# The epigenetic landscape of kinetoplastid parasites: From histone post-translational modifications to emerging therapeutic strategies

Inès Jacquet, Romain Paoli-Lombardo, Patrice Vanelle, Nicolas Primas

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Inès Jacquet, Romain Paoli-Lombardo, Patrice Vanelle, Nicolas Primas. The epigenetic landscape of kinetoplastid parasites: From histone post-translational modifications to emerging therapeutic strategies. Bioorganic and Medicinal Chemistry, 2025, 131, pp.118377. 10.1016/j.bmc.2025.118377. hal-05493565

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## Review article

# The epigenetic landscape of kinetoplastid parasites: From histone post-translational modifications to emerging therapeutic strategies

Inès Jacquet<sup>a</sup>, Romain Paoli-Lombardo<sup>a,b</sup>, Patrice Vanelle<sup>a,b,*</sup>, Nicolas Primas<sup>a,b ID,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

Epigenetics

Histone post-translational modifications

Histone modifiers

Bromodomain

Antikinetoplastid

Inhibitors

## ABSTRACT

Kinetoplastids are parasites which cause various neglected tropical diseases. A hallmark feature of their genetic composition is the presence of polycistronic transcription, a phenomenon that involves the transcription of multiple genes into a single mRNA molecule, along with unconventional modes of gene regulation. In these organisms, histone variants and post-translational modifications play pivotal roles in modulating chromatin structure and transcriptional activity. This review provides a comprehensive overview of histone variants and post-translational modifications identified across *Leishmania* spp., *Trypanosoma cruzi*, and *Trypanosoma brucei*, detailing both the diversity of modifications and their known functional roles. This review also focuses on the writers, erasers, and readers proteins, including available three-dimensional structural data, to better understand their contribution to chromatin regulation, cell cycle progression, and parasite adaptation. Concurrently, this review offers a synopsis of therapeutic endeavors that have targeted these pathways, emphasizing the outcomes of *in silico*, *in vitro* and *in vivo* studies. This comprehensive review underscores the potential of unraveling kinetoplastid epigenetic mechanisms as a promising avenue for developing innovative treatments against these major human pathogens.

## Contents

<table><tr><td>1.</td><td>Introduction</td><td>2</td></tr><tr><td></td><td>1.1. Presentation of the kinetoplastid</td><td>2</td></tr><tr><td></td><td>1.2. The kinetoplastid's genomic organization</td><td>2</td></tr><tr><td>2.</td><td>Post-translational modifications of histones</td><td>3</td></tr><tr><td></td><td>2.1. Post-translational modifications in Leishmania spp.</td><td>3</td></tr><tr><td></td><td>2.2. Post-translational modifications in T. cruzi</td><td>3</td></tr><tr><td></td><td>2.3. Post-translational modifications in Trypanosoma brucei</td><td>3</td></tr><tr><td>3.</td><td>Writers and Erasers of histone post-translational modifications</td><td>5</td></tr><tr><td></td><td>3.1. Histone acetyltransferases</td><td>5</td></tr><tr><td></td><td>3.2. Histone deacetylases</td><td>7</td></tr><tr><td></td><td>3.3. Histone methyltransferases</td><td>7</td></tr><tr><td>4.</td><td>Post-translational modifications readers</td><td>8</td></tr><tr><td></td><td>4.1. Acetylated lysine readers: Bromodomains</td><td>8</td></tr><tr><td></td><td>4.2. Other readers</td><td>8</td></tr><tr><td>5.</td><td>Therapeutics strategies</td><td>9</td></tr><tr><td></td><td>5.1. Inhibition of HDAC</td><td>9</td></tr><tr><td></td><td>5.1.1. Hydroxamic acid derivatives</td><td>10</td></tr><tr><td></td><td>5.1.2. Benzamides</td><td>12</td></tr><tr><td></td><td>5.1.3. Macrocycles</td><td>12</td></tr><tr><td></td><td>5.1.4. Other HDACi</td><td>13</td></tr></table>

<footnote><sup>a</sup>Aix Marseille Univ, CNRS, ICR UMR 7273, Team Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin, CS30064, CEDEX 05, 13385 Marseille, France</footnote>

<footnote><sup>b</sup>Service Central de la Qualité et de l'Information Pharmaceutiques, Hôpital de la Conception, AP-HM, 13005 Marseille, France</footnote>

<footnote>* Corresponding authors at: Aix Marseille Univ, CNRS, ICR UMR 7273, Team Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin, CS30064, CEDEX 05, 13385 Marseille, France.</footnote>

<footnote>E-mail addresses: patrice.vanelle@univ-amu.fr (P. Vanelle), nicolas.primas@univ-amu.fr (N. Primas).</footnote>
<table><tr><td>5.2.</td><td>Inhibition of bromodomain factors</td><td>15</td></tr><tr><td>5.3.</td><td>Other therapeutic strategies</td><td>16</td></tr><tr><td>6.</td><td>Therapeutic perspectives</td><td>16</td></tr><tr><td>7.</td><td>Conclusion</td><td>17</td></tr><tr><td></td><td>CRediT authorship contribution statement</td><td>18</td></tr><tr><td></td><td>Declaration of competing interest</td><td>18</td></tr><tr><td></td><td>Appendix A. Supplementary data</td><td>18</td></tr><tr><td></td><td>Data availability</td><td>18</td></tr><tr><td></td><td>References</td><td>18</td></tr></table>

# 1. Introduction

Neglected Tropical Diseases (NTDs) represent a heterogeneous group of 20 diseases, most of which are endemic to tropical regions and disproportionately impact the most impoverished populations in developing countries.¹ These conditions are caused by a variety of pathogens, including bacteria, viruses, protozoan parasites, and helminths. NTDs have a deleterious effect not only on health, but also on the social and economic environment of sufferers. Among the NTDs, three parasitic diseases are caused by flagellate protozoa of the kinetoplastid order: leishmaniasis, human African trypanosomiasis or sleeping sickness, and human American trypanosomiasis or Chagas disease. These diseases collectively affect approximately 20 million people annually, resulting in nearly 50,000 deaths reported each year according to the World Health Organization.

## 1.1. Presentation of the kinetoplastid

Leishmania spp., a dixenous parasite, is transmitted by female sand-flies. In its flagellated metacyclic and haptomonad promastigote form, it infects the mammalian host during the blood meal.² Following internalization by the host's macrophages, the parasite transforms into an intracellular amastigote stage, which is responsible for the manifestation of diverse clinical forms, including cutaneous, mucocutaneous, and visceral leishmaniasis (kala-azar). Kala-azar, the most severe form of the disease, is responsible for approximately 30,000 deaths per year. Leishmaniasis accounts for approximately one million new cases annually, predominantly among populations that are vulnerable and neglected.³ The treatment of this condition relies on the administration of toxic and poorly tolerated agents, such as pentavalent antimonials, and costly alternatives, including liposomal amphotericin B. This situation underscores the urgent need for the development of novel therapeutic molecules.

Chagas disease, a parasitic infection caused by *Trypanosoma cruzi*, affects an estimated 6 to 7 million people worldwide.⁴ The parasites are transmitted by triatomine bugs, which act as vectors. Within the triatomine bug, the parasites undergo different forms, including epimastigote and metacyclic trypomastigote. The disease progresses through several phases in humans. During the acute phase of infection, trypomastigotes present in the blood invade various tissues and transform into intracellular amastigotes. Following an asymptomatic chronic phase, approximately 30% of patients develop severe cardiac or digestive disorders. The treatment of this condition involves the administration of nifurtimox and benznidazole, two pharmaceutical agents with a long history of use, but which are associated with a multitude of adverse effects and a limited degree of efficacy.⁵

Sleeping sickness is a fatal parasitosis caused by *Trypanosoma brucei*. This parasite is found in the epimastigote form in its vector, the tsetse fly. The parasite then transforms into the amastigote form in human blood, the spinal cord, and cerebrospinal fluid.⁶ The development of two novel oral drugs, fexinidazole and acoziborole, has profoundly transformed the management of the disease, resulting in a decline in the number of new cases to below 2000 per year since 2018.⁷⁻⁹

## 1.2. The kinetoplastid's genomic organization

The organization of the kinetoplastid's genome exerts a significant influence on its gene expression regulation. Genetic information is encoded in DNA and adheres to the central principle of genetics, where DNA is transcribed into mRNA, which is subsequently translated into proteins. This transcription is carried out by three RNA polymerases (RNA Pol I-III), each with distinct functions.¹⁰⁻¹² RNA Pol I is responsible for the transcription of ribosomal RNAs (rRNAs), which are the structural elements of the ribosome. In addition to rRNAs, Pol I transcribes specific messenger RNAs (mRNAs) that encode Variant Surface Glycoproteins (VSG) and procyclins.¹³,¹⁴ RNA Pol II is responsible for the transcription of other mRNAs, which encode the majority of proteins. Pol II is also known to transcribe small nuclear RNAs (snRNAs), which play a crucial role in the maturation of RNA. RNA Pol III is the enzyme responsible for the transcription of transfer RNA, 5S rRNA, and some snRNAs.

