A multidisciplinary approach disclosing unexplored Aflatoxin B1 roles in severe impairment of vitamin D mechanisms of action.

Aflatoxin B1 (AFB1), produced by fungi of the genus Aspergillus, is the most toxic and carcinogenic mycotoxin among the classes of aflatoxins. Previous research showed that AFB1 affects vitamin D receptor (VDR) expression. In the present study, integrated computational and experimental studies were carried out to investigate how AFB1 can interfere with Vitamin D signalling. A competitive antagonism of AFB1 toward RXRα and VDR was hypothesized by comparing the docked complex of AFB1/RXRα and AFB1/VDR ligand-binding domain (LBD) with the X-ray structures of RXRα and VDR bound to known ligands. Accordingly, we demonstrated that AFB1 can affect vitamin D-mediated transcriptional activation of VDR by impairing the formation of protein complexes containing both VDR-RXRα and RXRα/RAR and affecting the subcellular localization of VDR and RXRα. As a whole, our data indicate that AFB1 can interfere with different molecular pathways triggered by vitamin D with an antagonistic mechanism of action.

Integrated assessment of personal monitor applications for evaluating exposure to urban stressors: A scoping review.

Urban stressors pose a health risk, and individual-level assessments provide necessary and fine-grained insight into exposure. An ever-increasing amount of research literature on individual-level exposure to urban stressors using data collected with personal monitors, has called for an integrated assessment approach to identify trends, gaps and needs, and provide recommendations for future research. To this end, a scoping review of the respective literature was performed, as part of the H2020 URBANOME project. Moreover, three specific aims were identified: (i) determine current state of research, (ii) analyse literature according with a waterfall methodological framework and identify gaps and needs, and (iii) provide recommendations for more integrated, inclusive and robust approaches. Knowledge and gaps were extracted based on a systematic approach, e.g., data extraction questionnaires, as well as through the expertise of the researchers performing the review. The findings were assessed through a waterfall methodology of delineating projects into four phases. Studies described in the papers vary in their scope, with most assessing exposure in a single macro domain, though a trend of moving towards multi-domain assessment is evident. Simultaneous measurements of multiple stressors are not common, and papers predominantly assess exposure to air pollution. As urban environments become more diverse, stakeholders from different groups are included in the study designs. Most frequently (per the quadruple helix model), civil society/NGO groups are involved, followed by government and policymakers, while business or private sector stakeholders are less frequently represented. Participants in general function as data collectors and are rarely involved in other phases of the research. While more active involvement is not necessary, more collaborative approaches show higher engagement and motivation of participants to alter their lifestyles based on the research results. The identified trends, gaps and needs can aid future exposure research and provide recommendations on addressing different urban communities and stakeholders.

Tetrasubstituted Pyrrole Derivative Mimetics of Protein-Protein Interaction Hot-Spot Residues: A Promising Class of Anticancer Agents Targeting Melanoma Cells.

A new series of tetrasubstituted pyrrole derivatives (TSPs) was synthesized based on a previously developed hypothesis on their ability to mimic hydrophobic protein motifs. The resulting new TSPs were endowed with a significant toxicity against human epithelial melanoma A375 cells, showing IC50 values ranging from 10 to 27 µM, consistent with the IC50 value of the reference compound nutlin-3a (IC50 = 15 µM). In particular, compound 10a (IC50 = 10 µM) resulted as both the most soluble and active among the previous and present TSPs. The biological investigation evidenced that the anticancer activity is related to the activation of apoptotic cell-death pathways, supporting our rational design based on the ability of TSPs to interfere with PPI involved in the cell cycle regulation of cancer cells and, in particular, the p53 pathway. A reinvestigation of the TSP pharmacophore by using DFT calculations showed that the three aromatic substituents on the pyrrole core are able to mimic the hydrophobic side chains of the hot-spot residues of parallel and antiparallel coiled coil structures suggesting a possible molecular mechanism of action. A structure-activity relationship (SAR) analysis which includes solubility studies allows us to rationalize the role of the different substituents on the pyrrole core.

Role of Stereochemistry on the Biological Activity of Nature-Inspired 3-Br-Acivicin Isomers and Derivatives.

