Asian Pacific Journal of Tropical Medicine

REVIEW ARTICLE
Year
: 2020  |  Volume : 13  |  Issue : 8  |  Page : 350--357

ATP gatekeeper of Plasmodium protein kinase may provide the opportunity to develop selective antimalarial drugs with multiple targets


Fauze Mahmud1, Ping Chin Lee2, Habibah A Wahab3, Khairul Mohd Fadzli Mustaffa4, Chiuan Herng Leow4, Azhar Rasul5, Ngit Shin Lai4,  
1 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang; Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Sabah, Malaysia
2 Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Sabah, Malaysia
3 Faculty of Pharmaceutical Science, Universiti Sains Malaysia, Penang, Malaysia
4 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia
5 Department of Zoology, Government College University, Faisalabad, Pakistan

Correspondence Address:
Ngit Shin Lai
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang
Malaysia

Abstract

Malaria is one of the most devastating infectious diseases that caused millions of clinical cases annually despite decades of prevention efforts. Recent cases of Plasmodium falciparum resistance against the only remaining class of effective antimalarial (artemisinin) in South East Asia may soon pose a significant threat. Hence, the identification of new antimalarial compounds with a novel mode of action is necessary to curb this problem. Protein kinase has been implicated as a valid target for drug development in diseases such as cancer and diabetes in humans. A similar approach is now recognized for the treatment of protozoan-related disease including malaria. Few Plasmodium protein kinases that are not only crucial for their survival but also have unique structural features have been identified as a potential target for drug development. In this review, studies on antimalarial drug development exploiting the size of Plasmodium protein kinase ATP gatekeeper over the past 15 years are mainly discussed. The ATP-binding site of Plasmodium protein kinases such as Pf CDPK1, Pf CDPK4, Pf PKG, Pf PK7, and Pf PI4K showed great potential for selective and multi-target inhibitions owing to their smaller or unique ATP-gatekeeper amino acid subunits compared to that of human protein kinase. Hence it is a feasible solution to identify a new class of active antimalarial agents with a novel mode of action and longer clinical life-span.



How to cite this article:
Mahmud F, Lee PC, Wahab HA, Fadzli Mustaffa KM, Leow CH, Rasul A, Lai NS. ATP gatekeeper of Plasmodium protein kinase may provide the opportunity to develop selective antimalarial drugs with multiple targets.Asian Pac J Trop Med 2020;13:350-357


How to cite this URL:
Mahmud F, Lee PC, Wahab HA, Fadzli Mustaffa KM, Leow CH, Rasul A, Lai NS. ATP gatekeeper of Plasmodium protein kinase may provide the opportunity to develop selective antimalarial drugs with multiple targets. Asian Pac J Trop Med [serial online] 2020 [cited 2020 Aug 13 ];13:350-357
Available from: http://www.apjtm.org/text.asp?2020/13/8/350/289439


Full Text



 1. Introduction



Since its first description in ancient Egypt texts, malaria remains one of the most life-threatening and widespread infectious diseases in the world. Currently, six Plasmodium species [Plasmodium (P.) falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri and P. knowlesi] are known to cause malaria in human, of which P. falciparum is the most virulent[1]. This species of malarial parasite caused almost half a million deaths annually, especially in the sub- Saharan region, while mild but prevalent malaria cases in Asia and South America are caused by P. vivax. The malarial protozoan is transmitted to humans by female Anopheles sp. mosquitoes, and its pathological symptoms start within one week to months after infection, depending on the Plasmodium species[2]. A global effort to eliminate malaria using both artemisinin-based combination therapy (ACT) and insecticide-treated bed nets since the 1950s has significantly reduced the malaria mortality rate[3],[4].