Unlike most eukaryotes, where genes are generally transcribed individually under the control of specific promoters, the kinetoplastid genome is organized into polycistronic gene clusters, meaning that several genes are sequentially aligned on a single strand of DNA. This organization also occurs in other eukaryotes, such as *Caenorhabditis elegans*, but remains limited (less than 25% of the genome), whereas it represents the dominant genomic organization in kinetoplastids.¹⁵

The transcription is also organized in Polycistronic Transcription Units (PTUs), which produce a single long precursor RNA. The transcription is bidirectional, starting on divergent Strand Switch Regions (dSSRs) at Transcription Start Regions (TSRs) and ending in convergent Strand Switch Regions (cSSRs) at Transcription Termination Sites (TTSs). The precursor RNA then undergoes a series of maturation processes, including trans-splicing and polyadenylation, resulting in the production of multiple functional RNAs. The addition of a poly(A) tail to the transcripts ensures their stability and efficient translation.¹⁰,¹²,¹⁶⁻¹⁹

The kinetoplastid genome is distributed over chromosomes, whose number and size vary according to species: *Leishmania* has 36 small chromosomes for a total of around 32.8 megabases,²⁰ *T. brucei* has 11 large chromosomes (26 megabases)²¹,²² and around 100 minichromosomes (30–150 kilobases),²³ while *T. cruzi* has a larger genome of 60.3 megabases spread over 41 chromosomes.²⁴,²⁵

Nuclear DNA is known to be compacted into chromatin, a dynamic structure composed of DNA and histones. The genome is organized into nucleosomes, the fundamental units of chromatin. Each nucleosome consists of an octamer of histones (two copies of the four core histones H2A, H2B, H3, and H4), around which approximately 147 base pairs of DNA are wound. Histone H1 interacts with linker DNA (around 80 base pairs between nucleosomes), contributing to the stabilization and compaction of chromatin.¹⁰,¹⁹

The degree of chromatin condensation can influence the accessibility of DNA to transcription factors. In general, compact chromatin is associated with a repressed state of transcription, while relaxed euchromatin tends to promote gene expression.¹⁰

These structural variations are key factors in regulating transcription in kinetoplastids. For instance, in *T. brucei*, they play a critical role in the regulation of Variant Surface Glycoproteins expression sites.
VSG coats the parasitic surface, thereby activating the host's immune response. However, their expression can be switched by homologous recombination, thus evading the host immune system.²²,²⁶ This antigenic variation is based on a repertoire of thousands of VSG genes and pro-genes, primarily located in minichromosomes, telomeres and sub-telomeric regions. Yet, at any given time, only one gene is actively transcribed by RNA Pol I. Epigenetic mechanisms, including alterations in nucleosome and chromatin structure orchestrate the translational regulation.²⁷,²⁸

The objective of this paper is to provide a comprehensive reference of the histone post-translational modifications (PTMs) and variants in the three kinetoplastid families that are responsible for NTDs. In addition, the paper will address the associated enzyme writers and erasers, as well as the readers of this histone code. The study will then proceed to investigate new therapeutic interventions targeting these epigenetic mechanisms.

Due to the high interspecific variability of histone H1, the studies devoted to it will not be covered. Additionally, the complexes involved in regulating transcription and translation will not be discussed to maintain a clear focus on the subject at hand.

The identification of genes and proteins associated with writers, erasers, and readers was facilitated by specialized databases, including TriTrypDB (genomic annotation, https://tritrypdb.org/tritrypdb/app/),²⁹ RCSB PDB (protein structures, https://www.rcsb.org/), and TrypTag (cellular localization in *T. brucei*, http://trytag.org/).³⁰ The complete list of gene access codes and the associated 3D structure code can be found in the supplementary materials.

## 2. Post-translational modifications of histones

Histones play a pivotal role in the variation of nucleosome structure and chromatin condensation.

This structure can be modified through the incorporation of variants, which are proteins that can be incorporated into the nucleosome in place of one of the four core histones (H2A, H2B, H3, and H4). The amino acid sequence of these variants differs from that of the core histones, thereby altering the conformation of the nucleosome and consequently impacting transcription.

Furthermore, the core histones and their variants can undergo modifications through the reversible addition of various chemical groups on the amino acids, such as methylations or acetylations on lysine or arginine residues. These PTMs contribute to the structural diversity of the nucleosome and affect the regulation of transcription in kinetoplastids. For example, PTMs at dSSRs have an impact on the regulation of PTU expression. They change the condensation of the chromatin at TSRs and control the initiation of transcription.

A list of all previously described post-translational modifications (PTM) and the associated studies can be found in the supplementary materials.

### 2.1. Post-translational modifications in *Leishmania* spp.

In addition to the four core histones, three histone variants were detected in *Leishmania*. Specifically, H2A.Z is incorporated in place of H2A, H2B.V in place of H2B, and H3.V in place of H3.

The incorporation of H2A.Z and H2B.V is essential for the viability of *L. major*.³¹ These variants are predominantly found at TSRs in *L. tarentolae*.³²

In contrast, the variant H3.V, while not essential, is present mainly in telomeres and at TTSs both in *L. major* and *L. tarentolae*.³¹,³²

Besides this diversity, their structure can be modulated through post-translational modifications (PTM). Despite the absence of a comprehensive global analysis, several PTM of the histones in *Leishmania* have been identified and examined. These PTM include methylations and acetylations found on H3 and H4 lysine (K) residues Fig. 1.

<figure><img src="image_4.png" /><figcaption>Fig. 1. Post-translational modifications of histones in *Leishmania* spp.</figcaption></figure>

The acetylations of histone H3, such as H3-K16ac and H3-K76ac, and the tri-methylation of H3-K4 are found on dSSRs and control the initiation of PTUs transcription in *L. major*, *L. infantum* and *L. tarentolae*.³²⁻³⁴

A study has examined the post-translational modifications (PTM) associated with rRNA genes in *L. major*.³⁵ Acetylations at H3-K14, H3-K23, and H3-K27 and tri-methylation at H3-K9 are enriched in the promoter region of rRNA genes, suggesting a role as transcriptional activator. Conversely, H4-K20me3 has been identified in the coding region of rRNA repeats.

Evidence suggests that the acetylation of H4-K10 regulates the transcription of genes coding for cyclins CYC4 and CYC9, this PTM being downregulated during the S phase of the cell cycle in *L. donovant*³⁶,³⁷ Furthermore, this PTM is subject to downregulation during the S phase of the cell cycle. This locus-specific transcriptional control is uncommon in the polycistronic transcription units of trypanosomids. It may allow the parasite to finely tune the expression of cyclin during at precise cell cycle stages, thereby ensuring proper coordination of DNA replication and mitosis.

The PTM localized at the centromeres help to maintain their structure and stability, thereby contributing to chromosomal segregation. The following PTM have been identified at this localization in *L. infantum* and *L. tarentolae*: H3-K20me2, H3-K4me3, H3-K36me3, K3-K16ac, and K3-K50ac.³²,³⁴

Some PTM may play a role in cell cycle regulation. For example, tri-methylation of H3-K73 is consistently present in *L. mexicana*, whereas its di-methylation is higher in cytokinetic or mitotic cells.³⁸

Finally, the role of H4-K4ac and H4-K14ac has not been described yet.³⁹⁻⁴²

### 2.2. Post-translational modifications in *T. cruzi*

Three histone variants can be incorporated in place of the core histones in *T. cruzi*: H2A.Z, H2B.V, and H3.V.⁴³ H2B.V is located on dSSRs.⁴⁴

The post-translational modifications (PTM) of *T. cruzi* histones were detected through comprehensive global analysis Fig. 2.⁴³,⁴⁵⁻⁴⁶

Studies have focused on some of these post-translational modifications (PTM), enabling their localization and associated roles to be determined. Among these, H3-K4me3 and the acetylations on histones H3 and H4 are often located on dSSRs and control the initiation of PTUs transcription.⁵⁵,⁵⁶

The variation of post-translational modifications (PTM) on histones during the cell cycle was the subject of extensive evaluation Fig. 3.⁵⁷ Specifically, the role of tri-methylation on H3-K76 has been studied, and it has been determined that this modification allows the progression of the cell cycle through cytokinesis after mitosis.⁵⁸,⁵⁹

### 2.3. Post-translational modifications in *Trypanosoma brucei*

The 3D structure of the nucleosome core particle of *T. brucei* with the four core histones was characterized Fig. 4.⁶⁰ This structure shows that this NCP is unstable overall and has weaker DNA binding
<figure><img src="image_5.png" /><figcaption>Fig. 2. Post-translational modifications of histones in *T. cruzi*.</figcaption></figure>

than model eukaryotes. Interestingly, the divergent H2A–H2B interface strengthens local DNA interactions. Additionally, major interaction surfaces, such as the acidic patch, are structurally altered, likely impairing recognition by canonical chromatin-binding proteins. These atypical features may reflect unique modes of chromatin regulation in *T. brucei*. Similar sequence divergences in other kinetoplastids suggest that these features may be evolutionarily conserved.

Four histone variants can be substituted for the core histones of *T. brucei*: H2A.Z, H2B.V, H3.V, and H4.V, replacing H2A, H2B, H3, and H4, respectively.⁶¹

H3.V is the first histone variant discovered in *T. brucei*, and is not essential for cell viability.⁶² It is predominantly found at the TSRs of RNA Pol II, where it would be a negative transcriptional regulator. Additionally, its presence in telomeric regions suggests a role in

antigenic variation and monoallelic transcription of telomeric antigenic genes.⁶³⁻⁶⁶

In contrast, H2A.Z and H2B.V co-localize at TSRs of RNA Pol II and are essential for cell viability.⁶⁷,⁶⁸

H4.V is found at TSRs of RNA Pol II and is a transcription termination mark. It also plays a vital role in antigenic variability.⁶⁵,⁶⁶

A series of exhaustive global analyses of post-translational modifications (PTM) on histones and their variants in *T. brucei* were conducted.⁶⁹⁻⁷⁴ These analyses were complemented by specific studies on particular modifications,⁷⁵,⁷⁶ thereby providing a comprehensive overview of the PTM of histones in *T. brucei* Fig. 5.

While a substantial number of PTMs have been identified, only a few have been extensively studied to date. These explorations have
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Fluctuation of histone variants and post-translational modifications expression in *T. cruzi* during the cell cycle. Arrows indicate changes in expression levels: ↗ represents an increase in PTM or histone variant expression, while ↘ indicates a decrease.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Structure of the nucleosome core particle of *T. brucei*. H2A in purple, H2B in yellow, H3 in green, H4 in pink, DNA double helix in red and blue (code 8COM).</figcaption></figure>

elucidated their functional roles and the dynamic of their expression throughout the cell cycle.

Notably, the acetylation of H4-K4 is increased in phase S of the cell cycle, but is not necessary for cell viability.⁷⁷ Recent functional mutagenesis experiments demonstrated that H4-K4ac directly contributes to transcriptional control at RNA Pol II promoters.⁷⁸ The same experiment also confirmed that H4-K14ac is dispensable for parasitic viability, although its function remains to be determined.

