Plasmodial Kinase Inhibitors Targeting Malaria: Recent Developments

molecules

Review Plasmodial Kinase Inhibitors Targeting Malaria: Recent Developments

Romain Mustière , Patrice Vanelle * and Nicolas Primas *

Aix Marseille Univ, CNRS, ICR UMR 7273, Equipe Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 13385 Marseille CEDEX 05, France; [email protected] * Correspondence: [email protected] (P.V.); [email protected] (N.P.)

Academic Editor: Pascal Marchand Received: 10 November 2020; Accepted: 11 December 2020; Published: 15 December 2020

Abstract: Recent progress in reducing malaria cases and ensuing deaths is threatened by factors like mutations that induce resistance to artemisinin derivatives. Multiple drugs are currently in clinical trials for malaria treatment, including some with novel mechanisms of action. One of these, MMV390048, is a plasmodial kinase inhibitor. This review lists the recently developed molecules which target plasmodial kinases. A systematic review of the literature was performed using CAPLUS and MEDLINE databases from 2005 to 2020. It covers a total of 60 articles and describes about one hundred compounds targeting 22 plasmodial kinases. This work highlights the strong potential of compounds targeting plasmodial kinases for future drug therapies. However, the majority of the Plasmodium kinome remains to be explored.

Keywords: kinase inhibitor; Plasmodium; malaria; SAR; medicinal chemistry

1. Introduction Malaria is the deadliest parasitic disease with an estimated 405,000 deaths and 213 million cases in 2018 [1], mainly in Africa. Of the estimated victims, 67% are children below ﬁve years old. The disease is caused by ﬁve Plasmodium species: P. falciparum (responsible for most of the deaths), P. vivax and three less important species, P. ovale, P. malariae and P. knowlesi. With the use of artemisinin-based combined therapies (ACT), combining an artemisinin derivate and another antimalarial drug, and vector control measures, the Word Health Organization hoped that 2020 would see a 40% reduction in malaria cases and deaths compared to 2015 [2]. However, this objective is not going to be achieved. The vector, mosquitoes from the Anopheles genus, is becoming resistant to pyrethroid insecticides (used in long-lasting insecticide nets) in Africa [3]. Moreover, mutations on the Pf kelch13 protein, plasmodial protein recently described to be involved in hemoglobin endocytosis mechanisms [4], mediate resistance to artemisinin and its derivates. Pf kelch13 mutations are linked [5]:

In vitro to a decreased susceptibility during a ring stage assay, • In vivo to a delayed parasitic clearance. • Increased prevalence of these mutations in South-East Asia is leading to clinical failures of ACT treatments [6]. Millions of people in Africa are threatened: recent researches indicate the appearance of in vitro artemisinin resistance in Rwanda [7], and the spread of city-dwelling A. stephensi in Africa [8]. Thus, research and development aimed at finding new drug therapies with novel mechanisms of action is a priority. Medicines for Malaria Venture, a non-profit organization, aims to develop new antimalarial drug therapies. To guide drug discovery, target candidate profiles (TCP) have been defined for antimalarial molecules setting clear goals for new therapy [9]. There are five TCP: blood-stage killer, hypnozoites killer, hepatic schizonts killer (chemoprotection), gametocytes killer, and transmission

Molecules 2020, 25, 5949; doi:10.3390/molecules25245949 www.mdpi.com/journal/molecules Molecules 2020, 25, 5949 2 of 35 blocker. The best compounds are the one following multiple or every TCP.One of these drug candidates, MMV390048, currently in phase II clinical trial, is the first plasmodial kinase inhibitor to reach this stage, opening the way for antimarial drugs targeting this type of protein. Kinases are proteins catalyzing the addition of a phosphate group on a substrate, like a simple molecule or a protein. In the latter, the kinase, designed under the term of “protein kinase”, will modified the activity of the targeted protein after addition of the phosphate group. The complete set of encoded protein kinases in the genome of an organism is defined as a kinome. The human kinome consists of 518 encoded kinases [10] that play a role in the regulation of cellular processes, and whose dysregulation is involved in numerous cancers [11]. The two first human kinase inhibitors approved by the Food and Drug Administration (FDA) were rapamycin in 1999 (indicated for immunosuppression) and imatinib in 2001. Importantly, the small molecule imatinib inhibits the BCR-Abl chimeric protein, found mainly in chronic myeloid leukemia. This finding accelerated research on human kinase inhibitors and as of 1 March 2019, 48 small molecules designed as human kinase inhibitors were approved by the FDA [12], the majority of them used in oncology. According to PKIDB (a website tracking human kinase inhibitors in clinical trials), as of July 2020, 253 human kinase inhibitors were currently in clinical trials [13]. Mapping of the P. falciparum kinome, depending on the literature source, indicates 85 (65 for eukaryotic protein kinases (ePKs) and 20 for ePKs-related proteins) [14] or 99 [15] encoding genes, and a significant number of these proteins do not possess a human ortholog [16]. These kinases are highly conserved among the different Plasmodium species. Moreover, 36 plasmodial kinases (in P. falciparum) are identified as likely to be essential for the asexual blood-stage [17], and 15 kinases are known to be involved in P. berghei development in mosquitoes [18]. Thus, developing selective plasmodial kinase inhibitors could lead to multiple new antimalarial drugs with new mechanisms of action. This review of the literature lists molecules proven to be inhibitors of plasmodial kinases and describes their properties.

2. Methods This systematic review complies with the PRISMA statement deﬁning the PRISMA checklist and PRISMA ﬂowchart [19].

2.1. Data Sources and Search Parameters Two databases were used: Sciﬁnder (http://sciﬁnder.cas.org, CAPLUS and MEDLINE database) and Pubmed (https://pubmed.ncbi.nlm.nih.gov/, MEDLINE database). In both databases, the keywords and Boolean operator “kinase inhibitor AND malaria” were used. The advanced search options were set to “journal” for document type and English or French for language. The research was conducted on articles published from 1 January 2005 to 1 August 2020.

2.2. Article Selection Articles were selected by one reviewer, with two rounds of selection. During the first round, for each database set, articles’ titles and abstracts were assessed to eliminate irrelevant material (outside medicinal chemistry or biological fields, non-malaria related, non-filtered reviews, duplicates). To be discussed in the following review, articles had to follow the inclusion and exclusion criteria listed in Table1. Molecules 2020, 25, 5949 3 of 35

Molecules 2020, 25, x FOR PEER REVIEWTable 1. Inclusion and exclusion criteria used. 3 of 35

ParameterTable Inclusion1. Inclusion and exclusion criteria used. Exclusion A plasmodial kinase clearly Target is not a plasmodial kinase or target is Compound’sParameter target Inclusion Exclusion defined as target undefined Compound’s A plasmodial kinase clearly Target is not a plasmodial kinase or target is target defined as target In silico studiesundefined without biological tests Medicinal chemistry studies Natural extract studies without active In silico studies without biological tests Biology studies listing compounds determined Study type Medicinalproperties chemistry of onestudies or more Natural extractBiological studies studies without on proteinactive Biology studies listing compounds determined Study type chemical compounds pathways/phenotypic changes using only properties of one or more Biologicalgenes disruptionstudies on orprotein polypeptides chemical compounds pathways/phenotypic changes using only genes disruption or polypeptides 3. Results Four3. Results hundred and fifty-six articles from Scifinder (after applying the “remove duplicates” tool) and 366 articlesFour hundred from Pubmed and fifty-six were articles obtained. from DuringScifinder the (after first applying round of the selection, “remove 348duplicates” Scifinder tool) articles and 286and Pubmed 366 articles articles from Pubmed were excluded. were obtained. During the first round of selection, 348 Scifinder articles Theand 286 two Pubmed sets were articles combined, were excluded. and duplicates removed, leaving 121 articles. During the second round of selection,The two sets 64 were articles combined, were excluded and duplicates leading removed, to 57 selected leaving 121 articles; articles. three During additional the second articles wereround added of in selection, the dataset 64 articles while were searching excluded for furtherleading to protein 57 selected information. articles; three additional articles Thiswere processadded in is the summarized dataset while in searching Scheme 1for in further accordance protein with information. the PRISMA flow diagram [19]. This process is summarized in Scheme 1 in accordance with the PRISMA flow diagram [19].

SchemeScheme 1. Flow 1. Flow diagram diagram of of article article selection selection (deriv (deriveded from from PRISMA PRISMA flow ﬂow diagram diagram [19]). [19 ]).

4. Discussion4. Discussion

4.1. Molecules4.1. Molecules Targeting Targeting P.falciparum P. falciparum Calcium-Dependant Calcium-Dependant Protein Kinases Kinases (PfCDPKs) (PfCDPKs) CDPKsCDPKs can becan found be found in plants in plants and and alveolate alveolate protists protists including includingApicomplexan Apicomplexanparasites parasites like likeT. T. gondii and Plasmodiumgondii and Plasmodium spp [20]. spp They [20]. belong They belong to the to calmodulin-dependent the calmodulin-dependent kinases kinases (CaMK) (CaMK) familyfamily [14]. The CDPK[14]. The proteins, CDPK proteins, possessing possessing seven members seven members (CDPK1 (CDPK1 to CDPK7), to CDPK7), are involved are involved in multiple in multiple parasitic parasitic stages [21], and constitute interesting targets as they are not found in humans. Only stages [21], and constitute interesting targets as they are not found in humans. Only inhibitors for inhibitors for PfCDPK1 and PfCDPK4 have been described in the literature. Pf CDPK1 and Pf CDPK4 have been described in the literature.

4.1.1. Molecules Targeting P. falciparum Calcium-Dependent Protein Kinase 1 (Pf CDPK1) As discovered by Zhao et al. [22], Pf CDPK1 is involved in the invasion of red blood cells (RBC) by merozoites and their egress through activation of a protein motor complex [23,24]. More recent studies Molecules 2020, 25, x FOR PEER REVIEW 4 of 35

4.1.1.Molecules Molecules2020, 25, 5949 Targeting P. falciparum Calcium-Dependent Protein Kinase 1 (PfCDPK1) 4 of 35 As discovered by Zhao et al. [22], PfCDPK1 is involved in the invasion of red blood cells (RBC) byindicate merozoites the role and of theirPf CDPK1 egress in through gametogenesis, activation mosquito of a protein infection motor [ 25complex], and regulation[23,24]. More of Pf recentPKA studies(P. falciparum indicate(cAMP)-dependent the role of PfCDPK1 protein in kinase)gametogenesis, [26]. Pf CDPK1 mosquito was assumedinfection to[25], be anand essential regulation protein of PfforPKAPlasmodium (P. falciparumsince knocking(cAMP)-dependent out the gene protein during kinase) blood-stages [26]. Pf wasCDPK1 impossible was assumed [24]. Bansal to be et an al., essentialstarting from protein a P. for falciparum Plasmodiumstrain since with knocking a mutant outPf CDPK1the gene having during reduced blood-stages activity, was recently impossible managed [24]. Bansalto knock et outal., completelystarting fromPf CDPK1a P. falciparum [25]. strain with a mutant PfCDPK1 having reduced activity, recentlyIn 2008,managed Green to etknock al. [ 23out] usedcompletely compound PfCDPK1 K252a [25]. (Figure 1, compound 1) to study the motor complexIn 2008, regulated Green byet Pfal.CDPK1. [23] used This compound indolopyrrolocarbazole K252a (Figure 1, displayed compound an 1 IC) to50 ofstudy 45 nM the towardmotor 50 complexPf CDPK1 regulated and was ableby Pf toCDPK1. reduce RBCThis invasionindolopyrrolocarbazole by merozoites with displayed an EC 50anof IC 348 of nM. 45 nM toward PfCDPK1 and was able to reduce RBC invasion by merozoites with an EC50 of 348 nM.

Figure 1. InhibitorsInhibitors of of PfPfCDPK1CDPK1 K252a K252a 11 andand purfalcamine purfalcamine 22..

KatoKato et al. screened screened a a library of 20,000 compounds on PfCDPK1CDPK1 [24]. [24]. The The most most active active class class of compoundscompounds waswas a seriesa series of 2,6,9-trisubstitued of 2,6,9-trisubstitued purines purines and the mostand potentthe most molecule, potent called molecule, purfalcamine called purfalcamine(Figure1, compound (Figure 21,), compound showed an 2 IC), 50showedvalue ofan 17 IC nM50 value on Pf ofCDPK1. 17 nM on EC Pf50CDPK1.in a parasitic EC50 in proliferation a parasitic proliferationassay on multiple assayP. falciparumon multiplestrains P. falciparum including astrains multi-resistant including W2 a strainmulti-resistant was determined, W2 strain as well was as determined,cytotoxic values as well on multipleas cytotoxic cell values lines. However,on multiple the cell compound lines. However, lacked activitythe compoundin vivo lackedin the four-day activity inPeter’s vivo in test the [27 four-day]: only a Peter’s delay intest onset [27]: ofonly parasitemia a delay in was onset observed. of parasitemia Other compoundswas observed. from Other the compounds2,6,9-trisubstituted from the purine 2,6,9-trisubst series lackeditutedPf purineCDPK1 series affinity lacked or P. Pf falciparumCDPK1 affinityin vitro oractivity P. falciparum whenthe in vitrofluorine activity atom when was replaced the fluorine by a atom carboxylic was replaced acid or a bytert a-butoxy carboxylic group. acidP. or falciparum a tert-butoxyin vitro group.activity P. falciparumwas also suppressed in vitro activity when was the4-aminocyclohexyl also suppressed when was replacedthe 4-aminocyclohexyl by a 4-hydroxycyclohexyl was replaced group. by a 4- hydroxycyclohexylIn 2009, Lemercier group. et al. identified two PfCDPK1 inhibitors after a screening of 54,000 compounds [28]: the indolizineIn 2009, 3Lemercierand the imidazopyridazine et al. identified4 (Figuretwo Pf2CDPK1). 3 displayed inhibitors a Ki ofafter 262 nMa screening on Pf CDPK1, of while54,0004 compoundspossessed a K[28]:i of the 37 nMindolizine on Pf CDPK1 3 and andthe imidazopyridazine an IC50 on P. falciparum 4 (Figureof 5.7 2).µM. 3 displayed Out of 46 humana Ki of 262 kinases nM ontested, PfCDPK1, compound while 34 inhibitedpossessed two a Ki humanof 37 nM kinases on PfCDPK1 and compound and an IC504, on five P. (atfalciparum 10 µM concentration).of 5.7 µM. Out ofModulation 46 human workkinases focusing tested, oncompound the phenolic 3 inhibited hydroxyl two group human of 4kinasesshowed and a tolerance compound on 4 the, five Ki (atvalue 10 µMwhen concentration). this 3-hydroxyl Modulation was replaced work with focusi H-bondng on acceptors the phenolic like 3,4-methylenedioxyl hydroxyl group of or 4 3-methoxyl, showed a tolerancebutMolecules in the 2020 on latter, 25 the, x accompanied FOR Ki PEERvalue REVIEW when with this a 4-hydroxyl 3-hydroxyl substitution. was replaced with H-bond acceptors like5 3,4-of 35 methylenedioxyl or 3-methoxyl, but in the latter accompanied with a 4-hydroxyl substitution.

FigureFigure 2.2. Inhibitors ofof PfCDPK1 discovered by Lemercier et al. [[28].28].

Screening of 35,000 compounds by Chapman et al. [29] displayed imidazopyridazine as a promising series, in line with the discovery of compound 4. An extensive Structure-Activity Relationship (SAR) work started on this scaffold (Table 2). Chapman et al. [30] explored the side chains at positions 3 and 6 of the imidazopyridazine core. Work on the R1 substituent largely targeted the introduction of cyclic amines, while work on R2 addressed para- or meta-functionalized phenyls, pyridines, or pyrimidines. Compound 5 (Table 2) displayed interesting results with an IC50 on PfCDPK1 of 13 nM, an IC50 on Pf3D7 of 400 nM, and the ability to reduce by 46% the parasitemia, in a P. berghei mouse model per os during a four-day Peter’s test. Finally, 5 showed inhibition toward 12 human kinases (out of 73 tested) ranging between 50% and 80% at 1 µM.

Table 2. Imidazopyridazine inhibitors of PfCDPK1.

In Vivo PfCDPK1 Pf3D7 Compound R1 R2 Parasitemia IC50 (nM) IC50 (nM) Reduction 1

5 13 400 46%

6 70 103 -

7 <10 12 4%

8 12 80 51%

1 Four-day Peter’s test [27] at an oral dose of 50 mg/kg/day.

Large et al. continued this work with the introduction of N-substituted imidazoles at position 3 of the imidazopyridazine core while modulating position 6 with substituents like aminocyclohexyl

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Molecules 2020, 25, 5949 5 of 35 Figure 2. Inhibitors of PfCDPK1 discovered by Lemercier et al. [28]. FigureFigureFigure Figure2.Figure Inhibitors 2.2. 2. Inhibitors2.InhibitorsInhibitors Inhibitors Inhibitors of PfCDPK1 ofofof of of PfPf Pf CDPK1PfCDPK1CDPK1 CDPK1discovered discovereddiscovered discovered discovered by Lemercier byby by by LemercierLemercier Lemercier Lemercier et al. [28]. etet et etal.al. al. al. [28].[28]. [28]. [28]. Screening of 35,000 compounds by Chapman et al. [29] displayed imidazopyridazine as a ScreeningScreeningScreeningScreeningScreening ofof 35,000 35,000 of of of of compounds35,000 35,000 35,00035,000compounds compounds compounds compoundscompounds by Chapmanby Chapman by by byby Chapman Chapman et ChapmanChapman al. [et29 ]al. displayed et et [29]et et al. al.al. al.al. displayed[29] [29][29] [29][29] imidazopyridazine displayed displayeddisplayed displayeddisplayed imidazopyridazine imidazopyridazine imidazopyridazineimidazopyridazine imidazopyridazineimidazopyridazine as a promising as a as asas as as a aa a a promising series, in line with the discovery of compound 4. An extensive Structure-Activity promisingseries,promisingpromisingpromising inpromising line series, with series, series, series, theseries,in line discovery in in in in withline line lineline with with of thewithwith compound discoverythe the thethe discovery discovery discoverydiscovery4 .of An compound extensiveof of of of compound compound compoundcompound Structure-Activity4. An 4 4 .4.. 4 extensive.An .AnAn AnAn extensive extensiveextensive extensiveextensive RelationshipStructure-Activity Structure-Activity Structure-ActivityStructure-Activity Structure-ActivityStructure-Activity (SAR) Relationship (SAR) work started on this scaffold (Table 2). Chapman et al. [30] explored the side workRelationshipRelationshipRelationship startedRelationshipRelationship on(SAR) this (SAR)(SAR) (SAR) scawork(SAR)ﬀold work workstarted workwork (Table startedstarted started started2on). Chapmanthis onon on on scaffold thisthis thisthis scaffold etscaffold scaffoldscaffold al. (Table [ 30 (Table](Table (Table explored2).(Table Chapman 2).2). 2).2). Chapman theChapman ChapmanChapman side et al. chains etet [30] et et al.al. al. atal. explored[30][30] positions [30][30] exploredexplored exploredexplored the 3 and side thethe the 6the sideside sideside 1 chainschains atat positionspositions 33 andand 66 ofof thethe imidazopyridazineimidazopyridazine core.core. WorkWork onon1 thethe R1R11 substituentsubstituent largelylargely targetedtargeted chainsof thechainschains imidazopyridazineatchains positions atat at positionspositions positions 3 and 33 6 andand core.3of and the 66 ofWorkof6 imidazopyridazine of thethe the onimidazopyridazineimidazopyridazine imidazopyridazine the R1 substituent core. core. Workcore. largelycore. WorkWork on Work the targeted onon Ron thethe substituent the R theR1 11R substituent substituentsubstituent1 introduction substituent largely largely largelylargely largelytargeted of cyclic targeted targetedtargeted targeted 2 thethe introductionintroduction ofof cycliccyclic amines,amines, whilewhile workwork2 onon R2R22 addressedaddressed parapara-- oror meta-meta-functionalizedfunctionalized phenyls,phenyls, theamines, introductionthethethethe while introductionintroductionintroduction introduction work of cyclic on ofofof R of cyclic2cycliccyclic amines, addressedcyclic amines,amines,amines, amines, whilepara whilewhilewhile work -while or meta work workworkon work R-functionalized onon onaddressed on RRR 222R addressedaddressedaddressed2 addressed para phenyls,- paraparaor para meta--- oror- pyridines, or meta-functionalizedmeta- meta-functionalizedfunctionalizedfunctionalized or pyrimidines. phenyls, phenyls,phenyls, phenyls, 50 pyridines,pyridines, oror pyripyrimidines.midines. CompoundCompound 55 (Table(Table 2)2) displayeddisplayed interestinginteresting resultsresults withwith 50anan ICIC505050 onon pyridines,Compoundpyridines,pyridines,pyridines, or5 (Tablepyri oror or midines. pyripyri2 )pyri displayedmidines.midines.midines. Compound Compound interestingCompound Compound 5 (Table results 55 5(Table(Table(Table (Table2)with displayed 2)2)2) an2) displayeddisplayeddisplayed displayed IC 50interestingon Pfinterestinginterestinginteresting CDPK1interesting results of resultsresultsresults 13 resultswith nM, withwithwithan anwith IC ananan50 an onICICIC IC 505050 50ononon on 50 PfPfCDPK1CDPK1 ofof 1313 nM,nM,50 anan ICIC505050 onon PfPf3D73D7 ofof 400400 nM,nM, andand thethe abilityability toto reducereduce byby 46%46% thethe parasitemia,parasitemia, inin PfCDPK13D7PfPf ofCDPK1PfCDPK1CDPK1 400of 13 nM, ofofnM, of 13 and13 13 an nM,nM, the nM,IC anan ability anon ICIC IC50Pf5050 on3D750on toon on reduce PfPf of3D7Pf3D7 3D7400 ofof by nM, of 400400 46% 400 and nM,nM, thenM, the andand parasitemia, and ability thethe the abilityability toability reduce in toto a to reduceP.reduce reduce bergheiby 46% byby mouseby the 46%46% 46% parasitemia, thethe modelthe parasitemia,parasitemia, parasitemia,per in os inin in a P. berghei mouse model per os during a four-day Peter’s test. Finally, 5 showed inhibition toward 12 aduring P. bergheiaa a P.P.a P. P. four-daybergheiberghei berghei bergheimouse mousemouse mouse Peter’smousemodel modelmodel model per test.model os Finally,per per duringper per osos os os duringduringduring during 5 duringashowed four-day aaa a four-dayfour-dayafour-day four-day four-day inhibition Peter’s Peter’sPeter’sPeter’s Peter’s Peter’stest. toward Finally, test.test.test. test. test. 12 Finally,Finally,Finally, Finally, humanFinally, 5 showed 55 5kinases showedshowed5showed showed showed inhibition (out inhibitioninhibitioninhibition inhibition inhibition of toward 73 tested) towardtowardtoward toward toward 12 121212 12 12 human kinases (out of 73 tested) ranging between 50% and 80% at 1 µM. humanranginghumanhumanhumanhuman kinases between kinaseskinases kinases kinases (out 50% of (out(out (out and(out73 ofoftested) 80%of of 7373 73 73 tested)tested) at tested) tested)ranging 1 µM. rangingranging ranging ranging between betweenbetween between between 50% and 50%50% 50% 50% 80% andand and and at 80%80% 80% 80%1 µM. atat at at 11 1 µM.µM.1 µM. µM.

