The proposed work plan leverages strategies, methodologies and experience gained during the previous project and exploits the available pool of HME inhibitors in an integrated approach. We will carry out phenotypic screens of Leishmania sp., T. cruzi and P. falciparum with these inhibitors, as well as performing high throughput and structure based screens on recombinant HMEs from these parasites. Moreover, inhibitors developed notably against P. falciparum and targeting PfHDAC1 (participants 11, 12) will also be tested against the other parasites. Lead to candidate development will be continued for the S. mansoni HME inhibitors. These approaches will be complemented by validation of the enzymes targeted in the different parasites, functional studies of the Leishmania and T. cruzi HME and transcriptomic determination of target selectivity. The workpackages (WP) of the programme and the participants involved (participant numbers in parentheses) in each are as follows:
- WP 1: Management and coordination. In order to achieve a high integration level and a timely management of the consortium, the coordinator (1) will be assisted by a project manager at Inserm Transfert (8). The tasks will include: contractual and financial management, knowledge and information management, periodic reporting, and support to meeting organisation.
- WP2: Identification of kinetoplastid HME and molecular cloning. The availability of assembled genome sequences for four Leishmania species and for T. cruzi, will enable the bioinformatics platform (7a) to identify all the HMEs of interest for the project. HDACs, HATs, HMTs and HDMs will be identified using protein motif searches and the creation of hidden Markov models of conserved (mainly catalytic) domains for mining the database. Sequences thus identified will allow the cloning of the corresponding full-length cDNA copies (7b and 15) and will also permit updating of the corresponding genome DB (SchistoDB, EuPathDB).
- WP3: Functional characterization and target validation. Available methods to generate null mutants for HME genes in T. cruzi will be carried out by participant 7b. Similar methods are not routinely applicable in Leishmania parasites, but generic inhibitors will be used to block HME function. Functional characterization of HME targets in kinetoplastids (7b and 15) and S. mansoni (4) will be carried out making use of inhibitors and transcriptome studies with the high-throughput RNASeq platform available in Sao Paolo (5) in order to define the signalling and metabolic pathways affected.
- WP4: Phenotypic screening using HME inhibitors. Platforms available in Lille (1) and Belo Horizonte (7a) will be used to screen the studied parasites (apoptosis and mortality phenotypes) with HME (with a major focus on HDAC) inhibitors derived from previous screening of S. mansoni (3, 6, 9) and P. falciparum (11, 12) HME, as well as inhibitors developed in participant laboratories (6, 13, 14). These platforms will also be used for testing the compounds derived from in silico screening and HTS to allow selection of the most promising inhibitors for lead optimization.
- WP5: Production of recombinant HME proteins, structural studies. The protein production and crystallography platform (2) will be used to produce recombinant HME, particularly from Leishmania and T. cruzi. Purified, enzymatically active proteins will be crystallized and their structures analysed by X-ray diffraction. We will focus here on the most promising proteins as judged by their pathophysiological relevance in conjunction with the possibility to produce high amounts of pure protein for crystallization and other use by the consortium.
- WP6: Target based synthesis, mode of action/enzyme testing. Enzyme assays will be optimized for the recombinant HME (6, 9, 11, 12) to permit inhibitor testing and screening. Target based synthesis will be carried out for HME inhibitors (6, 13, 14) both on compounds identified by virtual screening (3) or HTS (9). Testing of inhibitors on P. falciparum HME will be done by participants 11 and 12. Optimization of lead compounds to improve enzyme selectivity or bioavailability will be carried out by participants 9, 13 and 14. The main effort for S. mansoni will concern the optimization of previously identified lead compounds in terms of potency (while retaining selectivity) and pharmacokinetic properties to allow for animal testing.
- WP7: Modelling and virtual screening. Molecular modelling of HME (2,3) will allow virtual screening of inhibitors (3). These studies based either on the primary sequences and the known 3D structures of orthologues, or the 3D structures of the HME determined during the project, will allow the rational optimisation of inhibitors in order to render them specific for the parasite enzymes. In vitro testing of enzymes with compounds identified by virtual screening (supported by structural studies) will be carried out by participant 6. Bioguided optimization of HDAC inhibitors and inhibitors of other targets will also be performed (6, 9, 13, 14).
- WP8: High-throughput screening (HTS, 9). Recombinant HME will first be used to develop biochemical assays suitable for HTS. A carefully selected diversity set of 80,000 compounds will be screened followed by counterassays to filter out false positives. Following dose-response testing and compound quality control, the compounds will be clustered according to their structures, resulting in hit series and singletons of which the most promising will be selected for analogue searches and testing. Further development will be focused on compound series that are most promising in terms of potency and structure-activity relationships. Chemical synthesis for lead optimization and scale-up for in vivo testing will also be carried out, as well as in vitro ADMET (Absorption, Distribution, Metabolism, Excretion – Toxicology) assays.
- WP9: In vitro and in vivo testing, pharmacology, toxicology. In order to facilitate selection of compound families for such testing, upstream pharmacology and toxicology testing in vitro and in vivo will be carried out (10). This will allow us to weed out compound series with adverse effects on human cells or rodents. To assess potency, in vitro testing of lead compounds will be carried out on the targeted parasites (1, 7a, 7b, 11, 12, 15). In vivo studies will be carried out on lead compounds exhibiting both potent and selective in vitro activity as well as suitable ADMET and pharmacokinetic properties using mice infected with S. mansoni (1, 7a), Leishmania (10) T. cruzi (7b) or Plasmodium sp. (1, 11, 12).
- WP10: Dissemination and exploitation. The goal of this workpackage is to disseminate the knowledge created in A-PARADDISEwithin the consortium and to a broader audience (all). It will also deal with intellectual property monitoring and protection of the research results generated during the project (8).