Leishmania Protein Kinases: Important Regulators of the Parasite Life Cycle and Molecular Targets for Treating Leishmaniasis

Leishmania is a protozoan parasite of the trypanosomatid family, causing a wide range of diseases with different clinical manifestations including cutaneous, mucocutaneous and visceral leishmaniasis. According to WHO, one billion people are at risk of Leishmania infection as they live in endemic areas while there are 12 million infected people worldwide. Annually, 0.9–1.6 million new infections are reported and 20–50 thousand deaths occur due to Leishmania infection. As current chemotherapy for treating leishmaniasis exhibits numerous drawbacks and due to the lack of effective human vaccine, there is an urgent need to develop new antileishmanial therapy treatment. To this end, eukaryotic protein kinases can be ideal target candidates for rational drug design against leishmaniasis. Eukaryotic protein kinases mediate signal transduction through protein phosphorylation and their inhibition is anticipated to be disease modifying as they regulate all essential processes for Leishmania viability and completion of the parasitic life cycle including cell-cycle progression, differentiation and virulence. This review highlights existing knowledge concerning the exploitation of Leishmania protein kinases as molecular targets to treat leishmaniasis and the current knowledge of their role in the biology of Leishmania spp. and in the regulation of signalling events that promote parasite survival in the insect vector or the mammalian host.


Introduction Leishmaniases and Current Chemotherapy
Leishmania, a kinetoplastid protozoan parasite which belongs to the order of trypanosomatids together with Trypanosoma brucei and Trypanosoma cruzi, is the causative agent of leishmaniases, an umbrella term of diseases which includes cutaneous, mucocutaneous and visceral leishmaniasis [1].
Leishmania parasites are transmitted to the mammalian host after a bite of an infected female sandfly. Leishmania spp have a digenetic life cycle, involving a motile, extracellular promastigote stage that parasitizes the alimentary tract of a sandfly vector and an immotile amastigote stage that survives and replicates in the phagolysosomes of mononuclear phagocytes [2,3]. Apart from humans, primary hosts of Leishmania are vertebrates, including wild and domestic animals such as rodents and canines [4].
A main factor that contributes to the broad spectrum of disease manifestation is the diversity of Leishmania species [4,5]. More than twenty Leishmania species inflict disease with distinct clinical manifestations and are responsible for 10-12 million infected people worldwide. Annually, 0.9-1.6 million new infections and 20-50 thousand deaths are reported due to Leishmania infection [5]. Leishmania (L.) amazonensis, L. major, L. mexicana and L. tropica are associated with cutaneous leishmaniasis with reported cases in 92 countries [1]. Cutaneous leishmaniasis (CL), the most common form of the disease, causes self-curing activation loop is a complex domain in the kinase structure, and when reconstructed in its active form upon phosphorylation, it allows the substrate binding. The activation loop is therefore a part of the substrate binding site and is flexible in order to accommodate the ATP binding site [45]. Finally, a gatekeeper residue partially or fully blocks a hydrophobic region in the ATP binding pocket and is considered as a selectivity determinant of most ATP competitive kinase inhibitors [46].
Blocking the conserved ATP binding site is the most common mechanism to inhibit the kinase, however additional structures can be exploited for the inhibition of kinase activity. For instance, the fact that the substrate binding site can be blocked via intrasteric interactions or modulated by the conformation of the activation loop, can be useful for designing molecules interacting with those domains to block the activation of the kinase. Moreover, the flanking segments of the kinase can be responsible for autoinhibition by blocking the active site or by promoting conformational change in the kinase. Thus, flanking segments could also be targeted for modifying the kinase in its inactive structure [45].

Cyclin-Dependent Kinases (CDKs)
Cyclin-dependent kinases (CDKs) belong to the group CMGC kinases, and like their name reveals, possess a cyclin-binding region, for the binding of a cyclin partner which is required for kinase activation. CDKs are found in all eukaryotes and are key regulators of cell cycle progression and other processes, including transcription, mRNA processing and differentiation of nerve cells [47]. Animal cells contain at least 20 CDKs, four of which, namely CDK1, CDK2, CDK3 and CDK4, are directly involved in cell cycle regulation [48]. CDK1 was the first CDK identified and plays important role in mitosis, while it can drive S phase in the absence of CDK2. In higher eukaryotes, CDK2 and CDK3 are essential for the G1 phase of the cell cycle [49][50][51][52]. Kinases that belong to this group have been extensively studied and targeted for the treatment of various diseases including various types Helix C plays a crucial role in the modulation of the kinase activity as it is coupled to both the ATP binding site and the activation loop. Helix C can rotate in response to regulators and subsequently reconstitutes the ATP binding site promoting the active form of the kinase when there is simultaneously a phosphorylation of the activation loop [45]. The activation loop is a complex domain in the kinase structure, and when reconstructed in its active form upon phosphorylation, it allows the substrate binding. The activation loop is therefore a part of the substrate binding site and is flexible in order to accommodate the ATP binding site [45]. Finally, a gatekeeper residue partially or fully blocks a hydrophobic region in the ATP binding pocket and is considered as a selectivity determinant of most ATP competitive kinase inhibitors [46].
