Tropical Biomedicine 37(3): 822–841 (2020)

Review Article

Plasmodium falciparum protein kinase as a potential therapeutic target for antimalarial drugs development

Mahmud, F.1,3, Lee, P.C.3, Abdul Wahab, H.2, Mustaffa, K.M.F.1, Leow, C.H.1, Azhar, R.4 and Lai, N.S.1* 1Institute for Research in Molecular Medicine, Universiti Sains Malaysia 2School of Pharmaceutical Science, Universiti Sains Malaysia 3Faculty of Science and Natural Resources, Universiti Malaysia Sabah 4Department of Zoology, Government College University Faisalabad, Pakistan *Corresponding author e-mail: [email protected] Received 24 February 2020; received in revised form 17 June 2020; accepted 19 June 2020

Abstract. is one of the most dangerous infectious diseases due to its high infection and mortality rates, especially in the tropical belt. falciparum (P. falciparum), the most virulent malaria parasite in humans, was recently reported to develop resistance against the final efficient antimalarial drug, . Little is known about the resistance mechanisms, which further complicates the problem as a proper counteraction is unable to be taken. Hence, the understanding of drug mode of action and its molecular target is valuable knowledge that needs to be considered to develop the next generation of antimalarial drugs. P. falciparum protein kinase (Pf PK) is an attractive target for antimalarial chemotherapy due to its vital roles in all P. falciparum life stages. Moreover, overall structural differences and the presence of unique Pf PKs that are absent in human kinome, suggesting specific inhibition of Pf PK without affecting human cells is achievable. To date, at least 86 eukaryotic protein kinases have been identified in P. falciparum kinome, by which less than 40 were validated as potential targets at the erythrocytes stage. In this review, recent progress of the furthest validated Pf PKs; Pf Nek-1, Pf CDPK1, Pf CDPK4, Pf PKG, and Pf CLK-3 will be briefly discussed.

INTRODUCTION and 2.1% in the Mediterranean), by which P. falciparum causes 71% of the total (P. falciparum) is malaria infections. Malaria also accounted a protozoan parasite that causes the most for almost half a million deaths annually since virulent malaria cases in humans, followed 2010, mostly children aged under 5 (67% in by (P. vivax) and 2018) (WHO, 2019). . Meanwhile, mild or P. falciparum is an obligate parasite that moderate cases of malaria infections are requires two hosts, which are the invertebrate inflicted by and (female sp. mosquito as a vector) Plasmodium ovale (Rajahram et al., 2012; and vertebrate, to complete its sexual and Tougan et al., 2020). Malaria is currently asexual lifecycle, respectively (Singleton, listed as one of the top 3 infectious diseases 2020). In humans, Plasmodium asexual in the world due to its high infection and life cycle is divided into exoerythrocytic (in mortality rates (Osborn et al., 2020). As the liver) and intraerythrocytic (in the red reported by WHO in the latest Malaria report, blood cell, RBC) stages. Saliva injected by 228 million malaria cases were recorded in an infected mosquito will transfer sporozoites 2018 (93% in Africa, 3.4% in Southeast Asia, into the bloodstream and enter liver cells. The

822 entry of sporozoites into the hepatocytes is (Figure 1) (Cowman et al., 2012; Abdel- highly precise due to the presence of the Ghaffar et al., 2019). circumsporozoite protein that bears a Most of the antimalarial class introduced ligand that only binds specifically to the so far are targeting Plasmodium at the liver basolateral (hepatocyte cell RBC stage, such as 4-aminoquinolines membrane). The entry of sporozoites will (, amodiaquine, piperaquine), initiate the asexual reproduction, known as amino alcohols (quinine, lumefantrine, the pre-erythrocytic (PE) or primary , halofantrine) and antibiotics exoerythrocytic schizogony (EE) cycles. (tetracycline, doxycycline, clindamycin) Sporozoites then metamorphose into (Maerki et al., 2006; Wilson et al., 2013). The and are further transformed into RBC stage is an attractive target for drug schizont and merozoites through schizogony development owing to its apparent (Miller et al., 2013; Burda et al., 2017). importance for Plasmodium survival. In The erythrocytic cycle starts once addition, RBC stage screens also require a merozoites leave the liver cell to penetrate simple experimental setup for a vast RBC. The merozoites then once again turn compound library to be tested. Hence, asexual into trophozoites (early trophozoites or blood-stage will continue to be the main ring-stage and late trophozoites). Next, the target in drug development (Baragaña et al., late trophozoites developed into blood-stage 2015; Vos et al., 2015; Swann et al., 2016). schizont and released from ruptured RBC However, this approach causes most of together with metabolic waste and residual the antimalarial compounds were developed bodies (responsible for the characteristic without the knowledge of their mode of action symptoms of malaria). After an indeterminate as their activities were evaluated based on number of asexual cycles, some of the phenotypic assays. Their limited therapeutic merozoites will enter erythrocytes and window also causes quicker drug-resistance become macro- and microgamonts to be development. Plasmodium has been ingested by the mosquito for sexual described as unique compared with other reproduction and the cycle is repeated infectious diseases because they are capable

Figure 1. The life cycle of Plasmodium falciparum. (Created with BioRender.com)

