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Awarded Amount$982,436DiseaseNTD (Chagas disease / Leishmaniasis)InterventionDrugDevelopment StageTarget ValidationCollaboration PartnersRIKEN , The University of Melbourne , McGill University , Medicines for Malaria Venture (MMV) , Drugs for Neglected Diseases initiative , Structural Genomics Consortium at University of Toronto
Introduction and Background of the Project
The parasitic diseases malaria, Chagas disease, leishmaniasis and cryptosporidiosis cause death and sickness throughout the developing world. There is a lack of drugs to treat these diseases and resistance to existing drugs is emerging. Novel drugs with novel targets are a priority. During their development in the human host these parasites adopt different forms and drugs that can target these different forms of the same parasite are also a priority. For example, a single drug that kills asexual and sexual forms of malaria parasites can both cure disease and block parasite transmission. Furthermore, targeting the same protein family in all of the parasites may be an efficient and cheap way to find effective drugs for multiple diseases.
Parasites use proteins to package and modify their DNA into chromatin and the chromatin structure controls which genes are active. Bromodomains bind to acetylated lysines in chromatin and activate underlying genes. This interaction in humans can be inhibited by drug-like molecules, affecting gene activity and killing human cancer cells. Human and parasite bromodomains differ sufficiently such that selective drugs inhibiting bromodomains in parasites but not those in humans could be envisaged. Furthermore, bromodomains are expressed in multiple life cycle stages and regulate multiple cellular processes in parasites. One bromodomain protein from malaria parasites and two from Trypanosoma have already been shown to be essential for these parasites’ survival. Consequently, members of the superfamily of bromodomains in the parasites responsible for all four diseases hold strong promise as novel anti-parasitic drug targets.
This project aims to identify at least one potent bromodomain inhibitor that kills each of the following parasites: Plasmodium falciparum (malaria), Trypanosoma cruzi (chagas disease), Leishmania donovani (leishmaniasis) and Cryptosporidium parvum (cryptosporidiosis). The project also aims to identify the parasite bromodomains these inhibitors bind to in vitro and the molecular interactions involved in their binding. For P. falciparum (malaria), we aim to confirm that the bromodomain is targeted by the inhibitor by introducing mutations in the parasite bromodomain to alter parasite susceptibility to the inhibitor.
We will screen libraries of molecules containing potential bromodomain inhibitors against living parasites to identify compounds that inhibit parasite growth. These hit compounds will then be screened for binding to purified, recombinant parasite bromodomains. The structures of bromodomains with bound inhibitors will be resolved for a subset of molecules that are potent inhibitors of parasite growth and that bind strongly to recombinant bromodomains. Using the resolved structures, we can predict whether similar inhibitors (analogues) should be more or less potent. The inhibitor-bromodomain binding will then be chemically validated by testing the analogues for parasite growth inhibition and bromodomain binding. The bromodomains will be "chemically" validated as targets of the inhibitors if the observations confirm our predictions. We will also choose a P. falciparum bromodomain that binds strongly to a potent inhibitor and mutate parasites to express altered levels of the bromodomain and mutate the bromodomain to interfere with the molecular interactions with the inhibitor. We will then confirm that the inhibitor binds the P. falciparum bromodomain by testing for the mutants’ altered resistance to the inhibitor. For Trypanosoma, we will test inhibitors against a mutant cell line that either under-expresses or over-expresses a bromodomain of interest. The resulting change in inhibition potency may provide evidence of chemical validation.
How can your partnership (project) address global health challenges?
According to WHO, just under half a million people continue to die every year from malaria, with over 200 million new infections occurring annually in Africa, Asia and other endemic regions. In Latin America, there are over 7 million people living with Chagas disease. Concurrently, close to 2 million people are infected with visceral or subcutaneous leishmaniasis every year. Cryptosporidiosis is the second leading cause of diarrheal disease and mortality amongst infants. With the exception of malaria, resources dedicated to finding novel drug leads are not sufficiently adequate. In the case of malaria, the need for novel drug targets has not abated despite ongoing research because of emerging resistance to artemisinin. Therefore, an innovative approach is required to change the landscape for all of these parasitic diseases.
The superfamily of 37 bromodomains in the parasites of interest here represents an opportunity to find novel drug targets and drug leads because (i) some parasite bromodomains have been proven to be essential to parasite survival or are implicated in activating essential parasite genes (ii) the parasite bromodomains are different to human bromodomains and so potent inhibitors should not be cross-reactive with human bromodomains (iii) bromodomains have been successfully targeted by drugs in other species and (iv) the P. falciparum bromodomains are expressed throughout development in the human and are conserved across Plasmodium species and so present promising targets for a drug that could kill multiple malaria parasite species, cure malaria disease and prevent parasite transmission.