Similarly, H3-K76 methylations have been extensively studied: dimethylation regulates the mitotic cell cycle, while tri-methylation is essential for the transition between parasitic forms. The latter PTM is often located in nucleosomes bearing histone variant H2B.V.⁵⁹,⁷⁹,⁸⁰

Acetylation of H4-K10 is also essential. It is co-localized at dSSRs of RNA Pol II in a nucleosome bearing a tri-methylation in H3-K4 and the histone variant H2B.V, implying a role as transcription initiation

regulator.⁶¹,⁸⁰⁻⁸² This was recently confirmed by genome-wide lysine mutagenesis, in which no H4-K10 mutant strain could be generated.⁷⁸

Finally, H2A-T130 can undergo phosphorylation, a PTM that is also referred to as γH2A.⁸³ This mark is a conserved feature in eukaryotes and plays a central role in the cellular response to DNA double-strand breaks. γH2A accumulates during S and G2 phases, consistent with its function as a marker of DNA damage and ongoing repair. The detection of this PTM can be used for the evaluation of the parasitic genotoxicity of novel drug candidates.⁸⁴

### 3. Writers and Erasers of histone post-translational modifications

Histone post-translational modifications (PTM) are subject to dynamic regulation by two major classes of enzymes: writers, which add epigenetic marks, and erasers, which remove them. These enzymes play a pivotal role in regulating transcriptional processes and in adapting kinetoplastids to their environment, including during host infection. The nature of the post-translational modification (PTM) involved dictates the specific actions of the enzymes. In particular, acetylations and methylations are regulated by specific enzyme families, whose distribution and function differ between the three kinetoplastid groups.

#### 3.1. Histone acetyltransferases

Histone acetylation is a critical post-translational modification (PTM) that plays a pivotal role in regulating chromatin accessibility and gene expression. This process is catalyzed by histone acetyltransferases (HATs), which transfer an acetyl group from acetyl-CoA to histone lysines. In the context of kinetoplastids, the identification of HATs belonging to the MYST (MOZ, Ybf2, Sas2, and Tip60 family) and GNAT (Gcn5-related N-acetyltransferases) families has been undertaken.

MYST family HATs have been identified as the primary enzymes responsible for histone acetylation in kinetoplastids. The precise localization of these proteins, in conjunction with their functional consequences, has been a subject of extensive investigation, shedding light on their roles in specific critical parasite functions.²⁰,⁸¹
<figure><img src="image_8.png" /><figcaption>Fig. 5. Post-translational modifications of histones in *T. brucei*.</figcaption></figure>

* The presence of a chromodomain characterizes HAT1. In the case of *Leishmania*, it has been observed to be responsible for H4-K10 acetylation and exhibits a constant expression throughout the cell cycle in *L. donovani*.³⁷ In *T. brucei*, it is essential for cell viability and is located in the nucleoplasm. It plays a role in telomeric silencing, notably by acetylating H4-K2ac, but also numerous lysines on histones H2A.Z and H2B.V.⁷²

* Similar to HAT1, HAT2 has a chromodomain and has been little studied in *T. cruzi*. In *Leishmania donovani*, it has been observed to possess a Zinc finger. It is located in the nucleus and is essential for cell viability. It acetylates H4-K10ac and H4-K4ac,

and activates expression of the CYC4 and CYC9 cyclin genes. This activation promotes the switch between the S and G2/M phases.³⁶,⁴² In the case of *T. brucei*, it is found in the nucleoplasm and is also essential. It facilitates the acetylation of lysines on histones 4 (H4-K2, H4-K4 and H4-K10), H2A.Z (H2A.Z-K55) and H2B.V (H2B.V-K4 and H2B.V-K17).⁷²

* The research on HAT3 in *T. cruzi* is limited. In contrast, in both *Leishmania* and *T. brucei*, HAT3 localizes to the nucleus, where it acetylates H4-K4. This acetylation process has been shown to support the DNA damage response. However, it is not essential for cell viability. In *Leishmania donovani*, it has also been observed to facilitate progression through the cell cycle.⁴⁰ In *T*.
*brucei*, it induces repression of transcription by RNA Pol I, which plays a role in telomeric silencing.⁷⁷,⁸⁵

* HAT4 has not been identified in *T. brucei*. Its role in *T. cruzi* remains to be fully elucidated, and studies suggest that it is not essential for the survival of *Leishmania donovani* cells. Nevertheless, its presence increases parasite survival in macrophages. It has been observed in both the cytosol and the nucleus of post-mitotic cells. It acetylates H4-K14 and, to a lesser extent, H4-K2. It plays a role in the maintenance of euchromatin and regulates Cdc20, helping the transition to G2/M phases.³⁹,⁴¹

In addition to MYST family enzymes, other HATs belonging to the GNAT family have been identified in kinetoplastids. The genes corresponding to two enzymes of this family, namely ELP3a and ELP3b, have been detected. In the case of *T. brucei*, ELP3a is found in the nuclear envelope, and ELP3b is found in the nucleus, where it plays a role in repressing rDNA transcription.⁸⁶

Furthermore, a novel enzyme, designated Tb-KKT23, which contains a GNAT domain, has recently been identified in *T. brucei*. It is found in the cytoplasm and, to a lesser extent, in the nucleus and flagellum. It has been observed to facilitate acetylation of histone H2A, specifically at lysine residues 122, 125, and 128. The 3D structure of this enzyme has been determined.⁸⁷

## 3.2. Histone deacetylases

Histone deacetylases (HDACs) are the enzymes responsible for the removal of acetyl groups from histones. These enzymes are classified into two distinct classes: Class I and II HDACs, also referred to as “classic” HDACs, are zinc-dependent, while Class III HDACs, also known as silent information regulators (or sirtuins), are NAD-dependent.

Four “classic” HDACs (HDAC1-4) were identified in kinetoplastids.²⁰

* The study of these enzymes in *Leishmania* has been limited. However, recent modeling of the 3D structure of *L. donovani* L-HDACs has been completed.⁸⁸

* In *T. cruzi*, the enzymes in question were classified as follows: Tc-HDAC1 and Tc-HDAC4 were designated as part of class I, while Tc-HDAC2 and Tc-HDAC3 were classified as part of class II.⁵³ Among them, Tc-HDAC4 deacetylates several specific residues, namely H2A-T85ac, H4-K10ac, and H4-T78ac. Tc-HDAC4 may play a regulatory role in cell differentiation, as its expression increases in the epimastigotes and metacyclic trypomastigote forms. Concurrently, Tc-HDAC1 and Tc-HDAC2 have been identified as being essential for cell viability; however, their specific functions remain to be elucidated.⁸⁹ Furthermore, the 3D structure of Tc-HDAC2 has recently been elucidated.

* In *T. brucei*, Tb-HDAC1 and Tb-HDAC2 have been found to belong to class I, while Tb-HDAC3 and Tb-HDAC4 are part of class II.⁹⁰,⁹¹ Tb-HDAC1 is essential for cell viability and is found in both the nucleus and cytoplasm. It has been demonstrated to inhibit telomeric silencing. In contrast, Tb-HDAC4 is a nucleoplasmic essential enzyme that favors the silencing of VSG expression sites. Lastly, Tb-HDAC2 and Tb-HDAC3 are only cytoplasmic and are not necessary; however, Tb-HDAC3 has been observed to facilitate cell cycle progression.

In addition to these classic HDACs, kinetoplastid genomes possess genes coding for three isoforms of sirtuins, named SIR2-RP1, SIR2-RP2, and SIR2-RP3. It should be noted that *T. cruzi* lacks an ortholog for SIR2-RP2, in contrast to *T. brucei* and *Leishmania*.²⁰

* In *Leishmania infantum*, L-SIR2-RP2 and L-SIR2-RP3 are found in the mitochondria, while L-SIR2-RP1 is located in the cytosol.⁹² The latter is essential for cell viability and also possesses an ADP-ribosyltransferase domain. It deacetylates histone H4, and its 3D structure has been elucidated.⁹³⁻⁹⁶

* In *T. cruzi*, Tc-SIR2-RP1 is an essential cytosolic enzyme that facilitates parasitic differentiation in metacyclic trypomastigotes. Tc-SIR2-RP3 is also necessary for cell viability and favors epi-mastigote growth.⁸⁹,⁹⁷

* Tb-SIR2-RP1 is the sole sirtuin in *T. brucei* that is not localized to the mitochondria; rather, it is found in the nucleus on telomeres and minichromosomes.⁹⁸ While not essential for cell viability, Tb-SIR2-RP1 is known to deacetylate H2A and H2B histones. It also possesses an ADP-ribosyltransferase site and contributes to the DNA damage response. Furthermore, Tb-SIR2-RP1 represses RNA Pol I transcription and consequently plays a role in telomeric silencing.⁹⁹,¹⁰⁰

## 3.3. Histone methyltransferases

Histone methylations represent a significant category of post-translational modifications (PTM). The effect of these modifications varies depending on the specific residue and the extent of methylation. In kinetoplastids, three distinct families of enzymes have been identified as catalysts for methylations: Su(var)3-9 Enhancer-of-zeste and Trithorax domain containing lysine-specific proteins (SETs), non-SET domain containing lysine specific proteins, including Disruptor Of Telomeric silencing 1-like enzymes (DOT1), and Protein Arginine Methyl Transferases (PRMTs).²⁰

Kinetoplastid genomes contain genes encoding several enzymes of the SET family; however, their role in histone methylation has yet to be demonstrated. Nevertheless, a protein from this family has been the subject of study in *T. brucei*. This protein is known as Tb-SET27 and is a nuclear enzyme found at the level of RNA Pol II transcription start regions (TSRs). This enzyme is the key point of the SET27 Promoter-Associated Regulatory Complex (SPARC) and would play a role in transcription initiation.¹⁰¹

The presence of the DOT1A and DOT1B enzymes has been identified in kinetoplastids.²⁰ These enzymes have been observed to specifically methylate H3-K76 (or its equivalent H3-K73 in *Leishmania* spp.). The precise function of DOT1A lies in mono- or di-methylation of this lysine, whereas DOT1B demonstrates a capacity for tri-methylation. This PTM plays a role in mitosis and cell cycle regulation, but the role of DOT1B in kinetoplastid cell viability is non-essential.³⁸,⁵⁸

In the case of *T. cruzi*, Tc-DOT1B has been observed to play a role in cytokinesis.

In *T. brucei*, Tb-DOT1A and Tb-DOT1B are nuclear. Tb-DOT1B plays a role in cell differentiation, as well as in VSG silencing and switching.⁵⁹,⁷⁹,¹⁰²,¹⁰³ Tb-DOT1A is essential for cell viability and is regulated by ubiquitininylation of histone H2B. The level of Tb-DOT1A has been observed to increase significantly during mitosis, suggesting a potential role in regulating DNA replication.¹⁰⁴,¹⁰⁵ The 3D structural configuration of Tb-DOT1A has been determined.⁵⁹

PRMTs catalyze the methylation of arginine residues. They are often found in homodimers, and their importance was proved in other organisms, but data in kinetoplastids remain heterogeneous.