Blocking the conserved ATP binding site is the most common mechanism to inhibit the kinase, however additional structures can be exploited for the inhibition of kinase activity. For instance, the fact that the substrate binding site can be blocked via intrasteric interactions or modulated by the conformation of the activation loop, can be useful for designing molecules interacting with those domains to block the activation of the kinase. Moreover, the flanking segments of the kinase can be responsible for autoinhibition by blocking the active site or by promoting conformational change in the kinase. Thus, flanking segments could also be targeted for modifying the kinase in its inactive structure [45].

Cyclin-Dependent Kinases (CDKs)
Cyclin-dependent kinases (CDKs) belong to the group CMGC kinases, and like their name reveals, possess a cyclin-binding region, for the binding of a cyclin partner which is required for kinase activation. CDKs are found in all eukaryotes and are key regulators of cell cycle progression and other processes, including transcription, mRNA processing and differentiation of nerve cells [47]. Animal cells contain at least 20 CDKs, four of which, namely CDK1, CDK2, CDK3 and CDK4, are directly involved in cell cycle regulation [48]. CDK1 was the first CDK identified and plays important role in mitosis, while it can drive S phase in the absence of CDK2. In higher eukaryotes, CDK2 and CDK3 are essential for the G1 phase of the cell cycle [49][50][51][52]. Kinases that belong to this group have been extensively studied and targeted for the treatment of various diseases including various types of human cancers [53]. In the trypanosomatid kinome, CDKs are over-represented. The Leishmania genome encodes for 11 CDKs referred as CDC2-related kinases (CRKs) and 11 activating cyclins (CYCA, CYC2-CYC11) [35].
The most studied CRK in Leishmania is the leishmanial homologue of the mammalian CDK1, namely CRK3 (LmxM. 36.0550 in L. mexicana). Gene disruption studies carried out in L. mexicana demonstrated that both CRK3 alleles can be replaced, although ploidy changes occurred to allow retention of the wild-type copy of CRK3, suggesting but not confirming that CRK3 is an essential enzyme [54]. The essentiality of this kinase was confirmed later with the use of a rapamycin-inducible gene deletion system, based on a dimerized Cre recombinase (diCre) [55]. Induction of diCre activity in promastigotes with rapamycin resulted in G2/M growth arrest, confirming the essentiality of this kinase in mitotic transitions [56]. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thus genetically validating CRK3 as a drug target to treat leishmaniasis ( Figure 2) [56].
Microorganisms 2021, 9, x FOR PEER REVIEW 6 of 22 Figure 2. Leishmanial Protein kinases from CMGC family that could serve as drug targets. The ePKs displayed have been genetically and/or pharmacologically validated. The effects on their biological role and/or on their virulence upon pharmacological and/or genetic inhibition are also displayed in the diagram. Azapurine compounds NO Studies have highlighted that both the kinase and the cyclin binding partner of CRK3 are essential for its activity and thus are targeted as a complex by potential inhibitors [57]. In Leishmania, CRK3 is known to complex with CYC1, CYCA and CYC6 cyclins [33,[58][59][60]. Most inhibitor screens, however, are focused on the CRK3-CYC6 complex as it is thought to have homologous function to the cyclin-dependent kinase 1-cyclin B complex (CDK1-Microorganisms 2021, 9, 691 6 of 22 CYCB) in humans, which is essential for the proliferation and coordination of the eukaryotic cell cycle [61][62][63]. While CRK3 is required for the Leishmania G2/M transition possibly via CRK3-CYC6 complex formation, the CRK3-CYC1 is an S phase complex highlighting additional roles of CRK3 in the S phase of the cell cycle ( Figure 2). Although the importance and the role of CRK3-CYC1 substrates is yet to be determined, their identification could serve as a scaffold for generating inhibitors to screen the CRK3-CYC1 complex and to further investigate the role of CRK3 in Leishmania [64].
In addition to CRK3, CRK12 (LdBPK_090270) was chemically validated as a drug target for VL, with the use of a chemical series based on a pyrazolopyrimidine scaffold (Table 1), while CYC9 emerged as the definite partner of CRK12 [66]. A gene deletion mutant could not be generated without ectopic expression of CRK12, implying that CRK12 could be an essential Leishmania enzyme ( Figure 2). Moreover, the leading compound of the series could block the cell cycle in G1 and G2/M phases, suggesting that CRK12 is important for the cell cycle progression through these phases [66].
In addition to CRK12, attempts to generate LmxCRK1 (LmxM.21.1080) null mutants in L. mexicana promastigotes failed unless compensated with a T. brucei CRK1 homologue, thus strongly suggesting that this gene was essential for promastigote viability [67]. Recent data from the kinome-wide deletion screen identify, together with the aforementioned kinases, a number of CRKs refractory to deletion (CRK2-LmxM.05.0550, CRK9-LmxM. 27.1940 and CRK11-LmxM. 29.1780) and important CRK kinases for survival in the host [37]. Future enzymatic assays can be developed to further pharmacologically validate those results and to perform inhibitory screens for drug development.

