Orally Active and Selective Tubulin Inhibitors as Anti-Trypanosome Agents

Objectives

There is an urgent need to develop a safe, effective, orally active, and inexpensive therapy for African trypanosomiasis due to the drawbacks of current drugs. Selective tubulin inhibitors have the potential to be promising drug candidates for the treatment of this disease, which is based on the tubulin protein structural difference between mammalian and trypanosome cells. We propose to identify novel tubulin inhibitors from a compound library developed based on the lead compounds that selectively target trypanosomiasis.

Methods

We used Trypanosoma brucei brucei as the parasite model, and human normal kidney cells and mouse microphage cells as the host model. Growth rates of both trypanosomes and mammalian cells were determined as a means to screen compounds that selectively inhibit the proliferation of parasites. Furthermore, we examined the cell cycle profile of the parasite and compared tubulin polymerization dynamics before and after the treatment using identified compounds. Last, in vivo anti-parasite activities of these compounds were determined in T. brucei-infected mice.

Results

Three compounds were selected that are 100 fold more effective against the growth of T. brucei cells than mammalian cells. These compounds caused cell cycle progression defects in T. brucei cells. Western analyses indicated that these compounds decreased tubulin polymerization in T. brucei cells. The in vivo investigation revealed that these compounds, when admitted orally, inhibited T. brucei cell proliferation in mouse blood. However, they were not potent enough to clear up the infection completely.

Conclusions

These compounds are promising lead compounds as orally active agents for drug development of anti-trypanosome agents. A more detail structure activity relationship (SAR) was summarized that will be used to guide future lead optimization to improve the selectivity and potency of the current compounds.     

Statistical molecular design of a focused salicylidene acylhydrazide library and multivariate QSAR of inhibition of type III secretion in the Gram-negative bacterium Yersinia

A combined application of statistical molecular design (SMD), quantitative structure–activity relationship (QSAR) modeling and prediction of new active compounds was effectively used to develop salicylidene acylhydrazides as inhibitors of type III secretion (T3S) in the Gram-negative pathogen Yersinia pseudotuberculosis. SMD and subsequent synthesis furnished 50 salicylidene acylhydrazides in high purity. Based on data from biological evaluation in T3S linked assays 18 compounds were classified as active and 25 compounds showed a dose-dependent inhibition. The 25 compounds were used to compute two multivariate QSAR models and two multivariate discriminant analysis models were computed from both active and inactive compounds. Three of the models were used to predict 4416 virtual compounds in consensus and eight new compounds were selected as an external test set. Synthesis and biological evaluation of the test set in Y. pseudotuberculosis and the intracellular pathogen Chlamydia trachomatis validated the models. Y. pseudotuberculosis and C. trachomatis cell-based infection models showed that compounds identified in this study are selective and non-toxic inhibitors of T3S dependent virulence.     

Ultraviolet-induced amides and casbene diterpenoids from rice leaves

To discover new phytoalexins, an 80% MeOH extract of UV-irradiated rice leaves was analyzed using LC–MS, resulting in the detection of three unidentified compounds. We isolated the compounds from the UV-irradiated rice leaves using chromatographic methods and identified the compounds as N-benzoyltyramine (1), and two casbene-type diterpenes, 5-dihydro-ent-10-oxodepresssin (2) and 5-deoxo-ent-10-oxodepressin (3), using spectroscopic methods. Additionally, we compared the accumulation levels of major UV-inducible compounds in response to Magnaporthe oryzae inoculation and the antifungal activities of the compounds against M. oryzae colony growth. Although 1–3 showed negligible antifungal activity against M. oryzae, the compounds significantly accumulated in M. oryzae-inoculated rice leaves. Furthermore, we confirmed that N-benzoyltryptamine and N-cinnamoyltryptamine also accumulated after M. oryzae inoculation and have relatively high antifungal activity against M. oryzae to the same extent as phytocassanes. These results strongly support the hypothesis that the two amides are rice phytoalexins.     