Although the infection rate has steadily been decreasing by 48% in total over the past decade, malaria still represents a significant human and economic burden, with more than 1 billion malaria cases recorded worldwide from 2001-2015[3],[4]. The infection continues to put 3.2 billion people, especially children under the age of five, at risk as recent data indicate that 216 million malaria cases were reported in 2016, increasing by 5 million cases compared with 2015[3],[4]. Currently, only four classes of antimalarial compounds available in clinical use which comprises of artemisinin derivatives, quinine or other aminoquinolines, antifolates, and hydroxyl naphthoquinone atovaquone[5]. The recent confirmed development of artemisinin resistance in South East Asia further complicates this problem[6],[7]. As we are on the verge of yet another significant development of Plasmodium resistance strain since the emergence of chloroquine-resistance, the effort of finding new antimalarial drugs, especially with a novel mode of action or target has now become more critical than ever.

As in 2018, a total of 13 new lines of antimalarial drugs are now tested at phase I and beyond, including tafenoquine (analog of primaquine), which has been recently approved by US Food and Drug Administration (FDA)[8]. Commercialized as Krintafel, tafenoquine is used for the treatment of malaria caused by P. vivax during both blood and tissue stage besides acting as gametocytocidal. Other multi-stage acting drugs in the clinical phases include KAF156 (Phase II)[9],[10], and M5717 (Phase I)[11]. Despite their potential, the mechanisms of action of both KAF156 and M5717 are unknown[12]. Without the knowledge of the exact target, hit progression will become more challenging[13].

The use of antimalarial drugs with the unknown target is nothing new. Most antimalarial drugs in the market were developed without the knowledge of known drug target as their therapeutic potency have only been identified via cell-based assay[14]. Determining the exact target of a drug is valuable knowledge as it can prevent late-stage failures and increase the chances of drug approval. Information on the actual target also leads to better dosing, helps to monitor potential side effects of a drug, and stratify better clinical trials on suitable patients[15]. Thus, experimental design targeting selected targets such as protein or lipid, is an appropriate approach to solve this issue[16].

For this reason, MMV390048 is one of the most exciting new antimalarial drug lines as it is the first P. falciparum protein kinase (Pf PK) inhibitor reaching clinical validation, targeting Pf Phosphatidylinositol 4-kinase (Pf PI4K). This drug is acting as blood schizonticide that inhibits gametogenesis and oocyst formation[17]. Besides Pf PI4K, the only Pf PK validated clinically, few other protein kinase targets are now in development as potential drug targets such as Pf CDPKs (Pf CDPK1, Pf CDPK4), Pf PKG and Pf nek-1 (all genetically, phenotypically and in vivo validated), Pf MRK, Pf GSK-3, and Pf PI3K (all genetically and phenotypically validated), Pf FIKK8 (phenotypically validated) and Pf PKA (genetically validated) have also been identified[18].

All these protein kinases are verified to be essential for Plasmodium survival, and most of them are expressed at different stages of Plasmodium’s life cycle with varying importance. Infected mosquitos transfer the sporozoites into the bloodstream, and it travels to the liver (Pf PK7 and Pf PI4K are essential at this stage) (1)[19]. In the infected hepatocytes cells, sporozoites then matured into schizont, and finally, the merozoites released into the bloodstream and infect red blood cells for the asexual stage (essential Plasmodium protein kinases are Pf CDPK4 and Pf CDPK6) (2)[20],[21]. The merozoites then developed into ring-stage/early trophozoite, late/mature trophozoite, red blood cell (RBC) schizont, and finally merozoites are released to infect more RBC. At this stage, many Pf PK were identified to be involved such as Pf PI4K, Pf FIKK8, Pf CDPK4, and Pf GSK-3 (3)[22],[23]. Instead of progressing into RBC schizont, some of the trophozoites will develop into male and female gametocytes that will be taken up by the mosquito for the sexual stage, and the cycle is repeated (4) [Figure 1].{Figure 1}

Pf PK is also an emerging target for antimalarial drug development due to the success of protein kinase inhibitors in humans in which the US FDA has approved 33 human protein kinase inhibitors to date. Almost 100 eukaryotic protein kinase-related (ePK-related) enzymes have been identified in P. falciparum kinome, where most of them can be grouped into seven ePK families. Due to the long independent evolution of Plasmodium kinome, some Pf PK cannot be categorized into any ePK such as Pf PK6 and Pf PK7 and known as “orphan kinase”. Moreover, some of these orphan kinases have unique features with no orthologue in humans, such as Pf CDPKs (higher resemblance to plant protein kinase) and Pf FIKKs (can only be found in the phylum of Apicomplexa)[18],[24].