823 of inducing specific resistance in the cellular processes such as growth control, cell cycle target of the antimalarial drugs instead of regulation, signal transduction, and crucial random mutations (Goldberg et al., 2012). for parasite invasion into human RBC (Soni For instance, the emergence of resistance et al., 2017; Matthews et al., 2018; Wilde et against chloroquine (acting on al., 2019). and schizont) and mefloquine (acting on ring- Moreover, recombinant Pf PKs can be stage and merozoite) were reported in 12 and easily adapted into a high throughput 5 years after their introduction, respectively assay system against an unlimited number (Maerki et al., 2006; Wilson et al., 2013; of compounds. It leads to the identification WHO, 2014). Mutation of pfcrt and pfmdr1 of numerous new scaffolds of Pf PKs has been consistently reported as the source inhibitor, which was never reported as of resistance against blood schizonticides in antimalarial (Gamo et al., 2010; Hallyburton P. falciparum (Pulcini et al., 2015; Agrawal et al., 2017; Penzo et al., 2019). With a proper et al., 2017). target, Pf PKs may provide a new opportunity For this reason, artemisinin is widely for antimalarial drug development. accepted as the most effective antimalarial Bioinformatics analysis predicted that the drugs ever introduced due to its ability to P. falciparum genome is expressing at inhibit P. falciparum at all parasite life least 86 genes encoding for a stages. Although artemisinin significantly protein kinases (ePKs), following Hanks delays the development of resistance strain, kinase classification model, excluding the artemisinin-resistance strain was finally atypical protein kinases (aPKs) such as reported in Southeast Asia, 40 years after its phosphatidylinositol 3-kinase-related kinases introduction (1972-2009) (Dondorp et al., (PIKK) and right open reading frame (RIO) 2009; Su and Miller, 2015). Moreover, the kinase (Hanks and Hunter, 1995; Ward et al., actual target and mode of action of 2004; Anamika et al., 2005; Miranda- artemisinin are not well defined, which Saavedra et al., 2012). causes the identification of resistance Based on their structural features, these mechanisms in P. falciparum is yet to be ePKs superfamilies can be further divided fully understood. So far, the mutation of into those with and without human homologs. 3D7K13 and VPS34myc are described as the Plasmodium ePK with human homologs are possible sources of resistance in artemisinin- CMGC (named after the initials of its resistance strains (Bhattacharjee et al., members; CDK, MAPK, GSK3, CK2) (18 2018; Rocamora et al., 2018). Being the last kinases/21%), PKs A, G and C (AGC/AGC- efficient drug against P. falciparum, the related) (8 kinases/9%), never in mitosis gene recent issue associated with artemisinin is a (NIMA) (4 kinases/5%), casein kinase 1 the most significant problem in malaria (CK1/CK1-related) (2 kinases/2%), tyrosine control (Cui and Su, 2009; Ross and Fidock, kinase-like kinases (TKL/TKL-related) (5 2019). kinases/6%) and “sterile-phenotype” kinases (STE) (2 kinases/2%). Meanwhile, more than Plasmodium falciparum protein kinase 50% of Plasmodium ePKs are without human as a potential target homologs which comprise of calmodulin- Based on the current problems, the next line dependent protein kinase (CaMK) (17 kinase/ of antimalarial agents should be identified 20%), and orphan kinase (which include based on an assay with a specific target, eukaryotic initiation factor-2 (eIF2), and a paired with the result of phenotypic or in vivo novel protein family named after a conserved studies. Plasmodium falciparum protein Phe-Ile-Lys-Lys motif (FIKK)) (30 kinases/ kinase (Pf PK) is now gaining attention due 35%) (Sullivan et al., 2004; Ward et al., 2004; to the accumulated knowledge of human Doerig et al., 2008; Abdi et al., 2010; kinase and successful chemotherapy Solyakov et al., 2011; Talevich et al., 2012; application, targeting kinase protein Proellocks et al., 2016; Murungi and Kariithi, (Dondorp et al., 2010). In P. falciparum, Pf 2017; Zhang et al., 2018; Davies et al., 2020) PK plays a fundamental role in numerous (Table 1).

824 Table 1. The list of eukaryotic protein kinases in Plasmodium falciparum and their current status as potential targets at the asexual stage

Genetic No Gene ID Group Family Name validation (RBC stage)

1 PF3D7_1431500 CMGC MAPK Pf map-1 Dispensable (PF14_0294) 2 PF3D7_1113900 CMGC MAPK Pf map-2 Essential (PF11_0147) 3 PF3D7_1014400 CMGC CDK Pf mrk Essential (PF10_0141) 4 PF3D7_0417800 CMGC CDK Pf crk-1 Essential (PFD0865c) 5 PF3D7_0415300 CMGC CDK Pf crk-3 Essential (PFD0740w) 6 PF3D7_0317200 CMGC CDK Pf crk-4 Essential (PFC0755c) 7 PF3D7_1338900 CMGC CDK Pf crk-4-related Dispensable (MAL13P1.196) 8 PF3D7_0615500 CMGC CDK Pf crk-5 Dispensable (PFF0750w) 9 PF3D7_1356900 CMGC CDK Pf PK5 Essential (MAL13P1.279) 10 PF3D7_1337100 CMGC CDK Pf PK6 Essential (PF13_0206) 11 PF3D7_1445400 CMGC CDK-like Pf CLK-1/ Essential (PF14_0431) Pf Lammer 12 PF3D7_144300 CMGC CDK-like Pf CLK-2 Essential (PF14_0408) 13 PF3D7_1114700 CMGC CDK-like Pf CLK-3 Essential (PF11_0156) 14 PF3D7_0302100 CMGC CDK-like Pf CLK-4/ Essential (PFC0105w) Pf SRPK1 15 PF3D7_1108400 CMGC CK2 Pf CK2 Essential (PF11_0096) 16 PF3D7_0312400 CMGC GSK3 Pf GSK3 Essential (PFC0525c) 17 PF3D7_0821100 CMGC GSK3 Pf PK1 Dispensable (PF08_0044) 18 PF3D7_1316000 CMGC Pf GSK3-related Essential (MAL13P1.84)

