identified by transcriptomic studies by beneficiary 10.
3. Testing the drugs in cultures of larval and adult S. mansoni parasites.
4. Testing the drugs in vivo in schistosome-infected mice.
in addition, the exploitation of some methodologies developed by project beneficiaries, which cannot be specified here, is under study.
list of Websites:
http://SETTREND.cebio.org/
4.1.1 Executive summary
the overall aim of the SETTREND project was to develop new drug leads for the treatment of schistosomiasis. This disease, caused by blood-dwelling flatworm parasites of the genus Schistosoma, affects more than 200 million people worldwide, causing upwards of 200,000 deaths annually and its control currently depends on the use of the only available drug, praziquantel. The current use of this drug in mass-treatment campaigns in sub-Saharan Africa increases the probability of the development of resistance by the parasite. The development of new drugs is therefore imperative and our approach is to target enzymes involved in the epigenetic modification of the chromatin, the histone modifying enzymes (HME). In particular, we chose to focus our approach on enzymes catalyzing histone acetylation/deacetylation and methylation/demethylation. These enzymes are increasingly targeted in the therapy of several pathologies and in particular cancer, allowing us to benefit from the expertise and inhibitors already developed. The enzymes targeted in the project are the histone deacetylases (HDAC), histone acetyltransferases (HAT), histone methyltransferases (HMT) and histone demethylases (HDM) of Schistosoma mansoni the species responsible for intestinal schistosomiasis in Africa and South America.
the objective of the project was to identify novel inhibitors of HMEs that will be drug leads. To do this we used a holistic approach that enabled us to progress from the cloned target protein, via structural analysis, in silico and high-throughput screening, to the identification of new lead compounds validated by their toxicity for the parasite in vitro and in vivo.
the main achievements of the SETTREND strategy are as follows:
- The identification of the HMEs encoded in the schistosome genomes. Molecular cloning of the coding sequences of selected HMEs and the investigation of their expression during the parasite life-cycle.
- The elucidation of gene expression signatures of HDAC inhibition in parasite larvae, leading to the identification of key pathways and proteins.
- The implementation of a reverse chemical genetics strategy showing that inhibitors of all HME families induced apoptosis and death of schistosome larvae in culture.
- The validation of an HDAC (SmHDAC8) and two other HMEs (SmPRMT3 and SmKDM1) as therapeutic targets by RNAi.
- The production of SmHDAC8 as an enzymatically active recombinant protein and the solution of the structures of the apo-form of the protein, the protein bound to pan-HDAC inhibitors and to selective inhibitors generated during the project. Two other recombinant HME proteins, SmSirt2 and SmSirt6 were also produced.
- In silico screening of SmHDAC8 based on the solved crystal structure identified compounds with novel scaffolds and warheads with greater selectivity than reference inhibitors and that provoked apoptosis and death of schistosomula in culture. Treatment of schistosome-infected mice with one of these inhibitors, J1075, led to reduced worm burdens and egg loads.
- High-throughput inhibitor library screening of SmHDAC8 and bioguided synthetic chemistry optimization produced lead inhibitors that were extremely active against both schistosome larvae and adult worms in culture. High-throughput inhibitor screening was also carried out for SmSirt2.
project Context and Objectives:
4.1.2 Project context and objectives
the need for new drugs to combat schistosomiasis
schistosomiasis is a neglected tropical parasitic disease of major public health importance [1] caused by blood flukes of the Schistosoma spp. (class Trematoda, family Schistosomatidae). S. haematobium, S. japonicum and S. mansoni are the three main species parasitizing more than 200 million people worldwide, with 780 million at risk of infection [2]. S. mansoni, the agent of human intestinal schistosomiasis, is endemic in 54 countries, mostly in Africa, South America and the Middle-East [3]. There is currently no effective vaccine against schistosomiasis [4] and the following control strategies are recommended in a recent WHO report: preventive chemotherapy, intensified case management, vector control and provision of safe water, sanitation and hygiene. Unfortunately interruption of the transmission cycle through chemical or biological vector (snail) control and health education have generally proved insufficient on their own as control methods 5]. The treatment of the disease relies on a single drug, praziquantel (PZQ) [6]. In Africa, where the annual mortality rate is estimated to be as high as 280,000 [7], several control programs, such as those conducted by the Schistosome Control Initiative (SCI, see http://www3.imperial.ac.uk/schisto online), the United States Agency for International Development (USAID) and the Department for International Development (DFID), distribute PZQ on a large scale because PZQ is safe, cheap and effective against adult worms. Another recent input into the fight against schistosomiasis is Merck's commitment to a tenfold increase, from 25 million to 250 million, of its annual donation of PZQ tablets (see http://www.4-traders.com/MERCK-KGAA-436395/news/Merck-KGaA-Merck-to-Raise-Donation-Tenfold-to-Eliminate-Worm-Disease-13991828/ online). In Africa, PZQ is distributed generally to the whole population where overall prevalence is greater than 50% or to school-aged children in endemic areas. Treatment results in an immediate reduction in the prevalence and intensity of infection but because immunity to infection is weak, reinfection often brings the prevalence back to pre-treatment levels within 2 years [8].
epigenetics, the histone code and transcriptional regulation
epigenetic modifications, particularly those affecting DNA methylation or posttranslational modifications of histones (including acetylation and methylation of lysine or arginine residues), are known to be aberrant in a number of diseases, including cancer, diabetes, autoimmune disorders, asthma and neurological disorders (see [10] for review). In the case of cancer a global hypomethylation of DNA, leading to genetic instability, is accompanied by localized hypermethylation, in particular of the promoters of tumour suppressor genes. Additionally, histone hypoacetylation in malignant compared to the corresponding normal tissue has been found in lymphomas and colon cancers [11] characterized by the loss of acetylation at Lys16 of H4 (H4K16). This was also accompanied by the trimethylation of H4K20. Histone acetylation is generally associated with the activation of gene transcription via a relaxation of the chromatin structure, whereas deacetylation has the opposite effect. Methylation on the other hand can be associated both with transcriptional activation (H3K4 methylation) or repression (H3K9 or H3K27 for example) with nuanced effects depending on the degree (mono-, di- or tri-) of methylation. These modifications of the charged histone "tail" that extends from the nucleosome, along with others including phosphorylation, SUMOylation and ubiquitinylation, make up the "histone code" [12]. The different combinations of histone modifications enable the storage of a large amount of information that has a central role in the regulation of transcription, or its dysregulation in pathology. The enzymes that effect these modifications are consequently privileged targets for pharmacological intervention.
