**2. Challenging the target: phospholipid and ergosterol biosynthesis**

The first metabolically stable analogs derived from lysophosphatidylcholine were synthesized in the late 1960s. Two decades later, Eibl and Unger synthesized the first alkyl phospholipids (APLs), also called miltefosine, first administrated by an intravenous route to treat systemic tumors [10]. However, the treatment failed, and miltefosine was evaluated against topical cutaneous metastases from breast cancer [11]. In the late 1990s, miltefosine was assessed *in vitro* against the *Leishmania* genus [12, 13] and in murine models infected with *L. donovani* and *L. infantum* [14]. More recently, miltefosine was also evaluated against murine models of cutaneous leishmaniasis, alone or in combination with paromomycin [15–17].

After the evidence of the excellent anti-*Leishmania* activity of miltefosine demonstrated *in vitro* and *in vivo*, clinical tests began to be carried out immediately. Thus, miltefosine was the first oral drug approved for the treatment of visceral leishmaniasis, and for several years it was used as the first choice for the treatment of visceral leishmaniasis (VL), mainly in India [13, 18].

Miltefosine was also evaluated in patients infected with cutaneous leishmaniasis in Colombia, Guatemala, and Brazil [19, 20]. The efficacy in this clinical manifestation was variable, depending on the species. For patients infected with *L. panamensis* in Colombia, the cure rate was 91%, while in Guatemala, the cure rate was 53% for infections with *L. braziliensis* and *L. mexicana* [19]. Furthermore, in Brazil, in patients infected with *L. braziliensis*, the cure rate was 75%, compared to 53% cure achieved with pentavalent antimonials [20].

Several studies have demonstrated that the primary target of miltefosine is the cell membranes, affecting cellular processes such as signal transduction, lipid metabolism, and calcium homeostasis [21]. The selectivity for the plasma membrane is related to its chemical structure formed by a polar choline head bound to a long non-polar hydrocarbon chain, which easily inserts into the lipid bilayer, presenting detergent properties that lead to cell lysis in high concentrations [10]. In *L. donovani* and *T. cruzi*, miltefosine inhibits the phosphatidylcholine biosynthesis pathway (Greenberg pathway), being more selective for the protozoan parasites than mammalian cells, where the main route for phospholipid synthesis is the Kennedy pathway [22, 23]. Miltefosine also interfered with the ergosterol biosynthesis and promoted a disturbance in GPI synthesis [21, 24–26], leading to membrane permeability and fluidity changes. In addition, miltefosine interfered with the host immune response by inducing the production of interferon γ cytokine, leading to a biased immune response towards Th1, which would be a beneficial outcome for immunosuppressed patients [26, 27]. About the ultrastructure of *Leishmania* treated with miltefosine (**Figure 3A**), some studies revealed that it induced several alterations, mainly observed in the mitochondrion, in the plasma membrane, an increase of autophagic structures and phenotypes related to cell cycle arrest and apoptosis-like cell death [26, 28, 29].

With the success of miltefosine, several groups worldwide began to study new chemical routes to synthesize ether phospholipid derivatives in searching for novel molecules more active and selective against *Leishmania* [30]. Our group studied a novel hybrid derivative called alkyl phosphocholine-dinitroaniline, which presented a potent effect against *L. amazonensis* at least around 15-times better than miltefosine [31].

Another essential metabolic route for *Leishmania* and other protozoan parasites is the biosynthesis of ergosterol (or 24-methyl sterols) [32]. *Leishmania* has in these cell membranes three major sterol components that are absent in mammalian cells, 5-dehydroepisterol, episterol, and ergosterol. In mammals, cholesterol is the principal sterol present in cell membranes. Thus, the differences between some steps and enzymes in the biosynthetic route of the protozoan parasites and mammalian host cells have been exploited as targets to develop novel drugs as candidates to chemotherapeutic agents [32].

At least 20 metabolic steps are necessary to synthesize ergosterol, and several enzymes participate in these reaction sequences [32–34]. Furthermore, several works have shown that multiple classes of compounds targeting 24-methyl sterol biosynthesis exhibit suitable anti-trypanosomatid activities *in vitro* (**Figure 3B, C**) and *in vivo* [33–39].

#### **Figure 3.**

*Ultrathin sections of* L. amazonensis *promastigotes treated with miltefosine and two ergosterol biosynthesis inhibitors. (A) 30 μM miltefosine for 72 h; (B) 3 μM ravuconazole for 48 h; (C) 1 μM posaconazole for 48 h. Mitochondrion was the organelle more affected in all treatments, presenting alterations in the cristae and kDNA structure (asterisk, thin arrow, and arrowheads) and mitochondrial swelling and disorganization of its ultrastructure. In panel C, several lipid bodies appeared after treatment. K, kinetoplast; LB, lipid body; M, mitochondrion; N, nucleus.*

*Use of Cell Biology to Identify Cellular Targets in Drug Development Process... DOI: http://dx.doi.org/10.5772/intechopen.101662*

More than 30 drugs have been studied in the last 30 years. These drugs are included in large classes of inhibitors, such as 1) Statins, which inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), also evaluated as cholesterol-lowering drugs; 2) Bisphosphonates that act in the enzyme farnesyl pyrophosphate synthase and inhibit the isoprenoid pathway, mainly used to treat hypercalcemia; 3) Quinuclidines and zaragozic acid, developed to inhibit the squalene synthase, the enzyme that catalyzes the first committed step in the sterol biosynthesis pathway. This class of drugs was developed as an alternative to statin use because they do not inhibit the synthesis of the isoprenoids. 4) Allylamines, which include the known antifungal terbinafine that inhibits the squalene epoxidase; 5) Azoles, which are essential medicines to treat many fungal diseases and inhibit the C14α-demethylase. Several azoles were developed, always trying to find new tolerate and efficacy drugs, also searching to novel molecules to solve the problems with antifungal resistance; finally, 6) Azasterols, which inhibit the enzyme Δ24,25 sterol methyltransferase absent in mammalian cells, one enzyme that catalyzes the methylation of steroid nucleus of sterols, producing 24-methyl sterols, essential for *Leishmania*, *T. cruzi* and fungi (Reviewed in [32]).

In summary, several works have pointed to the importance of looking for the biochemical properties of each enzyme involved in the pathway and its relevance as an essential target for the parasite viability; this feature characterizes the enzyme as a promising target for the development of potential chemotherapeutic candidates for the treatment of leishmaniasis.