Glycogen Synthase Kinase 3 (GSK-3)
Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine kinase found in all eukaryotes. It is a member of the CMGC family and closely related to CDKs [86]. In higher eukaryotes, GSK-3 is important in many cellular processes (Wnt signaling, cell cycle, differentiation, apoptosis, neuronal functions, etc.), while its deregulation has been linked to many human diseases such as cancer, diabetes and Alzheimer's disease [86]. In trypanosomatids, two genes encode for two isoforms of GSK-3 (GSK-3s-short isoform and GSK-3l-long isoform). The short isoform in trypanosomatids has been validated pharmacologically or genetically as a potential drug target [26,27,87].
In Leishmania, GSK-3s has been validated by both pharmacological assessment [26,27] and genetic manipulation, where Leishmania promastigotes were refractory to GSK-3 deletion ( Figure 2) [37], and these studies suggest that GSK-3s is an essential gene. Chemical inhibition of the kinase resulted in cell cycle deregulation, namely G1 phase arrest that was followed by an apoptosis-like death in the parasites ( Figure 2) [26,27], suggesting that GSK-3s may have a direct or indirect role in cell cycle progression. Apart from the essentiality of the kinase, there are additional reasons that make this kinase a good drug target for antileishmanial drug design. Firstly, there are specific changes in the ATP binding pocket between the human and the parasite orthologues, i.e., the replacement of Gln185 hGSK-3β to His155 LdGSK-3s in the sugar-binding region, and the "gatekeeper" replacement of Leu132 hGSK-3β to Met100 LdGSK-3s [26] that can be exploited for selective inhibition. More specifically, the replacement of the leucine gatekeeper to a methionine which results in a larger entropic and desolvation cost upon inhibitor binding and the presence of the proton accepting His155 LGSK-3, could be exploited for designing bulkier inhibitors to enhance selectivity towards leishmanial over the human GSK-3 [26].
Secondly, the availability of the crystal structure of L. major GSK-3s (LmjF18.0270) is anticipated to advance drug discovery efforts [33,88]. Moreover, there is a large availability of known hGSK-3 inhibitors that can be tested against the leishmanial GSK-3s. Many studies have focused on the repositioning of hGSK-3 inhibitors against the leishmanial homologue [26,27,70,72] (Table 1). In this context, the anti-parasitic action of an in-house indirubin library known to inhibit mammalian GSK-3 and CDKs was evaluated. Amongst the indirubins displaying potent antileishmanial activity, were those with 6-Brsubstitutions on the indirubin backbone, a substitution known to greatly enhance the selectivity for mammalian GSK-3 over CDKs. Kinase assays revealed that the 5-Me-6-BIO was a potent inhibitor of LdGSK-3s (LdBPK_180270) (nM range), while it displayed potent anti-parasitic activity against L. donovani promastigotes and intracellular amastigotes (low micromolar range) [26]. In addition, 5-Me-6-BIO was several-fold more selective over the CRK3 homologue. Despite that selectivity, other 6-Br substituted analogues like 6-bromoindirubin-3 -oxime (6BIO), reversed selectivity from the respective mammalian kinases and targeted more potently CRK3 over GSK-3s. Thus, a new study was undertaken for understanding selectivity with new indirubin analogues and showed that the GSK-3s affinity of 6-BIO derivatives was enhanced upon 3 -substitution with bulky amino groups, namely piperazine or pyrrolidine [27]. This study confirmed that potent analogues (GSK-3s inhibitors, nM range) also possess important anti-parasitic activity against L. donovani promastigotes and intracellular L. donovani amastigotes at the low micromolar range [27].
Apart from indirubins, paullones which initially were characterised as CDK inhibitors were found to be excellent GSK-3 inhibitors [71]. For instance, Knockaert et al. showed that in vitro alsterpaullone inhibited the growth of L. mexicana promastigotes, while it prevented the parasite replication in macrophages [70]. The antileishmanial compounds maleimides [89,90] and lithium [91] were also found to inhibit the parasitic GSK-3s. In search for new scaffolds that inhibit GSK-3, another study that tested a group of compounds developed by GlaxoSmithKline known to possess antileishmanial action (Leishbox), and an in-house library of human GSK-3β inhibitors, revealed that compounds belonging to the families thiadiazolidindione, halomethylketone, maleimide, benzoimidazole, Nphenylpyrimidine-2-amine and oxadiazole, inhibited LGSK-3s. With respect to Leishbox, benzoimidazole, N-phenylpyrimidine-2-amine and oxadiazole were uncovered as new scaffolds for LdGSK-3s (Table 1) [72].
Although most of these compounds (Table 1) are also competitive inhibitors of the mammalian GSK-3, the selectivity index between Leishmania and human cell cytotoxicity appears in most cases to be high due to the tolerance of the host kinase inhibition in adult mammals [92,93]. This redundancy of the mammalian homologue kinase as well as the aforementioned arguments, highlights the kinase as an excellent candidate for targeted drug discovery.