New monoterpenes and benzylbutanoic acid from snowbell (Styrax japonica) honey and their quantitative analysis by LC-ESI-Q-TOF-MS/MS

Honey varies depending on plant origin and environmental conditions. Chemical constituents can be used to determine the origins, characteristics, and storage period of a specific honey. Styrax japonica (snowbell) honey has excellent flavor, but very few studies have been carried out on its chemical constituents. Therefore, the present study was aimed to identify chemical constituents containing in snowbell honey and to screen candidate marker compounds for predicting the storage period of snowbell honey using the identified compounds. Twelve compounds including three new compounds were purified and isolated from the snowbell honey. Three new compounds were elucidated as 1α,3β,5β-bornetriol, 7-hydroxy-2,2,6-trimethyl-1-oxaspiro[2.5]oct-5-en-4-one, and 2-benzyl-2,3-dihydroxybutanoic acid, based on ESI-MS and NMR experiments. 6,7-Dihydroxylinalool and 6,7-dihydroxygeraniol isolated as known compounds in this study were identified for the first time in snowbell honey as well as in other honeys. The isolated compounds were qualified and quantified by LC-ESI-Q-TOF-MS/MS analysis. All compounds were detected in snowbell honeys of different production years. The levels of tyrosol, 7-hydroxy-2,2,6-trimethyl-1-oxaspiro[2.5]oct-5-en-4-one, and (–)-dihydroxyphaseic acid were lower in snowbell honey with long storage period than in short storage period, which is suggested to be used as candidate marker compounds for predicting the storage period of snowbell honey.     

Semantic and prosodic effects of Dutch linking elements

The studies presented here investigate the contribution of linking -en- and rhythm to a compound’s conceptual plurality. Participants were asked to estimate the conceptual plurality of the modifier constituents of Dutch compounds. In the first study, pseudo-compounds (compounds composed of pseudo-words, burb+en+tijpis) and novel compounds (novel combinations of existing nouns, aap+en+leraar, ‘monkey teacher’) with linking -en- were investigated. In study two, we examined written existing compounds that occur with and without -en- (bloem+bak or bloem+en+bak, ‘flower box’) and present a stress clash or not at the constituent boundaries. Finally, study three investigated the same question as study two for spoken existing compounds. The results reveal that existing compounds written or spoken with linking -en- are considered to bear more plural meaning than the ones without linking element. Furthermore, an effect of rhythm on plurality was observed in pseudo-compounds and in spoken existing compounds. When these compounds contain linking -en- to prevent a stress clash, they are considered to bear less plural meaning than those with linking -en- in a neutral context. These studies demonstrate that rhythm can affect the interpretation of linking elements. The fact that we only find a rhythmic effect in pseudo-compounds and spoken existing compounds but not in written existing compounds is explained in terms of processing.     

Benzoate esters and flavonoids from Desmos pedunculosus

The genus Desmos (Annonaceae) is comprised of about thirty-three species of trees and shrubs found in southern Asia, Indonesia, and northern Australia. The chemistry of many of these species is largely unreported. Phytochemical investigation of a methanol extract of the aerial parts of D. pedunculosus led to the isolation of two flavonoids and eight benzoate ester derivatives. Three of these benzoate esters are new compounds, while two others are known synthetic compounds reported here as natural products for the first time. The compounds were characterized by mass spectrometry and 1D and 2D NMR spectroscopy. Based on a comparison of extracts of D. chinensis and D. pedunculosus from Vietnam, several of the compounds we report may serve as marker compounds to chemically distinguish these similar species.     

In silico–based combinatorial pharmacophore modelling and docking studies of GSK-3β and GK inhibitors of Hippophae

Type 2 diabetes is an inevitably progressive disease, with irreversible β cell failure. Glycogen synthase kinase and Glukokinase, two important enzymes with diverse biological actions in carbohydrate metabolism, are promising targets for developing novel antidiabetic drugs. A combinatorial structure-based molecular docking and pharmacophore modelling study was performed with the compounds of Hippophae salicifolia and H. rhamnoides as inhibitors. Docking with Discovery Studio 3.5 revealed that two compounds from H. salicifolia, viz Lutein D and an analogue of Zeaxanthin, and two compounds from H. rhamnoides, viz Isorhamnetin-3-rhamnoside and Isorhamnetin-7-glucoside, bind significantly to the GSK-3 β receptor and play a role in its inhibition; whereas in the case of Glucokinase, only one compound from both the plants, i.e. vitamin C, had good binding characteristics capable of activation. The results help to understand the type of interactions that occur between the ligands and the receptors. Toxicity predictions revealed that none of the compounds had hepatotoxic effects and had good absorption as well as solubility characteristics. The compounds did not possess plasma protein-binding, crossing blood–brain barrier ability. Further, in vivo and in vitro studies need to be performed to prove that these compounds can be used effectively as antidiabetic drugs.     

