Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.

Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma.

BACKGROUND: Immune checkpoint inhibitors targeting programmed cell death 1 (PD1) or its ligand (PD-L1) showed activity in several cancer types. METHODS: We performed immunohistochemistry for CD3, CD8, CD20, HLA-DR, phosphatase and tensin homolog (PTEN), PD-1, and PD-L1 and pyrosequencing for assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status in 135 glioblastoma specimens (117 initial resection, 18 first local recurrence). PD-L1 gene expression was analyzed in 446 cases from The Cancer Genome Atlas. RESULTS: Diffuse/fibrillary PD-L1 expression of variable extent, with or without interspersed epithelioid tumor cells with membranous PD-L1 expression, was observed in 103 of 117 (88.0%) newly diagnosed and 13 of 18 (72.2%) recurrent glioblastoma specimens. Sparse-to-moderate density of tumor-infiltrating lymphocytes (TILs) was found in 85 of 117 (72.6%) specimens (CD3+ 78/117, 66.7%; CD8+ 52/117, 44.4%; CD20+ 27/117, 23.1%; PD1+ 34/117, 29.1%). PD1+ TIL density correlated positively with CD3+ (P < .001), CD8+ (P < .001), CD20+ TIL density (P < .001), and PTEN expression (P = .035). Enrichment of specimens with low PD-L1 gene expression levels was observed in the proneural and G-CIMP glioblastoma subtypes and in specimens with high PD-L1 gene expression in the mesenchymal subtype (P = 5.966e-10). No significant differences in PD-L1 expression or TIL density between initial and recurrent glioblastoma specimens or correlation of PD-L1 expression or TIL density with patient age or outcome were evident. CONCLUSION: TILs and PD-L1 expression are detectable in the majority of glioblastoma samples but are not related to outcome. Because the target is present, a clinical study with specific immune checkpoint inhibitors seems to be warranted in glioblastoma.

Protein pocket and ligand shape comparison and its application in virtual screening.

Understanding molecular recognition is one major requirement for drug discovery and design. Physicochemical and shape complementarity between two binding partners is the driving force during complex formation. In this study, the impact of shape within this process is analyzed. Protein binding pockets and co-crystallized ligands are represented by normalized principal moments of inertia ratios (NPRs). The corresponding descriptor space is triangular, with its corners occupied by spherical, discoid, and elongated shapes. An analysis of a selected set of sc-PDB complexes suggests that pockets and bound ligands avoid spherical shapes, which are, however, prevalent in small unoccupied pockets. Furthermore, a direct shape comparison confirms previous studies that on average only one third of a pocket is filled by its bound ligand, supplemented by a 50 % subpocket coverage. In this study, we found that shape complementary is expressed by low pairwise shape distances in NPR space, short distances between the centers-of-mass, and small deviations in the angle between the first principal ellipsoid axes. Furthermore, it is assessed how different binding pocket parameters are related to bioactivity and binding efficiency of the co-crystallized ligand. In addition, the performance of different shape and size parameters of pockets and ligands is evaluated in a virtual screening scenario performed on four representative targets.

Comparing assays for the measurement of AhR ligand activity

In this thesis (TFG) the results of the comparison of three assays for the measurement of AhR ligand activity are exposed. This study was part of a collaborative project aiming at the characterization of the AhR signaling activities of known naturally occurring compounds to explore the potential of using non-toxic compounds to treat inflammatory diseases via oral administration. The first goal of this project was to find an assay able to measure AhR-activity, so the comparison of different assays has been done in order to find the most convenient one according to the efficiency, sensitivity and precision. Moreover, other elements with operational nature such as price, toxicity of components or ease of use has been considered. From the use of compounds known from the literature to be AhR ligands, three assays have been tested: (1) P450-GloTM CYP1A2 Induction/Inhibition assay, (2) quantitative Polymerase Chain Reaction (qPCR) and (3) DR. CALUX® Bioassay. Moreover, a different experiment using the last assay was performed for the study in vivo of the transport of the compounds tested. The results of the TFG suggested the DR. CALUX® Bioassay as the most promising assay to be used for the screening of samples as AhR-ligands because it is quicker, easier to handle and less expensive than qPCR and more reproducible than the CYP1A2 Induction/Inhibition assay. Moreover, the use of this assay allowed having a first idea of which compounds are uptaken by the epithelial barrier and in with direction the transport happens.