Chiral natural compounds are often biosynthesized in an enantiomerically pure fashion, and stereochemistry plays a pivotal role in biological activity. Herein, we investigated the significance of chirality for nature-inspired 3-Br-acivicin (3-BA) and its derivatives. The three unnatural isomers of 3-BA and its ester and amide derivatives were prepared and characterized for their antimalarial activity. Only the (5S, αS) isomers displayed significant antiplasmodial activity, revealing that their uptake might be mediated by the L-amino acid transport system, which is known to mediate the acivicin membrane's permeability. In addition, we investigated the inhibitory activity towards Plasmodium falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) since it is involved in the multitarget mechanism of action of 3-BA. Molecular modeling has shed light on the structural and stereochemical requirements for an efficient interaction with PfGAPDH, leading to covalent irreversible binding and enzyme inactivation. While stereochemistry affects the target binding only for two subclasses (1a-d and 4a-d), it leads to significant differences in the antimalarial activity for all subclasses, suggesting that a stereoselective uptake might be responsible for the enhanced biological activity of the (5S, αS) isomers.

Silybins are stereospecific regulators of the 20S proteasome.

A reduced proteasome activity tiles excessive amyloid growth during the progress of protein conformational diseases (PCDs). Hence, the development of safe and effective proteasome enhancers represents an attractive target for the therapeutic treatment of these chronic disorders. Here we analyze two natural diastereoisomers belonging to the family of flavonolignans, Sil A and Sil B, by evaluating their capacity to increase proteasome activity. Enzyme assays carried out on yeast 20S (y20S) proteasome and in parallel on a permanently "open gate" mutant (α3ΔN) evidenced that Sil B is a more efficient 20S activator than Sil A. Conversely, in the case of human 20S proteasome (h20S) a higher affinity and more efficient activation is observed for Sil A. Driven by experimental data, computational studies further demonstrated that the taxifolin group of both diastereoisomers plays a crucial role in their anchoring to the α5/α6 groove of the outer α-ring. However, due to the different stereochemistry at C-7" and C-8" of ring D, only Sil A was able to reproduce the interactions responsible for h20S proteasome activation induced by their cognate regulatory particles. The provided silybins/h20S interaction models allowed us to rationalize their different ability to activate the peptidase activities of h20S and y20S. Our results provide structural details concerning the important role played by stereospecific interactions in driving Sil A and Sil B binding to the 20S proteasome and may support future rational design of proteasome enhancers.

Investigating the Antiparasitic Potential of the Marine Sesquiterpene Avarone, Its Reduced form Avarol, and the Novel Semisynthetic Thiazinoquinone Analogue Thiazoavarone.

The chemical analysis of the sponge Dysidea avara afforded the known sesquiterpene quinone avarone, along with its reduced form avarol. To further explore the role of the thiazinoquinone scaffold as an antiplasmodial, antileishmanial and antischistosomal agent, we converted the quinone avarone into the thiazinoquinone derivative thiazoavarone. The semisynthetic compound, as well as the natural metabolites avarone and avarol, were pharmacologically investigated in order to assess their antiparasitic properties against sexual and asexual stages of Plasmodium falciparum, larval and adult developmental stages of Schistosoma mansoni (eggs included), and also against promastigotes and amastigotes of Leishmania infantum and Leishmania tropica. Furthermore, in depth computational studies including density functional theory (DFT) calculations were performed. A toxic semiquinone radical species which can be produced starting both from quinone- and hydroquinone-based compounds could mediate the anti-parasitic effects of the tested compounds.

Cooperative Binding of the Cationic Porphyrin Tris-T4 Enhances Catalytic Activity of 20S Proteasome Unveiling a Complex Distribution of Functional States.

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.

Exploring the Photodynamic Properties of Two Antiproliferative Benzodiazopyrrole Derivatives.