* In *Leishmania*, five PRMTs have been identified: L-PRMT1, L-PRMT3, L-PRMT5, L-PRMT6, and L-PRMT7. These enzymes have been demonstrated to be non-essential for cell viability, and their role as potential histone methylation enzymes remains to be thoroughly investigated.¹⁰⁶⁻¹⁰⁹

However, the presence of L-PRMT1 and L-PRMT3, localized in the nucleus and cytoplasm, respectively, has been demonstrated to favor the parasitic infection of macrophages.

Conversely, the nuclear enzyme L-PRMT6 acts as a negative regulator of *Leishmania major* infection *in vivo*, as its overexpression leads to a mild reduction in the number of viable parasites in lymph nodes.

Finally, L-PRMT7, exclusively found in the cytoplasm, is decreased during cell-phase transition. Its presence also facilitates parasitic infection of macrophages and is negatively correlated with pathogenicity.
* In *T. cruzi*, four PRMTs have been identified, including Tc-PRMT1, Tc-PRMT6, Tc-PRMT7, and one yet-to-be-categorized PRMT. The precise localization or functions of these enzymes have not been determined.

* Five PRMTs have been reported in *T. brucei*, namely Tb-PRMT1, Tb-PRMT3, Tb-PRMT5, Tb-PRMT6 and Tb-PRMT7, but none of them is essential for cell viability.¹⁰⁷

Tb-PRMT1 and its support prozyme Tb-PRMT3 are both nuclear and cytosolic. Tb-PRMT3 retains the canonical PRMT domains, but presents no intrinsic catalytic activity. Instead, its role is to stabilize Tb-PRMT1 and aid substrate recognition, forming a heterotetrameric complex. This obligatory association illustrates the prozyme paradigm, in which a low-activity enzyme (Tb-PRMT1) and an inactive paralog (Tb-PRMT3) assemble into a high-activity complex.⁹³,¹¹⁰ These enzymes have been found to play a regulatory role in energetic metabolism and the response to nutritional stress.¹¹¹,¹¹²

In contrast, the remaining Tb-PRMTs are found exclusively in the cytoplasm.¹¹¹ The functions of Tb-PRMT5 have not yet been elucidated; however, Tb-PRMT6 has been proven to methylate histones H3 and H4. Its role as a cell division helper has been elucidated, and its 3D structure has been determined.¹¹³⁻¹¹⁵

Finally, Tb-PRMT7 has been shown to methylate histone H4, specifically catalyzing H4-R3 di- and tri-methylations.¹¹⁶⁻¹¹⁸ Its 3D structure has also been determined.

## 4. Post-translational modifications readers

Histone post-translational modifications (PTM) form a “histone code” that is subsequently interpreted by specialized proteins called “readers”. These proteins possess specific recognition domains, that are capable of binding to specific PTM. This recognition triggers a cascade of cellular events, including euchromatin stabilization, chromatin compaction around the nucleosome, the activation or repression of transcription factors, and the addition or removal of other histone PTM. It is important to note that reader domains comprise a larger family of proteins capable of recognizing PTM on numerous other proteins in addition to histones. The study of readers in epigenetics represents a modest segment of a far more extensive research domain.

### 4.1. Acetylated lysine readers: Bromodomains

Bromodomain factors represent a class of reader proteins that recognize and bind to acetylated lysines, particularly on histones. Seven bromodomain factors have been identified in each kinetoplastid, designated as BDF1-8.²⁰,¹¹⁹⁻¹²²

Research has been conducted on the kinetoplastid's bromodomains; however, the data are still incomplete Table 1. Some subcellular localization and 3D structures have been determined, alongside their essentiality for cell viability. It is noteworthy that certain bromodomain factors exhibit additional functional motifs beyond the bromodomain itself.

The specific roles and functions of certain bromodomain factors have been studied.

In *Leishmania*, L-BDF5 has been the focus of recent extensive research.¹²¹,¹²³ It is mainly located on the dSSR of RNA Pol II and possess a double bromodomain. *In vitro* binding studies suggest its binding to acetylated lysine residues on histones H2B and H4, notably H2B-K19ac/K21ac and H4-K2ac/K4ac/K10ac/K14ac. In addition, a degenerate bromodomain factor, L-BDF8, was recently identified as part of the Conserved Regulators of Kinetoplastid Transcription complex (CRKT).¹²¹,¹³⁶

In *T. cruzi*, the bromodomain factors exhibit a range of functions. Tc-BDF1 supports trypomastigote infectivity,¹²⁴ while Tc-BDF3 favors cell differentiation and epimastigote growth by binding to acetylated alpha-tubulin.¹²⁷,¹²⁸ Furthermore, Tc-BDF2 recognizes acetylations of

H4-K10 and lysine residues on histone H2B.V. The expression of Tc-BDF2 is induced by UV irradiation and enhances the parasitic infection of the mammalian host.⁴⁴,¹²⁵,¹²⁶ Finally, the CRKT complex is conserved in *T. cruzi*, where an interaction between Tc-BDF8 and the MRG domain of Tc-BDF5 was demonstrated.¹³⁶

In *T. brucei*, the organization of bromodomain factors within protein complexes has been investigated.⁶¹,¹³²,¹³⁴ Tb-BDF3, Tb-BDF5, and Tb-BDF8 have been identified as components of the Conserved-Regulators of Kinetoplastid Transcription complex, which also encompasses enzymes such as Tb-HAT2.¹²¹,¹²²,¹³⁷ This complex has been shown to regulate RNA Pol II transcription. Tb-BDF7 is associated with NAPs (Nucleosome Assembly Proteins), and Tb-BDF6 has been identified as a significant interactor of Tb-HAT1. However, further analysis is required to elucidate the specific functions of these proteins.

The interaction of *T. brucei* bromodomain factors with histones and specific PTMs has been analyzed. TB-BDF2 has been shown to recognize the acetylations of lysines on histone variants H2A.Z and H2B.V, exhibiting a higher affinity for hyperacetylated N-ends of H2A.Z, where it has been observed to associate with Tb-HDAC3.¹³¹ In contrast, Tb-BDF3 has been observed colocalize with regions enriched in acetylated H4-K10, suggesting a potential interaction, although binding has not yet been proven.⁶¹

These studies imply that *T. brucei* bromodomain factors can regulate transcription. Furthermore, Tb-BDF1, Tb-BDF3, and Tb-BDF4 are localized on the TSRs of RNA Pol II. In addition, Tb-BDF2, Tb-BDF3, and Tb-BDF5 have been shown to play a role in the expression of VSGs. Finally, Tb-BDF3 would also control procyclin expression.

### 4.2. Other readers

A multitude of other readers can read about protein post-translational modifications (PTM). However, their role in the context of PTM in kinetoplastids remains to be fully elucidated.

Proteins containing the YEATS domain act as lysine acylation readers, with increased binding affinity for crotonylation.¹²⁰ Nuclear YEATS-domain proteins are found in all kinetoplastids, but two proteins have been further analyzed in *T. brucei*: Tb-YEA1 and Tb-YEA2.¹²²

Notably, Tb-YEA1 is also designated as Tb-SWRC1, owing to its association with the SWR1 complex. It has been determined that Tb-YEA1 is responsible for the incorporation of histone variant H2A.Z in the nucleosome.¹³⁷

Tb-YEA2, on the other hand, is part of a NuA4-like complex.

Chromodomains proteins, capable of binding methylated lysines, have been detected in the genome of kinetoplastids.²⁰,¹²⁰ Specifically, Tb-CRD1, a nuclear protein, is part of the SPARC with Tb-SET27, and is found on TSRs of RNA Pol II.¹⁰¹,¹²²

Tudor-domain proteins bind to proteins bearing methylations on lysine or arginine, and they are found in kinetoplastids. Notably, Tb-HAT1 (part 3.1) is a prime example, as it possesses a Tudor-domain for which a 3D structure has been elucidated. Subsequent studies have revealed that this domain is capable of binding to methylated lysine on histones, though it lacks the specificity to recognize methylated lysine on histones. However, it has been observed that this domain can bind to DNA in a non-specific manner.¹²⁰,¹²²

A protein containing a zinc finger-CW domain was identified in *T. brucei*, namely Tb-ZCW1.¹²² This protein has been observed to be associated with Tb-SET26 within the nucleus, and it is hypothesized to bind to methylated histones.

