Apicoplast-Resident Processes: Exploiting the Chink in the Armour of Plasmodium falciparum Parasites

The discovery of a relict plastid, also known as an apicoplast (apicomplexan plastid), that houses housekeeping processes and metabolic pathways critical to Plasmodium parasites' survival has prompted increased research on identifying potent inhibitors that can impinge on apicoplast-localised processes. The apicoplast is absent in humans, yet it is proposed to originate from the eukaryote's secondary endosymbiosis of a primary symbiont. This symbiotic relationship provides a favourable microenvironment for metabolic processes such as haem biosynthesis, Fe-S cluster synthesis, isoprenoid biosynthesis, fatty acid synthesis, and housekeeping processes such as DNA replication, transcription, and translation, distinct from analogous mammalian processes. Recent advancements in comprehending the biology of the apicoplast reveal it as a vulnerable organelle for malaria parasites, offering numerous potential targets for effective antimalarial therapies. We provide an overview of the metabolic processes occurring in the apicoplast and discuss the organelle as a viable antimalarial target in light of current advances in drug discovery. We further highlighted the relevance of these metabolic processes to Plasmodium falciparum during the different stages of the lifecycle.


Introduction
Te apicoplast (apicomplexan plastid) is a relict nonphotosynthetic plastid-like organelle present in apicomplexans such as Plasmodium species.It is an unusual organelle proposed to have evolved due to secondary and tertiary endosymbiosis, giving rise to a galactolipid-rich four-membrane organelle.Tis process involves the phagocytic assimilation of a plastid-containing eukaryote (which originally obtained its plastid via primary endosymbiosis of prokaryotes such as a red alga) by another eukaryote [1][2][3].Te outer membrane has features similar to a host's endomembrane as a result of phagocytosis.Te second outer membrane, also called the periplastid membrane (PPM), is believed to originate from the plasma membrane of the engulfed red alga, while the inner pair of membranes is analogous to the membrane of the chloroplast [3,4].Plastids have various metabolic functions in organisms in which they are resident and are mostly photosynthetically active; however, the apicoplast is nonphotosynthetic but plays crucial roles in the survival and development of apicomplexans [3].Plastids comprise chloroplasts, amyloplasts, chromoplasts, etioplasts, and leucoplasts [3].Te ancestry of apicoplastharbouring parasites has been linked to three origins: the clades Platyproteum, Chrompodellids, and Apicomplexa, according to phylogenetic and biological investigations [3,5,6].
Apicomplexans are obligate parasites classifed into fve groups: Piroplasmida, Coccidia, Haemosporidia, Cryptosporidium, and Gregarina.Tese groups of obligate parasites are intracellular parasites except Gregarina [6].Te more popular groups are Haemosporidia (e.g., Plasmodium spp.) and Coccidia (e.g., toxoplasma), which have been implicated in malaria and toxoplasmosis.Te apicomplexans are responsible for several diseases afecting humans, livestock, wild animals, and invertebrates [4].Obligate intracellular apicomplexans, including Plasmodium spp.and Toxoplasma gondii, depend heavily on the apicoplast for de novo synthesis of metabolites required for their normal development.Tis dependence is because, despite the parasite's ability to scavenge these apicoplast-derived metabolites from their hosts or environment, their complex lifecycle and changing environments pose a challenge to obtaining an adequate amount for their normal development at all stages of their lifecycle.Hence, the parasite could depend on the de novo synthetic property of the apicoplast for its survival in certain living environments [3,7].Tere is still an ongoing debate on the presence of plastid in the Gregarina; however, it is absent in the cryptosporidium lineage of apicomplexan.Te apicoplast is responsible for four major functions: fatty acid synthesis, isoprenoid biosynthesis, Fe-S cluster synthesis, and haem synthesis [8].
Te discovery of the apicoplast in malaria parasites has opened up novel prospects in antimalarial drug discovery and development because the organelle is absent in mammals, essential throughout the lifecycle of the parasite, and it possesses nonmammalian metabolic processes, which are critical for parasite growth and development [4,7].Several reports have indicated that the apicoplast is an excellent drug target, and more work is currently being done to develop therapeutic interventions with novel mechanisms of action or combination therapies to abrogate apicoplast-resident metabolic processes and diminish the current problem of antimalarial drug resistance [9][10][11].Here, we provide an overview of the metabolic processes occurring in the apicoplast and discuss the organelle as a viable antimalarial target in light of current advances.We further highlighted the relevance of these metabolic processes to Plasmodium falciparum during the diferent stages of the lifecycle.

