Antitrypanosomal Activity of 1,2,3-Triazole-Based Hybrids Evaluated Using In Vitro Preclinical Translational Models.

Chagas disease therapy still relies on two nitroderivatives, nifurtimox and benznidazole (Bz), which have important limitations and serious adverse effects. New therapeutic alternatives for this silent disease, which has become a worldwide public health problem, are essential for its control and elimination. In this study, 1,2,3-triazole analogues were evaluated for efficacy against T. cruzi. Three triazole derivatives, 1d (0.21 µM), 1f (1.23 µM), and 1g (2.28 µM), showed potent activity against trypomastigotes, reaching IC50 values 10 to 100 times greater than Bz (22.79 µM). Promising candidates are active against intracellular amastigotes (IC50 ≤ 6.20 µM). Treatment of 3D cardiac spheroids, a translational in vitro model, significantly reduced parasite load, indicating good drug diffusion and efficacy. Oral bioavailability was predicted for triazole derivatives. Although infection was significantly reduced without drug pressure in a washout assay, the triazole derivatives did not inhibit parasite resurgence. An isobologram analysis revealed an additive interaction when 1,2,3-triazole analogs and Bz were combined in vitro. These data indicate a strengthened potential of the triazole scaffold and encourage optimization based on an analysis of the structure-activity relationship aimed at identifying new compounds potentially active against T. cruzi.

An Update on the Therapeutic Potential of Antimicrobial Peptides against Acinetobacter baumannii Infections.

The rise in antibiotic-resistant strains of clinically important pathogens is a major threat to global health. The World Health Organization (WHO) has recognized the urgent need to develop alternative treatments to address the growing list of priority pathogens. Antimicrobial peptides (AMPs) rank among the suggested options with proven activity and high potential to be developed into effective drugs. Many AMPs are naturally produced by living organisms protecting the host against pathogens as a part of their innate immunity. Mechanisms associated with AMP actions include cell membrane disruption, cell wall weakening, protein synthesis inhibition, and interference in nucleic acid dynamics, inducing apoptosis and necrosis. Acinetobacter baumannii is a critical pathogen, as severe clinical implications have developed from isolates resistant to current antibiotic treatments and conventional control procedures, such as UV light, disinfectants, and drying. Here, we review the natural AMPs representing primary candidates for new anti-A. baumannii drugs in post-antibiotic-era and present computational tools to develop the next generation of AMPs with greater microbicidal activity and reduced toxicity.

Bioactivity of Novel Pyrazole-Thiazolines Scaffolds against Trypanosoma cruzi: Computational Approaches and 3D Spheroid Model on Drug Discovery for Chagas Disease.

Chagas disease, a century-old disease that mainly affects the impoverished population in Latin America, causes high morbidity and mortality in endemic countries. The available drugs, benznidazole (Bz) and nifurtimox, have limited effectiveness and intense side effects. Drug repurposing, and the development of new chemical entities with potent activity against Trypanosoma cruzi, are a potential source of therapeutic options. The present study describes the biological activity of two new series of pyrazole-thiazoline derivatives, based on optimization of a hit system 5-aminopyrazole-imidazoline previously identified, using structure−activity relationship exploration, and computational and phenotype-based strategies. Promising candidates, 2c, 2e, and 2i derivatives, showed good oral bioavailability and ADMET properties, and low cytotoxicity (CC50 > 100 µM) besides potent activity against trypomastigotes (0.4−2.1 µM) compared to Bz (19.6 ± 2.3 µM). Among them, 2c also stands out, with greater potency against intracellular amastigotes (pIC50 = 5.85). The selected pyrazole-thiazoline derivatives showed good permeability and effectiveness in the 3D spheroids system, but did not sustain parasite clearance in a washout assay. The compounds' mechanism of action is still unknown, since the treatment neither increased reactive oxygen species, nor reduced cysteine protease activity. This new scaffold will be targeted to optimize in order to enhance its biological activity to identify new drug candidates for Chagas disease therapy.

Structural Optimization and Biological Activity of Pyrazole Derivatives: Virtual Computational Analysis, Recovery Assay and 3D Culture Model as Potential Predictive Tools of Effectiveness against Trypanosoma cruzi.