Genes coding for proteins containing Plant Homeo-Domains (PHD) were detected in kinetoplastids. Within the *T. brucei* genome, these genes were designated Tb-PDH1-6. Specifically, Tb-PDH1 is a nuclear protein that is associated with a complex similar to the piccolo-NuA4 complex, which acetylates lysines on the histones of *Saccharomyces*.¹²² Tb-PHD6 is part of the nuclear SPARC, where it is associated with Tb-SET27, which regulates the initiation of RNA Pol II transcription.¹⁰¹
<table><caption>**Table 1**
Summary of Bromodomain factors found in kinetoplastids.</caption><thead><tr><th>Name</th><th>Gene ID<sup>a</sup></th><th>Other domains</th><th>Subcellular localization</th><th>PDB ID</th><th>likely essential</th><th>Reference</th></tr></thead><tbody><tr><td colspan="7"><strong>Leishmania donovani</strong></td></tr><tr><td>L-BDF1</td><td>LdBPK_367210.1</td><td></td><td></td><td></td><td>Yes<sup>b</sup></td><td>Refs. 20, 119-121</td></tr><tr><td>L-BDF2</td><td>LdBPK_363130.1</td><td></td><td></td><td>5C4Q</td><td>Yes<sup>b</sup></td><td>Refs. 20, 119-121</td></tr><tr><td>L-BDF3</td><td>LdBPK_363520.1</td><td></td><td></td><td>5FEA</td><td>Yes<sup>b</sup></td><td>Refs. 20, 119-121</td></tr><tr><td>L-BDF4</td><td>LdBPK_140360.1</td><td>Zinc-finger</td><td></td><td></td><td>Yes<sup>b</sup></td><td>Refs. 20, 119-121</td></tr><tr><td>L-BDF5</td><td>LdBPK_091320.1</td><td>MRG domain</td><td>Nucleus</td><td>5TCK, 5TCM, 6BYA, 8BPT</td><td>Yes<sup>b,c</sup></td><td>Refs. 119-121, 123</td></tr><tr><td>L-BDF6</td><td>LdBPK_120390.1</td><td></td><td></td><td></td><td>No<sup>b</sup></td><td>Refs. 119-121</td></tr><tr><td>L-BDF7</td><td>LdBPK_110910.1</td><td>ATPase</td><td></td><td></td><td>No<sup>b</sup></td><td>Refs. 119-121</td></tr><tr><td>L-BDF8</td><td>LdBPK_342070.1</td><td></td><td></td><td></td><td></td><td>Ref. 121</td></tr><tr><td colspan="7"><strong>T. cruzi</strong></td></tr><tr><td>Tc-BDF1</td><td>TcCLB.506247.80</td><td></td><td>Glycosome</td><td></td><td>Yes<sup>d</sup></td><td>Refs. 20, 119, 120, 124</td></tr><tr><td>Tc-BDF2</td><td>TcCLB.507769.30</td><td></td><td>Nucleus</td><td>6NP7, 6NIM</td><td>Yes<sup>e,f</sup></td><td>Refs. 20, 120, 125</td></tr><tr><td>Tc-BDF3</td><td>TcCLB.510719.70</td><td>Extraterminal domain</td><td>Cytoplasm</td><td>6NEY</td><td>Yes<sup>d,e</sup></td><td>Refs. 44, 119, 126</td></tr><tr><td>Tc-BDF4</td><td>TcCLB.508857.150</td><td></td><td>Nucleus</td><td></td><td></td><td>Refs. 20, 119, 120</td></tr><tr><td>Tc-BDF5</td><td>TcCLB.510359.130</td><td>Zinc-finger</td><td>Nucleus</td><td></td><td></td><td>Refs. 119, 120</td></tr><tr><td>Tc-BDF6</td><td>TcCLB.510889.330</td><td></td><td></td><td></td><td></td><td>Refs. 119, 120</td></tr><tr><td>Tc-BDF7</td><td>TcCLB.506297.110</td><td></td><td></td><td></td><td></td><td>Refs. 119, 120</td></tr><tr><td>Tc-BDF8</td><td>TcCLB.506559.310</td><td>ATPase</td><td></td><td></td><td></td><td>Ref. 121</td></tr><tr><td colspan="7"><strong>T. brucei</strong></td></tr><tr><td>Tb-BDF1</td><td>Tb927.10.8150</td><td></td><td>Nucleus</td><td>5KO4</td><td>No<sup>g</sup></td><td>Refs. 20, 119, 120</td></tr><tr><td>Tb-BDF2</td><td>Tb927.10.7420</td><td></td><td>Nucleus</td><td>2N9G, 4PKL, 5CZG</td><td>No<sup>g</sup></td><td>Refs. 20, 61, 119, 120</td></tr><tr><td>Tb-BDF3</td><td>Tb927.11.10070</td><td></td><td>Nucleus</td><td>5C8G</td><td>Yes<sup>h</sup></td><td>Refs. 122, 130-132</td></tr><tr><td>Tb-BDF4</td><td>Tb927.7.4380</td><td>Zinc-finger</td><td>Nucleus</td><td></td><td>Yes<sup>g</sup></td><td>Refs. 20, 61, 119-122</td></tr><tr><td>Tb-BDF5</td><td>Tb927.11.13400</td><td></td><td>Nucleus</td><td>5K29, 5WQZ, 6NEZ</td><td>Yes<sup>g</sup></td><td>Refs. 130, 132, 133</td></tr><tr><td>Tb-BDF6</td><td>Tb927.1.3400</td><td></td><td>Nucleus</td><td></td><td></td><td>Refs. 20, 119, 120</td></tr><tr><td>Tb-BDF7</td><td>Tb927.11.6350</td><td>ATPase</td><td>Nucleus</td><td></td><td></td><td>Refs. 122, 130</td></tr><tr><td>Tb-BDF8</td><td>Tb927.4.2340</td><td></td><td>Nucleus</td><td></td><td></td><td>Refs. 119-122</td></tr></tbody></table>

Finally, proteins containing PWWP-domains (Pro-Trp-Trp-Pro domains) have been identified in kinetoplastids.<sup>120,138</sup> Within the genome of *T. brucei*, two homologues of Transcription Factor IIS have been identified: Tb-TFIIS2-1 and Tb-TFIIS2-2. Notably, Tb-TFIIS2-1, despite its nuclear location, cannot bind to DNA or methylated histones.

Conversely, Tb-TFIIS2-2 has been observed to recognize tri-methylated lysines H3-K32 and H4-K17 at the TSRs of RNA Pol II in the nucleus. This protein has been observed to be associated with the PAF1 complex, which has been demonstrated to facilitate the elongation of RNA Pol II transcription and transcription-coupled histone modifications. The 3D structure of Tb-TFIIS2-2 has been elucidated.

## 5. Therapeutics strategies

A complete list of tested compounds, along with their 2D structures and results, can be found in the supplementary materials.

### 5.1. Inhibition of HDAC

The first class of treatments that disrupt the epigenetic mechanism is HDAC inhibitors (HDACi). Several treatments that had already received approval have been found to possess HDACi activity. Notably, valproic acid, marketed under the names Depakene® or Depakote®, is employed

in the treatment of epilepsy and bipolar disorder. This medication has been demonstrated to possess HDAC inhibitory activity.<sup>139</sup>

Since 2003, research has identified novel HDAC inhibitors (HDACi) with promising therapeutic applications, particularly in the field of oncology. Notably, several novel HDACi treatments have received FDA approval<sup>140</sup>:

* Vorinostat (or SAHA) was approved in 2006 as Zolina® for the treatment of cutaneous T-cell lymphoma. Additionally, it is currently undergoing clinical trials as an anti-HIV treatment.

* Romidepsin, approved in 2009 as Istodax®, is indicated for the treatment of cutaneous T-cell lymphoma and, since 2011, for other peripheral T-cell lymphomas.

* Panobinostat, also known as Farydak®, was approved in 2015 for the treatment of multiple myeloma.

* Belinostat was approved in 2014 as Beleodaq® against peripheral T-cell lymphoma.

* In 2024, givinostat received approval for the treatment of Duchenne muscular dystrophy under the brand name Duvyzat®.

This interest in HDACi extended to infectious diseases, such as HIV research with vorinostat. This has paved the way for exploring their potential against parasitic infections, including kinetoplastids. HDACi are known to exhibit a high degree of structural diversity. However, they are typically classified into three main distinct chemical classes.

<footnote>a TriTrypDB ID.</footnote>

<footnote>b From Cas9 gene deletion attempts on *L. mexicana* promastigotes.</footnote>

<footnote>c From DiCre inducible knock out analysis on *L. mexicana* murine infection model.</footnote>

<footnote>d From overexpression analysis of wild-type and mutated enzyme.</footnote>

<footnote>e From Cas9 gene deletion attempts.</footnote>

<footnote>f From dominant-negative analysis.</footnote>

<footnote>g From RNAi phenotyping data.</footnote>

<footnote>h From conditional knock-out analysis.</footnote>
<figure><img src="image_9.png" /><figcaption>Fig. 6. Structures of human-approved HDACi with hydroxamic acid group.</figcaption></figure>

### 5.1.1. Hydroxamic acid derivatives

The most represented chemical class is hydroxamic acid derivatives, which are capable of bonding with zinc ions (Zn⁺) from classes I and II HDAC. Thus, a majority of the approved HDACi contain a hydroxamic acid group Fig. 6.

The potential therapeutic interest of hydroxamic acid derivatives in treating kinetoplastid infections has been a subject of investigation since 2004. Initial studies conducted by Mai et al. demonstrated that aroyl-pyrrolyl-hydroxamides exhibit significant antileishmanial activity, with IC₅₀ values on *L. donovani* promastigotes < 10 µM for four compounds. They also showed high affinity for model HDAC (IC₅₀ (maize HD2) = 1.9–3.8 µM) and no detectable cytotoxicity on VERO cells. Overall, these results highlight promising hits, warranting further optimization to improve parasitic potency while maintaining selectivity.¹⁴¹,¹⁴²

In 2010, Patil et al. examined the potential antileishmanial activity of aryl-triazolyl-hydroxamates derivatives in *vitro*. Nine compounds showed good inhibition of HDAC (IC₅₀ < 10 nM, using HDAC fluorescent assay), yet their activity on *L. donovani* promastigotes was modest. Overall, only three compounds reached IC₅₀ < 20 µM, and cytotoxicity on VERO cells was frequently high, often greater than the antileishmanial activity, thereby limiting the interest for further development.¹⁴³

In 2012, Kelly et al. investigated numerous sub-classes of hydroxamic acid derivatives, including sulphone-amides, sulfone-piperazines, and heteroaromatic derivatives. Most compounds displayed potent activity against *T. brucei* bloodstream forms, with twenty compounds exhibiting sub-micromolar IC₅₀ values, and three compounds reaching IC₅₀ < 0.1 µM. HDAC inhibition was also strong, with nineteen derivatives showing IC₅₀ < 1 µM using fluorescent assay on HeLa cells lysates. However, cytotoxicity and selectivity were not evaluated, limiting conclusion regarding their therapeutic potential.¹⁴⁴

In 2013, Pavlik et al. studied Santacruzamate A Fig. 7, a natural hydroxamic acid derivative isolated from a Panamanian Marine Cyanobacterium. It showed only weak inhibition of *L. donovani* axenic amasitogte (7.9%) and *T. cruzi* intracellular amastigotes (9.2%) growth at 36 µM, limiting its interest for kinetoplastid drug development¹⁴⁵

In 2015, Carrillo et al. and Engel et al. tested HDACi undergoing clinical trials for oncology against *T. brucei* bloodstream forms Fig. 8. Quisinostat showed the highest potency with IC₅₀ = 29 nM, but their strong toxicity on human cells resulted in poor selectivity, limiting their potential for kinetoplastid therapy without redesigning these scaffolds.¹⁴⁶,¹⁴⁷

In the same year, Giannini et al. discovered several hydroxamic acids with amid-lactame moieties that exhibited µM to sub-µM activity against both *T. brucei rhodesiense* trypomastigotes and *T. cruzi* amastigotes, but no antileishmanial activity on *L. donovani* amastigotes. Among