19 PF3D7_0217500 CaMK CDPK Pf CDPK1 Essential (PFB0815w) 20 PF3D7_0610600 CaMK CDPK Pf CDPK2 Essential (PFF0520w) 21 PF3D7_0310100 CaMK CDPK Pf CDPK3 Essential (PFC0420w) 22 PF3D7_0717500 CaMK CDPK Pf CDPK4 Dispensable (PF11_0060) 23 PF3D7_1337800 CaMK CDPK Pf CDPK5 Essential (PF13_0211) 24 PF3D7_1122800 CaMK CDPK Pf CDPK6 Dispensable (PF11_0239) 25 PF3D7_1123100 CaMK CDPK Pf CDPK7 Dispensable (PF11_0242) 26 PF3D7_1423600 CaMK Pf CDPK6-related Dispensable (PF14_0227) 27 PF3D7_1450000 CaMK Pf CDPK6-related Dispensable (PF14_0476) 28 PF3D7_0715300 CaMK Pf CDPK6-related Dispensable (MAL7P1.73)

825 Table 1 continued...

29 PF3D7_0704500 CaMK Pf CDPK6-related Dispensable (MAL7P1.18) 30 PF3D7_1104900 CaMK Pf CDPK6-related Dispensable (PF11_0060) 31 PF3D7_0311400 CaMK Pf PKRP Dispensable (PFC0485w) 32 PF3D7_0214600 CaMK Pf STK2 Dispensable (PFB0665w) 33 PF3D7_1238900 CaMK Pf PK2 Essential (PFL1885c) 34 PF3D7_131500 CaMK Pf PK9 Essential (PF13_0085) 35 PF3D7_1454300 CaMK Pf KIN Essential (PF14_0516)

36 PF3D7_0203100 STE Pf PK8 Dispensable (PFB0150c) 37 PF3D7_0605300 STE-related Aurora Pf Ark-1 Essential (PFF0260w/MAL6P1.56)

38 PF3D7_0309200 AGC-related Aurora Pf Ark-2 Essential (PFC0385c) 39 PF3D7_1356800 AGC-related Aurora Pf Ark-3 Essential (MAL13P1.278)

40 PF3D7_0934800 AGC Pf PKA Essential (PFI1685w) 41 PF3D7_1246900 AGC Pf PKB Essential (PFL2250c) 42 PF3D7_1436600 AGC Pf PKG Essential (PF14_0346)

43 PF3D7_1145200 AGC-related Essential (PF11_0464) 44 PF3D7_1148000 AGC-related Essential (PF11_0488) 45 PF3D7_1441300 AGC-related Dispensable (PF14_0392)

46 PF3D7_0211700 TKL Pf TKL-1 Essential (PFB0520w) 47 PF3D7_1121300 TKL Pf TKL-2 Dispensable (PF11_0220) 48 PF3D7_1349300 TKL Pf TKL-3 Essential (PF13_0258) 49 PF3D7_0623800 TKL Pf TKL-4 Dispensable (PFF1145c/MAL6P1.191) 50 PF3D7_0926300 TKL-related Unknown (PFI1290w)

51 PF3D7_1228300 NIMA Pf nek-1 Essential (PFL1370w) 52 PF3D7_0525900 NIMA Pf nek-2 Dispensable (PFE1290w) 53 PF3D7_1201600 NIMA Pf nek-3 Dispensable (PFL0080c) 54 PF3D7_0719200 NIMA Pf nek-4 Dispensable (MAL7P1.100)

