Performance evaluation of a 3D-printed sharp-cut cyclone.

A sharp-cut cyclone with an aerodynamic cut-off diameter of 1 µm, when operated at a flow rate of 1 L min-1, was built by 3D-printing and tested against a metallic (aluminum) counterpart having the same design and dimensions. The penetration efficiency of both cyclones was experimentally determined using quasi-monodisperse aerosol particles having aerodynamic diameters from ca. 100 nm to 2 µm. The aerodynamic cut-off diameter for both cyclones was very similar and in accordance with the expected design value. The penetration efficiency curve of the 3D-printed cyclone was less steep compared to that of its metallic counterpart. This difference is most likely attributed to the higher surface roughness of the inner parts of the 3D-printed cyclone - as also indicated by the greater pressure drop it exhibits compared to the aluminum cyclone when operated at the same flow rate - and not by higher deviations from its design dimensions resulting from the tolerances of the 3D printer. Despite that, the substantially low cost, speed, and ease of manufacturing, make the 3D-printed cyclone a highly promising solution for applications in aerosol metrology.

New TRAP1 and Hsp90 chaperone inhibitors with cationic components: Preliminary studies on mitochondrial targeting.

TRAP1 (Hsp75) is the mitochondrial paralog of the Hsp90 molecular chaperone family. Due to structural similarity among Hsp90 chaperones, a potential strategy to induce apoptosis through mitochondrial TRAP1 ATPase inhibition has been envisaged and a series of compounds has been developed by binding the simple pharmacophoric core of known Hsp90 inhibitors with various appendages bearing a permanent cationic head, or a basic group highly ionizable at physiologic pH. Cationic appendages were selected as vehicles to deliver drugs to mitochondria. Indeed, masses of new derivatives were evidenced to accumulate in the mitochondrial fraction from colon carcinoma cells and a compound in the series, with a guanidine appendage, demonstrated good activity in inhibiting recombinant TRAP1 ATPase and cell growth and in inducing apoptotic cell death in colon carcinoma cells.

2'-Deoxyuridine 5'-monophosphate substrate displacement in thymidylate synthase through 6-hydroxy-2H-naphtho[1,8-bc]furan-2-one derivatives.

Thymidylate synthase (TS) is a target for antifolate-based chemotherapies of microbial and human diseases. Here, ligand-based, synthetic, and X-ray crystallography studies led to the discovery of 6-(3-cyanobenzoyloxy)-2-oxo-2H-naphto[1,8-bc]furan, a novel inhibitor with a Ki of 310 nM against Pneumocystis carinii TS. The X-ray ternary complex with Escherichia coli TS revealed, for the first time, displacement of the substrate toward the dimeric protein interface, thus providing new opportunities for further design of specific inhibitors of microbial pathogens.

Translational repression of thymidylate synthase by targeting its mRNA.

Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.

Ligand-based discovery of N-(1,3-dioxo-1H,3H-benzo[de]isochromen-5-yl)-carboxamide and sulfonamide derivatives as thymidylate synthase A inhibitors.

Phenolnaphthalein derivatives show potential for pharmacological activity as inhibitors of thymidylate synthase (TS) but difficulties in their synthesis and derivatization hinder their development. A deconstruction approach aimed at identifying a suitable new scaffold was proposed. A new scaffold was identified and two compound libraries based on this scaffold were designed. The carboxamide library (Library B) showed specific inhibition activity against Escherichia coli TS, whereas the sulfonamide library (Library C) showed a non-specific inhibition profile against hTS. N-(1,3-Dioxo-1H,3H-benzo[de]isochromen-5-yl)-sulfonamide derivatives, 1C and 9C, showed one order of magnitude improvement in inhibition constant against hTS with respect to the starting lead and represent potential compounds for further lead development.

Biochemical effects of riluzole on Leishmania parasites.