<figure><img src="image_10.png" /><figcaption>Fig. 7. Structure of Santacruzamate A.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Fig. 8. Structure of Quisinostat.</figcaption></figure>

them, ST8074AA1 (IC₅₀ = 0.85 µM on *T. cruzi* and 5.52 µM on *T. brucei*) and ST8078AA1 (IC₅₀ = 0.97 µM on *T. cruzi* and 4.16 µM on *T. brucei*) were the most potent Fig. 9. However, their high cytotoxicity on L6 cells (CC₅₀ = 1.23 and 0.64 µM respectively) resulted in poor selectivity, highlighting the need for further optimization before considering antikinetoplastid applications.¹⁴⁸

Concurrently, Wang et al. investigated the anti-parasitic properties of various hydroxamic acid derivatives, through both biological assays and *in silico* docking studies. Three compounds, MC2780, MC3031, and MC2126 Fig. 10, reached sub-nM potency on *T. brucei* brucei trypomastigotes, with IC₅₀ = 0.62 nM, 0.27 nM and 0.44 nM respectively. MC2780 was also the only compound displaying activity on *L. donovani* axenic amastigotes, with IC₅₀ = 0.47 nM. However, all compounds suffered from very high cytotoxicity on L929 cells, resulting in limited selectivity, and preventing therapeutic application without extensive structural redesign.¹⁴⁹

In 2017, Chua et al. conducted a study in which they examined clinically approved HDACi on two types of *Leishmania* parasites: *L. amazoniensis* promastigotes and *L. donovani* promastigotes, as well as *L. donovani* axenic amastigotes. The results demonstrated that romidepsin, belinostat, panobinostat, and vorinostat exhibited no antileishmanial
<figure><img src="image_12.png" /><figcaption>Fig. 9. Structure of ST8074AA1 and ST8078AA1.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>Fig. 10. Structure of MC2780, MC3031 and MC2126.</figcaption></figure>

activity at concentrations below 20 µM, indicating a lack of efficacy of these HDACi on these parasites.¹⁵⁰

In 2018, Loeuillet et al. examined derivatives in the amino-phenyl-hydroxamates and amino-benzyl-hydroxamates series for their antiparasitic activity Fig. 11. Although several compounds showed potent inhibition of HDAC (IC₅₀ down to 85 nM for compound 363 using fluorescent assay on HeLa cells lysates), no clear correlation was observed with antikinetoplastid activity. Most derivatives were inactive against *T. brucei* gambiense procyclic form and displayed only moderate activity on *Leishmania tropica*, *L. braziliensis* or *L. infantum* promastigotes. Compound 345 showed the highest potency on *Leishmania* spp., but this was offset by a high cytotoxicity on THP1 cells (CC₅₀ = 1.7 µM). Compound ST3 exhibited activity on *L. braziliensis* and *L. infantum* (IC₅₀ = 19.6 µM and 29.3 µM respectively) and low cytotoxicity (CC₅₀ (HFF) > 400 µM and CC₅₀ (THP1) = 271 µM) but lost almost ten fold its potency on the corresponding intracellular amastigote forms (IC₅₀ (*L. braziliensis*) = 140 µM and IC₅₀ (*L. infantum*) = 111 µM) and displayed no activity on *T. cruzi*. These results suggest that direct translation of HDAC inhibition on HeLa cells into anti-kinetoplastid activity is not a simple process and may vary due to parasitic-specific factors.¹⁵¹

In 2019, De Oliveira Santos et al. studied the effect of a natural hydroxamic acid against *T. cruzi* epimastigotes, trichostatin A Fig. 12. This compound showed limited *in vitro* antiparasitic activity (IC₅₀ = 60 µM) but marked effects on parasite cell biology, including histone and tubulin hyperacetylation, alterations in cell division, and microtubule cytoskeleton remodeling.¹⁵² In 2021, the same team investigates tubastatin A, a selective HDAC6 inhibitor Fig. 12. This compound also

exhibited low anti-*T. cruzi* activity (IC₅₀ = 30 µM) but induced profound perturbations in cell cycle progression and cytoskeletal organization.¹⁵³

In 2019 and 2021, Corpas-López et al. reported the synthesis of O-alkyl hydroxamic acid derivatives as potential inhibitors of L-SIR2-RP1, along with their nanodelivery via gold nanoparticles. Several derivatives displayed sub-µM activity against *L. infantum* intracellular amastigotes, with no detectable cytotoxicity on BMDM and L929 cells. The most potent was compound 6, also referred to as MDG, with an IC₅₀ = 0.44 µM against *L. infantum* and 0.81 µM against *L. donovani*. Formulation of MDG into gold nanoparticles significantly enhanced its *in vivo* efficacy in murine models of visceral leishmaniasis, without detectable cytotoxicity, highlighting nanodelivery as a promising approach to improve the therapeutic potential of hydroxamic acid derivatives Fig. 13.¹⁵⁴,¹⁵⁵

In 2020, Ángelo de Souza et al. investigated the inhibitory effect of several hydroxamic acid derivatives against *L. braziliensis*. Most compound showed only weak activity on promastigotes, but several compounds displayed IC₅₀ < 10 µM against intracellular amastigotes. TH60 was the most active (IC₅₀ = 4.38 µM on intracellular amastigotes) with low cytotoxicity on RAW 264.7 Fig. 14, highlighting a selective antileishmanial activity.¹⁵⁶

In a 2022 study, Di Bello et al. synthesized new hydroxamate derivatives and tested their activity against *T. cruzi* amastigotes and trypomastigotes, *L. donovani* promastigotes and axenic amastigotes and *L. amazoniensis* promastigotes. Overall, only two compounds, MC2780 and MC1575 displayed notable activity against *T. cruzi* Fig. 15. MC2780 also inhibited 95.7% of *L. amazoniensis* promastigotes and 61.4% of *L.*
<figure><img src="image_14.png" /><figcaption>Fig. 11. Structure of ST3, 345 and 363.</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>Fig. 12. Structures of trichostatin A and tubastatin A.</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>Fig. 13. Structure of MDG (O-alkyl derivative of vorinostat).</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>Fig. 14. Structure of TH60.</figcaption></figure>

donovani axenic amastigotes at 4 µM. However, this compound suffered from high cytotoxicity, with $CC_{50}$ = 5 µM on L929 cells.¹⁵⁷

### 5.1.2. Benzamides

The second chemical class of HDACi belongs to benzamides: the most well-known representative is sirtinol, a non-selective sirtuin inhibitor originally developed for cancer treatment Fig. 16. This compound has been shown to bind with L-SIR2-RP2, and this binding has been studied through *in silico* docking. While no antileishmanial activity was observed on promastigotes, sirtinol inhibited the proliferation of axenic amastigotes in a dose dependent manner above 15 µM. Interestingly, it has also been reported to overcome amphotericin B resistance in *L. donovani*.¹⁵⁸,¹⁵⁹ However, when evaluated against *T. cruzi*, sirtinol displayed no significant activity *in vitro*, with $IC_{50}$ > 100 µM on epimastigotes and > 25 µM on trypomastigotes.¹⁶⁰ As with many compounds initially designed for oncology, the lack of sirtuin selectivity raises concerns about cytotoxicity, particularly when targeting conserved host-like enzymes in parasitic systems.

Another representative of the benzamide class is salermide Fig. 17. Also originally developed in oncology, it was also identified as an

inhibitor of Tc-SIR2-RP3 in 2015. It showed promising *in vitro* activity ($IC_{50}$ = 10.6 µM on epimastigotes) and promising *in vivo* effects.¹⁶¹ Nonetheless, as with cirtinol, the off-target effect and associated cytotoxicity remains a limitation.

Recently, Di Bello et al. evaluated two benzamide derivatives, MC2392 and MS-275, for their antiparasitic activity. However, MC2392 showed only moderate inhibition of *L. donovani* axenic amastigotes (< 42% at 20 µM), *L. donovani* and *L. amazoniensis* promastigotes (< 10% up to 20 µM), and *T. cruzi* amastigotes and trypomastigotes ($IC_{50}$ = 14 µM). MS-275 remained largely inactive, with only low inhibition of *L. donovani* axenic amastigotes (< 30% at 20 µM). Both compounds exhibited significant cytotoxicity ($CC_{50}$ (L929) = 80 µM and 40 µM respectively), further stressing the need for parasite-selective epigenetic modulators.¹⁵⁷

### 5.1.3. Macrocycles

The third chemical class is that of macrocycles. Inside this class, a distinction is made between peptidic macrocycles, which include romidepsin Fig. 18, and non-peptidic macrocycles.

Romidepsin Fig. 18, a clinically approved cyclic peptide HDACi initially developed in oncology, was tested *in vitro* for its anti-*T. brucei* activity and showed remarkable potency, displaying an $IC_{50}$ = 35 nM on *T. brucei* brucei bloodstream forms.¹⁴⁷ However, further studies on *Leishmania* showed no antileishmanial activity, with $IC_{50}$ > 20 µM on *L. amazoniensis* and *L. donovani* promastigotes and *L. donovani* axenic amastigotes.¹⁵⁰

Non peptidic macrocycles were explored by Mwakwari et al. in 2010 as potential antileishmanial derivatives. Their inhibition of HDAC6 and 8 was on the micromolar level and only compound 15e displayed moderate antileishmanial activity ($IC_{50}$ = 5.0 µM on *L. donovani* promastigotes), but also high cytotoxicity ($CC_{50}$ > 5.0 µM on VERO cells) Fig. 19.¹⁶²

Similarly, Guerrant et al. tested new non peptide macrocycles against *L. donovani*. Some of these compounds exhibited nanomolar inhibition of *E. tenella* HDACs (for example, compound 5: $IC_{50}$ = 4.1 nM), but no correlation was observed with antileishmanial activity. The most active compound was compound 15 Fig. 20, displaying $IC_{50}$ = 3.2 µM on promastigotes and 4.7 µM on axenic amastigotes, but high cytotoxicity on VERO cells ($CC_{50}$ > 4.5 µM).¹⁶³
<figure><img src="image_18.png" /><figcaption>Fig. 15. Structure of TH60.</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>Fig. 16. Structure of sirtinol.</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>Fig. 17. Structure of Salermide.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>Fig. 18. Structure of Romidepsin, a macrocyclic peptide derivative.</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>Fig. 19. Structure of compound 15e, a macrocyclic non peptide derivative.</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>Fig. 20. Structure of compound 15, a macrocyclic non peptide derivative.</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>Fig. 21. Structure of Nicotinamide, a sirtuin inhibitor.</figcaption></figure>

#### 5.1.4. Other HDACi

Other HDACi that are of anti-kinetoplastid interest are not classified in these chemical categories.