55 PF3D7_1136500 CK1 Pf CK1 Essential (PF11_0377)

56 PF3D7_0926100 CK1-related Dispensable (PFI1280c)

826 Table 1 continued...

57 PF3D7_0107600 Orphan eIF2α kinase Pf eiK1 Dispensable (PFA0380w) 58 PF3D7_1444500 Orphan eIF2α kinase Pf eiK2 Dispensable (PF14_0423) 59 PF3D7_0628200 Orphan eIF2α kinase Pf PK4 Essential (PFF1370w) 60 PF3D7_1428500 Orphan Unknown (PF14_0264) 61 PF3D7_0718100 Orphan Pf EST Essential (MAL7P1.91) 62 PF3D7_0213400 Orphan Pf PK7 Dispensable (PFB0605w) 63 PF3D7_1247500 Orphan Dispensable (PFL2280w) 64 PF3D7_0102600 Orphan FIKK Pf FIKK1 Dispensable (PFA0130c) 65 PF3D7_0301200 Orphan FIKK Pf FIKK3 Dispensable (PFC0060c) 66 PF3D7_0424500 Orphan FIKK Pf FIKK4.1 Dispensable (PFD1165w) 67 PF3D7_0424700 Orphan FIKK Pf FIKK4.2 Dispensable (PFD1175w) 68 PF3D7_0500900 Orphan FIKK Pf FIKK5 Dispensable (PFE0045c) 69 PF3D7_0726200 Orphan FIKK Pf FIKK7.1 Dispensable (MAL7P1.144) 70 PF3D7_0731400 Orphan FIKK Pf FIKK7.2 Dispensable (MAL7P1.175) 71 PF3D7_0805700 Orphan FIKK Pf FIKK8 Essential (MAL8P1.203) 72 PF3D7_0902000 Orphan FIKK Pf FIKK9.1 Dispensable (PFI0095c) 73 PF3D7_0902100 Orphan FIKK Pf FIKK9.2 Dispensable (PFI0100c) 74 PF3D7_0902000 Orphan FIKK Pf FIKK9.3 Dispensable (PFI0105c) 75 PF3D7_0902300 Orphan FIKK Pf FIKK9.4 Dispensable (PFI0110c) 76 PF3D7_0902400 Orphan FIKK Pf FIKK9.5 Dispensable (PFI0115c) 77 PF3D7_0902500 Orphan FIKK Pf FIKK9.6 Dispensable (PFI0120c) 78 PF3D7_0902600 Orphan FIKK Pf FIKK9.7 Dispensable (PFI0125c) 79 PF3D7_1016400 Orphan FIKK Pf FIKK10.1 Dispensable (PF10_0160) 80 PF3D7_103900 Orphan FIKK Pf FIKK10.2 Dispensable (PF10_0380 R45) 81 PF3D7_1149300 Orphan FIKK Pf FIKK11 Dispensable (PF11_0510) 82 PF3D7_1200800 Orphan FIKK Pf FIKK12 Dispensable (PFL0040c) 83 PF3D7_1371700 Orphan FIKK Pf FIKK13 Dispensable (MAL13P1.109) 84 PF3D7_1476400 Orphan FIKK Pf FIKK14 Dispensable (PF14_0733/0734) 85 PF3D7_0928800 Orphan Unknown (PFI1415w) 86 PF3D7_1433900 Orphan Unknown (PF14_0320)

827 Few notable differences exist between phenotypic (Pf culture and enzymatic assay), P. falciparum and human kinome. In general, and in vivo levels (P. berghei infected or most Plasmodium kinase shares around 30- humanized P. falciparum-infected mouse 60% of sequence identity to human kinases model). (Doerig, 2004; Ruiz-Carrillo et al., 2018; Alam et al., 2019). Moreover, typical mammalian a) P. falciparum NIMA related protein kinase group such as tyrosine kinase (TyrK) kinase 1 (Pf nek-1) and (STE) are missing and exist in a low Pf nek-1 (along with Pf nek-2 to nek-4) is number in malaria kinome, respectively. a member of P. falciparum kinome that Instead, most malaria kinome contains shows the highest homology to the NIMA/ kinases such as FIKK (lacking the canonical NIMA-like kinase (Nek family). Pf nek-1 is glycine-rich motif in its ATP binding site, the only member of P. falciparum NIMA replaced with FIKK motif) and CDPKs that kinase whose mRNA is detectable throughout cannot be found in mammalian cells (Lin the RBC stage (hence, the only essential et al., 2017; Ghartey-Kwansah et al., 2020). member) and male (Dorin et Besides, the size of the ATP-gatekeeper in al., 2001; Dorin-Semblat et al., 2011). Plasmodium kinase is mostly guarded by Structurally, Pf nek-1 possesses an extension small amino acid subunits, exposing its on its C-terminal of the catalytic domain. In hydrophobic pocket in the ATP-binding comparison to mammalian homolog (such site (Huang et al., 2010; Tewari et al., 2010; as mouse nek-1 and human nek-2), the FXXT Bansal et al., 2016). It can be exploited to motif typically found in mammalian NIMA identify a class of ATP-competitive inhibitor kinase is substituted by SMAHS motif in Pf targeting this Pf PK hydrophobic pocket nek-1 (Dorin et al., 2001). This motif is unique as most human kinases possess a bulky against its mammalian homolog protein and ATP-gatekeeper, preventing interaction other Plasmodium kinases, making it an (Van Voorhis et al., 2017). Such differences attractive target (Kappes et al., 1999; Tanaka indicate that species-selective inhibition is and Nigg, 1999). possible for antimalarial chemotherapy. The importance of nek-1 protein on Plasmodium survival during the RBC stage The most studied P. falciparum was initially predicted from the failure to eukaryotic protein kinases develop the nek-1 knockout (KO) in P. berghei In general, the approach to target Pf PKs strain (Tewari et al., 2010). Pf nek-1 was then is still underexplored for antimalarial confirmed as an essential protein during the chemotherapy. However, continuous P. falciparum RBC stage based on a reverse progress made so far has enriched our genetic approach. Wild type (WT) and mutant understanding of the physiological P. falciparum 3D7 parasites bearing active importance, regulation, and structural and inactive Pf nek-1 (lacking in a subdomain features of Pf PK, which will soon pave for enzymatic activity), respectively, were a pathway for Pf PK inhibitor to reach subjected to blasticidin selection. PCR the clinical stage. This assumption is analysis was performed to detect the strengthened by the first Plasmodium presence of WT and disrupted pfnek-1 locus. lipid/protein kinase inhibitor reaching It was later confirmed that no integrated clinical test (MMV390048), targeting the fragment was amplified (mutated Pf nek-1), ATP-binding site of Pf PI4K (aPK), a general implying the importance of Pf nek-1 for feature shared by all kinases (Paquet et al., P. falciparum growth at the RBC stage 2017). So far, almost 40 Pf PKs have been (Dorin-Semblat et al., 2011). genetically validated as a druggable target Pf nek-1 was identified as a druggable at the RBC stage (Abdi et al., 2010; Solyakov target for antimalarial chemotherapy, as et al., 2011; Zhang et al., 2018; Davies et al., indicated by the moderate antimalarial 2020) (Table 1). However, only a total of five activity of xestoquinone (isolated from Pf PKs have been validated as drug targets Vanuatu marine sponge Xestospongia sp.) at genetic (knockout Plasmodium strain), on P. falciparum culture (Pf IC50=3 µM) and

