Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies.

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

Synthesis and Anti-Trypanosoma cruzi Activity of 3-Cyanopyridine Derivatives.

Chagas disease (CD) is a parasitic neglected tropical disease (NTD) caused by the protozoan Trypanosoma cruzi that affects 6 million people worldwide, often resulting in financial burden, morbidity, and mortality in endemic regions. Given a lack of highly efficient and safe treatments, new, affordable, and fit-for-purpose drugs for CD are urgently needed. In this work, we present a hit-to-lead campaign for novel cyanopyridine analogues as antichagasic agents. In a phenotypic screening against intracellular T. cruzi, hits 1 and 2 were identified and displayed promising potency combined with balanced physicochemical properties. As part of the Lead Optimization Latin America consortium, a set of 40 compounds was designed, synthesized, and tested against T. cruzi intracellular amastigotes and relevant human cell lines. The structural modifications were focused on three positions: cyanopyridine core, linker, and right-hand side. The ADME properties of selected compounds, lipophilicity, kinetic solubility, permeability, and liver microsomal stability, were evaluated. Compounds 1-9 displayed good potency (EC50T. cruzi amastigote <1 µM), and most compounds did not present significant cytotoxicity (CC50 MRC-5 = 32-64 µM). Despite the good balance between potency and selectivity, the antiparasitic activity of the series appeared to be driven by lipophilicity, making the progression of the series unfeasible due to poor ADME properties and potential promiscuity issues.

Design, synthesis, and lead optimization of piperazinyl-pyrimidine analogues as potent small molecules targeting the viral capping machinery of Chikungunya virus.

The worldwide re-emerge of the Chikungunya virus (CHIKV), the high morbidity associated with it, and the lack of an available vaccine or antiviral treatment make the development of a potent CHIKV-inhibitor highly desirable. Therefore, an extensive lead optimization was performed based on the previously reported CHVB compound 1b and the reported synthesis route was optimized - improving the overall yield in remarkably shorter synthesis and work-up time. Hundred analogues were designed, synthesized, and investigated for their antiviral activity, physiochemistry, and toxicological profile. An extensive structure-activity relationship study (SAR) was performed, which focused mainly on the combination of scaffold changes and revealed the key chemical features for potent anti-CHIKV inhibition. Further, a thorough ADMET investigation of the compounds was carried out: the compounds were screened for their aqueous solubility, lipophilicity, their toxicity in CaCo-2 cells, and possible hERG channel interactions. Additionally, 55 analogues were assessed for their metabolic stability in human liver microsomes (HLMs), leading to a structure-metabolism relationship study (SMR). The compounds showed an excellent safety profile, favourable physicochemical characteristics, and the required metabolic stability. A cross-resistance study confirmed the viral capping machinery (nsP1) to be the viral target of these compounds. This study identified 31b and 34 as potent, safe, and stable lead compounds for further development as selective CHIKV inhibitors. Finally, the collected insight led to a successful scaffold hop (64b) for future antiviral research studies.

Hit-to-lead optimization of a 2-aminobenzimidazole series as new candidates for chagas disease.

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. Because current treatments present several limitations, including long duration, variable efficacy and serious side effects, there is an urgent need to explore new antitrypanosomal drugs. The present study describes the hit-to-lead optimization of a 2-aminobenzimidazole hit 1 identified through in vitro phenotypic screening of a chemical library against intracellular Trypanosoma cruzi amastigotes, which focused on optimizing potency, selectivity, microsomal stability and lipophilicity. Multiparametric Structure-Activity Relationships were investigated using a set of 277 derivatives. Although the physicochemical and biological properties of the initial hits were improved, a combination of low kinetic solubility and in vitro cytotoxicity against mammalian cells prevented progression of the best compounds to an efficacy study using a mouse model of Chagas disease.

The translational challenge in Chagas disease drug development.

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. There is an urgent need for safe, effective, and accessible new treatments since the currently approved drugs have serious limitations. Drug development for Chagas disease has historically been hampered by the complexity of the disease, critical knowledge gaps, and lack of coordinated R&D efforts. This review covers some of the translational challenges associated with the progression of new chemical entities from preclinical to clinical phases of development, and discusses how recent technological advances might allow the research community to answer key questions relevant to the disease and to overcome hurdles in R&D for Chagas disease.

