PRO-Simat: Protein network simulation and design tool.

PRO-Simat is a simulation tool for analysing protein interaction networks, their dynamic change and pathway engineering. It provides GO enrichment, KEGG pathway analyses, and network visualisation from an integrated database of more than 8 million protein-protein interactions across 32 model organisms and the human proteome. We integrated dynamical network simulation using the Jimena framework, which quickly and efficiently simulates Boolean genetic regulatory networks. It enables simulation outputs with in-depth analysis of the type, strength, duration and pathway of the protein interactions on the website. Furthermore, the user can efficiently edit and analyse the effect of network modifications and engineering experiments. In case studies, applications of PRO-Simat are demonstrated: (i) understanding mutually exclusive differentiation pathways in Bacillus subtilis, (ii) making Vaccinia virus oncolytic by switching on its viral replication mainly in cancer cells and triggering cancer cell apoptosis and (iii) optogenetic control of nucleotide processing protein networks to operate DNA storage. Multilevel communication between components is critical for efficient network switching, as demonstrated by a general census on prokaryotic and eukaryotic networks and comparing design with synthetic networks using PRO-Simat. The tool is available at https://prosimat.heinzelab.de/ as a web-based query server.

Controlling Supramolecular Structures of Drugs by Light.

Controlling physicochemical properties of light-unresponsive drugs, by light, prima facie, a paradox approach. We expanded light control by ion pairing light-unresponsive salicylate or ibuprofen to photoswitchable azobenzene counterions, thereby reversibly controlling supramolecular structures, hence the drugs' physicochemical and kinetic properties. The resulting ion pairs photoliquefied into room-temperature ionic liquids under ultraviolet light. Aqueous solutions showed trans-cis-dependent supramolecular structures under a light with wormlike aggregates decomposing into small micelles and vice versa. Light control allowed for permeation through membranes of cis-ibuprofen ion pairs within 12 h in contrast to the trans ion pairs requiring 72 h. In conclusion, azobenzene ion-pairing expands light control of physicochemical and kinetic properties to otherwise light-unresponsive drugs.

Bioinspired Ion Pairs Transforming Papaverine into a Protic Ionic Liquid and Salts.

Microbial, mammalian, and plant cells produce and contain secondary metabolites, which typically are soluble in water to prevent cell damage by crystallization. The formation of ion pairs, for example, with carboxylic acids or mineral acids, is a natural blueprint to maintain basic metabolites in solution. Here, we aim at showing whether the mostly large carboxylates form soluble protic ionic liquids (PILs) with the basic natural product papaverine resulting in enhanced aqueous solubility. The obtained PILs were characterized by 1H-15N HMBC nuclear magnetic resonance (NMR) and in the solid state using X-ray powder diffraction, differential scanning calorimetry, and dissolution measurements. Furthermore, their supramolecular pattern in aqueous solution was studied by means of potentiometric and photometrical solubility, NMR aggregation assay, dynamic light scattering, zeta potential, and viscosity measurements. Thereby, we identified the naturally occurring carboxylic acids, citric acid, malic acid, and tartaric acid, as being appropriate counterions for papaverine and which will facilitate the formation of PILs with their beneficial characteristics, like the improved dissolution rate and enhanced apparent solubility.

Impurity profiling of l-aspartic acid and glycine using high-performance liquid chromatography coupled with charged aerosol and ultraviolet detection.

For the compendial related substances test of l-aspartic acid (Asp) and glycine (Gly), two separate reversed-phase ion-pair high-performance liquid chromatography methods coupled with charged aerosol and ultraviolet detection were developed. Separation of all putative impurities, in particular of the related carboxylic and amino acids, was achieved using volatile perfluorocarboxylic acids as ion-pairing reagents on a polar embedded C18 stationary phase. It was shown that an adjustment of the evaporation temperature of the charged aerosol detector (CAD) was an efficient strategy for meeting the required quantitation limits, when dealing with non-volatile analytes. It was also demonstrated that the usage of a two detector setup can be beneficial for extending the detection range and providing accurate quantitation of low level impurities (LOQs from 5 to 50 ng on column). Both methods were validated with accordance to ICH guideline Q2(R1) assessing specificity, linearity, accuracy, precision, and robustness. Several batches of Asp and Gly were tested for related substances using the developed methods. The purity of each sample was higher than 99.7 %. Coupled charged aerosol and UV detection proved to be a more simple, robust and selective alternative to established derivatization procedures such as the Amino-Acid-Analyser (AAA) for the impurity profiling of amino acids, and should thus be considered for implementation into pharmacopoeial monographs in the future.

