**2.5.3 Apicoplast-based targets**

330 Malaria Parasites

(Wegscheid-Gerlach et al., 2010). The cystein and aspartyl endoproteases involved in the essential pathway of haemoglobin degradation now known as falcipain-2, 2' and -3 and plasmepsin-I, -II, -III (or HAP, for histo-aspartyl protease) and –IV have first emerged as promising protease targets. Intensive research focused on these enzymes during the last decades has yielded inhibitors active down to the nM levels on the native or recombinant enzymes, inhibiting parasite growth in culture from the nM to µM levels and capable to cure *Plasmodium*-infected mice (McKerrow et al., 2008, Rosenthal, 2010). Gene disruption studies however clearly indicated that all of these enzymes besides falcipain-3 are highly redundant. In fact, falcipain-3 is the only for which gene disruption did not yield viable blood-stage parasites (Liu et al., 2006, Sijwali et al., 2006). The development of inhibitors targeting these enzymes but displaying improved properties in term of activity, selectivity, safety to the host nonetheless pertains, facilitated by their availability under recombinant active form, allowing both high-throughput screenings and experimental determination of their 3D-structures, including for the most recently described plasmepsin I (Bhaumik et al., 2011). A strong argument for maintaining such a development against these haemoglobinases is linked to the proposal to use combined protease inhibitor therapy based on the observation that cystein and aspartyl inhibitors demonstrated synergistic antimalarial

Fig. 2. Mechanisms of haemoglobin degradation and haeme detoxification in *Plasmodium* 

In parallel, the last decade has seen the emergence as promising targets of several new parasite proteases, among the ~100 that are encoded in the *P. falciparum* genome (Wu et al., 2003). Some are also involved in haemoglobin breakdown such as the cystein protease DPAP1 (Klemba et al., 2004), the metallo-endopeptidase falcilysin displaying however a

*falciparum*-infected red blood cell, after (Mambu & Grellier, 2008)

effects (Semenov et al., 1998).

The apicoplast is a vestigial, non-phostosynthetic, plastid-like organelle inherited from the prokaryotic world by secondary endosymbiosis and found in most apicomplexan parasites including *Plasmodium* that is essential to their biology. In *P. falciparum*, it maintains a 35-kb circular genome and several particular biochemical pathways that are present in bacteria and plants but are absent in humans, thus providing many attractive targets that are extensively investigated for drug development. These pathways include the type II fatty acid biosynthesis pathway, which involves 6 distinct enzymes in *Plasmodium* while in human the type I fatty acid biosynthesis pathway involves a multifunctional enzyme, the 1 deoxy D xylulose 5 phosphate (DOXP) isoprenoid biosynthesis pathway that is mevalonateindependent in the malaria parasite contrary to humans, and apicoplast replication, transcription and translation which involve enzymes of bacterial origins (Dahl & Rosenthal, 2008, Goodman & McFadden, 2007, Grawert et al., 2011, Jayabalasingham et al., 2010). Pioneering works led to the emergence of promising antimalarials such as triclosan (believed to target the NADH-dependent enoyl ACP reductase or FabI enzyme), thiolactomycin (targeting FabH and FabB enzymes) and fosmidomycin (targeting the DOXP reductoisomerase) to name the main ones. Also, antibiotics such as tetracyclines and clindamycin, targeting prokaryotic protein synthesis, or quinolone antibiotics and rifampicin, targeting the prokaryotic DNA and RNA machinery, were shown to inhibit *Plasmodium* growth in culture and *in vivo* (Seeber & Soldati-Favre, 2010).

These results stimulated efforts both to genetically validate these putative targets and to improve inhibitor discovery, by solving 3D structures and producing enzymes under recombinant active forms amenable to medium/high throughput screenings (Freundlich et al., 2007, Sato, 2011). In 2008, the genetic inactivation of FabI in *P. falciparum* and in *P. berghei* produced parasite blood stages that were growing normally and were still affected by triclosan (Yu et al., 2008). The FabI gene deletion, on the other hand, blocked the development of the parasite in the liver. Not long later, the genetic inactivation in murine

Advances in Antimalarial Drug Evaluation and New Targets for Antimalarials 333

of kinases may be performed by classical gene deletion in *P. falciparum* or rodent *Plasmodium*, but conditional expression systems have also been used as for example using the destabilizing domain and Shield ligand to show the involvement of PfCDPK5 in

Transporters are integral transmembrane proteins that enable the movement of solutes across biological membranes. In *Plasmodium,* they are currently considered as highly promising targets, due to their involvement, as carrier proteins, channels or pumps, in the movement of nutrients, metabolites and ions into and out of the parasite as well as between subcellular compartments within the parasite. They thus regulate essential nutrient uptake into the parasite, homeostasis and disposal of toxic wastes. Host cell transporters modified by the presence of the parasite may also be involved in these biological functions. Of note, malarial transmembrane proteins such as PfCRT, Pgh1, PfNHE1 and possibly PfATP6 are