The Plasmodium falciparum Apicoplast
Te Plasmodium falciparum apicoplast contains genetic material of approximately 50 genes, coding for 30 proteins, rRNAs, and tRNAs, with a size of about 35 kb long [6,12].Te apicoplast resident genes are responsible for genome maintenance, including processes of replication, transcription, and translation, except for iron-sulphur cluster assembly B (SufB), which plays roles in the sulphur mobilisation (SUF) pathway required for Fe-S cluster synthesis and the ATP-dependent Clp protease subunit C (clpC) gene, whose function is currently undefned but hypothetically believed to be responsible for protein import or as a chaperone hsp70 substitute [6,13].Te gene products encoded by the nuclear genome required for apicoplast function, called nucleus-encoded apicoplast-targeted (NEAT) proteins, are transcribed in the nucleus, translated in the cytosol, and exported to the apicoplast; however, membrane traversal occurs through a relatively obscure mechanism [3].Te trafcking of nuclear-encoded apicoplast-destined proteins is coordinated by a bipartite canonical signal located at the N-terminal of the protein, which comprises a canonical endoplasmic reticulum (ER)type secretory signal sequence followed sequel to a plant-like transit peptide [14].Te predominant transport hypothesis for the apicoplast involves the Golgi-independent model, whereby the vesicles emanating from the endoplasmic reticulum convey proteins directly to the apicoplast [14][15][16].
Apicoplast division and inheritance are important for parasite survival.Further studies on the apicoplast have revealed that it is maternally inherited similarly to the mitochondrial genome (6 kb).Te disparity in genome size and lack of copurifcation during subcellular fractionation diferentiated the apicoplast as a distinct organelle from the mitochondria [8].Nonetheless, both organelles are closely associated during all the stages of the parasite's lifecycle, indicating a strong metabolic reliance between the apicoplast and mitochondria [4,8].
Recent studies employing exogenous IPP supplementation have been used to study the molecular mechanism involved in apicoplast fssion [17].Autophagy-related proteins have been implicated in autophagy-independent roles, which are critical for apicoplast inheritance and parasite replication during the blood stage of infection [17][18][19].During nutrient depletion, Plasmodium falciparum ATG8bounded autophagosomes are destined for the lysosome, where the cargo inherent in the autophagosome is degraded for nutrients.However, during replication, ATG8 has been reported to localise to the apicoplast during the liver and blood stages of parasite development [3,18].Walczak and colleagues reported a novel function of ATG8 in Plasmodium falciparum in the apicoplast biogenesis [17].A study by Bansal et al. revealed the importance of the interaction between ATG8 and another autophagy-related protein ATG18.Tis study showed a novel nonautophagy-related role of ATG18 in controlling ATG8's localisation to the apicoplast and its membrane conjugation [18].Phosphoinositide kinases (PIKs) phosphorylate phosphatidylinositol to yield phosphatidylinositol 3-phosphate (PI3P), which is required for the cellular localisation of ATG18.However, as the interaction between ATG18 and ATG8 has not yet been validated, it was proposed that PI3P-PfATG18 is important for PfATG8 lipidation and may trafc it to the apicoplast [18].Te fssion of the apicoplast presents interesting and excellent opportunities for drug development against malaria, though the exact mechanism is still obscure.Recent fndings have been encouraging, and elucidating the processes and molecular mechanisms involved will be important in understanding apicoplast biology.

Apicoplast-Localised Housekeeping Metabolic Processes in
Plasmodium falciparum.Housekeeping metabolic processes are essential in the apicoplast, allowing basic cellular function maintenance and continuity [20].Because apicoplast division happens over four lipid bilayers, it difers from how bacteria and chloroplasts divide.When the apicoplast separates, it is exactly divided among the merozoite cells that 2 Advances in Pharmacological and Pharmaceutical Sciences result from this process [21].Most of the genes in the apicoplast genome are responsible for the transcription and translation machinery of the organelle; however, no DNA replication or organisation genes are present.Te apicoplast does not regulate the translation of nuclear-encoded proteins needed, and recent studies reported the accumulation of these proteins in vesicle-like structures in the cytoplasm in apicoplast-negative parasites [22].Replication begins in the late trophozoite phase and continues into the schizogony phase of the parasite's life cycle, utilizing the D-loop/bidirectional ori mechanism [23].Proteins associated with replication possess a bipartite N-terminal leader sequence, which destines nuclearencoded proteins to be post-translationally targeted to the apicoplast via a secretory pathway [24].Plastidic DNA replication/repair enzyme complex (Prex) is directed to the apicoplast for DNA replication and repair.Primase, helicase, and apicoplast DNA polymerase (apPol) required for template creation, priming, and elongation, respectively, are encoded by the same open reading frame of the Prex gene [25].Tis gene is cotranslated as a single polyprotein into the ER lumen and then transported as a polyprotein into the apicoplast [26].Te type II topoisomerases gyrase A (GyrA) and gyrase B (GyrB), which introduce negative supercoils in DNA that are necessary for replication and transcription, are present in the apicoplast [27].Tis reduces the strain during the replication process by employing the C-terminal domains of GyrA (which forms a six-bladed propeller) to wrap the DNA with positive handedness and a "DNA clamp" at the N terminus of GyrB (which dimerises when ATP is bound), which seizes another chunk of the same doublestranded DNA (dsDNA) [28].
In contrast with plant chloroplasts with multiple RNA polymerases, apicoplasts only have one RNA polymerase responsible for the transcription of all the genes in the apicoplast genome [29].Te schizont stage is when its expression peaks, coinciding with the replication of the apicoplast genome [30].Te RNA polymerase comprises fve subunits: two α (rpoA), β (rpoB), β′ (rpoC), and ω (rpoD), of which only the rpoB and rpoC subunits are apicoplastencoded.Te Plasmodium falciparum apicoplast employs the polycistronic mode of transcription, and its transcripts (both sense and antisense) exhibit several cleavage sites connected to a UUAUA motif [29].Te apicoplast gene expression machinery is prokaryote-like due to polycistronic mRNA transcripts and 70S ribosomes.Te apicoplast genome contains all the rRNA and tRNA genes and several necessary ribosomal proteins, transcription factors, and translation factors [8].It encodes the open reading frames for the caseinolytic protease clpC, RNA polymerase subunits rpoB, rpoC1, and rpoC2, [Fe-S] cluster protein SufB, and the translation elongation factor (EF-Tu) [31].Te coordinated action of translation initiation, elongation, and release factors is necessary to translate proteins encoded by the apicoplast genome to facilitate the various steps of peptide synthesis in the ribosomes [32].
In the apicoplast, translation starts with forming a ternary initiation complex, consisting of a ribosome with a protein-coding mRNA whose initiation codon (AUG) is base-paired to an aminoacylated initiator tRNA and controlled by the action of initiation factors (IFs) [33].Elongation begins with the entry of the initiator tRNA into a P-site, which causes a conformational shift and opens the A-site for binding a new aminoacyl-tRNA [30].Te P. falciparum apicoplast has UAA as the predominant stop codon [34].