Table 2. Imidazopyridazine inhibitors of PfCDPK1. TableTable Table2.2.TableTable Imidazopyridazine 2.2. 2. Imidazopyridazine2.ImidazopyridazineImidazopyridazine Imidazopyridazine Imidazopyridazine inhibitorsinhibitors inhibitorsinhibitorsinhibitors inhibitors inhibitors ofof PfCDPK1. ofofof of of PfPf Pf CDPK1.PfCDPK1.CDPK1.CDPK1.

In Vivo In VivoInInInIn VivoIn VivoVivo VivoVivo PfCDPK1PfPfPfCDPK1CDPK1CDPK1 Pf 3D7PfPfPf Pf3D73D73D73D7 IC 50 InIn Vivo Vivo CompoundCompound RR1 R2 PfPfPfCDPK1CDPK1CDPK1 PfPfPf3D73D73D7 ParasitemiaParasitemia CompoundCompoundCompoundCompound R1 RR1R1 1 R2 RR2R2 2 ParasitemiaParasitemiaParasitemiaParasitemia CompoundCompoundCompound RR11R 1 RRR22R 2 ICIC5050(nM) (nM) IC(nM)50 (nM) ParasitemiaParasitemiaParasitemia1 IC50 (nM)ICICICIC505050 (nM) (nM) (nM)(nM)IC 50 (nM)ICICICIC505050 (nM) (nM) (nM)(nM) Reduction 1 ICICIC5050 (nM)50(nM) (nM) ICICIC5050 (nM)50(nM) (nM) Reduction1 11 11 ReductionReductionReductionReductionReduction 11 1

5 5 1313 400 46 46% 5 55 5 5 13 13131313 400 400400400400 46% 46% 46% 46% 46% 5 13 400 46%

6 70 103 - 6 666 6 6 70 7070707070 103 103103103 103103 ------6 70 103 -

7 <10 12 4% 7 777 7 7 <10 <10<10<<1010<10 12 121212 1212 4% 4% 4% 4% 4% 4% 7 <10 12 4%

8 12 80 51% 8 888 8 8 12 1212121212 80 808080 8080 51% 51% 51% 51% 51% 51% 8 12 80 51%

1 1 11 1 Four-day Peter’s test [27] at an oral dose of 50 mg/kg/day. Four-day11 Four-day Four-day1Four-day Four-day Peter’s Peter’sPeter’s Peter’stest Peter’s [27] testtest test testat [27][27] an[27] [27] oralatat at atanan an dosean oraloral oral oral of dosedose dose 50dose mg/kg/day. ofof of of 5050 50 50 mg/kg/day.mg/kg/day. mg/kg/day. mg/kg/day. 1 Four-day Peter’s test [27] at an oral dose of 50 mg/kg/day. Large et al. continued this work with the introduction of N-substituted imidazoles at position 3 LargeLargeLarge etLargeLarge al. etcontinuedet et et al.al. al. al. continuedcontinued continued continued this work thisthis this this work work withwork work withthewith with with introduction thethe the the introductionintroduction introduction introduction of N-substituted ofof of of NN N -substituted-substitutedN-substituted-substituted imidazoles imidazolesimidazoles imidazoles imidazoles at position atat at at positionposition position position 3 33 3 3 Largeof the et imidazopyridazine al. continued this work core with while the modulating introduction po ofsitionN-substituted 6 with substituen imidazolests like at position aminocyclohexyl 3 of of theofof ofimidazopyridazine of thethe the the imidazopyridazineimidazopyridazine imidazopyridazine imidazopyridazine core whilecorecore core core whilewhile modulatingwhile while modulatingmodulating modulating modulating position popo po positionsition 6sitionsition with 66 6 with6substituenwith with with substituensubstituen substituen substituents liketststs ts aminocyclohexyl ts likelikelike like like aminocyclohexylaminocyclohexylaminocyclohexyl aminocyclohexyl aminocyclohexyl the imidazopyridazine core while modulating position 6 with substituents like aminocyclohexyl or

N-methylpiperidine [31]. This resulted in the synthesis of compound 6 (Table2) showing an improved activity on Pf 3D7 compared to compound 5 and improved ligand-lipophilicity eﬃciency (LLE) with a score of 6, compared to 4.5 for compound 5. Inhibition assays on human kinases were not performed on compound 6, but other compounds from this series showed lower selectivity than compound 5. The last work on this series was done by Chapman et al. [32] using docking studies on the T. gondii CDPK1 ortholog structure to guide them. Starting from 5, they started by modulating R2 with N-substituted 2-aminopyrimidines, hoping for improved in vivo results. Compound 7 (Table2) emerged as the best compound with improved in vitro parameters. However, low PAMPA (parallel Molecules 2020, 25, x FOR PEER REVIEW 6 of 35

or N-methylpiperidine [31]. This resulted in the synthesis of compound 6 (Table 2) showing an improved activity on Pf3D7 compared to compound 5 and improved ligand-lipophilicity efficiency (LLE) with a score of 6, compared to 4.5 for compound 5. Inhibition assays on human kinases were Moleculesnot2020 performed, 25, 5949 on compound 6, but other compounds from this series showed lower selectivity than6 of 35 compound 5. The last work on this series was done by Chapman et al. [32] using docking studies on the T. 1 artificialgondii membrane CDPK1 ortholog permeability structure assay)to guide permeability them. Starting (4from Papp 5, they/nms started− ) led by to modulating poor results R2 within vivo, withN-substituted only 4% reduction 2-aminopyrimidines, of parasitemia hoping in the for four-day improved Peter’s in vivo test results. [27]. Compound Out of 66 human7 (Table kinases2) testedemerged at 1 µM as concentration, the best compound7 inhibited with improved nine human in vitro kinasesparameters. by at However, least 80%. low Additional PAMPA (parallel work was −1 thenartificial done: the membrane side chain permeabi at positionlity assay) 6 was permeability modulated (4 withPapp/nms different) led to cyclic poor amines,results in and vivo, at with position only 4% reduction of parasitemia in the four-day Peter’s test [27]. Out of 66 human kinases tested at 3, the fluorine atom position on both cycles was explored along with the nature of the two cycles 1 µM concentration, 7 inhibited nine human kinases by at least 80%. Additional work was then done: (phenyls or pyridines). This led to compound 8 (Table2) with slightly lower activity in vitro than 7. the side chain at position 6 was modulated with different cyclic amines, and at position 3, the fluorine The reductionatom position of parasitaemia on both cycles in was P. berghei explored mouse along model with the was nature slightly of the better two atcycles 51% than(phenyls that or of 5 at 46% (Tablepyridines).2). This led to compound 8 (Table 2) with slightly lower activity in vitro than 7. The reduction Inof parasitaemia 2016, Crowther in P. etberghei al. screened mouse model 14,000 was compounds slightly better on at multiple 51% than plasmodial that of 5 at 46% kinases (Table including 2). Pf CDPK1In [33 2016,]. One Crowther hundred et al. and screened eighty-one 14,000 molecules compounds showed on multiple a sub-micromolar plasmodial kinases IC50 includingon Pf CDPK1 and 12PfCDPK1 compounds [33]. One grouped hundred in four and chemicaleighty-one series molecules displayed showed an a IC sub-micromolar50 equal to or below IC50 on 20 Pf nM.CDPK1 The best compoundsand 12 compounds from each grouped series are in represented four chemical in series Figure displayed3. an IC50 equal to or below 20 nM. The best compounds from each series are represented in Figure 3.

FigureFigure 3. Best 3. Best compounds, compounds, from from each each chemical chemical seriesseries having a amolecule molecule with with an anIC50 IC equal50 equal to or tobelow or below 20 nM20 towardnM towardPf CDPK1 PfCDPK1 in in the the screening screening realizedrealized by Crowther Crowther et et al. al. [33]. [33 ].

4.1.2.4.1.2. Molecules Molecules Targeting TargetingPlasmodium Plasmodium falciparum falciparum Calcium-Dependent Protein Protein Kinase Kinase 4 (Pf 4CDPK4) (Pf CDPK4) Pf CDPK4PfCDPK4 is ais key a key enzyme enzyme for for the the exﬂagellationexflagellation of of gametocytes, gametocytes, a mechanism a mechanism leading leading to the to the formationformation of microgametocytes, of microgametocytes, the the male male gametocytes, gametocytes, in in the the mosquito mosquito midgut midgut [34–36]. [34–36 ]. In 2012, Ojo et al. described the properties of BKI-1 (compound 13), a disubstituted pyrazolo[3,4- In 2012, Ojo et al. described the properties of BKI-1 (compound 13), a disubstituted d]pyrimidin-4-amine [36] (Figure 4). 13 displayed an IC50 on PfCDPK4 of 4.1 nM, an IC50 on PfCDPK1 pyrazolo[3,4-d]pyrimidin-4-amine [36] (Figure4). 13 displayed an IC50 on Pf CDPK4 of 4.1 nM, of 136 nM, an EC50 toward asexual stages of 2 µM (unsurprisingly since CDPK4 is important in the an IC on Pf CDPK1 of 136 nM, an EC toward asexual stages of 2 µM (unsurprisingly since CDPK4 is sexual50 stage) and was able to inhibit 50P. falciparum exflagellation with an EC50 of 35 nM. 13′s selectivity importantwas assessed in the sexual with IC stage)50 measures and was on Hs ableABL to inhibitand HsSRC,P. falciparum two humanexﬂagellation kinases, who with were an superior EC50 of to35 nM. 130s selectivity50 and 20 µM, was respectively. assessed with Lastly, IC50 onmeasures a P. berghei on-infectedHsABL andmouseHs model,SRC, two 13 was human able kinases,to completely who were superiorinhibit to the 50 formation and 20 µM, of oocysts respectively. in mosquitoes Lastly, at on the a intraperitonealP. berghei-infected dose mouseof 10 mg/kg. model, 13 was able to completely inhibit the formation of oocysts in mosquitoes at the intraperitoneal dose of 10 mg/kg. Ojo et al. then carried out minor pharmacomodulations on 13 [37] including the replacement of the piperidine with a N-methylpiperidine, morpholine or pyrane heterocycle, replacement of the naphthalene by a quinoline and replacement of the methoxy group by an ethoxy group. Activities on the enzyme and the parasite were maintained for all molecules except the quinoline analog without a methoxy group. Compound 14, the N-methypiperidine analog of 13 (Figure4) showed similar in vitro activity parameters to 13 but clear improvements in in vitro and in vivo absorption-distribution-metabolism-elimination-toxicology (ADMET) parameters (Table3). Selectivity against human kinases was conserved as only one protein out of the 80 assessed was found to be inhibited. The only downside to 14 was its hERG channel activity, which was reduced, from 0.767 µM to more than 10 µM, by replacing the N-methylpiperidine with a pyrane. Molecules 2020, 25, 5949 7 of 35

Molecules 2020, 25, x FOR PEER REVIEW 7 of 35

Figure 4. 4-Aminopyrazolopyrimidine and 4-aminoimidazopyrazine inhibitors of PfCDPK4. 1 On Figure 4. 4-Aminopyrazolopyrimidine and 4-aminoimidazopyrazine inhibitors of Pf CDPK4. mammalian fibroblast cells. 2 On HepG2 cells. 1 On mammalian ﬁbroblast cells. 2 On HepG2 cells. Ojo et al. then carried out minor pharmacomodulations on 13 [37] including the replacement of the piperidine with aTable N-methylpiperidine, 3. ADMET parameters morpholine of compound or pyrane13 andheterocycle,14 [37]. replacement of the naphthalene by a quinoline and replacement of the methoxy group by an ethoxy group. Activities on the enzyme and the parasite wereIn Vitro maintained for all molecules except the In quinoline Vivo analog without a methoxy group. Compound 14, the N-methypiperidine AUCanalog1 of 13 (FigureAUC 4)1 showed similar in Solubility t (min) t (h) at vitro activity parameters to 13 but clear improvements1/2 in(µ inM vitromin) and at in vivo(µM absorption-distribution-min) at 1/2 Compound (µM) in Buﬀer Human Liver 100 mg/kg metabolism-elimination-toxicology (ADMET) parameters10 mg · /(Tablekg per 3). Selectivity10 mg· /kg against human pH 6.5 Microsome per os kinases was conserved as only one protein out of the 80 assessedos was foundIntraperitoneal to be inhibited. The only downside13 to 14 was its 47hERG channel activity, - which was re 57duced, from 0.767 317 µM to more than - 10 µM, by14 replacing the N-82methylpiperidine >with60 a pyrane. 430 863 13.5 1 AUC = Area Under Curve. Table 3. ADMET parameters of compound 13 and 14 [37].

This work was followedby In Vitro a massive medicinal chemistry study In by Vivo Vidadala et al. focused on the modulation of the lateral chains of the pyrazolo[3,4-d]pyrimidin-4-amine core [38]. ATP bindingt1/2 (h) at sites of Solubility t1/2 (min) AUC 1 AUC1 (µM·min) Pf Pf T. gondii Tg100 both CompoundCDPK1 and (µM)CDPK4 in are veryHuman similar Liver to the(µM·min) one found at in10 at 10CDPK1. mg/kg Thus CDPK1 was mg/kg used for docking studiesBuffer with pH 6.5 previously Microsome described Pfmg/kgCDPK4 per inhibitors os Intraperitoneal to guide pharmacomodulations. per os The C3 side-chain was the first to be modulated while keeping an isopropyl or tert-butyl group as 13 47 - 57 317 - N-substituent. The best C3 group was then used during the modulation of the N-substituent. A scaffold 14 82 >60 430 863 13.5 hopping strategy was also carried out, changing the pyrazolopyrimidine to an imidazopyrazine core. 1 AUC = Area Under Curve. This led to compound 15 with an interesting dual activity on Pf CDPK1 and Pf CDPK4 (Figure4) and a conservedThis selectivity work was followed versus Hs bySRC a massive (IC50 medicinal> 10 µM). chemistry The scaff studyold hopping by Vidadala strategy et al. focused was successful, on as illustratedthe modulation by compound of the lateral16. chains The authors of the pyrazolo[3,4- stated that theyd]pyrimidin-4-amine are now seeking core to enhance[38]. ATP the binding metabolic stabilitysites of of their both compoundsPfCDPK1 andin Pf vivoCDPK4, since are they very need similar to remainto the one in thefound bloodstream in T. gondii for CDPK1. a period Thus of three to fourTgCDPK1 weeks towas have used their for edockingffect on studies gametocytes. with previously described PfCDPK4 inhibitors to guide pharmacomodulations.In 2016, Huang et al. The used C3 on side-chain 5-aminopyrazole-4-carboxamides, was the first to be modulated while known keeping to work an isopropyl on Toxoplasma gondiior andtert-butylCryptosporidium group as N-substituent. parvum CDPK1The best C (orthologs3 group was of thenPf usedCDPK4), during to the obtain modulation new ofPf theCDPK4 N-substituent. A scaffold hopping strategy was also carried out, changing the pyrazolopyrimidine to inhibitors [39]. They synthesized 28 compounds; most of which have IC toward Pf CDPK4 below an imidazopyrazine core. This led to compound 15 with an interesting dual activity50 on PfCDPK1 and 100 nM. The best compound regarding Pf CDPK4 inhibition was 17, while 18 was interesting with PfCDPK4 (Figure 4) and a conserved selectivity versus HsSRC (IC50 > 10 µM). The scaffold hopping a dualstrategyPf CDPK1 was successful,/4 inhibition as illustrated (Figure5). by These compound two compounds 16. The authors did stated not possess that they any are activity now seeking on hERG channels.to enhance17 was the selective metabolic as itstability possessed of their an IC compounds50 superior in to vivo 30 µ, Msince against they HsneedSRC. to Workremain on in this theseries is currentlybloodstream focused for a on periodC. parvum of threetherapy to four weeks [40]. to have their effect on gametocytes.

Molecules 2020, 25, x FOR PEER REVIEW 8 of 35 Molecules 2020, 25, x FOR PEER REVIEW 8 of 35 In 2016, Huang et al. used on 5-aminopyrazole-4-carboxamides, known to work on Toxoplasma gondii andIn 2016,Cryptosporidium Huang et al. parvum used on CDPK1 5-aminopyr (orthologsazole-4-carboxamides, of PfCDPK4), to obtainknown new to work PfCDPK4 on Toxoplasma inhibitors gondii and Cryptosporidium parvum CDPK1 (orthologs of PfCDPK4), to obtain new PfCDPK4 inhibitors [39]. They synthesized 28 compounds; most of which have IC50 toward PfCDPK4 below 100 nM. The best[39]. compound They synthesized regarding 28 compPfCDPK4ounds; inhibitionmost of which was have 17, ICwhile50 toward 18 wasPfCDPK4 interesting below 100with nM. a Thedual PfCDPK1/4best compound inhibition regarding (Figure Pf 5).CDPK4 These inhibition two compounds was 17, whiledid not 18 possesswas interesting any activity with ona dualhERG PfCDPK1/4 inhibition (Figure 5). These two compounds did not possess any activity on hERG Moleculeschannels.2020 17, 25 was, 5949 selective as it possessed an IC50 superior to 30 µM against HsSRC. Work on8 ofthis 35 channels. 17 was selective as it possessed an IC50 superior to 30 µM against HsSRC. Work on this series is currently focused on C. parvum therapy [40]. series is currently focused on C. parvum therapy [40].

FigureFigureFigure 5.5. 5.Aminopyrazole-carboxamide Aminopyrazole-carboxamide andand indoloneindolone inhibitorsinhibitors of ofof Pf PfPfCDPK4.CDPK4. In the Crowther et al. screening [33], 55 compounds showed an activity below the micromolar In theIn the Crowther Crowther et etal. al. screening screening [33], [33], 55 55 compou compoundsnds showed anan activityactivity below below the the micromolar micromolar rangerange against againstPf CDPK4.PfCDPK4. The The best best compound compound ((1919)) displayeddisplayed an an IC IC5050 ofof 21 21 nM nM (Figure (Figure 5).5). range against PfCDPK4. The best compound (19) displayed an IC50 of 21 nM (Figure 5). TheThe only only thing thing that that prevents preventsPf PfCDPK4CDPK4 inhibitorsinhibitors fromfrom entering more more advanced advanced pre-clinical pre-clinical The only thing that prevents PfCDPK4 inhibitors from entering more advanced pre-clinical studiesstudies is theis the long-term long-term bioavailability bioavailability they they would would needneed to be active agains againstt gametocytes. gametocytes. Moreover, Moreover, studies is the long-term bioavailability they would need to be active against gametocytes. Moreover, findingfinding from from Vidadala Vidadala et al. et [38 al.] and [38] data and from data screening from screening by Crowther by Crowther et al. [33 ],et identified al. [33], compoundsidentified finding from Vidadala et al. [38] and data from screening by Crowther et al. [33], identified providingcompounds dual inhibitionproviding ofdualPf CDPK1 inhibition and PfofCPDK4, PfCDPK1 off eringand thePfCPDK4, prospect offering of compounds the prospect with action of compounds providing dual inhibition of PfCDPK1 and PfCPDK4, offering the prospect of on twocompounds stagesout with of action three on of thetwoPlasmodium stages out ofcycle. three of the Plasmodium cycle. compounds with action on two stages out of three of the Plasmodium cycle. 4.2.4.2. Molecules Molecules Targeting Targeting Plasmodium Plasmodium falciparum falciparum Choline Choline KinaseKinase (PfCK)(PfCK) 4.2. Molecules Targeting Plasmodium falciparum Choline Kinase (PfCK) CholineCholine kinase kinase is anis enzymean enzyme involved involved in the in synthesisthe synthesis of phosphatidylcholine; of phosphatidylcholine; phospholipid phospholipid most Choline kinase is an enzyme involved in the synthesis of phosphatidylcholine; phospholipid frequentlymost frequently found in P.found falciparum in P.. falciparum It transforms. It cholinetransforms into choline phosphocholine, into phosphocholine, and is also involved and is also in the most frequently found in P. falciparum. It transforms choline into phosphocholine, and is also synthesisinvolved of in phosphatidylethanolamine, the synthesis of phosphatidylethanolamine, the second most the common second most phospholipid common phospholipid [41]. Inhibition [41].of involved in the synthesis of phosphatidylethanolamine, the second most common phospholipid [41]. Pf CKInhibition has been of Pf shownCK has to been reduce shown parasite to reduce growth parasitein vitro grow[42th, 43in ].vitro The [42,43]. active The sites active of Pf sitesCK haveof PfCK 69% Inhibitionhave 69% of similarityPfCK has withbeen the shown human to reduceenzyme parasite HsCKα 1grow [44].th in vitro [42,43]. The active sites of PfCK similarity with the human enzyme HsCKα1 [44]. have 69%In similarity2007, Choubey with theet al. human [45] studied enzyme hexadecyltrimethylammonium HsCKα1 [44]. bromide (compound 20, In 2007, Choubey et al. [45] studied hexadecyltrimethylammonium bromide (compound 20, FigureIn 2007, 6). It Choubey displayed etin al.vitro, [45] at 10studied µM and hexadecyltrimethylammonium 20 µM, parasitic growth inhibition bromide of 62% (compound and 81%, and 20 , Figure6). It displayed in vitro, at 10 µM and 20 µM, parasitic growth inhibition of 62% and 81%, Figurereduced 6). It phosphocholine displayed in vitro, synthesis at 10 µMby 57% and at 20 10 µM, µM. parasitic In a P. yoelii growth mouse inhibition model, it of was 62% able and to 81%, reduce and and reduced phosphocholine synthesis by 57% at 10 µM. In a P. yoelii mouse model, it was able to reducedparasitemia phosphocholine by almost 50% synthesis after four by 57%days at of 10 intravenous µM. In a P. doses yoelii of mouse 5 mg/kg. model, it was able to reduce reduce parasitemia by almost 50% after four days of intravenous doses of 5 mg/kg. parasitemia by almost 50% after four days of intravenous doses of 5 mg/kg.

Figure 6. Hexadecyltrimethylammonium bromide 20 structure.