The translational challenge in Chagas disease drug development

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. There is an urgent need for safe, effective, and accessible new treatments since the currently approved drugs have serious limitations. Drug development for Chagas disease has historically been hampered by the complexity of the disease, critical knowledge gaps, and lack of coordinated R&D efforts. This review covers some of the translational challenges associated with the progression of new chemical entities from preclinical to clinical phases of development, and discusses how recent technological advances might allow the research community to answer key questions relevant to the disease and to overcome hurdles in R&D for Chagas disease.

Chagas Disease Drug Discovery in Latin America-A Mini Review of Antiparasitic Agents Explored Between 2010 and 2021.

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi that endangers almost 70 million people worldwide. The only two drugs that are currently approved for its treatment, benznidazole and nifurtimox, have controversial efficacy in adults and restricting safety issues, leaving thousands of patients without a suitable treatment. The neglect of Chagas disease is further illustrated by the lack of a robust and diverse drug discovery and development portfolio of new chemical entities, and it is of paramount importance to build a strong research and development network for antichagasic drugs. Focusing on drug discovery programs led by scientists based in Latin America, the main endemic region for this disease, we discuss herein what has been published in the last decade in terms of identification of new antiparasitic drugs to treat Chagas disease, shining a spotlight on the origin, chemical diversity, level of characterization of hits, and strategies used for optimization of lead compounds. Finally, we identify strengths and weaknesses in these drug discovery campaigns and highlight the importance of multidisciplinary collaboration and knowledge sharing.

2-aminobenzimidazoles for leishmaniasis: From initial hit discovery to in vivo profiling.

Leishmaniasis is a major infectious disease with hundreds of thousands of new cases and over 20,000 deaths each year. The current drugs to treat this life-threatening infection have several drawbacks such as toxicity and long treatment regimens. A library of 1.8 million compounds, from which the hits reported here are publicly available, was screened against Leishmania infantum as part of an optimization program; a compound was found with a 2-aminobenzimidazole functionality presenting moderate potency, low metabolic stability and high lipophilicity. Several rounds of synthesis were performed to incorporate chemical groups capable of reducing lipophilicity and clearance, leading to the identification of compounds that are active against different parasite strains and have improved in vitro properties. As a result of this optimization program, a group of compounds was further tested in anticipation of in vivo evaluation. In vivo tests were carried out with compounds 29 (L. infantum IC50: 4.1 µM) and 39 (L. infantum IC50: 0.5 µM) in an acute L. infantum VL mouse model, which showed problems of poor exposure and lack of efficacy, despite the good in vitro potency.

Structure-activity relationship of 4-azaindole-2-piperidine derivatives as agents against Trypanosoma cruzi.

The structure-activity relationship of a 4-Azaindole-2-piperidine compound selected from GlaxoSmithKline's recently disclosed open-resource "Chagas box" and possessing moderate activity against Trypanosoma cruzi, the parasite responsible for Chagas disease, is presented. Despite considerable medicinal chemistry efforts, a suitably potent and metabolically stable compound could not be identified to advance the series into in vivo studies. This research should be of interest to those in the area of neglected diseases and in particular anti-kinetoplastid drug discovery.

Hit-to-lead optimization of a benzene sulfonamide series for potential antileishmanial agents.

A series of benzene sulphonamides with good potency and selectivity against Leishmania spp. intracellular amastigotes was identified by high-throughput screening. Approximately 200 compounds were synthesized as part of a hit-to-lead optimization program. The potency of the series appears to be strongly dependent on lipophilicity, making the identification of suitable orally available candidates challenging due to poor pharmacokinetics. Despite not identifying a clinical candidate, a likely solvent exposed area was found, best exemplified in compound 29. Ongoing detailed mode-of-action studies may provide an opportunity to use target-based medicinal chemistry to overcome the issues with the current series.

Chemical Constituents and Pharmacology properties of Aristolochia triangularis: a south brazilian highly-consumed botanical with multiple bioactivities.