Characterization of tyrosinase inhibitory constituents from the aerial parts of Humulus japonicus using LC-MS/MS coupled online assay

In the screening of natural products for the development as cosmetic ingredients, the EtOAc-soluble fraction of Humulus japonicus showed tyrosinase inhibitory activity. HPLC-MS/MS coupled online tyrosinase assay of EtOAc-soluble fraction of H. japonicus characterized the twenty-eight constituents including two unknown ones and their tyrosinase inhibitory activity. Fractionation of H. japonicus using various chromatographic techniques yielded thirty-eight compounds. The chemical structures of isolated compounds were identified by spectroscopic analysis. As characterized by HPLC-MS/MS analysis, we isolated twenty-four predicted compounds and further identified two unknown ones, named humulusides A (1) and B (2). Additional ten compounds were also identified by purification. Tyrosinase inhibitory activity of isolated compounds were evaluated, which was closely correlated with the results from HPLC-MS/MS coupled online tyrosinase assay. Consistent with predicted data, two major compounds, trans-N-coumaroyltyramine (14) and cis-N-coumaroyltyramine (15) showed tyrosinase inhibition with IC₅₀ values of 40.6 and 36.4?μM. Taken together, H. japonicus is suggested as whitening ingredient in cosmetic products. In addition, HPLC-MS/MS coupled tyrosinase assay is powerful tool for predicting active compounds with short time and limited amounts, although identification of new compounds and verification of predicted data are also needs to be demonstrated by further experiment.     

Human acidic mammalian chitinase as a novel target for anti-asthma drug design using in silico screening

Human acidic mammalian chitinase (hAMCase) was recently shown to be involved in the development of asthma, suggesting a possible application for hAMCase inhibitors as novel therapeutic agents for asthma. We therefore initiated drug discovery research into hAMCase using a combination of in silico methodologies and a hAMCase assay system. We first selected 23 candidate hAMCase inhibitors from a database of four million compounds using a multistep screening system combining Tripos Topomer Search technology, a docking calculation and two-dimensional molecular similarity analysis. We then measured hAMCase inhibitory activity of the selected compounds and identified seven compounds with IC₅₀ values ?100 μM. A model describing the binding modes of these hit compounds to hAMCase was constructed, and we discuss the structure–activity relationships of the compounds we identified, suggested by the model and the actual inhibitory activities of the compounds.     

Promising antiparasitic agents from marine sponges

Parasitic diseases especially those prevail in tropical and subtropical regions severely threaten the lives of people due to available drugs found to be ineffective as several resistant strains have been emerged. Due to the complexity of the marine environment, researchers considered it as a new field to search for compounds with therapeutic efficacy, marine sponges represents the milestone in the discovery of unique compounds of potent activities against parasitic infections. In the present article, literatures published from 2010 until March 2021 were screened to review antiparasitic potency of bioactive compounds extracted from marine sponges. 45 different genera of sponges have been studied for their antiparasitic activities. The antiparasitic activity of the crude extract or the compounds that have been isolated from marine sponges were assayed in vitro against Plasmodium falciparum, P. berghei, Trypanosoma brucei rhodesiense, T. b. brucei, T. cruzi, Leishmania donovani, L. tropica, L. infantum, L. amazonesis, L. major, L. panamesis, Haemonchus contortus and Schistosoma mansoni. The majority of antiparastic compounds extracted from marine sponges were related to alkaloids and peroxides represent the second important group of antiparasitic compounds extracted from sponges followed by terpenoids. Some substances have been extracted and used as antiparasitic agents to a lesser extent like steroids, amino acids, lipids, polysaccharides and isonitriles. The activities of these isolated compounds against parasites were screened using in vitro techniques. Compounds' potent activity in screened papers was classified in three categories according to IC₅₀: low active or inactive, moderately active and good potent active.     