A screening byFigure Crowther 6. Hexadecyltrimethylammonium et al. on around 5000 commercially bromide 20available structure. small molecules led to the discovery of three hits against PfCK [46] (compounds 21, 22 and 23, Figure 7). A screeningscreening byby CrowtherCrowther et et al. al. on on around around 5000 5000 commercially commercially available available small small molecules molecules led led to the to discoverythe discovery of three of three hits hits against againstPf CK PfCK [46] [46] (compounds (compounds21, 2221,and 22 and23, Figure23, Figure7). 7). While studying Pf CK metabolic activities, Serrán-Aguilera et al. used two compounds (24 and 25, Table4) to inhibit the enzyme [ 41], which possessed a Ki between 30 and 40 µM (when assessed for choline conversion to phosphocholine). To explore the activity of these pyridinium salts on Pf CK, Schiaﬁno-Ortega et al. assessed 1,2-diphenoxyethane salts with a symmetrical structure [47] close to 24 and 25. Bisquinolinium bromide salts derivatives were the most potent with submicromolar activity on P. falciparum. However, their activity on the Pf CK enzyme was found to be above 2 µM for all this series, raising doubts about their real mechanism of action. The best compound of this series was compound 26, with an improved Pf CKCho IC50 (formation of phosphocholine is measured) reduced from 103 to 2.4 µM, but it no longer showed P. falciparum in vitro activity (Table4). Compound 26 was not selective as it also inhibited the human HsCKα1 enzyme. Molecules 2020, 25, x FOR PEER REVIEW 9 of 35 MoleculesMolecules 2020 2020, 25,, x25 FOR, x FOR PEER PEER REVIEW REVIEW 9 of 935 of 35

Molecules 2020, 25, 5949 9 of 35 Molecules 2020, 25, x FOR PEER REVIEW 9 of 35

Figure 7. Hits from Crowther et al. against PfCK [46]. FigureFigure 7. Hits 7. Hits from from Crowther Crowther et al. et againstal. against PfCK Pf CK[46]. [46]. While studying PfCK metabolic activities, Serrán-Aguilera et al. used two compounds (24 and WhileWhile studying studying PfCK PfCK metabolic metabolic activities, activities, Serrán-Agu Serrán-Aguilerailera et al.et al.used used two two compounds compounds (24 ( and24 and 25, Table 4) to inhibit the enzyme [41], which possessed a Ki between 30 and 40 µM (when assessed 25, 25Table, Table 4) to4) inhibitto inhibit the the enzyme enzyme [41], [41], which which possessed possessed a K ai betweenKi between 30 and30 and 40 µM40 µM (when (when assessed assessed for choline conversion to phosphocholine). To explore the activity of these pyridinium salts on PfCK, for forcholine choline conversion conversion to phosphocholine).to phosphocholine). To Toexpl exploreore the the activity activity of theseof these pyridinium pyridinium salts salts on onPfCK, PfCK, Schiafino-Ortega et al. assessed 1,2-diphenoxyethane salts with a symmetrical structure [47] close to Schiafino-OrtegaSchiafino-Ortega et al.et assessedal. assessed 1,2-diphenoxyethane 1,2-diphenoxyethane salts salts with with a symmetrical a symmetrical structure structure [47] [47] close close to to 24 and 25. Bisquinolinium bromide salts derivatives were the most potent with submicromolar 24 24and and 25 . 25Bisquinolinium. Bisquinolinium bromide bromide salts salts derivatives derivatives were were the the most most potent potent with with submicromolar submicromolar activity on P. falciparum. However, their activity on the PfCK enzyme was found to be above 2 µM activityactivity on onP. falciparumP. falciparum. However,. However, their their activity activity on onthe the PfCK PfCK enzyme enzyme was was found found to beto beabove above 2 µM 2 µM for all this series, raising doubts about their real mechanism of action. The best compound of this for forall allthis this series, series, raising raising doubts doubts about about their their real real mechanism mechanism of acoftion. action. The The best best compound compound of thisof this series was compound 26, with an improved PfCKCho IC50 (formation of phosphocholine is measured) seriesseries was was compound compound 26, 26with, with an animproved improved PfCK PfCKCho ChoIC50 IC (formation50 (formation of phosphocholineof phosphocholine is measured) is measured) reduced from 103 to 2.4 µM, but it no longer showed P. falciparum in vitro activity (Table 4). reducedreduced from from 103 103 to to2.4 Figure2.4µM, µM, but7. HitsHitsbut it fromno fromit nolonger Crowther longer showed et showed al. against againstP. P.falciparum PfPffalciparumCKCK [46]. [46 in]. invitro vitro activity activity (Table (Table 4). 4). Compound 26 was not selective as it also inhibited the human HsCKα1 enzyme. CompoundCompound 26 was26 was not not selective selective as itas also it also inhibited inhibited the the human human Hs CKHsCKα1 αenzyme.1 enzyme. While studying PfCK metabolic activities, Serrán-Aguilera et al. used two compounds (24 and TableTableTable 4. 4.Bispyridinium 4.Bispyridinium Bispyridinium bromide bromide bromide salt salt salt and and and bisquinolinium bisquinolinium bisquinolinium bromide bromide bromide salt salt salt inhibitors inhibitors inhibitors of ofPf of PfCK. PfCK.CK. 25, Table 4)Table to inhibit 4. Bispyridinium the enzyme bromide [41], which salt and possessed bisquinolinium a Ki between bromide 30salt and inhibitors 40 µM of (when PfCK. assessed for choline conversion to phosphocholine). To explorePfP CKthefCKCho activityCho IC IC50 50 of thesePPffP pyridinium3D73D7f3D7 IC IC50 50 saltsHsHsCK onHsCKα CKPf1αCK,1α 1 CompoundCompoundCompound PfCKPfCKChoChoIC 50IC(50µ M) Pf3D7 IC50 HsCKα1 Schiafino-Ortega etCompound al. assessed 1,2-diphenoxyethane salts(µM) with a symmetrical(nM)(nM) structure [47]IC IC50close50 to (µM)(µM) (nM)(nM) IC50IC 50 24 and 25. Bisquinolinium bromide salts derivatives were the most potent with submicromolar activity on P. falciparum. However, their activity on the PfCK enzyme was found to be above 2 µM for all this series, raising doubts about their real mechanism276 of action. The 3best compound ofthis 276276 3 33 -- - series was compound 26, with an improved PfCKCho IC50 (formation of phosphocholine is measured) reduced from 103 to 2.4 µM, but it no longer showed P. falciparum in vitro activity (Table 4).

Compound 26 was not selective as it also inhibited the human HsCKα1 enzyme.

Table 4. Bispyridinium bromide salt and bisquinolinium103 bromide salt inhibitors3 of PfCK. - 103103 3 33 -- - PfCKCho IC50 Pf3D7 IC50 HsCKα1 Compound (µM) (nM) IC50

276 3 - 2.4 142 1.7 2.4 2.4 142 142142 1.71.7 1.7

103 3 - MostMost of of the the molecules molecules presented presented above above (from (from 21 21 to to 25 25) probably) probably target target more more than than just just Pf PfCK,CK, as as Most of the molecules molecules presented presented above above (from (from 2121 toto 2525) probably) probably target target more more than than just just PfCK,Pf CK, as enzymeenzyme IC IC50 50values values not not in in line line IC IC50 50on on the the parasite: parasite: 24 24 is isthe the best best example example of of such such behavior. behavior. asenzyme enzyme IC50 IC valuesvalues not not in inline line IC IC50 onon thethe parasite: parasite: 2424 is isthe the best best example example of of such such behavior. behavior. Furthermore,Furthermore,50 the the design design of of these these compounds compounds50 needs needs to to take take into into account account the the human human choline choline kinase. kinase. Furthermore, the design of these compounds needs to take into account the human choline kinase. Thus,Thus, Pf PfCKCK inhibitors inhibitors are are potentially potentially valuable valuable compounds, compounds, they they are are still still in in the the early early stages stages of of Thus, PfCK inhibitors are potentially valuable compounds, they they are are still in the early stages of developmentdevelopment and and promising promising hits hits have have yet yet to to be be discovered. discovered. development and promising hitshits havehave yetyet toto bebe discovered.discovered.

2.4 142 1.7 4.3. Molecules Targeting Plasmodium falciparum Casein Kinase 2 (PfCK2) Casein kinases are serine/threonine protein kinases found in eukaryotic organisms. Two casein

kinases can be found in P. falciparum: CK1 and CK2. Pf CK2 is thought to be a key enzyme during the asexual blood-stage of the parasite. It has been shown that many proteins are possibly phosphorylated, Most of the molecules presented above (from 21 to 25) probably target more than just PfCK, as and thus have their activity regulated, via Pf CK2 during the asexual blood-stage [48]. Both Pf CK1 and enzyme IC50 values not in line IC50 on the parasite: 24 is the best example of such behavior. Pf CK2 have been demonstrated to be essential for the asexual blood-stage [17,49]. Furthermore, the design of these compounds needs to take into account the human choline kinase. While studying the Pf CK2 catalytic domain, Ruiz-Carillo et al. used CX-4945 (Figure8, compound Thus, PfCK inhibitors are potentially valuable compounds, they are still in the early stages of 27)[50], a human CK2 inhibitor currently in multiple phase II clinical trials in oncology [51], to inhibit development and promising hits have yet to be discovered. Pf CK2 with an IC50 of 13.2 nM.

Molecules 2020, 25, x FOR PEER REVIEW 10 of 35

4.3. Molecules Targeting Plasmodium falciparum Casein Kinase 2 (PfCK2)

MoleculesMoleculesCasein 2020 2020, ,kinases25 25, ,x x FOR FOR PEER PEERare serine/threoniREVIEW REVIEW ne protein kinases found in eukaryotic organisms.1010 of of Two 35 35 casein kinases can be found in P. falciparum: CK1 and CK2. PfCK2 is thought to be a key enzyme during the 4.3.4.3. MoleculesMolecules TargetingTargeting PlasmodiumPlasmodium falciparumfalciparum CaseinCasein KinaseKinase 22 (PfCK2)(PfCK2) asexual blood-stage of the parasite. It has been shown that many proteins are possibly phosphorylated,CaseinCasein kinaseskinases and areare thus serine/threoniserine/threoni have theirnene proteinactivityprotein kinaseskinases regulated, foundfound via inin eukaeukaPfCK2ryoticryotic during organisms.organisms. the asexual TwoTwo caseincasein blood-stage [48].kinaseskinases Both cancan Pf bebeCK1 foundfound and inin PPfP.. CK2falciparumfalciparum have:: CK1 CK1been andand demonstrated CK2.CK2. PfPfCK2CK2 isis thoughttothought be essential toto bebe aa keykey for enzymeenzyme the asexual duringduring theblood-stagethe [17,49].asexualasexual blood-stageblood-stage ofof thethe parasite.parasite. ItIt hashas beenbeen shownshown thatthat manymany proteinsproteins areare possiblypossibly phosphorylated,phosphorylated, andand thusthus havehave theirtheir activityactivity regulated,regulated, viavia PfPfCK2CK2 duringduring thethe asexualasexual blood-stageblood-stage While studying the PfCK2 catalytic domain, Ruiz-Carillo et al. used CX-4945 (Figure 8, [48].[48]. BothBoth PfPfCK1CK1 andand PfPfCK2CK2 havehave beenbeen demonstrateddemonstrated toto bebe essentialessential forfor thethe asexualasexual blood-stageblood-stage compoundMolecules 2020 27, 25), 5949[50], a human CK2 inhibitor currently in multiple phase II clinical trials in oncology10 of 35 [17,49].[17,49]. [51], toWhileWhile inhibit studyingstudying PfCK2 thewiththe PfPf anCK2CK2 IC 50catalyticcatalytic of 13.2 domain,nM.domain, Ruiz-CarilloRuiz-Carillo etet al.al. usedused CX-4945CX-4945 (Figure(Figure 8,8, compoundcompound 2727)) [50],[50], aa humanhuman CK2CK2 inhibitorinhibitor currentlycurrently inin multiplemultiple phasephase IIII clinicalclinical trialstrials inin oncologyoncology [51],[51], toto inhibitinhibit PfPfCK2CK2 withwith anan ICIC5050 ofof 13.213.2 nM.nM.

Figure 8. CX-4945,CX-4945, an HsCK2 inhibitor in clinical trials, also inhibits PfCK2.CK2. 4.4. Molecules TargetingFigureFigure 8.8. PlasmodiumCX-4945,CX-4945, anan HsHs falciparumCK2CK2 inhibitorinhibitor Cyclin-Dependent-Like inin clinicalclinical trials,trials, alsoalso inhibitsinhibits Kinase PfPfCK2.CK2. (PfCLK) 4.4. Molecules Targeting Plasmodium falciparum Cyclin-Dependent-Like Kinase (PfCLK) 4.4.4.4. MoleculesTheMolecules CLK TargetingTargeting family includes PlasmodiumPlasmodium four falcipfalcip enzymesarumarum Cyclin-Dependent-LikeCyclin-Dependent-Like (Pf CLK1 to Pf CLK KinaseKinase 4) involved (PfCLK)(PfCLK) in the phosphorylation The CLK family includes four enzymes (PfCLK1 to PfCLK 4) involved in the phosphorylation (and the activity modulation) mainly of serine-arginine-rich proteins found in spliceosomes [14,52,53]. (and TheThethe CLKCLKactivity familyfamily modulation) includesincludes fourfour mainlyenzymesenzymes of ((PfPf CLK1CLK1serine-arginine-rich toto PfPfCLKCLK 4)4) involvedinvolved proteins inin thethe foundphosphorylationphosphorylation in spliceosomes Spliceosomes(and the activity are complexesmodulation) of mainly proteins of involvedserine-arginine-rich in the removal proteins of intronsfound in pre-messengerspliceosomes RNA. [14,52,53].(and the activitySpliceosomes modulation) are complexesmainly of ofserine-arginine-rich proteins involved proteins in the found removal in spliceosomes of introns in pre- All[14,52,53].[14,52,53].Pf CLKs SpliceosomesSpliceosomes are considered areare complexes essentialcomplexes for ofof theproteinsproteins asexual involvedinvolved blood-stage inin thethe removal [removal17]. Inhibition ofof intronsintrons of inin CLKs pre-pre- was first messenger RNA. All PfCLKs are considered essential for the asexual blood-stage [17]. Inhibition of linkedmessengermessenger to a schizonticideRNA.RNA. AllAll PfPfCLKsCLKs and areare gametocytocidal consideredconsidered essentialessential effect forfor [53 thethe]. A asas moreexualexual recentblood-stageblood-stage study [1[1 on7].7]. PfInhibitionInhibitionCLK3 showed ofof that CLKs was first linked to a schizonticide and gametocytocidal effect [53]. A more recent study on inhibitionCLKsCLKs waswas of firstfirst this linkedlinked protein toto aa ffschizonticideschizonticideected all three andand stages gametocytocidalgametocytocidal of the malaria effecteffect cycle [53].[53]. [A54A more].more recentrecent studystudy onon PfCLK3 showed that inhibition of this protein affected all three stages of the malaria cycle [54]. PfPfCLK3CLK3In 2014, showedshowed Kern thatthat et inhibitioninhibition al. studied ofof thisthis the proteinprotein effect of affeaffe inhibitingctedcted allall threethreePf CLKs stagesstages [ ofof53 the].the The malariamalaria authors cyclecycle did [54].[54]. not manage to InInIn 2014, 2014,2014, KernKernKern et etet al. al.al. studiedstudied studied thethe the effecteffect effect ofof inhibiting inhibitingof inhibiting PfPfCLKsCLKs PfCLKs [53].[53]. The The[53]. authorsauthors The authors diddid notnot did managemanage not tomanageto to disrupt the genes directly and therefore turned to chemical compounds. They screened 63 HsCLK disruptdisruptdisrupt the thethe genesgenes directlydirectlydirectly andand and thereforetherefore therefore turnedturned turned toto chemicalchemical to chemical compounds.compounds. compounds. TheyThey screened screenedThey screened 6363 HsHsCLKCLK 63 HsCLK inhibitors against Pf CLKs. Two compounds, 28 and chlorhexidine (29) showed interesting results inhibitorsinhibitorsinhibitors againstagainst PfPfPfCLKs.CLKs.CLKs. TwoTwo Two compounds,compounds, compounds, 2828 andand28 and chlorhexidinechlorhexidine chlorhexidine ((2929)) showedshowed (29) showed interestinginteresting interesting resultsresults results regarding Pf CLK inhibition and Pf 3D7 growth inhibition; results are summarized in Table5. regardingregardingregarding PfPfCLKCLK inhibitioninhibition andand and PfPf 3D7Pf3D73D7 growthgrowth growth inhibition;inhibition; inhibition; resultsresults results areare summarizedsummarized are summarized inin TableTable in 5.5. Table 5.

TableTable 5.5. 5. SummarySummarySummary ofof ofinin vitroinvitro vitro propertiesproperties properties ofof PfPfCLK ofCLKPf inhibitorsCLKinhibitors inhibitors fromfrom KernKern from etet Kernalal.. [53].[53]. et al. [53]. Table 5. Summary of in vitro properties of PfCLK inhibitors from Kern et al. [53]. 11 PfPf3D73D7 EIAEIA 1 %% InhibitionInhibition% Inhibition PfPf3D73D7 EIAEIAIC 501 % Inhibition Compound 50 50 (at Pf503D7 IC50 Concentration) CompoundCompound ICICIC5050( µ M) ICIC50( µ M) (at(at PfPf3D73D7 ICIC50 Concentration)Concentration) Compound (µM)(µM)IC 50 (µM)(µM)IC 50PP ffCLK1CLK1Pf CLK1 P(atPffCLK2CLK2 Pf3D7Pf CLK2P PICffCLK3CLK350 Concentration)Pf CLK3PPffCLK4CLK4 PfCLK4 (µM) (µM) PfCLK1 PfCLK2 PfCLK3 PfCLK4

4.44.44.4 13.8 13.8 13.8 73% 73% 73% 45.6% 45.6% 45.6%35.1%35.1% 35.1%71.6%71.6% 71.6% 4.4 13.8 73% 45.6% 35.1% 71.6%

0.60.60.6 19.8 19.8 19.8 68.4% 68.4% 68.4% 76.4%76.4% 76.4%74.2%74.2% 74.2%76.1%76.1% 76.1%

0.6 19.8 68.4% 76.4% 74.2% 76.1% 11 Exflagellation Exflagellation1 Exﬂagellation InhibitoryInhibitory Inhibitory Assay.Assay. Assay.

Molecules 2020, 25, x FOR PEER REVIEW 11 of 35 In 2017, Bendjeddou et al. tested1 an imidazopyridazine series on multiple human and parasitic Exflagellation Inhibitory Assay. kinases, includingIn 2017,Pf BendjeddouCLK1 [55 et]. al. Two tested compounds an imidazopyridazine (30 and series31, Figure on multiple9) showed human and activity parasitic on Pf CLK1 kinases, including PfCLK1 [55]. Two compounds (30 and 31, Figure 9) showed activity on PfCLK1 below 100 nM but lacked selectivity as they also targeted human kinases, in some cases with IC50 below 100 nM but lacked selectivity as they also targeted human kinases, in some cases with IC50 below 100below nM. 100 nM.

Figure 9. Imidazopyridazines inhibitors of PfCLK1. Figure 9. Imidazopyridazines inhibitors of Pf CLK1. More recently, Alam et al. screened 30,000 compounds on PfCLK1 and PfCLK3 [54]. TCMDC- 135793 (compound 32) and TCMDC-135051 (compound 33) were among the most potent and selective compounds on PfCLK1 and PfCLK3, respectively (Figure 10). Target was confirmed for 33 with PfDd2 strain possessing a mutant PfCLK3 that showed an increased IC50 value. Compund 33 was active at all stages, inhibiting: • Schizont development in blood-stage, • Sporozoite invasion and development in the liver stage, • Gametocyte development, • Exflagellation in the mosquito midgut.

Figure 10. Molecules inhibiting PfCLK1 or PfCLK3.

SAR work associated with 33 was carried out by Mahindra et al. [56]. Modulations targeting the substituents of the phenyl ring at position 2 of the pyrrolo[2,3-b]pyridine core led to analogs possessing an IC50 against PfCLK3 below 100 nM. However, micromolar activity was found against Pf3D7 when the diethylamine was changed to dimethylamine, primary amine or morpholine substituents. The position of the methoxy substituent appeared to be important for activity. Similar modifications were carried out on the other phenyl ring: removing the isopropyl substituent or replacing it by methyl led to compounds with micromolar activities against Pf3D7, and removing the carboxylic acid or changing it to an ethyl ester resulted in coumpounds with micromolar activity on PfCLK3. Replacing the carboxylic acid to a tetrazole, a known bioisoster, led to compound 34 showing a slight improvement in PfCLK3 activity, with an IC50 of 19 nM but with an increased Pf3D7 IC50 to 270 nM. 34 stability was conserved in vitro in mouse liver microsomes with an intrinsic clearance at 2.32 mL/min/g of liver, compared to 33 with 1.33.

Molecules 2020, 25, x FOR PEER REVIEW 11 of 35

In 2017, Bendjeddou et al. tested an imidazopyridazine series on multiple human and parasitic kinases, including PfCLK1 [55]. Two compounds (30 and 31, Figure 9) showed activity on PfCLK1 below 100 nM but lacked selectivity as they also targeted human kinases, in some cases with IC50 below 100 nM.

Molecules 2020, 25, 5949 11 of 35

More recently, Alam et al. screened 30,000 compounds on Pf CLK1 and Pf CLK3 [54]. TCMDC- Figure 9. Imidazopyridazines inhibitors of PfCLK1. 135793 (compound 32) and TCMDC-135051 (compound 33) were among the most potent and selective compoundsMore onrecently,Pf CLK1 Alam and etPf al.CLK3, screened respectively 30,000 compounds (Figure 10). on Target PfCLK1 was and conﬁrmed PfCLK3 for[54].33 TCMDC-with Pf Dd2 strain135793 possessing (compound a mutant 32) andPf CLK3TCMDC-135051 that showed (compound an increased 33) ICwere50 value. among Compund the most33 potentwas active and at allselective stages, inhibiting:compounds on PfCLK1 and PfCLK3, respectively (Figure 10). Target was confirmed for 33 with PfDd2 strain possessing a mutant PfCLK3 that showed an increased IC50 value. Compund 33 Schizont development in blood-stage, • was active at all stages, inhibiting: Sporozoite invasion and development in the liver stage, • • Schizont development in blood-stage, Gametocyte development, • • Sporozoite invasion and development in the liver stage, Exﬂagellation in the mosquito midgut. • • Gametocyte development, • Exflagellation in the mosquito midgut.

FigureFigure 10.10. Molecules inhibiting inhibiting PfPfCLK1CLK1 or or PfPfCLK3.CLK3.

SARSAR work work associated associated withwith 3333 was carried out out by by Mahindra Mahindra et et al. al. [56]. [56 ].Modulations Modulations targeting targeting the the substituentssubstituents of of the the phenyl phenyl ring ring at position at position 2 of the2 of pyrrolo[2,3-b]pyridine the pyrrolo[2,3-b]pyridine core led core to analogsled to analogs possessing anpossessing IC50 against an ICPf50CLK3 against below PfCLK3 100 below nM. 100 However, nM. However, micromolar microm activityolar activity was foundwas found against againstPf 3D7 whenPf3D7 the when diethylamine the diethylamine was changed was tochanged dimethylamine, to dimethylamine, primary amineprimary or morpholineamine or morpholine substituents. Thesubstituents. position of The the position methoxy of substituent the methoxy appeared substituen tot beappeared important to be for important activity. for Similar activity. modiﬁcations Similar weremodifications carried out were on the carried other phenylout on ring:the other removing phenyl the ring: isopropyl removing substituent the isopropyl or replacing substituent it by methyl or ledreplacing to compounds it by methyl with led micromolar to compound activitiess with micromolar against Pf activities3D7, and against removing Pf3D7, the and carboxylic removing acid the or changingcarboxylic it to acid an ethylor changing ester resulted it to an in ethyl coumpounds ester resulted with in micromolar coumpounds activity with onmicromolarPf CLK3. activity Replacing on the PfCLK3. Replacing the carboxylic acid to a tetrazole, a known bioisoster, led to compound 34 showing carboxylic acid to a tetrazole, a known bioisoster, led to compound 34 showing a slight improvement a slight improvement in PfCLK3 activity, with an IC50 of 19 nM but with an increased Pf3D7 IC50 to in Pf CLK3 activity, with an IC of 19 nM but with an increased Pf 3D7 IC to 270 nM. 34 stability 270 nM. 34 stability was conserved50 in vitro in mouse liver microsomes with an50 intrinsic clearance at was conserved in vitro in mouse liver microsomes with an intrinsic clearance at 2.32 mL/min/g of liver, 2.32 mL/min/g of liver, compared to 33 with 1.33. compared to 33 with 1.33.