Aristolochia triangularis Cham., is one of the most frequently used medicinal plant in Southern Brazil. Preparations containing the leaves and/or stems are traditionally used as anti-inflammatory, diuretic, as well as antidote against snakebites. This study screened A. triangularis extracts, fractions and isolated compounds for different bioactivities. A weak antiproliferative activity against human lung cancer cell line (A549) was observed only for chloroform fraction obtained from stems (CFstems - CC50: 2.93 µg/mL). Also, a moderate antimicrobial activity against Staphylococcus aureus was detected just for chloroform fraction obtained from leaves (CFleaves -13-16 mm inhibition zone). Additionally, two semi-purified fractions (CFstems-4 and CFleaves-4) selectively inhibited HSV-1 replication (IC50 values of 0.40 and 2.61 µg/mL, respectively), while only CFleaves showed promising results against Leishmania amazonensis. Fractionation of extracts resulted in the isolation of one neolignan (-) cubebin and one lignan (+) galbacin. However, these compounds are not responsible for the in vitro bioactivities herein detected. The presence of aristolochic acid I and aristolochic acid II in the crude ethanol extract of stems (CEEstems) and leaves (CEEleaves) was also investigated. The HPLC analysis of these extracts did not display any peak with retention time or UV spectra comparable to aristolochic acids I and II.

In Silico Workflow for the Discovery of Natural Products Activating the G Protein-Coupled Bile Acid Receptor 1.

The G protein-coupled bile acid receptor (GPBAR1) has been recognized as a promising new target for the treatment of diverse diseases, including obesity, type 2 diabetes, fatty liver disease and atherosclerosis. The identification of novel and potent GPBAR1 agonists is highly relevant, as these diseases are on the rise and pharmacological unmet therapeutic needs are pervasive. Therefore, the aim of this study was to develop a proficient workflow for the in silico prediction of GPBAR1 activating compounds, primarily from natural sources. A protocol was set up, starting with a comprehensive collection of structural information of known ligands. This information was used to generate ligand-based pharmacophore models in LigandScout 4.08 Advanced. After theoretical validation, the two most promising models, namely BAMS22 and TTM8, were employed as queries for the virtual screening of natural product and synthetic small molecule databases. Virtual hits were progressed to shape matching experiments and physicochemical clustering. Out of 33 diverse virtual hits subjected to experimental testing using a reporter gene-based assay, two natural products, farnesiferol B (27) and microlobidene (28), were confirmed as GPBAR1 activators reaching more than 50% receptor activation at 20 µM with EC50s of 13.53 µM and 13.88 µM, respectively. This activity is comparable to that of the endogenous ligand lithocholic acid (1). Seven further virtual hits showed activity reaching at least 15% receptor activation either at 5 or 20 µM, including new scaffolds from natural and synthetic origin.

Natural products modulating the hERG channel: heartaches and hope.

Covering: 1996-December 2016The human Ether-à-go-go Related Gene (hERG) channel is a voltage-gated potassium channel playing an essential role in the normal electrical activity in the heart. It is involved in the repolarization and termination of action potentials in excitable cardiac cells. Mutations in the hERG gene and hERG channel blockage by small molecules are associated with increased risk of fatal arrhythmias. Several drugs have been withdrawn from the market due to hERG channel-related cardiotoxicity. Moreover, as a result of its notorious ligand promiscuity, this ion channel has emerged as an important antitarget in early drug discovery and development. Surprisingly, the hERG channel blocking profile of natural compounds present in frequently consumed botanicals (i.e. dietary supplements, spices, and herbal medicinal products) is not routinely assessed. This comprehensive review will address these issues and provide a critical compilation of hERG channel data for isolated natural products and extracts over the past two decades (1996-2016). In addition, the review will provide (i) a solid basis for the molecular understanding of the physiological functions of the hERG channel, (ii) the translational potential of in vitro/in vivo results to cardiotoxicity in humans, (iii) approaches for the identification of hERG channel blockers from natural sources, (iv) future perspectives for cardiac safety guidelines and their applications within phytopharmaceuticals and dietary supplements, and (v) novel applications of hERG channel modulation (e.g. as a drug target).