828 recombinant Pf nek-1 (Pf nek-1 IC50=1 µM). in humans but more related to the kinases of This compound also displayed specific some algae, plant, and (Doerig et activity as it showed weak to no activity al., 2005; Kadian et al., 2017; Ghartey- on other P. falciparum and human (Homo Kwansah et al., 2020). CDPKs are composed sapiens, Hs) kinases such Pf PK5, Pf PK7, of four separate regions with distinct Pf GSK-3, Hs GSK-3α/β, Hs CDK1 Hs CDK5 functions. The N-terminal plays two critical and Hs PKA. Although xestoquinone was roles; variable regions (responsible for shown to inhibit P. berghei in vivo, its substrate recognition, localization, and relation with nek-1 inhibition was not interaction with target proteins) and the confirmed. Besides, total parasitemia catalytic kinase domain (catalyzes the clearance was not achievable at 5 mg/kg phosphorylation of serine/threonine residues (47% clearance), and the mice did not survive in the substrate). Meanwhile, the C-terminal at 20 mg/kg dose (Laurent et al., 2006). (a calmodulin-like domain (CLD)) is crucial Subsequently, alisiaquinones A (related for calcium-binding assisted by the EF-hand to xestoquinone, also purified from marine motifs and the J-domain (junction domain sponges), was shown to inhibit both that is also referred as a calcium activation chloroquine-sensitive and -resistance P. domain (CAD)) leading to the enzyme falciparum strains with the IC50 of <10 µM. activation (Wernimont et al., 2010). A total This compound was also shown to com- of seven members of CDPKs have been pletely inhibited Pf nek-1 activity at 50 µM. identified so far (CDPK1-7), by which Pf Although alisiaquinones A showed slightly CDPK1 and Pf CDPK4 are the most better parasitemia in vivo clearance (63% progressed as both have been validated up clearance at 5 mg/kg) than xestoquinone, its to in vivo studies (Zhang and Choi, 2001; mortality rate at 20 mg/kg was also 100%. Harper and Harmon, 2005; Nagamune et al., Due to its toxic activity, alisiaquinones A was 2008). not further pursued as an antimalarial agent Pf CDPK1 is mainly involved in the (Desoubzdanne et al., 2008). motility of parasite invasion of the host RBC Numerous studies reported other Pf by acting as a Ca2+-dependent effector for nek-1 inhibitors such as roscovitine (Pf microneme secretion and critical in the nek-1 IC50=10 µM, Pf 3D7 IC50=10.48 µM), formation of both male and female gametes compound 97 (purvalanol derivatives) (Pf (Kumar et al., 2017; Bansal et al., 2018). Pf nek-1 IC50=0.2 µM), hymenialdisine 01 (Pf CDPK1 is validated as a druggable target nek-1 IC50=4 µM) (Dorin et al., 2001; Houzé based on failed initial attempts to produce a et al., 2014), methyl (2,4-dibromo-3,6- Pf CDPK1 KO strain, indicating its essential dihydroxyphenyl) acetate (Pf nek-1 IC50= roles for parasite survival at the RBC stage 1.8 µM, Pf FcB1 IC50=12 µM) (Lebouvier et (Kato et al., 2008; Solyakov et al., 2011; al., 2009) and recently compound 17/18 Zhang et al., 2018). Numerous studies (both with Pf nek-1 IC50=0.05 µM) (Penzo indicated successful Plasmodium clearance et al., 2019). However, none of this study by Pf CDPK1 inhibitors based on in vitro managed to established the connection phenotypic and enzymatic assays. For between Plasmodium nek-1 inhibition and instance, 2,6,9-trisubstituted purines which parasitemia clearance in vivo. More inhibited the growth of P. falciparum 3D7 research is needed as this enzyme is an culture (Pf 3D7 IC50=230 nm) and attractive target for antimalarial chemo- recombinant Pf CDPK1 (Pf CDPK1 IC50= therapy, given its unique structure and 17 nm) (Kato et al., 2008). Besides, importance for Plasmodium survival. imidazopyridazine was also reported to show antimalarial activity (Pf 3D7 IC50=0.034 b) P. falciparum calcium-dependent µM) mediated by the inhibition of Pf CDPK1 kinase 1 (Pf CDPK1) (Pf IC50=0.008 µM) (Green et al., 2016). Calcium-dependent kinases (CDPKs) Based on the phenotypic and recom- represent a unique class of Plasmodium ePK binant assays, the inhibition of Pf CDPK1 for drug target since they have no homologs causes accumulation of P. falciparum at late