4.5. Molecules Targeting Coenzyme A Synthesis Pathway Kinases: Plasmodium falciparum Pantothenate Kinase and Dephospho-Coenzyme A Kinase (PfPanK & PfDPCK)

Coenzyme A is a molecule found in many metabolic pathways in eukaryotic life forms. In P. falciparum, coenzyme A is synthesized in ﬁve steps starting from pantothenate; two of these steps involve kinases:

the ﬁrst step, transforming pantothenate into 4 -phosphopantothenate, is catalyzed by Pf PanK, • 0 the last step, transforming dephospho-coenzyme A into coenzyme A, is catalyzed by Pf DPCK. • While the synthesis and the function of coenzyme A are well preserved around all organisms that possess it [57], the enzymes’ coding sequences can diﬀer. Studies on the inhibition of Pf PanK focused on the synthesis of pantothenate (Table6, compound 35) analogs able to competitively inhibit 40-phosphopantothenate synthesis. Spry et al. synthesized ten MoleculesMolecules 2020 2020, ,25 25, ,x x FOR FOR PEER PEER REVIEW REVIEW 1212 of of 35 35

4.5.4.5. MoleculesMolecules TargetingTargeting CoenzymeCoenzyme AA SynthesisSynthesis PathwayPathway Kinases:Kinases: PlasmodiumPlasmodium falciparumfalciparum PantothenatePantothenate KinaseKinase andand Dephospho-CoenzymeDephospho-Coenzyme AA KinaseKinase (PfPanK(PfPanK && PfDPCK)PfDPCK) CoenzymeCoenzyme AA isis aa moleculemolecule foundfound inin manymany metabolicmetabolic pathwayspathways inin eukaryoticeukaryotic lifelife forms.forms. InIn P.P. falciparumfalciparum,, coenzymecoenzyme AA isis synthesizedsynthesized inin fivefive stepssteps ststartingarting fromfrom pantothenapantothenate;te; twotwo ofof thesethese stepssteps involveinvolve kinases:kinases: • •• thethe firstfirst step,step, transformingtransforming pantothenatepantothenate intointo 44′-phosphopantothenate,′-phosphopantothenate, isis catalyzedcatalyzed byby PfPfPanK,PanK, • •• thethe lastlast step,step, transformingtransforming dephospho-coenzydephospho-coenzymeme AA intointo coenzymecoenzyme A,A, isis catalyzedcatalyzed byby PfPfDPCK.DPCK. WhileWhile thethe synthesissynthesis andand thethe functionfunction ofof coenzycoenzymeme AA areare wellwell preservedpreserved aroundaround allall organismsorganisms thatthat possesspossess itit [57],[57], thethe enzymeenzymes’s’ codingcoding sequencessequences cancan differ.differ. Molecules 2020, 25, 5949 12 of 35 StudiesStudies on on the the inhibition inhibition of of Pf PfPanKPanK focused focused on on the the synthesis synthesis of of pantothenate pantothenate (Table (Table 6, 6, compound compound 3535)) analogsanalogs ableable toto competitivelycompetitively inhibitinhibit 44′-phosphopantothenate′-phosphopantothenate synthesisynthesis.s. SprySpry etet al.al. synthesizedsynthesized analogstenten analogs analogs with various with with various various chains chains chains on the on nitrogenon the the nitrog nitrog atomenen of atom atom the amideof of the the amide groupamide [groupgroup58]. Compound [58]. [58]. Compound Compound36 was 36 36 the was was best the the bestbest ofof thethe series,series, withwith anan ICIC5050 ofof 7676 µMµM onon PfPf3D73D7 (at(at 11 µMµM pantothenatepantothenate concentration).concentration). IncreasingIncreasing ofbest the series,of the withseries, an with IC50 anof IC 7650µ ofM 76 on µMPf 3D7 on Pf (at3D7 1 µ M(at pantothenate1 µM pantothenate concentration). concentration). Increasing Increasing the thethe pantothenatepantothenate concentrationconcentration increasedincreased thisthis ICIC5050 suggestingsuggesting aa mechanismmechanism ofof actionaction relatedrelated toto thethe pantothenatethe pantothenate concentration concentration increased increased this ICthis50 ICsuggesting50 suggesting a mechanism a mechanism of actionof action related related to to the the pantothenatepantothenatepantothenate metabolism. metabolism.metabolism. Changing ChangingChanging the thethe terminal terminalterminal group grougroup ofp ofof the thethe chain chainchain or oror its itsits length lengthlength did diddid not notnot aﬀ affectectaffect the thethe activity.activity.activity. Spry SprySpry et al. etet recently al.al. recentlyrecently investigated investigatedinvestigated the synthesis thethe synthesissynthesis of analogs ofof analogs ofanalogs CJ-15,801 ofof CJ-15,801CJ-15,801 (37)[59], a((37trans37)) [59],[59],enamide aa transtrans analogenamideenamide of pantothenate. analoganalog ofof pantothenate.pantothenate. Modulations MoMo weredulationsdulations focused werewere on focusedfocused the ester onon and thethe ester modifyingester andand modifyingmodifying the diol side thethe dioldiol chain. sideside chain.chain. CompoundCompound 3838 showedshowed anan improvedimproved ICIC5050 of of 1313 µMµM onon PfPf3D73D7 comparedcompared toto 3636 µMµM forfor 3737 butbut Compoundchain. Compound38 showed 38 anshowed improved an improved IC50 of 13 ICµ50M of on13 PfµM3D7 on compared Pf3D7 compared to 36 µ Mto for36 µM37 but for most37 but mostmost importantlyimportantly aa 100-fold100-fold decreaseddecreased ofof thethe PfPfPanKPanK ICIC5050 value.value. InIn 2017,2017, ChiuChiu etet al.al. describeddescribed importantlymost importantly a 100-fold a decreased100-fold decreased of the Pf PanK of the IC 50PfPanKvalue. IC In50 2017, value. Chiu In et2017, al. described Chiu et al. compound described compoundcompound 3939 asas aa pantothenatepantothenate kinasekinase inhibitorinhibitor withwith anan ICIC5050 ofof 3030 nMnM onon PfPfW2W2 [60].[60]. AddingAdding moremore 39compoundas a pantothenate 39 as a kinasepantothenate inhibitor kinase with aninhibitor IC50 of with 30 nM an on ICPf50 W2of 30 [60 nM]. Adding on PfW2 more [60]. pantothenate Adding more pantothenate to the growing medium increased this value. The authors also demonstrated that 39 topantothenate thepantothenate growing medium toto thethe growinggrowing increased mediummedium this value. increasedincreased The authors ththisis value. alsovalue. demonstrated TheThe authorsauthors that alsoalso39 demonstrateddemonstratedwas able to inhibit thatthat 3939 thewaswasPf PanK ableable toto homolog inhibitinhibit the oftheS. PfPf cervisiaePanKPanK homologhomolog, Cab1. ofof S.S. cervisiaecervisiae,, Cab1.Cab1.

TableTableTable 6. Pantothenate6. 6. Pantothenate Pantothenate and and and pantothenate pantothena pantothena analogtete analog analog inhibitors inhibitors inhibitors of Pfof of PanK.Pf PfPanK.PanK. PantothenatePantothenate P. falciparum IC50 PantothenatePantothenate PfPanK P.P.P. falciparumfalciparum falciparum ICIC5050 PfPfPanKPanK CompoundCompoundCompound 50 PhosphorylationPhosphorylationPhosphorylation InhibitionInhibitionPfPanK IC Compound 1,21,21,2 Phosphorylation Inhibition 50 50 (µM)(µM)(µM) 1,2 ICIC5050 Inhibition %%

------

767676 >85% >85% >85% (at 1 mM) (at(at 11 mM)mM) - - -

363636 --- 14 µM 1414 µMµM

131313 >60% >60% >60% (at 10 (at(atµM) 1010 µM)µM) 140 nM 140 140 nMnM

0.030.030.03 ------

1 2 1 Pf3D711 Pf3D7 Pf3D7strain strain strain except except except for 39for for, where 39 39, ,where where the value the the value shownvalue shown shown is on Pf is isW2 on on strain. Pf PfW2W2 2strain. strain.Pantothenate 2 2 Pantothenate Pantothenate concentration concentration concentration in growth in in medium was 1 µM except for 39, where it was 1.14 µM. growthgrowth medium medium was was 1 1 µM µM except except for for 39 39, ,where where it it was was 1.14 1.14 µM. µM.

Concerning Pf DPCK, work by Fletcher et al. identified some potent compounds [61]. The activity of assessed compounds on P. falciparum was counterbalanced by adding coenzyme A to the culture medium to identified compounds active on coenzyme A synthesis. Similar rescue tests were then done with Pf PanK or Pf DPCK substrates. Four compounds (40–43, Figure 11) showed possible activity on Pf DPCK, affecting the blood-stage and gametocytes. Molecules 2020, 25, x FOR PEER REVIEW 13 of 35

MoleculesConcerning 2020, 25 Pf, xDPCK, FOR PEER work REVIEW by Fletcher et al. identified some potent compounds [61]. The13 of activity 35 of assessed compounds on P. falciparum was counterbalanced by adding coenzyme A to the culture Concerning PfDPCK, work by Fletcher et al. identified some potent compounds [61]. The activity medium to identified compounds active on coenzyme A synthesis. Similar rescue tests were then of assessed compounds on P. falciparum was counterbalanced by adding coenzyme A to the culture done with PfPanK or PfDPCK substrates. Four compounds (40–43, Figure 11) showed possible medium to identified compounds active on coenzyme A synthesis. Similar rescue tests were then Molecules 2020, 25, 5949 13 of 35 activitydone on with PfDPCK, PfPanK affecting or PfDPCK the blood-stagesubstrates. Four and compoundsgametocytes. (40 –43, Figure 11) showed possible activity on PfDPCK, affecting the blood-stage and gametocytes.

FigureFigureFigure 11.11. Inhibitors Inhibitors11. Inhibitors ofof PfofPf DPCKPfDPCK from from FletcherFletcher Fletcher etet al.al. [[61].[61].61]. 1 1At1 At 40 40 µM. µµM.M. EG EGEG = Early-stage == Early-stage Gametocytes, Gametocytes, LGLG ==LG Late-stage = Late-stage Gametocytes. Gametocytes.

4.6.4.6. Molecules4.6. Molecules Targeting Targeting PlasmodiumPlasmodium Plasmodium falciparumfalciparum falciparum FIKKFIKK KinasesKinases (PfFKks) (PfFKks)(PfFKks) FIKKFIKKFIKK kinaseskinases kinases are are namedare named named after after after a shareda ashared shared sequence sequencesequence of of fourof four four amino amino amino acids acids acids (Phe-Ile-Lys-Lys(Phe-Ile-Lys-Lys (Phe-Ile-Lys-Lys or FIKK) FIKK) or FIKK) and and are an important group of kinases in malaria parasites [14]. While P. vivax and P. berghei possess areand an are important an important group group of kinases of kinases in malaria in malaria parasites parasites [14]. [14]. While WhileP. vivax P. vivaxand andP. berghei P. bergheipossess possess one one FIKK kinase, P. falciparum differs completely, possessing 20 FIKK kinases. The FIKK kinase gene FIKK kinase, P. falciparum diﬀers completely, possessing 20 FIKK kinases. The FIKK kinase gene may one FIKKmay be kinase, essential P. falciparum for the parasite differs [18], completely, yet the role possessing of these proteins 20 FIKK remains kinases. unclear. The FIKK PfFKks kinase are gene be essential for the parasite [18], yet the role of these proteins remains unclear. Pf FKks are exported may exportedbe essential into thefor RBC the cytosolparasite (except [18], PfyetFKk8), the whererole of they these possibly proteins interact remains with RBC’s unclear. cytoskeletal PfFKks are intoexported theproteins RBC into and cytosol the membrane RBC (except cytosol [62,63].Pf (exceptFKk8), wherePfFKk8), they where possibly they interactpossibly with interact RBC’s with cytoskeletal RBC’s cytoskeletal proteins andproteins membrane andThe membraneonly [62 studies,63]. [62,63]. on inhibitors of FKks were performed by Lin et al. [64,65], who screened ThecompoundsThe onlyonly studiesstudies on PfFKk8 onon and inhibitorsinhibitors P. vivax FKk ofof FKksFKks (catalytic werewere domain performedperformed only for bybythe LinLinlatter). etet In al.al. both [[64,65],64 ,articles,65], whowho emodin screenedscreened compoundscompounds(44, Figure on on 12),PfPfFKk8 FKk8an anthraquinone, and and P. P.vivax vivax FKkshowedFKk (catalytic (catalytican IC50 domainof 1.9 domain µM only and onlyfor 2 µM the for against latter). the latter).these In both tested Inarticles, bothproteins, articles,emodin respectively. emodin(44, Figure (44 ,12), Figure an anthraquinone, 12), an anthraquinone, showed showedan IC50 of an 1.9 IC µM50 of and 1.9 2µ MµM and against 2 µM these against tested these proteins, tested proteins,respectively. respectively.

Figure 12. Chemical structure of emodin.

4.7. Molecules Targeting Plasmodium falciparum Guanylate Kinase (PfGK) FigureFigure 12.12. Chemical structure ofof emodin.emodin. Guanylate kinase catalyzes the transformation of (deoxy)guanosine-monophosphate into 4.7.4.7. Molecules(deoxy)guanosine-diphosphate. Targeting PlasmodiumPlasmodium However, falciparumfalciparum this GuanylateGuanylate transformation KinaseKinase (PfGK) (PfGK)can potentially be bypassed by P. falciparum thymidylate kinase, which can also catalyze the PfGK reaction [66]. In their screening [46], Guanylate kinase catalyzes the transformation of (deoxy)guanosine-monophosphate into CrowtherGuanylate et al.kinase included catalyzes guanylate the kinase transformation as one of their screenedof (deoxy)guanosine-monophosphate proteins but used PvGK (possibly into (deoxy)guanosine-diphosphate.(deoxy)guanosine-diphosphate.because guanylate kinase expression However, However, is greater this this tratansformationtransformation liver stage [67]). can canThree potentially potentially compounds be be bypassedemerged bypassed as by byP. P.falciparum falciparumpotential thymidylatethymidylate hits (45 to 47 kinase,, kinase, Figure which 13), which but can canas thealso also authors catalyze catalyze stated, the the stPfPfructuralGKGK reaction reaction features [66]. [66 do]. notIn In their theircurrently screening screening make [46], [46], CrowtherCrowtherthem goodetet al.al. candidates included guanylatefor further medicinal kinase as chemistry one of their studies. screened proteins but used PvGK (possibly becausebecause guanylateguanylate kinasekinase expressionexpression isis greatergreater atat liverliver stagestage [[67]).67]). ThreeThree compoundscompounds emergedemerged asas potentialpotential hitshits ((4545 toto 4747,, FigureFigure 1313),), butbut asas thethe authors stated, structuralstructural featuresfeatures dodo notnot currentlycurrently makemake themthem goodgood candidatescandidates forfor furtherfurther medicinalmedicinal chemistrychemistry studies.studies.

Molecules 2020, 25, x FOR PEER REVIEW 14 of 35

MoleculesMoleculesMolecules 2020 20202020, ,25, 2525, ,x, x xFOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 141414 of ofof 35 3535

Molecules 2020, 25, 5949 14 of 35

Molecules 2020, 25, x FOR PEER REVIEW 14 of 35

Figure 13. Inhibitors of PvGK.

4.8. Molecules Targeting Plasmodium falciparum Glycogen Synthase Kinase 3 (PfGSK3) PfGSK3 is possibly an essential FigureenzymeFigureFigure 13. 13.13. Inhibitors InhibitorsInhibitorsfor the of ofofparasite Pv PvPvGK.GK.GK. asexual blood-stage [17] but its implication in biological pathways is not clear. One of its phosphorylation targets, the apical 4.8.4.8. Molecules Molecules Targeting Targeting Plasmodium Plasmodium falcip falciparumarum Glycogen Glycogen Synthase Synthase Kinase Kinase 3 3(PfGSK3) (PfGSK3) membrane4.8. Molecules antigen Targeting1 (AMA1) Plasmodium [68], is required falciparum for Glycogen RBC invasion Synthase Kinaseby merozoites. 3 (PfGSK3) Pf GSK-3 possesses a human ortholog,PfPfPfGSK3GSK3GSK3 Hs isisis GSK3. possiblypossiblypossibly Hs ananGSK3an essentialessentialessential hyperactivation enzymeenzymeenzyme forforfor thisthth elinkede e parasiteparasiteparasite to asexualAlzheimer’sasexualasexual blood-stageblood-stageblood-stage disease, [17][17][17] diabetes, butbutbut itsitsits or implicationimplication in in biological biological pathways pathways is is not not clear. clear. One One of of its its phosphorylation phosphorylation targets, targets, the the apical apical cancersimplication and many incompounds biological pathwaysinhibiting is Hs notGSK3 clear. have One been of itsdescribed phosphorylation [69]. targets, the apical membranemembrane antigen antigen 1 1(AMA1) (AMA1) [68], [68], is is required required for for RBC RBC invasion invasion by by merozoites. merozoites. Pf PfGSK-3GSK-3 possesses possesses a a Inmembrane 2013, Fugel antigen et al. 1 realized (AMA1) [68],a high-thrFigure is required 13.oughput Inhibitors for RBC screening of invasion PvGK. of by 10,000 merozoites. molecules PfGSK-3 on possessesPfGSK3 [70].a humanhuman ortholog, ortholog, Hs HsGSK3.GSK3. Hs HsGSK3GSK3Figure hyperactivation hyperactivation 13. Inhibitors is ofis linked PvlinkedGK. to to Alzheimer’s Alzheimer’s disease, disease, diabetes, diabetes, or or Multiplehuman molecules ortholog, with Hs GSK3.a thieno[2,3-b]pyridine HsGSK3 hyperactivation core were is linked found to Alzheimer’sto be hits against disease, Pf diabetes,GSK3. Forty- or cancerscancerscancers and andand many manymany compounds compoundscompounds inhibiting inhibitinginhibiting Hs HsHsGSK3GSK3GSK3 have havehave been beenbeen described describeddescribed [69]. [69].[69]. 4.8.4.8.three Molecules Molecules analogs Targeting from Targeting four Plasmodium Plasmodiumdifferent thieno[2,3-b]pyrid falcip falciparumarum Glycogen ines Synthase scaffolds Kinase were 3 synthesized. (PfGSK3) The 3,6-diamino- InInIn 2013, 2013,2013, Fugel FugelFugel et etet al. al.al. realized realizedrealized a a ahigh-thr high-thrhigh-throughputoughputoughput screening screeningscreening of ofof 10,000 10,00010,000 molecules moleculesmolecules on onon Pf PfPfGSK3GSK3GSK3 [70]. [70].[70]. 4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyridPfPfMultipleGSK3MultipleGSK3Multiple isis molecules possiblymoleculesmoleculespossibly anwith withwithan essential a essential a athieno[2,3-b]pyridine thieno[2,3-b]pyridinethieno[2,3-b]pyridine enzyme enzyme forine-5-nitriles the for parasitecore corecoreth ewere werewereparasite asexualshowed found foundfound asexualto blood-stage totothe be bebe hits best hitshits blood-stage against againstagainstresults [17] butPf Pf PfonGSK3.GSK3.GSK3. its [17]Pf implicationGSK3 Forty- Forty- Forty-but andits implicationinSus biological scrofathreethreethree GSK3 analogs analogsinanalogs pathways biological (used from fromfrom for four is fourfour selectivity notpathways different differentdifferent clear. One thieno[2,3-b]pyrid comparison). thieno[2,3-b]pyridthieno[2,3-b]pyridis not of its clear. phosphorylation Hs OneinesGSK3inesines scaffoldsof scaffoldsscaffolds ICits50 and targets,phosphorylation were werewere Pf NF54LUCsynthesized. synthesized.synthesized. the apical EC targets,The membrane TheThe50 tests3,6-diamino- 3,6-diamino-3,6-diamino- thewere antigenapical done 4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyrid4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyridine-5-nitrilesine-5-nitriles showed showed the the best best results results on on Pf PfGSK3GSK3 and and membrane1on (AMA1) the 4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyridbest [antigencompound,68], is required 1 (AMA1) 48 (Table for [68], RBC 7). is invasionrequired byforine-5-nitriles merozoites. RBC invasion showedPf GSK-3by merozoites.the possessesbest results PfGSK-3 a on human PfGSK3 possesses ortholog, and a SusSus scrofa scrofa GSK3 GSK3 (used (used for for selectivity selectivity comparison). comparison). Hs HsGSK3GSK3 IC IC5050 and and Pf PfNF54LUCNF54LUC EC EC5050 tests tests were were done done humanHsGSK3.Based Susortholog,Hs scrofa onGSK3 a GSK3 dockingHs hyperactivationGSK3. (used study forHs selectivityGSK3 of 48 ishyperactivation linkedon comparison). an analogy to Alzheimer’s Hs modelisGSK3 linked IC of disease,50 toPfand GSK3,Alzheimer’s PfNF54LUC diabetes, Masch EC ordisease,el 50 cancersal. tests synthesized werediabetes, and done many or23 analogsononon ofthe thethe 48best bestbest with compound, compound,compound, different 48 4848 (Table (Tablesubstituents(Table 7). 7).7). (halogens, ethers, alkylated amines, cyclic amines) at cancerscompounds andBased Basedmany inhibiting on on compounds a adocking dockingHsGSK3 study studyinhibiting have of of been48 48 on Hson described anGSK3 an analogy analogy have [69 model modelbeen]. ofdescribed of Pf PfGSK3,GSK3, [69].Masch Masch el el al. al. synthesized synthesized 23 23 position 4 ofBased the 2-chlorophenylon a docking study cycle of [71]48 on (Table an analogy 7). Compound model of Pf49GSK3, showed Masch lower el al. activity synthesized on Pf GSK323 InInanalogs 2013,analogs 2013, Fugel of of Fugel 48 48 etwith with al. et realizeddifferent al.different realized asubstituents substituentshigh-thr aoughput high-throughput (halogens, (halogens, screening ethers, ethers, screeningalkylated ofalkylated 10,000 amines, moleculesamines, of 10,000 cyclic cyclic on molecules amines) Pfamines)GSK3 at [70].at on but its potencyanalogs ofon 48in vitrowith parasitesdifferent substituentswas improved (halogens, compared ethers, to 48 alkylated. Aqueous amines, solubility cyclic was amines) improved, at MultiplePf GSK3positionposition [molecules70]. 4 Multiple 4of of the the with 2-chlorophenyl 2-chlorophenyl molecules a thieno[2,3-b]pyridine withcycle cycle [71] a [71] thieno[2,3-b]pyridine (Table (Table core 7). 7). Compound Compoundwere found 49 49 core showedto showed be were hits lower lower found against activity activity to Pf be GSK3.on on hits Pf PfGSK3GSK3 againstForty- going fromposition 1.5 4 µM of the for 2-chlorophenyl 48 to 4.8 µM cyclefor 49 [71]. The (Table authors 7). Compound explored 49 the showed possible lower axial activity chirality on PfGSK3 created threePf GSK3. analogsbutbutbut Forty-threeits itsits potency potencypotencyfrom four on onon analogs in indifferentin vitro vitrovitro parasites fromparasitesparasites thieno[2,3-b]pyrid four was waswas di improved improvedﬀimprovederent thieno[2,3-b]pyridines inescompared comparedcompared scaffolds to toto 48 4848 were. .Aqueous. AqueousAqueous synthesized. sca solubility solubilitysolubilityﬀoldswere Thewas waswas improved,3,6-diamino- improved, synthesized.improved, by the 2-chlorophenyl cycle. They isolated the two isomers of 50 and compared their activity: (+)-50 4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyridThe 3,6-diamino-4-(2-halophenyl)-2-benzoylthieno[2,3-b]pyridine-5-nitrilesgoinggoinggoing from fromfrom 1.5 1.51.5 µM µMµM for forfor 48 4848 to toto 4.8 4.84.8 µM µMµM for forfor 49 4949. .ine-5-nitriles.The TheThe authors authorsauthors explored explored exploredshowed the the thethe possible possiblepossible best showed resultsaxial axialaxial chirality chirality thechirality on best Pf GSK3created created resultscreated and on was activebyby the the but 2-chlorophenyl 2-chlorophenyl not (−)-50. cycle. Accordingcycle. They They isolated isolatedto the theauthors, the two two isomers isomers this diff of of 50erence 50 and and compared comparedarose from their their the activity: activity: chlorine (+)- (+)- 50atom50 SusPf GSK3 scrofaby and GSK3the Sus2-chlorophenyl (used scrofa forGSK3 selectivity cycle. (used They forcomparison). isolated selectivity the twoHs comparison).GSK3 isomers IC 50of and 50Hs and GSK3PfNF54LUC compared IC50 and theirECPf50 activity:testsNF54LUC were (+)- done50 EC 50 ontestsposition the were wasbestwaswas in doneactive compound,active(active−)-50 on but ,but butwhich the not notnot best48 ( prevented−(( −(Table)-−)-)-50 compound,5050. .. According AccordingAccording 7). it from48 to toto(Table fitting the thethe authors, authors,authors,7 ).into the this thisthis protein diff diffdifferenceerenceerence binding arose arosearose pocket. from fromfrom the thethe chlorine chlorinechlorine atom atomatom positionposition in in ( −()-−)-5050, which, which prevented prevented it it from from fitting fitting into into the the protein protein binding binding pocket. pocket. Basedposition on ain docking (−)-50, which study prevented of 48 on it froman analogy fitting into model the protein of PfGSK3, binding Masch pocket. el al. synthesized 23 Table 7. Thieno[2,3-b]pyridine inhibitors of PfGSK3. analogs of 48 with differentTable substituents 7. Thieno[2,3-b]pyridine (halogens, inhibitorsethers, alkylated of Pf GSK3. amines, cyclic amines) at TableTableTable 7. 7.7. Thieno[2,3-b]pyridine Thieno[2,3-b]pyridineThieno[2,3-b]pyridine inhibitors inhibitorsinhibitors of ofof Pf PfPfGSK3.GSK3.GSK3. position 4 of the 2-chlorophenyl cycle [71] (Table 7). Compound 49 showed lower activity on PfGSK3 but its potency on in vitro parasites was improved compared to 48. Aqueous solubility was improved, going from 1.5 µM for 48 to 4.8 µM for 49. The authors explored the possible axial chirality created by the 2-chlorophenyl cycle. They isolated the two isomers of 50 and compared their activity: (+)-50 was active but not (−)-50. According to the authors, this difference arose from the chlorine atom % % %Inhibition% Inhibition Inhibition PPfPGSK3fGSK3fGSK3 CCCCCC50CC50 50 position in (−)-50, which prevented it from% InhibitionInhibition fitting into PthefPfGSK3GSK3 protein HsHsHs bindingGSK3GSK3HsGSK3GSK3 pocket.PfPfPfNF54LUCPfNF54LUCNF54LUCNF54LUC CC5050 Compound R 505050 HsGSK3 PfNF54LUC (HEK293, CompoundCompoundCompound R RR PfNF54LUCPfPfNF54LUCNF54LUC ICICIC50 (HEK293(HEK293(HEK293 Compound R PfPfNF54LUCNF54LUC ICIC50 (µ M) IC50IC50 50(µM)(µM) ICIC505050 (µM)(µM) (HEK293 ICICIC5050 (µM) (µM) ICICIC50 (µM)50 (µM) (µM) µM) Table 7. Thieno[2,3-b]pyridineat at3atat µM3at 3 3µM 3µMµMµ M (µM) (µM)inhibitors(µM)(µM) of PfGSK3. , ,µM), µM)µM), µM)