Dual-Specificity Tyrosine-Regulated Kinases (DYRKs)
The dual-specificity tyrosine-regulated kinases, namely DYRKs, belong to the CMGC group of kinases [94] and as their name reveals, they possess dual specificity. More specifically, DYRKs demonstrate an autophosphorylation activity of a tyrosine residue in their activation loop, and a serine/threonine kinase activity towards their substrates [95]. DYRKs are expressed in all eukaryotes [96]. YAKs and minibrain kinases are DYRK family members expressed in lower eukaryotes (yeast) and nematodes, respectively [95,[97][98][99]. In higher eukaryotes, DYRK family members are categorized into three subfamilies based on their kinase domain homology: the HIPKs (homeodomain interacting protein kinase) that are regulators of a variety of stress signals [100], the pre-mRNA processing protein 4 kinases (PRP4s) that are involved in transcriptional regulation [101] and the DYRKs [96]. DYRKs are further divided to class I (DYRK1A and DYRK1B) and class II (DYRK2, DYRK3 and DYRK4) [96]. DYRK subfamily members are multifunctional kinases involved in a variety of cellular processes in mammalian cells, such as stasis regulation, differentiation, proteosomal degradation, chromatic remodeling, gene expression and others [96]. The most studied members in mammalian cells are the two closely related and multifaceted DYRK1 kinases (DYRK1A and DYRK1B). DYRK1A is implicated in Down syndrome pathology and neurodegenerative disorders, whereas DYRK1B deregulation is associated with metabolic syndrome and cancer [102][103][104][105][106][107].
Moreover, the role of L. infantum DYRK1 (LinJ.15.0180) has been recently investigated [108]. The deletion of DYRK1 by gene replacement rendered viable L. infantum promastigotes, showing that parasites can compensate for its loss [108]. Furthermore, LinDYRK1 deletion mutants displayed defects in the stationary phase and in metacyclogenesis, followed by significant reduction in survival within peritoneal macrophages ( Figure 2) [108]. Another study, however, in L. mexicana using CRISPR-CAS9 technology failed to isolate a gene deletion mutant for DYRK1 (LmxM.15.0180) [37]. This could be due to differences between species or compensatory mutations or because of the background expression levels of other DYRK kinases that may be able to compensate for the loss of DYRK1. In addition, it was shown that LinDYRK1 over-expressing parasites displayed a decrease in proliferation and in cell cycle re-entry, suggesting a role as a stasis regulator similar to its mammalian homologues. LinDYRK1 has a distinct localization in the cytoplasm, flagellar pocket area and the endolysosome [108]. The latter localization suggests that DYRK1 is a multifaceted kinase, while its presence in the endosomal compartment is reminiscent of a known role for mammalian DYRK1A in endocytosis and vesicle recycling [109,110]. A similar localization to DYRK1 was also shown in L. mexicana for another DYRK family member, namely LmxM.14.0 70 (LinJ.14.1140 orthologue of L. infantum), suggesting that the two kinases could participate in the same cellular processes. In addition, the most closely related homologue of DYRK1, LmxM.14.830 was refractory to deletion, suggesting that this kinase could be essential for viability and thus could be a potential drug target. Another important DYRK-like kinase, namely PK4 (LmxM.21.1650), was shown to be required for differentiation and/or survival in the mammalian host only [37]. Overall, Leishmania DYRKs are interesting kinases as potential drug targets, and the study of these kinases will provide important information on to the life-cycle of the parasite. The above data together suggest that DYRK1 and other family members like LmxM.14.830 could serve as potential drug target candidates.

Mitogen-activated Protein Kinases
Mitogen-activated protein kinases (MAPKs) are members of the CMGC of serine/threonine kinases [38] found in all eukaryotes, and are known to mediate signal transduction from external stimuli, such as mitogens, pro-inflammatory cytokines, heat shock and other stress stimuli [111]. The mammalian MPK family of kinases includes three subfamilies: the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs) and the p38 mitogen-activated protein kinases (p38s). ERKs are known to be activated by growth factors and mitogens, whereas cellular stresses and inflammatory cytokines activate JNKs and p38s [112]. Classical MAPKs become activated by phosphorylation in their activation loops, which contain a characteristic TxY motif [113]. This phosphorylation is normally part of a signaling cascade with upstream kinases belonging to the STE family ("sterile"), referring from null mutants that resulting in sterile yeast. This cascade starts by the stimulation of MAP kinase kinase kinases (M3Ks), that phosphorylate and activate MAP kinase kinases (M2Ks), which finally phosphorylate and activate MAP kinases (MAPKs), for the regulation of various cellular activities like cell proliferation, differentiation, stress response, infectivity and apoptosis [111,114].
