In silico identification of a biarylamine acting as agonist at human ß3 adrenoceptors and exerting BRL37344-like effects on mouse metabolism.

Human ß3-adrenoceptor (ß3AR) agonists were considered potential agents for the treatment of metabolic disorders. However, compounds tested as ß3AR ligands have shown marked differences in pharmacological profile in rodent and human species, although these compounds remain attractive as they were successfully repurposed for the therapy of urinary incontinence. In this work, some biarylamine compounds were designed and tested in silico as potential ß3AR agonists on 3-D models of mouse or human ß3ARs. Based on the theoretical results, we identified, synthesized and tested a biarylamine compound (polibegron). In CHO-K1 cells expressing the human ß3AR, polibegron and the ß3AR agonist BRL 37344 were partial agonists for stimulating cAMP accumulation (50 and 57% of the response to isoproterenol, respectively). The potency of polibegron was 1.71- and 4.5-fold higher than that of isoproterenol and BRL37344, respectively. These results indicate that polibegron acts as a potent, but partial, agonist at human ß3ARs. In C57BL/6N mice with obesity induced by a high-fat diet, similar effects of the equimolar intraperitoneal administration of polibegron and BRL37344 were observed on weight, visceral fat and plasma levels of glucose, cholesterol and triglycerides. Similarities and differences between species related to ligand-receptor interactions can be useful for drug designing.

Consensus Virtual Screening Protocol Towards the Identification of Small Molecules Interacting with the Colchicine Binding Site of the Tubulin-microtubule System.

Modification of the tubulin-microtubule (Tub-Mts) system has generated effective strategies for developing different treatments for cancer. A huge amount of clinical data about inhibitors of the tubulin-microtubule system have supported and validated the studies on this pharmacological target. However, many tubulin-microtubule inhibitors have been developed from representative and common scaffolds that cover a small region of the chemical space with limited structural innovation. The main goal of this study is to develop the first consensus virtual screening protocol for natural products (ligand- and structure-based drug design methods) tuned for the identification of new potential inhibitors of the Tub-Mts system. A combined strategy that involves molecular similarity, molecular docking, pharmacophore modeling, and in silico ADMET prediction has been employed to prioritize the selections of potential inhibitors of the Tub-Mts system. Five compounds were selected and further studied using molecular dynamics and binding energy predictions to characterize their possible binding mechanisms. Their structures correspond to 5-[2-(4-hydroxy-3-methoxyphenyl) ethyl]-2,3-dimethoxyphenol (1), 9,10-dihydro-3,4-dimethoxy-2,7-phenanthrenediol (2), 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-1-benzopyran-4-one (3), 13,14-epoxyparvifoline-4',5',6'-trimethoxybenzoate (4), and phenylmethyl 6-hydroxy-2,3-dimethoxybenzoate (5). Compounds 1-3 have been associated with literature reports that confirm their activity against several cancer cell lines, thus supporting the utility of this protocol.

Microscopy-based cellular contractility assay for adult, neonatal, and hiPSC cardiomyocytes.

This protocol provides instructions to acquire high-quality cellular contractility data from adult, neonatal, and human induced pluripotent stem cell-derived cardiomyocytes. Contractility parameters are key to unravel mechanisms underlying cardiac pathologies, yet difficulties in acquiring data can compromise measurement accuracy and reproducibility. We provide optimized steps for microscope and camera setup, as well as cellular selection criteria for different cardiomyocyte cell types, aiming to obtain robust and reliable data. Moreover, we use CONTRACTIONWAVE software to analyze and show the optimized results. For complete details on the use and execution of this profile, please refer to Scalzo et al. (2021).

Tubulin Inhibitors: A Chemoinformatic Analysis Using Cell-Based Data.

Inhibiting the tubulin-microtubules (Tub-Mts) system is a classic and rational approach for treating different types of cancers. A large amount of data on inhibitors in the clinic supports Tub-Mts as a validated target. However, most of the inhibitors reported thus far have been developed around common chemical scaffolds covering a narrow region of the chemical space with limited innovation. This manuscript aims to discuss the first activity landscape and scaffold content analysis of an assembled and curated cell-based database of 851 Tub-Mts inhibitors with reported activity against five cancer cell lines and the Tub-Mts system. The structure-bioactivity relationships of the Tub-Mts system inhibitors were further explored using constellations plots. This recently developed methodology enables the rapid but quantitative assessment of analog series enriched with active compounds. The constellations plots identified promising analog series with high average biological activity that could be the starting points of new and more potent Tub-Mts inhibitors.

Cytosolic Ca2+ measurements by ratiometric fluorescence microscopy in melanoma cells.

Cytosolic Ca2+ levels are maintained at low nanomolar concentrations, and disruption of Ca2+ homeostasis is associated with cell/tissue damage. Thus, methods have been developed to accurately assess cellular Ca2+ levels, each with intrinsic advantages and disadvantages. Here, we present in detail a ratiometric fluorometric method for cytosolic Ca2+ measurement in cultured melanoma cells using Fura 2-AM cell loading and fluorescence microscopy imaging. For complete details on the use and execution of this protocol, please refer to Esteves et al. (2020).