1 1 48 484848 n.d.n.d.n.d.n.d. 1 11 0.240.240.240.24 9.089.089.089.08 5.5 5.5 5.5 5.5 35.2 35.2 35.2 35.2 48 n.d. 0.24 9.08 5.5 35.2

4949 99.999.999.9 0.720.72 0.72 40.240.2 40.2 1.2 1.2 1.2 5.56 5.56 5.56 49 49 99.999.9 0.720.72 40.240.2 1.2 1.2 5.56 5.56

% Inhibition PfGSK3 CC50 HsGSK3 PfNF54LUC (+(+)-)-5050 73.073.0 n.d. n.d. n.d. n.d. 1.1 1.1 n.d. n.d. CompoundMolecules(+)-(+)- 5050 2020 , 25, x FORR PEER REVIEWPf NF54LUC73.073.0 ICn.d.n.d.50 n.d.n.d. 1.11.1 n.d.(HEK293 n.d.15 of 35 (+)-50 73.0 n.d. IC50n.d. (µM) IC50 1.1(µM) n.d. at 3 µM (µM) , µM)

48 n.d. 1 0.24 9.08 5.5 35.2 ( ()-−)-5050 −24.424.4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. − −

49 99.9 0.72 40.2 1.2 5.56 1 n.d. = not determined 1 n.d. = not determined 4.9. Molecules Targeting Plasmodium falciparum Hexokinase (PfHK) (+)-50 Hexokinase is the first enzyme implicated73.0 in glycolysis,n.d. catalyzingn.d. the transformation1.1 of glucose n.d. into glucose 6-phosphate. Studies showed that PfHK inhibition (directly or indirectly by inhibiting glucose transporters) is linked to parasite death during the asexual blood-stage [72,73]. PfHK only possesses 24% similarity with human glucokinase, suggesting the possibility of designing selective PfHK inhibitors [74]. In 2013, Harris et al. screened a small library of compounds on PfHK [74]: three simple benzoisothiazolinones (51–53, Table 8) and the closely-related selenium compound ebselen (54) showed activity against PfHK below 300 nM. These values were not completely correlated regarding EC50 against Pf3D7, as stated by the authors, who suggested that cell permeability and off-target interaction problems might explain the difference.

Table 8. Isobenzothiazolinones and ebselen as inhibitors of PfHK.

Compound X R1 R2 PfHK IC50 (nM) Pf3D7 EC50 (µM) 51 S F Cl 160 0.62 52 S H Cl 270 1.1 53 S H H 230 5.2 54 Se H H 10 6.8

In another screening from the same team, Davis et al. screened 50,000 compounds on PfHK [75]: two compounds (55 and 56, Table 9) displayed a micromolar activity on PfHK but lacked activity on Pf3D7 in vitro.

Table 9. Dimethyl 2-amido-cyclopenta[b]thiophene-3,4-dicarboxylates as inhibitors of PfHK.

Compound R PfHK IC50 (µM)

55 1.09

56 1.94

Molecules 2020, 25, 5949 15 of 35

MoleculesMoleculesBased 20202020,, on 2525,, xax FORFOR docking PEERPEER REVIEW studyREVIEW of 48 on an analogy model of Pf GSK3, Masch el al. synthesized1515 ofof 233535 MoleculesMolecules 20202020,, 2525,, xx FORFOR PEERPEER REVIEWREVIEW 1515 ofof 3535 analogs of 48 with diﬀerent substituents (halogens, ethers, alkylated amines, cyclic amines) at position 4 of the 2-chlorophenyl cycle [71] (Table7). Compound 49 showed lower activity on Pf GSK3 but its potency on in vitro parasites was improved compared to 48. Aqueous solubility was improved, going ((−−)-)-5050 −−24.424.4 n.d.n.d. n.d.n.d. n.d.n.d. n.d. n.d. from((−−)-)- 1.55050 µM for 48 to 4.8 µM for 49. The−−24.424.4 authors exploredn.d.n.d. the possiblen.d.n.d. axial chiralityn.d.n.d. created n.d. n.d. by the 2-chlorophenyl cycle. They isolated the two isomers of 50 and compared their activity: (+)-50 was

active but not ( )-50. According to the authors, this difference arose from the chlorine atom position in − 11 n.d. n.d. == notnot determineddetermined ( )-50, which prevented it from fitting into11 n.d. the = not protein determined binding pocket. − n.d. = not determined 4.9.4.9. MoleculesMolecules TargetingTargeting PlasmodiPlasmodiumum falciparumfalciparum HexokinaseHexokinase (PfHK)(PfHK) 4.9.4.9.4.9. MoleculesMolecules Molecules TargetingTargeting Targeting PlasmodiPlasmodi Plasmodiumumum falciparumfalciparum falciparum HexokinaseHexokinase Hexokinase (PfHK)(PfHK) (PfHK) HexokinaseHexokinase isis thethe firstfirst enzymeenzyme implicatedimplicated inin glycolysis,glycolysis, catalyzingcatalyzing thethe transformationtransformation ofof glucoseglucose HexokinaseHexokinase is is the the first first enzyme enzyme implicated implicated in in glycolysis, glycolysis, catalyzing catalyzing the the transformation transformation of of glucose glucose intointo Hexokinaseglucoseglucose 6-phosphate.6-phosphate. is the first StudiesStudiesenzyme showed showedimplicated thatthat in PfPf glycolysis,HKHK inhibitioninhibition catalyzing (directly(directly the or ortransformation indirectlyindirectly byby of inhibitinginhibiting glucose intointo glucose glucose 6-phosphate. 6-phosphate. Studies Studies showed showed that that PfPfHKHK inhibition inhibition (directly (directly or or indirectly indirectly by by inhibiting inhibiting intoglucoseglucose glucose transporters)transporters) 6-phosphate. isis linkedlinked Studies toto parasiteshowedparasite deathatdeathth Pf duringduringHK inhibition thethe asexualasexual (directly blood-stageblood-stage or indirectly [72,73].[72,73]. by Pf PfinhibitingHKHK onlyonly glucoseglucose transporters) transporters) is is linked linked to to parasite parasite dea deathth during during the the asexual asexual blood-stage blood-stage [72,73]. [72,73]. PfPfHKHK only only glucosepossessespossesses transporters) 24%24% similaritysimilarity is linked withwith humanhumanto parasite glucokinase,glucokinase, death during suggestingsuggesting the asexual thethe possibilitypossibility blood-stage ofof designing[72,73].designing Pf HK selectiveselective only possessespossesses 24% 24% similarity similarity with with human human glucokinase, glucokinase, suggesting suggesting the the possibility possibility of of designing designing selective selective possessesPfPfHKHK inhibitorsinhibitors 24% similarity [74].[74]. with human glucokinase, suggesting the possibility of designing selective PfPfHKHK inhibitors inhibitors [74]. [74]. PfHKInIn inhibitors 2013,2013, HarrisHarris [74]. etet al.al. screenedscreened aa smallsmall librarylibrary ofof compoundscompounds onon PfPfHKHK [74]:[74]: threethree simplesimple InIn 2013, 2013, Harris Harris et et al. al. screened screened a asmall small library library of of compounds compounds on on PfPfHKHK [74]: [74]: three three simple simple benzoisothiazolinonesbenzoisothiazolinonesIn 2013, Harris et ( (5151al.–– 5353screened,, TableTable 8)a8) smallandand thethelibrary closely-relatclosely-relat of compoundseded seleniumselenium on Pf compoundcompoundHK [74]: three ebselenebselen simple ((5454)) benzoisothiazolinonesbenzoisothiazolinones ((5151––5353,, TableTable8) and8) and the closely-relatedthe closely-relat seleniumed selenium compound compound ebselen ebselen ( 54) showed (54) benzoisothiazolinonesshowedshowed activityactivity againstagainst ( 51PfPfHK–HK53, belowbelowTable 3003008) andnM.nM. ThesetheThese closely-relat valuesvalues werewereed not notselenium completelycompletely compound correlatedcorrelated ebselen regardingregarding (54) showedshowedactivity activityactivity against againstagainstPf HK below PfPfHKHK below 300below nM. 300300 These nM.nM. TheseThese values valuesvalues were notwerewere completely notnot completelycompletely correlated correlatedcorrelated regarding regardingregarding EC50 ECEC5050 againstagainst PfPf3D7,3D7, asas statedstated byby thethe authors,authors, whowho suggestsuggesteded thatthat cellcell permeabilitypermeability andand off-targetoff-target ECagainst50 againstPf 3D7, Pf3D7, as stated as stated by the by authors, the authors, who suggested who suggest that celled that permeability cell permeability and off-target and interactionoff-target ECinteractioninteraction50 against problemsproblems Pf3D7, as mightmight stated explainexplain by the thethe authors, difference.difference. who suggested that cell permeability and off-target interactioninteractionproblems might problemsproblems explain mightmight the explainexplain difference. thethe difference.difference. TableTable 8.8. IsobenzothiazolinonesIsobenzothiazolinones andand ebselenebselen asas inhibitorsinhibitors ofof PfPfHK.HK. TableTableTable 8.8. 8. IsobenzothiazolinonesIsobenzothiazolinonesIsobenzothiazolinones andand and ebselenebselen ebselen asas as inhibitorsinhibitors inhibitors ofof of PfPfPfHK.HK.HK.

µ CompoundCompoundCompound X XX RR R11 1 RR22 PRPf2fHKHK ICICPf5050HK (nM) (nM) IC 50 P(nM)Pff3D73D7 ECECPf5050 3D7 (µM)(µM) EC 50 ( M) Compound X R1 R2 PfHK IC50 (nM) Pf3D7 EC50 (µM) Compound51 5151 SXSS RFF1 F RClCl2 P ClfHK 160IC 16050 (nM) 160 Pf3D7 0.62EC 0.6250 (µM) 0.62 51 S F Cl 160 0.62 52 515252 SSS HFH H ClCl Cl 160270 270 270 0.62 1.1 1.1 1.1 53 52 SS H H Cl H 270 230 1.1 5.2 525353 SS HH ClHH 270230 230 1.15.2 5.2 54 53 SS H H H H 230 10 5.2 6.8 535454 SeSeS HH HH 230 10 10 5.26.8 6.8 5454 SeSe HH HH 10 10 6.8 6.8 InIn anotheranother screeningscreening from from the the same same team, team, Davis Davis et et al. al. screened screened 50,00050,000 compoundscompounds onon PfPfHKHKHK [75]:[75]: [75]: InIn anotheranother screeningscreening fromfrom thethe samesame team,team, DavisDavis etet al.al. screenedscreened 50,00050,000 compoundscompounds onon PfPfHKHK [75]:[75]: twotwo compoundscompounds (( 5555 andand 5656,, Table Table 9 9)9)) displayed displayeddisplayed a aa micromolar micromolarmicromolar activity activityactivity on onon PfPfHKHK butbut lackedlacked activityactivity onon twotwo compoundscompounds ((5555 andand 5656,, TableTable 9)9) displayeddisplayed aa micromolarmicromolar activityactivity onon PfPfHKHK butbut lackedlacked activityactivity onon PfPf3D73D73D7 inin in vitro.vitro. PfPf3D73D7 inin vitro.vitro. TableTable 9. 9. DimethylDimethyl 2-amido-cyclopenta[ 2-amido-cyclopenta[bb]thiophene-3,4-dicarboxylat]thiophene-3,4-dicarboxylates]thiophene-3,4-dicarboxylateses as as inhibitors inhibitors of of PfPfHK.HK. TableTable 9.9. DimethylDimethyl 2-amido-cyclopenta[2-amido-cyclopenta[bb]thiophene-3,4-dicarboxylat]thiophene-3,4-dicarboxylateses asas inhibitorsinhibitors ofof PfPfHK.HK.

CompoundCompound RR PPffHKHK ICIC5050 (µM) (µM) Compound R PfHK IC50 (µM) CompoundCompound RR PPffHKHK ICIC5050 (µM)(µM) 5555 1.091.09 55 5555 1.091.09 1.09

5656 1.941.94 56 5656 1.941.94 1.94

4.10.4.10. MoleculesMolecules TargetingTargeting PlasmodiumPlasmodium falciparumfalciparum Mitogen-ActivatedMitogen-Activated ProteinProtein KinaseKinase 22 (PfMAP2)(PfMAP2) 4.10.4.10. MoleculesMolecules TargetingTargeting PlasmodiumPlasmodium falciparumfalciparum Mitogen-ActivatedMitogen-Activated ProteinProtein KinaseKinase 22 (PfMAP2)(PfMAP2) Mitogen-activatedMitogen-activated proteinprotein kinaseskinases (MAPK)(MAPK) areare protproteinseins involvedinvolved inin signalsignal transductiontransduction andand areare Mitogen-activated protein kinases (MAPK) are proteins involved in signal transduction and are thethe keykeyMitogen-activated toto manymany cellularcellular protein processesprocesses kinases inin eukaeuka (MAPK)ryoticryotic are organisms.organisms. proteins TwoinvolvedTwo MAPKsMAPKs in signal areare foundfound transduction inin P.P. falciparumfalciparum and are:: the key to many cellular processes in eukaryotic organisms. Two MAPKs are found in P. falciparum: thePfPfMAP1MAP1 key to andand many PfPfMAP2. MAP2.cellular VeryVery processes recently,recently, in euka HitzHitzryotic etet al.al. organisms. defineddefined thth Twoee rolerole MAPKs ofof thesethese are twotwo found proteins:proteins: in P. falciparum neitherneither isis: PfMAP1 and PfMAP2. Very recently, Hitz et al. defined the role of these two proteins: neither is PfessentialessentialMAP1 andforfor asexualPfasexualMAP2. development,development, Very recently, butbut Hitz PfPfMAP2 MAP2et al. definedisis essentialessential the roleforfor exflagellation,exflagellation,of these two proteins: aa mechanismmechanism neither thatthat is essentialessential forfor asexualasexual development,development, butbut PfPfMAP2MAP2 isis essentialessential forfor exflagellation,exflagellation, aa mechanismmechanism thatthat

Molecules 2020, 25, 5949 16 of 35

4.10. Molecules Targeting Plasmodium falciparum Mitogen-Activated Protein Kinase 2 (PfMAP2) Mitogen-activated protein kinases (MAPK) are proteins involved in signal transduction and are the key to many cellular processes in eukaryotic organisms. Two MAPKs are found in P. falciparum: Pf MAP1 and Pf MAP2. Very recently, Hitz et al. deﬁned the role of these two proteins: neither is Molecules 2020, 25, x FOR PEER REVIEW 16 of 35 essentialMolecules 2020 for, asexual25, x FORdevelopment, PEER REVIEW but Pf MAP2 is essential for exﬂagellation, a mechanism that appears16 of 35 to be directly linked to this protein without the involvement of Pf MAP1 [76]. Brumlik et al. screened appears to be directly linked to this protein without the involvement of PfMAP1 [76]. Brumlik et al. someappearsHs p38to beα (adirectly human linked MAPK) to this inhibitors protein to without assess their the activityinvolvement on P. falciparumof PfMAP1[77 [76].]. One Brumlik compound et al. screened some Hsp38α (a human MAPK) inhibitors to assess their activity on P. falciparum [77]. One (screened57) displayed some sub-micromolarHsp38α (a human activity MAPK) on inhibitorsPf W2 (Figure to assess 14). their activity on P. falciparum [77]. One compound (57) displayed sub-micromolar activity on PfW2 (Figure 14). compound (57) displayed sub-micromolar activity on PfW2 (Figure 14).

FigureFigure 14.14. InhibitorInhibitor ofofPf Pfmap-2map-2 discovereddiscovered byby BrumlikBrumlik etet al.al. [[77].77]. Figure 14. Inhibitor of Pfmap-2 discovered by Brumlik et al. [77]. 4.11.4.11. MoleculesMolecules TargetingTargeting PlasmodiumPlasmodium falciparumfalciparum MO15-Related MO15-Related Protein Protein Kinase Kinase (PfMRK) (PfMRK) 4.11. Molecules Targeting Plasmodium falciparum MO15-Related Protein Kinase (PfMRK) PfPfMRKMRK isis a a cyclin-dependent cyclin-dependent kinase kinase located located in thein the nucleus. nucleus. It interacts It interacts with with two two other other proteins proteins [78]: PfMRK is a cyclin-dependent kinase located in the nucleus. It interacts with two other proteins CDK-activating[78]: CDK-activating kinase kinase assembly assembly factor factor (Pf MAT1) (PfMAT1) and and cyclin cyclin 1 ( Pf1 (cyc-1).Pfcyc-1). These These three three proteins proteins areare [78]: CDK-activating kinase assembly factor (PfMAT1) and cyclin 1 (Pfcyc-1). These three proteins are consideredconsidered likely likely to to be be important important for for gene gene expression expression and and DNA DNA replication, replication, and andPf MRKPfMRK is believedis believed to considered likely to be important for gene expression and DNA replication, and PfMRK is believed beto essentialbe essential for for the the asexual asexual blood-stage blood-stage [17 [17].]. Pf PfMRKMRK shares shares the the most most properties, properties, regarding regarding humanhuman to be essential for the asexual blood-stage [17]. PfMRK shares the most properties, regarding human proteins,proteins, withwithHs HsCDK7.CDK7. proteins, with HsCDK7. WoodardWoodard et et al. al. screenedscreened 1212 isoquinolineisoquinoline andand naphthalenenaphthalene sulfonamides,sulfonamides, knownknown forfor beingbeing kinasekinase Woodard et al. screened 12 isoquinoline and naphthalene sulfonamides, known for being kinase inhibitors,inhibitors, on onPf PfMRKMRK [[79].79]. Only 5858 displayed aa sub-micromolarsub-micromolar activityactivity ononPf PfMRKMRK butbut waswas notnot activeactive inhibitors, on PfMRK [79]. Only 58 displayed a sub-micromolar activity on PfMRK but was not active ononP. P. falciparum falciparumin in vitro vitro.. TheThe samesame teamteam laterlater designeddesigned somesome chalconeschalcones basedbased onon anan in-silicoin-silico modelmodel ofof on P. falciparum in vitro. The same team later designed some chalcones based on an in-silico model of HsHsCDK7CDK7 [[80].80]. OneOne ofof thesethese chalcones,chalcones, 5959,, displayeddisplayed aa 1.31.3µ µMMactivity activityagainst againstPf PfMRKMRK associatedassociated withwith HsCDK7 [80]. One of these chalcones, 59, displayed a 1.3 µM activity against PfMRK associated with ananin in vitrovitroactivity activity of of 4.6 4.6µ MµM on onPf PfW2.W2. Caridha Caridha et et al. al. tested tested 27 27 sulfonamide sulfonamide compounds compounds substituted substituted by an in vitro activity of 4.6 µM on PfW2. Caridha et al. tested 27 sulfonamide compounds substituted phenylby phenyl or thiophene or thiophene cycles cycles [81]. [81]. Ten Ten molecules, molecules, including including compound compound60 (Figure 60 (Figure 15), possessed15), possessed a sub- a by phenyl or thiophene cycles [81]. Ten molecules, including compound 60 (Figure 15), possessed a micromolarsub-micromolar activity activity on Pf onMRK, PfMRK, but nonebut none was activewas active on Pf onW2. Pf CytotoxicityW2. Cytotoxicity of compound of compound60 on 60 ﬁve on sub-micromolar activity on PfMRK, but none was active on PfW2. Cytotoxicity of compound 60 on difiveﬀerent different cell lines cell lines was assessed,was assessed, and all and the all values the values were were around around or above or above 25 µM. 25 µM. five different cell lines was assessed, and all the values were around or above 25 µM.