Furthermore, recombinant Pf PKs are adaptable for high-throughput screening (HTS) format in which a screening using thousands of compounds has become a powerful and robust tool to identify potential new antimalarial compounds. One of the largest and most diverse compound libraries screening performed to date utilized 1.7 million compounds in which fully integrated and automated high-throughput fluorescence-based phenotypic assay (1 536-well format) was applied. This study identified approximately 6 000 small molecules (with more than 500 distinct scaffolds) that showed potent antimalarial activity (<1.25 μM). Over 95% of the identified active scaffolds were not previously reported as having antimalarial activity[25].

Another study was performed in which almost 2 million compounds in GlaxoSmithKline’s library were screened against P. falciparum 3D7 and Dd2 culture in 384-well. The antimalarial potential was measured based on the LDH activity, of which almost 14 000 compounds were confirmed with more than 70% parasite clearance at 2 μM concentration. Also, more than 8 000 compounds showed potent activity against the multidrug-resistant strain Dd2. More than 80% of the identified active compounds were never previously described with antimalarial activity[25],[26].

Both studies postulated that Pf PK might be the possible target based on the mechanism of action (MOA) historical data or structure-activity relationship (SAR) analysis of compounds with known activity. For instance, SAR analysis revealed that out of 6 000 active compounds, 48 active ligands sharing the same scaffold as staurosporine, a well-known protein kinase inhibitor[22]. Meanwhile, MOA analysis indicated that almost 50% of the active compounds are targeting protein kinase. At this point, Pf PKs are strongly suggested as a potential yet unexploited antimalarial drug target as its inhibitor not only could potentially inhibit multiple Pf PKs but also distantly related enzymes due to structural similarities of the binding sites. However, this may also pose a risk to humans as it might allow the inhibition of related human protein kinases[23].

As a follow up to the HTS cell-based assay, the first step typically is to identify the actual protein kinase targeted by the inhibitor that caused antimalarial activity observed in the phenotypic assay. Next, is to identify druggable Pf PK that has unique structural features for specific inhibition over human protein kinase[18]. Thus, the critical structural difference between Pf and human protein kinases could be identified to minimize specificity concern due to interspecies structural similarities. ATP- and substrate binding sites of the protein kinase are usually the primary target for ligand binding as they directly control protein kinase activation and activity. Some studies reported the differences between human and Plasmodium protein kinase, especially on the ATP-binding pocket of glycogen synthase kinase-3 (GSK-3). It was revealed that subtle structural differences which may provide a certain degree of specific Plasmodium protein kinase inhibition[27-29].

 2. ATP-binding site of P. falciparum protein kinase provides a potential target for drug development



Sequencing of human/Homo sapiens GSK-3 (Hs GSK-3) and P. falciparum GSK-3 (Pf GSK-3) revealed low conservation in both domain identity and similarity, 56% and 76% similarities, respectively. Computational analysis of Pf GSK-3 with Hs GSK-3 using SYBYL further revealed that the ATP-biding site of Pf GSK-3 is slightly smaller and less extensive compared with Hs GSK-3β. Also, the hydrophobic sub-pocket at the bottom of the Pf GSK-3 ATP binding site is protected by Met137 (Leu135 in Hs GSK-3). Four out of 11 probes were shown to have different binding strength in Pf- and Hs GSK-3 ATP-binding sites. OC1 probe (H-bond acceptor probe) is shifted towards the inner region of the Pf GSK-3 ATP-binding site as no salt bridge formed between Gln162 and Lys166 to arrest its confirmation (formed in human)[27].

