Mining the Cryptosporidium kinome: using kinobead technology to reveal novel biology and potential therapeutic targets

Diarrhoeal disease is a leading cause of morbidity and mortality for young children. The WHO estimates that 10% of the deaths of children under the age of five globally are due to diarrheal disease. In 2013 the Global Enteric Multicentre Study identified the pathogens responsible for deadly diarrhoeal disease. Surprisingly, cryptosporidiosis caused by infection with Cryptosporidium parasites, was revealed as second only to Rotavirus in causing deadly disease. Diarrhoea from cryptosporidiosis is severe, but self-limiting in immune-competent people. However, for young, malnourished children and the immunocompromised, the disease can be fatal. Annually there are an estimated 7.5 million cases of cryptosporidiosis, resulting in >200,000 deaths. Recurrent diarrhoeal episodes associated with cryptosporidiosis alters gut morphology, leading to growth stunting, delayed development, and impaired cognitive function. The greatest impact of the disease occurs in Low and Middle-Income Countries. However, waterborne outbreaks threaten public health in High-Income Countries including the USA and UK. Waterborne outbreaks also carry significant economic impact. Unfortunately, there is no vaccine and no effective treatment for cryptosporidiosis. New drugs are desperately needed. Phosphorylation, the addition of a phosphate group to a molecule or protein, is mediated by kinases. Kinases are key regulators of cell signalling and play a crucial role in modulating almost all cellular events. Therefore, it is no surprise that kinase inhibitors have become one of the pharmaceutical industry’s most important drug target classes with ~30% of drug discovery efforts worldwide directed at the kinase superfamily. Despite the fact that little is known about the function of kinases in Cryptosporidium, they are gaining traction as exploitable drug targets. The two compounds currently in development for cryptosporidiosis specifically target kinases. In addition, compounds known to inhibit kinases kill Cryptosporidium parasites, although the details about how the compounds do this are unknown. In order to leverage these promising compounds as treatments for cryptosporidiosis, it is critical to identify which specific kinase, or kinases they target (Cryptosporidium have >100 kinases). This information will strengthen current drug discovery projects and help initiate new ones. Unfortunately, many of the tools and reagents available to identify the targets of kinase inhibitors are optimised for human kinases and are considerably less effective for the study of kinases in other organisms. Similarly, compared to other related parasites, like Plasmodium and Toxoplasma (which cause malaria and toxoplasmosis respectively), our understanding of the basic biology of Cryptosporidium is limited. To address this knowledge gap, we will adopt an interdisciplinary approach that combines chemistry, proteomics (the study of proteins), biology, and genetics. To better understand the role of kinases in Cryptosporidium biology we will: 1] develop Cryptosporidium-centric chemical tools; 2] use these tools, in combination with proteomics, to identify the protein targets of kinase inhibitors capable of killing Cryptosporidium parasites and 3] interrogate the biology and function(s) of these biologically significant Cryptosporidium kinases. This research will improve our fundamental understanding of Cryptosporidium biology and will lay the foundations for the future exploitation of kinases for anti-cryptosporidial drug discovery. Effective drugs for cryptosporidiosis will lead to improvements to the health and welfare of children and immune-compromised patients world-wide.