We have previously shown that riluzole (6-(trifluoromethoxy)benzothiazol-2-amine), an agent used to treat CNS disorders, possesses inhibitory activity against pteridine reductase (PTR1) in pathogenic protists at low micromolar concentrations. Therefore, the potential use of this drug in anti-parasitic chemotherapy deserves evaluation. In this study, we report the effect of this compound on cell cultures of Leishmania mexicana and L. major. The anti-parasitic activity of riluzole was confirmed, with the largest effect observed when the drug was administered to cells during their exponential growth phase. Moreover, a remarkable decrease in PTR1 activity was observed in the lysates of cells pretreated with the compound, which is due to impairment of the enzyme's preferential reaction with biopterin as a cofactor. In addition, the treatment increased the parasites' susceptibility to oxidative stress, affecting the ability of Leishmania to survive under severe oxidative conditions. These results suggest that the inhibitory effect of riluzole on PTR1 is not the only mechanism through which it induces the death of Leishmania parasites.

Inhibitor of ovarian cancer cells growth by virtual screening: a new thiazole derivative targeting human thymidylate synthase.

Human thymidylate synthase (hTS) was targeted through a virtual screening approach. The most optimal inhibitor identified, 2-{4-hydroxy-2-[(2-hydroxybenzylidene)hydrazono]-2,5-dihydrothiazol-5-yl}-N-(3-trifluoromethylphenyl)acetamide (5), showed a mixed-type inhibition pattern, with a K(i) of 1.3 µM and activity against ovarian cancer cell lines with the same potency as cisplatin. X-ray studies revealed that it binds the inactive enzyme conformation. This study is the first example of a nonpeptidic inhibitor that binds the inactive hTS and exhibits anticancer activity against ovarian cancer cells.

Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase.

Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.

Identification of the binding modes of N-phenylphthalimides inhibiting bacterial thymidylate synthase through X-ray crystallography screening.

To identify specific bacterial thymidylate synthase (TS) inhibitors, we exploited phenolphthalein (PTH), which inhibits both bacterial and human enzymes. The X-ray crystal structure of Lactobacillus casei TS (LcTS) that binds PTH showed multiple binding modes of the inhibitor, which prevented a classical structure-based drug design approach. To overcome this issue, we synthesized two phthalimidic libraries that were tested against TS enzymes and then we performed X-ray crystallographic screening of the active compounds. Compounds 6A, 8A, and 12A showed 40-fold higher affinity for bacterial TS than human TS. The X-ray crystallographic screening characterized the binding mode of six inhibitors in complexes with LcTS. Of these, 20A, 23A, and 24A showed a common unique binding mode, whereas 8A showed a different, unique binding mode. A comparative analysis of the LcTS X-ray complexes that were obtained with the pathogenic TS enabled the selection of compounds 8A and 23A as specific compounds and starting points to be exploited for the specific inhibition of pathogen enzymes.

Virtual screening identification of nonfolate compounds, including a CNS drug, as antiparasitic agents inhibiting pteridine reductase.

Folate analogue inhibitors of Leishmania major pteridine reductase (PTR1) are potential antiparasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new molecules with specificity for PTR1, we carried out a virtual screening of the Available Chemicals Directory (ACD) database to select compounds that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like molecules with low micromolar affinities and high in vitro specificity profiles. Their efficacy against Leishmania species was studied in cultured cells of the promastigote stage, using the compounds both alone and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compounds showed efficacy only in combination. In toxicity tests against human fibroblasts, several compounds showed low toxicity. One compound, 5c (riluzole; 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine), a known drug approved for CNS pathologies, was active in combination and is suitable for early preclinical evaluation of its potential for label extension as a PTR1 inhibitor and antiparasitic drug candidate.

Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer.

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43' with an excitation energy donor/acceptor pair. The dimer-monomer equilibrium of the enzyme is then characterized through steady-state fluorescence determination of the intersubunit resonance energy transfer efficiency.

Homodimeric enzymes as drug targets.