The identification of molecules whose biological activity can be properly modulated by light is a promising therapeutic approach aimed to improve drug selectivity and efficacy on the molecular target and to limit the side effects compared to traditional drugs. Recently, two photo-switchable diastereomeric benzodiazopyrrole derivatives 1RR and 1RS have been reported as microtubules targeting agents (MTAs) on human colorectal carcinoma p53 null cell line (HCT 116 p53-/-). Their IC50 was enhanced upon Light Emitting Diode (LED) irradiation at 435 nm and was related to their cis form. Here we have investigated the photo-responsive behavior of the acid derivatives of 1RR and 1RS, namely, d1RR and d1RS, in phosphate buffer solutions at different pH. The comparison of the UV spectra, acquired before and after LED irradiation, indicated that the trans→cis conversion of d1RR and d1RS is affected by the degree of ionization. The apparent rate constants were calculated from the kinetic data by means of fast UV spectroscopy and the conformers of the putative ionic species present in solution (pH range: 5.7-8.0) were modelled. Taken together, our experimental and theoretical results suggest that the photo-conversions of trans d1RR/d1RS into the corresponding cis forms and the thermal decay of cis d1RR/d1RS are dependent on the presence of diazonium form of d1RR/d1RS. Finally, a photo-reaction was detected only for d1RR after prolonged LED irradiation in acidic medium, and the resulting product was characterized by means of Liquid Chromatography coupled to High resolution Mass Spectrometry (LC-HRMS) and Nuclear Magnetic Resonance (NMR) spectroscopy.

Antiplasmodial Activity of p-Substituted Benzyl Thiazinoquinone Derivatives and Their Potential against Parasitic Infections.

Malaria is a life-threatening disease and, what is more, the resistance to available antimalarial drugs is a recurring problem. The resistance of Plasmodium falciparum malaria parasites to previous generations of medicines has undermined malaria control efforts and reversed gains in child survival. This paper describes a continuation of our ongoing efforts to investigate the effects against Plasmodium falciparum strains and human microvascular endothelial cells (HMEC-1) of a series of methoxy p-benzyl-substituted thiazinoquinones designed starting from a pointed antimalarial lead candidate. The data obtained from the newly tested compounds expanded the structure-activity relationships (SARs) of the thiazinoquinone scaffold, indicating that antiplasmodial activity is not affected by the inductive effect but rather by the resonance effect of the introduced group at the para position of the benzyl substituent. Indeed, the current survey was based on the evaluation of antiparasitic usefulness as well as the selectivity on mammalian cells of the tested p-benzyl-substituted thiazinoquinones, upgrading the knowledge about the active thiazinoquinone scaffold.

Exploring the antimalarial potential of the methoxy-thiazinoquinone scaffold: Identification of a new lead candidate.

A small library of antiplasmodial methoxy-thiazinoquinones, rationally designed on the model of the previously identified hit 1, has been prepared by a simple and inexpensive procedure. The synthetic derivatives have been subjected to in vitro pharmacological screening, including antiplasmodial and toxicity assays. These studies afforded a new lead candidate, compound 9, endowed with higher antiplasmodial potency compared to 1, a good selectivity index when tested against a panel of mammalian cells, no toxicity against RBCs, a synergistic antiplasmodial action in combination with dihydroartemisinin, and a promising inhibitory activity on stage V gametocyte growth. Computational studies provided useful insights into the structural requirements needed for the antiplasmodial activity of thiazinoquinone compounds and on their putative mechanism of action.

Computer-Aided Drug Discovery from Marine Compounds: Identification of the Three-Dimensional Structural Features Responsible for Antimalarial Activity.

An integrated computational approach, based on molecular dynamics/mechanics, semi-empirical, and DFT calculations as well as dynamic docking studies, has been employed to gain insight into the mechanism of action of new antimalarial agents characterized by the scaffold of the marine compounds plakortin and aplidinone. The results of this approach show that these molecules, after interaction with Fe(II), likely coming from the heme molecule, give rise to the formation of radical species, that should represent the toxic intermediates responsible for subsequent reactions leading to plasmodium death. The three-dimensional structural requirements necessary for the activity of these new classes of antimalarial agents have been identified and discussed throughout the chapter.

Insight into the Mechanism of Action of Marine Cytotoxic Thiazinoquinones.