829 schizogony. It was also suggested that Pf organism as it encodes all members of Pf CDPK1 inhibition might affect P. falciparum CDPKs except for Pf CDPK2. The Pb CDPK4 during gametocytes that were later gene disruption was shown to cause sexual confirmed based on the gamete abnormality impair and altered the process of sporozoite observed in the recent development of P. invasion of hepatocyte cells (Billker et al., falciparum CDPK1 KO strains. This KO strain 2004; Govindasamy et al., 2016). was generated using the CDPK1 T145M Hence, CDPK4 in P. falciparum is likely parasite, a mutant P. falciparum strain. This to be essential to maintain normal malaria strain showed a slower growth rate than WT life cycle, transmission, crucial for the and the unaltered CDPK1 T145M strains. transition of gametocytes into microgamete, In addition, the CDPK1 KO parasite also male exflagellation in the produced an abnormal gamete and failed to mosquito midgut and cell invasion (Billker infect the mosquitoes. This finding validated et al., 2004; Tewari et al., 2010; Bansal et al., Pf CDPK1 as an essential enzyme in both the 2018; Fang et al., 2018). As most of the sexual and asexual stages (Bansal et al., known antimalarial drugs are targeting P. 2018). falciparum during the RBC stage, Pf CDPK4 A total of 47 imidazopyridazine series is an exciting target for drug development were synthesized from imidazopyridazine- (Ahmed et al., 2012). To identify Pf CDPK4 compound 1 (as an early lead), to improve inhibitors, a series of kinase inhibitors that its potency against Pf 3D7 parasite culture, showed no activity on humans proteins recombinant Pf CDPK1 and P. berghei (in known as “Bumped kinase inhibitor” (BKI) vivo). Compound 10 and 41 were sub- were tested (Ojo et al., 2012). sequently identified as the most potent BKIs are compounds with pyrazolo- compounds as both compounds showed Pf pyrimidine backbone that was designed with 3D7 EC50 of <0.1 µm and Pf CDPK1 IC50 of a large R1 and R2 groups so that it will show <15 nm, compared with compound 1 Pf 3D7 no or little effect on human cells. The ATP EC50 of <0.1 µm and Pf CDPK1 IC50 of <50 binding pocket of human kinases is mostly nm. However, a clear connection was not guarded by bulky ATP-gatekeepers, thus established between in vitro and in vivo preventing large compounds from binding studies since compound 41 showed moderate (Uo et al., 2018). Based on the prior P. berghei clearance in vivo tested at 50 screening of BKIs on CDPK1 of Toxoplasma mg/kg (about 50% reduction, which is gondii (T. gondii) and similar to compound 1). It might be parvum (C. parvum), few selective contributed by the differences in CDPK1 inhibitors were identified that showed no importance and biology between P. activity on human kinases. This selectivity falciparum and P. berghei (Chapman et al., is due to a small ATP-gatekeeper (glycine) 2014). of these enzymes (Ojo et al., 2010; Tewari et al., 2010). Since Pf CDPK4 also possesses c) P. falciparum calcium-dependent a small gatekeeper, many of these BKIs kinase 4 (Pf CDPK4) also inhibited Pf CDPK4 activity, by which Pf CDPK4 was shown to interact with Pf PKG BKI-1 being the most potent (IC50=4 nM) (Ojo at the asexual erythrocyte stage, especially et al., 2012). at the final stage of the schizont or invasion As expected, BKI-1 did not inhibit the of the new RBC. However, the knockdown growth of parasite at the RBC stage, but of Pf CDPK4 alone does not affect parasite showed potent in vivo inhibition of P. growth, which indicates its disposable role falciparum exflagellation (EC50=35 nm), during the RBC stage (Fang et al., 2018). possibly mediated by Pf CDPK4 inhibition. Subsequently, the role of CDPK4 during the BKI-1 was then tested on P. berghei infected Plasmodium sexual phase was deduced mice at 10 mg/kg, and it exerted no effect on based on the knockout of P. berghei CDPK4 asexual parasitemia or gametocyte rates, (Pb CDPK4). Mice malaria is a suitable model but the dose adequate to block exflagellation.

