1,3,4-oxadiazoles as inhibitors of the atypical member of the BET family bromodomain factor 3 from (BDF3).

Chagas disease, caused by the protozoan parasite , affects millions globally, with increasing urban cases outside of Latin America. Treatment is based on two compounds, namely, benznidazole (BZ) and nifurtimox, but chronic cases pose several challenges. Targeting lysine acetylation, particularly bromodomain-containing proteins, shows promise as a novel antiparasitic target.

Identification of novel bromodomain inhibitors of bromodomain factor 2 (BDF2) using a fluorescence polarization-based high-throughput assay.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones.

Deciphering Divergent Trypanosomatid Nuclear Complexes by Analyzing Interactomic Datasets with AlphaFold2 and Genetic Approaches.

Acetylation signaling pathways in trypanosomatids, a group of early branching organisms, are poorly understood due to highly divergent protein sequences. To overcome this challenge, we used interactomic datasets and AlphaFold2 (AF2)-multimer to predict direct interactions and validated them using yeast two and three-hybrid assays. We focused on MORF4 related gene (MRG) domain-containing proteins and their interactions, typically found in histone acetyltransferase/deacetylase complexes.

Essential Bromodomain BDF2 as a Drug Target against Chagas Disease.

is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin.

Update on the Biological Relevance of Lysine Acetylation as a Novel Drug Target in Trypanosomatids.

The number of acetylated proteins identified from bacteria to mammals has grown exponentially in the last ten years, and it is now accepted that acetylation is a key component in most eukaryotic signaling pathways and is as important as phosphorylation. The enzymes involved in this process are well described in mammals; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids, several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase.

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN).

Alpha-Tubulin Acetylation in : A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression.

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body.

Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity.

Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro.

Aim for the Readers! Bromodomains As New Targets Against Chagas' Disease.

Bromodomains recognize and bind acetyl-lysine residues present in histone and non-histone proteins in a specific manner. In the last decade they have raised as attractive targets for drug discovery because the miss-regulation of human bromodomains was discovered to be involved in the development of a large spectrum of diseases. However, targeting eukaryotic pathogens bromodomains continues to be almost unexplored.

Discovery of a Biologically Active Bromodomain Inhibitor by Target-Directed Dynamic Combinatorial Chemistry.

Target-directed dynamic combinatorial chemistry (DCC) has emerged as a strategy for the identification of inhibitors of relevant therapeutic targets. In this contribution, we use this strategy for the identification of a high-affinity binder of a parasite target, the bromodomain-containing protein BDF3. This protein is essential for viability of , the protozoan parasite that causes Chagas disease.

A Bioactive Trypanosoma cruzi Bromodomain Inhibitor from Chemically Engineered Extracts.

A set of chemically engineered extracts enriched in compounds including N-N and N-O fragments in their structures was prepared. Bromodomain binding screening and bioguided fractionation led to the identification of one oxime hit that interacts with TcBDF3 with affinity in the submicromolar range and that shows interesting antiparasitic properties against the different life cycle stages of T. cruzi.

Antitrypanosomal and antileishmanial activity of prenyl-1,2,3-triazoles.

A series of prenyl 1,2,3-triazoles were prepared from isoprenyl azides and different alkynes. The dipolar cycloaddition reaction provided exclusively primary azide products as regioisomeric mixtures that were separated by column chromatography and fully characterized. Most of the compounds displayed antiparasitic activity against and .

Development and in vitro/in vivo evaluation of a novel benznidazole liquid dosage form using a quality-by-design approach.

Objectives: To develop an alcohol-free solution suitable for children of benznidazole, the drug of choice for treatment of Chagas disease.

Methods: In a quality-by-design approach, a systematic optimisation procedure was carried out to estimate the values of the factors leading to the maximum drug concentration. The formulations were analysed in terms of chemical and physical stability and drug content.

Overexpression of bromodomain factor 3 in Trypanosoma cruzi (TcBDF3) affects differentiation of the parasite and protects it against bromodomain inhibitors.

