Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 9 January 2017 doi:10.20944/preprints201701.0040.v1 Peer-reviewed version available at Pathogens 2017, 6, 12; doi:10.3390/pathogens6010012 Article Sarcocystis neurona Protein Kinases: Computational Identification, Evolutionary Analysis and Putative Inhibitor Docking Edwin K. Murungi 1 & Henry M. Kariithi 2 1 Department of Biochemistry and Molecular Biology, Egerton University, P.O. Box 536, 20115, Njoro, Kenya; [email protected] 2 Biotechnology Research Institute, Kenya Agricultural and Livestock Research Organization, P.O Box 57811, 00200, Kaptagat Rd, Loresho, Nairobi, Kenya; [email protected] * Correspondence: [email protected]; Tel: (+254)-789-716059 Abstract: The apicomplexan parasite, Sarcocystis neurona causes the degenerative neurological equine protozoal myeloencephalitis (EPM) disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs) have been implicated in a myriad of critical functions such as host cell invasion, cell cycle progression and host immune responses evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. Further, three-dimensional (3D) homology models for selected S. neurona putative PKs were constructed and evaluated for inhibitor docking. We identified 92 putative PKs clustering within the AGC, CAMK, CK1, CMGC, STE, TKL, aPK and OPK groups. Although containing the universally conserved PKA (AGC group), S. neurona kinome was devoid of PKB and PKC, but contained the six apicomplexan conserved CDPKs (CAMK group). The OPK group was represented by ROPKs 19A, 27, 30, 33, 35 and 37, but was devoid of the virulence-associated ROPKs 5, 6, 18 and 38. Two out of the three S. neurona CK1 enzymes had high sequence similarities to T. gondii TgCK1-α and TgCK1-β and the Plasmodium PfCK1. Docking of four inhibitors onto homology models of putative ROP27 and PKA indicated that inhibition of S. neurona PKs is feasible, but needs to be experimentally tested. The essentiality of apicomplexan PKs makes the elucidation of S. neurona kinome a key milestone for development of novel therapeutics for EPM. Keywords: Sarcocystis neurona; EPM; Docking; Apicomplexans; Phylogeny; Homology modeling. 1. Introduction Equine protozoal myeloencephalitis (EPM) is an infectious, progressive, degenerative neurological disease of horses caused by the apicomplexan parasite, Sarcocystis neurona [1]. To complete its life cycle, this heteroxenous parasite requires a reservoir host (i.e. opossums; Didelphis virginiana, D. albiventris) and an aberrant (horses) or intermediate host (cats, skunks, raccoons and sea otters) [2]. Opossums become infected upon ingestion of sarcocysts containing hundreds of bradyzoites, which undergo gametogony that sporulate into mature oocysts, which are then shed in the faeces. Upon ingestion by the intermediate or aberrant hosts, the oocysts release the environmentally resistant sporozoites, which chronically parasitize the neural and inflammatory cells of the host’s central nervous system (CNS). Clinical EPM symptoms depend on which part of the CNS is parasitized; parasitization generally results in abnormal gait, dysphagia and muscle atrophy in affected horses [3]. The intracellular nature of S. neurona and its ability to evade the host's immune surveillance [4] makes EPM treatment expensive, lengthy and challenging. Traditionally, clinical treatment of EPM Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 9 January 2017 doi:10.20944/preprints201701.0040.v1 Peer-reviewed version available at Pathogens 2017, 6, 12; doi:10.3390/pathogens6010012 2 of 34 involved inhibitors of folate synthesis and metabolism (sulphonamides/pyrimethamine combination) over a prolonged period [5]. More recently, triazines derivatives (diclazuril, ponazuril) that target the parasite’s apicoplast [6], nitazoxanide, a pyruvate:ferridoxin oxidoreductase analogue that inhibits the parasite’s anaerobic metabolism [7], and anti-inflammatory agents and immune stimulants [8] have been used with variable success in eliminating clinical signs. Despite the availability of these drugs, EPM treatment is complicated by the emergence of drug-resistance (due to intermittent or periodic treatments), cost of therapies and drug toxicity (e.g. diarrhea and/or anemia) and infection relapses due to re-growth of residual parasites after the treatment regimes [2]. As such, discovery and development of novel therapeutics for EPM is imperative. To successfully invade the host cells, apicomplexans utilize three specialized exocytic organelles (micronemes, rhoptries and dense-granules) [9]. The microneme is used for host cell recognition, binding, penetration and gliding along the