Enzymatic synthesis of ascorbyl ester derived from linoleic acid.

Antioxidants are substances that defend cells against damage, kidnapping and destroying free radicals. They have been largely used in the food industry due the possibility to control the oxidation process, aimed to increase shelf life. Thus, esterification reaction to obtain ascorbyl linoleate catalyzed by Novozym 435 lipase assisted by ultrasound bath was investigated. In this work, molecular sieve (4 Å) was added to the reaction medium to remove the water formed during the esterification reaction to improve the process performance. According to the results, ascorbyl linoleate production up to 90 % was reached after 1 h of reaction time carried out using ultrasound bath, 1:9 molar ratio of substrates L-ascorbic acid to linoleic acid, 20 mL of tert-butanol as organic solvent, 5 wt% of Novozym 435 lipase, 10 wt% of molecular sieve at 70 °C.

Solid dispersions enhance solubility, dissolution, and permeability of thalidomide.

Thalidomide (THD) is a BCS class II drug with renewed and growing therapeutic applicability. Along with the low aqueous solubility, additional poor biopharmaceutical properties of the drug, i.e. chemical instability, high crystallinity, and polymorphism, lead to a slow and variable oral absorption. In this view, we developed solid dispersions (SDs) containing THD dispersed in different self-emulsifying carriers aiming at an enhanced absorption profile for the drug. THD was dispersed in lauroyl macrogol-32 glycerides (Gelucire® 44/14) and α-tocopherol polyethylene glycol succinate (Kolliphor® TPGS), in the presence or absence of the precipitation inhibitor polyvinylpyrrolidone K30 (PVP K30), by means of the solvent method. Physicochemical analysis revealed the formation of semicrystalline SDs. X-ray diffraction and infrared spectroscopy analyses suggest that the remaining crystalline fraction of the drug in the SDs did not undergo polymorphic transition. The impact of the solubility-enhancing formulations on the THD biopharmaceutical properties was evaluated by several in vitro techniques. The developed SDs were able to increase the apparent solubility of the drug (up to 2-3x the equilibrium solubility) for a least 4 h. Dissolution experiments (paddle method, 75 rpm) in different pHs showed that around 80% of drug dissolved after 120 min (versus 40% of pure crystalline drug). Additionally, we demonstrated the enhanced solubility obtained via SDs could be translated into increased flux in a parallel artificial membrane permeability assay (PAMPA). In summary, the results demonstrate that SDs could be considered an interesting and unexplored strategy to improve the biopharmaceutical properties of THD, since SDs of this important drug have yet to be reported.

Pharmacokinetics of hERG Channel Blocking Voacangine in Wistar Rats Applying a Validated LC-ESI-MS/MS Method.

Herbal preparations from Voacanga africana are used in West and Central African folk medicine and are also becoming increasingly popular as a legal high in Europe. Recently, the main alkaloid voacangine was found to be a potent human ether-à-go-go-related gene channel blocker in vitro. Blockage of this channel might imply possible cardiotoxicity. Therefore, the aim of this study was to characterise voacangine in vivo to assess its pharmacokinetics and to estimate if further studies to investigate its cardiotoxic risk are required. Male Wistar rats received different doses of voacangine as a pure compound and as a hydro-ethanolic extract of V. africana root bark with a quantified amount of 9.71 % voacangine. For the obtained data, a simultaneous population pharmacokinetics model was successfully developed, comprising a two-compartment model for i. v. dosing and a one-compartmental model with two first-order absorption rates for oral dosing. The absolute bioavailability of voacangine was determined to be 11-13 %. Model analysis showed significant differences in the first absorption rate constant for voacangine administered as a pure compound and voacangine from the extract of V. africana. Taking into account the obtained low bioavailability of voacangine, its cardiotoxic risk might be neglectable in healthy consumers, but may have a serious impact in light of drug/drug interactions and impaired health conditions.

hERG Channel Blocking Ipecac Alkaloids Identified by Combined In Silico - In Vitro Screening.