histone modifying enzymes (HME)
the enzyme families involved in epigenetic modification of histones are conserved in eukaryotes and the most studied as drug targets are those catalyzing acetylation/deacetylation and methylation/demethylation. Acetylation of lysine residues in histone tails and of other proteins is carried out by a large number of proteins with histone acetyltransferase (HAT) activity of which it is possible to distinguish at least six families [13]: the GNAT superfamily (PCAF/GCN5), the MYST family, the CBP/p300 family, the nuclear receptor coactivator family, the TAFII 250 family and the GNAT-related family (TFIIIC).
HME inhibitors as drug candidates
inhibitors of epigenetic targets have been under active investigation for decades, particularly as potential cancer drugs [reviewed in 20]. Although the two HME inhibitors already approved for use, as well as 17 others currently in clinical trials [20], are all inhibitors of HDACs or sirtuins, other enzyme classes are also being targeted, such as the lysine methyltransferase EZH2 for which inhibitors have been shown to have potential in the treatment of lymphoma [21].
strategy and objectives of the SETTREND project
we adopted a holistic approach to drug discovery with schistosome HMEs as the targets. The targeting of histone acetylation and methylation (to the exclusion of other types of modification) was deliberate since they are the two crucial modifications that impact directly on gene transcription and are the subject of intense investigation, both in the context of cancer research and, increasingly, in the search for parasiticidal drugs.
the main elements in our strategy were as follows:
- The identification of the members of HDAC classes I, II and III (sirtuins) HAT, HMT and HDM encoded in the genome of S. mansoni. Protein motifs and hidden Markov models of conserved, mainly catalytic, domains were used to search the databases.
- Molecular cloning of the coding sequences of selected HMEs in order to allow production of the corresponding recombinant proteins. The biological roles of selected HMEs were investigated in order to give insights into the potential action of inhibitors.
- A reverse chemical genetics approach using generic inhibitors of HME subclasses available within the consortium in cultures of schistosome larvae was employed to identify those classes that are bona fide drug targets. This allowed the earliest indications about crucial epigenetic targets in Schistosoma.
- Validation of these enzymes as therapeutic targets individually or collectively using RNA interference (RNAi) to knockdown the transcription of the corresponding genes. Individual HMEs, chosen using the criteria of structural divergence from human orthologues and/or the phenotypic effects of inhibitors of HME classes, were validated as targets using RNAi
- Production of enzymatically active, recombinant HME proteins and structural analysis by X-ray crystallography in the presence or absence of inhibitors. HMEs selected due to their divergence (catalytic domain) from the corresponding human orthologues and/or shown to be vital for worm survival in the phenotypic screen will be produced as recombinant proteins. Selected proteins were to be crystallized for structural analysis by X-ray diffraction.
- Target based synthesis and enzyme testing. Enzyme assays were to be optimized for the recombinant S. mansoni HMEs. Target based synthesis of inhibitors was initiated with HDAC inhibitors. In vitro testing of compounds identified by virtual screening (supported by structural studies) and synthetic bioguided optimization of HDAC inhibitors and inhibitors of other targets that originate from virtual screening were also performed.
- In silico screening for potential inhibitors (HDACi, HATi, HMTi, HDMi) was carried out by docking to the modelled catalytic domains (based on the crystal structures) of the enzymes. Bioguided optimization of inhibitors was also performed.
- High-throughput screening, hit to lead optimisation, synthetic optimisation of lead structures. After initial screening of a large set of compounds for their ability to inhibit the activity of the corresponding recombinant proteins in high-throughput assays, collections of analogues were tested both in biochemical assays and phenotypic assays on the parasite. Bioguided optimization of inhibitors was performed.
- The establishment of gene expression profiles corresponding to inhibition using drug candidates in cultured larval stages (schistosomula) was carried out to enable the determination of metabolic and signalling pathways affected.
- In vivo testing of the best lead candidates was done in infected mice. In this way, during the study period we aimed to develop a series of candidate molecules that could progress to clinical trials.
project Results:
4.1.3 Main S/T Results/Foregrounds
identification and cloning of S. mansoni HMEs
the predicted proteome of S. mansoni was screened using protein motif searches and Hidden Markov Models (by beneficiary 7) and a total of 55 HMEs were identified, as well as a number of alternatively spliced products. Molecular phylogeny shows that all are orthologues of human proteins. The most abundant group are the histone methyltransferases (23 representatives) that include both lysine and arginine methyltransferases. The data generated during this search was used to update the database SchistoDB. Moreover, the corresponding sequences and gene identifications were used to devise strategies for cloning the cDNAs encoding these proteins. In addition, since the sequence data for the Schistosoma japonicum and the Schistosoma haematobium genomes became available after the start of the project, the same methods were applied to determine the complement of HMEs in the genomes of the related parasites. Although the fragmentary nature of the S. japonicum genome meant that fewer HMEs were identified, the sequences obtained allow us in particular to compare those of proteins of interest, such as HDAC8. The genome of S. haematobium was much more comparable to that of S. mansoni. Importantly, no further HMEs were detected in addition to those already identified in the S. mansoni genome. This study will be instrumental in determining whether HME inhibitors can be developed that will be active against both species and in this context it should be noted that the catalytic domain of S. japonicum HDAC8 possesses the same modifications in relation to the human orthologue as does SmHDAC8. This indicates that inhibitor specificity should be similar for the two helminth enzymes and hence that broadband anti-schistosomal drugs could be envisaged.
schistosome HMEs as drug targets: phenotypic screening and RNAi
A reverse chemical genetics approach was used in order to identify HMEs or HME classes that are potential therapeutic targets. For this we used an in vitro assay in which schistosomula larvae or adult worms were incubated in the presence of different concentrations of HME inhibitors. For schistosomula, mortality was assessed by appearance (dead larvae are opaque) and motility and the induction of apoptosis was measured using the TUNEL assay, and in some cases the measurement of caspase 3/7 activity and Annexin V staining. For adult worms, the separation of worm pairs and in vitro egg laying were measured and in some cases worm morphology, particularly of the genital apparatus, was examined.