The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein

The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. Although it is clear that the monosaccharides require binding to a periplasmic binding protein before they can interact with the sensor VirA protein, it is not certain whether the phenolic compounds also interact with a binding protein or directly interact with the sensor protein. To shed light on this question, we tested the vir-inducing abilities of several different phenolic compounds using two wild-type strains of A. tumefaciens, KU12 and A6. We found that several compounds such as 4-hydroxyacetophone and p-coumaric acid induced the vir of KU12, but not A6. On the other hand, acetosyringone and several other phenolic compounds induced the vir of A6, but not KU12. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic sensing determinant is associated with the Ti plasmid. Subcloning of the Ti plasmid indicated that the virA locus determines which phenolic compounds can function as vir inducers. These results suggest that VirA directly senses the phenolic compounds for vir activation.     

Identification of Antifungal Compounds Active against Candida albicans Using an Improved High-Throughput Caenorhabditis elegans Assay

Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans–C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay.     

Design,synthesis, biological and in silico evaluation of coumarin-hydrazone derivatives as tubulin targeted antiproliferative agents

Coumarin-based different series of hydrazone derivatives were synthesized and evaluated for anticancer activity against four different human cancer cell lines. The activity of the compounds were compared with doxorubicin as a standard drug and all the compounds exhibited good to moderate cytotoxicity with IC₅₀ values ranging from 6.07 to 60.45 µM against all the examined cancer cell lines. Based on the screening results, it was concluded that the compounds 12a and 18a were the most promising medicinal entities. In vitro tubulin polymerisation inhibition assay was performed for the compounds 12a and 18a and these two compounds displayed good potency when compared with colchicine as the standard drug. The interaction of these compounds with tubulin protein was also studied with the help of molecular docking technique using Discovery studio software. Furthermore, the molecular and ADMET properties of the compounds were computed with Osiris property software and PreADMET server. The compounds exhibited exciting in vitro and in silico results. Hence we propose that the compounds 12a and 18a could be developed as tubulin targeted potential antiproliferative agents.     

Derivatives of aryl amines containing the cytotoxic 1,4-dioxo-2-butenyl pharmacophore

Several series of compounds containing the 1,4-dioxo-2-butenyl moiety have been prepared as candidate cytotoxins, including the methyl N-arylmaleamates, methyl N-arylfumaramates, and N-arylmaleimides. In addition, the N-arylisomaleimides were synthesized which are the structural isomers of N-arylmaleimides. These compounds were evaluated against human Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 cells. Methyl N-arylfumaramates showed the highest cytotoxic potencies and, in particular, methyl N-(3,4-dichlorophenyl)fumaramate is six times more potent than melphalan towards L1210 cells and is equipotent with this drug in the Molt 4/C8 assay. Electrophilicity of compounds under investigation was demonstrated by carrying out thiolation using model benzyl mercaptan on representative compounds. Methyl N-(3,4-dichlorophenyl)fumaramate and methyl N-(4-chlorophenyl)maleamate inhibited human N-myristoyltransferase, a possible molecular target, in high micromolar range. QSAR and molecular modeling revealed some correlations between different structural features of a number of the molecules and cytotoxic potencies. Methyl N-arylfumaramates were well tolerated in mice in comparison to the analogs in other series of compounds tested. The data obtained in this investigation affords guidelines for preparing new series of molecules with greater potencies.     

Algicidal metabolites produced by Bacillus sp. strain B1 against Phaeocystis globosa

The bloom of Phaeocystis globosa has broken out frequently in the coastal areas of China in recent years, which has led to substantial economic losses. This study shows that Bacillus sp. strain B1, which was previously identified by our group, is effective in regulating P. globosa by excreting active metabolites. Heat stability, pH stability and molecular weight range of the algicidal compounds from strain B1 were measured and the results demonstrated that the algicidal activities of these compounds were not affected by pH or temperature variation. The algicidal compounds extracted with methanol were isolated and purified by ODS-A column chromatography and HPLC. The algicidal compounds corresponding to peaks 2–5 eluted from HPLC were further analysed by quadrupole time-of-flight mass spectrometry (Q-TOF–MS). PeakView? Software determined the compounds corresponding to peaks 2–5 to be l-histidine, o-tyrosine, N-acetylhistamine and urocanic acid on the basis of the accurate mass information, the isotopic pattern and MS–MS spectra. Furthermore, these compounds were also able to eliminate Skeletonema costatum, Prorocentrum donghaiense and Heterosigma akashiwo. This is the first report of bacteria-derived algicidal compounds being identified only by Q-TOF–MS and PeakView? Software, and these compounds may be used as the constituents of algicides in the future.     

