Research review paperAtypical (RIO) protein kinases from Haemonchus contortus — Promise as new targets for nematocidal drugs
Introduction
Protein kinases are a group of enzymes that are crucial for the regulation of a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, and thus play functional roles in reversible protein phosphorylation (Hanks et al., 1988). Based on their structure, protein kinases can be classified into eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs) (Manning et al., 2002). The aPKs have kinase activity and share limited sequence similarity to the majority of eukaryotic protein kinases. Of the 518 kinases known to be encoded in the human genome, for instance, 40 are aPKs. These molecules have been classified into 13 families or groups, one of which represents the RIO kinases (designated here as RIOKs) (Manning et al., 2002). These kinases are considered essential for life, but almost nothing is known about them for multicellular organisms (metazoans), including nematodes (LaRonde-LeBlanc and Wlodawer, 2005a, LaRonde-LeBlanc and Wlodawer, 2005b).
Although the primary structures (amino acid sequences) of RIOKs are divergent from those of other protein kinase families, their conformation is similar to those of known canonical protein kinases. In yeast (Saccharomyces cerevisiae), RIOK-1 has been shown to have protein kinase activity in vitro, which is dependent upon amino acid residues recognised as being essential for kinase function (Angermayr et al., 2002). Other research has demonstrated an important role for RIOK-1 in cell-cycle progression (G1 to S transition), the regulation of the onset of anaphase and mitotic chromosome stability (Angermayr et al., 2002) as well as the processing of 20S precursor ribosomal RNA (rRNA) to the 18S species (Vanrobays et al., 2001). RIOK-2 of S. cerevisiae is also required for 20S rRNA processing. However, in contrast to RIOK-1, RIOK-2 appears to be localised predominantly to the nucleus (Vanrobays et al., 2001). Although RIOK-1 and RIOK-2 are known to be associated with the 20S precursor rRNA in yeast, the biological activities of both kinases do not overlap (Vanrobays et al., 2001, Geerlings et al., 2003). Recently, RIOK-2 has also been identified as an essential, late-acting 40S ribosome synthesis factor (Granneman et al., 2010). Interestingly, an RNA interference (RNAi) screen of human kinase genes identified that the knock-down of riok-1 and riok-2 decreased cell viability and accelerated epithelial cell migration, respectively, whereas no effect was detected for riok-3 which is specific to metazoans (Simpson et al., 2008). A recent report (Kimmelman et al., 2008) suggests that human RIOK-3 might be involved in human tumour cell motility and invasion, possibly through the modulation of the Rho family of GTPases. Taken together, this information indicates that RIOKs are involved in diverse and crucial biological processes in eukaryotes, but their precise roles remain to be elucidated in multicellular organisms.
RIOKs are encoded in the genome of the best-characterised metazoan, the free-living nematode Caenorhabditis elegans (see Manning, 2005). RNAi, which decreases messenger RNA (mRNA) levels of the targeted C. elegans gene, has been shown to affect predominantly embryonic and larval growth and/or development (Fraser et al., 2000, Ashrafi et al., 2003, Simmer et al., 2003, Rual et al., 2004, Sonnichsen et al., 2005). In spite of the functional importance of this molecule, there is no published information on aPKs for any related, parasitic nematodes, with the exception of Trichostrongylus vitrinus (order Strongylida) (see Hu et al., 2008). In the present article, we elucidate the full-length complementary cDNAs and genes of three RIOKs from Haemonchus contortus (an economically important blood-feeding strongylid nematode of small ruminants) and compare them with related molecules encoded in other organisms, infer the three-dimensional structures of RIOKs by comparison with known crystal structures of homologues, and assess the potential of these kinases as novel drug targets in parasitic helminths.
This article provides new insights into three different atypical (RIO) kinases in H. contortus, one of the economically most important parasites of livestock. Based on bioinformatic and phylogenetic analyses, these kinases are proposed to be novel drug targets. Currently, computational approaches (e.g., Krasky et al., 2007, Caffrey et al., 2009, Doyle et al., 2010) are increasingly being used to assess the potential of key genes/gene products as novel drug targets in parasitic worms. In addition, structure-based virtual screening has been proven to be useful in the identification of compounds able to inhibit the activity of molecules whose three dimensional structure had been established using homology models (reviewed by Villoutreix et al., 2007). For instance, in silico docking for ~ 200,000 compounds (i.e., ChemDiv) into the binding site of an homology model of a human BCR-ABL tyrosine kinase (which is known to play a crucial role in the pathogenesis of chronic myeloid leukaemia; Deininger et al., 2000) led to the identification of 15 compounds selected for biological testing, eight of which were demonstrated to significantly inhibit tumour cell growth (Peng et al., 2003). In another study (Vangrevelinghe et al., 2003), novel and selective inhibitors of protein casein kinase II were identified by in silico docking of an homology model of CK2 with a subset of 400,000 molecules available in the Novartis database (Vangrevelinghe et al., 2003). Although numerous examples of protein-ligand interaction studies and drug design using in silico approaches are described in the literature (cf. Villoutreix et al., 2007, Cavasotto and Phatak, 2009, Hammami and Fliss, 2010), and computational structure prediction methods are cost- and time-effective in the absence of experimental structures, the success of these approaches depends on the accuracy of the model predicted and on the sequence similarities between the protein used as a template and the homologous sequence(s) (see Cavasotto and Phatak, 2009).