A focused library of HME inhibitors (including 21 novel compounds supplied by beneficiary 6) of all classes of HME (class I and II histone deacetylases, sirtuins, histone acetyltransferases, histone methyltransferases (protein arginine methyltransferases) and histone demethylases) were tested by beneficiary 1. All induced TUNEL positivity and larval death to varying degrees in the assay. One (LH 19N, a PRMT inhibitor) showed a very high level of activity (85% TUNEL positivity) at a concentration of only 5μM, whereas CS13, a sirtuin inhibitor, gave only 14% positivity at 100μM. Results of these assays showed notably that inhibitors of HDACs and in particular potential inhibitors of SmHDAC8 (e.g. Pe 11/5) efficiently induce parasite death via apoptosis.
gene expression signatures
another means of determining the roles of HME in the parasite is to identify with microarrays the Gene Expression Signatures (GES) of S. mansoni treated with candidate HME inhibitors in order to elucidate the downstream molecular pathways engaged to mediate their effects. Work was carried out by beneficiary 10 with the pan-HDAC inhibitor TSA with the aim of identifying such pathways and gene networks that might reveal new therapeutic targets.
total RNA was extracted from schistosomula (LE strain) treated for 12, 24, 48 and 72 hours either with 1 μM of Trichostatin A (TSA) or with ethanol (vehicle of the drug) as a control. The integrity of RNA samples was evaluated using microfluidic electrophoresis in the Bioanalyser equipment (Agilent Technologies). The 4x180k oligoarray platform used in this work was previously designed by beneficiary 10, which has single-strand oligonucleotide probes representing essentially all known S. mansoni transcripts [29], and it was extensively re-annotated according to the newly available S. mansoni genome sequence [30]. Each sample (biological and technical replicates) was labeled with either Cy3 or Cy5 and hybridized to the oligoarrays. For data analysis a Log2 ratio was calculated between gene intensities of TSA Treated/Control, and Significance Analysis of Microarray (SAM) was used as the statistical test to identify significantly (p-value = 0.05) differentially expressed genes. For further functional analysis, only significantly affected genes with a minimal fold change cut-off of 2 were used, allowing for a more stringent analysis of expression change. Ingenuity Pathway Analysis software (IPA) was used to identify significantly enriched gene networks among the differentially expressed S. mansoni genes.
in order to identify the gene expression signature of schistosomula treated with TSA, microarray experiments were performed in three biological replicates (each one with a technical replicate). Gene expression analyses show a set of genes regulated by inhibition of histone deacetylases, either induced or repressed by the effect of TSA over time.
recombinant protein production and X-ray crystallography
while active inhibitors can be discovered using phenotypic screening, for a rational optimization of drugs towards parasite selectivity it was vital to be able to produce recombinant HME proteins to allow structural analyses, enzyme assay development, comparative studies with human enzymes and high-throughput inhibitor screening. The first target chosen was SmHDAC8, based on its dissimilarity to and phylogenetic distance from its human orthologue and initial results from the phenotypic assay showing that inhibitors of human HDAC8 induced apoptosis and death of schistosomula in culture.
the cDNA encoding SmHDAC8 was cloned into E. coli expression vectors by beneficiary 2 and although initially the expression of soluble protein was difficult to obtain, subsequent efforts were highly successful and high yields (1.5 mg per litre of culture) of pure protein were obtained. The enzyme has been supplied (almost gram quantities throughout the duration of the project) for high-throughput inhibitor screens, tests of its catalytic activity and used for the generation of specific antibodies, as well as being crystallized for structural studies (see below).
of the 58 HMEs identified by genome data-mining, many were eliminated as candidates for drug screening since they were too conserved (e.g. HDAC1 and HDAC3), too large to permit efficient protein production (a number of the lysine methyltransferases) or enzymatically active only when in complexes with partner proteins. Eight cDNA clones encoding HMEs (Sirtuins 1, 2 and 6, PRMT 1 and 4, the HATs Tip60 and GCN5 and the HMT SET8) were cloned into expression vectors and produced in E. coli. Only two (Sirt2 and Sirt6) could be expressed as soluble proteins in sufficient quantity and although the clones producing insoluble proteins were also tested in the baculovirus expression system, this has so far proved unsuccessful. An initial protocol was set up which yielded large amounts of these proteins (more than 5 mgs per litre of culture). The protocol was further modified to prevent degradation of the purified proteins, notably in the case of SmSirt2. During the project, tens of milligrams of SmSirt2 and smSirt6 have been provided to beneficiaries 6 and 9 for their use in inhibitor assays and high-throughput screening, respectively. Sirt6 has not shown activity in standard sirtuin assay systems up to now. The setup of the purification of SmSirt2 has been carried out successfully and the target has undergone inhibitor screening. However, all attempts to obtain well-diffracting crystals of these two sirtuins have failed so far.
as the major initial target of inhibitor screening in the project, the crystallization of the SmHDAC8 protein was initiated by beneficiary 2 as a priority. Crystals were successfully obtained and the structure of the apo form (the first time for an HDAC8) was solved at 1.8 Å resolution. Interestingly, comparison of this structure to the inhibitor-bound structure of human (h) HDAC8 revealed only few gross structural changes between both proteins. Major changes concern the different insertions that are observed at the sequence level in the SmHDAC8 enzyme. Structural comparison shows that these insertions form loops at the surface of the protein, possibly providing S. mansoni specific protein/protein interaction surfaces.