Figure 15. Inhibitors of PfMRK. Figure 15.15. Inhibitors of PfMRK. 4.12. Molecules Targeting Plasmodium falciparum NIMA-Related Kinase 1 (PfNEK1) 4.12. Molecules Targeting Plasmodium falciparumfalciparum NIMA-RelatedNIMA-Related KinaseKinase 11 (PfNEK1)(PfNEK1) Never-in-mitosis/Aspergillus (NIMA) related protein kinases are found in multiple eukaryotic Never-in-mitosisNever-in-mitosis/Aspergillus/Aspergillus (NIMA) related protein kinases are found in multiple eukaryotic organisms and are involved in the cell division process. Four NEK proteins have been discovered in organisms and are involved in the cell division process.process. Four NEK proteins have been discovered in Plasmodium [82]: Plasmodium [[82]:82]: • PfNEK2 and PfNEK4 are related to sexual stages of the parasite, • PfNEK2 and PfNEK4NEK4 are are related related to to sexual sexual stages stages of of the the parasite, •• PfNEK1 and PfNEK3 can interact with PfMAP2 and, • PfNEK1 and PfNEK3 can interact with PfMAP2 and, • PfNEK1 is also expressed during schizogony (it is the only NEK essential in the blood-stage) and • PfNEK1 is also expressed during schizogony (it is the only NEK essential in the blood-stage) and in male gametocytes. in male gametocytes. Laurent et al. and Desoubzdanne et al. found two natural compounds targeting this protein Laurent et al. and Desoubzdanne et al. found two natural compounds targeting this protein (Figure 16): xestoquinone 61 and alisiaquinone 62 [83,84] displayed interesting in vitro parameters (Figure 16): xestoquinone 61 and alisiaquinone 62 [83,84] displayed interesting in vitro parameters and an in vivo activity on a mouse model at 5 mg/kg during a four-day Peter’s test [27]. Xestoquinone and an in vivo activity on a mouse model at 5 mg/kg during a four-day Peter’s test [27]. Xestoquinone 61 was found to be selective of PfNEK1 versus other plasmodial and human kinases. 61 was found to be selective of PfNEK1 versus other plasmodial and human kinases.

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Pf NEK1 and Pf NEK3 can interact with Pf MAP2 and, • Pf NEK1 is also expressed during schizogony (it is the only NEK essential in the blood-stage) and • in male gametocytes.

Laurent et al. and Desoubzdanne et al. found two natural compounds targeting this protein (Figure 16): xestoquinone 61 and alisiaquinone 62 [83,84] displayed interesting in vitro parameters and an in vivo activity on a mouse model at 5 mg/kg during a four-day Peter’s test [27]. Xestoquinone 61 was found to be selective of Pf NEK1 versus other plasmodial and human kinases. Molecules 2020, 25, x FOR PEER REVIEW 17 of 35

Figure 16. 16. NaturalNatural quinone quinone inhibitors inhibitors of of PfPfnek-1.nek-1. 1 4-day1 4-day Peter’s Peter’s test test at at5 mg/kg/4 5 mg/kg/ 4days days intraperitoneal. intraperitoneal.

4.13. Molecules Targeting Plasmodium falciparum Phosphatidyl-Inositol 4-Kinase (PfPI4K) 4.13. Molecules Targeting Plasmodium falciparum Phosphatidyl-Inositol 4-Kinase (PfPI4K) Phosphatidyl-inositol 4-kinase 4-kinase catalyzes the tr transformationansformation of phosphatidyl-inositolphosphatidyl-inositol into phosphatidyl-inositol 4 4 phosphate (PI4P). (PI4P). PI4P PI4P is a key secondary messenger involved in multiple Pf Plasmodium cellular pathways. Inhibition of PfPI4KPI4K led led to to activity activity on on every every stage stage of of the the Plasmodium cyclecycle [85] [85] with with inhibitioninhibition of: • Development of schizonts and hypnozoites in the liver stage, • Development of schizonts and hypnozoites in the liver stage, • Asexual growth and gametocytogenesis in blood-stage, • • Reduction of oocysts in mosquito midgut in the sexual stage. • PfPI4KPI4K inhibitioninhibition phenotypic phenotypic consequences consequences were foundwere byfound McNamara by McNamara et al. using et four al. compounds using fouras compoundstools (three are as showedtools (three in Table are 10showed) [85]. Thein Tabl moste 10) assessed [85]. The compound, most assessed KDU691 compound, (63) an imidazopyrazine KDU691 (63), anshowed imidazopyrazine, promising in showed vivo activity promising on luciferase-expressing in vivo activity onP. luciferase-expressing berghei-infected mice: P. with berghei a single-infected dose mice:of 7.5 mgwith/kg, a single63 was dose able of to protect7.5 mg/kg, mice 63 from was what able wouldto protect be otherwise mice from a fatalwhat infection. would be63 otherwiseis selective a fatalof Pf infection.PI4K versus 63 is other selective human of Pf kinasesPI4K versus including otherHs huPI4Kman (IC kinases50 = 7.9 includingµM). Other HsPI4K tested (IC compounds50 = 7.9 µM). Otherincluded tested the compounds imidazopyridazine included KAI715 the imidazopyridazine (64) and the quinoxaline KAI715 BQR695(64) and ( 65the). quinoxaline Their in vitro BQR695results (are65). summed Their in upvitro in Tableresults 10 are. Pf summedPI4K was up confirmed in Table 10. as thePfPI4K target was of theseconfirmed compounds as the target by analysis of these of compoundsresistant clones by andanalysis artificial of resistant modifications clones of andPf PI4K artificial gene weremodifications also associated of PfPI4K with gene drug were resistance. also associatedPf PI4K with inhibitors drug resistance. are the most advanced plasmodial kinase inhibitors in terms of drug development. One compound, MMV390048 (66), is currently in phase II of clinical trials and is the only plasmodial kinase inhibitorTable currently 10. Three in clinical of the inhibitors trial (Figure of Pf 17PI4K)[86 described]. MMV390048 by McNamara PI4K inhibitory et al. [85]. effects, with the consequence of a multistage activity,P. were yoelii demonstrated liver by Paquet et al. using chemoproteomicsPfNF54 studies [87]. This inhibition is selective, as HsPI4KP. falciparumα and HsPI4K Asexualβ were not targeted by 66. Compound stage IC50 Gametocyte III Numerous in vivo experiments were performed (onBlood-Stage mice, rats, dogs,IC50 (nM) and monkeys): multistage (nM) IC50 (nM) potency was conserved in in vivo models and 66 displayed very good pharmacokinetic parameters including bioavailability of 74% and a half-time of 66 h at an oral dose of 5 mg/kg on monkeys. All these parameters pointed 66 as a strong lead compound candidate for clinical studies for single exposure radical cure; it could also be promising for36 chemoprevention.29.9 Compound 66 originally 220 emerged from a SAR study by Younis et al. which, starting from compound 67, focused on modulation of the methoxyphenol group [88].

9 3.9 n.d. 1

n.d. 71 n.d.

Molecules 2020, 25, x FOR PEER REVIEW 17 of 35 MoleculesMolecules 2020 2020, 25, 25, x, FORx FOR PEER PEER REVIEW REVIEW 1717 of of 35 35

Figure 16. Natural quinone inhibitors of Pfnek-1.1 1 4-day Peter’s test at 5 mg/kg/4 days intraperitoneal. FigureFigure 16. 16. Natural Natural quinone quinone inhibitors inhibitors of of Pf Pfnek-1.nek-1. 4-day 1 4-day Peter’s Peter’s test test at at 5 mg/kg/45 mg/kg/4 days days intraperitoneal. intraperitoneal.

4.13.4.13.4.13. Molecules Molecules Molecules Targeting Targeting Targeting Plasmodium Plasmodium Plasmodium falcipar falcipar falciparumumum Phosphatidyl-Inositol Phosphatidyl-Inositol Phosphatidyl-Inositol 4-Kinase 4-Kinase 4-Kinase (PfPI4K) (PfPI4K) (PfPI4K) Phosphatidyl-inositolPhosphatidyl-inositolPhosphatidyl-inositol 4-kinase 4-kinase4-kinase catalyzes catalyzescatalyzes the thethe tr trtransformationansformationansformation of ofof phosph phosphphosphatidyl-inositolatidyl-inositolatidyl-inositol into intointo phosphatidyl-inositolphosphatidyl-inositolphosphatidyl-inositol 4 4 4phosphate phosphate phosphate (PI4P). (PI4P). (PI4P). PI4P PI4P PI4P is is is a a akey key key secondary secondary secondary messenger messenger messenger involved involved involved in in in multiple multiple multiple cellularcellularcellular pathways. pathways. pathways. Inhibition Inhibition Inhibition of of of Pf Pf PfPI4KPI4KPI4K led led led to to to activity activity activity on on on every every every stage stage stage of of of the the the Plasmodium Plasmodium Plasmodium cycle cycle cycle [85] [85] [85] with with with inhibitioninhibitioninhibition of: of: of: •• • DevelopmentDevelopmentDevelopment of of of schizonts schizonts schizonts and and and hypnozoites hypnozoites hypnozoites in in in the the the liver liver liver stage, stage, stage, •• • AsexualAsexualAsexual growth growth growth and and and gametocytogenesis gametocytogenesis gametocytogenesis in in in blood-stage, blood-stage, blood-stage, •• • ReductionReductionReduction of of of oocysts oocysts oocysts in in in mosquito mosquito mosquito midgut midgut midgut in in in the the the sexual sexual sexual stage. stage. stage. PfPfPfPI4KPI4KPI4K inhibition inhibitioninhibition phenotypic phenotypicphenotypic consequences consequencesconsequences we wewererere found foundfound by byby McNamara McNamaraMcNamara et etet al. al.al. using usingusing four fourfour compoundscompoundscompounds as as as tools tools tools (three (three (three are are are showed showed showed in in in Tabl Tabl Tablee e10) 10) 10) [85]. [85]. [85]. The The The most most most assessed assessed assessed compound, compound, compound, KDU691 KDU691 KDU691 ( 63( 63(63) ) ) ananan imidazopyrazine, imidazopyrazine, imidazopyrazine, showed showed showed promising promising promising in in in vivo vivo vivo activity activity activity on on on luciferase-expressing luciferase-expressing luciferase-expressing P. P. P. berghei berghei berghei-infected-infected-infected mice:mice:mice: with with with a a asingle single single dose dose dose of of of 7.5 7.5 7.5 mg/kg, mg/kg, mg/kg, 63 63 63 was was was able able able to to to protect protect protect mice mice mice from from from what what what would would would be be be otherwise otherwise otherwise a a a fatal infection. 63 is selective of PfPI4K versus other human kinases including HsPI4K (IC5050 = 7.9 µM). fatalfatal infection. infection. 63 63 is is selective selective of of Pf PfPI4KPI4K versus versus other other hu humanman kinases kinases including including Hs HsPI4KPI4K (IC (IC 50= =7.9 7.9 µM). µM). OtherOtherOther tested tested tested compounds compounds compounds included included included the the the imidazopyridazine imidazopyridazine imidazopyridazine KAI715 KAI715 KAI715 ( 64( 64(64) )and )and and the the the quinoxaline quinoxaline quinoxaline BQR695 BQR695 BQR695 (65(65(65).). ).Their Their Their in in in vitro vitro vitro results results results are are are summed summed summed up up up in in in Table Table Table 10. 10. 10. Pf Pf PfPI4KPI4KPI4K was was was confirmed confirmed confirmed as as as the the the target target target of of of these these these compoundscompoundscompounds by byby analysis analysisanalysis of ofof resistant resistantresistant clones clonesclones and andand artificial artificialartificial modifications modificationsmodifications of ofof Pf PfPfPI4KPI4KPI4K gene genegene were werewere also alsoalso Moleculesassociatedassociatedassociated2020, 25 with with, with 5949 drug drug drug resistance. resistance. resistance. 18 of 35

Table 10. Three of the inhibitors of PfPI4K described by McNamara et al. [85]. TableTable 10. 10. Three Three of of the the inhibitors inhibitors of of Pf PfPI4KPI4K described described by by McNamara McNamara et et al. al. [85]. [85]. Table 10. Three of the inhibitors of Pf PI4K described by McNamara et al. [85]. P.P. yoelii yoelii liver liver PPfNF54fNF54 P. yoelii liver P.P. falciparum falciparum Asexual Asexual PfNF54 Compound stage IC5050 P. falciparum Asexual Gametocyte III Compound P.stage yoelii ICliver 50 stage P. falciparum Asexual GametocytePfNF54 Gametocyte III CompoundCompound stage IC Blood-Stage IC5050 (nM) Gametocyte III Blood-Stage IC 50(nM) IC(nM)50 (nM) Blood-StageBlood-Stage IC IC (nM)50 (nM) ICIII5050 IC(nM)50 (nM) (nM)(nM) ICIC50(nM) (nM)

363636 36 29.929.929.9 29.9 220 220 220 220 Molecules 2020, 25, x FOR PEER REVIEW 18 of 35

1 n.d. = not determined

PfPI4K inhibitors are the most advanced plasmodial kinase inhibitors in terms of drug development. One compound, MMV390048 (66), is currently in phase II of clinical trials11 1 and is the 99 9 93.93.93.9 3.9 n.d. n.d. n.d.n.d. 1 only plasmodial kinase inhibitor currently in clinical trial (Figure 17) [86]. MMV390048 PI4K inhibitory effects, with the consequence of a multistage activity, were demonstrated by Paquet et al. using chemoproteomics studies [87]. This inhibition is selective, as HsPI4Kα and HsPI4Kβ were not targeted by 66. Numerous in vivo experiments were performed (on mice, rats, dogs, and monkeys): multistage potency was conserved in in vivo models and 66 displayed very good pharmacokinetic n.d.n.d.n.d.n.d. 717171 71 n.d. n.d. n.d. n.d. parameters including bioavailability of 74% and a half-time of 66 h at an oral dose of 5 mg/kg on monkeys. All these parameters pointed 66 as a strong lead compound candidate for clinical studies for single exposure radical cure; it could also be promising for chemoprevention. Compound 66 1 originally emerged from a SAR study by Younisn.d. = not et determinedal. which, starting from compound 67, focused on modulation of the methoxyphenol group [88].

Figure 17. Starting from from 6767,, medicinal medicinal chemistry chemistry work work led led to to 6666 whichwhich was was further further improved improved to to 6868.. EG = Early-stageEarly-stage gametocytes, gametocytes, LG LG == Late-stageLate-stage gametocytes. gametocytes. 1 1mousemouse model, model, 20 20 mg/kg mg/kg per per os. os.

Initial human clinical studies on 66 “showed high variability in exposure, which was attributed to low low aqueous aqueous solubility” solubility” [89]. [89]. To To solve, solve, this this problem, problem, Brunshwig Brunshwig et al et., synthesized al., synthesized UCT943 UCT943 (68), (an68 ),analog an analog of 66 of where66 where thethe methylsulfone methylsulfone was was replaced replaced by by a apiperazinyl piperazinyl carboxamide carboxamide and and the aminopyridine by an aminopyrazine to improve aqueousaqueous solubility [[89].89]. This second generation of PfPI4K inhibitorinhibitor showed showed improved improved parameters parameters on every on every stage ofstage the malariaof the cycle.malaria ADMET cycle. properties ADMET wereproperties impacted were by impacted the modiﬁcation by the modification but the in vivo but the activity in vivo was activity conserved. was conserved. Continuing onon this this new aminopyrazinenew aminopyrazine scaﬀold, scaffold, Gibhard etGibhard al., synthesized et al., two synthesized aminopyrazines two (Figureaminopyrazines 18) to explore (Figure a potential18) to explore prodrug a potential strategy pr byodrug conversion strategy ofby a conversion sulfoxide intoof a asulfoxide sulfone into [90]. aCompounds sulfone [90].69 Compounds(pyrazine analog 69 (pyrazine of 66) and analog70 showed of 66) and nanomolar 70 showed activities nanomolarin vitro activities. Solubility in vitro. was Solubilitybetter at physiological was better at pHphysiological for the sulfoxide pH for derivative the sulfoxide compared derivative to the compared sulfone. toIn the vivo sulfone., the 90% In vivo, the 90% effective dose (ED90) per os was at 0.12 mg.kg−1 for both compounds compared to 0.57 mg.kg−1 for 66 and 0.25 mg.kg-1 for 68 (on Pf3D7 infected mouse model). As expected by the authors, 70 was found to be rapidly converted in vivo into 69, validating the sulfoxide prodrug approach for this series of compounds.

Molecules 2020, 25, 5949 19 of 35

1 1 eﬀective dose (ED90) per os was at 0.12 mg.kg− for both compounds compared to 0.57 mg.kg− for 66 and 0.25 mg.kg-1 for 68 (on Pf 3D7 infected mouse model). As expected by the authors, 70 was found to be rapidly converted in vivo into 69, validating the sulfoxide prodrug approach for this series of compounds. Molecules 2020, 25, x FOR PEER REVIEW 19 of 35 Molecules 2020, 25, x FOR PEER REVIEW 19 of 35

Figure 18. AminopyrazinesAminopyrazines derivatives derivatives used to study a sulfoxide-to sulfone produg approach. Figure 18. Aminopyrazines derivatives used to study a sulfoxide-to sulfone produg approach. Other compounds with original scaffolds scaﬀolds have have been been discovered discovered since since the the development development of of 6666.. KandepeduOther compounds et al. described described with originala a series series scaffoldsof of 1,5-naph 1,5-naphthyridines, havethyridines, been discovered most most of sincethem the having development submicromolar of 66. activitiesKandepedu on etPf NF54NF54al. described [91]. [91]. Starting Starting a series from from of MMV024101 MMV0241011,5-naphthyridines, ( (7171),), a a SAR SARmost study study of them was was havingperformed submicromolar on on the two activitiessubstituents on of Pf NF54the naphthyridine [91]. Starting core from (Figure MMV024101 1919)) and (71 48), analogs analogsa SAR study were synthesized.synthesized.was performed Compound on the two 73 substituents of the naphthyridine core (Figure 19) and 48 analogs were synthesized. Compound 73 showed good activity on on PfNF54NF54 with with an an IC IC5050 ofof 63 63 nM nM and and good good metabolic metabolic stability stability (t (t1/21 /2= =3333 h hat at5 mg/kgshowed5 mg/kg per good per os) os) activitybut but limited limited on PforalNF54 oral bioavailability bioavailability with an IC50 (39%) of (39%) 63 onnM on a and amouse mouse good model. model. metabolic stability (t1/2 = 33 h at 5 mg/kg per os) but limited oral bioavailability (39%) on a mouse model.

Figure 19. 1,5-naphthyridines as inhibitors of PfPI4K. Figure 19. 1,5-naphthyridines as inhibitors of PfPI4K.PI4K. Liang et al. described a bipyridine series targeting PfPI4K from screening in their compound libraryLiang [92]. et Compound al. described 74 displayed a bipyridine an activity series targeting around 4 PfµMPI4KPI4K on Pffrom from3D7 screening but was able in to their inhibit compound PfPI4K withlibrary an [92]. [ 92IC].50 Compoundof Compound 7.7 nM (Figure 74 displayed 20). An anin-silico activity homology around 4 model µµMM on of Pf Pf3D7PI4K but was was then able used to inhibit to rationally PfPI4KPI4K withguide an modulations IC50 ofof 7.7 7.7 nM nM on (Figure (Figure74: the 20).2-chloro-3-sulfonamide20). An An in-silico in-silico homology homology pyridine model model coreof of PfPf PI4KwasPI4K kept was was andthen then modulations used used to to rationally rationally were performedguide modulations on the two on 74side: the the chains. 2-chloro-3-sulfonamide 2-chloro-3-sulfonamide Compound 75 displayed pyridine pyridine the core core best was activity kept and on modulationsPf3D7 of 25.1 were nM togetherperformed with on an the IC two50 on side PfPI4K chains. of 0.9 Compound nM. This activity75 displayed was shown the best to activity be selective on Pf of3D73D7 PfPI4K of 25.1 versus nM 50 togetherhuman kinases with an including IC50 onon Pf HsPI4KPI4KPI3K of of or 0.9 0.9 Hs nM. nM.PI4K. This This With activity oral bioavailabilitywas shown to of be 80% selective and a of t 1/2Pf ofPI4K 3.6 versus h on a mousehuman modelkinases (at including including 10 mg/kg Hs HsperPI3KPI3K os), or 75 or HswasHsPI4K.PI4K. able With Within vivo oral oral to bioavailability cure bioavailability blood-stage of of 80%P. 80% yoelii and and at a 80 at1/2 t 1mg/kg/7 /of2 of 3.6 3.6 h days hon on a mouseanda mouse to stopmodel model liver-stage (at (at 10 10 mg/kg mg infection/kg per per os), by os), P.7575 berghei waswas able able sporozoites inin vivo vivo toto at cure curea single blood-stage blood-stage dose of P.1P. mg/kg yoelii yoelii at atin 80 80a mousemg/kg/7 mg/kg model./7 days and to stop stop liver-stage liver-stage infection by P.P. berghei sporozoites at a single dose of of 1 1 mg/kg mg/kg in in a a mouse mouse model. model.

Figure 20. 2-chloro-3-sulfonamide pyridines as inhibitors of PfPI4K. Figure 20. 2-chloro-3-sulfonamide pyridines as inhibitors of PfPI4K. 4.14. Molecules Targeting Plasmodium falciparum Protein Kinase 5, 6, 7 and 9 (PfPK5, PfPK6, PfPK7 and PfPK9)4.14. Molecules Targeting Plasmodium falciparum Protein Kinase 5, 6, 7 and 9 (PfPK5, PfPK6, PfPK7 and PfPK9)

Molecules 2020, 25, x FOR PEER REVIEW 19 of 35

Figure 18. Aminopyrazines derivatives used to study a sulfoxide-to sulfone produg approach.

Other compounds with original scaffolds have been discovered since the development of 66. Kandepedu et al. described a series of 1,5-naphthyridines, most of them having submicromolar activities on PfNF54 [91]. Starting from MMV024101 (71), a SAR study was performed on the two substituents of the naphthyridine core (Figure 19) and 48 analogs were synthesized. Compound 73 showed good activity on PfNF54 with an IC50 of 63 nM and good metabolic stability (t1/2 = 33 h at 5 mg/kg per os) but limited oral bioavailability (39%) on a mouse model.

Figure 19. 1,5-naphthyridines as inhibitors of PfPI4K.

Liang et al. described a bipyridine series targeting PfPI4K from screening in their compound library [92]. Compound 74 displayed an activity around 4 µM on Pf3D7 but was able to inhibit PfPI4K with an IC50 of 7.7 nM (Figure 20). An in-silico homology model of PfPI4K was then used to rationally guide modulations on 74: the 2-chloro-3-sulfonamide pyridine core was kept and modulations were performed on the two side chains. Compound 75 displayed the best activity on Pf3D7 of 25.1 nM together with an IC50 on PfPI4K of 0.9 nM. This activity was shown to be selective of PfPI4K versus human kinases including HsPI3K or HsPI4K. With oral bioavailability of 80% and a t1/2 of 3.6 h on a mouseMolecules model2020, 25 (at, 5949 10 mg/kg per os), 75 was able in vivo to cure blood-stage P. yoelii at 80 mg/kg/720 days of 35 and to stop liver-stage infection by P. berghei sporozoites at a single dose of 1 mg/kg in a mouse model.

FigureFigure 20. 20. 2-chloro-3-sulfonamide2-chloro-3-sulfonamide pyri pyridinesdines as as inhibitors inhibitors of of PfPfPI4K.PI4K.