Many enzymes and proteins are regulated by their quaternary structure and/or by their association in homo- and/or hetero-oligomer complexes. Thus, these protein-protein interactions can be good targets for blocking or modulating protein function therapeutically. The large number of oligomeric structures in the Protein Data Bank (http://www.rcsb.org/) reflects growing interest in proteins that function as multimeric complexes. In this review, we consider the particular case of homodimeric enzymes as drug targets. There is intense interest in drugs that inhibit dimerization of a functionally obligate homodimeric enzyme. Because amino acid conservation within enzyme interfaces is often low compared to conservation in active sites, it may be easier to achieve drugs that target protein interfaces selectively and specifically. Two main types of dimerization inhibitors have been developed: peptides or peptidomimetics based on sequences involved in protein-protein interactions, and small molecules that act at hot spots in protein-protein interfaces. Examples include inhibitors of HIV protease and HIV integrase. Studying the mechanisms of action and locating the binding sites of such inhibitors requires different techniques for different proteins. For some enzymes, ligand binding is only detectable in vivo or after unfolding of the complexes. Here, we review the structural features of dimeric enzymes and give examples of inhibition through interference in dimer stability. Several techniques for studying these complex phenomena will be presented.

Design and characterization of a mutation outside the active site of human thymidylate synthase that affects ligand binding.

Owing to its central role in DNA synthesis, human thymidylate synthase (hTS) is a well-established target for chemotherapeutic agents, such as fluoropyrimidines. The use of hTS inhibitors in cancer therapy is limited by their toxicity and the development of cellular drug resistance. Here, with the aim of shedding light on the structural role of the A-helix in fluoropyrimidine resistance, we have created a fluoropyrimidine-resistant mutant by making a single point mutation, Glu30Trp. We postulated that residue 30, which is located in the A-helix, close to but outside the enzyme active site, could have a long-range effect on inhibitor binding. The mutant shows 100 times lower specific activity with respect to the wild-type hTS and is resistant to the classical inhibitor, FdUMP, as shown by a 6-fold higher inhibition constant. Circular dichroism experiments show that the mutant is folded. The results of molecular modeling and simulation suggest that the Glu30Trp mutation gives rise to resistance by altering the hydrogen-bond network between residue 30 and the active site.

[The impact of obesity in the management and evolution of diabetes mellitus]. / El impacto de la obesidad en el manejo y evolución de la diabetes mellitus.

OBJECTIVES: To assess both management and evolution of diabetes mellitus type 2 (DM2) in Primary Care centers in Spain and the related factors, especially obesity. METHODOLOGY: Epidemiological, cross-sectional, multicenter, retrospective study. PATIENTS: Patients suffering from DM2, over 20 years of age, were consecutively enrolled from 30 Primary Care centers in 16 autonomous communities. Métodos. Data was collected on age, gender, educational level, DM2 duration, HbA1c, treatment and body measurement index (BMI). RESULTS: A total of 294 patients, 50% male, with a mean age (SD) of 67.5 years (10.2) and BMI 28.9 (4.5) kg/m(2) were included. Of them, 58.16% had HbA1c levels >6.5%, 38% being obese or severely obese. A total of 93.9% were under drug treatment for DM2. Significant differences in the mean value of HbA1c were shown between the over-weight and severely obese groups (Tukey-Kramer test). Differences were observed in the presence of macrovascular complications between patients with normal weight and patients with obesity (p=0.006). Patients with low educational level had 3.39 more probability of being obese or severely obese than patients with secondary school or university studies (p=0.0041; 95% CI 1.47-7.80), and patients with primary school 2.22 more probability (p= 0.038; 95% CI 1.04-4.73). A total of 47.8% reported high compliance. Obese and severely obese patients showed 2.2 more probability of having low or mild compliance than non-obese patients (p=0.002; 95% CI 1.31-3.74). CONCLUSIONS: Results obtained in this population suggest that obesity is related with more macro-vascular complications, worst metabolic control and worst compliance.