Nicotinamide is a known sirtuin inhibitor Fig. 21. Its potential in kinetoplastids has been a subject of study since 2005. Sereno et al. reported an $IC_{50}$ < 2.5 mM in L. infantum intracellular amastigotes.164 The same study reported a lack of activity against T. brucei gambiense, suggesting a species selectivity. Subsequent in silico docking studies by Kadam et al. in 2008 enabled the synthesis and in vitro evaluation of nicotinamide derivatives, which exhibited sub-mM activity on L. infantum axenic amastigotes and improved predicted selectivity for parasitic over human sirtuins.165 The first tests of nicotinamide against T. cruzi were performed by Soares et al. in 2012, and showed submicromolar activity on T. cruzi epimastigotes ($IC_{50}$ = 100–126 nM, strain-dependant).166 Subsequent studies by Sacconnay et al. in 2013 and 2014 employed docking in silico techniques to investigate the potential of nicotinamide as Tc-SIR2 inhibitor,167,168 a hypothesis that was later confirmed by in vivo experiments conducted by Moretti et al. in 2015.161 However, their study revealed a high $IC_{50}$ for nicotinamide (25.8 mM)on T. cruzi epimastigotes.

In 2014, Sodji et al. studied a novel chemical class of inhibitors of HDAC6 and 8, the 3-hydroxypyridine-2-thione (3HPT) derivatives Fig. 22.169 Among them, several compounds displayed promising antileishmanial activity despite moderate in vitro HDAC inhibition. Compound 3b was the most potent, with $IC_{50}$ (HDAC6) = 847 nM and inhibition of L. donovani on several forms ($IC_{50}$ (promastigotes) = 0.34 µM, $IC_{50}$
<figure><img src="image_25.png" /><figcaption>Fig. 22. 3b, a 3-hydroxypyridine-2-thione derivative.</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>Fig. 23. KH-TFMDI, a sirtuin inhibitor.</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>Fig. 24. 2, a polyphenol derivative and sirtuin inhibitor.</figcaption></figure>

(axenic amastigotes) = 21.5 µM and IC₅₀ (intramacrophagic amastigotes) = 2.0 µM), as well as low cytotoxicity on THP1 cells (CC₅₀ > 34.1 µM).

The anti-kinetoplastid potential of KH-TFMDI, a novel sirtuin inhibitor, has been demonstrated Fig. 23. In 2014, Veiga-Santos and colleagues reported its good *in vitro* activity *T. cruzi* (IC₅₀ (amastigotes) = 0.5 µM, LC₅₀ (bloodstream form, 37 °C) = 2.5 µM, LC₅₀ (trypomastigotes, 37 °C) = 1.6 µM), as well as low cytotoxicity on peritoneal macrophages (CC₅₀ = 81 µM).¹⁷⁰ Verçoza et al. investigated the activity of this inhibitor on *L. amazonensis* in 2017, observing IC₅₀ = 1.98 µM on promastigotes and 1.15 µM on intracellular amastigotes.¹⁷¹ These results highlight how rationally designed inhibitors can achieve both potent antiparasitic activity and good selectivity.

In 2019, Matutino Bastos et al. investigated the anti-sirtuin potential of natural compounds.¹⁷² The investigation revealed that polyphenolic derivatives extracted from cashew nuts (*Anacardium occidentale*) exhibited the ability to inhibit Tc-SIR2-RP1 and Tc-SIR2-RP3 Fig. 24. Compound 2 was the most potent, which inhibited Tc-SIR2-RP1 (IC₅₀ = 31.40 µM) and displayed anti-T. cruzi activity (IC₅₀ (trypomastigotes) = 12.25 µM and IC₅₀ (amastigotes) = 14.70 µM) and moderate cytotoxicity on hFIB cells (CC₅₀ = 53.70 µM). Despite overall low cytotoxicity, antiparasitic activity remained modest.

The determination of the 3D structure of kinetoplastids HDAC and sirtuins facilitates the application of computational approaches, including molecular docking and structure-based design.

As early as 2010, Tavares et al. identified the L-SIR2-RP1 inhibitory activity of bisnaphthalimido-propyl (BNPID) derivatives Fig. 25.¹⁷³ Several analogs showed a selective inhibition of L-SIR2-RP1 over human SIRT1, leading to interesting *in vitro* antileishmanial activity. Compound BNIPD-anon was the most potent, with IC₅₀ (L-SIR2-RP1) = 5.7 µM, IC₅₀ (human SIRT1) = 97.4 µM and IC₅₀ (*L. infantum* intracellular amastigotes) = 6.03 µM. Docking studies further characterized the binding interactions within the sirtuin pocket, guiding future design.

In 2015, Melesina et al. employed *in silico* methods to analyze the binding of previously reported HDACi in the pocket of kinetoplastids HDAC.¹⁷⁴

<figure><img src="image_28.png" /><figcaption>Fig. 25. BNIPD-anon, a sirtuin inhibitor.</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>Fig. 26. 17, a cambinol analog and Tc-SIR2-RP1 inhibitor.</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>Fig. 27. CDMS-01, a sirtuin inhibitor.</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>Fig. 28. Fragment 1, a sirtuin inhibitor.</figcaption></figure>

In 2020, Matutino Bastos et al. conducted *in vitro* experiments to assess the inhibitory effects of human sirtuin inhibitors on Tc-SIR2-RP1 and Tc-SIR2-RP3 of cambinol derivatives Fig. 26.¹⁷⁵ These tests were completed by *in vitro* trypanocidal activity assays and docking simulations. The findings demonstrated the presence of anti-*T. cruzi* activity within the µM range, accompanied by selective inhibition of one of the two enzymes. For example, compound 17 displayed IC₅₀ = 25.5 µM on Tc-SIR2-RP1 and moderate trypanocidal activity (IC₅₀ (*T. cruzi* trypomastigotes) = 5.7 µM) and cytotoxicity on L929 cells (CC₅₀ = 19.3 µM).

Ferreira et al. employed virtual screening in 2023 to identify potential inhibitors of *T. cruzi* sirtuins Fig. 27.¹⁷⁶ This *in silico* study was subsequently validated *in vitro* using inhibition and antitrypanosomal activity assays. Among the candidates, CDMS-01 showed *in vitro* Tc-SIR2 inhibition (IC₅₀ = 39.98 µM), yet poor antitrypanosomal activity (IC₅₀ = 76.7 µM on intracellular amastigotes). The study highlighted a disconnect between enzymatic inhibition and antikinetoplastid efficacy.
<figure><img src="image_32.png" /><figcaption>**Fig. 29.** Structure of human BET inhibitor with its kinetoplastid target and activity.</figcaption></figure>

Concurrently, Prasanna et al. evaluated valproic acid derivatives as potential leishmanial HDACi.<sup>139</sup> They identified four promising compounds through *in silico*-based molecular docking, molecular dynamic simulations, and pharmacokinetics studies, namely Valproic acid glucuronide, Rac 5-hydroxy valproic acid-d7 sodium Salt, Rac 5-Hydroxy Valproic Acid Sodium Salt and 4-Hydroxy Valproic Acid Sodium Salt.

Finally, Gomes et al. employed fragment screening against Tb-SIR2-RP1 in 2024 to identify novel potential inhibitor structures Fig. 28.<sup>177</sup> This *in silico* study was completed by an *in vitro* inhibition assay and molecular docking and allowed the discovery of fragment 1, which inhibited 99% of Tb-SIR2-RP1 at 500 µM and displayed an IC₅₀ = 17.8 µM against this sirtuin.

<figure><img src="image_33.png" /><figcaption>**Fig. 30.** Structure of I-BET151.</figcaption></figure>

## 5.2. Inhibition of bromodomain factors

The second primary class of therapies targeting epigenetic regulation is Bromodomain and Extra-Terminal Motif (BET) inhibitors. They have emerged in recent years as attractive targets for human drugs, particularly in oncology and cardiovascular disease.<sup>178</sup> The best-known BET inhibitors are chemical derivatives of triazolo-diazepines, such as JQ1 and molibresib. However, a wide range of chemically diverse compounds have also been identified as BET inhibitors. BET inhibitors have also been investigated as potential treatments against kinetoplastid parasites, with drug repositioning being the first approach explored.

A range of human inhibitors was assessed for their activity against kinetoplastids, with results showing minimal to no *in vitro* activity against these parasites. However, the ability to bind certain kinetoplastid BDF was observed Fig. 29.<sup>123,126,128,131</sup>

The most well-known human BET inhibitor active against kinetoplastids is I-BET151 Fig. 30.

In 2015, Schultz et al. conducted a study in which this compound was tested against *T. brucei*, demonstrating its binding to Tb-BDF2 and Tb-BDF3.<sup>132</sup> The crystallographic structure of I-BET151 in complex

with Tb-BDF2 was determined, facilitating the identification of the binding pocket. Furthermore, the compound was found to disrupt *T. brucei*'s immune evasion by replacing the VSG with procyclins. In addition, the *in vivo* efficacy of this inhibitor was demonstrated by a decrease in virulence in a murine model. In 2023, Poli et al. synthesized

<figure><img src="image_34.png" /><figcaption>**Fig. 31.** Structure of SGC-CBP30.</figcaption></figure>
<figure><img src="image_35.png" /><figcaption>Fig. 32. Structure and *in vitro* activity on *T. cruzi* and cytotoxicity on VERO cells of acylhydrazone and 1,3,4-oxadiazoles, a potential BET inhibitor.</figcaption></figure>

I-BET151 derivatives by modifying its 3,5-dimethyl-isoazole or the im-
dazolone moiety.¹⁷⁹ Compounds AP-08-188 and AP-09-008 exhibited
good anti-T. brucei activity on bloodstream form (IC₅₀ = 1.74 µM and
1.27 µM respectively, compared to I-BET151 IC₅₀ = 31.71 µM). AP-09-
008 also displayed a moderate selectivity (K₉ (Tb-BDF2) = 1.68 µM, K₉
(Tb-BDF3) = 1.43 µM and K₉ (human BRD4) = 6.1 µM). *In vivo* efficacy
of these compounds was also encouraging.