compound screening and molecular modelling
the collection of a focused library of about 300 HME inhibitors led to the supply of subsets for phenotypic screening (see above) and in vitro screening assays were successfully developed for the two HMEs so far produced as recombinant proteins, SmHDAC8 and SmSirt2. The reference inhibitors of SmHDAC8 were further supplied for the phenotypic assay. These have been used to screen the candidates from the focused library, and also from the virtual screening strategy.
assay development
the development of biochemical assays for the targeted HMEs was necessary for the filters downstream of virtual screening. Furthermore, acquired knowledge was shared with beneficiary 9 who also carried out assay development for high-throughput screening. As SmHDAC8 was chosen as the initial target, assays were first developed for this parasite deacetylase subtype. Besides this, an in vitro assay for sirtuin 2 from S. mansoni (SmSirt2) was also set up.
it was already known that small acetylated substrates are not well accepted by human HDAC8 (hHDAC8), which we found out to be true for smHDAC8 as well. Thus, the well-established deacetylase assay that was developed by beneficiary 6 was adapted by using a small peptidic substrate with a trifluoroacetyl moiety. In cooperation with beneficiary 9, also a tripeptidic substrate carrying a trifluoracetyl group was used in the same assay. It could be shown that both substrates were converted by SmHDAC8 to a comparable degree. Therefore, our small substrate was used for further testing.
being aware of the fact that using a trifluoroacetyl group instead of the endogenously converted acetyl moiety might lead to false negatives or the discovery of false positives, a commercially available assay setup was adapted that uses an acetylated substrate derived from a sequence of the deacetylase substrate p53.
most of the compounds tested in both in vitro assay systems (trifluoroacetyl substrate and acetylated peptide) led to comparable results, but in some cases the resulting potency of the tested compounds depended on the assay (and substrate) that was used. Hence, it was decided that the use of one assay system would be sufficient for pre-screening of library compounds. Identified hits, however, were then countertested in the other assay setup. The influence of pH on activity (around 6-7) and also mutants provided by beneficiary 2 that highlight the importance of a cysteine near the catalytic site were also tested.
screening for inhibitors of SmHDAC8
virtual screening of inhibitors for SmHDAC8 was initially done by beneficiary 3 using 3D structural models based on known structures of human orthologues and used for docking of known HDAC inhibitors. This pointed out the similarity of the residues of the active site between the human and schistosome HDAC8 enzymes and suggested a similar interaction pattern with inhibitors, but also indicated a notable difference at the rim of the active site (Met/His substitution). The in silico screen for inhibitors was refined with the contribution of the 3D structure of SmHDAC8 determined by X-ray crystallography. A virtual database of ~2 million compounds was focused on compounds with zinc-chelating groups. Of 4300 compounds with such moieties, 3209 were docked into the SmHDAC8 active site. About 150 compounds selected for stability and availability were tested as inhibitors in the fluorescence-based assays. We identified as novel smHDAC8 inhibitor the internal hydroxamate J1036 from the Ibscreen compound library. This compound and analogues represent a class of compounds with a scaffold not previously described for HDAC inhibitors. Similarity-based screening and docking identified ten further analogues that were sent to beneficiary 6 for phenotypic screening. For J1036 beneficiary 2 obtained a low-resolution X-ray structure with SmHDAC8. Two compounds, J1036 and J1060 were identified as the most potent SmHDAC8 inhibitors from this series (IC50 values around 5 μM). The similarity-based screening identified also J1075 as an SmHDAC8 inhibitor active in the low micromolar range. Biological characterization was carried out and a crystal structure in complex with SmHDAC8 was obtained by beneficiary 2 that confirmed the docking results.
screening for inhibitors of SmSirt2
although it proved impossible to produce well-diffracting crystals of SmSirt2, a homology model was generated using the MODELLER approach and analyzed by means of molecular dynamics simulations using the AMBER software. The SmSirt2 model was compared with the human Sirt2 X-ray structure and was used for docking studies of in house sirtuin inhibitors. Superposition of SmSirt2 with hSirt2-inhibitor complex reveals that the residues in the active site are similar and therefore will have similar interaction pattern with inhibitor/substrate. However a notable difference was found at the peptide substrate binding site in which I169, I232, F234, and L239 in human Sirt2 are mutated into V132, V193, L195, and M200 in SmSirt2.
high-throughput inhibitor library screening
SmHDAC8
recombinant SmHDAC8 was first used by beneficiary 9 to develop a biochemical assay suitable for high-throughput screening (HTS). A carefully designed set of 80,000 diverse compounds was implemented, eliminating promiscuous compounds and plated. After screening, counterassays were performed to filter out false positives. The 66 compounds obtained in this screen were subjected to quality control and then clustered according to their structures, resulting in 9 hit series and 21 singletons. Three of the hit series and 3 singletons were then selected for analogue searches, resulting in the identification of 122 analogous compounds. Dose response testing allowed us to concentrate on 2 series that are most promising in terms of potency, but with a large spread in activity.
two novel SmHDAC8 lead series originating from the HTS campaign have been generated in the course of the project. The main series consists of greater than100 novel compounds with potencies in the biochemical assay ranging from 43 nM to inactive. The second (backup) series consists of 19 novel compounds with potencies in the biochemical assay ranging from 0.8 to 12 μM.
the main SmHDAC8 inhibitor series: Considerable efforts have been invested in establishing chemical routes for the synthesis of different types of these compounds. About 75 compounds have been synthesized and tested in the biochemical assay leading to the establishment of a clear structure/activity relation (SAR). The activities of five selected compounds were confirmed by beneficiary 6 using two alternative substrates. Testing of compounds against human HDAC8 has been performed and showed that there is no selectivity of the tested compounds against human HDAC8. Ligand-detected NMR binding experiments (transverse relaxation filtered experiments with equimolar amounts of protein and compound) have confirmed direct binding of representative compounds to SmHDAC8. Information on the binding mode of the main series of compounds was first obtained from a modelling/docking study (beneficiary 3) and later from the crystal structure determination of two highly potent compounds in complex with SmHDAC8 (beneficiary 2), including one of the selected lead compounds. The crystal structures have allowed a deeper understanding of the SAR and provided hypotheses on how to obtain selectivity against human HDAC8. Compounds from the main inhibitor series have been tested in the phenotypic assay at different times during the project. The first set of main series compounds tested did not show any significant effect on the schistosomula, only a compound from the backup series clearly induced apoptosis in the schistosome larvae. Since the main series compounds were potent SmHDAC8 inhibitors we hypothesized that the compounds did not pass the double tegument membrane of the larvae. Following this, synthesized compounds were tested in a membrane affinity assay in order to direct the chemistry towards compounds with higher membrane affinity and thus higher passive diffusion over a membrane. A second batch of compounds in the main series with higher membrane affinities did indeed induce apoptosis in the schistosome larvae. In order to further improve the permeability of selected lead compounds, a pro-drug strategy has been employed.
