There is need for a more efficient cell-based assay amenable to high-throughput drug screening against Giardia lamblia. Here, we report the development of a screening method utilizing G. lamblia engineered to express red-shifted firefly luciferase. Parasite growth and replication were quantified using D-luciferin as a substrate in a bioluminescent read-out plateform. This assay was validated for reproducibility and reliability against the Medicines for Malaria Venture (MMV) Pathogen Box compounds. For G. lamblia, forty-three compounds showed ≥ 75% inhibition of parasite growth in the initial screen (16 μM), with fifteen showing ≥ 95% inhibition. The Pathogen Box was also screened against Nanoluciferase expressing (Nluc) C. parvum, yielding 85 compounds with ≥ 75% parasite growth inhibition at 10 μM, with six showing ≥ 95% inhibition. A representative set of seven compounds with activity against both parasites were further analyzed to determine the effective concentration that causes 50% growth inhibition (EC50) and cytotoxicity against mammalian HepG2 cells. Four of the seven compounds were previously known to be effective in treating either Giardia or Cryptosporidium. The remaining three shared no obvious chemical similarity with any previously characterized anti-parasite diarrheal drugs and offer new medicinal chemistry opportunities for therapeutic development. These results suggest that the bioluminescent assays are suitable for large-scale screening of chemical libraries against both C. parvum and G. lamblia.

Here we study the role of Nek8445 in regulating cell division and microtubule array organization in Giardia. Depletion of Nek8445 results in 87% of cells being stalled or blocked in cytokinesis. Nek8445 regulates ventral disk organization, funis formation, axoneme exit, and cell shape, all of which contribute to the observed cytokinesis defects.

10Giardia has 189 Nek kinases whereas humans have only 11. We previously identified Nek8445 11 as a potential drug target due to susceptibility to a specific class of kinase inhibitors and 12 essential function in parasite proliferation and attachment. Giardia has a complex tubulin 13 cytoskeleton that includes eight flagella and several unique microtubule arrays that are utilized 14 for parasite attachment and facilitation of rapid mitosis and cytokinesis. The need to regulate 15 these structures may explain the parallel expansion in the number of Nek family kinases. Here 16 we use live and fixed cell imaging to uncover the role of Nek8445 in regulating Giardia cell 17 division. We demonstrate that Nek8445 localization is cell cycle regulated and this kinase has a 18 role in regulating overall microtubule organization. Nek8445 depletion results in short flagella, 19 aberrant ventral disc organization, loss of the axostyle (funis), defective flagella exit and altered 20 cell shape. The flagella exit defect is specific to the caudal flagella, which exit from the posterior 21 of the cell, and this defect correlates with rounding of the cell posterior and loss of the axostyle.22 This correlation implicates a role for the axostyle in establishing Giardia's cell shape and guiding 23 flagella docking. Axostyle biogenesis can now be included in the list of functions regulated by 24 Nek family kinases. 25 26 29 kinases from their homologs, in humans has been identified as an opportunity for the 30 development of new drugs (Rotella, 2012). The Giardia kinome (strain WB) contains 278 protein 31 kinases, 80 of which constitute the core kinome from 49 different classes of kinases while the 32 remainder are all Nek Kinase homologs (Manning et al., 2011). The highly expanded Giardia 33Nek family contains 198 kinases, compared to only one in budding yeast, seven in Arabidopsis, 34 11 in humans, and 13 in Chlamydomonas (Manning et al., 2011;Takatani et al., 2015). 35Nek kinases are named for their homology to the Aspergillus nidulans NIMA (never in 36 mitosis A) kinase. Nek kinases have been shown to control mitotic entry and flagella length in 37 other eukaryotes (O'Connell et al., 2003;Bradley and Quarmby, 2005), but may also have a 38 more general role in regulating microtubule organization (Takatani et al., 2015). Nek kinases are 39 typically more abundant in the genomes of eukaryotes that build cilia or flagella (Parker et al., 40 2007). Notably, Giardia possesses four pairs of flagella that undergo a complex developmental 41 cycle (Nohynkova et al., 2006) (See Figure 1 for diagram of Giardia structures). As pairs of 42 flagella do not share paired basal bodies, maintaining the identity of each flagella is complex 43 and the regulatory mechanism remains uncharacterized (Nohynkova et al., 2006). The 44 complexity of maintaining eight flagella identities could explain the development of a greatly 45 expanded set of Nek kinases.46 Flagella positioning and identity are essential for Giardia cellular processes including 47 patho...