**5. 5-Aminolevulinic Acid (ALA): A new antimalarial candidate targeting the mitochondrion**

ALA is a precursor used in the biosynthesis of tetrapyrroles such as chlorophyll and heme. The heme is an iron-containing complex macrocycle that plays a fundamental role in several cellular processes, including oxygen transport and storage, mitochondrial respiratory chain, and detoxification [84]. Generally, in mammalian cells, heme biosynthesis begins with ALA formation by ALA synthase in the mitochondria from glycine and succinyl-CoA [85]. The next four steps and three final steps occur in the cytosol and mitochondria, respectively. In cancer cells, the uptake of a high concentration of ALA results in elevated levels of its metabolites, particularly protoporphyrin IX (PPIX), due to insufficient activity of ferrochelatase [86]. The PPIX accumulates in the mitochondria and consequently acts as a photosensitizer releasing singlet oxygen and other reactive oxygen species (ROS), resulting in induction of cell death in cancer. ALA therefore has been applied to the development of photodynamic diagnosis and photodynamic therapy (PDT) of various cancers [87, 88].

Recently, all the enzymes of *de novo* heme-biosynthetic pathway have been characterized in the human malaria parasite, *P. falciparum* [89-91]. In contrast to the mammalian enzymes of heme biosynthesis, the parasite enzymes have unique localizations (Figure 5). The first enzyme, ALA synthase, and the final two enzymes, protoporphyrinogen IX oxidase and ferrochelatase (FC), localize to the mitochondrion. The enzymes that catalyze the intermediate three steps—ALA dehydratase, porphobilinogen deaminase, and uroporphyrinogen decar‐ boxylase (UROD)—localize to the apicoplast, a nonphotosynthetic plastid. The next enzyme, coproporphyrinogen III oxidase, is cytosolic. In addition, the catalytic efficiency of these enzymes of the erythrocytic stages differs from that of mammalian enzymes: the enzymes localizing to the apicoplast have very low catalytic efficacy [92]. Altogether, the properties of the heme-biosynthetic pathway are remarkably different between malaria parasites and their hosts. Hence, the heme-biosynthetic pathway has been recognized as a novel chemotherapeu‐ tic target in *Plasmodium*. Smith and Kain attempted PDT for human malaria parasites by adding ALA to an *in vitro* culture [93]. The growth of *Plasmodium* was completely inhibited by 0.2 mM ALA, followed by exposure to white light or by a higher concentration (2 mM) of ALA alone without light exposure. This use of PDT is, unfortunately, clinically unrealistic because white light cannot illuminate the inside of a malaria-infected patient's body and the concentration of 2 mM is extremely high to apply clinically.

**Figure 5.** Heme biosynthesis of malaria parasites. The enzymes in the pathway are localized in the mitochondrion, api‐ coplast, and cytosol [89-91].

Our recent study resolved this issue: in the presence of ferrous ion, ALA efficiently inhibited the *in vitro* growth of *Plasmodium* even without light exposure [94]. Because there was a previous report on protection from malaria by elevated zinc protoporphyrin, which binds to heme crystals to inhibit further crystallization to form hemozoin [95], we first investigated effects of metal ions on growth inhibition by ALA using an *in vitro* culture system of *P. falciparum*. Our results showed that treatment with 10 μM sodium ferrous citrate (SFC) and 0.2 mM ALA increased the growth inhibition to more than 50% when compared with that of 0.2 mM ALA alone. Notably, no other metal ions (e.g., zinc, lead, and copper) had such a syner‐ gistic effect, indicating that only ferrous compounds are synergistic with ALA.

Next, to determine heme intermediate, we analyzed the cell extract of the parasite using HPLC. The extract contained three major intermediates: coproporphyrin I, coproporphyrin III (CPIII), and PPIX. Unlike in cancer cells, CPIII was majorly accumulated in the apicoplast. Although its contribution to the parasite growth inhibition remains unknown, we believe that these differences are due to the complicated heme-biosynthetic pathway (Figure 5) and life cycle of *Plasmodium* (Figure 1). Moreover, PPIX, as is the case with cancer cells, accumulated mainly in the mitochondrion. In PDT of cancer cells, PPIX acts as a photosensitizer releasing ROS, resulting in extensive cellular damage and cell death [96]. This suggests that PPIX accumulated in the parasite mitochondria is a factor contributing to the inhibition of parasite growth. Thus, the parasite heme-biosynthetic pathway in the mitochondrion and apicoplast may be a potential target of an antimalarial drug.

Recently, to confirm the efficacy of the combination of ALA and SFC (ALA/SFC) in treating malaria using an animal model, we performed a preclinical drug evaluation of orally admin‐ istered ALA/SFC for the treatment of mice infected with the malaria parasite. ALA/SFC cured 50% of the Py17XL-infected mice, and the cured mice showed long-lasting humoral immune responses to the same parasite strain and protection from homologous malarial infections [97]. ALA can be safe compound because a phase I clinical study has been successfully completed. Considering the safety and mild antimalarial activities of ALA/SFC, a combination with an available antimalarial drug, such as artemisinin or chloroquine, would be applicable for the treatment of malaria.