El impacto de la obesidad en el manejo y evolución de la diabetes mellitus / The impact of obesity in the management and evolution of diabetes mellitus

Objetivo. Estudiar el manejo y evolución de ladiabetes mellitus tipo 2 (DM2) en Atención Primaria(AP) en España y los factores implicados,especialmente la obesidad.Diseño. Estudio epidemiológico, transversal,multicéntrico, retrospectivo.Participantes. Se incluyeron pacientes con DM2mayores de 20 años seleccionadosconsecutivamente en 30 centros de AP, en16 comunidades autónomas (CCAA).Métodos. Se recogió información sobre edad, sexo,nivel educativo, duración de DM2, HbA1c,tratamiento e índice de masa corporal (IMC).Resultados. De un total de 294 pacientes, 50%hombres, con edad media (DE) 67,5 años (10,2) eIMC 28,9 (4,5) kg/m2, el 58,16% presentabanniveles de HbA1c >6,5%, el 38% era obeso oseveramente obeso. El 93,9% seguía tratamientofarmacológico para su diabetes. Se mostrarondiferencias significativas en el valor medio deHbA1c entre el grupo con sobrepeso y el grupocon obesidad severa (test de Tukey-Kramer).Se observaron diferencias en la presencia decomplicaciones macrovasculares entrepacientes con peso normal y pacientes obesos(p=0,006). Pacientes con menor grado dealfabetización mostraron 3,39 más probabilidad deser obesos o severamente obesos que pacientescon estudios secundarios o universitarios(p=0,0041; 95% intervalo de confianza [IC] 1,47-7,80), y los pacientes con estudios primarios 2,22veces más (p=0,038; 95% IC 1,04-4,73). Un47,8% refirieron un cumplimiento elevado. Losobesos y severamente obesos presentaron 2,2veces más probabilidad de presentar cumplimientobajo o moderado que los no obesos (p=0,002;95% IC 1,31-3,74 ).Conclusiones. Los resultados obtenidos en estapoblación sugieren que la variable obesidad serelaciona con más complicaciones macrovasculares,peor control metabólico y peor cumplimiento

Objectives. To assess both management andevolution of diabetes mellitus type 2 (DM2) inPrimary Care centers in Spain and the relatedfactors, especially obesity.Methodology. Epidemiological, cross-sectional,multicenter, retrospective study.Patients. Patients suffering from DM2, over 20years of age, were consecutively enrolled from 30Primary Care centers in 16 autonomouscommunities.Métodos. Data was collected on age, gender,educational level, DM2 duration, HbA1c, treatmentand body measurement index (BMI).Results. A total of 294 patients, 50% male, with amean age (SD) of 67.5 years (10.2) and BMI 28.9(4.5) kg/m2 were included. Of them, 58.16% hadHbA1c levels >6.5%, 38% being obese or severelyobese. A total of 93.9% were under drug treatmentfor DM2. Significant differences in the mean valueof HbA1c were shown between the over-weight andseverely obese groups (Tukey-Kramer test).Differences were observed in the presence ofmacrovascular complications between patients withnormal weight and patients with obesity (p=0.006).Patients with low educational level had 3.39 moreprobability of being obese or severely obese thanpatients with secondary school or university studies(p=0.0041; 95% CI 1.47-7.80), and patients withprimary school 2.22 more probability (p= 0.038;95% CI 1.04-4.73). A total of 47.8% reported highcompliance. Obese and severely obese patientsshowed 2.2 more probability of having low or mildcompliance than non-obese patients (p=0.002; 95%CI 1.31-3.74).Conclusions. Results obtained in this populationsuggest that obesity is related with more macrovascularcomplications, worst metabolic control andworst compliance

Comprehensive mechanistic analysis of hits from high-throughput and docking screens against beta-lactamase.

High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate "hit lists"; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against beta-lactamase using quantitative HTS (qHTS). Of the 1,274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting beta-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 microM. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens.

Synthesis of N-(5,7-diamino-3-phenyl-quinoxalin-2-yl)-3,4,5-substituted anilines and N-[4[(5,7-diamino-3-phenylquinoxalin-2-yl)amino]benzoyl]-l-glutamic acid diethyl ester: evaluation of in vitro anti-cancer and anti-folate activities.