830 Anopheles mosquitoes were then allowed to identified (type I and type II) that exist as feed on the infected mice, 30 minutes after homodimers (Pearce et al., 2010). PKG BKI-1 administration (10 mg/kg). Complete ortholog in apicomplexan parasites, including inhibition of oocyst production in the P. falciparum, was first identified in early mosquito midgut was observed. At this point, 2000 (Gurnett et al., 2002). In general, P. falciparum exflagellation inhibition was apicomplexan PKG exists as three cGMP- strongly suggested due to the inhibition of subunit. However, Pf PKG showed striking Pf CDPK4 by BKI-1. However, it was not structural features as it has the fourth confirmed as the generation Pf CDPK4 cGMP binding site known as the “degenerate mutant for further study was failed. In fourth site” (Deng et al., 2003). Besides, Pf addition, BKI-1 bioavailability needs to be PKG also equipped with a smaller ATP- prolonged for better parasite gametocytes gatekeeper subunit (threonine) than human clearance (Ojo et al., 2012). PKGs (Huang et al., 2010). To improve the activity of BKI-1, Pf PKG is an attractive drug target due compound 1294 (BKI-1294) was synthesized to its role as a regulator of other Pf PKs that showed a reduced rate of microsomal such as Pf CDPK5, suggesting its function metabolism while retaining comparable at various P. falciparum lifecycle phases inhibitory to BKI-1. BKI-1294 blocked P. such as asexual (RBC, liver) (Deng and falciparum gametocyte exflagellation with Baker, 2002; Falae et al., 2010) and sexual the EC50 of 35 nm (BKI-1, EC50=47 nm) and stages (gametogenesis and ookinete motility) anti-Pf CDPK4 activity with the IC50 of 10 nm (McRobert et al., 2008; Brochet et al., 2014; (BKI-1, IC50=4 nm). BKI-1294 was also Alam et al., 2015). The importance of Pf PKG confirmed to exert transmission-blocking during the asexual and sexual stage was first activity, evaluated with untransfected WT recognized based on its high level of mRNA NF54 P. falciparum gametocytes grown in and protein expression at these phases (Diaz human blood (EC50=23 nm). In this study, et al., 2006). Moreover, the Pf PKG knockout NF54 P. falciparum expressing mutant Pf strain was unable to be generated, which CDPK4 (Pf CDPK4S147M), resulting in a further indicates its crucial role in P. bulky ATP-gatekeeper (Ser147JMet). Hence, falciparum survival during the erythrocytes it restricted the space to accommodate cycle (Taylor et al., 2010). Pf PKG was also bulky R1-group of BKI-1294 (ethoxynapthtyl), indicated as indispensable during the RBC making it insensitive. BKI-1294 and BKI-1 stage based on a mutagenesis index score were confirmed to inhibit Pf CDPK4 as that was calculated according to the number these compounds showed low activity of piggyBac insertions relative to the number against Pf CDPK4 S147M recombinant of TTAA target sites in Pf PKG gene. In enzyme, both with the IC50 of >2 µM. general, the gene with low MIS is likely to be Moreover, BKI-1294 also exerted lower essential (Zhang et al., 2018). activity on the exflagellation of the NF54 Besides, a chemical genetic approach S147M mutant (EC50=0.292 µM). In vivo exploiting the size of Pf PKG ATP-gatekeeper, study later confirmed bioavailability of generating an insensitive P. falciparum BKI-1294 was improved, and higher dosage mutant (T618Q) strain, was constructed to to stop transmission exerted no adverse confirm the importance of this enzyme for effect on mice (Ojo et al., 2014). P. falciparum survival during RBC stage. This mutant strain is expressing Pf PKG d) P. falciparum cGMP-dependent with glutamine (bulky amino acid) as its protein kinase (Pf PKG) ATP-gatekeeper instead of threonine (small). In mammalian cells, PKG is known as an Potent anticoccidial agents, trisubstituted essential regulator of the cGMP signaling pyrrole (compound 1), and imidazopyridine pathway that mediates numerous physio- (Compound 2 or ML1), targeting coccidian logical processes such as apoptosis and cell PKGs ATP-binding site were tested against differentiation (Haas et al., 2009; Soh et al., the WT and T618Q mutant strains in vitro 2001). In humans, two isomers of PKG were (Donald et al., 2006). It was concluded that

831 the antimalarial activity of both compounds related to the serine-arginine protein kinase on WT P. falciparum growth is mediated by (SPRK) family in mammalian cells. In the inhibition of Pf PKG, as mutant T618Q general, SPRK mediates the phosphorylation P. falciparum strain was unaffected (Taylor of serine-arginine-rich (SR) proteins, for et al., 2010). accurate assembly and catalytic activity of Recently, a total of 9 imidazopyridine spliceosomes, hence pre-mRNA processing. derivatives (ML2-ML10) was synthesized Due to its possible role in the regulation of from ML1. Each derivative was tested against SR proteins, Pf CDK-like gain attention as a WT P. falciparum culture/recombinant Pf druggable target since mRNA expression is PKG and mutant P. falciparum T618Q required at all Plasmodium life cycle (Ward culture/recombinant Pf PKG T618Q. ML10 et al., 2004; Kern et al., 2014). exerted the most potent activity against WT Pf CLK-3 was recently validated up to P. falciparum in vitro with the EC50 of 2.1 the in vivo level as a druggable target for nm. This compound also showed a significant antimalarial drug development by which reduction (1000-fold) in inhibitory activity TCMDC-135051 was identified as its potent against P. falciparum mutant (T618Q) with inhibitor. This compound was also previously the EC50 of 2430 nm. Besides, Enzymatic reported to exert antimalarial activity against assay demonstrates the importance of the P. falciparum culture with the EC50 of 320 size of Pf PKG ATP-gatekeeper as the WT nM (Gamo et al., 2010). In a more recent study, Pf PKG is 100 000-fold more sensitive this compound was identified from a high- towards ML10 with the IC50 of 0.16 nm, in throughput screening program utilizing 13 comparison with mutant Pf PKG T618Q 533 compounds against recombinant Pf (IC50=29 540 nm) (Baker et al., 2017). CLK-3. TCMDC-135051 showed a high degree In a further study, the in vivo effect of of selectivity as it showed no activity on ML10 was evaluated in an animal model using kinase Pf PKs such as Pf CLK-1 (closely the P. falciparum-infected SCID mouse related), Pf PKG, Pf CDPK1, and Pf CLK3. model engrafted with human RBC (infected TCMDC-135051 also exerted no activity on with P. falciparum 3D70087/N9). ML10 was closely related human kinases, pre-mRNA administered at two doses, 50 and 100 mg/kg processing factor 4B (PRPF4B) (tested up to daily for four days (oral administration). Both 50 µM), and CLK2 (29% sequence identity) treatments significantly reduced the infection (Alam et al., 2019). rate with >90% parasitemia clearance, by To confirm the molecular target of which a higher dosage completely eliminate TCMDC_135051, strain with reduced the parasite. ML10 also tested against a panel sensitivity (TM051C strain) was produced of human kinases consisting of 80 kinase by exposing P. falciparum D2d to an proteins representing all main families, increasing concentration of TCMDC_135051. including 14 human kinases with a small TM051C strain containing H529P ATP-gatekeeper. The only significant (His259JPro) mutation in its Pf CLK-3. The inhibitory activity of ML10 was observed phenotypic assay revealed that the mutant against human MLK3 (40% inhibition, tested strain is 12-fold less sensitive than the WT at 100 nM). In addition, ML10 was also with the EC50 of 1.25 µM and 0.1 µM, tested against four human cell lines to respectively. Further genetic validation of reveal a very high selectivity window Pf CLK-3 was performed using recombinant (>4500 fold) (Baker et al., 2017). mutant Pf CLK-3, G449P (Gly449JPro, at the C-terminus). This mutant enzyme was e) P. falciparum Cyclin-dependent-like generated by swapping the residues between kinase 3 (Pf CLK-3) Pf CLK-1 (not inhibited by TCMDC_135051) In Plasmodium kinome, the member of the and Pf CLK-3. Recombinant Pf CLK-3 G449P CDK-like family consists of four members; is almost 550-fold less sensitive towards Pf CLK1/Pf Lammer, Pf CLK-2, Pf CLK-3, and TCMDC_135051 (IC50=21.57 µM) than the Pf CLK-4/Pf SRPK1. Pf CDK-like is closely WT (IC50=0.04 µM) (Alam et al., 2019).