Leishmania parasites encode for 17 mitogen-activated protein kinases (MAPKs), 5 putative MAP2Ks (STE) and around 25 MAP3Ks (STE) [35,115]. Of all leishmanial protein kinase families, MAPKs are the most commonly studied, and their known role in coupling extracellular signals with cell survival has put them in the spotlight as an important source of potential drug targets. In this context, some members of MAPK family have already been validated as potential targets. Amongst these, is LmxMPK1 (LmxM.20_36.6470), a kinase required for intracellular parasite survival [116]. LdMPK1 phosphorylates HSP90 and HSP70, thus may regulate the stability and activity of the foldosome [117,118] specific to the Leishmania infectious stage [119]. Thus, deregulation and inhibition of its activity may result in increased thermo-sensitivity and decreased intracellular parasite viability ( Figure 2). It also appears that MPK1 negatively regulates the expression of P-glycoproteintype efflux pumps in L. donovani promastigotes [120], thus this kinase activity maybe associated with drug resistance. In addition to LmxMPK1, LmjMPK2 (LmjF.36.0720), which is required to establish infection in mice, seems to be essential in amastigotes and could serve as a potential drug target [121]. LmjMPK2 also controls osmoregulation and Sb(III) uptake by phosphorylating and stabilizing aquaporin [121]. Thus, the activity of Leishmania MPK2, like MPK1, is inversely associated with the acquisition of drug resistance [121]. Confirmation about the importance of LmxMPK1 and LmxMPK2 in intracellular survival, came by the study of Baker et al., who showed that these kinases are important for differentiation in both insect vector and mammalian host [37]. Moreover, it was shown that an arginine deprivation response induced during macrophage infection is mediated through an MPK2-dependent signaling cascade, thus providing evidence for its mode of action in intracellular parasites [122].
In addition to Leishmania MPK1 and MPK2, reverse genetics approaches revealed a role for LmxMPK3 (LmxM. 10.0490) and LmxMPK9 (LmxM. 19.0180) in the promastigote flagellum length regulation [123,124]. LmxMPK9 deletion mutants had a long flagellum whereas LmxMPK9 over-expressing parasites had a shorter paraflagellar rod protein PFRA [124]. Moreover, it was shown that LmxMPK9 is important for the parasites' colonization in the sandfly [37]. On the other hand, LmxMPK3 deletion mutants had a shorter flagellum (Figure 2) [123], a similar phenotype to null mutants of LmxMPKK1, the activating kinase of LmxMPK3 [125]. Although LmxMPK3 is not essential for parasite viability, small molecule inhibitors have been identified, as this kinase is important for Leishmania transmission [80]. Shweta Raj et al. showed that the plant-based natural compounds genistein and chrysin (Table 1) inhibited the kinase, and at the same time, minimized the parasitic load of infected macrophages (Figure 2) [80]. However, genistein and chrysin may act on enzymes of the macrophage or other parasitic enzymes, and hence further work is necessary to ascertain that the reduction of infectivity is due to the inhibitory effect of LdMPK3. Furthermore, as L. donovani MPK3 (LdBPK_100540) crystal structure is available from the Protein Data Bank (PDB), drug design against MPK3 is more feasible [126,127].
Genetic analyses of L. major MAPKs (termed LmaMPKs) revealed amastigote-specific phosphorylation and activity of LmaMPK4, LmaMPK7 and LmaMPK10 [128] and established a role of LmaMPK7 in translational control of amastigote proliferation [129]. Wang et al. generated LmxMPK4 null mutants only in the presence of an extrachromosomal copy [130], while Dacher et al. performed a facilitated approach of MPK4 deletion mutants in the presence of a plasmid susceptible for negative selection that expresses MPK4, which confirmed that this kinase is essential for viability [131] and hence validated MPK4 as a potential drug target. In addition, a mutant expressing MPK4 with an altered ATP binding pocket (K59R) showed defects in metacyclogenesis (Figure 2), linking MPK4 activity with parasite differentiation [131]. Located primarily in the lysosome [37], LmxMPK4 is activated upon phosphorylation by the STE7 LmxMKK5. The finding of LmxMPK4 s in vitro activator formed the basis on which screens for identifying small molecule LmxMPK4 inhibitors can be established [132]. Despite the knowledge of the activator kinase, only in silico screening has been performed. In this context, Shweta Raj et al. performed an in silico study to identify potent LdMPK4 inhibitors among 110 natural inhibitors of Leishmania parasite and demonstrated that genistein and chrysin are potential lead molecules for targeting the kinase [133]. Moreover, Saravanan et al. virtually screened 2654 compounds from an NCI Diversity set against the human ERK2 and the LmxMPK4 and identified potential inhibitors with higher affinity against LmxMPK4 [134]. The authors also demonstrated that the LmxMPK4 ATP binding domain, although highly conserved, possesses minor but potentially important structural differences to the homologous human ERK2. More specifically, ligands bind to the LmxMPK4 hydrophobic inner part of the cavity to the residues Val26, Leu91, Met93, and Cys155 and form, at the same time, hydrogen bonds with anchoring key residues such as Tyr96 which surround the ATP binding cavity [134]. Taking into consideration the above characteristics, LmxMPK4 is an important candidate for designing new scaffolds of targeted drugs with high affinity against the specific kinase.