N-Coronafacoyl-L-isoleucine and N-coronafacoyl-L-alloisoleucine,potential biosynthetic intermediates of the phytotoxin coronatine

Two new naturally-occurring analogues of the phytotoxin coronatine have been isolated from liquid cultures of Pseudomonas syringae pv. glycinea. These have been identified as N-coronafacoyl-L-isoleucine and N-coronafacoyl-L-alloisoleucine by mass spectrometry and by studies of the products of acid hydrolysis of the two compounds. The compounds were purified as a mixture of ca 2:1 composition, but the two parent components were not preparatively separated. The possible significance of the two compounds, to the biosynthesis of coronatine, is discussed.     

A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs

Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as K_D, k_on, k_off, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand–p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in T_m values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.     

English cum, a borrowed coordinator turned complex-compound marker

This article takes issue with the traditional view of English compounds such as governess-cum-piano-teacher, according to which the medial morpheme -cum- is insignificant. The study is first centered on the appearance of the linking element in the English language. New insight into its distribution and function is then provided by scrutinizing a list of 259 compounds extracted from a present-day newspaper corpus. It is found that -cum- appears exclusively in non-institutionalized coordinate nominal and adjectival compounds and that it plays a distinctive role which sets -cum- compounds apart from asyndetic compounds: the linking element is predominantly used in complex compounds to simultaneously mark the internal boundary (boundaries) within the construction and the coordinate relation that holds between the compounding elements. The discussion finally focuses on the status of -cum-, which appears to be a hybrid syntactic-morphological unit of present-day English.     

Repurposing approach identifies new treatment options for invasive fungal disease

Drug repositioning is the process of discovery, validation and marketing of previously approved drugs for new indications. Our aim was drug repositioning, using ligand-based and structure-based computational methods, of compounds that are similar to two hit compounds previously selected by our group that show promising antifungal activity. Through the ligand-based method, 100 compounds from each of three databases (MDDR, DrugBank and TargetMol) were selected by the Tanimoto coefficient, as similar to LMM5 or LMM11. These compounds were analyzed by the scaffold trees, and up to 10 compounds from each database were selected. The structure-based method (molecular docking) using thioredoxin reductase as the target drug was performed as a complementary approach, resulting in six compounds that were tested in an in vitro assay. All compounds, particularly raltegravir, showed antifungal activity against the genus Paracoccidioides. Raltegravir, an antiviral drug, showed promising antifungal activity against the experimental murine paracoccidioidomycosis, with significant reduction of the fungal burden and decreased alterations in the lung structure of mice treated with 1 mg/kg of raltegravir. In conclusion, the combination of two in silico methods for drug repositioning was able to select an antiviral drug with promising antifungal activity for treatment of paracoccidioidomycosis.     

Phenolic compounds from Bursera simaruba Sarg. bark: Phytochemical investigation and quantitative analysis by tandem mass spectrometry

Phytochemical investigation of the methanolic extract of Bursera simaruba bark led to the isolation of 11 compounds, including lignans yatein, β-peltatin-O-β-d-glucopyranoside, hinokinin and bursehernin, and three natural compounds namely 3,4-dimetoxyphenyl-1-O-β-d-(6-sulpho)-glucopyranoside, 3,4,5-trimetoxyphenyl 1-O-β-d-(6-sulpho)-glucopyranoside and 3,4-diidroxyphenylethanol-1-O-β-d-(6-sulpho)-glucopyranoside. Their structures were established by NMR and ESI/MS experiments. Additionally, an LC-ESI/MS qualitative study on the phenolic compounds and an LC-ESI/MS/MS quantitative study on the lignans found in the methanolic extract of B. simaruba bark were performed to give value to the plant as source of these biological active compounds. Quantitative analyses results confirmed that compounds yatein, β-peltatin-O-β-d-glucopyranoside, hinokinin and bursehernin are major compounds in the bark and, in particular, β-peltatin-O-β-d-glucopyranoside appears to be the most abundant.