Recent applications of the Charged Aerosol Detector for liquid chromatography in drug quality control.

High performance liquid chromatography (HPLC) methods with UV/vis detection are the most widespread analytical procedures in modern pharmaceutical applications, but reach their limitations when it comes to non-chromophore molecules. Hence, instead of using tiresome derivatization procedures, many liquid chromatography methods make use of the so-called aerosol-based universal detectors, namely the evaporative light scattering detector (ELSD), the condensation nucleation light scattering detector (CNLSD) and the charged aerosol detector (CAD). Amongst these, the CAD, being the youngest (introduced in 2005) of these three options, is often described as the most easy-to-use detector and is stated to exhibit sufficient sensitivity and good linearity of signal in a dedicated range of concentration. Therefore, this review sets its focus on the recent applications of the CAD for active pharmaceutical ingredients, excipient analysis as well as botanical applications. Alongside the post-column solvent addition techniques, the new CAD's ability to adjust the evaporation temperature and the possibility to use an integrated power function for signal linearization are reviewed as previously unavailable, new parameters for optimization.

Quantitative structure-property relationship modeling of polar analytes lacking UV chromophores to charged aerosol detector response.

In this study, a quantitative structure-property relationship model was built in order to link molecular descriptors and chromatographic parameters as inputs towards CAD responsiveness. Aminoglycoside antibiotics, sugars, and acetylated amino sugars, which all lack a UV/vis chromophore, were selected as model substances due to their polar nature that represents a challenge in generating a CAD response. Acetone, PFPA, flow rate, data rate, filter constant, SM5_B(s), ATS7s, SpMin1_Bh(v), Mor09e, Mor22e, E1u, R7v+, and VP as the most influential inputs were correlated with the CAD response by virtue of ANN applying a backpropagation learning rule. External validation on previously unseen substances showed that the developed 13-6-3-1 ANN model could be used for CAD response prediction across the examined experimental domain reliably (R2 0.989 and RMSE 0.036). The obtained network was used to reveal CAD response correlations. The impact of organic modifier content and flow rate was in accordance with the theory of the detector's functioning. Additionally, the significance of SpMin1_Bh(v) aided in emphasizing the often neglected surface-dependent CAD character, while the importance of Mor22e as a molecular descriptor accentuated its dependency on the number of electronegative atoms taking part in charging the formed particles. The significance of PFPA demonstrated the possibility of using evaporative chaotropic reagents in CAD response improvement when dealing with highly polar substances that act as kosmotropes. The network was also used in identifying possible interactions between the most significant inputs. A joint effect of PFPA and acetone was shown, representing a good starting point for further investigation with different and, especially, eco-friendly organic solvents and chaotropic agents in the routine application of CAD.

Comparing Human Factors for Augmented Reality Supported Single-User and Collaborative Repair Operations of Industrial Robots.

In order to support the decision-making process of industry on how to implement Augmented Reality (AR) in production, this article wants to provide guidance through a set of comparative user studies. The results are obtained from the feedback of 160 participants who performed the same repair task on a switch cabinet of an industrial robot. The studies compare several AR instruction applications on different display devices (head-mounted display, handheld tablet PC and projection-based spatial AR) with baseline conditions (paper instructions and phone support), both in a single-user and a collaborative setting. Next to insights on the performance of the individual device types for the single mode operation, the study is able to show significant indications on AR techniques are being especially helpful in a collaborative setting.

Influence of charged aerosol detector instrument settings on the ultra-high-performance liquid chromatography analysis of fatty acids in polysorbate 80.

The analysis of polysorbate 80 is a challenge because all components lack a chromophore. Here, an ultra-high-performance liquid chromatography system equipped with a charged aerosol detector (UHPLC-CAD) was used to study the effect of systematic variation of the CAD settings, namely evaporation temperature, filter constant and power function value (PFV), on the detector response of fatty acid standards and manufacturing batches of polysorbate. Evaporation temperature and filter constant strongly affect the detection limits described by signal-to-noise (S/N) ratios. Although evaporation temperature can be increased to improve signal to noise ratios, analyte volatility at higher temperatures is an important limiting factor. The PFV was found to be a strong tool for optimizing response linearity, but the optimal PFV differed depending on analyte volatility. Because PFV optimization required some additional measurement time and because double-logarithmic transformation at the default PFV of 1.0 yielded satisfying universal results with less measurement time over a range of two orders of magnitude for every homologue fatty acid from C14 to C18, use of the log-log transformation is the favored linearization strategy. Possible optimization procedures for semi volatile substances are presented. Overall, this new UHPLC method method offers improved detection limits, as well as time savings of over 75% and eluent savings of more than 40% compared to the previously published HPLC-CAD method for polysorbate analysis.

