Selenosugars targeting the infective stage of Trypanosoma brucei with high selectivity.

Earlier evidences showed that diglycosyl diselenides are active against the infective stage of African trypanosomes (top hits IC50 0.5 and 1.5 µM) but poorly selective (selectivity index <10). Here we extended the study to 33 new seleno-glycoconjugates with the aim to improve potency and selectivity. Three selenoglycosides and three glycosyl selenenylsulfides displayed IC50 against bloodstream Trypanosoma brucei in the sub-µM range (IC50 0.35-0.77 µM) and four of them showed an improved selectivity (selectivity index >38-folds vs. murine and human macrohages). For the glycosyl selenylsulfides, the anti-trypanosomal activity was not significantly influenced by the nature of the moiety attached to the sulfur atom. Except for a quinoline-, and to a minor extent a nitro-derivative, the most selective hits induced a rapid (within 60 min) and marked perturbation of the LMWT-redox homeostasis. The formation of selenenylsulfide glycoconjugates with free thiols has been identified as a potential mechanism involved in this process.

The indole motif is essential for the antitrypanosomal activity of N5-substituted paullones.

Severe infections with potentially fatal outcomes are caused by parasites from the genera Trypanosoma and Leishmania (class Kinetoplastea). The diseases affect people of remote areas in the tropics and subtropics with limited access to adequate health care. Besides insufficient diagnostics, treatment options are limited, with tenuous developments in recent years. Therefore, new antitrypanosomal antiinfectives are required to fight these maladies. In the presented approach, new compounds were developed and tested on the target trypanothione synthetase (TryS). This enzyme is crucial to the kinetoplastids' unique trypanothione-based thiol redox metabolism and thus for pathogen survival. Preceding studies have shown that N5-substituted paullones display antitrypanosomal activity as well as TryS inhibition. Herein, this compound class was further examined regarding the structure-activity relationships (SAR). Diverse benzazepinone derivatives were designed and tested in cell-based assays on bloodstream Trypanosoma brucei brucei (T. b. brucei) and intracellular amastigotes of Leishmania infantum (L. infantum) as well as in enzyme-based assays on L. infantum TryS (LiTryS) and T. b. brucei TryS (TbTryS). While an exchange of just the substituent in the 9-position of paullones led to potent inhibitors on LiTryS and T. b. brucei parasites, new compounds lacking the indole moiety showed a total loss of activity in both assays. Conclusively, the indole as part of the paullone structure is pivotal for keeping the TryS inhibitory and antitrypanosomal activity of this substance class.

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro.

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence. Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy -performed in a large and diverse chemolibrary- complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening. Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 µM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12-20 µM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7-45 µM). Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known "garbage in, garbage out" machine learning principle.

Discovery of Antitrypanosomal Indolylacetamides by a Deconstruction-Optimization Strategy Applied to Paullones.

The parasitic kinetoplastid diseases Leishmaniasis, Chagas disease and Human African Trypanosomiasis constitute serious threats for populations throughout the (sub-)tropics. Most available drugs to treat these diseases possess inadequate properties and candidates to fill the drug pipeline are urgently needed. Paullone-N5 -acetamides inhibit trypanothione synthetase (TryS), an essential kinetoplastid enzyme, and exhibit antiparasitic activity in the low micromolar range, but lack the desired selectivity against mammalian cells (selectivity index (SI):<10). With the aim to identify the paullones' moieties responsible for TryS inhibition and bioactivity, we applied molecular simplification and ring disconnection approaches. The new indolylacetamides lost activity against the expected molecular target (TryS) compared to the reference paullone MOL2008 (Leishmania infantum TryS IC50 : 150 nM; Trypanosoma brucei bloodstream form EC50 : 4.3 µM and SI: 2.4). However, several of them retained potency (T. b. brucei EC50 : 2.4-12.0 µM) and improved selectivity (SI: 5 to >25).

A Simple, Robust, and Affordable Bioluminescent Assay for Drug Screening Against Infective African Trypanosomes.

This chapter introduces a simple and robust in vitro viability assay to screen bioactive small molecules (e.g., natural, synthetic) against the monomorphic and infective (bloodstream) form of Trypanosoma brucei brucei. The assay relies on a bioluminescent transgenic parasite harboring a genetically encoded copy of a thermostable redshifted firefly luciferase from Photinus pyralis.The major advantages of the assay are simplicity and cost efficiency, along with excellent quality parameters. The bioassay allows estimating parasite numbers and viability (and metabolic state) as a function of bioluminescence (BL) signal. Parasites are grown in the presence of the molecules of interest in a 96-well microplate, and 24 h later, BL is determined with a simple protocol lacking washing steps, using cost-efficient reagents with a reasonable readout time for high-throughput applications.

Iboga Inspired N-Indolylethyl-Substituted Isoquinuclidines as a Bioactive Scaffold: Chemoenzymatic Synthesis and Characterization as GDNF Releasers and Antitrypanosoma Agents.

The first stage of the drug discovery process involves the identification of small compounds with biological activity. Iboga alkaloids are monoterpene indole alkaloids (MIAs) containing a fused isoquinuclidine-tetrahydroazepine ring. Both the natural products and the iboga-inspired synthetic analogs have shown a wide variety of biological activities. Herein, we describe the chemoenzymatic preparation of a small library of novel N-indolylethyl-substituted isoquinuclidines as iboga-inspired compounds, using toluene as a starting material and an imine Diels-Alder reaction as the key step in the synthesis. The new iboga series was investigated for its potential to promote the release of glial cell line-derived neurotrophic factor (GDNF) by C6 glioma cells, and to inhibit the growth of infective trypanosomes. GDNF is a neurotrophic factor widely recognized by its crucial role in development, survival, maintenance, and protection of dopaminergic neuronal circuitries affected in several neurological and psychiatric pathologies. Four compounds of the series showed promising activity as GDNF releasers, and a leading structure (compound 11) was identified for further studies. The same four compounds impaired the growth of bloodstream Trypanosoma brucei brucei (EC50 1-8 µM) and two of them (compounds 6 and 14) showed a good selectivity index.

A simple, robust, and affordable bioluminescent assay for drug discovery against infective African trypanosomes.

African trypanosomiasis is a major problem for human and animal health in endemic countries, where it threatens millions of people and affects economic development. New drugs are needed to overcome the toxicity, administration, low efficacy, and resistance issues of the current chemotherapy. Robust, simple, and economical high-throughput, whole-cell-based assays are required to accelerate the identification of novel chemical entities. With this aim, we generated a bioluminescent cell line of the bloodstream stage of Trypanosoma brucei brucei and established a screening assay. Trypanosomes were stably transfected to constitutively express a thermostable red-shifted luciferase. The growth phenotype and drug sensitivity of the reporter cell line were essentially identical to that of the parental cell line. The endogenous luciferase activity, measured by a simple bioluminescence assay, proved to be proportional to parasite number and metabolic status. The assay, optimized to detect highly potent compounds in a 96-well-plate format, was validated by screening a small compound library (inter-assay values for Z' factor and coefficient variation were 0.77 and 5.8%, respectively). With a hit-confirmation ratio of ~97%, the assay was potent enough to identify several hits with EC50 ≤ 10 µM. Preliminary tests indicated that the assay can be scaled up to a 384-well-plate format without compromising its robustness. In summary, we have generated reporter trypanosomes and a simple, robust, and affordable bioluminescence screening assay with great potential to speed up the early-phase drug discovery against African trypanosomes.