**3. Challenging the target: histone deacetylases**

Histone deacetylase (HDACs) inhibitors are a relatively new class of potential agents in treating neurodegenerative diseases, various types of cancer, and parasitic infections. HDACs have broad importance in the cellular environment. They regulate histone and non-histone proteins affecting the cell cycle, energy metabolism, and inducing cell death. Some HDAC inhibitors were already approved by the FDA (Food and Drug Administration) to treat lymphoma and myeloma, such as vorinostat, romidepsin, belinostat, and panobinostat, in combination with bortezomib and dexamethasone [40]. Given the results obtained *in vitro* and *in vivo* in several disease models, the advancement of clinical trials in tumors, and the transposition of drugs as an old ally in the treatment of leishmaniasis, HDAC inhibitors are a promising approach in the understanding of cell biology of the parasite, especially concerning its chemotherapy.

There are 18 histone deacetylases in humans, which can be grouped according to cell location and the molecule used as a cofactor for its enzymatic action. These HDACs are divided into 1) zinc-dependent HDACs, also called "classical" histone deacetylases; and 2) nicotinamide and adenine dinucleotide [NAD+ ]-dependent HDACs. The first one comprises class I (HDACs 1–3, 8), IIa (HDACs 4, 5, 7, and 9), IIb (HDACs 6 and 10), and IV (HDAC 11). While the second one, the NAD<sup>+</sup> -HDAC, belongs to class III and is also known as sirtuins (SIRT 1–7). HDACs are still poorly understood and characterized in *Leishmania*. So far, four Zn2+-dependent histones deacetylases and three NAD<sup>+</sup> -dependent histones deacetylases were discovered in the parasite [41, 42]. Although four homologs of classical HDACs were identified, none was functionally characterized. Among the information in the literature, a *L. major* HDAC (gene LmjF21.0680) was shown to be expressed during the differentiation of promastigotes to amastigotes, with a possible role in chromatin structure and impacts on gene transcription [43]. Furthermore, Prasanna et al. [44] managed to isolate, express, and purify an *L. donovani* histone deacetylase

(LD\_HDAC), with less than 40% identity with class I human HDACs. Information about classical HDACs in the *Leishmania* genus is still very early and deserves further development.

Unlike classical HDACs, there are several studies about NAD<sup>+</sup> -dependent HDACs in the *Leishmania* genus. Three sirtuins were already found in *Leishmania*, SIR2RP1, SIR2RP2, and SIR2RP3 [45]. These sirtuins were not found in the nucleus, as described for *Saccharomyces cerevisiae* [45]. SIR2RP1 is expressed from a single copy gene in *L. amazonensis* (LaSIR2RP1), resulting in a monomeric protein with NAD+ -dependent deacetylase action immunodetected in its glycosylated form [46]. LaSIR2RP1 has dispersed localization in the cytosol or cytoplasmic granules [47] and is secreted in lesions derived from intracellular amastigotes [48]. In addition, the *L. donovani* SIR2RP1 was 46% similar to the human SIRT2 [49]. In *L. infantum* and *L. major*, the SIR2RP1 proteins present two functional sites for NAD+ dependent deacetylation activity and ADP-ribosyl transferase activity [49].

In *L. major* SIR2RP1, removing the acetyl group of lysine 40 from α-tubulin was demonstrated *in vitro* and *in vivo* [50]. However, in these parasites, the ribosylation function of α-tubulin ADP was also shown, resulting in its depolymerization or even inhibiting its assembly [51]. Thus, sirtuins in *Leishmania* have demonstrated an essential role in cytoskeleton dynamics and may have significant implications for remodeling the parasite's morphology and its interaction with the host cell. LmSIR2RP1 also showed a close relationship with the HSP83 protein, an orthologous chaperone of the human HSP90 chaperone, although the intracellular levels of LmSIR2RP1 do not influence the acetylation status of HSP83 [52]. The use of geldanamycin, an HSP90 inhibitor, induced alterations in the cytodifferentiation of promastigotes to intracellular amastigotes [53]; the same was observed for protozoa overexpressing or knockout for the LmSIR2RP1 gene [52]. This study confirms that the SIR2RP1/HSP83 interaction may play an essential role in the differentiation of the parasite.

The work in [54] demonstrated that *L. major* sirtuins may be related to the success of infection through interaction with macrophage surface proteins and, therefore, play a regulatory role in immune responses [55]. This work also showed the capacity of *L. major* sirtuins to trigger the effector response of B cells, promoting a robust humoral response with the secretion of specific antibodies, such as IgG1 and IgG2a [55]. Meanwhile, the overexpression of *L. major* sirtuin, also observed for *L. infantum* sirtuin [48], revealed that it would be involved in the proliferation rate, besides participating in the regulation of death factors, thus preventing death by apoptosis.