832 In addition, changes in gene transcription CONCLUSION in response to TCMDC_135051 was also evaluated. The genome-wide transcriptional P. falciparum protein kinase represents an pattern showed that the inhibition of Pf excellent opportunity for the development of CLK-3 leads to the down-regulation/up- a new line of antimalarial drugs with a well- regulation of 799/155 genes in WT strain in defined mode of action and cellular target. comparison to 88/6 genes in mutant strains, Such information may prevent late-stage respectively. These genes are involved in cell failure in the drug approval process since egress and invasion, cytoadherence, sexual compound targeting Pf PKs will have little or stages, metabolism, lipid modification, and no toxic effect on human kinases and cells. mitochondrial function (Alam et al., 2019). Besides, this approach may lead to the Hence, proven the assumption of Pf CLK-3 as identification of antimalarial drugs with a the regulator of mRNA processing (Kern et longer clinical span as some inhibitors al., 2014). mimicking the interaction of ATP with ATP- Given the proven roles of Pf CLK-3 in binding sites. For example, imidazopyridine the mRNA processing, the effect of derivatives were shown to inhibit multiple Pf TCMDC_135051 at multiple stages of the PKs at the same time, contributed by small Plasmodium life cycle was characterized. ATP-gatekeeper subunits of most Pf PKs. In the asexual RBC stage, TCMDC_135051 Besides, the size of ATP-gatekeeper is causes rapid Plasmodium clearance, which generally conserved among the other human prevented the transition of parasite from malaria and apicomplexan parasites, ring-stage to trophozoites and trophozoites including P. vivax, C. parvum, and T. to schizont. In the sexual stage, gondii. It suggests the possible application TCMDC_135051 was first tested on the P. of antimalarial agents targeting Pf PKs to falciparum 2004 strain that produces a cure the other infectious diseases as well. high-level of gametocytes and was shown to However, this approach is not without exert potent parasiticidal activity with the its challenges. As discussed in this review, EC50 of 0.26 µM (Brancucci et al., 2015). some Pf PKs were unable to be studied due Subsequently, the mosquito membrane to the failure to produce knockout strains. feeding assay was performed to study the Moreover, structural differences do exist effect of TCMDC_135051 on the transmission between protein kinases across Plasmodium of P. falciparum to the mosquito vector. Stage parasites, which hinder a proper evaluation II gametocytes were treated with the of the tested drugs at an in vivo level that compound, and the parasites were allowed prevents their progression. Recent updates to develop to stage V, revealing a significant on malaria research approaches such as the reduction of gametocyte number (EC50=0.8 combination of P. falciparum metabolomic µM) (Alam et al., 2019). profiling and phenotypic study may have Finally, TCMDC_135051 was also shown widened the identification of Pf PKs (or the to inhibit recombinant CLK-3 enzymes from other cell components) targeted by the different malaria parasites, especially P. tested compounds, which was previously berghei CLK-3 (IC50=0.013 µM), which unidentifiable via the conventional methods warranted in vivo study. The P. berghei (Muruthi et al., 2020). It is no doubt that infected mice were treated with daily such techniques may eventually introduce intraperitoneal dosing of TCMDC_135051 Pf PKs inhibitor into the clinical trials. for five days (maximal dose of 50 mg/kg) resulted in almost total parasite clearance. Acknowledgment. This work was supported Inspection on P. berghei sporozoites in the by a research grant from the Ministry of liver showed potent activity with the EC50 Higher Education under the Fundamental of 0.40 µM (Alam et al., 2019). Overall, Pf Research Grant Scheme (FRGS) (203/ CLK-3 is currently one of the most convincing CIPPM/6711582). The first author is the Pf PK to be targeted for antimalaria receiver of SLAB scholarship from the chemotherapy. Ministry of Higher Education, Malaysia.

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