Apicoplast-Localised
Nonhousekeeping Metabolic Processes in Plasmodium falciparum.Te apicoplast is an intracellular organelle that is the target of about 400 nucleiencoded gene products responsible for several metabolic pathways, including haem synthesis needed for the mitochondrial electron transport chain, the SUF mobilisation pathway for the FE-S cluster synthesis, the fatty acid synthesis II pathway, and the methylerythritol phosphate (MEP) pathway [35].In addition, the apicoplast performs genome replication, transcription, translation, post-translational modifcation, and protein turnover [36].Each of the metabolic functions of the apicoplast is described as follows.

Shemin Pathway.
Te synthesis of haem occurs via the Shemin pathway, which takes place in three intracellular locations, mitochondria, apicoplast, and the cytosol, and is catalysed by eight enzymes.Following the acquisition of the secondary endosymbiont, the eukaryote was challenged with redundancy in the Shemin pathway, leading to the evolutionary rationalisation of redundancy.Te rationalisation of redundancy involved eliminating and substituting similar steps within the pathway, facilitating the collaboration between the mitochondria and apicoplast in haem synthesis [4].Te pathway is initiated by the condensation of glycine and succinyl-CoA in the mitochondria, resulting in the formation of aminolevulinic acid (ALA) by the enzyme ALA synthase, followed by the traversal of ALA to the apicoplast.Four apicoplast-localised enzymes, including δ-aminolevulinate dehydratase, porphobilinogen deaminase, uroporphyrinogen III synthase, and uroporphyrinogen III decarboxylase, are involved in the synthesis of coproporphyrinogen III [4,37].Te subsequent activity of cytosolic coproporphyrinogen oxidase on coproporphyrinogen III leads to the formation of protoporphyrinogen IX, which translocates into the mitochondria and is acted upon by protoporphyrinogen oxidase (PfPPO) and ferrochelatase (FC), resulting in the eventual synthesis of haem (Figure 1) [37].
Haem is an important moiety of biological structures such as chlorophyll and haemoglobin required for binding and carrying small molecules or electrons.Te biosynthesis of haem is essential to the malaria parasite, as it is needed as the prosthetic group of cytochromes during the electron transport chain [38].De novo haem biosynthesis is not required in the blood stage but rather during the mosquito stage, making it useful in mosquito transmission [37].Tis is possible because the parasite can scavenge haem, and some Shemin pathway enzymes can be scavenged from the hosts.Furthermore, two routes were proposed for haem acquisition from the host during the blood stage, including Advances in Pharmacological and Pharmaceutical Sciences sequestration and transportation of haem by haem-binding proteins following haemoglobin degradation in the food vacuole or scavenging free haem present in the cytosol of the invaded erythrocyte [37].Te dispensable nature of de novo haem synthesis during the intraerythrocytic blood stage of falciparum malaria decreases the viability of the Shemin pathway as a potential target for antimalarial therapy.