Furthermore, I-BET151 was assessed for its potential against *T. cruzi*. In 2016, Alonso et al. demonstrated the binding of I-BET151 to Tc-BDF3 and its *in vitro* activity on epimastigotes with an IC₅₀ value of 6.35 µM.¹²⁸ Subsequent studies by Pezza et al. in 2022 further elucidated the interaction of I-BET151 with Tc-BDF2, providing a more comprehensive understanding of its mechanisms of action, and showing a K₉ = 14.56 µM.¹²⁶

Among the BET inhibitors tested, SGC-CBP30 stood out as the most
promising candidate for *Leishmania* BDF5 targeting Fig. 31. It demon-
strated submicromolar binding affinity for L-BDF5 first bromodomain,
with K<sub>D</sub> = 281 nM (using ITC) and 369 nM (using MST), and induced a
significant thermal shift of +1.88 °C at 10 µM. SGC-CBP30 inhibited *in vitro* growth of *L. mexicana* and *L. donovani* promastigotes, with IC<sub>50</sub> =
7.16 µM and 6.16 µM, respectively.<sup>123</sup> Although its activity against *T. cruzi* was limited (IC<sub>50</sub> = 25.72 µM on epimastigotes),<sup>128</sup> SGC-CBP30
represents a valuable chemical tool for further development of selective
BDF5 inhibitors in *Leishmania*.

In addition to drug repositioning, various strategies have been em-
ployed to identify novel BET inhibitors with enhanced activity against
kinetoplastid parasites. These approaches are designed to overcome the
limitations observed with repurposed human BET inhibitors by identify-
ing compounds with improved affinity and selectivity for kinetoplastid
bromodomain factors.

In 2018, Garcia et al. employed target-based drug design on Tc-
BDF3 and identified A1B4, an acylhydrazone, as a potential inhibitor
Fig. 32.¹⁸⁰ This compound displayed moderate activity against *T. cruzi*
epimastigotes, trypomastigotes, and amastigotes, and low cytotoxicity
on VERO cells. This study was subsequently completed in 2024 by
Alonso et al. who tested 1,3,4-oxadiazoles derivatives of A1B4 to
improve potency and selectivity Fig. 32.¹²⁹ These results highlight
the potential of rational design approaches to optimize bromodomain-
targeting scaffolds for *T. cruzi*, and support further development of
oxadiazole-based BET inhibitors in kinetoplastid drug design.

In 2018, Ramallo et al. identified a new Tc-BDF3 inhibitor, (E)-5-(ethoxymethyl)furan-2-carbaldehyde oxime Fig. 33.¹⁸¹ They used a methodology based on chemically engineered extracts, which are semisynthetic mixtures of compounds produced by diversification of a natural product extract. The compound exhibited promising activity against *T. cruzi*, particularly on trypomastigotes and amastigotes, although cytotoxicity on VERO cells remained important, limiting its selectivity. Nevertheless, the study highlights the potential of hybrid natural product-based strategies for identifying novel BET inhibitors.

In 2023, Araujo et al. employed *in silico* screening methodologies
to identify inhibitors of Tc-BDF2.¹⁸² Four compounds exhibited sub-
stantial virtual affinity for the enzyme and were subjected to further

<figure><img src="image_36.png" /><figcaption>Fig. 33. (E)-5-(ethoxymethyl)furan-2-carbaldehyde oxime IC₅₀ epi. = 50 µM, IC₅₀ tryp. = 8.18 µM, IC₅₀ ama. = 12.37 µM, CC₅₀ = 57.66 µM.</figcaption></figure>

study using *in silico* docking, molecular dynamics simulation, and *in vitro* anti-*T. cruzi* activity on trypomastigotes and *in vitro* cytotoxicity on L929 cells Fig. 34. Among them, compound 72 showed the best selectivity profile, with an IC₅₀ around 10 µM and low cytotoxicity.

In 2021, Laurin et al. employed fragment-based drug design to identify novel Tc-BDF3 inhibitors.¹⁸³ The newly created compounds demonstrated the capacity to bind Tc-BDF3, with compound 58 displaying K<sub>D</sub> = 3.9 µM. However, all active fragments retained a high affinity for the human bromodomain protein, with compound 58 displaying K<sub>D</sub> (human BRD4(1)) = 9.9 nM, highlighting a poor selectivity.

Lastly, Tavernelli et al. conducted a screening in 2024 by fluo-
rescence polarization on Tc-BDF2.¹⁸⁴ Despite strong binding proteins
(K<sub>D</sub> = 1.3–3.5 µM), none of the compounds displayed *in vitro* activity
against *T. cruzi* epimastigotes, nor cytotoxicity on VERO cells.

5.3. *Other therapeutic strategies*

Beyond HDACi and BET inhibitors, therapeutic strategies targeting epigenetic regulation in kinetoplastids have received limited attention yet. While several approaches have already been investigated, given the complexity and number of proteins involved in epigenetic regulation, many possibilities remain to be explored.

One such approach involves activating HDAC instead of inhibiting
it. In 2016, an investigation by Campo et al. explored the potential of
resveratrol, a human histone deacetylase (HDAC) activator, against *T.
cruzi* Fig. 35. This compound induced a reduction in surface protein
expression and demonstrated *in vitro* trypanocidal activity.<sup>160</sup>

Another strategy that has been explored involves inhibiting histone
methyltransferases. In 2017, Zuma et al. demonstrated the anti-*T. cruzi*
potential of Chaetocin, a human histone methyltransferase inhibitor
Fig. 36.¹⁸⁵

Finally, the last explored strategy is the inhibition of histone acetyl-
transferase (HAT). In 2025, Bortolami et al. studied the effect of
three natural HAT inhibitors on *T. cruzi*, namely anacardic acid and
triptolide.¹⁸⁶ Notably, triptolide exhibited promising trypanocidal ac-
tivity, highlighting the potential of these inhibitors Fig. 37.

**6. Therapeutic perspectives**

The elucidation of epigenetic mechanisms in kinetoplastids is open-
ing the way to innovative and new therapeutic strategies. Several
<figure><img src="image_37.png" /><figcaption>Fig. 34. Structure and *in vitro* activity on *T. cruzi* trypomastigotes and cytotoxicity on L929 cells of Tc-BDF2 inhibitors discovered through *in silico* screening.</figcaption></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /><figcaption>Fig. 35. Resveratrol $IC_{50}$ epi. = 250 µM, $IC_{50}$ tryp. = 50.3 µM.</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>Fig. 36. Chaetocin $IC_{50}$ epi. = 2 µM.</figcaption></figure>

emerging approaches enables the targeted and specific treatment of epi-
genetic marks and their associated proteins, leading to the development
of novel potential therapies.

Currently, no vaccines for kinetoplastid infections are available for
humans, despite their high prophylactic potential. In addition to ther-
apeutic approaches, vaccination can provide long-term immunity and
reduce disease burden. Furthermore, kinetoplastids, particularly *Leish-
mania*, have been observed to manipulate the host immune response
to establish chronic infections. One potential strategy for stimulating a
prolonged immune reaction involves targeting the epigenetic proteins
associated with these parasites.

Although studies remain scarce, some studies have yielded promis-
ing preliminary results for the treatment of visceral leishmaniasis. For
instance, in 2014, Baharia et al. tested a combination of H2A, H2B,
H3, and H4 recombinant proteins in an *in vivo* hamster model.187
The vaccine induced a robust Th1-type immune response, leading to
significant protection against experimental visceral leishmaniasis. In a
subsequent study, the same research team investigated the potential
of a recombinant sirtuin in a hamster model, which also promoted a
Th1-type immune response.188

These studies suggest that epigenetic regulators such as histones and
enzymes could serve as potential vaccine targets. Further research is
needed to explore their potential and optimize formulations for human
use fully.

7. Conclusion

The field of epigenetics in kinetoplastid parasites poses distinc-
tive challenges, primarily due to the unique characteristics of their
polycistronic transcription, which differs considerably from classical
eukaryotic models. While numerous histone PTMs and histone variants
have been identified, their precise roles remain largely unknown.

In recent years, significant progress has been made in characteriz-
ing histone post-translational modifications (PTM) writers and erasers;
however, many gaps still need to be filled. Furthermore, recent re-
search has identified and analyzed several epigenetic readers, including
bromodomain-containing proteins, which offer valuable insights into
the functional dynamics of these regulatory mechanisms.

These findings have led to the exploration of novel therapeutic
avenues. However, despite the growing list of validated enzymatic
activities and structural data, most inhibitors were originally designed
for human orthologues, and thus display poor selectivity for kinetoplas-
tid enzymes. This is evidenced by the high level of toxicity exhibited
against mammalian cells, thereby limiting their translational potential.
<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>**Fig. 37.** Structure and *in vitro* activity on *T. cruzi* epimastigotes and cytotoxicity on LLC-MK2 cells of natural HAT inhibitors.</figcaption></figure>

Compared to clinical lead candidates under development by DNDi, such as proteasome, CRK12 kinase, or cytochrome b inhibitors, most epigenetic-targeting compounds exhibit substantially higher IC₅₀ values, often in the low-to-mid micromolar range. In contrast, most clinical leads display IC₅₀ values typically below 1 µM, and frequently approaching 100 nM or lower. In drug discovery for kinetoplastids, an initial “hit” is generally expected to display a selectivity index above 10. Progression to a “lead” compound requires a selectivity index above 100 combined with favorable pharmacokinetic properties. Many currently reported epigenetic inhibitors, developed primarily for human targets, show limited or no selectivity for parasite enzymes. This results in suboptimal selectivity, precluding direct advancement in the development pipeline.

Nevertheless, recent studies have begun to characterize inhibitor binding to kinetoplastid epigenetic proteins in detail, enabling the first docking simulations and in silico structure-activity analyses. These advances in target-based drug design pave the way for the synthesis of novel, parasite-selective inhibitors that are more potent, less cytotoxic to hosts, and have an optimized pharmacological profile.

### CRediT authorship contribution statement

**Inès Jacquet:** Writing – original draft, Investigation, Data curation.
**Romain Paoli-Lombardo:** Writing – review & editing, Data curation.
**Patrice Vanelle:** Writing – review & editing, Validation, Supervision, Project administration.
**Nicolas Primas:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Conceptualization.

### Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or or-for-profit sectors.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Appendix A. Supplementary data

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.bmc.2025.118377.

### Data availability

Data will be made available on request.

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