Section snippets
Nucleic acids
Genomic DNA was extracted from 50 mg of pooled H. contortus using a small-scale sodium dodecyl-sulphate (SDS)/proteinase K extraction procedure, followed by purification over a mini-column (Wizard Clean-Up, Promega) (Gasser et al., 2006). The specific identity and mono-specificity of the parasite material was verified by PCR-coupled, automated sequencing of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA from genomic DNA (see Bott et al., 2009). Total RNA was extracted
Three riok genes and their inferred gene products for Haemonchus contortus
The full-length cDNAs (designated Hc-riok-1, Hc-riok-2 and Hc-riok-3) were 1842, 1590 and 1434 nucleotides (nt) in length, respectively (GenBank accession nos. HQ198854.1-HQ198857.1 and HQ207527.1-HQ207528.1.; Table 1). The transcripts representing Hc-riok-1, Hc-riok-2 and Hc-riok-3 were reproducibly detected by reverse transcription PCR in all developmental stages examined, except for the exsheathed L3 (Fig. 1, Conder and Johnson, 1998). Transcription was usually greatest in eggs, L4 female
Inference of gene function and interactions based on information available for C. elegans and other eukaryotic organisms
In C. elegans, riok-1 is involved in biological processes essential for nematode viability and fertility as well as endocytosis and fat storage (Fraser et al., 2000, Ashrafi et al., 2003, Simmer et al., 2003, Rual et al., 2004, Sonnichsen et al., 2005, Balklava et al., 2007, Ceron et al., 2007; cf. www.wormbase.org; Table 2). In contrast, knowledge of the functions of the C. elegans riok-2 and riok-3 is limited to the observation that gene perturbation by RNAi results in lethality and
Three-dimensional structural modelling
In this section, all residue numbers refer to the sequence of Hc-RIOK-1 (see Fig. 4). Topologically, the three RIOKs differ in the N- and C-terminal domains that flank the central kinase domain. RIOK-1 usually possesses an N-terminal domain of ~ 100 aa residues, with few predicted secondary structure elements. Af-RIOK-1 from A. fulgidus, the only RIOK-1 for which an experimental three-dimensional structure is available (PDB accession code 1ztf) (LaRonde-LeBlanc et al., 2005b), seems to be rather
Application of the RIOK-1 model for the prediction of drugs in silico
In a first attempt to probe the active site of RIOKs, with a view toward drug discovery, we conducted an in silico screen using the homology model of Hc-RIOK-1 employing the SPECS database. The top 12 binding compounds identified from this screen are listed in Table 3. Interestingly, four of these 12 compounds possess a carbohydrate moiety. For the compounds ranked third and tenth, a second binding mode was observed and ranked in positions 8 and 11, respectively, indicating an increased
Conclusions and future prospects
Based on modelling, structural comparison of the three RIOKs shows that the RIOK domain harbouring the catalytic site is a well-conserved fold among parasitic nematodes, in particular between H. contortus and T. vitrinus. However, despite this fold, there are several aa substitutions in functionally important, conserved secondary structure elements (Supplementary Fig. 4), whose impact can only be assessed from three-dimensional structures determined experimentally. Future structural studies
Acknowledgements
Funding from the Australian Research Council (ARC) is gratefully acknowledged (RBG). PRB is supported by funding from the National Health and Medical Research Council (NHMRC).
References (76)
- et al.
The mar regulon: multiple resistance to antibiotics and other toxic chemicals
Trends Microbiol
(1999) - et al.
Rio1, an extraordinary novel protein kinase
FEBS Lett
(2002) - et al.
The many faces of the helix-turn-helix domain: transcription regulation and beyond
FEMS Microbiol Rev
(2005) - et al.
A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep
Int J Parasitol
(2009) - et al.
Homology modeling in drug discovery: current trends and applications
Drug Discov Today
(2009) - et al.
Natural compounds as a source of protein phosphatase inhibitors; application to the rational design of small-molecule derivatives
Biochemie
(2006) - et al.
Rio2p, an evolutionarily conserved, low abundant protein kinase essential for processing of 20S Pre-rRNA in Saccharomyces cerevisiae
J Biol Chem
(2003) - et al.
Current trends in antimicrobial agent research: chemo- and bioinformatics approaches
Drug Discov Today
(2010) A contig assembly program based on sensitive detection of fragment overlaps
Genomics
(1992)- et al.
Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase
Bioorg Med Chem Lett
(2008)
A combined bioinformatics and chemoinformatics approach for the development of new antiparasitic drugs
Genomics
A family portrait of the RIO kinases
J Biol Chem
The Rio kinases: an atypical protein kinase family required for ribosome biogenesis and cell cycle progression
Biochim Biophys Acta
Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings
Adv Drug Deliv Rev
HcSTK, a Caenorhabditis elegans PAR-1 homologue from the parasitic nematode, Haemonchus contortus
Int J Parasitol
Identification of novel inhibitors of BCR-ABL tyrosine kinase via virtual screening
Bioorg Med Chem Lett
Comparative protein modelling by satisfaction of spatial restraints
J Mol Biol
Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop
Structure
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
Nucleic Acids Res
Yeast Rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression
Mol Microbiol
Gene ontology: tool for the unification of biology. The Gene Ontology Consortium
Nat. Genet.
Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes
Nature
The specificities of protein kinase inhibitors: an update
Biochem J
Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic
Nat Cell Biol
The Pfam protein families database
Nucleic Acids Res
Organization and expression of eucaryotic split genes coding for proteins
Annu Rev Biochem
Protein structure prediction servers at University College London
Nucleic Acids Res
The mouse genome database (MGD): mouse biology and model systems
Nucleic Acids Res
A comparative chemogenomics strategy to predict potential drug targets in the metazoan pathogen, Schistosoma mansoni
PLoS One
Massively parallel sequencing and analysis of the Necator americanus transcriptome
PLoS Negl Trop Dis
Large-scale RNAi screens identify novel genes that interact with the C. elegans retinoblastoma pathway as well as splicing-related components with synMuv B activity
BMC Dev Biol
BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009
Nucleic Acids Res
Genetic and physical maps of Saccharomyces cerevisiae
Nature
Ribosomal S6 kinase 2 is a key regulator in tumor promoter induced cell transformation
Cancer Res
Viability of infective larvae of Haemonchus contortus, Ostertagia ostertagi, and Trichostrongylus colubriformis following exsheathment by various techniques
J Parasitol
Isoflavonoids III — constituents of Cotoneaster species
Aust J Chem
Mechanism of CYP2C9 inhibition by flavones and flavonols
Drug Metab Dispos
BCR-ABL Tyrosine kinase activity regulates the expression of multiple genes implicated in the pathogenesis of chronic myeloid leukemia
Cancer Res
Cited by (26)
Yellow catfish RIO kinases (RIOKs) negatively regulate fish interferon-mediated antiviral response
2023, Developmental and Comparative ImmunologyFunctional genomic exploration reveals that Ss-RIOK-1 is essential for the development and survival of Strongyloides stercoralis larvae
2017, International Journal for ParasitologyHaemonchus contortus: Applications in Drug Discovery
2016, Advances in ParasitologyCitation Excerpt :An alternative approach identifies potential H. contortus drug targets through bioinformatics searches, using a variety of strategies (eg, Campbell et al., 2011a,b; Doyle et al., 2010; Kotze, 2012; Krasky et al., 2007; Preston et al., 2015b; Taylor et al., 2013a,b; Wang et al., 2015a,b). Cases in which some chemical validation of the target was obtained by assays using H. contortus include the identification of norcantharadin analogues as protein phosphatase inhibitors, some of which were active in an LDA, albeit with relatively low potency (Campbell et al., 2011b). A screen of a set of known protein kinase inhibitors identified three compounds that were active in an L3 motility assay (Taylor et al., 2013a), and screening a different set of kinase inhibitors in a larval motility assay identified two active compounds with modest potency (Preston et al., 2015b).
Bioinformatic exploration of RIO protein kinases of parasitic and free-living nematodes
2014, International Journal for ParasitologyCitation Excerpt :The benefits of these resources can now be realised through a collective effort of mining and curating smaller datasets, such as particular gene families involved in essential biological processes. Protein kinases (PKs) are one of the largest gene families of metazoans and regulate a wide range of cellular processes including cell-cycle progression, transcription, DNA replication and metabolic processes (Vanrobays et al., 2001; LaRonde-LeBlanc et al., 2005; LaRonde-LeBlanc and Wlodawer, 2005a; Granneman et al., 2010; Campbell et al., 2011; Widmann et al., 2012; Read et al., 2013). PKs can activate/inactivate target proteins by catalysing the transfer of phosphate groups to specific residues (i.e. His/Asp, Ser/Thr/Tyr and Arg) on their target proteins and thus play a regulatory role in nearly all cell signalling pathways (Hanks et al., 1988).
Insights into the immuno-molecular biology of Angiostrongylus vasorum through transcriptomics-Prospects for new interventions
2013, Biotechnology AdvancesCitation Excerpt :As these atypical kinases appear to diverge from mammalian RIOKs, particularly in regions that may complex with ligands, future structural studies are warranted to reveal the details of such molecular interactions, for example, in complex with the phosphate-donating nucleotides, to provide a solid basis for structure-based drug design. To date, the mechanistic aspects of RIOKs are poorly understood, and our current working hypothesis assumes that the two flexible elements in the RIOK domain, the hinge and the flexible loop, serve as docking points for the substrate and might undergo conformational change in the substrate-bound state (Campbell et al., 2011). Such a process might be further aided by phosphorylation of Hc-RIOK-1 Ser165 (corresponding to Av-RIOK-1-Ser170), which is located in the flexible loop and seems to be a conserved residue for RIOKs.
- 1
Equal contributions.