LmxMPK5 (LmxM.29.2910) and LmxMPK10 (LmxM.10.0200) also seem to play a crucial role in the amastigote form of the parasite and thus could serve as potential drug targets in order to stop the parasites from hijacking the macrophages and infecting the host. Mpk5 gene was successfully deleted in L. mexicana and the generated null mutants although persisted in the inoculation site upon infection of BALB/c mice, they displayed decreased ability to cause lesions (Figure 2) [135]. Moreover, LmxMPK10 has been shown to be implicated in amastigote differentiation, while it also displays stage-specific activity important in promastigote and amastigote differentiation (Figure 2) [136]. Interestingly, it was also shown that LmaMPK10 possesses a potential C-terminal autoinhibitory mechanism which could be exploited for the discovery of molecules interfering with MPK10 [81]. This autoinhibitory mechanism seems crucial for the stage-regulated activity and parasite viability [136]. The stage-regulated activity of Leishmania MPK10 was confirmed for the L. mexicana orthologue [37]. A crystal structure of LmaMPK10 is available, highlighting the significant differences compared to human p38 kinase in the catalytic pocket, as well as the potentially regulatory sites in the N-terminal lobe [81].
Moreover, Baker et al. showed that MPK15 (LmxM.32.2070) is likely important in the differentiation and/or survival in the mammalian host [37]. The same study also revealed that L. mexicana parasites were refractory for the deletion of 8 STE genes, including MPKK4 and MPKK5 [37]. In summary, the above results show that members of these groups of kinases (MPKs and STEs) merit in-depth investigation for drug discovery efforts and for their role in differentiation and environmental sensing.

Casein Kinases
The term "casein kinase" (CK) applies to three unrelated categories of protein kinases, denoted by the acronyms CK1, CK2 (casein kinase 1 and 2) and G-CK (Golgi-Casein kinase or Fam20C), sharing the ability to readily phosphorylate casein in vitro [137]. CK1 is a family of monomeric, Ser/Thr protein kinases ubiquitously expressed and present in all eukaryotes. Seven isoforms of human CK1 exist (α, β, γ1, γ2, γ3, δ and ε), displaying highly conserved kinase domains [137]. CK1s are involved in various cellular processes, such as in Wnt and Hedgehog signalling, circadian rhythm, nucleo-cytoplasmic shuttling, DNA repair, DNA transcription and others [138][139][140]. CK1s are known to have a wide range of substrates [138], while they are involved in hierarchical phosphorylation events, acting either as a priming or as a processive kinase [141,142]. Thus, deregulation of CK1 isoforms is implicated in several diseases, such as neurodegenerative, sleeping disorders, and neoplasia [137]. CK2 is one of the most pleiotropic protein kinases, with dual specificity, which phosphorylates either serine/threonine or tyrosine residues and is composed of two catalytic (CKA1, CKA2) and two regulatory subunits (CKB1 and CKB2), respectively. CK2, like CK1, is responsible for the generation of a large proportion of the phosphoproteome, phosphorylating plethora of substrates. CK2 is involved in many processes like cell proliferation and survival, apoptosis, angiogenesis and other activities [143]. The third member of the CKs, FAM20C is an atypical kinase that also generates a high number of the phosphoproteome and a pleotropic enzyme [137]. As human casein kinases are implicated in many processes, they have become the focus of drug discovery efforts, usually against cancer and neurodegenerative diseases [137].
Out of the three families of casein kinases, only CK1 and CK2 are found in Leishmania, while FAM20C is not present. Leishmania parasites have an expanded CK1 family relative to their kinome, with 6 members (CK1.1-LmxM. 34 [35,37,73]. Sacerdoti-Sierra and Jaffe showed that released ectoproteins from L. major promastigotes displayed CK1 activity [144] and later on the finding were confirmed as at least one member of the family, namely CK1.2, was found in the secretome [145,146]. It has been demonstrated that the Leishmania CK1 phosphorylated human IFNAR1, a receptor implicated in interferon signalling and hence it was suggested that it may be implicated in macrophage function and immune response [147]. Furthermore, Rachidi et al. with the use of a specific CK1 inhibitor D4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide) (Table 1), pharmacologically validated CK1.2 as a potential drug, as the inhibitor blocked promastigote growth, compromised axenic amastigote viability and decreased the number of intracellular L. donovani and L. amazonensis amastigotes in infected macrophages (Figure 3) [73]. In addition, the study of Baker et al. showed that L. mexicana promastigotes were refractory to CK1.2 deletion, suggesting that CK1.2 maybe essential for viability [37]. It was also shown that Leishmania CK1 has the ability to phosphorylate Leshmania HSP83, an important protein for stage regulation and Leishmania viability [118]. The fact that Leishmania CK1.2 is important for viability highlights this kinase as an important drug target candidate to treat leishmaniasis, and several CK1.2 inhibitors have been identified (Table 1). Apart from D4476, inhibition of the kinase by trisubstituted pyrrole, blocked promastigote growth [148]. Durieu et al. screened 5018 compounds to assess their antileishmanial activity against promastigotes and intracellular amastigotes, as well as their activity against CK1.2 [74]. They identified two compounds, PP2 (1-tert-butyl-3-(4-chlorophenyl)-1 h-pyrazolo [3,4-d]pyrimidin-4-amine) and an indirubin analogue (compound 42), that targeted the kinase and displayed potent antileishmanial activity with a high selectivity index (>10) against murine macrophages (Table 1) [74]. Thus, targeted inhibition of the parasites' essential kinase CK1.2 could have dual activity for treating leishmaniasis, as it would lead to parasite death and it would cease the establishment of the infection in the mammalian host.