Impurity profiling of l-asparagine monohydrate by ion pair chromatography applying low wavelength UV detection.

l-asparagine is a non-essential amino acid being used for a variety of pharmaceutical applications. The compound may be produced following synthetic or fermentative pathways leading to the formation of distinct impurities such as organic acids, other amino acids, dipeptides, or cyclic amino acid derivatives. Analysis of the respective analytes is challenging due to the lack of a chromophore, thus the monograph of the European Pharmacopoeia describes a thin layer chromatographic test for detection of other amino acids. Thus, a sensitive and robust liquid chromatographic method was developed and validated applying detection at 210nm for determining the related substances. Separation and quantification of the analytes was achieved on a reversed phase C18 column using a mobile phase composed of a mixture of a phosphate buffer, sodium octanesulfonate, and acetonitrile in an isocratic elution mode. In contrast to the currently used thin layer chromatographic test, the method is capable of separating and quantitatively assessing expected ninhydrin-positive and -negative impurities from synthetic and enzymatic production pathways.

Control of a HexaPOD treatment couch for robot-assisted radiotherapy.

Moving tumors, for example in the vicinity of the lungs, pose a challenging problem in radiotherapy, as healthy tissue should not be irradiated. Apart from gating approaches, one standard method is to irradiate the complete volume within which a tumor moves plus a safety margin containing a considerable volume of healthy tissue. This work deals with a system for tumor motion compensation using the HexaPOD® robotic treatment couch (Medical Intelligence GmbH, Schwabmünchen, Germany). The HexaPOD, carrying the patient during treatment, is instructed to perform translational movements such that the tumor motion, from the beams-eye view of the linear accelerator, is eliminated. The dynamics of the HexaPOD are characterized by time delays, saturations, and other non-linearities that make the design of control a challenging task. The focus of this work lies on two control methods for the HexaPOD that can be used for reference tracking. The first method uses a model predictive controller based on a model gained through system identification methods, and the second method uses a position control scheme useful for reference tracking. We compared the tracking performance of both methods in various experiments with real hardware using ideal reference trajectories, prerecorded patient trajectories, and human volunteers whose breathing motion was compensated by the system.

Selectivity of propeptide-enzyme interaction in cathepsin L-like cysteine proteases.

Cathepsin L-like endopeptidases of the papain family are synthesized as proenzymes. N-terminal proregions are essential for folding and latency of the enzyme unit. While selectivity has been reported for the inhibitory function of papain-family propeptides, there is no systematic investigation of the selectivity of their chaperone-like function to date. The chaperone-like cross-reactivity between the cathepsins S, K, and L were thoroughly quantified in trans-experiments, i.e., with isolated propeptides and mature enzymes, and compared to the inhibitory cross-reactivity. The three endopeptidases have been chosen due to only minimal evolutionary distance and nearly identical X-ray structures of their zymogenes. The intramolecular chaperone function of the proregion was found to be more selective than the inhibitory activity and significant differences were found between the selectivity profiles, underlining the assumption that the inhibitory and the chaperone-like propeptide functions are autonomous. Considering the data published on the intramolecular chaperone-like propeptide function within other protease classes as well, our data suggest that intrinsically structured propeptides are more selective than intrinsically unstructured propeptides, i.e., those adopting tertiary structure elements only in complex with their maternal enzyme.

Tumor tracking and motion compensation with an adaptive tumor tracking system (ATTS): system description and prototype testing.