The electrochemical response of four natural cytotoxic thiazinoquinones isolated from the Aplidium species was studied using conventional solution-phase and solid-state techniques, based on the voltammetry of immobilized particles methodology. The interaction with O2 and electrochemically generated reactive oxygen species (ROS) was electrochemically monitored. At the same time, a molecular modeling study including density functional theory (DFT) calculations was performed in order to analyze the conformational and electronic properties of the natural thiazinoquinones, as well as those of their reduced intermediates. The obtained electrochemical and computational results were analyzed and correlated to cytotoxic activity of these compounds, highlighting some features possibly related to their mechanism of action.

(18)F-FET and (18)F-FCH uptake in human glioblastoma T98G cell lines.

BACKGROUND: Despite complex treatment of surgery, radiotherapy and chemotherapy, high grade gliomas often recur. Differentiation between post-treatment changes and recurrence is difficult. (18)F-methyl-choline ((18)F-FCH) is frequently used in staging and detection of recurrent prostate cancer disease as well as some brain tumours; however accumulation in inflammatory tissue limits its specificity. The (18)F-ethyl-tyrosine ((18)F-FET) shows a specific uptake in malignant cells, resulting from increased expression of amino acid transporters or diffusing through the disrupted blood-brain barrier. (18)F-FET exhibits lower uptake in machrophages and other inflammatory cells. Aim of this study was to evaluate (18)F-FCH and (18)F-FET uptake by human glioblastoma T98G cells. MATERIAL AND METHODS: Human glioblastoma T98G or human dermal fibroblasts cells, seeded at a density to obtain 2 × 10(5) cells per flask when radioactive tracers were administered, grew adherent to the plastic surface at 37°C in 5% CO2 in complete medium. Equimolar amounts of radiopharmaceuticals were added to cells for different incubation times (20 to 120 minutes) for (18)F-FCH and (18)F-FET respectively. The cellular radiotracer uptake was determined with a gamma counter. All experiments were carried out in duplicate and repeated three times. The uptake measurements are expressed as the percentage of the administered dose of tracer per 2 × 10(5) cells. Data (expressed as mean values of % uptake of radiopharmaceuticals) were compared using parametric or non-parametric tests as appropriate. Differences were regarded as statistically significant when p<0.05. RESULTS: A significant uptake of (18)F-FCH was seen in T98G cells at 60, 90 and 120 minutes. The percentage uptake of (18)F-FET in comparison to (18)F-FCH was lower by a factor of more than 3, with different kinetic curves.(18)F-FET showed a more rapid initial uptake up to 40 minutes and (18)F-FCH showed a progressive rise reaching a maximum after 90 minutes. CONCLUSIONS: (18)F-FCH and (18)F-FET are candidates for neuro-oncological PET imaging. (18)F-FET could be the most useful oncological PET marker in the presence of reparative changes after therapy, where the higher affinity of (18)F-FCH to inflammatory cells makes it more difficult to discriminate between tumour persistence and non-neoplastic changes. Additional studies on the influence of inflammatory tissue and radionecrotic cellular components on radiopharmaceutical uptake are necessary.

(99m)Tc-human serum albumin nanocolloids: particle sizing and radioactivity distribution.

Several parameters affect the biodistribution of administered nanocolloids (NC) for Sentinel Lymph Node (SLN) detection: particle size distribution, number of Tc atoms per particle and specific activity (SA). Relatively few data are available with frequently conflicting results. (99m)Tc-NC-human serum albumin (HSA) Nanocoll®, Nanoalbumon® and Nanotop® were analysed for particles' dimensional and radioactivity distribution, and a mathematical model was elaborated to estimate the number of particles involved. Commercially available kits were reconstituted at maximal SA of 11 MBq/µg HSA. Particles size distribution was evaluated by Dynamic Light Scattering. These data were related to the radioactivity distribution analysis passing labelled NC through three polycarbonate filters (15-30-50-nm pore size) under vacuum. Highest radioactivity was carried by 30-50 nm particles. The smallest ones, even though most numerous, carried only the 10% of (99m)Tc atoms. Nanocoll and Nanotop are not significantly different, while Nanoalbumon is characterized by largest particles (>30 nm) that carried the most of radioactivity (80%). Smallest particles could saturate the clearing capacity of macrophages; therefore, if the tracer is used for SLN detection, more node tiers could be visualized, reducing accuracy of SLN mapping. Manufacturers could implement technical leaflets with particle size distribution and could improve the labelling protocol to provide clinicians useful information.