Andean Prumnopitys Andina (Podocarpacae) Fruit Extracts: Characterization of Secondary Metabolites and Potential Cytoprotective Effect.

The fruits from the Chilean Podocarpaceae Prumnopitys andina have been consumed since pre-Hispanic times. Little is known about the composition and biological properties of this fruit. The aim of this work was to identify the secondary metabolites of the edible part of P. andina fruits and to assess their antioxidant activity by means of chemical and cell-based assays. Methanol extracts from P. andina fruits were fractionated on a XAD7 resin and the main compounds were isolated by chromatographic means. Antioxidant activity was determined by means of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), ferric reducing power (FRAP), trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. The cytoprotective activity of the extract against oxidative and dicarbonyl stress was evaluated in human gastric epithelial cells (AGS). The total intracellular antioxidant activity (TAA) of the extract was determined in AGS cells. The inhibition of meat lipoperoxidation was evaluated under simulated gastric digestion conditions. Rutin, caffeic acid ß-glucoside and 20-hydroxyecdysone were identified as major components of the fruit extract. Additional compounds were identified by high-performance liquid chromatography diode-array detector mass spectrometry (HPLC-DAD-MSn) and/or co-injection with standards. Extracts showed dose-dependent cytoprotective effects against oxidative and dicarbonyl-induced damage in AGS cells. The TAA increased with the pre-incubation of AGS cells with the extract. This is the first report on the composition and biological activity of this Andean fruit.

Phytochemical profile and biological activities of 'Ora-pro-nobis' leaves (Pereskia aculeata Miller), an underexploited superfood from the Brazilian Atlantic Forest.

Pereskia aculeata Miller, known worldwide as ora-pro-nobis, is a highly nutritive species of the Cactaceae family from the Brazilian Atlantic Forest. In this work, we report inedited information on the phenolic profile of P. aculeata leaves, besides a broad study of their antioxidant potential using a set of five different methods. A total of ten phenolic compounds were identified, such as two phenolic acids (caffeic acid derivatives) and eight flavonoids (quercetin, kaempferol and isorhamnetin glycoside derivatives). Caftaric acid was the extract's major phenolic constituent, accounting for more than 49% of the phenolic content, followed by quercetin-3-O-rutinoside (14.99%) and isorhamnetin-O-pentoside-O-rutinoside (9.56%). Overall, the ora-pro-nobis leaf extract showed relevant values of antioxidant capacity, with higher activities than the Trolox in the DPPH and ABTS trials. The antimicrobial activity exhibited by the extract against both Gram-positive and Gram-negative bacteria suggests the presence of a broad spectrum of phytochemicals with antibiotic activity.

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform.

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.

Biological Activity and Physicochemical Properties of Dipeptidyl Nitrile Derivatives Against Pancreatic Ductal Adenocarcinoma Cells.

BACKGROUND: Pancreatic cancer is one of the most aggressive types with high mortality in patients. Therefore, studies to discover new drugs based on cellular targets have been developed to treat this disease. Due to the importance of Cysteine Protease (CP) to several cellular processes in cancer cells, CP inhibitors have been studied as novel alternative approaches for pancreatic cancer therapy. OBJECTIVE: The cytostatic potential of new CP inhibitors derived from dipeptidyl nitriles is analyzed in vitro using pancreatic cancer (MIA PaCa-2) cells. METHODS: The cytotoxic and cytostatic activities were studied using MTT colorimetric assay in 2D and 3D cultures. Colony formation, migration in Boyden chamber and cell cycle analysis were applied to further study the cytostatic activity. The inhibition of cysteine proteases was evaluated with Z-FR-MCA selective substrate, and ROS evaluation was performed with DCFH-DA fluorophore. Permeability was investigated using HPLC-MS to obtain log kw. Combination therapy was also evaluated using the best compound with gemcitabine. RESULTS: The inhibition of intracellular CP activity by the compounds was confirmed, and the cytostatic effect was established with cell cycle retention in the G1 phase. CP inhibitors were able to reduce cell proliferation by 50% in the clonogenic assay, and the same result was achieved for the migration assay, without any cytotoxic effect. The Neq0554 inhibitor was also efficient to increase the gemcitabine potency in the combination therapy. Physicochemical properties using an artificial membrane model quantified 1.14 ≥ log Kw ≥ 0.75 for all inhibitors (also confirmed using HPLC-MS analysis) along with the identification of intra and extracellular metabolites. Finally, these dipeptidyl nitrile derivatives did not trigger the formation of reactive oxygen species, which is linked to genotoxicity. CONCLUSION: Altogether, these results provide a clear and favorable picture to develop CP inhibitors in pre-clinical assays.

Novel anti-prostate cancer scaffold identified by the combination of in silico and cell-based assays targeting the PI3K-AKT-mTOR pathway.

Phenotypic assays were performed in prostate cancer cell lines to describe the biological activity of PI3K-AKT-mTOR pathway inhibitors retrieved from the virtual screening initiative. These novel chemicals share in common the aminopyridine scaffold, hitting PC-3 cells in macromolar range, with selectivity index over fibroblast cell lines. Moreover, a preliminary study of the mode of action by flow cytometry assay pointed out that these compounds had a rapamycin-like response for the PI3K-AKT-mTOR pathway modulation.