Jamal KHALIFE

Molecular Signaling in the Control of Parasite Growth and Differentiation (MSCPGD)

Malaria and schistosomiasis are the two most important parasitic diseases in terms of the mortality and morbidity that they cause. Both, but particularly schistosomiasis, can be considered as “neglected diseases” since the public and private investment in measures to control or eradicate them is incommensurate with their enormous impact on the affected populations.

Malaria is caused by protozoan parasites of the genus Plasmodium transmitted by Anopheles mosquitoes that infected about 219 million people and caused an estimated 660000 deaths in 2010 (WHO estimate), mostly among African children.

Schistosomiasis (or bilharzia) is a water-borne disease caused by five species of flatworm of the genus Schistosoma that have fresh-water snail intermediate hosts. More than 200 million people are infected by the parasites and, in addition to significant mortality (around 280000 deaths annually in sub-Saharan Africa) schistosomiasis also causes, particularly in children, anemia, growth stunting and reduced school performance.

For both diseases there is a clear and present need for new strategies and drugs for their treatment and control. Currently, no effective vaccines are available. Moreover, drug resistance is a recurring problem with malaria and, since there is only one drug available for treatment of schistosomiasis, is anticipated to become one for this disease.

The ultimate aim of our research is to contribute to the development of new drugs to treat malaria and schistosomiasis. Our approach is to study the molecular signaling mechanisms involved in parasite growth and differentiation within the human host, of which the main actors are promising therapeutic targets. We also participate in international research consortia with the specific aim of developing new drugs that target these signaling mechanisms. The highlights of our research over the last few years are as follows:

Protein kinases of Schistosoma mansoni (Colette Dissous)

We have studied several important protein kinases associated with the reproductive organs of S. mansoni and demonstrated their essential functions in gametogenesis. First, we have characterized the two polo-like kinases SmPlk1 and SmPlk4. In the Xenopus oocyte model, we demonstrated respectively their functions in the entry of mitosis (G2/M transition) and centriole multiplication. More interestingly, we showed for the first time that Plk1 and Plk4 proteins are able to interact and cross-activate, leading to resumption of meiosis in G2-blocked oocytes. The implication of this process in mitosis and cancer cell proliferation is currently studied.

In parallel, we have characterized a novel family of Receptor Tyrosine Kinases named VKR for Venus Kinase Receptors, present only in invertebrates and formed by an intracellular kinase domain, similar to that of Insulin Receptors (IR), linked to an extracellular Venus Flytrap two-lobed domain that binds amino-acids. In S. mansoni worms, SmVKRs are abundantly expressed in female ovaries and their targeting by RNA interference or by the use of the potent IR kinase inhibitor, tyrphostin AG1024, provokes a complete disorganization of the ovary as well as the absence of egg production. SmVKR therefore constitute attractive targets to control the egg-dependent pathology of schistosomiasis and the VKR-dependent signaling pathways (particularly the JNK and Akt pathways) are currently under investigation.

Absence of a viable egg in the ootype (arrow) of a schistosome treated (right panel) with the IR kinase inhibitor AG1024. An untreated worm with an egg in the ootype is shown in the left panel.

Schistosome histone modifying enzymes as drug targets (Ray Pierce, Emmanuel Roger, Stéphanie Caby)