What sort of innovation are you bringing in your project?
This project will investigate a totally novel class of parasite proteins as drug targets. It will also screen a novel library of compounds derived from natural products. The project will innovate through the integration of expertise in high-throughput screens, parasite biology, structural biology, protein chemistry and molecular genetics. The structural biological component will use bespoke and sophisticated assays of inhibitor/bromodomain binding integrated with advanced crystal structure analysis to rapidly assess the molecular probability that parasite killing compounds are targeting bromodomains. This project will also use recent advances in CRISPR Cas9 targeted mutagenesis to modify malaria parasites in response to structural binding and growth inhibition data.
Role and Responsibility of Each Partner
RIKEN (Prof. Hiroyuki Osada and Dr Nobumoto Watanabe) will supply their natural compound library, combining this with compound libraries from SGC for use in their high throughput in vitro Plasmodium assay.
SGC (Dr. Raymond Hui) will assemble a library of compounds known to bind bromodomains or inhibit parasite growth and provide this to other partners for high-throughput screens. SGC will also continue to express recombinant parasite bromodomains and resolve their crystal structures. SGC will use their purified proteins in binding assays to identify potent inhibitors of parasite bromodomains. SGC will identify analogues of potent inhibitors for use in validation assays. The SGC will use resolved P. falciparum bromodomain structures to predict molecular interactions with the potent inhibitors to guide mutations at the University of Melbourne.
McGill University (Prof. Armando Jardim and Prof. Momar Ndao) will use provided by SGC and RIKEN in growth inhibition assays of T. cruzi, L. donovani, and C. parvum. Prof. Jardim will also develop transgenic parasites expressing altered levels of one essential bromodomain to help validate at least one Trypanosoma bromodomain as a drug target.
University of Melbourne (Dr Michael Duffy) will provide their established mutant P. falciparum parasite lines expressing altered levels of bromodomain proteins and develop new ones. They will use structural data from SGC to direct mutations of P. falciparum bromodomains that will interfere with inhibitor binding. All of these mutant parasites will be used in assays to test for altered susceptibility to inhibitors and thus confirm inhibitor-bromodomain interactions in the parasite.
1. Project objective
This project had two objectives. The first was to validate bromodomains as drug targets for treatment of several parasitic diseases. Bromodomains are protein-protein interaction domains that are often important for gene regulation. The parasitic diseases investigated were malaria, Chagas disease, leishmaniasis and cryptosporidiosis, these are caused by the parasites Plasmodium falciparum, Trypanosoma cruzi, Leishmania donovani and Cryptosporidium parvum. The second objective was to identify molecules capable of inhibiting parasite bromodomains.
2. Project design
For target validation we used genetic engineering to identify which bromodomains were essential for survival of P. falciparum, L. donovani and T. brucei. In parallel, the structures of purified parasite bromodomains were determined by x-ray crystallography to provide information that would inform inhibitor design. To identify inhibitors we screened a curated library of compounds known to inhibit bromodomains or related proteins and an additional library of natural product compounds. Compounds were screened for inhibition of parasite growth and for binding to purified bromodomains. In some cases the crystal structure of the bromodomain with bound inhibitor was determined.
3. Results, lessons learned
This research revealed that five of the eight P. falciparum bromodomains analysed and two of the four L. donovani bromodomains analysed were essential for survival in the human host and all four L. donovani bromodomains were important for normal insect stage growth. These findings validated these bromodomains as drug targets for Plasmodium and Leishmania. Crystal structures were solved for; four P. falciparum bromodomains, two with bound inhibitors; five L. donovani bromodomains, four with bound inhibitors; seven for Trypanasoma, three with bound inhibitors; and three for C. parvum, two with bound inhibitors. These structures will provide invaluable information for further chemical optimisation of inhibitors that bind to the bromodomains in the parasites. Screening of compound libraries identified three compounds that killed P. falciparum and that bound purified P. falciparum bromodomains, one of these shows promise as a bromodomain specific inhibitor. Eleven compounds were identified that inhibited growth of insect infective forms of L. donovani and T. cruzi, three of these compounds also inhibited growth of human infective forms however the specificity of these compounds for bromodomains has not been established. Overall the project has successfully validated multiple parasitic bromodomains as drug targets and provided critical structural data that can be utilised in targeted drug design. It has also generated several promising candidate inhibitors with anti-parasitic effects but their specificity for bromodomains remains unproven.