Aurora Kinases
Aurora kinases are known mediators of the cell division cycle in eukaryotes [154]. Since they are considered important mitotic serine/threonine kinases, Aurora kinases are well studied in a variety of organisms. In L. mexicana, three Aurora kinases have been identified (AUK3-LmxM. 26.2440, AUK1/AIRK-LmxM. 28.0520, AUK2-LmxM.08_29.1330) [37], while in L. major, the role of one Aurora kinase member (LmAIRK-AUK1 homologue) has been investigated [155][156][157]. A computational analysis identified the putative Auroralike kinase in L. donovani (LdAIRK), the LmAIRK homologue, as a potential drug target [78]. Knockout LdAIRK parasites could not be generated, a fact that strengthens the hypothesis that the kinase is important for viability [78]. Another important feature of the kinase is that although in mammalian cells, Aurora kinases play a role in mitosis, cytokinesis and chromosome segregation, in L. major, one gene of Aurora kinase, namely LmAIRK, may regulate all these processes [156]. With a main nuclear localization throughout the cell-cycle, LdAIRK seems to temporarily migrate from the cytoplasm to the nuclear In addition to CK1.2, LmxCK1.4 isoform was also shown to be essential for parasitic viability, as no CRISPR-cas9 mutants could be generated (Figure 3) [37]. CK1.4 is the only CK1 isoform that is unique to Leishmania [35,37,149] and therefore it is considered a potential chemotherapeutic target. Although CK1.4 is localized in the flagellar pocket of L. mexicana promastigotes, it is also secreted like CK1.2 and is considered a potential virulence factor [37,73,149]. This theory is verified by the fact that overexpression of the kinase in L. donovani promastigotes resulted in significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites [149]. Subsequently, CK1.4 isoform has to be further investigated, as it plays a crucial role in both the parasitic survival and the ability to infect the host macrophages [149].
Another Leishmania isoform, CK1.1 was not found to be essential for Leishmania survival or axenic amastigote differentiation. It was demonstrated that CK1.1 was a lowabundance protein present in promastigotes and in amastigotes. In the same study, however, there was a link between CK1.1 and stationary phase, as CK1.1 null parasites reached lower cell density (Figure 3) [150].
Along with CK1 kinases, Leishmania parasites encode for two CK2 isoforms. Many studies have described CK2 activity in Leishmania species, such as L. tropica [76], L. major [144], L. amazonensis [151] and L. donovani [152]. CK2 isoforms have been reported to be extensively distributed in Leishmania from the surface of the parasitic cells to the cytoplasm, the nucleus and as a secreted kinase, possessing ecto-CK2, intracellular CK2 and secreted CK2 activity, respectively [37,75,76,151,152]. It actually seems that CK2 is located in different cell compartments and it responds to different extracellular protein targets, suggesting that CK2, although proven dispensable for the parasite's viability, can serve as a chemotherapeutic target due to its multifunctionality. It was shown that CK2 is vital for the growth of the virulent parasites, as CK2 inhibition with heparin, DRB (5,6dichlorobenzimidazone-1-β-D-ribofuranoside) and TBB (4,5,6,7-tetrabromobenzotriazole) resulted in growth inhibition only in a virulent parasite strain (Table 1, Figure 3) [75]. Moreover, in L. major and L. amazonensis, the kinase seems to influence not only the growth and morphology of the parasites, but also the infection and/or survival within macrophages in vitro and in vivo, in a BALB/c animal model (Figure 3) [76,151]. It has been observed that secreted CK2 seems to play an important role during the host infection. In L. chagasi (or L. infantum), mRNA levels of the kinase differ between the different life stages of the parasite, a fact that also indicates its importance of the parasite survival in the host. There is also an indication that LcCK2A controls the gene expression during the parasite's life cycle. Highly virulent L. braziliensis promastigotes secrete increased levels of CK2 whose substrates are presented by macrophages or human serum, while iNOS and arginase, two mammalian enzymes responsible for the parasite survival in the macrophages, seem to activate LCK2 [144,152]. Dutra et al. demonstrated that platelet activating factor (PAF) also activates the secreted and the membrane-bound CK2 [76] and induces parasite differentiation in L. tropica [153]. All the above advocate that CK2 activation promotes parasite-host interactions. Moreover, CK2 inhibitors TBB and DRB inhibited the stimulation of macrophage infection, suggesting that CK2 can also serve as a target in order to cease parasite invasion and differentiation in the macrophages [76].