4.14. Molecules Targeting Plasmodium falciparum Protein Kinase 5, 6, 7 and 9 (PfPK5, PfPK6, 4.14. Molecules Targeting Plasmodium falciparum Protein Kinase 5, 6, 7 and 9 (PfPK5, PfPK6, PfPK7 and MoleculesPfPK7 and 2020 PfPK9), 25, x FOR PEER REVIEW 20 of 35 PfPK9) PfPfPK5PK5 is is a acyclin-dependent-like cyclin-dependent-like kinase kinase [93], [93 esse], essentialntial for the for parasite the parasite blood-stage blood-stage [17], with [17], functions with functions still unclear. still unclear. Because Because of its homology of its homology with Hs withCDK2Hs CDK2[94], Pf [PK594], couldPf PK5 be could linked be to linked cell divisionto cell division regulation regulation [95]. PfPK6 [95]. isPf anotherPK6 is anothercyclin-dependent cyclin-dependent like kinase like most kinase likely most essential likely essential for the asexualfor the asexualblood-stage blood-stage [17,18,96]. [17 Pf,18PK7,96 is]. anPf PK7orphan is an kinase orphan (it does kinase not (it cluster does notwith cluster defined with eukaryotic deﬁned kinaseeukaryotic groups) kinase distantly groups) related distantly to the related MAPKK to theproteins MAPKK (mitogen-activated proteins (mitogen-activated protein kinase) protein [97]. Disruptingkinase) [97 ].Pf DisruptingPK7 showedPf thatPK7 it showed was not that essential it was notfor essentialthe asexual for blood-stage the asexual blood-stageand was linked and to was a reducedlinked to growth a reduced rate growth during ratethe asexual during theblood-stage asexual blood-stage and a lower and number a lower of numberoocysts in of the oocysts mosquito in the midgutmosquito [98]. midgut PfPK9 [98 is]. anotherPf PK9 is orphan another kinase, orphan essential kinase, essential for the blood-stage for the blood-stage [17], with [17], currently with currently only oneonly known one known phosphorylation phosphorylation target: target: the E2 the ubiquitin-conjugating E2 ubiquitin-conjugating enzyme enzyme 13 (Pf 13UBC13) (Pf UBC13) [99]. The [99]. humanThe human homolog, homolog, HsUBC13,HsUBC13, is involved is involved in DNA in DNA repair repair and immune and immune responses responses [100]. [100 ].

4.14.1.4.14.1. Molecules Molecules Targeting Targeting PlasmodiumPlasmodium falciparum falciparum ProteinProtein Kinase 5 ( PfPK5)PK5) CompoundCompound 7676 isis a a known known HsHsCDK2CDK2 and and PfPfPK5PK5 inhibitor, inhibitor, but but is is 1000 1000 times times more more potent potent toward toward thethe human human protein protein (Figure (Figure 21).21). Euba Eubanksnks et et al. al. synthesized synthesized analogs analogs of of 7676 toto try try to to obtain obtain selective selective compoundscompounds against against PfPfPK5PK5 but but their their modifications modiﬁcations failed [95]. [95]. Since Since PfPfPK5PK5 crystal crystal structure structure was was available,available, authors authors also also realized realized an an in-silico in-silico screening screening of 35,000 compounds against PfPK5PK5 followed followed byby an an inin vitro high throughput binding screening.screening. A A chromen-2-one-ba chromen-2-one-basedsed compound compound was was found found interestinginteresting and and analogs analogs of of this this compound compound were were purchased purchased and and tested. tested. Compound Compound 7777 waswas the the most most interestinginteresting analog, analog, with with a a K Kd(app)d(app) ofof 3.8 3.8 µMµM and and 5 5 µMµM against against PfPfPK5PK5 and and HsHsCDK2CDK2 respectively respectively but but lackedlacked activity activity against against PfPfDd2Dd2 in in vitro vitro (Figure (Figure 21).21).

FigureFigure 21. 21. InhibitorsInhibitors of of PfPfPK5PK5 discovered discovered by by Eubanks Eubanks et et al. al. [95]. [95].

4.14.2.4.14.2. Molecules Molecules Targeting Targeting PlasmodiumPlasmodium falciparum falciparum ProteinProtein Kinase 6 ( PfPK6)PK6) PfPfPK6PK6 inhibitors inhibitors have have only only been been identified identiﬁed as as hi hitt compounds compounds in in the the screening screening by by Crowther Crowther et et al. al. Figure 22 displays two compounds (78 and 79) having an IC against Pf PK6 of around 60 nM but Figure 22 displays two compounds (78 and 79) having an IC50 against PfPK6 of around 60 nM but lackedlacked selectivity selectivity toward toward PfPfPK6.PK6.

Figure 22. Molecules inhibiting PfPK6 discovered by Crowther et al. [33].

4.14.3. Molecules Targeting Plasmodium falciparum Protein Kinase 7 (PfPK7) Starting from hit compound 80 identified by a high-throughput screening campaign, Bouloc et al. synthesized a series of 3-amino-6-phenyl-imidazopyridazines (Figure 23) [101]. Thirty-five

Molecules 2020, 25, x FOR PEER REVIEW 20 of 35

PfPK5 is a cyclin-dependent-like kinase [93], essential for the parasite blood-stage [17], with functions still unclear. Because of its homology with HsCDK2 [94], PfPK5 could be linked to cell division regulation [95]. PfPK6 is another cyclin-dependent like kinase most likely essential for the asexual blood-stage [17,18,96]. PfPK7 is an orphan kinase (it does not cluster with defined eukaryotic kinase groups) distantly related to the MAPKK proteins (mitogen-activated protein kinase) [97]. Disrupting PfPK7 showed that it was not essential for the asexual blood-stage and was linked to a reduced growth rate during the asexual blood-stage and a lower number of oocysts in the mosquito midgut [98]. PfPK9 is another orphan kinase, essential for the blood-stage [17], with currently only one known phosphorylation target: the E2 ubiquitin-conjugating enzyme 13 (PfUBC13) [99]. The human homolog, HsUBC13, is involved in DNA repair and immune responses [100].

4.14.1. Molecules Targeting Plasmodium falciparum Protein Kinase 5 (PfPK5) Compound 76 is a known HsCDK2 and PfPK5 inhibitor, but is 1000 times more potent toward the human protein (Figure 21). Eubanks et al. synthesized analogs of 76 to try to obtain selective compounds against PfPK5 but their modifications failed [95]. Since PfPK5 crystal structure was available, authors also realized an in-silico screening of 35,000 compounds against PfPK5 followed by an in vitro high throughput binding screening. A chromen-2-one-based compound was found interesting and analogs of this compound were purchased and tested. Compound 77 was the most interesting analog, with a Kd(app) of 3.8 µM and 5 µM against PfPK5 and HsCDK2 respectively but lacked activity against PfDd2 in vitro (Figure 21).

Figure 21. Inhibitors of PfPK5 discovered by Eubanks et al. [95].

4.14.2. Molecules Targeting Plasmodium falciparum Protein Kinase 6 (PfPK6) PfPK6 inhibitors have only been identified as hit compounds in the screening by Crowther et al. Molecules 2020, 25, 5949 21 of 35 Figure 22 displays two compounds (78 and 79) having an IC50 against PfPK6 of around 60 nM but lacked selectivity toward PfPK6.

FigureFigure 22.22. MoleculesMolecules inhibitinginhibiting PfPfPK6 discovered by Crowther etet al.al. [[33].33].

4.14.3.4.14.3. Molecules Targeting PlasmodiumPlasmodium falciparumfalciparumProtein Protein KinaseKinase 77 ((PfPfPK7)PK7) StartingStarting fromfrom hithit compound compound80 80identiﬁed identified by by a a high-throughput high-throughpu screeningt screening campaign, campaign, Bouloc Bouloc et al.et Moleculessynthesizedal. synthesized 2020, 25 a, series x FORa series PEER of 3-amino-6-phenyl-imidazopyridazines REVIEWof 3-amino-6-phenyl-imidazopyridazines (Figure (Figure 23)[101 23)]. Thirty-ﬁve [101]. Thirty-five analogs21 of 35 Molecules 2020, 25, x FOR PEER REVIEW 21 of 35 were synthesized with modulations targeting the substitution of the amine at position 3 and the nature analogsof the para were-substituent synthesized on the with phenyl modulations at position targeting 6. Compound the substitution81 was the of best the compoundamine at position of this series,3 and analogs were synthesized with modulations targeting the substitution of the amine at position 3 and withthe nature an IC of ofthe 0.13 paraµ-substituentM and 1.09 µ onM the against phenylPf PK7 at position and Pf 3D7 6. Compound respectively. 81 was the best compound the nature 50of the para-substituent on the phenyl at position 6. Compound 81 was the best compound of this series, with an IC50 of 0.13 µM and 1.09 µM against PfPK7 and Pf3D7 respectively. of this series, with an IC50 of 0.13 µM and 1.09 µM against PfPK7 and Pf3D7 respectively.

Figure 23. Imidazopyridazine inhibitors of PfPK7. Figure 23. ImidazopyridazineImidazopyridazine inhibitors inhibitors of of PfPK7.PK7. Merckx etet al.al. realizedrealized aa screeningscreening of of compounds compounds against againstPf PfPK7,PK7, where where two two molecules molecules (82 (and82 and83, Merckx et al. realized a screening of compounds against PfPK7, where two molecules (82 and 83Figure, Figure 24) with24) with an imidazopyridazine an imidazopyridazine or pyrazolopyrimidine or pyrazolopyrimidine core displayed core displayed moderate moderate activity activity on both 83, Figure 24) with an imidazopyridazine or pyrazolopyrimidine core displayed moderate activity onkinase both and kinasePlasmodium and inhibitionPlasmodium assays inhibition [102]. Kleinassays et al.[102]. tested Klein their et series al. oftested pyrazolopyrimidines their series of on both kinase and Plasmodium inhibition assays [102]. Klein et al. tested their series of pyrazolopyrimidinesagainst Pf PK7 [103]. against Compound PfPK784 [103].was shownCompound to inhibit 84 wasPf PK7shown at 10to µinhibitM by approximatelyPfPK7 at 10 µM 60% by pyrazolopyrimidines against PfPK7 [103]. Compound 84 was shown to inhibit PfPK7 at 10 µM by (Figureapproximately 24). 60% (Figure 24). approximately 60% (Figure 24).

Figure 24. Hits inhibiting PfPK7 discovered by Merckx et al. and Klein et al. [102,103]. FigureFigure 24. 24. HitsHits inhibiting inhibiting PfPfPK7PK7 discovered discovered by by Merckx Merckx et et al. al. and and Klein Klein et et al. al. [102,103] [102,103].. 4.14.4. Molecules Targeting Plasmodium falciparum Protein Kinase 9 (PfPK9) 4.14.4. Molecules Targeting Plasmodium falciparum Protein Kinase 9 (PfPK9) PfPK9 inhibitors have only been explored by Raphemot et al. [100]. Starting from a screening of PfPK9 inhibitors have only been explored by Raphemot et al. [100]. Starting from a screening of 3,200 molecules, authors discovered that taketinib (85), an inhibitor of HsTAK1, was an inhibitor of 3,200 molecules, authors discovered that taketinib (85), an inhibitor of HsTAK1, was an inhibitor of PfPK9 (Table 11). 85 used at its EC50 on P. berghei infected HuH7 cells (7.3 µM) was able to increase PfPK9 (Table 11). 85 used at its EC50 on P. berghei infected HuH7 cells (7.3 µM) was able to increase the size of intracellular parasites but not their numbers, suggesting a deregulation of growth the size of intracellular parasites but not their numbers, suggesting a deregulation of growth pathways by inhibition of PbPK9. Analogs of taketinib were synthesized to obtain selective pathways by inhibition of PbPK9. Analogs of taketinib were synthesized to obtain selective compounds against PfPK9: analog 86 displayed a decreased affinity toward PfPK9 at 4.1 µM but was compounds against PfPK9: analog 86 displayed a decreased affinity toward PfPK9 at 4.1 µM but was unable to interact with HsTAK1 (Table 11). unable to interact with HsTAK1 (Table 11). Table 11. Taketinib and its analog as inhibitors of PfPK9. Table 11. Taketinib and its analog as inhibitors of PfPK9.

Compound R Kd(app) PfPK9 (µM) HuH7 P.berghei Infected Cell Load EC50 (µM) Compound R Kd(app) PfPK9 (µM) HuH7 P.berghei Infected Cell Load EC50 (µM) 85 (taketinib) NH2 0.46 7.3 85 (taketinib) NH2 0.46 7.3 86 OH 4.1 43 86 OH 4.1 43

Molecules 2020, 25, x FOR PEER REVIEW 21 of 35 analogs were synthesized with modulations targeting the substitution of the amine at position 3 and the nature of the para-substituent on the phenyl at position 6. Compound 81 was the best compound of this series, with an IC50 of 0.13 µM and 1.09 µM against PfPK7 and Pf3D7 respectively.

Figure 23. Imidazopyridazine inhibitors of PfPK7.

Merckx et al. realized a screening of compounds against PfPK7, where two molecules (82 and 83, Figure 24) with an imidazopyridazine or pyrazolopyrimidine core displayed moderate activity on both kinase and Plasmodium inhibition assays [102]. Klein et al. tested their series of pyrazolopyrimidines against PfPK7 [103]. Compound 84 was shown to inhibit PfPK7 at 10 µM by approximately 60% (Figure 24).

Molecules 2020, 25, 5949 22 of 35 Figure 24. Hits inhibiting PfPK7 discovered by Merckx et al. and Klein et al. [102,103].

4.14.4. Molecules Molecules Targeting Targeting Plasmodium falciparum Protein Kinase 9 (PfPK9) PfPK9PK9 inhibitors inhibitors have have only only been been explored explored by Raphem Raphemotot et al. [100]. [100]. Starting Starting from from a a screening screening of 3,200 molecules, authors discovered discovered that that taketinib taketinib ( (8585),), an an inhibitor inhibitor of of HsTAK1,TAK1, was an inhibitor of PfPK9 (Table 11). 85 used at its EC50 on P. berghei infected HuH7 cells (7.3 µM) was able to increase Pf PK9 (Table 11). 85 used at its EC50 on P. berghei infected HuH7 cells (7.3 µM) was able to increase the thesize size of intracellular of intracellular parasites parasites but not but their not numbers, their numbers, suggesting suggesting a deregulation a deregulation of growth pathways of growth by pathwaysinhibition ofbyPb inhibitionPK9. Analogs of Pb ofPK9. taketinib Analogs were of synthesized taketinib were to obtain synthesized selective compoundsto obtain selective against compoundsPf PK9: analog against86 displayed PfPK9: analog a decreased 86 displayed aﬃnity a towarddecreasedPf PK9 affinity at 4.1 towardµM but PfPK9 was at unable 4.1 µM to but interact was unablewith Hs toTAK1 interact (Table with 11 ).HsTAK1 (Table 11).

Table 11. Taketinib and its analog as inhibitors of PfPK9.

µ µ CompoundCompound R RKd(app) P Kfd(app)PK9 Pf(µM)PK9 ( HuH7M) P.berghei HuH7 P. berghei InfectedInfected Cell Load Cell Load EC50 EC (µM)50 ( M)

8585 (taketinib) (taketinib) NH2 NH2 0.46 0.46 7.3 7.3 8686 OH OH4.1 4.1 43 43

4.15. Molecules Targeting cGMP Cyclin-Dependent Protein Kinase (PfPKG) Pf PKG is a serine/threonine kinase involved in mechanisms at all the stages of parasite life: parasite motility, hepatocyte invasion, asexual blood-stage development, and gametocytogenesis [104–108]. Compared to HsPKG, Pf PKG possesses a smaller gatekeeper (the amino acid at the entrance of the catalytic domain), making it easier to develop molecules selective toward Pf PKG. Inhibition of Pf PKG led to multistage activity [109,110] with inhibition of:

sporozoite invasion during the liver-stage, • development of asexual blood-stage, • development of gametocytes, • exflagellation in the mosquito midgut. • Two different scaffolds were explored for Pf PKG inhibitors: imidazopyridines and trisubstituted five-membered aromatic cycles which could be viewed as ring-simplified analogs of the first series.

4.15.1. Imidazopyridines Targeting Pf PKG Starting from compound 87 (Table 12), designed originally as a PKG inhibitor against Eimeria tenella in chickens [111], Baker et al. realized a SAR study and synthesized nine analogs [110]. This work led to compound 88 with an IC50 against Pf PKG reduced from 3.1 to 0.16 nM and IC50 against Pf 3D7 strain reduced from 395 to 2.1 nM. 88 was also able to reduce blood-stage growth of P. berghei in a mouse model during a four-day Peter’s test by around 60% (25 mg/kg/twice per day per os)[27]. Additional work to better understand the properties of the imidazopyridine series was carried out by Large et al., leading to compound 89 [112] which showed lower potency on P. falciparum than 87 but better selectivity and metabolic stability. Molecules 2020, 25, x FOR PEER REVIEW 22 of 35 MoleculesMolecules 2020 2020, 25,, 25, x,, xxFOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 2222 of of 35 35 4.15. Molecules Targeting cGMP Cyclin-Dependent Protein Kinase (PfPKG) 4.15.4.15. Molecules Molecules Targeting Targeting cGMP cGMP Cyclin-Dependent Cyclin-Dependent Protein Protein Kinase Kinase (PfPKG) (PfPKG) PfPKG is a serine/threonine kinase involved in mechanisms at all the stages of parasite life: PfPKG is a serine/threonine kinase involved in mechanisms at all the stages of parasite life: parasitePfPf PKGmotility,PKG is is a ahepatocyteserine/threonine serine/threonine invasion, kinase kinase asexua involved involvedl blood-stage in in mechanisms mechanisms development, at at all all the the stages andstages gametocytogenesis of of parasite parasite life: life: parasiteparasite motility, motility, hepatocyte hepatocyte invasion, invasion, asexua asexual lblood-stage blood-stage development, development, and and gametocytogenesis gametocytogenesis [104–108].parasite Comparedmotility, hepatocyte to HsPKG, invasion, PfPKG asexua possessesl blood-stage a smaller development, gatekeeper and(the gametocytogenesisamino acid at the [104–108].[104–108]. Compared Compared to to Hs HsPKG,PKG, Pf PfPKGPKG possesses possesses a asmaller smaller gatekeeper gatekeeper (the (the amino amino acid acid at at the the entrance[104–108]. of the Compared catalytic todomain), HsPKG, making PfPKG itpossesses easier to a developsmaller gatekeepermolecules selective(the amino toward acid atPf PKG.the entranceentrance of of the the catalytic catalytic domain), domain), making making it it easier easier to to develop develop molecules molecules selective selective toward toward Pf PfPKG.PKG. Inhibition of PfPKG led to multistage activity [109,110] with inhibition of: InhibitionInhibitionInhibition of ofof Pf PfPKGPKG led led to to multistage multistage activity activity [109,110] [109,110] with with inhibition inhibition of: of: • • sporozoite invasion during the liver-stage, •• sporozoitesporozoite invasion invasion during during the the liver-stage, liver-stage, • • development of asexual blood-stage, •• developmentdevelopment of of asexual asexual blood-stage, blood-stage, • • •• developmentdevelopmentdevelopment of of ofgametocytes, gametocytes, gametocytes, • • •• exflagellationexflagellationexflagellation in in inthe the the mosquito mosquito mosquito midgut. midgut. midgut. TwoTwoTwo different different different scaffolds scaffolds scaffolds were were were explored explored explored for for Pf PfPKGPKG inhibitors:inhibitors: inhibitors: imidazopyridines imidazopyridines and and and trisubstituted trisubstituted trisubstituted five-memberedfive-memberedfive-memberedfive-membered aromatic aromatic aromaticaromatic cycles cycles cyclescycles which which whichwhich couldcould couldcould be bebe viewed viewedviewed as as as ring-simplified ring-simplifiedring-simplified analogs analogsanalogs of of of the the the first first first series. series. series.

4.15.1.4.15.1.4.15.1. Imidazopyridines Imidazopyridines Imidazopyridines Targeting Targeting Targeting Pf Pf PfPKGPKGPKG StartingStartingStarting from from from compound compound compound 87 87 87 (Table(Table (Table(Table 12),12), 12),12), designed designeddesigned originally originallyoriginallyoriginally as asas a a aPKG PKGPKG inhibitor inhibitorinhibitorinhibitor against againstagainst against Eimeria Eimeria Eimeria tenellatenellatenellatenella in in chickensinin chickens chickenschickens [111], [111], [111],[111], Baker Baker BakerBaker et et et et al.al. al.al. realizedrealized realizedrealized a a aSAR SARSAR study study study and andand synthesized synthesizedsynthesized nine ninenine analogs analogs analogs [110]. [110]. [110]. This This This work led to compound 88 with an IC50 against PfPKG reduced from 3.1 to 0.16 nM and IC50 against workworkwork led led led to to compoundto compound compound 88 88 88 with with withwith an an anan ICIC ICIC5050 5050 againstagainst againstagainst Pf PfPKGPKG reduced reducedreduced from fromfrom 3.1 3.1 to toto 0.16 0.160.16 nM nM nM and and and IC IC IC505050 against 50 againstagainst against Pf3D7PfPf3D73D7 strain strain strain reduced reduced reduced from from from 395 395 395 to to to 2.1 2.1 2.1 nM.nM. nM. 8888 88 was waswas alsoalso alsoalso ableable ableable to tototo reduce reducereducereduce blood-stage blood-stageblood-stage growth growthgrowth growth of ofof ofP. P. P.berghei berghei berghei in inainin mousea a amouse mousemouse model model modelmodel during during duringduring a a four-daya afour-day four-dayfour-day Peter’sPeter’s Peter’sPeter’s test testtest by byby aroundaround aroundaround 60% 60%60%60% (25 (25(25 mg/kg/twice mg/kg/twicemg/kg/twice per perper per day dayday day per per per os os )os ))[27]. [27].[27].) [27]. Additional work to better understand the properties of the imidazopyridine series was carried out AdditionalAdditionalAdditional work work work to to tobetter better better understand understand understand thethe the properties properties of of ofof the thethethe imidazopyridine imidazopyridineimidazopyridineimidazopyridine series seriesseriesseries was waswas was carried carriedcarried carried out outout out by Large et al., leading to compound 89 [112] which showed lower potency on P. falciparum than 87 byby Largeby Large Large et et alet al. ,al .leading,. .,,leading leadingleading to to totocompound compound compoundcompound 8989 89 [112][112] [112][112] which whichwhich showedshowed showedshowed lower lowerlowerlower potency potencypotency on ononon P. P. P. falciparum falciparumfalciparum falciparum than thanthan than 87 87 87 Moleculesbutbut better better2020, selectivity25 selectivity, 5949 and and metabolic metabolic stability. stability. 23 of 35 butbut better better selectivity selectivity and and metabolic metabolic stability. stability.

TableTable 12. 12. Imidazopyridines ImidazopyridinesImidazopyridines as asas inhibitors inhibitorsinhibitors of ofof Pf PfPKG.PKG. Table 12.12. ImidazopyridinesImidazopyridines asas inhibitorsinhibitors ofof PfPfPKG.