Type II Fatty Acid Synthesis (FAS II) Pathway.
Lipids are essential to all living organisms, as they function as building blocks for membranes, for cell growth and diferentiation, for maintaining cellular homeostasis, as energy storage molecules, and in post-translational modifcation processes such as palmitoylation and myristoylation, which control membrane localisation and function of proteins [39,40].In light of recent studies, the FAS II pathway is indispensable during the late liver and mosquito stages of the life cycle of Plasmodium.However, environmental stress leading to lipid starvation during in vitro growth or physiological stress has suggested lipid metabolic plasticity, leading to reliance on the de novo fatty acid synthesis pathway to produce necessary lipid components [6,[41][42][43].Malaria parasites obtain the required fatty acids from their host (vertebrate host or mosquito vector) or through de novo synthesis (FAS II pathway).Te multi-enzyme apicoplast-localised FAS II pathway (also called the dissociative pathway) is distinct from the mammalian FAS I pathway (also called the associative pathway), which takes place in the cytosol and is catalysed by a single giant multidomain enzyme called fatty acid synthase [40,44].
Te FAS II pathway employs six enzymes and the acyl carrier protein (PfACP), and the process can be partitioned into three stages: preparation, initiation, and elongation (Figure 2).Te frst phase involves the shuttle of phosphoenolpyruvate (PEP) into the apicoplast from the cytosol via two transporters known as plastidic phosphate transporters (pPTs) resident in the innermost and outermost apicoplast membranes.PEP is then converted to pyruvate via the activity of pyruvate kinase II, which is localised to the apicoplast [9,45].Te terminal step of the preparation phase involves pyruvate dehydrogenase (PDH), leading to the formation of acetyl-CoA and ATP and reducing the equivalents required for downstream processes [45] Ensuing is the initiation of the elongation step by β-ketoacyl-ACP synthase III (KAS III; FabH), which condenses malonyl-ACP with acetyl-ACP, leading to the formation of 3-oxoacyl-ACP.Next, 3-hydroxyacyl-ACP is synthesised following the reduction of 3-oxoacyl-ACP by OAR (3-oxoacyl-ACP reductase; FabG).3-hydroxyacyl-ACP is then dehydrated to form enoyl-ACP by β-hydroxyacyl-ACP dehydratase (HAD; FabZ), which is then reduced to a saturated acyl-ACP species catalysed by enoyl-ACP reductase (FabI).Te chain length of acyl-ACP is elongated by Claisen condensation with malonyl-CoA, albeit with a diferent enzyme (FabBF) this time, following subsequent actions of FabG, FabZ, and FabI, ultimately resulting in the synthesis of fatty acids, thus efectively completing the elongation cycle [40,45].
Tere are several hypotheses regarding the fate of fatty acids produced via the FAS II pathway, such as a source of the lipid moiety for the glycosylphosphatidylinositol (GPI) anchor required to anchor circumsporozoite protein and other sporozoite proteins to the parasite's plasma membrane, for sporoblast development.However, no study has elucidated the exact fate of FAS II pathway products beyond the apicoplast to date [42,46].Recently, Kloehn et al. suggested that the apicoplast-synthesised fatty acid and lysophosphatidic acid were precursors for the bulk lipid synthesis in the endoplasmic reticulum [47].Nevertheless, two pathways have been expounded as possible fates for the FAS II products within the apicoplast, including the synthesis of lipoate, a cofactor, and fatty acid precursors during membrane lipid synthesis [45].Lipoate synthesis within the apicoplast is especially important because it is required for its antioxidant properties during the intraerythrocytic stage as there is no apicoplast membrane-resident transporter of lipoate, even if it is scavenged from the host [9,48].By targeting the FAS II pathway, drugs can disrupt the synthesis of essential fatty acids necessary to maintain the integrity of the parasite's cell membranes and impede its ability to replicate and survive within host cells [47].Inhibiting this pathway is, therefore, a promising strategy for developing antimalarial drugs to combat the devastating efects of Plasmodium falciparum infection and truncate parasite migration from the liver to the blood asexual stage.