Aurora Kinases
Aurora kinases are known mediators of the cell division cycle in eukaryotes [154]. Since they are considered important mitotic serine/threonine kinases, Aurora kinases are well studied in a variety of organisms. In L. mexicana, three Aurora kinases have been identified (AUK3-LmxM. 26.2440, AUK1/AIRK-LmxM.28.0520, AUK2-LmxM.08_29.1330) [37], while in L. major, the role of one Aurora kinase member (LmAIRK-AUK1 homologue) has been investigated [155][156][157]. A computational analysis identified the putative Aurora-like kinase in L. donovani (LdAIRK), the LmAIRK homologue, as a potential drug target [78]. Knockout LdAIRK parasites could not be generated, a fact that strengthens the hypothesis that the kinase is important for viability [78]. Another important feature of the kinase is that although in mammalian cells, Aurora kinases play a role in mitosis, cytokinesis and chromosome segregation, in L. major, one gene of Aurora kinase, namely LmAIRK, may regulate all these processes [156]. With a main nuclear localization throughout the cell-cycle, LdAIRK seems to temporarily migrate from the cytoplasm to the nuclear periphery and ultimately to the spindle poles during the early mitosis and post mitosis [78]. Based on the importance of the role that the kinase plays in the life cycle of the parasite, it emerged as a potential chemotherapeutic target. The advantage of repositioning mammalian Aurora kinase inhibitors in order to evaluate their antileishmanial activity and to assess their in-hibitory activity against the leishmanial homologue, provides an advantage for discovering new treatment that targets the Aurora kinase in Leishmania. Among the repositioned Aurora inhibitors, hesperadin (Table 1) was found to possess a strong antileishmanial activity, as parasites incubating with the inhibitor displayed an accumulation of cells in G2/M phase that finally led to the loss of cellular and cytoskeletal integrity (Figure 3). The above results imply that LdAIRK has a crucial role in cytokinesis and cell-cycle progression [78,79].

Other Kinases
Apart from the parasite kinases mentioned above, an increasing number of studies are shedding light on the role of new kinases outside the aforementioned groups. For instance, a new gene was recently identified in L. donovani, namely Ld-RAC/Akt-like gene (and its L. panamensis orthologue Lp-RAC/Akt-like gene), which expresses a protein with major mammalian Akt hallmarks, including the typical pleckstrin homology domain, protein kinase and AGC kinase domains, but with only 26.5% identity with mammalian Akt1 [158,159]. This kinase is considered a putative molecular target to treat leishmaniasis, and there is evidence that is essential for parasitic survival [82]. Thus far, Devki Nandan et al. showed that Miransertib, a mammalian Akt inhibitor, possesses inhibitory activity against both Leishmania promastigotes and intracellular amastigotes and indicated that the homologue leishmanial Akt is the probable target of the inhibitor (Table 1) [82].
Moreover, the L. donovani eukaryotic initiation factor 2 alpha (eIF2α) kinase (LdeK1), which regulates translation under stress conditions (nutrient starvation, acidic pH, high temperature) through the phosphorylation of eIF2α, has emerged as a candidate for vaccine development against leishmaniasis [160,161]. The above hypothesis was supported by the fact that LdeK1 modulates the immune response towards host protection as in vitro and in vivo infection, with LdeK1 negative mutants resulting in reduced parasite burden in macrophages and in splenic/hepatic load, respectively (Figure 3), and in enhanced pro-inflammatory cytokines and nitric oxide levels with parallel reduced levels of TH 2 and Treg populations [160].
The Ser/Thr kinase LmjF. 22.0810 has been characterized as a likely essential protein for viability and seems to be involved in the drug response of Leishmania. More specifically, its sensitivity towards aminoglycosides such as paromomycin (Table 1) is potentially correlated to the mechanism of drug resistance in Leishmania parasites [83]. Another kinase (Ld_f1), a homologue of bacterial-zeta-toxin was recently identified in L. donovani possessing UNAG kinase and ATP-binding activity. Ld_f1 is tightly correlated with cell death and is a putative target candidate for development novel antileishmanial chemotherapeutics [162].

Future Directions-Concluding Remarks
On the basis of the drawbacks of the current chemotherapy for the treatment of leishmaniasis (emerging resistance, cost, toxicity), the discovery of new antileishmanial drugs and the development of new treatments may be urgent, but appears challenging. Protein kinases serve as ideal targets for rational drug design. In general, eukaryotic protein kinases play a fundamental role in the survival and/or virulence of Leishmania parasites, and this fact places them in the center of attention for designing novel antileishmanial small molecule inhibitors. The understanding of the role of each specific parasitic kinase can expand our knowledge on the mechanisms used by Leishmania parasites to adapt in the host and to establish infection, and can be used as an exploitable tool to fight the disease. Although there are new studies that focus on leishmanial ePKs and in their potential role as molecular targets for rational drug design, more efforts are needed in the field. The availability of the crystal structures of certain leishmanial kinases could speed up the discovery of molecules inhibiting their activity, with relevance to antileishmanial drug development. Moreover, more efforts are needed to establish screening platforms of important kinases, and technical issues need to be addressed, including the knowledge of substrates, the development of purification protocols enabling the isolation of active form of the protein. All the above knowledge will aid towards the discovery of novel and specific antileishmanials, and in combination with new technologies such as nanotechnology approaches for optimal drug delivery, it is expected to provide us with a new perspective in the battle against leishmaniasis.
Funding: This work was supported by the Action "KRIPIS I" (MIS 450598), which was co-financed by European Union and the National Ministry of Education and Religion Affairs under the Operational Strategic Reference Framework (NSRF 2014-2020) and the General Secretariat of Research and Technology (GSRT,) and Hellenic Pasteur Institute.

Conflicts of Interest:
The authors declare no conflict of interest.