cytoskeletal structures. Rhoptry proteins are discharged into the host cell during parasite internalization and are crucial in the formation of the parasitophorous vacuoles (PVs). Developing zoites contain non-pedunculated condensing vesicles that synthesize and package inactive rhoptry proteins, which are proteolytically activated when the rhoptry contents are condensed [10]. The PVs facilitate parasite development by allowing nutrient transport from the host cell and by blocking lysosomal fusion, which would otherwise kill the parasites [11]. Upon internalization, zoites use the dense-granules to remodel the PVs into functionally active organelles. The virulence of the apicomplexans is highly dependent on protein kinases (PKs) that are involved in the invasion and modification of host cell structure and function. PKs are classified into various groups, comprising of many families and sub-families [12-14]. The major apicomplexans PK groups are the PKs A, G and C (AGCs), which are regulated by cyclic nucleotides and other second messengers; the calmodulin/calcium-dependent PKs (CAMKs); the casein kinase 1 (CK1) and close relatives, the PKs involved in cell-cycle control and signal transduction (containing cyclin-dependent, mitogen-activated and cell division cycle [CDC]-like PKs; or CMGCs); the receptor guanylate cyclase (RGC); the “sterile” serine/threonine kinases (STEs, including several kinases functioning in the MAPK cascades); the tyrosine kinases (TKs); and the tyrosine kinase-like kinases (TKLs) [15-17]. In addition, there is a sizeable proportion of other PKs that do not fit into any of the above-mentioned eight major PK groups (i.e. the “Other” kinases [OPKs]), and another smaller set of atypical PKs (aPKs) that have no clear sequence similarity with the conventional PKs [13, 18, 19]. Each of the PK families have vital roles in parasite’s survival. For instance, PfPK-B (AGC family), PfTKL3 (TKL family), and four of the seven CDPKs (CAMK family) are required by Plasmodium parasites to complete their asexual cycle [20-22]. In a recent study, deletion of TgCK1α (CK1 family) resulted in defective replication of Toxoplasma gondii in vitro [23]. The P. falciparum CKL and SRPK1 (CMGC family) complement each other in the regulation of mRNA splicing (via phosphorylation of splicing factors [24]). Since apicomplexans lack typical MAPK cascades, the STEs are not well studied. However, in the parasites that do have the MAPK pathways, STEs are essential for parasite growth (e.g. in the human parasites, Schistosoma mansoni [25]). Further, for parasites without the conventional MAPK cascades, aPKs can activate the signaling pathways, for instance the Plasmodium Pfnek3 [26]. Finally, some of the notable OPKs include aurora kinases, rhoptry kinases (ROPKs) and parasite-specific eukaryotic initiation factor-2 (elF2) kinases (elF2K), which are important in parasite virulence and differentiation [27-29]. The differences between the parasite’s and their host’s PK homologs can be used to develop anti-parasitic strategies via specific inhibition of the parasite’s PKs [30]. This is feasible due to the essential roles of PKs in the parasite’s growth and development. Proof of principle of this approach has been demonstrated by the inhibition of human PKs using chemical ligands to treat cancers and other diseases [31, 32]. Recently, Ojo et al., [33] provided evidence that PKs can be targeted for rationally designed drugs that can potently inhibit growth of S. neurona. The technology is available and approved for therapeutic intervention, thus offering a unique prospect of repurposing chemical ligands to manage S. neurona infections [34]. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 9 January 2017 doi:10.20944/preprints201701.0040.v1 Peer-reviewed version available at Pathogens 2017, 6, 12; doi:10.3390/pathogens6010012 3 of 34 Here, we used a genome-wide approach to define the kinome of S. neurona and determined the relatedness of the putative PKs to those reported in other apicomplexans. We also tested the docking of some specific inhibitors to a selection of some of the putative PKs. Defining the S. neurona kinome is not only important in providing insights into the parasite’s biology, but also identification of potential novel drug targets that can be used to clear chronic S. neurona infections, reduce parasite survival, augment the host's anti-parasitic immune responses, or as candidates for development of EPM vaccines. 2. Results 2.1 Sarcocystis neurona encodes 92 putative kinases To date, at least 15 apicomplexan genomes (coccidians, gregarines, hemosporidians and piroplasmids) have either been fully sequenced