SUF Pathway.
Iron is essential to many life processes and acts as an inorganic cofactor in redox and nonredox catalytic processes, including haem and biotin synthesis, repair of DNA, transcriptional control, substrate binding, and nitrogen fxation [49][50][51][52].Despite their numerous functions in living cells, iron is toxic; as such, mechanisms have evolved to obtain, store, and mobilise iron in a less toxic and readily accessible form.Malaria parasites independently partition the sulphur mobilisation pathway for Fe-S cluster synthesis between the apicoplast and mitochondria [50].Iron and sulphur can coordinate in several forms to form rhombic [2Fe-2S] as found in ferredoxin and cubic [4Fe-4S] Fe-S clusters, or more complex mixed-metal clusters, including [Mo-7Fe-9S] clusters found in nitrogenase [51].Te genes coding for the SUF system are largely located in the nucleus (SufA, C, D, E, and S) and SufB, which is encoded in the apicoplast genome [49].A recent study was conducted on the indispensability of the FE-S cluster during the Malonyl-CoA is converted to malonyl-CoA: ACP by malonyl-CoA: ACP transacylase (FabD).β-Ketoacyl-ACP synthase III (FabH) catalyses the initiation of the elongation step that leads to the formation of a butyryl-acyl carrier protein (ACP), which is then elongated by condensation with malonyl-ACP to form acetoacetyl-ACP. Acetoacetyl-ACP is reduced to β-hydroxyacyl-ACP, which is then dehydrated to trans-2-acyl-ACP and ultimately to acyl-ACP.Tese steps are catalysed by β-ketoacyl-ACP synthase II (FabB/FabF), β-ketoacyl-ACP reductase (FabG), β-hydroxyacyl-ACP dehydratase (FabZ), and enoyl-ACP reductase (FabI), respectively.Te n + 2 depicts the condensation of two carbons into the nascent fatty acid chain.Repeated cycles of these steps lead to the synthesis of C 14 -ACP.mosquito stage of the lifecycle [49].Also, it has been shown to be relevant for apicoplast maintenance during the blood stage of infection, and disruption of SUF C, D, E, and S functions during the blood stage abrogates infection [50,53,54].
Te biosynthesis of the Fe-S cluster requires three different groups of enzymes that make up the scafold protein, including NIF (nitrogen fxation), ISC (iron-sulphur cluster formation), and SUF (sulphur mobilisation) pathways to assemble Fe-S clusters [55].Te ISC pathway provides Fe-S clusters to mitochondria and cytoplasm-resident proteins in Plasmodium falciparum.However, the SUF pathway is responsible for the Fe-S cluster biosynthesis in the apicoplast [51].Te biogenesis of the FE-S cluster is not a spontaneous process, as shown in Figure 3. Te initial step involves the transference and assembly of iron and sulphur onto a scaffold protein to form a Fe-S complex.Tis involves the activity of cysteine desulfurase (SufS), which mobilises sulphur from L-cysteine.Charan et al. showed that SufS works in a complex with SufE, which catalyses the speedy mobilisation of the sulphur from SufS to the scafold protein (SufBCD complex) [49].Tough the exact source of iron is still obscure, it is proposed that an iron-binding homolog YfhJ, in Plasmodium spp., is the iron donor instead of frataxin, the proposed iron donor in bacteria [51,52].Te Fe-S complex forms on the SufBCD complex following the liberation of sulphur from the SufSE complex alongside the presence of iron [51].
Te second step involves mobilising the nascent Fe-S cluster to a target apoprotein, which employs the activity of several accessory shuttle proteins, including the homodimeric A-type carrier SufA and NfU-like domain-containing protein (NfU).Hausig et al. reported that SufA is dispensable throughout the lifecycle of Plasmodium berghei, and Charan et al. independently reported NfU being the dominant Fe-S cluster transfer protein from the scafold protein complex in Plasmodium falciparum [49,50].SufA and NfU have now been reported to be dispensable during the blood stage, leading to conditions compatible with cell death without exogenous supplementation of mevalonate [35].Te SUF pathway is important to the malaria parasite as it provides the Fe-S cluster necessary for the activity of several proteins, including dihydrolipoyl dehydrogenase, glyoxalase I-like protein, ferredoxin, antioxidant protein, glutathione reductase, lipoic acid synthase (LipA), (dimethylallyl)adenosine tRNA methylthiotransferase, and also the methylerythritol phosphate (MEP) pathway enzymes ((E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) and (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (IspH)) [35,55].Te Fe-S synthetic pathway is important during the sexual stage of the parasite's development; hence, discovering new and potent inhibitors against this stage is important to arrest the parasite's lifecycle and reduce malaria transmission [56].

Methylerythritol Phosphate (MEP)/Rohmer Pathway.
Isoprenoids are a signifcant class of biomolecules derived from two 5-carbon isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), forming isoprene units, which can undergo modifcation including cyclisation, oxidation, reduction, and addition, resulting in a repertoire of more than 40,000 naturally found isoprenoids [7,57].Plasmodium spp.metabolise glyceraldehyde 3-phosphate and pyruvate as precursors to synthesise IPP and DMAPP through the MEP pathway in seven enzymatic steps, as depicted in Figure 4 [58].Reported evidence on glyceraldehyde 3-phosphate and pyruvate transporters resident in the apicoplast has strongly indicated the apicoplast as the site of isoprenoid biosynthesis [58,59].IPP and DMAPP are building blocks for the synthesis of isoprenoids, which are important in Plasmodium falciparum in the synthesis of sesquiterpenes and diterpenes for protein prenylation [60], vitamin E synthesis [61], sterols for membrane stability [57,62], carotenoids [57], ubiquinone as an electron acceptor during pyrimidine synthesis [57], and dolichols for protein modifcation processes including dolichylation, GPI anchors, and O-and N-linked glycosylation [57].During the MEP pathway, two ATPs and three NADPH molecules are required to convert glucose to the isoprenoid synthesis precursors [63].
Te mevalonate pathway was frst reported in 1958 as the classical biosynthetic route for precursors (IPP and DMAPP) of isoprenoid derivatives, and it was initially thought to be the single route to IPP and DMAPP synthesis until a published review by Lichen proposed the possibility of an alternative pathway [64].However, later studies reveal an alternate pathway in eubacteria and later apicomplexans that bypass mevalonate and employ glyceraldehyde 3-phosphate and pyruvate as starting materials instead of 3-hydroxy-3-methylglutaryl coenzyme A used by the mevalonate pathway [57,65].Of all the anabolic pathways that have been associated with the apicoplast, recent discoveries have suggested that the MEP pathway, hence, isoprenoid biosynthesis, is the primary function of the apicoplast during the blood stage of P. falciparum lifecycle [3,47].Furthermore, Wiley et al. reported that IPP and DMAPP are critical for the development of gametocytes and mosquito stages oocysts and sporozoites [7].More recently, a novel role of isoprenoid biosynthesis in apicoplast biogenesis via the activity of polyprenyl synthetase, which synthesises long-chain polyprenols necessary for maintaining apicoplast membrane integrity, was reported [66].