PPfPKGfPKG PPf3D7f3D7 PfPKG Pf3D7 % MLM PfPKG Pf3D7 %% MLM MLM% MLM CompoundCompound RR1 11 RRR2 22 RR3 33 RR4 44 PICfICPKG505050 ICPICf503D75050 Compound R1 R2 R3 R4 1 11 1 IC50 (nM) IC50 (nM)RemainingRemainingRemaining% MLM Compound R1 R2 R3 R4 (nM)(nM)(nM)IC50 (nM)(nM)(nM)IC50 Remaining 1 (nM) (nM) 878787 HH H HH H H H H 3.13.1 3.1 395395 395 52 52 52

87 H H H 3.1 395 52

888888 MeMeMe -NHSO-NHSO-NHSO2Me22Me2Me 0.160.160.16 2.12.1 2.1 - - -

88 Me -NHSO2Me 0.16 2.1 -

898989 HH H HH H Me Me Me 2.82.8 2.8 525525 525 93 93 93

89 1 H H Me 2.8 525 93 1 1%1 % remaining remaining after after 30 30 min min incubation incubation with with mouse mouse liver liver microsomes. microsomes. 1 % remaining after 30 min incubation with mouse liver microsomes. 1 % remaining after 30 min incubation with mouse liver microsomes. AnotherAnother study study by by Large Large et et al. al. involved involved modifying modifying the the imidazopyridine imidazopyridine core core using using a a scaffold scaffold hoppinghopping strategy strategy [113]. [113].[113]. Compound CompoundCompound 90 90, ,,a a apyrazolopyridine pyrazolopyridinepyrazolopyridine analog analoganalog of ofof 87 87, ,,displayed displayeddisplayed activities activitiesactivities on onon both bothboth Another study by Large et al. involved modifying the imidazopyridine core using a scaffold PfPfPKGAnotherPKG and and Pf studyPf3D73D7 similar bysimilar Large to to 87 87 et but al.but with involvedwith lower lower modifyingmetabolic metabolic stability stability the imidazopyridine (22% (22% of of compound compound core remaining remaining using a sca after afterﬀold hopping strategy [113]. Compound 90, a pyrazolopyridine analog of 87, displayed activities on both hopping3030 min min strategyincubation incubation [113 with with]. Compound mouse mouse liver liver90 microsome) microsome), a pyrazolopyridine due due to to a a more more analog lipophilic lipophilic of 87 ,molecule displayedmolecule (Figure (Figure activities 25). 25). on both PfPKG and Pf3D73D7 similar similar to to 8787 butbut with with lower lower metabolic metabolic stability (22% of compound remaining after 30 min incubation with mouse liverliver microsome)microsome) duedue toto aa moremore lipophiliclipophilic moleculemolecule (Figure(Figure 25 25).). Molecules 2020, 25, x FOR PEER REVIEW 23 of 35

FigureFigure 25.25. PyrazolopyridinePyrazolopyridine90 90as as inhibitorinhibitor ofofPf PfPKG.PKG.

4.15.2.4.15.2. TrisubstituedTrisubstitued Five-MemberedFive-Membered AromaticAromatic CyclesCycles TargetingTargetingPf PfPKGPKG WorkWork onon trisubstitutedtrisubstituted ﬁve-memberedfive-membered aromaticaromatic cyclescycles startedstarted withwith DiazDiaz etet al.,al., whowho testedtested compoundcompound91 91on onP. falciparumP. falciparum(Figure (Figure 26)[114 26)]. 91[114].was 91 designed was designed initially asinitially an anticoccidial as an anticoccidial compound targetingcompoundEimeria targeting tenella EimeriaPKG [tenella115]. 91 PKGdisplayed [115]. 91 nanomolar displayed activity nanomolar against activity multiple against recombinant multiple PfrecombinantPKG and an Pf ICPKG50 of and 0.49 anµ MIC against50 of 0.49P. µM falciparum againstNF54 P. falciparum strain. Activity NF54 strain.in vivo Activityon a mouse in vivo model on a infectedmouse model by P. berghei infectedwas by also P. assessed. berghei was At 50 also mg /assessed.kg/7 days byAt intraperitoneal50 mg/kg/7 days injections, by intraperitoneal91 was able toinjections, delay the 91 death was ofable all to the delay mouse the groupsdeath of by all 12 the days. mouse groups by 12 days.

Figure 26. Trisubstituted pyrrole as inhibitor of PfPKG.

Tsagris et al. decided to change the pyrrole core in 91 into a thiazole while keeping key substituents from both compounds 87 and 91 [116]. The authors started from compound 92 which displayed nanomolar inhibition of PfPKG but lacked good potency in vitro against Pf3D7 (Figure 27). 15 analogs were synthesized, with modifications targeting all three substituents, leading to compound 93. Using the same substituents as 87 led to improved potency but 93 displayed an hERG IC50 of 1.3 µM (Figure 27). 93 was assessed against human kinases and inhibited all the tested kinases below 70% at 100 nM. Starting from 93, Matralis et al. continued pharmacomodulations, mainly on the N-substituent of aminopyrimidine and the replacement of N-methypiperidine [117]. Multiple compounds showed improved potency, including compound 94 (Figure 27). 94 was able to kill parasites rapidly, like artemisinin. Thus, the authors decided to assess 94 in a binding assay against other plasmodial kinases and found that it was also able to bind PfCLK2. This dual activity was linked to the fast-killing profile, since compounds only targeting PfPKG possessed a slow killing profile. Moreover, 94 also showed an affinity toward PfCDPK1 and PfCDPK4.

Molecules 2020, 25, x FOR PEER REVIEW 23 of 35

Figure 25. Pyrazolopyridine 90 as inhibitor of PfPKG.

4.15.2. Trisubstitued Five-Membered Aromatic Cycles Targeting PfPKG Work on trisubstituted five-membered aromatic cycles started with Diaz et al., who tested compound 91 on P. falciparum (Figure 26) [114]. 91 was designed initially as an anticoccidial compound targeting Eimeria tenella PKG [115]. 91 displayed nanomolar activity against multiple recombinant PfPKG and an IC50 of 0.49 µM against P. falciparum NF54 strain. Activity in vivo on a Moleculesmouse 2020model, 25, 5949infected by P. berghei was also assessed. At 50 mg/kg/7 days by intraperitoneal24 of 35 injections, 91 was able to delay the death of all the mouse groups by 12 days.

FigureFigure 26.26. Trisubstituted pyrrolepyrrole asas inhibitorinhibitor ofof PfPfPKG.

Tsagris etet al.al. decided decided to to change change the the pyrrole pyrrole core core in 91 into a thiazolethiazole whilewhile keepingkeeping keykey substituentssubstituents from bothboth compoundscompounds 87 andand 9191 [[116].116]. TheThe authorsauthors startedstarted fromfrom compoundcompound 92 whichwhich displayed nanomolarnanomolar inhibitioninhibition ofof PfPfPKG but lacked good potencypotency in vitro against Pf3D73D7 (Figure (Figure 27).27). 1515 analogsanalogs were were synthesized, synthesized, with with modifications modification targetings targeting all three all substituents, three substituents, leading to compoundleading to 93compound. Using the 93. sameUsing substituents the same substituents as 87 led toas improved87 led to improved potency butpotency93 displayed but 93 displayed an hERG an IC h50ERGof 1.3IC50µ ofM 1.3 (Figure µM (Figure 27). 93 27).was 93 was assessed assessed against against human human kinases kinases and an inhibitedd inhibited all all the the tested tested kinaseskinases below 70%70% atat 100100 nM.nM. StartingStarting fromfrom 9393,, MatralisMatralis etet al.al. continuedcontinued pharmacomodulations,pharmacomodulations, mainlymainly onon thethe NN-substituent-substituent ofof aminopyrimidineaminopyrimidine andand thethe replacementreplacement ofof NN-methypiperidine-methypiperidine [[117].117]. MultipleMultiple compoundscompounds showed improved potency,potency, includingincluding compoundcompound 9494 (Figure(Figure 2727).). 94 was able to killkill parasitesparasites rapidly,rapidly, likelike artemisininartemisinin.. Thus,Thus, thethe authorsauthors decideddecided toto assessassess 9494 inin aa bindingbinding assayassay againstagainst other plasmodialplasmodial kinaseskinases andand foundfound thatthat itit waswas alsoalso ableable toto bindbindPf PfCLK2. This dual activity was linked toto thethe fast-killingfast-killing profile,profile, sincesince compoundscompounds only targeting PfPKG possessed a a slow killing profile. profile. Moreover, 9494 alsoalso showedshowed anan aaffinityffinity towardtowardPf PfCDPK1CDPK1 andand PfPfCDPK4. Molecules 2020, 25, x FOR PEER REVIEW 24 of 35

Figure 27. TrisubstitutedTrisubstituted thiazoles as inhibitors of PfPKG.PKG.

FromFrom three isoxazole isoxazole-based-based hits hits on on PfPfPKGPKG of of their their own own compounds’ compounds’ library, library, Ul Ul Mahmood Mahmood et et al. al. exploredexplored the three positions on the isoxazole cycle while keeping key features features like aminopyrimidine fromfrom previously previously described described PfPfPKGPKG inhibitors inhibitors [118]. [118 29]. analogs 29 analogs were synthesized were synthesized and tested and on tested PfPKG: on Pf 95 sixPKG: of them six displayed of them displayed an IC50 below an IC 5050 nM,below including 50 nM, compound including compound95, the most potent, the most one (Figure potent 28). one Finally,(Figure 28MMV030084). Finally, MMV030084 (compound (compound 96, Figure96 ,28), Figure a trisubstituted 28), a trisubstituted imidazole, imidazole, was wasdescribed described by Vanaershotby Vanaershot et al. et al.as a as multistage a multistage active active co compoundmpound (no (no gametocytocidal gametocytocidal effect) eﬀect) targeting targeting PfPfPKGPKG [109]. [109]. UsingUsing knockout studies, PfPKGPKG was was confirmed conﬁrmed to to be be the the main main target target of 96..

Figure 28. Trisubstituted isoxazole and imidazole as inhibitors of PfPKG.

4.16. Molecules Targeting Plasmodium falciparum Thymidylate Kinase (PfTMK) PfTMK is the enzyme catalyzing the phosphorylation of thymidine monophosphate to thymidine diphosphate. PfTMK is involved in the de novo synthesis of purine bases and is the only way for the parasite to create these bases [119]. This pathway also includes major drug targets such as dihydrofolate reductase (PfDHFR, targeted by sulfadoxine) and more recently, dihydroorotate dehydrogenase (PfDHODH, targeted by DSM265 [120]). PfTMK is also able to catalyze the phosphorylation of deoxyguanosine monophosphate into deoxyguanosine diphosphate. Research on PfTMK inhibitors has been centered on thymidine analogs. Cui et al. synthesized a large number of thymidine analogs bearing a urea side chain [121]. Compound 97 was used as a starting point for pharmacomodulations which led to compound 98 (Figure 29). Modulations showed several structural features important for potency: α-anomers were more potent than β, thiourea decreased potency whereas hydrophobic para-substituent on the phenyl urea increased potency. 98 displayed nanomolar activity on Pf3D7 but with low microsomal stability. Surprisingly, no inhibitory assays were performed on the most potent compounds of the series, meaning no clear conclusion can be drawn on the target of these compounds.

Molecules 2020, 25, x FOR PEER REVIEW 24 of 35

Figure 27. Trisubstituted thiazoles as inhibitors of PfPKG.

From three isoxazole-based hits on PfPKG of their own compounds’ library, Ul Mahmood et al. explored the three positions on the isoxazole cycle while keeping key features like aminopyrimidine from previously described PfPKG inhibitors [118]. 29 analogs were synthesized and tested on PfPKG: six of them displayed an IC50 below 50 nM, including compound 95, the most potent one (Figure 28). Finally, MMV030084 (compound 96, Figure 28), a trisubstituted imidazole, was described by VanaershotMolecules 2020 ,et25 ,al. 5949 as a multistage active compound (no gametocytocidal effect) targeting PfPKG 25[109]. of 35 Using knockout studies, PfPKG was confirmed to be the main target of 96.

Figure 28. TrisubstitutedTrisubstituted isoxazole and imidazole as inhibitors of PfPKG.

4.16. Molecules Molecules Targeting Targeting Plasmodium falciparumfalciparum Thymidylate Kinase (PfTMK) PfTMKTMK isis thethe enzyme enzyme catalyzing catalyzing the phosphorylationthe phosphorylation of thymidine of thymidine monophosphate monophosphate to thymidine to thymidinediphosphate. diphosphate.Pf TMK is involvedPfTMK is in involved the de novo in the synthesis de novo of synthesis purine bases of purine and is ba theses only and wayis the for only the wayparasite for tothe create parasite these to bases create [119 these]. This bases pathway [119]. Th alsois includespathway major also includes drug targets major such drug as dihydrofolate targets such asreductase dihydrofolate (Pf DHFR, reductase targeted (PfDHFR, by sulfadoxine) targeted by and sulfadoxine) more recently, and dihydroorotatemore recently, dehydrogenasedihydroorotate dehydrogenase(Pf DHODH, targeted (PfDHODH, by DSM265 targeted [120 ]).by PfDSM265TMK is [120]). also able PfTMK to catalyze is also the able phosphorylation to catalyze the of phosphorylationdeoxyguanosine monophosphateof deoxyguanosine into monophos deoxyguanosinephate into diphosphate. deoxyguanosine diphosphate. Research on PfPfTMKTMK inhibitors inhibitors has has been been centered centered on on thym thymidineidine analogs. analogs. Cui Cui et et al. al. synthesized synthesized a largea large number number of ofthymidine thymidine analogs analogs bearing bearing a urea a urea side side chain chain [121]. [121 ].Compound Compound 9797 waswas used used as asa startinga starting point point for forpharmacomodulations pharmacomodulations which which led to led compound to compound 98 (Figure98 (Figure 29). Modulations 29). Modulations showed severalshowed structural several structural features important features important for potency: for potency:α-anomersα -anomerswere more were potent more than potent β, thiourea than β, decreasedthiourea decreased potency whereas potency hydrophobic whereas hydrophobic para-substituentpara-substituent on the phenyl on theurea phenyl increased urea potency. increased 98 displayedpotency. 98 nanomolardisplayed activity nanomolar on Pf activity3D7 but onwithPf 3D7low microsomal but with low stability. microsomal Surprisingly, stability. no Surprisingly, inhibitory assaysno inhibitory were performed assays were on performedthe most potent on the compounds most potent of compoundsthe series, meaning of the series, no clear meaning conclusion no clear can beconclusion drawn on can the be target drawn of onthese the compounds. target of these compounds. Molecules 2020, 25, x FOR PEER REVIEW 25 of 35

FigureFigure 29. 29. αα-thymidine-thymidine urea urea analogs analogs as as inhibitors inhibitors of of PfTMK. PfTMK.

SimplerSimpler structures structures were were explored explored by by Kato Kato et et al., al., who who replaced replaced the the tetrahydrofuran tetrahydrofuran by bya cyclopentenea cyclopentene [122]. [122 This]. This led to led compound to compound 99, showing99, showing a weak a weakactivity activity with a withKi of a20 K µMi of on 20 PfµMTMK on (FigurePf TMK 30). (Figure 99 was 30). then99 was used then by usedNoguchi by Noguchi et al. as eta starting al. as a startingpoint for point pharmacomodulations for pharmacomodulations on the sideon the chain side of chain the cyclopentene of the cyclopentene core [123]. core Compound [123]. Compound 100 with a100 fluoroethanolwith a ﬂuoroethanol substituent substituent displayed betterdisplayed potency better than potency 99, with than a K99i ,of with 14 µM a K (Figurei of 14 µ 30).M (Figure 30).

Figure 30. Cyclopentene analogs of thymidine as inhibitors of PfTMK.

5. Conclusions Plasmodial kinases appear to be promising targets for new drug therapies against malaria. In the best cases, their inhibition can impact every stage of the parasitic cycle. This is due to the involvement of kinases in most of the cellular mechanisms. Zhang et al. created a mutagenesis index score (MIS) for each gene of the P. falciparum asexual blood-stage, illustrating the essentialness of each of these genes (Figure 31) [124]: only five of the kinases described in this review are considered dispensable (MIS close to 1) but two of them (PfCDPK4 and PfPK7) are known to be key proteins during exflagellation.

Molecules 2020, 25, x FOR PEER REVIEW 25 of 35

Figure 29. α-thymidine urea analogs as inhibitors of PfTMK.

Simpler structures were explored by Kato et al., who replaced the tetrahydrofuran by a cyclopentene [122]. This led to compound 99, showing a weak activity with a Ki of 20 µM on PfTMK (Figure 30). 99 was then used by Noguchi et al. as a starting point for pharmacomodulations on the sideMolecules chain2020 of, 25 the, 5949 cyclopentene core [123]. Compound 100 with a fluoroethanol substituent displayed26 of 35 better potency than 99, with a Ki of 14 µM (Figure 30).

Figure 30. CyclopenteneCyclopentene analogs analogs of of thymidi thymidinene as as inhibitors of PfTMK.

5. Conclusions Conclusions Plasmodial kinases appearappear toto be be promising promising targets targets for for new new drug drug therapies therapies against against malaria. malaria. In theIn thebest best cases, cases, their their inhibition inhibition can impact can impact every stage every of stage the parasitic of the cycle.parasitic This cycle. is due This to the is involvementdue to the involvementof kinases in mostof kinases of the in cellular most of mechanisms. the cellular Zhangmechanisms. et al. created Zhang a et mutagenesis al. created a index mutagenesis score (MIS) index for scoreeach gene(MIS) of for the eachP. falciparum gene of theasexual P. falciparum blood-stage, asexual illustrating blood-stage, the illustrati essentialnessng the of essentialness each of these of genes each of(Figure these 31 genes)[124 (Figure]: only ﬁve31) [124]: of the only kinases five described of the kinases in this described review are in considered this review dispensable are considered (MIS close to 1) but two of them (Pf CDPK4 and Pf PK7) are known to be key proteins during exﬂagellation. dispensableMolecules 2020 (MIS, 25, x FORclose PEER to REVIEW1) but two of them (PfCDPK4 and PfPK7) are known to be key 26proteins of 35 during exflagellation.

Figure 31. Mutagenesis Index Score graph with protein kinases described in the review highlighted. Figure 31. Mutagenesis Index Score graph with protein kinases described in the review highlighted. All P. falciparum genes were ordered from the lowest to the highest score and then attributed an All P. falciparum genes were ordered from the lowest to the highest score and then attributed an arbitrary arbitrary rank number to obtain this curve. Data from Zhang et al. [124]. rank number to obtain this curve. Data from Zhang et al. [124].

MMV390048MMV390048 (66 (66),), targeting targetingPf PfPI4KPI4K is is currently currently the the only only plasmodial plasmodial kinase kinase inhibitor inhibitor in in clinical clinical trial. trial. However, this could quickly change as the way is opened to other PfPI4K inhibitors. This makes However, this could quickly change as the way is opened to other Pf PI4K inhibitors. This makes Pf PI4K PfPI4K inhibitors the most advanced plasmodial kinase inhibitors in the drug development process inhibitors the most advanced plasmodial kinase inhibitors in the drug development process (Table 13). (Table 13). PfCDPK4 inhibitors are getting close to a pre-clinical stage while PfCDPK1, PfCLK3, Pf CDPK4 inhibitors are getting close to a pre-clinical stage while Pf CDPK1, Pf CLK3, Pf GSK3 and PfGSK3 and PfPKG inhibitors are promising compounds awaiting further studies. This represents Pf PKG inhibitors are promising compounds awaiting further studies. This represents only six out only six out of the 22 kinases described in this review. Many are still in a hit discovery stage, ofsometimes the 22 kinases without described progress in for this more review. than Many ten years. are still in a hit discovery stage, sometimes without progressThe for development more than ten of plasmodial years. kinase inhibitors is slowed by a lack of X-ray structural data, challenges regarding selectivity against human kinases, and loss of potency going from protein to parasite activity. Moreover, new antiplasmodial hit molecules without a defined target usually have their mechanism of action explored based on commercial antiplasmodial drugs. If these compounds possess multistage activity, they should be tested on plasmodial kinases.

Table 13. Plasmodial kinases targeted by inhibiting compounds described in this review.

Development Stage of Role of Protein—Consequence(s) of Studies Protein the Most Advanced Inhibition 1 Reviewed Compound(s) Egress and invasion processes regulator— CDPK1 Inhibition of asexual blood-stage growth and Hit to lead [23,24,28–33] gametogenesis, reduced mosquito infection Exflagellation regulator—Reduced mosquito CDPK4 Lead optimization [33,36–39] infection Transformation of choline into Hit discovery—Target CK [41,45–47] phosphocholine validation needed Serine/threonine kinase, possibly Hit discovery—In vitro CK2 phosphorylating many proteins during activity to be [50] asexual blood-stage determined

Molecules 2020, 25, 5949 27 of 35

Table 13. Plasmodial kinases targeted by inhibiting compounds described in this review.

Development Stage of Protein Role of Protein—Consequence(s) of Inhibition 1 the Most Advanced Studies Reviewed Compound(s) Egress and invasion processes regulator—Inhibition of asexual blood-stage CDPK1 Hit to lead [23,24,28–33] growth and gametogenesis, reduced mosquito infection Exflagellation regulator—Reduced mosquito CDPK4 Lead optimization [33,36–39] infection Hit discovery—Target CK Transformation of choline into phosphocholine [41,45–47] validation needed Serine/threonine kinase, possibly phosphorylating Hit discovery—In vitro CK2 [50] many proteins during asexual blood-stage activity to be determined Phosphorylation of spliceosomes Hit discovery—In vitro CLK1 proteins—Inhibition of asexual and sexual [53,55] activity to be determined blood-stage growth Phosphorylation of spliceosomes CLK3 Hit to lead [53,54,56] proteins—All-stage activity Transformation of pantothenate into PanK Hit discovery [58–60] 40-phosphopantothenate Transformation of dephospho-coenzyme A into DPCK Hit discovery [61] coenzyme A FKk8 Not defined Hit discovery [64,65] Transformation of (deoxy) Hit discovery—Target GK guanosine-monophosphate into (deoxy) [46] validation needed guanosine-diphosphate GSK3 Not defined Hit to lead [70,71] Transformation of glucose into HK Hit discovery [74,75] glucose-6-phosphate Involved in many key cellular Hit discovery—Target MAP2 processes—Inhibition of asexual blood-stage and [77] validation needed exflagellation MRK Regulation of gene expression and DNA replication Hit discovery [79–81] NEK1 Regulation of cell division processes Hit discovery [83,84] Transformation of phosphatidyl-inositol into PI4K phosphatidyl-inositol 4 phosphate—All-stage Phase II clinical trial [85,87,89–92] activity Cyclin-dependent like kinase, possibly regulating PK5 Hit discovery [95] cell division Hit discovery - In vitro PK6 Cyclin-dependent like kinase, role not defined [33] activity to be determined Orphan kinase, role not defined—Inhibition of PK7 asexual blood-stage and reduced mosquito Hit to lead [101–103] infection Orphan kinase, role unclear, able to phosphorylate PK9 Pf UBC13—Deregulation of asexual hepatic stage Hit discovery [100] growth [109,110,112–114,116– PKG Serine/threonine kinase—All-stage activity Hit to lead 118] Transformation of thymidine monophosphate into TMK Hit to lead [121–123] thymidine diphosphate 1 When not specified, each of these proteins is assumed to at least inhibit asexual blood-stage growth. Molecules 2020, 25, 5949 28 of 35

The development of plasmodial kinase inhibitors is slowed by a lack of X-ray structural data, challenges regarding selectivity against human kinases, and loss of potency going from protein to parasite activity. Moreover, new antiplasmodial hit molecules without a deﬁned target usually have their mechanism of action explored based on commercial antiplasmodial drugs. If these compounds possess multistage activity, they should be tested on plasmodial kinases. There is still plenty of room for new kinase inhibitors: only 22 out of 86 (or 99) estimated plasmodial kinases were found to be targeted by the compounds in this review. In addition to plasmodial kinases inhibitors, other new promising drugs in development targeting plasmodial proteins such as Pf DHODH or Pf ATP4 will be important for the creation of new powerful drug combinations to reduce numbers of malaria cases and deaths in years to come.

Author Contributions: Conceptualization, N.P.; methodology, R.M. and N.P.; formal analysis, R.M.; data curation, R.M.; writing—original draft preparation, R.M.; writing—review and editing, N.P. and P.V.; supervision, N.P. and P.V.; funding acquisition, N.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by “Fondation pour la Recherche Médicale (FRM)”, project code DCM20181039565. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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