A novel system for real-time tumor tracking and motion compensation with a robotic HexaPOD treatment couch is described. The approach is based on continuous tracking of the tumor motion in portal images without implanted fiducial markers, using the therapeutic megavoltage beam, and tracking of abdominal breathing motion with optical markers. Based on the two independently acquired data sets the table movements for motion compensation are calculated. The principle of operation of the entire prototype system is detailed first. In the second part the performance of the HexaPOD couch was investigated with a robotic four-dimensional-phantom capable of simulating real patient tumor trajectories in three-dimensional space. The performance and limitations of the HexaPOD table and the control system were characterized in terms of its dynamic behavior. The maximum speed and acceleration of the HexaPOD were 8 mm/s and 34.5 mm/s2 in the lateral direction, and 9.5 mm/s and 29.5 mm/s2 in longitudinal and anterior-posterior direction, respectively. Base line drifts of the mean tumor position of realistic lung tumor trajectories could be fully compensated. For continuous tumor tracking and motion compensation a reduction of tumor motion up to 68% of the original amplitude was achieved. In conclusion, this study demonstrated that it is technically feasible to compensate breathing induced tumor motion in the lung with the adaptive tumor tracking system.

Optimized folding and activation of recombinant procathepsin L and S produced in Escherichia coli.

Large scale production of the recombinant human cathepsins L and S was optimized. Final purity was nearly 100%, yield 65% and 41%, respectively. Cost-effective expression in Escherichia coli, inclusion body purification and solubilization followed modified standard protocols. Most contribution to the remarkable increase in over-all efficiency came from the subsequent steps: folding by dilution, selective HIC-capturing of the folded proenzymes, and auto-activation. The effort to optimize the process parameters for folding and activation was greatly reduced by central composite fractional factorial experimental design, considering curved responses as well as factor interactions. Theoretical and practical features of this powerful tool for experimental design are given. Yield in procathepsin S folding could be further increased by addition of an excess of its own native propeptide with known intramolecular chaperone function. This corroborates literature data on proenzyme folding and is broadly discussed in the light of the lower conformational stability of the prodomain compared to the catalytic unit. Auto-activation kinetics was largely different between the two related proenzymes; from its time course contribution of uni- and bimolecular events in proregion hydrolysis and rate constants were estimated.

Specificity of human cathepsin S determined by processing of peptide substrates and MHC class II-associated invariant chain.

Cathepsin S (CatS) is a lysosomal cysteine protease of the papain family, the members of which possess relatively broad substrate specificities. It has distinct roles in major histocompatibility complex (MHC) class II-associated peptide loading and in antigen processing in both the MHC class I and class II pathways. It may therefore represent a target for interference with antigen presentation, which could be of value in the therapy of (auto)immune diseases. To obtain more detailed information on the specificity of CatS, we mapped its cleavage site preferences at subsites S3-S1' by in vitro processing of a peptide library. Only five amino acid residues at the substrate's P2 position allowed for cleavage by CatS under time-limited conditions. Preferences for groups of amino acid residues were also observed at positions P3, P1 and P1'. Based on these results, we developed highly CatS-sensitive peptides. After processing of MHC class II-associated invariant chain (Ii), a natural protein substrate of CatS, we identified CatS cleavage sites in Ii of which a majority matched the amino acid residue preference data obtained with peptides. These observed cleavage sites in Ii might be of relevance for its in vivo processing by CatS.

The crystal structure of a Cys25 -> Ala mutant of human procathepsin S elucidates enzyme-prosequence interactions.

The crystal structure of the active-site mutant Cys25 --> Ala of glycosylated human procathepsin S is reported. It was determined by molecular replacement and refined to 2.1 Angstrom resolution, with an R-factor of 0.198. The overall structure is very similar to other cathepsin L-like zymogens of the C1A clan. The peptidase unit comprises two globular domains, and a small third domain is formed by the N-terminal part of the prosequence. It is anchored to the prosegment binding loop of the enzyme. Prosegment residues beyond the prodomain dock to the substrate binding cleft in a nonproductive orientation. Structural comparison with published data for mature cathepsin S revealed that procathepsin S residues Phe146, Phe70, and Phe211 adopt different orientations. Being part of the S1' and S2 pockets, they may contribute to the selectivity of ligand binding. Regarding the prosequence, length, orientation and anchoring of helix alpha3p differ from related zymogens, thereby possibly contributing to the specificity of propeptide-enzyme interaction in the papain family. The discussion focuses on the functional importance of the most conserved residues in the prosequence for structural integrity, inhibition and folding assistance, considering scanning mutagenesis data published for procathepsin S and for its isolated propeptide.

Interaction of papain-like cysteine proteases with dipeptide-derived nitriles.