the backup SmHDAC8 inhibitor series: The chemical route for synthesis has been worked out and 19 novel compounds have been synthesized and quality controlled. The compounds have been tested in dose-response in the biochemical assay against both SmHDAC8 and hHDAC8. An early SAR is emerging but there is no clear selectivity against human HDAC8. Two compounds from this series has been tested in the in vitro schistosomula assay by beneficiary 1, both compounds showing toxicity against the schistosomula at both 5 and 20 μM compound concentration. Further, one of the two tested compounds also showed effects in the adult worm pairing assay.
three potent compounds (denoted compounds 1, 2 and 3 below) from the main inhibitor series were selected as leads; their IC50s in the biochemical assay were respectively 43, 45 and 350 nM. Most efforts have been invested in the two first compounds that are quite similar and the most potent in the biochemical assay. The crystal structure of SmHDAC8 in complex with the most potent compound shows a perfect fit of the lead compound in the active site and the optimization of the lead compounds have been focused on increasing their permeability.
the three lead compounds have been subjected to a pro-drug strategy in order to further improve and optimize the permeability of the compounds. The strategy takes advantage of the fact that the concentration of reducing agent (GSH) inside the worm (or cell) is on the order of 1,000 times higher compared to the outside. The pro-drugs are designed so that they stay intact outside of the worm (cell) and upon entering the worm (cell) are reduced to the active compound. A large variety of different pro-drugs have been synthesized starting from the lead compounds. The synthesized compounds have been subjected to QC analyses and tested in the biochemical assay in presence and absence of reducing agent to mimic the situation inside and outside the worm (cell), respectively. Also, a smaller number of pro-drugs have been synthesized starting from a few other parent compounds and tested in the biochemical assay and cell assays.
before sending compounds to be tested in the in vitro phenotypic assays, as a first filter a much larger number of SmHDAC8 inhibitors have been tested in a 48 hour cell toxicity assay. This has been done in order to test if the prodrug strategy is working on cells and thus to select compounds for the phenotypic assays with a higher probability to be active against the schistosomes. A high-throughput cellular assay in 384-well format has been developed which, apart from testing of compounds on cell lines, also allows the testing of compounds on primary cells due to the small volumes needed. Effects of active SmHDAC8 inhibitors have been tested in dose-response (11 concentrations from 20 μM down to 20 nM, in duplicate) on the standard HepG2 cell line as well as primary fibroblasts and a few cancer cell lines (osteosarcoma, neuroblastoma, pancreatic cancer). The lead compounds show low toxicity while most of the pro-drugs based on the lead compounds show a much higher cell toxicity demonstrating that the prodrug is indeed cleaved intracellularly and that the prodrug strategy is working. Further, the prodrug versions of the lead compounds are more toxic against the cancer cell lines (several compounds with low μM IC50s) compared to the primary fibroblasts and the HepG2 cell line (no IC50 values, i.e. IC50 greater than 20 μM).
ten compounds that showed a good effect in the cell assays were selected for the last round of testing in the in vitro phenotypic assays. The selected compounds were five prodrugs based on the lead compound 1, two prodrugs based on the lead compound 2, one prodrug based on lead compound 3, and two compounds from the backup series. All ten compounds induced mortality of schistosomula at 20 μM compound concentration, and a few of them a clear effect also at 5 μM. Further, an adult worm pairing assay was performed on the ten compounds. Four of them showed a clear effect by inducing separation of worm pairs at 20 μM compound concentration in this assay.
SmSirt2
recombinant SmSirt2 was supplied by beneficiary 2. A biochemical assay with fluorescence read-out suitable for high-throughput screening (HTS) of SmSirt2 was successfully developed and formatted by beneficiary 9. During the assay development several different substrates were tested, among them a substrate supplied in small amounts by beneficiary 6. This latter substrate was selected for the HTS and was synthesized in large amounts at iNovacia. In order to increase the sensitivity of the assay with respect to protein consumption, an extensive buffer optimisation was performed resulting in an assay that required reasonable amounts of protein and thus making an HTS possible to perform.
the HTS of a carefully designed screening set consisting of ~80,000 diverse compounds was performed at 10 μM compound concentration. 875 positives (hit rate 1.08%) were identified in the primary SmSirt2 HTS. 27 positives that have been identified as hits in 4 or more previous HTS campaigns were removed from the hit list, and dose-response curves (11 concentrations from 100 μM and down to 1.5 nM, in duplicate) were performed for 848 compounds. Dose-dependent SmSirt2 inhibitory activity was confirmed for 764 compounds. Of these 9 compounds showed an IC50 less than 0.1 μM; 111 compounds showed an IC50 less than 1 μM and 646 compounds showed an IC50 less than 10 μM. Several counterassays were employed to filter out compounds active in the biochemical assay by non-desired mechanisms. These assays included detection of compounds that interfere with the fluorescence readout (quenching the substrate fluorescence) and detection of compounds that are redox-active. Further, the activities of selected hits were determined in the biochemical assay also in the absence of buffer components such as reducing agent and detergent. The hits were subjected to quality control (QC, checked for identity, purity, chemical stability and solubility in buffer). The confirmed hits were then clustered with respect to their structures and the resulting 165 hit series and 159 singletons were evaluated by the beneficiary 9 medicinal chemistry group. The evaluation aimed at identifying the most promising hit series and singletons from a medicinal chemistry perspective (e.g. lead likeness, synthetic and library expansion tractability, emerging structure-activity relationship (SAR) and IP space). Six hit series and four singletons were given the highest prioritization and a further six series and nine singletons were selected as backups.