The recent publication of the theoretical permeome of *P. falciparum* indicated that at least 2.5% of the parasite genome encode transporters, channels and pumps (Martin et al., 2009). Experimental localizations, that have been achieved by using either specific antibodies or epitope-tagged or fluorescent recombinant fusion proteins transfected into parasites, concern few proteins of the parasite surface (PfHT1 or the hexose transporter, PfENT1 or PfNT1, PfMRP, PfAQP), the mitochondrial membrane (ATP/ADP transporter), or the foodvacuole membrane (PfCRT, Pgh1, V-type H+ ATPase and ATP-driven H+ pump). Substrate specificities are also often ill-defined. Even if these could be predicted by orthology for about half of the candidates, experimental validations are still necessary to confirm or discover which they are. Extensive experimental work therefore needs to be done to further exploit such a rich source of potential targets (Staines et al., 2010). Some promising results nonetheless emerged in this field. First, the challenging functional expression of recombinant proteins, which is necessary for the biochemical testing of substrates and inhibitors, has been achieved successfully in *Xenopus laevis* oocytes for several *Plasmodium* transporters. Alternative heterologous systems have also been used such as yeast, *Dictyostelium discoideum* and mammalian cells (Martin et al., 2009). In some cases, cell-free transporter assay systems are used. Recently, recombinant expression of codon-optimized PfHT1 in yeast was performed to permit high-throughput screening of inhibitors (Blume et al., 2010). The P-type ATPases, PfATP4 and PfATP6, and the drug-resistance involved PfCRT and Pgh1 have also been expressed in such heterologous system, opening the road for functional studies (Martin et al., 2009). The number of known 3D-structures remains however extremely low, limited to PfAQP although a model has been proposed for PfHT1

Transfection methods have been also critical in this field, in particular to assess the role of PfCRT and Pgh1 in drug resistance, by transfecting *P. falciparum* resistant alleles into sensitive parasites and vice-versa, or by transfecting these alleles in various heterologous expression systems such as yeast (Martin et al., 2009). Gene deletion studies have been also undertaken, but so far for a limited number of candidates. While several genes could be deleted with none or limited impact on parasite asexual growth such as those encoding PfENT1, PfMRP, the *P. berghei* orthologue of the PfAQP, PfKch1 (PfK1) and PfACα, the

merozoite egress (Dvorin et al., 2010).

involved in drug-resistance mechanisms.

based on the structure of *E. coli* permease (Staines et al., 2010).

**2.5.5 Transporters** 

models of other enzymes belonging to the FasII metabolism such as FabB/F confirmed that the FasII pathway was dispensable through the entire parasite development to the exception of the hepatic-blood stage transition phase. These results raised the question of the nature of the parasitic target of triclosan in blood stages. Recently five laboratories reported their failed attempts to inhibit rodent *P. berghei* and *P. chabaudi* proliferation in mice by using similar doses of triclosan (Baschong et al., 2011).

Conversely, recent works focusing on the DOXP non-mevalonate isoprenoid pathway confirmed it appears as a relevant target for antimalarial drug development. Key results include the recent failure to delete the DOXP reductoisomerase gene (Odom & Van Voorhis, 2009). The DOXP metabolic pathway is active in all erythrocytic stages but problems with fosmidomycin, which has long been known to inhibit *Plasmodium* growth *in vitro* and *in*  mice (Jomaa et al., 1999), emerged. Due to its short half life that may lead to parasite relapses and/or facilitate selection of resistant parasites, analogues with improved half-lives are actively looked for against this enzyme and the other enzymes of this metabolic pathway (Seeber & Soldati-Favre, 2010). The recent discovery that fosmidomycin is little effective against *Theileria*, *Eimeria* and *Toxoplasma* also raised the question of its bioavailability (Seeber & Soldati-Favre, 2010). This led to the proposal that fosmidomycin and its more active derivative FR900098, both active against *Plasmodium* erythrocytic but not hepatocytic stages, would enter the parasite through new permeation pathways called NPPs (Baumeister et al., 2011). Recently also two triose-phosphate transporters present on the apicoplast membranes have been characterized, PfiTPT/PfAPT1 and PfoTPT/PfATP2, and are being considered as putative targets since they are believed to import from the cytosol into the apicoplast key metabolites to fuel the DOXP pathway (Lim & McFadden, 2010).

#### **2.5.4 Kinases**

Malarial kinases but also host cell kinases involved in host-parasite interactions are also currently considered as promising targets (Doerig et al., 2010). The *P. falciparum* theroretical kinome was predicted to comprise 85 to 99 enzymes, depending on the stringency of the computational methods used to identify them, and phylogenetic studies have revealed interesting differences with the human kinome that may be exploited for kinase-targeted drug discovery (Leroy & Doerig, 2008). For example, *P. falciparum* possesses kinases and kinase families such as calcium-dependent protein kinases (CDPK) and the apicomplexan specific FIKK kinase families, that are absent in humans. Important advances in this field have been provided by the fact that many *P. falciparum* kinases have been successfully produced as recombinant active enzymes, allowing medium/high troughput studies, many structures are known or can be deduced by modelling (see http://www.thesgc.com) and several chemical libraries, developed for other pathologies such as cancer may be screened on *P. falciparum* putative targets and parasites. For example, PfCDPK1 that has no ortholog in humans was produced as an active recombinant enzyme and used to screen a library of ~20,000 molecules developed against kinases, from which a purine derivative called purfalcamine active to the low nM range on the enzyme was isolated (Kato et al., 2008). This molecule, able to prevent the *P. falciparum* development in culture had however a low *in vivo* activity on *P. berghei*, possibly due to poor pharmacokinetic parameters. Importantly, not only protein kinases but kinases phosphorylating other substrates such as choline kinase and pantothenate kinases could also be targeted (Leroy & Doerig, 2008). Genetic validation of kinases may be performed by classical gene deletion in *P. falciparum* or rodent *Plasmodium*, but conditional expression systems have also been used as for example using the destabilizing domain and Shield ligand to show the involvement of PfCDPK5 in merozoite egress (Dvorin et al., 2010).