Several diamino quinoxalines were designed, synthesized and evaluated as anti-tumor agents. Two compounds showed the most potent cytotoxic activities against the leukemia CCRF-CEM cell line (GI(50)<0.01microM) and the ovarian cancer cell line OVCAR-4 (GI(50)=0.03microM), respectively, with comparable/better activities than Methotrexate (MTX). Docking calculations of the complexes of hDHFR with the most active compounds identified the binding mode of the described molecules with respect to MTX.

Optimizing cell permeation of an antibiotic resistance inhibitor for improved efficacy.

Benzo[b]thiophene-2-ylboronic acid, 1, is a 27 nM inhibitor of the class C beta-lactamase AmpC and potentiates the activity of beta-lactam antibiotics in bacteria that express this and related enzymes. As is often true, the potency of compound 1 against the enzymes is much attenuated in cell culture against Gram negative bacteria, where the minimum inhibitor concentration of compound 1 is in the mid-micromolar range. Here, we modulated the properties of this lead to enhance its ability to cross the membrane, using a combination of X-ray crystallography, structure-based design, and application of physical models of outer membrane crossing. This strategy led us to derivatives with substantially improved permeability. Also, the greater solubility of these compounds allowed us to measure their efficacy at higher concentrations than with the lead 1, leading to higher maximum potentiation of the antibiotic effect of ceftazidime on resistant bacteria.

New thymidylate synthase inhibitors induce apoptosis in melanoma cell lines.

Malignant melanoma is particularly resistant to conventional chemotherapy and radiotherapy. For this reason in the past years a huge variety of new compounds has been developed with potential chemotherapeutic activity which needs to be tested in vitro and in vivo. We investigated the in vitro action of three new experimental antifolate substances (MR7, MR21 and MR36) with a critical target for thymidylate synthase (TS), an essential enzyme for DNA synthesis. The response of two melanoma cell lines (SK-MEL-2 derived from malignant melanoma metastasis and SK-MEL-28 derived from primary malignant melanoma) was examined after treatment with these substances. The antifolate agents induced apoptosis in SK-MEL-2 and SK-MEL-28 cells as confirmed by the TUNEL technique and Comet Assay. Western-blot analysis showed a down-regulation of Bcl-2 protein level and PARP cleavage, otherwise p53 and Bax expressions were not modulated. Moreover, these antifolate-induced apoptosis was accompanied by both pro-caspase-9 and -8 activations. These results were supported by the use of the pan-caspases inhibitor Z-VAD-FMK that almost completely decreased the amount of apoptosis in both the melanoma cell lines treated with antifolate. In conclusion our results show that TS inhibitors are able to induce apoptosis through a caspase-mediated pathway, but without the involvement of the p53/Bax signalling.

Interação social no autismo em ambientes digitais de aprendizagem / Social interaction in austim in learning virtual environment

O objetivo deste artigo é discutir sobre o desenvolvimento da interação social em ambientes digitais de aprendizagem, com sujeitos com Autismo. A revisão de literatura baseia-se na Teoria Sócio-Histórica e em teóricos do Autismo. Para tanto, parte-se de uma discussão mais ampla sobre Interação Social e seu desenvolvimento, assim como as características apresentadas no autismo, considerando a intencionalidade de comunicação como um dos principais construtos teóricos para análise da interação social de pessoas com autismo em ambientes digitais. Finalmente, apresentamos e discutimos dados coletados num estudo de caso com quatro sujeitos com diferentes níveis da síndrome que foi realizado ao longo de dois anos de pesquisa utilizando ambientes digitais diversos, assim como os resultados alcançados no estudo.(AU)

The goal of this paper is to discuss the development of social interactions in virtual environments, specially considering the autism. The literature review is based on Sociohistorical Theory and main theoretical works about this condition. We start from a wide discussion about social interaction, its development and relation to autism characteristics, considering that the concept of intentional communication is fundamental to the analysis of social interaction of autistic subjects in virtual environments. Furthermore, we present and discuss the data obtained from a case of study carried out with four subjects that suffer from different levels of autism. The study was developed as a two years of research applying several virtual environments. We also discuss the main results of this research.(AU)