the six inhibitor series and four singletons with the highest prioritization were selected for analogue searching. 551 analogous compounds were identified and tested in dose-response followed by counterassays and compound QC. The analogue testing further increased the number of active compounds in the highest prioritized inhibitor series and six of these series contain a large number of active compounds. In addition to these ten series there are 159 series and 155 singletons containing active compounds that have been given a lower priority. The abundance of active compounds that have passed all counterassays and the emerging SAR in the highest prioritized inhibitor series constitutes a very good foundation for the further development of SmSirt2 inhibitors.
conclusions
the aim of the SETTREND project was to develop new drugs for use in treating schistosomiasis, the second most important parasitic disease after malaria and involves steps to bring drugs to the pre-clinical testing stage, determining their activity in vitro and in vivo in infected mice. The rationale for the project is the existence of only one effective drug against schistosomes, praziquantel, and the increasing likelihood of the development of strains of parasite resistant to this drug, given its intensive use, particularly in the context of the Schistosomiasis Control Initiative (see http://www3.imperial.ac.uk/schisto online). We have attempted to fulfil this aim using the most comprehensive strategy yet assembled to resolve such a problem. We have provided a number of lead compounds for development as drugs (or at least valuable tool compounds for cellular and animal studies) that could either proceed to clinical trials or be further optimized. Our strategy, combining high-throughput and rational approaches has proved its worth and novel compounds with potent activity against the parasite in vitro and in vivo have been generated by both. Therefore, the proof of concept of the strategy has already been delivered. Moreover, the comparison of the HME targets from S. mansoni and the related species Schistosoma japonicum, added to the project, shows that, in the case of schistosome HDAC8, drugs developed against the enzyme from the former species are likely to be active against the latter. As we stated in our submission, we do not consider that clinical trials (mentioned as a possible aim of the original call) represent a reasonable aim within the project duration. However, the development of optimized lead compounds will lay the foundations for such trials.
summary of main scientific achievements
- The identification of the HMEs encoded in the schistosome genomes. Molecular cloning of the coding sequences of selected HMEs and the investigation of their expression during the parasite life-cycle.
- The elucidation of gene expression signatures of HDAC inhibition in parasite larvae, leading to the identification of key pathways and proteins.
- The implementation of a reverse chemical genetics strategy showing that inhibitors of all HME families induced apoptosis and death of schistosome larvae in culture. This validated both the SETTREND approach and the targeted enzymes.
- The validation of an HDAC (SmHDAC8) and two other HMEs (SmPRMT3 and SmKDM1) as therapeutic targets by RNAi. This showed that single HMEs (and not just whole families) can be therapeutic targets and validated our target-based approach to develop selective inhibitors for individual HMEs.
- The production of SmHDAC8 as an enzymatically active recombinant protein and the solution of the structures of the apo-form of the protein, the protein bound to pan-HDAC inhibitors and to selective inhibitors generated during the project. Two other recombinant HME proteins, SmSirt2 and SmSirt6 were also produced.
- In silico screening of SmHDAC8 based on the solved crystal structure identified compounds with novel scaffolds and warheads with greater selectivity than reference inhibitors and that provoked apoptosis and death of schistosomula in culture. Treatment of schistosome-infected mice with one of these inhibitors, J1075, led to reduced worm burdens and egg loads.
- High-throughput inhibitor library screening of SmHDAC8 and bioguided synthetic chemistry optimization produced lead inhibitors that were extremely active against both schistosome larvae and adult worms in culture. High-throughput inhibitor screening was also carried out for SmSirt2.
- These results justify our strategy and suggests that further lead optimization will generate highly selective and potent compounds that could provide the basis for clinical trials.
Overall, the objectives set out for the project have been achieved with minor exceptions. Potential pitfalls in our strategy have been avoided and problems solved. No major changes in strategy have been necessary during the project. The main aim, to develop a series of candidate molecules with a future potential for clinical trials, has been achieved.
references
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[23] Dubois F, Caby S, Oger F et al. Histone deacetylase inhibitors induce apoptosis, histone hyperacetylation and up-regulation of gene transcription in Schistosoma mansoni. Mol Biochem Parasitol 2009; 168: 7-15.
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potential Impact:
4.1.4 Potential Impact
the impact of the work done is assessed in terms of the criteria set out in the original project application.
strategic Impact of the SETTREND project
development of new drugs for treating schistosomiasis
the strategy for drug development used in the SETTREND project was the most comprehensive yet assembled in anti-parasitic drug discovery. The combination of both in silico and high-throughput screening, both rigorously target-based, allowed the identification of a large number of inhibitors of HMEs, particularly of HDACs.
proof of concept was provided early on in the project with:
- Phenotypic screening showing that HME inhibitors were capable of inducing apoptosis and mortality in schistosomes.
- The validation of SmHDAC8 as a therapeutic target, the production of the active recombinant enzyme, structural studies and initial screening results identifying potent inhibitors
subsequent and parallel work showed that the approach could represent a new paradigm for drug discovery for parasitic diseases in general, but also for other pathologies.
- Optimized lead compounds that are nM range inhibitors of SmHDAC8 have been produced from both in silico and high-throughput screens that are also highly toxic for schistosomes in culture. In one case the compound was also effective in vivo on schistosome-infected mice.
- Potent inhibitors of another HME, SmSirt2 have also been produced by both screening methods.
- The screening approaches merge in the phase of lead optimization with the docking of compounds into the solved crystal structure of the target enzyme or after co-crystallization with the enzyme.