Al-Bīrūnī's Kitāb Sānk and Kitāb Pātanğal:A Historical and Textual Study

The historical pole of this research distinguishes differing historical and cultural contexts in which the scholar al-Bïrûnî evolved. Between the years 973 and 1017, he lived in Khwarezm (Kät and JürjänTya), Ray, and Jürjän. He also dwelt in Kabul and Ghazna, both situated on a passage between Persia and India, and travelled to some parts of early medieval India between the years 1017 and 1030. Evidence pointing to him having made actual direct observations beyond the abode of Islam remains scanty. According to his writings, only five locales emerge as having been visited by him, all situated in today's Afghanistan and Pakistan. When al-BTrunl visited these places, he encountered the society of the Indian Shähis, who followed a form of Brahmanism. Al-Bïrûnï's knowledge of Sanskrit was the result of a long process that lasted at least 30 years (1000-1030). In order to reach the level of Sanskrit that enabled him to translate several works from Sanskrit into Arabic, he needed to work with literate people well-versed in Sanskrit, who may also have had some comprehension of Arabic, and/or Persian. The textual pole of this dissertation examines the question of the relationship between al- Bïrûnï's Arabic Kitab Sank and Kitäb Pätangal - two works related to Sämkhya-Yoga - and their possible Sanskrit sources. A philological survey based on these Arabic translations and on Sämkhya-Yoga Sanskrit literature highlights that al-Bïrûnï's translations, both, are related to the classical phase in the development of these two Indian philosophical systems. Despite the early spread of Yoga and Sämkhya ideas through Sanskrit literature, it seems that between the early 11th and 16th centuries they lost vitality amongst Indian scholars. Therefore, al-Bïrûnï's translation of works related to these specific Indian philosophies in the early 11th century CE deserves attention. The second pole of this study also demonstrates that al-BTrünl's hermeneutics played an important part in his transmission of these two Indian schools of thought, as he highly transformed his source in both form and substance. This dissertation considers the question of the relationship between al-Bïrûnï's Arabic translations and their possible Sanskrit sources from the viewpoint of Translation Studies; which makes it possible to point out potential candidates for being al-Bïrûnï's original Sanskrit sources with some confidence. Overall, the Kitäb Sank and the Kitäb Pätangal represent original works of Sämkhya and Yoga, as viewed and transmitted by a Perso-Muslim scholar, rather than pure translations of Sanskrit work.

Estimating the value of the metal-ligand bond dissociation enthalpy<D> (M-L) for adducts using empirical equations supported by TG data

In this work is presented and tested (for 106 adducts, mainly of the zinc group halides) two empirical equations supported in TG data to estimate the value of the metal-ligand bond dissociation enthalpy for adducts: <D> (M-O) = t i / g if t i < 420 K and <D> (M-O) = (t i / g ) - 7,75 . 10-2 . t i if t i > 420 K. In this empirical equations, t i is the thermodynamic temperature of the beginning of the thermal decomposition of the adduct, as determined by thermogravimetry, andg is a constant factor that is function of the metal halide considered and of the number of ligands, but is not dependant of the ligand itself. To half of the tested adducts the difference between experimental and calculated values was less than 5%. To about 80% of the tested adducts, the difference between the experimental (calorimetric) and the calculated (using the proposed equations) values are less than 15%.

Analysis, improvement, and development of new firmware for the smart citizen kit ambient board

The objective of this project was to develop firmware for both the Arduino-compatible boards of the Smart Citizen initiative, and for the RTX4100 low-power WiFi.

Role of PheE15 gate in ligand entry and nitric oxide detoxification function of Mycobacterium tuberculosis truncated hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

The saccharinate anion: a versatile and fascinating ligand in coordination chemistry

The saccharinate anion, obtained by deprotonation of the N-H moiety of saccharin (o-sulfobenzimide) is a very versatile and polyfunctional ligand in coordination chemistry. In this review the different forms of metal-to-ligand interactions involving this anion and some other coordination peculiarities are briefly discussed on the basis of some selected examples.

Imobilização de enzimas a partir de "kit" comercial: determinação de parâmetros metabólicos em sangue animal empregando multicomutação em fluxo

Automatic flow procedures based on the multicommutation concept, dedicated to the determination of 3-hydroxybutyrate, glucose and cholesterol are proposed. The enzymes were immobilized on glass beads and packed into mini-columns that were coupled to a flow system. Sampling throughputs of 55, 40 and 40 determinations per hour, linear response from 10 to 150, 50 to 600, 25 to 125 mg L-1, detection limits of 1.5, 14 and 4 mg L-1 and relative standard deviations of 1, 2 and 2% for 3-hydroxybutyrate, glucose and cholesterol, respectively, were achieved.

Building complexity in O2-binding copper complexes : site-selective metalation and intermolecular O2-binding at dicopper and heterometallic complexes derived from an unsymmetric ligand

A novel unsymmetric dinucleating ligand (LN3N4) combining a tridentate and a tetradentate binding sites linked through a m-xylyl spacer was synthesized as ligand scaffold for preparing homo- and dimetallic complexes, where the two metal ions are bound in two different coordination environments. Site-selective binding of different metal ions is demonstrated. LN3N4 is able to discriminate between CuI and a complementary metal (M′ = CuI, ZnII, FeII, CuII, or GaIII) so that pure heterodimetallic complexes with a general formula [CuIM′(LN3N4)]n+ are synthesized. Reaction of the dicopper(I) complex [CuI 2(LN3N4)]2+ with O2 leads to the formation of two different copper-dioxygen (Cu2O2) intermolecular species (O and TP) between two copper atoms located in the same site from different complex molecules. Taking advantage of this feature, reaction of the heterodimetallic complexes [CuM′(LN3N4)]n+ with O2 at low temperature is used as a tool to determine the final position of the CuI center in the system because only one of the two Cu2O2 species is formed

Electrocatalytic oxidation of methanol by the [Ru3O(OAc)6(py)2(CH3OH)]3+cluster: improving the metal-ligand electron transfer by accessing the higher oxidation states of a multicentered system

The [Ru3O(Ac)6(py)2(CH3OH)]+ cluster provides an effective electrocatalytic species for the oxidation of methanol under mild conditions. This complex exhibits characteristic electrochemical waves at -1.02, 0.15 and 1.18 V, associated with the Ru3III,II,II/Ru3III,III,II/Ru 3III,III,III /Ru3IV,III,III successive redox couples, respectively. Above 1.7 V, formation of two RuIV centers enhances the 2-electron oxidation of the methanol ligand yielding formaldehyde, in agreement with the theoretical evolution of the HOMO levels as a function of the oxidation states. This work illustrates an important strategy to improve the efficiency of the oxidation catalysis, by using a multicentered redox catalyst and accessing its multiple higher oxidation states.

Preconcentration of copper from natural water samples using ligand-less in situ surfactant-based solid phase extraction prior to FAAS determination

In the present work, a simple and rapid ligand-less, in situ, surfactant-based solid phase extraction for the preconcentration of copper in water samples was developed. In this method, a cationic surfactant (n-dodecyltrimethylammonium bromide) was dissolved in an aqueous sample followed by the addition of an appropriate ion-pairing agent (ClO4-). Due to the interaction between the surfactant and ion-pairing agent, solid particles were formed and subsequently used for the adsorption of Cu(OH)2 and CuI. After centrifugation, the sediment was dissolved in 1.0 mL of 1 mol L-1 HNO3 in ethanol and aspirated directly into the flame atomic absorption spectrometer. In order to obtain the optimum conditions, several parameters affecting the performance of the LL-ISS-SPE, including the volumes of DTAB, KClO4, and KI, pH, and potentially interfering ions, were optimized. It was found that KI and phosphate buffer solution (pH = 9) could extract more than 95% of copper ions. The amount of copper ions in the water samples varied from 3.2 to 4.8 ng mL-1, with relative standard deviations of 98.5%-103%. The determination of copper in water samples was linear over a concentration range of 0.5-200.0 ng mL-1. The limit of detection (3Sb/m) was 0.1 ng mL-1 with an enrichment factor of 38.7. The accuracy of the developed method was verified by the determination of copper in two certified reference materials, producing satisfactory results.

KIT COLORIMÉTRICO PARA DETECÇÃO DE METANOL EM ETANOL COMBUSTÍVEL PARA O MONITORAMENTO DA QUALIDADE DE COMBUSTÍVEIS

A colorimetric kit for methanol detection in ethanol-containing fuels and ease of use in the field was developed and tested. The analysis can detect the presence of methanol in fuels when exceeding specification (0.5% v/v) in about 20 min and its simple instrumentation does not require a specialist. The kit method was successfully validated at gas stations located in São Paulo State and the Federal District.