Human ether-a-go-go-related gene channel blocking is associated with QT interval prolongation and increased risk of potentially fatal arrhythmias. As natural products keep increasing in popularity, there is an urgent need for studies assessing human ether-a-go-go-related gene channel-related cardiotoxic risks. We selected 49 plant species based on the results of a pharmacophore-based virtual screening campaign, in parallel with a literature data survey concerning highly consumed herbal medicines with reported cardiac liabilities. Lead-like enhanced extracts were prepared, an initial in vitro screening was performed at 100 µg/mL by voltage clamp on Xenopus oocytes, and five human ether-a-go-go-related gene channel blocking extracts were identified. In accordance to the six virtually predicted alkaloids, the root extract of Carapichea ipecacuanha inhibited human ether-a-go-go-related gene channel currents by 32.5 %. A phytochemical workflow resulted in the isolation and identification of five out of the six virtually predicted alkaloids. All isolates blocked human ether-a-go-go-related gene channel currents to different extents. The major ipecac constituents emetine (1) and cephaeline (2) showed IC50 values of 21.4 and 5.3 µM, respectively, measured by whole-cell patch clamp in HEK293 cells. This is the first report on human ether-a-go-go-related gene channel blockers from C. ipecacuanha. Its roots and rhizomes are used to produce different pharmacopeial ipecac preparations that are mainly used as emetics for poisoning treatment. Our findings raise further questions regarding the safety and over-the-counter appropriateness of these herbal products.

Rosmarinic Acid--Pharmaceutical and Clinical Aspects.

The biosynthesis and biotechnological production of Rosmarinic acid, a phenolic ester that is widespread in the plant kingdom, has been widely investigated. This compound has shown many remarkable biological and pharmacological activities, which have led to its pharmaceutical and analytical development, as well as clinical studies, which are summarized and analyzed here for the first time. This review compiles data from the Pubmed, Scopus, Scifinder, Web Of Science, and Science Direct databases published between 1990 and 2015, restricting the search to works with the keywords "Rosmarinic acid" in the title. The initial search identified more than 800 articles; after an initial screening and removal of duplicate works, the search was further refined, resulting in approximately 300 articles that were scrutinized and comprise this review. The articles were organized to describe extraction and isolation, analytical methods, pharmaceutical development, and biological and pharmacological activities [divided into nonclinical (in vitro, in vivo) and clinical studies], pharmacokinetic studies, and stability studies.

Exploration and pharmacokinetic profiling of phenylalanine based carbamates as novel substance p 1-7 analogues.

The bioactive metabolite of Substance P, the heptapeptide SP1-7 (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), has been shown to attenuate signs of hyperalgesia in diabetic mice, which indicate a possible use of compounds targeting the SP1-7 binding site as analgesics for neuropathic pain. Aiming at the development of drug-like SP1-7 peptidomimetics we have previously reported on the discovery of H-Phe-Phe-NH2 as a high affinity lead compound. Unfortunately, the pharmacophore of this compound was accompanied by a poor pharmacokinetic (PK) profile. Herein, further lead optimization of H-Phe-Phe-NH2 by substituting the N-terminal phenylalanine for a benzylcarbamate group giving a new type of SP1-7 analogues with good binding affinities is reported. Extensive in vitro as well as in vivo PK characterization is presented for this compound. Evaluation of different C-terminal functional groups, i.e., hydroxamic acid, acyl sulfonamide, acyl cyanamide, acyl hydrazine, and oxadiazole, suggested hydroxamic acid as a bioisosteric replacement for the original primary amide.

Experimentally validated HERG pharmacophore models as cardiotoxicity prediction tools.

The goal of this study was to design, experimentally validate, and apply a virtual screening workflow to identify novel hERG channel blockers. The hERG channel is an important antitarget in drug development since cardiotoxic risks remain as a major cause of attrition. A ligand-based pharmacophore model collection was developed and theoretically validated. The seven most complementary and suitable models were used for virtual screening of in-house and commercially available compound libraries. From the hit lists, 50 compounds were selected for experimental validation through bioactivity assessment using patch clamp techniques. Twenty compounds inhibited hERG channels expressed in HEK 293 cells with IC50 values ranging from 0.13 to 2.77 µM, attesting to the suitability of the models as cardiotoxicity prediction tools in a preclinical stage.