N2 (H-bond donor probe) interact with both carbonyl groups in Pf-/ Hs GSK-3. However, significant molecular interaction fields (MIFs) difference still can be observed due to differences in nearby residues. N1 probe has stronger MIFs for Pf GSK-3 as its binding is affected by the backbone carbonyl of Ala106/Ala83 residues near gatekeeper at a distance of 3.34 Å (3.17 Å in human). Iodine (I) (van der Waals radius of 2.15 Å) shows strong interaction with Pf GSK-3 as it faced less steric hindrance at the bottom of Pf GSK-3 ATP-binding site due to a greater distance of MIF to the sulfur of Cys224/Cys199 (Pf-/ Hs GSK-3) in Pf GSK-3 (4.2 Å) compared with Hs GSK-3 (2.4 A). Hence, this study managed to demonstrate the effect of subunit differences between Pf- and Hs GSK-3 on the binding of a possible inhibitor[27].

Another bioinformatics study on human and parasite GSK-3 managed to demonstrate inhibitor selectivity differences due to the size of the ATP-gatekeeper[28]. The activity of paullone (ATP-competitive inhibitor) and its derivatives were found to have a higher affinity towards Hs GSK-3 by 30 to 300-fold compared with Pf GSK-3. As previously indicated, it is likely caused by methionine gatekeeper in Pf GSK-3 that is longer and more flexible than the leucine subunit of Hs GSK-3[30]. It created a steric effect on paullone and its derivatives. These amino acids are termed as “ATP gatekeeper” because they guard the access to the unexploited hydrophobic pocket[31].

In addition, 3,6-diamino-4-(2-halophenyl)-2-benzoylthienol[2,3-b] pyridine-5-carbonitriles (5v) was identified as a new class of Pf GSK-3 inhibitor. Its derivatives with 2-ClPhe and 3-ClPh on R1 and R2 respectively showed the most potent activity (Pf IC50 is 5.5 μM). The IC50 of 5v on Pf GSK-3, Hs GSK-3 , and Hs GSK-3P is 0.48 μM, >100 μM, and 3.0 μM, respectively. In silico study showed that better inhibition of Pf GSK-3 achieved by 5v was due to hydrophobic interaction between the thiophene of 5v with ATP-gatekeeper subunit of Pf GSK-3 (methionine). This interaction was not formed in Hs GSK-3 as its gatekeeper subunit (Leucine) is located further[29]. Hence, the size of Pf PK ATP-gatekeeper is a compelling feature to be exploited for drug discovery as it can control which inhibitor to bind in the ATP-binding site[32].

Currently, not many Pf PKs ATP binding site has been described. However, most of the Pf PKs have small or very small ATP gatekeepers, as approximately 30 Pf PKs (of 86 to 99 known Pf Pks) have threonine or serine ATP gatekeeper subunit[21],[33]. Besides that, some Pf PKs possessing bulky yet with unique amino acid subunits (tyrosine) that is very rare in humans[18],[34],[35],[36]. In contrast, human protein kinase usually has bulky gatekeeper residues such as methionine, leucine, and phenylalanine. Only 19% of human protein kinase has a small ATP gatekeeper (threonine)[37],[38]. This structural difference is significant as a bulky gatekeeper blocks the hydrophobic pocket located behind ATP-binding sites, and a small gatekeeper exposes this pocket to inhibitors[36]. Thus, it can be exploited for the identification of specific inhibitors termed as “bumped kinase inhibitor” that relatively has low off-target effects on the human kinome[34],[39].

To target Pf PK ATP-binding sites, scaffolds such as purine that are structurally related to ATP (natural substrate of all protein kinase) were indicated as a potential inhibitor[27]. Purfalcamine (2,6,9-trisubstituted purine) was identified as a Pf CDPK1 inhibitor that caused P. falciparum to accumulate in the late schizogony and failed to egress from the merozoite stage. In the ATP-binding site of Pf CDPK1, nonconserved residues such as Arg60, Glu149, and Lys202 can be targeted to establish a salt bridge for better selectivity. Moreover, Pf CDPK1 has small-sized ATP gatekeeper residue (threonine) protecting the bottom of the ATP-binding site. Interestingly, purfalcamine was also indicated to inhibit Pf CDPK5 (highest homology with Pf CDPK1, but with bulky ATP gatekeeper) with lower affinity (IC50 of 17 nm in Pf CDPK1 and 3.5 μM in Pf CDPK5, respectively). Further test on four mammalian cell lines in which the EC50 (230 nm) suggested the therapeutic window of purfalcamine was 23- to 26- fold[40].