The bromodomain is the only protein domain known to bind acetylated lysine. In the last few years many bromodomain inhibitors have been developed in order to treat diseases such as cancer caused by aberrant acetylation of lysine residues. We have previously characterized Trypanosoma cruzi bromodomain factor 3 (TcBDF3), a bromodomain with an atypical localization that binds acetylated α-tubulin.

Glycosomal bromodomain factor 1 from Trypanosoma cruzi enhances trypomastigote cell infection and intracellular amastigote growth.

Acetylation is a ubiquitous protein modification present in prokaryotic and eukaryotic cells that participates in the regulation of many cellular processes. The bromodomain is the only domain known to bind acetylated lysine residues. In the last few years, many bromodomain inhibitors have been developed in order to treat diseases caused by aberrant acetylation of lysine residues and have been tested as anti-parasitic drugs.

Overexpression of cytoplasmic TcSIR2RP1 and mitochondrial TcSIR2RP3 impacts on Trypanosoma cruzi growth and cell invasion.

Background: Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives.

Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid.

Trypanosoma cruzi bromodomain factor 3 binds acetylated α-tubulin and concentrates in the flagellum during metacyclogenesis.

Bromodomains are highly conserved acetyl-lysine binding domains found mainly in proteins associated with chromatin and nuclear acetyltransferases. The Trypanosoma cruzi genome encodes at least four bromodomain factors (TcBDFs). We describe here bromodomain factor 3 (TcBDF3), a bromodomain-containing protein localized in the cytoplasm.

Lysine acetylation: elucidating the components of an emerging global signaling pathway in trypanosomes.

In the past ten years the number of acetylated proteins reported in literature grew exponentially. Several authors have proposed that acetylation might be a key component in most eukaryotic signaling pathways, as important as phosphorylation. The enzymes involved in this process are starting to emerge; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions.

Efficacy of novel benznidazole solutions during the experimental infection with Trypanosoma cruzi.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. About 8 million people throughout Latin America are infected causing approximately 10,000 deaths annually. Benznidazole, available as unique 100 mg tablets in many of the endemic countries, is currently the drug of choice for the specific treatment of this condition.

Characterization of TcHMGB, a high mobility group B family member protein from Trypanosoma cruzi.

High mobility group B (HMGB) proteins are highly abundant non-histone chromatin proteins that play important roles in the execution and control of many nuclear functions. Based on homology searches, we identified the coding sequence for the TcHMGB protein, an HMGB family member from Trypanosoma cruzi. TcHMGB has two HMG box domains, similar to mammalian HMGBs, but lacks the typical C-terminal acidic tail.

Benznidazole treatment attenuates liver NF-κB activity and MAPK in a cecal ligation and puncture model of sepsis.

Recent studies have shown that Benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and nitric oxide (NO) release in activated macrophages by blocking NF-κB through inhibition of IKK in vitro. As so far, little is known about the mechanism by which BZL provokes the inhibition of inflammatory response in sepsis in vivo, we aimed to delineate the possible role of BZL as a modulator in liver inflammation in mice with sepsis induced by cecal ligation and puncture (CLP). Specifically, we analyzed leukocytes, liver production of TNF-α and NO and the intracellular pathways modulated by these mediators, including NF-κB and MAPKs, in the liver of mice 24 h post-CLP.

Cloning, characterization and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids.

Over the last years an expanding family of small non-coding RNAs (sRNA) has been identified in eukaryotic genomes which behave as sequence-specific triggers for mRNA degradation, translation repression, heterochromatin formation and genome stability. To achieve their effectors functions, sRNAs associate with members of the Argonaute protein family. Argonaute proteins are segregated into three paralogous groups: the AGO-like subfamily, the PIWI-like subfamily, and the WAGO subfamily (for Worm specific AGO).

Benznidazole blocks NF-kappaB activation but not AP-1 through inhibition of IKK.

Previously, we demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and NO release in macrophages by inhibiting NF-kappaB. We now proceeded to elucidate the molecular mechanisms by which BZL exerts its inhibitory action on NF-kappaB. We demonstrated that the inhibitory effect of BZL is not extended to other macrophage responses, since it did not inhibit other typical hallmarks of macrophage activation such as phagocytosis, MHC-II molecules expression or production of reactive oxygen species (ROS) by NADPH oxidase.