Exploiting the Apicoplast-Resident Processes as an Antimalarial Drug Target
Te prokaryotic origin of the apicoplast makes it vulnerable to antibiotics, which have been exploited against Plasmodium malaria [40].Te apicoplast houses pathways that are parallel to bacterial pathways, including the FAS II, MEP, and "housekeeping" pathways, which are distinct from analogous mammalian pathways; therefore, antibiotics, including ciprofoxacin, azithromycin, chloramphenicol, clindamycin, tetracycline, fuoroquinolones, aminocoumarin, and rifampicin, have been reported to target this pathway and, hence, have been employed to treat malaria [8,36,70].However, the kinetics of these drugs have made them unattractive in treating acute/complicated malaria due to a phenomenon known as delayed death, in which the introduction of the drug invokes cell death following subsequent merozoite egress and reinvasion of the erythrocyte.Hence, these drugs do not clear parasitaemia immediately except after the second asexual cycle following the introduction of the drug [70].In addition, repurposing antibiotics for malaria treatment can lead to prolonged use or overreliance, which increases the potential for antibiotic resistance, as observed for folate inhibitors [71][72][73].
3.1.Targeting the Apicoplast Housekeeping Processes.All the apicoplast-resident processes, including housekeeping processes such as DNA replication, transcription, and translation, can be potential antimalarial targets and have been exploited in drug discovery against malaria.Te DNA replication of the circular apicoplast genome requires the nuclear-encoded tetrameric DNA gyrase, an ATP-dependent type II topoisomerase that helps resolve topological problems during replication and transcription [28].Earlier studies reporting bacterial gyrase as a potent antibiotic target have propelled studies into the malaria parasite DNA gyrase as a novel antimalarial target.Several successes have been recorded in targeting gyrase and inhibiting parasite development, including fuoroquinolones (e.g., ciprofoxacin), purpurogallin (PPG), aminocoumarin (e.g., novobiocin), and clindamycin [28,36,74].Also, nuclear-encoded apicoplast DNA polymerase (apPOL) replicates and repairs the apicoplast genome.Recent studies have validated apPOL as a viable antimalarial drug target, with the Malaria Box compound MMV666123 showing promising results [75].Furthermore, nine small molecules (KU0263501, KU0036696, KU0260920, KU0007309, KU0241474, KU0177470, KU0261556, KU0001071, and KU0271653) have been reported to be potential inhibitors against apPOL [76].

Advances in Pharmacological and Pharmaceutical Sciences
Te apicoplast genome codes for RNA polymerase subunits, RpoB, RpoC1, and RpoC2, which make up the RNA polymerase required for DNA transcription to RNA and share homology with bacterial RNA polymerase [36].Inhibition of this transcription machinery blocks the transcription of the apicoplast genome, which codes for genes responsible for apicoplast maintenance, ultimately leading to cell death.Inhibitors, including rifampin, doxycycline, and spiramycin, have been implicated in abrogating transcription [8,36].
Most inhibitors targeting the apicoplast's housekeeping processes are antibiotics that exhibit slow-acting delayed death, characteristic of their use against malaria.Nevertheless, some antibiotics that act immediately are believed to have an of-target efect, resulting in the observed rapid clearance of parasitaemia [70].

Targeting the Apicoplast Metabolic
Processes.Te apicoplast provides an ideal microenvironment responsible for four anabolic processes such as the MEP pathway for the building blocks for isoprenoid biosynthesis, the Shemin pathway for the synthesis of haem, the SUF pathway for the Fe-S cluster biosynthesis, and the FAS II pathway for fatty acid synthesis.Tese pathways have presented excellent opportunities for developing antimalarials because they are critical at diferent time points in the parasite's lifecycle and distinct from similar pathways in their mammalian host.In contrast with inhibitors of the housekeeping processes that seem initially lackadaisical in action against malaria parasites, inhibitors against the nonhousekeeping processes act promptly to impinge parasite survival [85].Druggable targets, recent advances, and novel inhibitors targeting these nonhousekeeping processes are discussed as follows.