A series of 44 dipeptide nitriles with various amino acids at the P2 position and glycine nitrile at position P1 were prepared and evaluated as inhibitors of cysteine proteinases. With respect to the important contribution of the P2-S2 interaction to the formation of enzyme-inhibitor complexes, it was focused to introduce structural diversity into the P2 side chain. Nonproteinogenic amino acids were introduced, and systematic fluorine, bromine, and phenyl scans for phenylalanine in the P2 position were performed. Moreover, the N-terminal protection was varied. Kinetic investigations were carried out with cathepsin L, S, and K as well as papain. Changes in the backbone structure of the parent N-(tert-butoxycarbonyl)-phenylalanyl-glycine-nitrile (16), such as the introduction of an R-configured amino acid or an azaamino acid into P2 as well as methylation of the P1 nitrogen, resulted in a drastic loss of affinity. Exemplarily, the cyano group of 16 was replaced by an aldehyde or methyl ketone function. Structure-activity relationships were discussed with respect to the substrate specificity of the target enzymes.

Functions of propeptide parts in cysteine proteases.

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at the wrong time and location may be lethal. Two principal mechanisms to control the activity of proteases have been developed during evolution. The first is the co-evolution of endogenous inhibitors, typically occurring in cellular compartments separated from those containing active enzymes. The second is the fact that proteases are synthesized as inactive or less active precursor molecules. They are activated, in some cases, upon an appropriate signal like acidification, Ca(++) -binding or, in other cases, by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize experimental data concerning three functions of propeptides of clan CA family C1 cysteine peptidases (papain family), namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Cysteine peptidases of the CA-C1 family include members from the plant kingdom like papain as well as from the animal kingdom like the lysosomal cathepsins L and B. As it will be shown, the functions are determined by certain structural motifs conserved over millions of years after the evolutionary trails have diverged. The function of propeptides of two other important classes of cysteine peptidases - the calpains, clan CA family C4, and the caspases, clan CD family C 14 - are not considered in this review.

An unfolding/refolding step helps in the crystallization of a poorly soluble protein.

Proteins that are unstable or poorly soluble often elude crystallization. Here, a novel strategy is presented that leads to the crystallization of the isolated N-terminal propeptide of human procathepsin S, a proteinase belonging to the cathepsin L-like endopeptidases of the clan CA1 cysteine peptidases. Being very hydrophobic, the propeptide is extremely poorly soluble in aqueous solvents at neutral pH. Solubility is much better at acidic pH, but the native structure is destroyed under these conditions. A novel approach to the crystallization of this poorly soluble protein is presented in which it is first unfolded in an acidic buffer (pH 4.5) and then mixed with a nearly neutral crystallization buffer (pH 6.75) in which the native conformation should form spontaneously. Crystals were grown at a high concentration of MES (1.14 M) with 10% 2-propanol as precipitant. They belong to a tetragonal space group, with unit-cell parameters a = b = 151.1, c = 75.8 A. Diffraction data to a resolution of 3.5 A were obtained.

Parasite-specific immunomodulatory functions of filarial cystatin.

Cystatins of parasitic nematodes are well-described pathogenicity factors which contribute to downregulation of T-cell proliferation of their hosts and induce anti-inflammatory cytokine responses. We compared the immunomodulatory effects of two cystatins of the filarial nematodes Onchocerca volvulus and Acanthocheilonema viteae with two homologous proteins of the free-living nematode Caenorhabditis elegans. Like filarial cystatins, the C. elegans cystatins (rCysele1 and rCysele2) possessed domains relevant for inhibition of papain-like proteases and were biologically active inhibitors of human cathepsins B, L, and S. However, the inhibition of cathepsin B by C. elegans cystatin was much stronger. C. elegans cystatins lacked a domain involved in inhibition of legumain-like proteases that was present in O. volvulus cystatin. Filarial cystatins suppressed the proliferation of human peripheral blood mononuclear cells (PBMC) and murine spleen cells, while the C. elegans cystatins had this effect to a much lesser extent. Whereas filarial cystatins markedly increased the production of interleukin (IL)-10, C. elegans cystatins increased the production of IL-12 and gamma interferon (IFN-gamma) by human PBMC. The cystatins of both the filariae and C. elegans induced an upregulation of inducible nitric oxide by IFN-gamma-stimulated murine macrophages. These data suggest that filarial cystatins but not the C. elegans cystatins downregulate proliferative responses of host cells due to characteristics which might reflect an adaptation of filariae to their parasitic life style.