- Testing of compounds in phenotypic assays to judge the effects of compounds on the parasite is applied at each step of the hit to optimized lead process.
the candidate drugs effective against S. mansoni are likely to be effective against the two other main species of schistosome infecting humans Phylogenetic and structural studies confirm that candidate drugs effective against S. mansoni are very likely to be effective against the other two main species of schistosome infecting humans. For example, the catalytic site differences of SmHDAC8 compared to its human orthologue are conserved in both S. haematobium and S. japonicum HDAC8. This is very probably the case for other HMEs.
the candidate drugs developed against schistosomes will serve as lead compounds for the treatment of other helminth diseases. The lack of effective treatments for hydatid disease caused by Echinococcus granulosus (cystic hydatid disease) and E. multilocularis (alveolar hydatid disease) suggests that the exploration of the possible application of inhibitors developed against S. mansoni HMEs should be explored. As a first step the exploration of the HME content and orthologies in the E. multilocularis genome will be possible once the reference quality genome has been produced (see http://www.sanger.ac.uk/resources/downloads/helminths/echinococcus-multilocularis.html online).
candidate drugs developed against schistosomes could provide novel lead compounds for cancer therapy. Our strategy for drug development targeting histone modifying enzymes was directly inspired by emerging approaches to develop treatments for cancer. Moreover, two HDAC inhibitors are currently approved for the treatment of cutaneous T-cell lymphoma and many others are in clinical trials. Beneficiary 9 has tested compounds developed from hits obtained for SmHDAC8 on cancer cell lines and shown that some are highly toxic with low μM IC50 values. It is clear that a number of the novel inhibitor scaffolds identified by screening the schistosome HMEs will prove of interest as leads for the development of anti-cancer drugs.
european dimension of the project
the project required a combination of expertise and capacity not available at the national level in any of the beneficiary countries, whether in Europe or Brazil. Moreover, the project required the participation of beneficiaries working on aspects of parasite molecular biology and with specific expertise and access to material not available in Europe, but only in Brazil, a country endemic for schistosomiasis. Funding for collaborations between Europe and Brazil is not available on the necessary scale via any other mechanism. The financial support of the EC is essential.
one substantial impact of the project has been to develop and encourage scientific contacts between the Brazilian and European partners and between the different European partners. This has been reflected in the exchanges of students between these partners.
this collaboration has kept Europe at the forefront of a domain (the search for drugs against parasitic diseases) that is highly competitive, with a strong representation in the USA. The model character of our approach could be the seeding point for other similar campaigns aimed at other infectious diseases. This is in fact already the case, since the SETTREND project has formed the basis of a new project, A-PARADDISE, submitted under FP7, which will apply the same strategy to drug development against schistosomes and three protozoan parasites.
the SETTREND project has been the starting point to move toward the production of solid industrial standard-based candidate drugs that could be taken over by a larger European pharmaceutical company. Thus, on top of the humanitarian objective, the economics will drive a larger interest into these diseases by the medium and large European companies,
european competitiveness
impact on industrial activity within the project. The consortium included an industrial participant, the SME iNovacia (beneficiary 9), as well as a company involved in intellectual property management and dissemination (beneficiary 8). The project undoubtedly contributed to their credentials in the domain of drug development, although these are already well-established, and involvement in this collaborative project will have increased awareness of their expertise, within and outside Europe. Moreover, the SME iNovacia was a full partner in the project and had a major influence on the direction taken by the research. More specifically, for the SME in the project, working within the Life Science sector in a comparatively small country like Sweden, it is important to reach out and extend the customer base. Participation in an FP7 project could provide new contacts throughout Europe that may lead to business opportunities. However, this is an objective, not an achieved impact. Similarly, an impact on the biotech industry by a bottom-up approach to innovation and funding has not yet had any tangible effects, and the progression towards an industrial exploitation of the compounds developed during the SETTREND project via partnerships with pharmaceutical industry remains a future aim.
innovation impact
the scientific impact of the project within parasitology research has been considerable, both in terms of innovation and dissemination (see below). The major innovation impacts predicted in the submitted project have been achieved:
- The identification and characterization of schistosome HMEs has been completed. The improved annotation is available in SchistoDB and a manuscript describing the main conclusions is in preparation (beneficiary 7), in particular concerning the phylogenetic relationships of schistosome HMEs to their orthologues in other species and the implications for the evolution of these enzyme families in invertebrates. Very divergent HMEs were not found in S. mansoni or the two other schistosome species investigated, demonstrating the overall conservation of both these enzymes and the epigenetic marks they generate. In addition, a study of the schistosome histones was carried out making use of the newly available RNASeq data newly available (beneficiary 10) and which allowed the correction of some of the genomic assemblies and annotations in the databases. Moreover, it allowed the confirmation of the structures of schistosome histones and the conservation of the sites of epigenetic marks. The more detailed characterization of the functional properties of HMEs demonstrated the role of HATs in the transcriptional control of a gene encoding a major eggshell protein, confirming HAT inhibition as a therapeutic goal (beneficiary 4). Furthermore, the main HME target studied during the project, SmHDAC8, was found to interact with proteins involved in cell division (beneficiary 1). This underlines that many HMEs, in addition to functions in transcriptional control, are also involved in other processes and have substrates other than histones.
- Comparative structural studies were carried out between SmHDAC8 and its human orthologue (beneficiary 2). These studies (manuscript submitted for publication) were central to the demonstration of differences in catalytic pocket architecture that can be exploited to identify and synthesize inhibitors that are selective for the schistosome enzyme. The studies allowed precise modelling of inhibitor-enzyme interactions (beneficiary 3) and the identification of inhibitors that exploited the specific characteristics of the SmHDAC8 catalytic site. Although we focused on the crystal structure of SmHDAC8 and small molecule interactions, modelling studies of SmSirt2, for which it proved impossible so far to obtain crystals, also flagged up catalytic pocket differences with respect to the human orthologue which will again allow the identification of selective inhibitors.
- Gene expression signatures. The studies of gene expression signatures did not allow the definition of drug specificity via the comparison of expression profiles of parasites treated with a drug or knocked down for the transcription of the HME drug target by RNAi (for reasons elaborated in the second periodic report). However, the profiles generated by HDAC inhibition and the analysis of the pathways and interaction networks of the genes for which expression was modulated (beneficiary 10) have already allowed the identification of the lysine methyltransferase EZH2 as a therapeutic target, which was confirmed using a specific inhibitor.