Inhibitors of the Fatty Acid Synthesis. Malaria parasites
were previously thought to depend on the host for their fatty acid requirements; however, following the discovery of the FAS II pathway for fatty acid synthesis in the apicoplast in 1998, several earlier research groups worked on developing drugs that impinge on this pathway.Genetic studies, however, elucidated the dispensable nature of the FAS II pathway during the blood stage of infection [45].Tis discovery attenuates previous claims of viable inhibitors targeting this pathway and suggests parasite growth inhibition via of-target action.Abrogation of the FAS II pathway is incompatible with parasite cell death during the blood stage.Still, it is critical during the liver stage as the FAS II pathway enzymes in this stage can be targeted as prophylaxis [9].Although it has faced challenges over the years in its validation as a druggable pathway resident in the apicoplast, promising successes have still been reported (Table 2).Te FAS II pathway can be grouped into preparatory, initiation, and elongation phases.Te preparatory phase involves pPT, pyruvate kinase II (PK2), and PDH.Te preparatory phase of the FAS II pathway involves the import of PEP and subsequent conversion to acetyl-CoA and ATP.It is required for the initiation stage of the pathway by employing PK2, PDH, and pPT [45].Tese agents of the preparatory stage present promising antimalarial targets.pPT is the only transporter of PEP into the apicoplast resident in the inner and outer membranes of the apicoplast membrane.Te inner and outer membrane pPTs have been reported to be critical to parasite growth during the blood, mosquito, and liver stages of the lifecycle, respectively [45].Acetyl-CoA carboxylase (ACC), the enzyme that catalyses the frst committed step of the FAS II pathway, is a known target of herbicides such as clodinafop, butroxydim, and cyclohexanedione (Figure 5) [9,40].
KAS III/FabH, the enzyme responsible for the Claisen condensation of malonyl-CoA ACP and acetyl-CoA ACP from β-ketoacyl-ACP is a target of thiolactomycin (Figure 5).Te earlier discovery of the inhibitory activity of thiolactomycin against E. coli KAS III prompted studies into their role against the analogous Plasmodium KAS III [9].Interestingly, thiolactomycin has been reported to act against PfKAS III, although with low activity [86].More importantly, thiolactomycin has greater activity against FabBF, which catalyses the Claisen condensation step during the later elongation step.Tiolactomycin acts as a competitive inhibitor of FabBF by actively competing for the active site with malonyl-ACP, and further studies have led to the discovery of thiolactomycin analogues with greater inhibitory activity [9].Cerulenin is another important inhibitor of FabBF that has been reported [87].It acts as an irreversible inhibitor by binding to the cysteine residue of the Cys/His/His catalytic triad of the FabBF enzyme [9].It can, however, bind to the type I FAS active site, which limits its specifcity as an antimalarial [88].Te next enzyme following FabH in the FAS II pathway, FabG, has been reported to be inhibited by a triclosan derivative hexachlorophene, which is very active in inhibiting parasite growth during the hepatocyte stage in vitro [40].FabZ, the enzyme responsible for the synthesis of enoyl-ACP from β-hydroxyacyl-ACP via a dehydration reaction, is inhibited by two competitive inhibitors, NAS-75, NAS-79, NAS-91, and NAS-21, in addition to (−)-catechin gallate, which prevents substrate: enzyme interaction by blocking the substrate-binding tunnel [9,40].
Several inhibitors have been identifed against FabI, including coumarin and triclosan, which act as uncompetitive inhibitors (Figure 5).Several advances have been made in modifying triclosan to be a more potent inhibitor, but progress has been slow, and there is still no derivative more potent than triclosan against RabI (reviewed in [9]).Flavonoids have also been reported to be active Responsible for replicating and repairing the apicoplast genome MMV666123, KU0263501, KU0036696, KU0260920, KU0007309, KU0241474, KU0177470, KU0261556, KU0001071, KU0271653 [75,76] Apicoplast ribosome RNA translation Tetracycline, chloramphenicol, fusidic acid, azithromycin [70,82] Aminoacyl-tRNA synthetases (aaRS) Aminoacyl-tRNA synthesis Phenylalanine analogue (e.g., ochratoxin A), halofuginone [17,84] Advances in Pharmacological and Pharmaceutical Sciences   .Recently, computational studies employing Petri net analysis on FabD, FabG, and FabF revealed that impinging their activities leads to deadlock in the FAS II pathway [98].Tis is important as similar studies could help unravel the complexities of metabolic networks and pinpoint key druggable enzymes for developing new antimalarial drugs.

Inhibitors
Targeting the MEP Pathway.Te methylerythritol phosphate pathway comprises seven enzymes that catalyse the conversion of glyceraldehyde 3-phosphate and pyruvate to the building blocks of isoprenoids (IPP and DMAPP).Te MEP pathway is responsible for the biosynthesis of isoprenoids, which play critical roles in the parasite's development [99].Humans biosynthesise isoprenoids via the mevalonate pathway, thereby making the MEP pathway a promising target for novel antimalarials as it is peculiar to the parasite and critical to its survival [57].Te seven MEP enzymes are potential drug targets, and the essential role of isoprenoid biosynthesis in the intraerythrocytic stage of the malaria parasite's lifecycle has prompted research to develop potent inhibitors capable of arresting parasite growth [58].Currently known inhibitors against this pathway are shown and discussed as follows (Figure 6 and Table 3).Te antibiotics, fosmidomycin (FSM) and FR900098, obtained from Streptomyces lavendulae and Streptomyces rubellomurinus, respectively, are popular inhibitors of IspC.Te inhibition of IspC efectively terminates the availability of downstream precursory metabolites for isoprenoid biosynthesis [58].Although FSM has poor drug-like properties, clinical trials have shown promising results when coadministered with other known antimalarials, including clindamycin and artesunate [100].A detailed review of the activity of fosmidomycin and its derivatives is discussed by Patrick and Wang [9,101].A new lipid kinase called prenol kinase (PfPolK) has been recently discovered.It is responsible for phosphorylating unphosphorylated isoprenoids (farnesol and geranylgeraniol) [102].Tis process helps the parasite survive in an environment where isoprenoids are limited, such as when de novo isoprenoid biosynthesis is inhibited by a drug like FSM, and the parasite can scavenge unphosphorylated isoprenoids from the host to supplement its needs in such limited conditions [102].Although PfPolK is not critical for parasite survival, inhibitors against it could be relevant in developing combination therapy with a novel mechanism of action that could have enhanced efcacy against malaria.Also, in silico studies revealed fve new compounds, Z-2, Z-3, Z-10, Z-13, and Z-14, and three FDA-approved drugs, aliskiren, ceftolozane, and ombitasvir, that target IspC and have been proposed to be potential antimalarial candidates [103].