A similar strategy of using a pyridine motif and another aromatic linker such as pyrimidine (class 1) and fluoropyridine (class 2) attached to imidazopyridazine was recently applied. Class 1 compound was found to be active against both Pf CDPK1 and Pf PKG. The ATP-binding site of Pf CDPK1 and Pf PKG is closely related and shared the same sequence homology that includes gatekeeper residues, threonine at Thr145 (Pf CDPK1) and Thr618 (Pf PKG), which may explain dual inhibition exerted by class 1 inhibitor. The substitution of Pf CDPK1 gatekeeper with bulky residue such as glutamine significantly reduced the sensitivity of Pf CDPK1 to these inhibitors as it blocks the access to the ATP-binding site. A series of class 1 inhibitors were tested in which most of them showed a higher affinity towards Pf PKG, notably class 1-compound A (IC50 Pf PKG: 0.002 μM and IC50 Pf CDPK1: 0.008 μM, respectively). This compound also almost 6 000 more potent on wild type Pf PKG than mutant protein (Pf PKG with glutamine ATP- gatekeeper)[38].

A recent study on the potential of Pf PKG as a druggable protein was explored using the imidazopyridine series. Imidazopyridine (with the addition of cyclopropylmethylene group) (ML10) was identified as the most potent compound (160 pM). ML10 caused merozoite failed to egress and block transmission of P. falciparum gametocytes to Anopheles sp. Until today, Pf PKG crystallization with or without inhibitor is still unable to be achieved. Thus, the interaction between ML10 on the Plasmodium ATP-binding site was studied based on the co-crystal structure of P. vivax PKG (Pv PKG). Amino-pyrimidine of ML10 formed a hydrogen bond with the backbone of Val614 (Val621 in Pf PKG), mimicking ATP. Also, the sulfonamide group of ML10 formed a hydrogen bond with Asp675 (Asp682 in Pf PKG) and Phe676 (Phe683 in Pf PKG) of the DFG triad for stronger binding. Remarkably, the fluorophenyl group can interact with the hydrophobic pocket of Pv PKG that is guarded by threonine (Thr611 in Pv PKG and Thr618 in Pf PKG), a small gatekeeper[39],[41].

Moreover, Pf PKG has been recognized to have distinct properties than human PKG[42]. Hence, such interaction is blocked by a large subunit that making human PKG insensitive against ML10. Interestingly, ML10 also showed very little inhibitory activity against 80 human protein kinase panels representing all kinase families including human kinase protein with small ATP gatekeeper (tested at 100 μM)[41]. Besides, Pf CDPK1 was found dispensable for the survival of Plasmodium during the red blood stage. Instead, the antimalarial activity of class 1 was found due to the inhibition of Pf PKG during red blood stage. Although Pf CDPK1 was suggested as not a suitable target for blood schizonticide development, it is the potential target for antimalarial acting during the gametocytes stage[43],[44]. Hence, a compound such as class 1-compound A may provide selective antimalarial that can act on different stages of Plasmodium life-cycle.

Pf PKs with bulky but unique ATP gatekeepers notably, Pf PK7 and Pf PI4K, also showed potential as drug targets. A series of pyrimidine and pyridazine class of compounds were identified to inhibit the activity of Pf PK7, in which the most potent compound is (S)-4-[6-(1-hydroxy-3-methylbutan-2-ylamino)imidazo[1,2-b] pyridazin-3-yl] benzonitrile (K510). K510 was shown to mimic ATP by forming a hydrogen bond with Met120 (backbone amide group), and heterocyclic ring established interactions with Leu34 and Leu179. The interaction is further strengthened by the dipolar interaction between K510 nitrile moiety and the hydroxyl group of Pf PK7 gatekeeper (Tyr117)[34]. Another study described imidazopyridazine derivative-34 with antimalarial activity (Pf 3D7 IC50 of 1.03 μM) and a selectivity index of 23.32. Although the binding site of derivative-34 is unknown, the replacement of larger substituent (amine, in derivative-22 to 24), resulted in significant antimalarial activity decrease[45].