Chagas disease, a chronic and silent disease caused by Trypanosoma cruzi, is currently a global public health problem. The treatment of this neglected disease relies on benznidazole and nifurtimox, two nitroheterocyclic drugs that show limited efficacy and severe side effects. The failure of potential drug candidates in Chagas disease clinical trials highlighted the urgent need to identify new effective chemical entities and more predictive tools to improve translational success in the drug development pipeline. In this study, we designed a small library of pyrazole derivatives (44 analogs) based on a hit compound, previously identified as a T. cruzi cysteine protease inhibitor. The in vitro phenotypic screening revealed compounds 3g, 3j, and 3m as promising candidates, with IC50 values of 6.09 ± 0.52, 2.75 ± 0.62, and 3.58 ± 0.25 µM, respectively, against intracellular amastigotes. All pyrazole derivatives have good oral bioavailability prediction. The structure-activity relationship (SAR) analysis revealed increased potency of 1-aryl-1H-pyrazole-imidazoline derivatives with the Br, Cl, and methyl substituents in the para-position. The 3m compound stands out for its trypanocidal efficacy in 3D microtissue, which mimics tissue microarchitecture and physiology, and abolishment of parasite recrudescence in vitro. Our findings encourage the progression of the promising candidate for preclinical in vivo studies.

Nicastrin-Like, a Novel Transmembrane Protein from Trypanosoma cruzi Associated to the Flagellar Pocket.

Nicastrin (NICT) is a transmembrane protein physically associated with the polytypical aspartyl protease presenilin that plays a vital role in the correct localization and stabilization of presenilin to the membrane-bound γ-secretase complex. This complex is involved in the regulation of a wide range of cellular events, including cell signaling and the regulation of endocytosed membrane proteins for their trafficking and protein processing. Methods: In Trypanosoma cruzi, the causal agent of the Chagas disease, a NICT-like protein (Tc/NICT) was identified with a short C-terminus orthologous to the human protein, a large ectodomain (ECD) with numerous glycosylation sites and a single-core transmembrane domain containing a putative TM-domain (457GSVGA461) important for the γ-secretase complex activity. Results: Using the Spot-synthesis strategy with Chagasic patient sera, five extracellular epitopes were identified and synthetic forms were used to generate rabbit anti-Tc/NICT polyclonal serum that recognized a ~72-kDa molecule in immunoblots of T. cruzi epimastigote extracts. Confocal microscopy suggests that Tc/NICT is localized in the flagellar pocket, which is consistent with data from our previous studies with a T. cruzi presenilin-like protein. Phylogenetically, Tc/NICT was localized within a subgroup with the T. rangeli protein that is clearly detached from the other Trypanosomatidae, such as T. brucei. These results, together with a comparative analysis of the selected peptide sequence regions between the T. cruzi and mammalian proteins, suggest a divergence from the human NICT that might be relevant to Chagas disease pathology. As a whole, our data show that a NICT-like protein is expressed in the infective and replicative stages of T. cruzi and may be considered further evidence for a γ-secretase complex in trypanosomatids.

Small Angle X-ray Scattering, Molecular Modeling, and Chemometric Studies from a Thrombin-Like (Lmr-47) Enzyme of Lachesis m. rhombeata Venom.

INTRODUCTION: Snakebite envenomation is considered a neglected tropical disease, and SVTLEs critical elements are involved in serious coagulopathies that occur on envenoming. Although some enzymes of this group have been structurally investigated, it is essential to characterize other proteins to better understand their unique properties such as the Lachesis muta rhombeata 47 kDa (Lmr-47) venom serine protease. METHODS: The structure of Lmr-47 was studied in solution, using SAXS, DLS, CD, and in silico by homology modeling. Molecular docking experiments simulated 21 competitive inhibitors. RESULTS: At pH 8.0, Lmr-47 has an Rg of 34.5 ± 0.6 Å, Dmax of 130 Å, and SR of 50 Å, according to DLS data. Kratky plot analysis indicates a rigid shape at pH 8.0. Conversely, the pH variation does not change the center of mass's intrinsic fluorescence, possibly indicating the absence of fluorescent amino acids in the regions affected by pH variation. CD experiments show a substantially random coiled secondary structure not affected by pH. The low-resolution model of Lmr-47 presented a prolate elongated shape at pH 8.0. Using the 3D structure obtained by molecular modeling, docking experiments identified five good and three suitable competitive inhibitors. CONCLUSION: Together, our work provided insights into the structure of the Lmr-47 and identified inhibitors that may enhance our understanding of thrombin-like family proteins.

Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay.

Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4-13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

The combination therapy of meglumine antimoniate and oxiranes (epoxy-α-lapachone and epoxymethyl-lawsone) enhance the leishmanicidal effect in mice infected by Leishmania (Leishmania) amazonensis.

Current treatment of cutaneous leishmaniasis includes pentavalent antimonials as first-line drugs, but this therapy has shown severe adverse effects. An alternative to minimize this issue is based on combination therapy scheme with other drugs. In this study we analyzed the potential of the association of meglumine antimoniate (MA) with the oxiranes epoxy-α-lapachone (LAP) or epoxymethyl-lawsone (LAW). Results demonstrated that association between these drugs enhanced leishmanicidal activity on Leishmania (Leishmania) amazonensis infection. The compounds were tested in monotherapy or in combinations (3:1; 1:1 and 1:3) and reduced intracellular parasite numbers, measured by the endocytic index, in all tested conditions. The most effective combination regimens were MA/LAP or MA/LAW in 3:1 ratio, which achieved a reduction of 98.3% and 93.6% in the endocytic index, respectively. BALB/c mice challenged with L. (L.) amazonensis showed significant reduction in lesion size and parasite load in both footpad and lymph nodes, after four weeks of treatment. Although, MA, LAP or LAW monotherapy were able to control the evolution of lesions when compared to untreated animals (30%, 40% and 40% of reduction, respectively), the combination of MA/LAP and LAW in 3:1 ratio showed better results reducing 61.7 and 54.4%, respectively. The results indicate that the association of meglumine antimoniate to oxiranes lead to an increment in the antileishmanial activity and represent a promising approach for the cutaneous leishmaniasis treatment.

Heme metabolism as a therapeutic target against protozoan parasites.

Neglected tropical diseases caused by protozoan parasites affect the life of millions of people worldwide, causing mortality, morbidity and high economic and social burden. The search for new drug targets and therapeutic strategies to fight these pathogens are necessary, since many current drugs have limited effects, cause severe side effects and their use has resulted in pathogen resistance. Heme (iron protoporphyrin IX) is a ubiquitous molecule important in many biological processes, including the homeostasis, growth and development of human pathogens such as trypanosomatids (Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) and Plasmodium spp. In this review, several chemotherapy approaches and strategies are discussed that target heme transport, catabolism, crystallization and hemeproteins.

Interactions between 4-aminoquinoline and heme: Promising mechanism against Trypanosoma cruzi.

Chagas disease is a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. The current drugs used to treat this disease have limited efficacy and produce severe side effects. Quinolines, nitrogen heterocycle compounds that form complexes with heme, have a broad spectrum of antiprotozoal activity and are a promising class of new compounds for Chagas disease chemotherapy. In this study, we evaluated the activity of a series of 4-arylaminoquinoline-3-carbonitrile derivatives against all forms of Trypanosoma cruzi in vitro. Compound 1g showed promising activity against epimastigote forms when combined with hemin (IC50<1 µM), with better performance than benznidazole, the reference drug. This compound also inhibited the viability of trypomastigotes and intracellular amastigotes. The potency of 1g in combination with heme was enhanced against epimastigotes and trypomastigotes, suggesting a similar mechanism of action that occurs in Plasmodium spp. The addition of hemin to the culture medium increased trypanocidal activity of analog 1g without changing the cytotoxicity of the host cell, reaching an IC50 of 11.7 µM for trypomastigotes. The mechanism of action was demonstrated by the interaction of compound 1g with hemin in solution and prevention of heme peroxidation. Compound 1g and heme treatment induced alterations of the mitochondrion-kinetoplast complex in epimastigotes and trypomastigotes and also, accumulation of electron-dense deposits in amastigotes as visualized by transmission electron microscopy. The trypanocidal activity of 4-aminoquinolines and the elucidation of the mechanism involving interaction with heme is a neglected field of research, given the parasite's lack of heme biosynthetic pathway and the importance of this cofactor for parasite survival and growth. The results of this study can improve and guide rational drug development and combination treatment strategies.