Inhibitors Targeting the SUF and Shemin Pathway.
Recent studies have strengthened the SUF pathway as a promising antimalarial drug target, which is important during the blood and mosquito stages.Nonetheless, the identifcation of potent inhibitors against this process is still Advances in Pharmacological and Pharmaceutical Sciences a work in progress [49,50,55].Table 3 shows a study that reported moderate activity of D-cycloserine against SufS during the asexual blood stage [55].Given that P. falciparum SufS (PfSufS) is not yet fully characterized, it shares a high degree of protein sequence and active site similarity with Bacillus subtilis SufS (BsSufS).BsSufS was employed as a model system to elucidate D-cycloserine's reaction mechanism and probe for stronger SufS inhibitors [112].L-Cycloserine, an enantiomer of D-cycloserine, was reported to be a higher afnity inhibitor of SufS than D-cycloserine [112].
Research targeting the development of inhibitors against Shemin has not progressed into product development despite the druggable targets and earlier reports of potent inhibitors.Tis is because the Shemin pathway has been discarded as relevant during the clinically relevant phase, i.e., the blood stage of malaria infection; thus, the inhibitors probably showed positive inhibitory activity during this stage due to the of-target efect [70].

Conclusion
Plasmodium falciparum is responsible for most malariarelated deaths, developing an array of molecular machinery that mediates host-parasite interaction, tropism, and invasion of the human red blood cells [113,114].Over the last century, malaria eradication strategies and interventions have been fairly successful, but importantly, it has shown how resilient the malaria parasite can be, as it has co-evolved with each new intervention, and eradication is still a work in progress [115].Te fact that both the housekeeping and nonhousekeeping processes in the apicoplast are distinct from analogous mammalian processes them attractive druggable antimalarial targets.Maximising this potential to develop potent antimalarials has been met with limitations, including of-target activity of inhibitors [70], poor drug-like properties of potent inhibitors such as fosmidomycin [100], delayed death [116], drug resistance [117],  Ortho-bis-sulphonamide [108] IspG and IspH (MEP pathway) Catalyse the penultimate and fnal steps of the MEP pathway, resulting in IPP and DMAPP Alkyl phosphate [110,111] SufS (SUF pathway) Catalyses the mobilisation of the sulphur from SufS to the scafold protein (Suf BCD complex) D-Cycloserine L-cycloserine [55,112] 12 Advances in Pharmacological and Pharmaceutical Sciences and an increase in antibiotic resistance [71].Furthermore, the nuclear genome of Plasmodium falciparum is predicted to contain about 5-10% apicoplast targeting signal.However, 70% of these apicoplast gene products are of no known function [118,119].Te difculty of developing and implementing reliable screening assays that are adapted to apicoplast biology is one of the primary obstacles to identifying and verifying potential therapeutic targets in the apicoplast pathways.Furthermore, thorough functional characterization of potential drug targets to evaluate their precise role within the apicoplast can be difcult [84,120].Despite these limitations, progress has been made in elucidating the apicoplast biology that has played important roles in apicoplast-targeted drug discovery.Chemical rescue using IPP supplementation has been an important tool for studying apicoplast biology [121].Recent studies employing this tool have led to the discovery of autophagy-related proteins, ATG8 and ATG18, which have been implicated in apicoplast biogenesis, and caseinolytic proteases, ClpC and ClpP, needed for apicoplast segregation, the ATPbinding cassette protein ABCF1, and the membrane transporter DMT2, in addition to studying apicoplast activities in response to treatment responses [17,18,70,[122][123][124]. Furthermore, DNA aptamers are gaining increasing prominence as an alternative therapeutic option against malaria.Recent studies have employed aptamer technology to develop oligonucleotide aptamer-based synthetic antibodies targeting critical apicoplast proteins, in addition to its use as a biomarker for apicoplast-positive parasites and potentials in diagnostic and therapeutic purposes [125,126].In light of advancing technologies in bioinformatics, genomics, proteomics, structural biology, and medicine, there has been a signifcant increase in the identifcation of inhibitors targeting apicoplast-resident processes; more research is still required to elucidate better the apicoplast biology that will help in novel drug discovery and combination therapies against malaria.

Figure 3 :
Figure3: Te SUF pathway in Plasmodium apicoplast.Cysteine desulfurase (SufS) acts in complex with SufE to mobilise sulphur from L-cysteine to form the Fe-S cluster on the scafold protein (Suf BCD complex).Te Fe-S cluster formed is then transferred via a carrier protein to an apoprotein, ultimately forming a holoenzyme.

Figure 6 :
Figure 6: Structures of some MEP pathway inhibitors.

Table 1 :
Druggable housekeeping targets localised in the apicoplast, their function, and inhibitors.

Table 2 :
FAS II pathway druggable targets, their function, and inhibitors.