Specific inhibition of Pf PK despite having a bulky ATP gatekeeper subunit was confirmed achievable, using imidazopyrazines and quinoxalines scaffolds against Pf PI4K. Imidazopyrazines KDU691 and KAI407 were identified to target Pf PI4K ATP-binding site, but both displayed excellent selectivity over human lipid and protein kinases. In silico analysis revealed that the N1 of KAI407 imidazole ring formed a hydrogen bond mimicking hydrogen bonds made by the adenine of ATP[18],[46],[47]. The activity of these compounds on PI4K was further confirmed based on enzymatic assay against recombinant Pv PI4K. Their inhibitory activities are ATP concentration-dependent, which indicates the ATP-competitive mode of action. Also, KA1407 showed no effect on a panel of human protein kinases consisting of Hs PI4KHfα and Hs PI4Kmβ[46].

Recently, aminopyridine was identified as another potential class of compounds that act as potent and selective Pf PI4K inhibitor resulting in the identification of 2-aminopyridine MMV390048, the first Pf PK inhibitor reaching the clinical stage. This compound was recognized to act as an ATP-competitive inhibitor, as well[48]. Its efficacy targeting Pf PI4K was described in-depth from the screening of a 2-aminopyridine class of small molecule. MMV390048 potency was defined based on a humanized mouse model, mouse-to-mouse transmission, and monkeys. This inhibitor is indicated to be active against all Plasmodium life-cycles except for late hypnozoites in the liver. Pf PI4K was confirmed as its molecular target from genomic and chemoproteomic studies. Interestingly, the kinobeads analysis revealed that MMV390048 was shown to binds only on the ATP-binding site of Pf PI4K but not to its human analog (both PI4Kα and PI4Kβ) indicating selective inhibition[17].

Based on these findings, the ATP gatekeeper of Pf PK is a potential structural feature to be exploited for the development of antimalarial agents as it provides selective inhibition by specific chemical scaffolds towards Plasmodium protein kinase that reduce unwanted risk in human[49]. Pf PKs with small gatekeeper was also shown to be inhibited by the same active compound such as Pf CDPK1 and Pf PKG both inhibited by imidazopyridazine, and compound BKI 1294 inhibited Pf CDPK1 and Pf CDPK4 [Table 1][33],[44]. Interestingly, some Pf PKs with small and bulky gatekeepers (Pf PK7, Pf PKG, and Pf CDPK1) were also reported as the targets of imidazopyridazines derivatives, owing to the overall similarities of their ATP-binding sites[44],[45],[52]. A similar finding was reported in which various scaffolds able to inhibit Pf CDPK1, Pf CDPK4, Pf PK6, and Pf PK7 at the same time[53].{Table 1}

Besides, the exploration of Pf PKs with very small ATP gatekeepers (especially the Pf FIKK family) might shed more light on their potential to develop a new line of antimalarial agents[18],[24]. In all, protein kinase inhibitor targeting the ATP-binding site of Pf PKs may not only work well for specific inhibition but also with multiple targets in Plasmodium as well, partly contributed by the size of Pf PKs ATP gatekeepers that are divergent than human protein kinase[54-57].

 3. Conclusions



The size of the ATP-gatekeeper has been indicated as potential structural features to provide specific Plasmodium inhibition as they are generally smaller than that of human protein kinase. The main advantage of this approach is the selective inhibition of Pf PKs over human protein kinase. ATP-binding may also lead to the identification of antimalarial drugs with multiple targets as Pf PKs generally conserved among them. Hence, it may eventually delay the development of drug resistance strain, an assumption based on slow resistance development against artemisinin (almost 40 years), that is believed to act on multiple targets as well (all stages of malaria)[58],[59]. Overall, a compound that showed a high affinity towards Plasmodium that is influenced by the size of its Pf PKs ATP gatekeeper is probably one of the best options for the development of the next antimalarial agent.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Authors’ contributions

F.M. conceived the idea, drafting, and editing the presented article. L.P.C, H.A.W, K.M.F.M, L.C.H, and A.R. were involved in the critical revision of the article. L.N.S is the project leader, contributed to the focus and critical revision of the manuscript.

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