Our previous work on the characterization of schistosome histone deacetylases (HDACs) both confirmed the potential of their inhibitors as novel drugs against schistosomiasis and allowed the initiation of the dissection of the epigenetic mechanisms involved in the control of the growth and differentiation of the parasites. Moreover, our results provided the basis for the SEtTReND project, coordinated by the team, which was financed by the EC (FP7-Health) for the period 2010-2012. This project aimed at the development of selective inhibitors of schistosome histone modifying enzymes (including HDACs) using a target and structure-based strategy. With our project partners we were notably able to show using RNAi transcript knockdown that schistosome HDAC8 is a valid therapeutic target. This was reinforced by our finding that SmHDAC8 interacts with the cohesin complex and thereby plays a central role in cell division. The production of the recombinant protein and the resolution of its crystal structure by X-ray diffraction confirmed that structural differences in its active site would allow the development of selective inhibitors, of which a number were obtained by both high-throughput and in silico screening. We were able to show that these lead compounds induced apoptosis and death in schistosome larvae and that one of them effectively reduced the parasite burden of infected mice. Similar work on inhibitors of the class III HDAC, S. mansoni Sirt2 has also led to the development of lead compounds. Finally, in a new EC project (A-PARADDISE) funded for 2014-2017 we will extend and expand this strategy to other major human parasitic diseases including malaria.

S.  mansoni larvae undergoing apoptosis (pink stain) after treatment with an HDAC inhibitor.

Immunology and biology of malaria infection (Jamal Khalife, Christine Pierrot)

We identified new steps involved in the age-dependent susceptibility to malaria after a primary infection of young rats and were able to show that the adequate activation of these steps could protect young infected hosts. Recent published work from human studies confirmed the involvement of some factors we identified in the rat malaria experimental model, underlining its usefulness. Thanks to the solid experience we acquired in the use of rat model to study experimental infections, (including S. mansoni), strong international links have been created, allowing to the group to actively participate in an EU program to develop vaccines for helminth infections.

In a study of the role of Protein Phosphatase type 1 in P.falciparum (PfPP1), one of the major Ser/Thr phosphatases, in the growth and differentiation of the parasite, we characterized two nuclear proteins that interact with and regulate the enzyme. Reverse genetic approaches indicated that these regulators are essential for blood stage parasites and seem to play a role in the cell cycle. In addition, peptides derived from these regulators were shown to exhibit an antiplasmodial activity by blocking parasite growth in vitro. More recent studies using yeast two hybrid screening identified several new potential nuclear regulators of PfPP1, including histones 2A and 2B. Our work on antiplasmodials has also allowed the development of fruitful collaborations to study their mode of action, particularly Ferroquine. Our expertise in this field allowed us to participate in the A-PARADDISE FP7-funded project devoted to the search for new drugs against P. falciparum.

Schizont stage of P. falciparum expressing a protein phosphatase regulator fused to green fluorescent protein (the blue DAPI counterstain shows the nucleus of the parasite).

Strategy and Perspectives

During the next few years the research of the team will continue to focus on the processes of molecular signaling involved in controlling the growth and differentiation of S. mansoni and P. falciparum. The two parasites studied are evidently very different and we do not draw inappropriate parallels between such evolutionarily distant organisms. However, their differences provide an opportunity since they carry out molecular and cellular processes that are not present in their hosts, nor in the “model organisms” most often studied. The choice of continuing to study both P. falciparum and S. mansoni is motivated by their importance as pathogens and the knowledge of essential, fundamental cellular processes that can be gained from studying signaling pathways in which the main actors are conserved, but which may have different biological outcomes. The cross-fertilization between the two models will shed light on the molecular mechanisms involved in each organism and suggest strategies for the development of novel therapeutics. We intend to nourish the progression from basic science to therapeutic applications through continuing collaborations with both French and foreign laboratories and participation in international projects. Notably the team will coordinate an EC funded project, A-PARADDISE, with the aim of producing novel lead compounds and drug precursors against schistosomiasis, malaria and two other neglected parasitic diseases by targeting histone modifying enzymes.

Major publications 2010-2012

Baeza Garcia A, Pierce RJ, Gourbal B, Werkmeister E, Colinet D, Reichhart JM, Dissous C, Coustau C. 2010. Involvement of the cytokine MIF in the snail host immune response to the parasite Schistosoma mansoni. PLoS Pathogens 6: e1001115.