Endoperoxide polyketides from a Chinese Plakortis simplex: further evidence of the impact of stereochemistry on antimalarial activity of simple 1,2-dioxanes.

Chemical investigation of the organic extract obtained from the sponge Plakortis simplex collected in the South China Sea afforded five new polyketide endoperoxides (2 and 4-7), along with two known analogues (1 and 3). The stereostructures of these metabolites have been deduced on the basis of spectroscopic analysis and chemical conversion. The isolated endoperoxide derivatives have been tested for their in vitro antimalarial activity against Plasmodium falciparum strains, showing IC50 values in the low micromolar range. The structure-activity relationships were analyzed by means of a detailed computational investigation and rationalized in the light of the mechanism of action proposed for this class of simple antimalarials. The relative orientation of the atoms involved in the putative radical generation and transfer reaction was demonstrated to have a great impact on the antimalarial activity. The resulting 3D pharmacophoric model can be a useful guide to design simple and effective antimalarial lead compounds belonging to the class of 1,2-dioxanes.

Using small molecules to overcome drug resistance induced by a viral oncogene.

We used small molecule screening to discover compounds and mechanisms for overcoming E6 oncogene-mediated drug resistance. Using high-throughput screening in isogenic cell lines, we identified compounds that potentiate doxorubicin's lethality in E6-expressing colon cancer cells. Such compounds included quaternary ammonium salts, protein synthesis inhibitors, 11-deoxyprostaglandins, and two additional classes of compounds-analogs of 1,3-bis(4-morpholinylmethyl)-2-imidazolidinethione (a thiourea) and acylated secondary amines that we named indoxins. Indoxins upregulated topoisomerase IIalpha, the target of doxorubicin, thereby increasing doxorubicin lethality. We developed a photolabeling strategy to identify targets of indoxin and discovered a nuclear actin-related protein complex as a candidate indoxin target.

Discovery of a spirocyclic 3-bromo-4,5-dihydroisoxazole covalent inhibitor of hGAPDH with antiproliferative activity against pancreatic cancer cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme, plays a crucial role in the energy metabolism of cancer cells and has been proposed as a valuable target for the development of anticancer agents. Among a series of 5-substituted 3-bromo-4,5-dihydroisoxazole (BDHI) derivatives, we identified the spirocyclic compound 11, which is able to covalently inactivate recombinant human GAPDH (hGAPDH) with a faster reactivity than koningic acid, one of the most potent hGAPDH inhibitors known to date. Computational studies confirmed that conformational rigidification is crucial to stabilize the interaction of the inhibitor with the binding site, thus favoring the subsequent covalent bond formation. Investigation of intrinsic warhead reactivity at different pH disclosed the negligible reactivity of 11 with free thiols, highlighting its ability to selectively react with the activated cysteine of hGAPDH with respect to other sulfhydryl groups. Compound 11 strongly reduced cancer cell growth in four different pancreatic cancer cell lines and its antiproliferative activity correlated well with the intracellular inhibition of hGAPDH. Overall, our results qualify 11 as a potent hGAPDH covalent inhibitor with a moderate drug-like reactivity that could be further exploited to develop anticancer agents.

The Insulin-Degrading Enzyme from Structure to Allosteric Modulation: New Perspectives for Drug Design.

The insulin-degrading enzyme (IDE) is a Zn2+ peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the ß-amyloid (Aß) peptide. Therefore, a role for the IDE in the cure of diabetes and Alzheimer's disease (AD) has been long envisaged. Anyway, its role in degrading amyloidogenic proteins remains not clearly defined and, more recently, novel non-proteolytic functions of the IDE have been proposed. From a structural point of view, the IDE presents an atypical clamshell structure, underscoring unique enigmatic enzymological properties. A better understanding of the structure-function relationship may contribute to solving some existing paradoxes of IDE biology and, in light of its multifunctional activity, might lead to novel therapeutic approaches.

Electronic Circular Dichroism Detects Conformational Changes Associated with Proteasome Gating Confirmed Using AFM Imaging.

Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and ß-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and ß-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators.