The identification of new ATAD2 bromodomain inhibitors: the application of combined ligand and structure-based virtual screening

Ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches were used to identify new inhibitors for ATAD2 bromodomain. The LBVS approach was used to search 23,129,083 clean compounds to identify compounds similar to an active compound with reported pIC₅₀ equal to 7.2. Based on LBVS results, 19 compounds were selected. To perform SBVS, by applying nine filters on 23,129,083 clean compounds, 1,057,060 compounds were selected. After performing SBVS on these selected compounds with idock software, 16 compounds with the lowest binding energies were selected. More accurate molecular docking analysis was performed on these 35 selected compounds by using iGEMDOCK software and six of them with the lowest binding energies were selected as hit compounds. These compounds were zinc36647229, zinc77969074, zinc13637358, zinc77971540, zinc12991296 and zinc19374204.     

VSDMIP: virtual screening data management on an integrated platform

A novel software (VSDMIP) for the virtual screening (VS) of chemical libraries integrated within a MySQL relational database is presented. Two main features make VSDMIP clearly distinguishable from other existing computational tools: (i) its database, which stores not only ligand information but also the results from every step in the VS process, and (ii) its modular and pluggable architecture, which allows customization of the VS stages (such as the programs used for conformer generation or docking), through the definition of a detailed workflow employing user-configurable XML files. VSDMIP, therefore, facilitates the storage and retrieval of VS results, easily adapts to the specific requirements of each method and tool used in the experiments, and allows the comparison of different VS methodologies. To validate the usefulness of VSDMIP as an automated tool for carrying out VS several experiments were run on six protein targets (acetylcholinesterase, cyclin-dependent kinase 2, coagulation factor Xa, estrogen receptor alpha, p38 MAP kinase, and neuraminidase) using nine binary (actives/inactive) test sets. The performance of several VS configurations was evaluated by means of enrichment factors and receiver operating characteristic plots. ángel R. Ortiz deceased on May 5, 2008.     

Identification of Novel TRPC5 Inhibitors by Pharmacophore-Based and Structure-Based Approaches

Canonical transient receptor potential-5 (TRPC5), which belongs to the subfamily of transient receptor potential (TRP) channels, is a non-selective cation channel mainly expressed in the central nervous system and shows more restricted expression in the periphery. TRPC5 plays a crucial role in human physiology and pathology, for instance, anxiety, depression, epilepsy, pain, memory and chronic kidney disease (CKD). However, due to lack of the effective and selective inhibitors, its physiological and pathological mechanism remains so far unknown. It is therefore pivotal to identify potential TRPC5 inhibitors. We have applied ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) methods. The pharmacophore models of TRPC5 antagonists generated by using the HypoGen and HipHop algorithms were used as a query model for the screening of potential inhibitors against the Specs database. The resultant hits from LBVS were further screened by SBVS. SBVS was carried out based on the homology model generation of human TRPC5, binding site identification, molecular dynamics optimization and molecular docking studies. In our systematic screening approaches, we have identified 7 hits compounds with comparable dock score after Lipinski and Veber rules, ADMET, PAINS analysis, cluster analysis, and similarity analysis. In conclusion, the current research provides novel backbones for the new-generation of TRPC5 inhibitors.     

Virtual screening methods that complement HTS

In this review, we discuss a number of computational methods that have been developed or adapted for molecule classification and virtual screening (VS) of compound databases. In particular, we focus on approaches that are complementary to high-throughput screening (HTS). The discussion is limited to VS methods that operate at the small molecular level, which is often called ligand-based VS (LBVS), and does not take into account docking algorithms or other structure-based screening tools. We describe areas that greatly benefit from combining virtual and biological screening and discuss computational methods that are most suitable to contribute to the integration of screening technologies. Relevant approaches range from established methods such as clustering or similarity searching to techniques that have only recently been introduced for LBVS applications such as statistical methods or support vector machines. Finally, we discuss a number of representative applications at the interface between VS and HTS.     

Mesophilic Digestion Kinetics of Manure Slurry

Anaerobic digestion kinetics study of cow manure was performed at 35°C in bench-scale gas-lift digesters (3.78 l working volume) at eight different volatile solids (VS) loading rates in the range of 1.11–5.87 g l⁻¹ day⁻¹. The digesters produced methane at the rates of 0.44–1.18 l l⁻¹ day⁻¹, and the methane content of the biogas was found to increase with longer hydraulic retention time (HRT). Based on the experimental observations, the ultimate methane yield and the specific methane productivity were estimated to be 0.42 l CH₄ (g VS loaded)^–1 and 0.45 l CH₄ (g VS consumed)^–1, respectively. Total and dissolved chemical oxygen demand (COD) consumptions were calculated to be 59–17% and 78–43% at 24.4–4.6 days HRTs, respectively. Maximum concentration of volatile fatty acids in the effluent was observed as 0.7 g l^–1 at 4.6 days HRT, while it was below detection limit at HRTs longer than 11 days. The observed methane production rate did not compare well with the predictions of Chen and Hashimoto’s [1] and Hill’s [2] models using their recommended kinetic parameters. However, under the studied experimental conditions, the predictions of Chen and Hashimoto’s [1] model compared better to the observed data than that of Hill’s [2] model. The nonlinear regression analysis of the experimental data was performed using a derived methane production rate model, for a completely mixed anaerobic digester, involving Contois kinetics [3] with endogenous decay. The best fit values for the maximum specific growth rate (μ _m) and dimensionless kinetic parameter (K) were estimated as 0.43 day^–1 and 0.89, respectively. The experimental data were found to be within 95% confidence interval of the prediction of the derived methane production rate model with the sum of residual squared error as 0.02.     

Rationalizing the role of SAR tolerance for ligand-based virtual screening

It is well appreciated that the results of ligand-based virtual screening (LBVS) are much influenced by methodological details, given the generally strong compound class dependence of LBVS methods. It is less well understood to what extent structure-activity relationship (SAR) characteristics might influence the outcome of LBVS. We have assessed the hypothesis that the success of prospective LBVS depends on the SAR tolerance of screening targets, in addition to methodological aspects. In this context, SAR tolerance is rationalized as the ability of a target protein to specifically interact with series of structurally diverse active compounds. In compound data sets, SAR tolerance articulates itself as SAR continuity, i.e., the presence of structurally diverse compounds having similar potency. In order to analyze the role of SAR tolerance for LBVS, activity landscape representations of compounds active against 16 different target proteins were generated for which successful LBVS applications were reported. In all instances, the activity landscapes of known active compounds contained multiple regions of local SAR continuity. When analyzing the location of newly identified LBVS hits and their SAR environments, we found that these hits almost exclusively mapped to regions of distinct local SAR continuity. Taken together, these findings indicate the presence of a close link between SAR tolerance at the target level, SAR continuity at the ligand level, and the probability of LBVS success.     

Extending the kinetic solution of the classic Michaelis–Menten model of enzyme action

The principal aim of studies of enzyme-mediated reactions has been to provide comparative and quantitative information on enzyme-catalyzed reactions under distinct conditions. The classic Michaelis–Menten model (Biochem Zeit 49:333, 1913) for enzyme kinetic has been widely used to determine important parameters involved in enzyme catalysis, particularly the Michaelis–Menten constant (K _M ) and the maximum velocity of reaction (V _max ). Subsequently, a detailed treatment of the mechanisms of enzyme catalysis was undertaken by Briggs–Haldane (Biochem J 19:338, 1925). These authors proposed the steady-state treatment, since its applicability was constrained to this condition. The present work describes an extending solution of the Michaelis–Menten model without the need for such a steady-state restriction. We provide the first analysis of all of the individual reaction constants calculated analytically. Using this approach, it is possible to accurately predict the results under new experimental conditions and to characterize and optimize industrial processes in the fields of chemical and food engineering, pharmaceuticals and biotechnology.     

Characterization of unstable enzyme systems which evolve according to a three-exponential equation

The time course of an enzyme catalyzed reaction is normally followed either by monitoring the instantaneous concentration or velocity of an enzyme species or a product. In many enzyme catalyzed reactions these time variations are multi-exponential. The accurate fit of the relevant curves to obtain the kinetic parameters involved can be difficult using conventional methods (Galvez et al. in J Theor Biol 89:37–44, 1981; Garcia-Canovas et al. in Biochim Biophys Acta 912:417–423, 1987; Tudela et al. in Biochim Biophys Acta 912:408–416, 1987; Teruel et al. in Biochim Biophys Acta 911:256–260, 1987; Garrido del Solo et al. in Biochem J 294:459–464, 1993; Varon et al. in Int J Biochem 25:1889–1895, 1993; Garrido del Solo et al. in An Quim 89:319–324, 1993; Varon et al. in J Mol Catal 83:273–285, 1993; Garrido del Solo et al. in Biochem J 303(Pt 2):435–440, 1994; Garrido del Solo and Varon in An Quim 91:13–18, 1995; Garrido del Solo et al. in Biosystems 38:75–86, 1996; Garrido del Solo et al. in Int J Biochem Cell Biol 28:1371–1379, 1996; Garrido del Solo et al. in Int J Biochem Cell Biol 30:735–743, 1998; Varon et al. in J Mol Catal 59:97–118, 1990). In order to circumvent such difficulties Arribas et al. (J Math Chem 44:379–404, 2008) proposed an evaluation method which is applicable regardless of the complexity of the kinetic equation. This procedure is based on the numerical determination of statistical moments from experimental time progress curves. The fitting of these experimentally obtained moments to the corresponding theoretical symbolic expressions allows, in most cases, all the individual rate constants involved to be evaluated. In this paper we perform a general analysis that can be applied to any unstable enzyme system described by a three-exponential equation and apply it to a substrates induced enzyme inactivation process that is described by this type of equation. To verify the goodness of the method we have simulated time progress curves and applied the suggested procedure to these curves, obtaining kinetic parameters values very close to those used to obtain simulated curves. Finally, we compare our results with those obtained in previous contributions in which other procedures were used.     

Solid-state mechanical properties of crystalline drugs and excipients

Thermal mechanical analysis (TMA) of crystalline drugs and excipients in their pre-melt temperature range performed in this study corroborate their newly found linear dielectric conductivity properties with temperature. TMA of crystalline active pharmacy ingredients (APIs) or excipients shows softening at 30–100 °C below the calorimetric melting phase transition, which is also observed by dielectric analysis (DEA). Acetophenetidin melts at 135 °C as measured calorimetrically by DSC, but softens under a low mechanical stress at 95 °C. At this pre-melting temperature, the crystals collapse under the applied load, and the TMA probe shows rapid displacement. The mechanical properties yield a softening structure and cause a dimensionally slow disintegration resulting in a sharp dimensional change at the melting point. In order to incorporate these findings into a structure–property relationship, several United States Pharmacopeia (USP) melting-point standard drugs were evaluated by TMA, DSC, and DEA, and compared to the USP standard melt temperatures. The USP standard melt temperature for vanillin (80 °C) [1], acetophenetidin (135 °C) [2], and caffeine (235 °C) [3] are easily verified calorimetrically via DSC. The combined thermal analysis techniques allow for a wide variety of the newly discovered physical properties of drugs and excipients.     

Thermal investigation of ‘molten starch’

Detailed thermal analysis studies have shown that a ‘molten starch’ phase is obtained during controlled heating of starch. Before the ‘molten’ stage, depolymerisation of starch produces lower molecular weight compounds like dextrins, oligo, di- and monosaccharides, as well as other types of compounds. These compounds should have ideal properties for plasticizing starches because of the similarities of the molecules, helping lower phase changes in collaboration with molecular weight decrease. Interestingly, it was found previously that these materials only act as adhesives in a narrow temperature range around 523 K (250 °C) (Shuttleworth et al. J Mater Chem 19(45):8589–8593, 2009). Materials were investigated using thermal and mechanical analyses of single lap joints.     

Towards more fair and effective doping tests based on chromatography with mass spectrometric detection

Van Eenoo and Delbeke in Accred Qual Assur (2009) have criticized Faber (in Accred Qual Assur, 2009) for not taking “all factors under consideration when making his claims”. Here, it is detailed that their criticism is based on a misunderstanding of examples that were merely intended to be illustrative. Motivated by this criticism, further discussion is provided that may help in the pursuit of more fair and effective doping tests, here exemplified by chromatography with mass spectrometric detection. Surely, any doping test can only be improved or even optimized if the risks of false positives and false negatives are well defined. This requirement is consistent with a basic principle concerning mathematical approximations (Parlett in “The symmetric eigenvalue problem”, Prentice-Hall, Englewood Cliffs, 1980): apart from just being good, they should be known to be good. Author’s reply to the response on “Regulations in the field of residue and doping analysis...” Papers published in this section do not necessarily reflect the opinion of the Editors, the Editorial Board and the Publisher.     

Silver electrodeposition from water–acetonitrile mixed solvents. Part III—an in situ investigation by optical second harmonic generation spectroscopy

In this work, we report an investigation based on silver electrodeposition from water–acetonitrile mixed solvents onto a polycrystalline Au electrode, based on in situ optical second harmonic generation (SHG) spectroscopy. This paper is the last one of a series attacking the same topic by cyclic voltammetry and potentiostatic current transients (Mele et al., J Solid State Electrochem in press, 1) and in situ surface-enhanced Raman scattering (Mele et al., J Solid State Electrochem in press, 2). SHG intensity transients following the application of potentiostatic cathodic steps have been measured in order to obtain detailed information on the formation of Ag clusters and nuclei during the electrodeposition process. Our SHG data have been rationalised in terms of a simple optical model accounting for SHG enhancement brought about by Ag cluster formation.     

Structure and Reactivity of the <Emphasis Type="BoldItalic">N</Emphasis>-Acetyl-Cysteine Radical Cation and Anion: Does Radical Migration Occur?

The structure and reactivity of the N-acetyl-cysteine radical cation and anion were studied using ion-molecule reactions, infrared multi-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations. The radical cation was generated by first nitrosylating the thiol of N-acetyl-cysteine followed by the homolytic cleavage of the S–NO bond in the gas phase. IRMPD spectroscopy coupled with DFT calculations revealed that for the radical cation the radical migrates from its initial position on the sulfur atom to the α-carbon position, which is 2.5 kJ mol^–1 lower in energy. The radical migration was confirmed by time-resolved ion-molecule reactions. These results are in contrast with our previous study on cysteine methyl ester radical cation (Osburn et al., Chem. Eur. J. 2011 , 17, 873–879) and the study by Sinha et al. for cysteine radical cation (Phys. Chem. Chem. Phys. 2010 , 12, 9794–9800) where the radical was found to stay on the sulfur atom as formed. A similar approach allowed us to form a hydrogen-deficient radical anion of N-acetyl-cysteine, (M – 2H) ^•– . IRMPD studies and ion-molecule reactions performed on the radical anion showed that the radical remains on the sulfur, which is the initial and more stable (by 63.6 kJ mol^–1) position, and does not rearrange.     

Optimization of TRPV6 Calcium Channel Inhibitors Using a 3D Ligand‐Based Virtual Screening Method

Herein, we report the discovery of the first potent and selective inhibitor of TRPV6, a calcium channel overexpressed in breast and prostate cancer, and its use to test the effect of blocking TRPV6‐mediated Ca²⁺‐influx on cell growth. The inhibitor was discovered through a computational method, xLOS, a 3D‐shape and pharmacophore similarity algorithm, a type of ligand‐based virtual screening (LBVS) method described briefly here. Starting with a single weakly active seed molecule, two successive rounds of LBVS followed by optimization by chemical synthesis led to a selective molecule with 0.3 μM inhibition of TRPV6. The ability of xLOS to identify different scaffolds early in LBVS was essential to success. The xLOS method may be generally useful to develop tool compounds for poorly characterized targets.     

Molecular orientation in the Nematic Ordered Cellulose film using polarized FTIR accompanied with a vapor-phase deuteration method

Previously, the authors reported “Nematic Ordered Cellulose (NOC)” that is a well-ordered state of β-1,4-glucan chains without exhibiting typical X-ray diffraction patterns of any cellulose polymorphs (Togawa and Kondo 1999; Kondo et al. 2001; Kondo 2007). The NOC was prepared by stretching water-swollen gel-like films at the draw ratio of 2.0 to provide highly oriented β-1,4-glucan molecular chains of cellulose, which was proved by the high resolution TEM observation. In this paper, a detailed study of the unique ordered state of the NOC was attempted to characterize orientation of the main chains as well as the OH groups of molecules using polarized FTIR accompanied with a vapor-phase deuteration method. The dichroic analysis suggested that the main chains were fairly oriented in the stretching direction whereas the OH groups remained unoriented. The disordered state of the OH groups regardless of the oriented state for the main chain may hinder the oriented crystallization during the preparation of NOC films.     

Initial Experimental Characterization of a New Ultra-High Resolution FTICR Cell with Dynamic Harmonization

A new Fourier transform ion cyclotron resonance (FTICR) cell based on completely new principles of formation of the effective electric potential distribution in Penning type traps, Boldin and Nikolaev (Proceedings of the 58th ASMS Conference, 2010), Boldin and Nikolaev (Rapid Commun Mass Spectrom 25:122–126, 2011) is constructed and tested experimentally. Its operation is based on the concept of electric potential space-averaging via charged particle cyclotron motion. Such an averaging process permits an effective electric force distribution in the entire volume of a cylindrical Penning trap to be equal to its distribution in the field created by hyperbolic electrodes in an ideal Penning trap. The excitation and detection electrodes of this new cell are shaped for generating a quadratic dependence on axial coordinates of an averaged (along cyclotron motion orbit) electric potential at any radius of the cyclotron motion. These electrodes together with the trapping segments form a cylindrical surface like in a conventional cylindrical cell. In excitation mode this cell being elongated behaves almost like an open cylindrical cell of the same length. It is more effective in ion motion harmonization at larger cyclotron radii than a Gabrielse et al.-type (Int J Mass Spectrom Ion Processes 88:319–332, 1989) cylindrical cell with four compensation sections. A mass resolving power of more than twenty millions of reserpine (m/z 609) and more than one million of highly charged BSA molecular ions (m/z 1357) has been obtained in a 7T magnetic field.     

Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva

We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 μm wide with barriers as narrow as 250 μm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318–1320, 1; Martinez et al., Anal Chem 10:3699–3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606–19611, 3; Lu et al., Electrophoresis 9:1497–1500, 4; Abe et al., Anal Chem 18:6928–6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 μM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 μM) were generated. The time of device fabrication to the time of test results was about 25 min.     

Counting minimal reactions with specific conditions in $${\mathbb{R} ^4}$$

We give the sharp lower bound on the number of minimal reactions when no “parallel” species (isomers or multiples) are allowed and all the species are built up from at most four kinds of atoms in Theorem 16. This continues the investigations in Kumar and Pethő (Intern Chem Eng 25:767–769, 1985) through Szalkai and Laflamme (Electr J Comb 5(1), 1998) which results we briefly summarize in the first Section.