Daher W, Pierrot C, Kalamou H, Pinder JC, Margos G, Dive D, Franke-Fayard B, Janse CJ, Khalife J. 2010. Plasmodium falciparum dynein light chain 1 interacts with actin/myosin during blood stage development. J Biol Chem 285: 20180-20191.

Long T, Cailliau K, Beckmann S, Browaeys E, Trolet J, Grevelding CG, Dissous C. 2010. Schistosoma mansoni Polo-like kinase 1: A mitotic kinase with key functions in parasite reproduction. Int J Parasitol 40: 1075-1086.

Dissous C, Grevelding CG. 2011. Piggy-backing the concept of cancer drugs for schistosomiasis treatment: a tangible perspective? Trends Parasitol 27: 59-66.

Pierrot C, Acroute dit Vampouille A, Vandomme A, Lafitte S, Pierce RJ, Hot D, Khalife J. 2011. Gene profiling analysis reveals the contribution of CD24 and P2Y6R to the susceptibility of young rats to Plasmodium berghei infection. Cell Microbiol 13: 752-763.

Freville A, Landrieu I, Garcia-Gimeno MA, Vicogne J, Montbarbon M, Bertin B, Verger A, Kalamou H, Sanz P, Werkmeister E, Pierrot C, Khalife J. 2012. Plasmodium falciparum inhibitor-3 homolog increases protein phosphatase type 1 activity and is essential for parasitic survival. J Biol Chem 287: 1306-1321.

Khalife J. 2012. Current strategies for drug development against major human parasites: malaria and schistosomiasis. Curr Pharm Des 18: 3453.

Long T, Vanderstraete M, Cailliau K, Morel M, Lescuyer A, Gouignard N, Grevelding CG, Browaeys E, Dissous C. 2012. SmSak, the second Polo-like kinase of the helminth parasite Schistosoma mansoni: conserved and unexpected roles in meiosis. PLoS One 7: e40045.

Pierce RJ, Dubois-Abdesselem F, Lancelot J, Andrade L, Oliveira G. 2012. Targeting schistosome histone modifying enzymes for drug development. Curr Pharm Des 18: 3567-3578.

Pierrot C, Freville A, Olivier C, Souplet V, Khalife J. 2012. Inhibition of protein-protein interactions in Plasmodium falciparum: future drug targets. Curr Pharm Des 18: 3522-3530.

Vanderstraete M, Gouignard N, Ahier A, Morel M, Vicogne J, Dissous C. 2013. The venus kinase receptor (VKR) family: structure and evolution. BMC Genomics 14: 361.

Vanderstraete M, Gouignard N, Cailliau K, Morel M, Lancelot J, Bodart JF, Dissous C. 2013. Dual targeting of insulin and Venus Kinase Receptors of Schistosoma mansoni for novel anti-schistosome therapy. PLoS Negl Trop Dis 7: e2226.

Freville A, Cailliau-Maggio K, Pierrot C, Tellier G, Kalamou H, Lafitte S, Martoriati A, Pierce RJ, Bodart J-F, Khalife J. 2013. Plasmodium falciparum encodes a conserved active inhibitor-2 Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy. BMC Biol 11:80.

Lancelot, J., Caby, S., Dubois-Abdesselem, F., Vanderstraete, M., Trolet, J., Oliveira, G., Bracher, F., Jung, M., Pierce, R.J. 2013 Schistosoma mansoni sirtuins: characterization and potential as chemotherapeutic targets. PLoS Negl. Trop. Dis. 7(9):e2428.

Marek, M., Kannan, S., Hauser, A.T., Mourao, M.M., Caby, S., Cura, V., Stolfa, D.A., Schmidtkunz, K., Lancelot, J., Andrade, L., Renaud, J.P., Oliveira, G., Sippl, W., Jung, M., Cavarelli, J., Pierce, R.J*., Romier, C*. 2013 Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni. PLoS Pathogens, 9(9) :e1003645. (*co-corresponding authors).

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