The SIR2RP2 present in *L. infantum* mitochondrion is related to the growth rates of promastigote forms with a direct relationship with NAD<sup>+</sup> homeostasis [56]. In 2017, a study revealed that the *L. donovani* SIR2RP2 also has a mitochondrial location [49]. The deletion of this gene led to a reduction in the proliferation rate, further resulting in the interruption of the cell cycle in the G2/M phase. Furthermore, the deletion of LdSIR2RP2 resulted in greater susceptibility of the parasite to commercially available sirtuin inhibitors and a reduction in the mitochondrial membrane potential, resulting in a low concentration of ATP in the mitochondrion. Interestingly, in this SIR2RP2 knockout *Leishmania*, there was an intensification of glycolysis that could be an attempt to compensate for the disbalance of the mitochondrial metabolism. LdSIR2RP2 showed NAD<sup>+</sup> -dependent ADP-ribosyl transferase activity with 39% similarity to SIRT4 from humans [49].

The third sirtuin, SIR2RP3, still has few descriptions in the literature. The *L. donovani* SIR2RP3 was 37% similar to the human SIRT5 [49], so that molecular coupling assays, using known inhibitors and SIR2RP3, revealed a strong analogy with SIRT5, which refers to the form of interaction with inhibitors. However, the *Use of Cell Biology to Identify Cellular Targets in Drug Development Process... DOI: http://dx.doi.org/10.5772/intechopen.101662*

interaction of these inhibitors with SIR2RP3 also demonstrated significant molecular differences compared to SIRT5 in humans, which could act as selective targets for the treatment of leishmaniasis [57].

A recent study with a histone deacetylase inhibitor *in L. amazonensis* revealed the sirtuins' potential to develop novel molecules with anti-*Leishmania* activity [58]. Furthermore, this study from our group demonstrated the potent inhibition of parasite proliferation that is probably related to essential functions for HDACs in *Leishmania*, which include the control of the cell cycle and the induction of cell death [58]. Other effects already observed with HDAC inhibitors are different levels of chromatin compaction, increased number of lipid bodies randomly distributed throughout the cytosol, increased production of reactive oxygen species, changes in *Leishmania* morphology, and increased expression of acetylated α-tubulin (**Figure 4**) [58].

However, the parasite's ability to modulate the histone deacetylases of the mammalian host to establish the infection has already been observed. For example, the upregulation of the macrophage HDAC1 was observed during infection with *L. amazonensis*, resulting in deacetylation of the histone tail of the gene's promoter region responsible for producing nitric oxide. This deacetylation prevents the access of transcription factors, which culminates in the repression of nitric oxide production, which allows the establishment of intracellular amastigote forms in the parasitophorous vacuole [59].

Besides, HDAC inhibitors have also been used in combination therapy to treat antimony-resistant *L. donovani* infection, where the upregulation of the multiresistant protein depends on IL-10 production. Thus, the imipramine antidepressant positively regulates HDAC11, inhibiting the acetylation of the IL-10 promoter, which leads to a decrease in its production [60]. This imipramine-mediated lowering of the IL-10 level reduces MDR-1 expression and aids in the elimination of the parasite.

Thus, histone deacetylase inhibitors belong to a class of compounds with potential application to develop novel molecules with anti-*Leishmania* activity and for the treatment of leishmaniasis, alone or in combination with other medications already established. Furthermore, the World Health Organization (WHO) has already recommended the therapeutic combination to reduce the doses and toxicity and develop a more effective and safe treatment. Finally, HDAC inhibitors seem to be an exciting tool for a better understanding of the Cell Biology of *Leishmania* and enabling the knowledge of new routes for the development of novel drug candidates.

#### **Figure 4.**

*Ultrathin sections of* L. amazonensis *promastigotes treated with HDAC inhibitors for 48 h. (A) 1.5 μM NIH119; (B) 1 μM NIH119; (C) 15 μM tubastina A. NIH119 and tubastatin a induced different ultrastructural alterations such as 1) increase in the number of lipid bodies, some of them presenting different morphologies (A, B); 2) presence of membrane protrusions (A, B – Arrow); 3) alterations in the kinetoplast (A); 4) decondensation of chromatin (B, C); and, 5) changes in mitochondrial ultrastructure. FP, flagellar pocket; K, kinetoplast; LB, lipid body; M, mitochondrion; N, nucleus.*