- New HME inhibitors available for functional studies. In addition to the focused inhibitor library generated by beneficiary 6, which supplied inhibitors already used in functional studies of HATs and sirtuins, the project has generated several hundred new compounds that are hits from high-throughput and in silico screening of SmHDAC8 and SmSirt2, analogues or optimized leads. Many of these have novel scaffolds or warheads for HDAC or sirtuin inhibitors and promise to be extremely useful, not only for functional studies in schistosomes, but more particularly as lead compounds for other human pathologies. Moreover, these novel structures are candidates for IP protection (see below).
societal impact
schistosomiasis remains a neglected tropical disease (in terms of the financial resources devoted to its prevention and treatment) and is a leading cause of chronic disability and poverty, in addition to the direct mortality that can be attributed to it in the 75 endemic tropical and sub-tropical countries. The ultimate aim of the work contained in this project, to contribute to reducing the impact of schistosomiasis on the affected populations, will have a dual impact on both health and living standards. This future impact is predicated on the development of an effective drug that is relatively cheap to produce. The results and foreground generated by the SETTREND project show this outcome is attainable based on the strategy that we have employed.
training and technology transfer impact
direct training activities during the project concerned PhD students from beneficiaries 1, 4 and 7 who underwent training in beneficiary laboratories (1, 2, and 6) for recombinant protein production, enzyme inhibition assays, gene transcription techniques or RNAi (in a laboratory outside the consortium, expert in the domain).
in addition to these activities, technology transfer between beneficiaries has concerned:
- Exchanges and discussions between beneficiaries 1 and 7 concerning the invalidation of HME gene transcription by RNAi and the phenotypic screen using HME inhibitors on parasite larvae in vitro.
- Transfer of technology concerning the functional characterization of schistosome HMEs between beneficiaries 1 and 4, specifically the chromatin immunoprecipitation methodology.
- Development and standardisation of enzyme assay methods between beneficiaries 2, 6 and 9. This has specifically concerned the assays used for SmHDAC8 and SmSirt2.
- Molecular modelling and virtual screening approaches between beneficiaries 2 and 3.
plan for the use and dissemination of foreground
knowledge generated by the project has been made available to the public respecting the general rules laid down in the Consortium Agreement and according to the European Commission guidelines. Participants planning dissemination informed the consortium via e-mail and by posting the proposed communication or article on the consortium website (consortium intranet section) in order to obtain approval, or to allow for objections if it was felt that certain data required protection before dissemination. In practice, however, this latter proviso did not prove necessary.
dissemination to the general public and media
the aims and strategies of the project were disseminated in seven press releases or articles published in the popular press, as well as in the project brochure, which was widely distributed.
publications and communications
to date the project results have been published in nine full journal articles. This number is destined to rise significantly in the coming months. One reason for this is that the publication of a certain number of articles in preparation will be subsequent to that of the major article concerning the structural characterization of SmHDAC8 and the characterization of selective inhibitors, which will be submitted after finalization of the reporting procedure.
A large number of presentations (29) were given by project beneficiaries in international conferences and workshops. These included international conferences on parasitology, specifically on schistosomiasis or on epigenetics. In addition to these conferences, 19 communications and posters were also presented at such meetings. A further seven presentations were given to more select (single country) audiences.
databases
the results of the project concerning the identification of HMEs have permitted the improved annotation of the corresponding genes in SchistoDB (see http://schistodb.net/schisto/ online) which is part of the EuPathDB project (see http://eupathdb.org/eupathdb/ online). Furthermore, cloned cDNA copies of schistosome HMEs have led to the correction of some assemblies and the elucidation of splicing isoforms. Similarly, the results of RNASeq studies have led to the correction of histone gene predictions in SchistoDB. Fully characterized transcript sequences have also been submitted to the NCBI database (see http://www.ncbi.nlm.nih.gov/ online)
consortium website
the SETTREND website (see section 4.1.5) as well as being used for consortium business (posting proposed articles and abstracts for approval by consortium members) also posted news of activities of consortium members, in addition to basic information about the project.
exploitation of foreground
the plan is to develop the current foreground to pharmaceutical agents for treatment of schistosomiasis. Most of the compounds under consideration were identified first as inhibitors of SmHDAC8. Further research is necessary to optimize these compounds, primarily medicinal chemistry work and iterative testing of compounds in several different assays such as the primary biochemical assay, the in vitro assays on schistosome larvae and adult worms, ADMET assays, and finally schistosome-infected mice. Optimized compounds or candidate drugs are possible to exploit once such work has been done. Novel scaffolds and warheads of HDAC inhibitors were identified that are potentially patentable. However, further research by the SETTREND consortium members is necessary to validate the in vivo activity of a number of these. Moreover, some of the inhibitors still have to be tested on human enzymes.
some compounds have been tested against human HDACs and show activity. A number have also been tested on human cancer cell lines and show toxicity. These compounds could therefore potentially be developed into pharmaceutical agents for treatment of human cancers and other human diseases.
this is also true for the sirtuin inhibitors that it may be possible to develop some of them into pharmaceutical agents for treatment of human cancers, neurodegenerative diseases and other human diseases.
in addition to the novel HDAC8 inhibitors active on human and schistosome enzymes, the consortium has identified potential drugs that interfere with parasite sexual development.
this foreground can be exploited with further research as follows:
1. Cloning S. mansoni HMEs in mammalian vectors to study the molecular mechanisms involved in the action of these HMEs in parasite biology.
2. Screening new drugs to interfere with any of the signaling pathways identified by transcriptomic studies by beneficiary 10.
3. Testing the drugs in cultures of larval and adult S. mansoni parasites.
4. Testing the drugs in vivo in schistosome-infected mice.
in addition, the exploitation of some methodologies developed by project beneficiaries, which cannot be specified here, is under study.
list of Websites:
http://SETTREND.cebio.org/